mAb Targeting Research Articles

LXR inhibitor SR9243-loaded immunoliposomes modulate lipid metabolism and stemness in colorectal cancer cells

Reprogrammed metabolism and active stemness contribute to cancer stem cells' (CSCs) survival and tumorigenesis. LXR signaling regulates the metabolism of different cancers. A selective LXR inhibitor, SR9243 (SR), can target and eradicate non-CSC tumor cells. CD133 is a stem marker in solid tumors-associated CSCs within the active lipogenesis, and anti-CD133 mAb targeting liposomal drug delivery systems expected to increase drug internalization and improve the therapeutic efficacy of poor-in water solubility drugs, e, g., SR. In this study, anti-CD133 mAbs-targeted Immunoliposomes (ILipo) were developed for specific delivery of SR into MACS-enriched CD133 + CSCs and induce their functional effects. Results have shown that ILipo having an average size of 64.79nm can encapsulate SR in maximum proportion, and cell association studies have shown cationic ILipo and targeting CD133 provide the CSCs binding. Also, FCM analysis of RhoB has demonstrated that the ILipo uptake was higher in CD133 + CSCs than in the non-targeted liposomes. ILipo-SR was significantly more toxic in CD133 + CSCs compared to the free SR and non-targeted ones. More efficient than Lipo-SR, ILipo-SR improved the reduction of clonogenicity, stemness, and lipogenesis in CD133 + CSCs in vitro, boosted ROS generation, and induced apoptosis. Our study revealed the dual targeting of CD133 and LXR appears to be a promising strategy for targeting CD133 + CSCs-presenting dynamic metabolism and self-renewal potentials.

Preclinical Evaluation of ON203, A Novel Bioengineered mAb Targeting Oxidized Macrophage Migration Inhibitory Factor as an Anticancer Therapeutic.

High levels of macrophage migration inhibitory factor (MIF) in patients with cancer are associated with poor prognosis. Its redox-dependent conformational isoform, termed oxidized MIF (oxMIF), is a promising tumor target due to its selective occurrence in tumor lesions and at inflammatory sites. A first-generation anti-oxMIF mAb, imalumab, was investigated in clinical trials in patients with advanced solid tumors, where it was well tolerated and showed signs of efficacy. However, imalumab has a short half-life in humans, increased aggregation propensity, and an unfavorable pharmacokinetic profile. Here, we aimed to optimize imalumab by improving its physicochemical characteristics and boosting its effector functions. Point mutations introduced into the variable regions reduced hydrophobicity and the antibodies' aggregation potential, and increased plasma half-life and tumor accumulation in vivo, while retaining affinity and specificity to oxMIF. The introduction of mutations into the Fc region known to increase antibody-dependent cellular cytotoxicity resulted in enhanced effector functions of the novel antibodies in vitro, whereas reduced cytokine release from human peripheral blood mononuclear cells in the absence of target antigen by the engineered anti-oxMIF mAb ON203 versus imalumab reveals a favorable in vitro safety profile. In vivo, ON203 mAb demonstrated superior efficacy over imalumab in both prophylactic and established prostate cancer (PC3) mouse xenograft models. In summary, our data highlight the potential of the second-generation anti-oxMIF mAb ON203 as a promising immunotherapy for patients with solid tumors, warranting clinical evaluation.

A quantitative systems pharmacology model of the pathophysiology and treatment of COVID-19 predicts optimal timing of pharmacological interventions

A quantitative systems pharmacology (QSP) model of the pathogenesis and treatment of SARS-CoV-2 infection can streamline and accelerate the development of novel medicines to treat COVID-19. Simulation of clinical trials allows in silico exploration of the uncertainties of clinical trial design and can rapidly inform their protocols. We previously published a preliminary model of the immune response to SARS-CoV-2 infection. To further our understanding of COVID-19 and treatment, we significantly updated the model by matching a curated dataset spanning viral load and immune responses in plasma and lung. We identified a population of parameter sets to generate heterogeneity in pathophysiology and treatment and tested this model against published reports from interventional SARS-CoV-2 targeting mAb and antiviral trials. Upon generation and selection of a virtual population, we match both the placebo and treated responses in viral load in these trials. We extended the model to predict the rate of hospitalization or death within a population. Via comparison of the in silico predictions with clinical data, we hypothesize that the immune response to virus is log-linear over a wide range of viral load. To validate this approach, we show the model matches a published subgroup analysis, sorted by baseline viral load, of patients treated with neutralizing Abs. By simulating intervention at different time points post infection, the model predicts efficacy is not sensitive to interventions within five days of symptom onset, but efficacy is dramatically reduced if more than five days pass post symptom onset prior to treatment.

Abstract 4263: Anti-Siglec 7 antibody displays potent anti-tumor immunity and demonstrates improved tumor control in combination with anti-PD1 in ovarian cancer

Abstract High grade serous ovarian cancer (OC) and ovarian carcinosarcoma, referred as “Cold” tumors have restricted treatment choices and being associated with high mortality. Current immune checkpoint inhibitors (CPIs) have had modest impact for OC treatment; with anti PD-1/PD-L1 single therapies reported response rates of 4-15%. Accordingly, additional approaches are important for these patients. Natural killer (NK) cells represent an important subset of effector lymphocytes with strategic approaches for maximizing NK reactivity to target OC are likely important. Of interest, an inhibitory receptor and glyco immune checkpoint- Siglec 7 is expressed on 100% of peripheral blood and umbilical cord NK cells. We have generated antibodies against Siglec 7 through an optimized human Siglec 7 DNA/protein immunization approach in humanized next generation transgenic mice. By high throughput flow screening, we identified several Siglec 7 antibodies and down selected relevant clones. A unique clone DB7.2 was highly specific for Siglec 7. We demonstrated the ability of DB7.2 (human IgG1) to activate NK cells and induce OC cell killing using xCELLigence RTCA for in vivo OC challenge studies. DB7.2 (1μg/ml) bound to >90% of NK cells including both CD56dim and CD56bright subsets, evaluated in the PBMCs of multiple donors. It showed strong binding to recombinant Siglec 7 and Siglec 7 transduced HEK293T but not to wild type 293T cells supporting specificity. DB7.2 induced specific killing of multiple OC lines (OVCAR3&10,TOV21G, CaOV3, OVISE, PEO4) carrying different mutations; BRCA1&2, AKT, TP53, PIK3CA, BRAF etc. and resistant against a wide array of cancer drug targets; HSP90, HDAC, MTORC, DNA alkylating agents, EGFR, PARP, PI3K, and WEE1. Tumor killing was indicated to be mediated via enhanced secretion of soluble Fas, perforin, granulysin as well as granzyme A. Of note, OVISE (BRCA1mutated) and PEO4 (BRCA2 mutated, PARPi resistant) were susceptible to DB7.2 killing with EC50- 82.67 and 68.67 nM respectively. A single dose of in vivo expressed DB7.2 significantly reduced the tumor burden in an OVISE challenged humanized mice model enhancing median survival by 57 days. As OCs are highly diverse in nature and likely to require combinatorial approaches for simultaneous targeting of immune pathways; we combined DB7.2 with anti-PD1 for further investigation. In xCELLigence assay, anti-PD1 demonstrated killing of PEO4 cells with EC50 680 nM. The combination of anti PD-1 with DB7.2 showed further enhancement of OC killing in the presence of human PBMCs. This is the first demonstration of the impact of Siglec 7 targeting mAb alone as well as in combination with anti-PD1 (NK and T cell CPIs) studied for targeting OC or any human tumor. These studies have important implications for tumor therapy and provide a novel non T cell CPI potential approach to augment current immune therapy strategies. Citation Format: Devivasha Bordoloi, Abhijeet J. Kulkarni, Opeyemi S. Adeniji, M Betina Pampena, Pratik S. Bhojnagarwala, Shushu Zhao, Elizabeth M. Parzych, Rugang Zhang, Michael R. Betts, Mohamed Abdel-Mohsen, David B. Weiner. Anti-Siglec 7 antibody displays potent anti-tumor immunity and demonstrates improved tumor control in combination with anti-PD1 in ovarian cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4263.

Abstract 6300: Therapeutic potential of EO-3021/SYSA1801, a Claudin18.2 antibody-drug conjugate, for the treatment of CLDN18.2-expressing cancers

Abstract Background: Claudin18.2 (CLDN18.2), a tight junction protein normally expressed only on gastric mucosa, is overexpressed in gastric, pancreatic, esophageal, ovarian, lung and other solid tumors. Unlike in normal tissue, CLDN18.2 is exposed on epithelial surfaces in malignancy. There are no approved targeted therapies for CLDN18.2-expressing cancers. An antibody-drug conjugate (ADC) composed of a monoclonal antibody (mAb) targeting CLDN18.2 with a monomethyl auristatin E (MMAE) payload site-specifically conjugated via a cleavable linker at a drug-antibody ratio (DAR) of 2, EO-3021/SYSA1801, was developed to target CLDN18.2-expressing cancer cells, minimize toxicities and maximize therapeutic index. Methods: HEK293-CLDN18.2 cells were used to evaluate CLDN18.2-dependent EO-3021 endocytosis, MMAE payload release, inhibition of proliferation and antibody dependent cellular cytotoxicity (ADCC). Cell cycle distribution and caspase-3/7 activity were measured 24 hours after treating BxPC3-CLDN18.2 pancreatic cells with EO-3021 or the unconjugated antibody, EO-3021 mAb. Inhibition of proliferation by EO-3021 was measured on cell lines ectopically expressing medium to high CLDN18.2 and on cell lines with endogenous low to medium CLDN18.2 expression. Xenograft studies evaluating tumor growth inhibition by EO-3021, EO3021 mAb, and cisplatin or gemcitabine were done in gastric (NUGC4; NUGC4-CLDN18.2), pancreatic (Patu8988S; BxPC3-CLDN18.2), and lung (NCI-H460) cancer models. Results: EO-3021 binding to cancer cells, endocytosis, MMAE release, and inhibition of proliferation were dependent on CLDN18.2 expression. EO-3021 (EC50: 172 ng/ml) and EO-3021 mAb (EC50: 130 ng/ml) demonstrated similar levels of ADCC. EO-3021 but not EO-3021 mAb promoted G2/M cell cycle arrest and apoptosis and exhibited potent activity across cell lines with low, medium, and high CLDN18.2 expression (IC50: 7-456 ng/mL). EO-3021 induced tumor regressions with a single dose across low, medium, and high CLDN18.2-expressing in vivo models derived from pancreatic (2-10 mg/kg), gastric (0.5-10 mg/kg), and lung cancers (4 mg/kg), respectively. In contrast, standard of care (SOC) chemotherapies and EO-3021 mAb did not induce tumor regressions across in vivo models. Conclusions: EO-3021 demonstrated profound in vivo antitumor activity and outperformed SOC in gastric, pancreatic, and lung cancer models. Results from in vitro and in vivo studies highlight the promising therapeutic potential of EO-3021/SYSA1801 for patients with CLDN18.2-expressing cancers. A Phase 1 study is ongoing to evaluate the safety, tolerability, pharmacokinetics and preliminary anti-tumor activity of SYSA1801 in patients with CLDN18.2 positive advanced solid tumors (NCT05009966). Citation Format: Mo Dan, Xiwu Hui, Yancui Wang, Can Yuan, Thomas O'Hare, Valerie Malyvanh Jansen, Shawn M. Leland, Yang Zhang, David Dornan, Xiaoyan Wang. Therapeutic potential of EO-3021/SYSA1801, a Claudin18.2 antibody-drug conjugate, for the treatment of CLDN18.2-expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6300.

Abstract 465: The preclinical characterization of TST003, a first-in-class mAb targeting gremlin-1 overexpressed by cancer-associated fibroblasts and tumor cells

Abstract Cancer associated fibroblast (CAF) is a rich source of factors for immune suppression and promotes cancer progression and metastasis via epithelial to mesenchymal transition (EMT). It is well known that tumors with mesenchymal phenotype have significantly poor prognosis and reduced response to checkpoint inhibitors. Targeting CAF becomes an increasingly attractive cancer therapeutic approach. Gremlin-1 is a member of the TGFβ superfamily, expressed by CAFs and tumor cells, and known to play a key role in EMT transition, cancer cell proliferation and stroma maintenance. Gremlin-1 overexpression in CAF or tumor cells often correlates with poor clinical prognosis in multiple cancers including prostate, pancreatic, gastric cancer etc. Here we describe the characterization of TST003 (14E3), a novel humanized IgG1 monoclonal antibody targeting Gremlin-1. TST003 bound to human Gremlin-1 with high affinity and high selectivity, and blocked Gremlin-1 binding to BMP2/4 with an EC50 of 3.68 nM to BMP2 and 4.53 nM to BMP4 respectively. TST003 could dose-dependently reverse Gremlin-1 inhibition of the BMP4-mediated Smad1/5/9 phosphorylation in tumor cells as measured by Western blot. In an ex vivo assay using prostate cancer patient-derived organoids (PDOs), TST003 inhibited the growth of 9 out of 12 human PDOs significantly. TST003 also demonstrated its single agent tumor growth inhibition (TGI>50%) at 10 mg/kg in a human prostate cancer PC3 xenograft model on castrated male nude mice and a human colorectal cancer patient derived xenograft (PDX) mouse model in the presence of human PBMC. Both tumor models expressed Gremlin-1 as detected by IHC analysis. In addition, a significantly better anti-tumor activity was observed when TST003 combined with an anti-VEGFR2 antibody in this CRC PDX (MSS, Kras G12D, PD-L1 negative) model. The safety profiles of TST003 were characterized in the single and repeated dose toxicology studies integrated with safety pharmacology, local tolerance, and in vitro hemolysis study, TCR study, and cytokine release study. TST003 was well tolerated in cynomolgus monkeys without noteworthy abnormalities following a single dose or repeated doses. In summary, TST003 is a first-in-class therapeutic mAb targeting Gremlin-1 overexpressed by CAFs and tumor cells. Our preclinical characterization results provided the rationale for on-going clinical evaluation of TST003 in patients with advanced solid tumors with high unmet medical need either as monotherapy or in combination with SoC. Citation Format: Di Sun, Huanhuan Guo, Chaping Cheng, Hongjun Li, Xinlai Yao, Shuang Lu, Jie Ding, Shenjie Zhang, Shijie Zhou, Steven Yu, Yi Gu, Xueming Qian. The preclinical characterization of TST003, a first-in-class mAb targeting gremlin-1 overexpressed by cancer-associated fibroblasts and tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 465.

Abstract 696: Sensitizing breast cancers to immune checkpoint inhibitors through CD27 agonism and vaccination against tumor-associated antigen

Abstract Project objective: Despite the recent revolution in immune checkpoint inhibitors (ICIs), only modest improvement in overall survival and likely caused by not enough potent cellular immunity among BC patients. Our lab has been focus on inducing cellular immunity against HER2+ BC through vaccination against the tumor-associated antigen HER2. Approximately 20 years ago, we performed an experimental pilot study by administrating HER2 peptide and recombinant protein pulsed dendritic cells (DC vaccine) to six patients with refractory HER2+ advanced or metastatic (stage II (≥ 6 +LN), III, or stage IV) BC. We followed the patients on 2019 found that all of the six patients were still alive, 18 years after vaccination. Their blood sample were analyzed with cytometry by time-of-flight (CyTOF) and found there is a significantly increased presence of CD27 expressing memory T cells in response to HER2 peptide stimulation. Recent report on the SARS-CoV2 mRNA vaccine also suggested that CD27 expressing memory T cells plays a critical role in long-lasting cellular immunity against SARS-CoV2 infection. Therefore, we hypothesized that CD27 plays a critical role in cellular immunity against BC, and the stimulation of CD27 expressing T cells with mAb targeting CD27 significantly increase the cellular immunity triggered by vaccination against tumor-associated antigen. Results: We recapitulate the rise of CD27+ antigen specific T cells among the vaccinated patients using a transgenic mouse model expressing human CD27. When combined the adenoviral-vector based HER2 (Ad-HER2) vaccination with a single dose of human aCD27 antibody (Varlilumab), we found there is a robust increase in the HER2 specific T cells compared to vaccination alone, especially CD27+CD44+ memory CD4 T cells, even after 120 days post vaccination. Using an ICI-insensitive syngeneic HER2+ BC models, we found 50% of mice in the combination group of aCD27 antibody plus Ad-HER2 showed total tumor regression by the end of study. When combined with anti-PD1 antibody, the combination of AdHER2 and Varlilumab leads to total tumor regression in 90% of tumor bearing mice with syngeneic HER2+ BC, indicating that the vaccination against tumor associated antigen HER2 plus anti-CD27 antibody sensitized ICI-insensitive HER2+ BC toward ICI. Conclusions: Our data demonstrates that the administration of anti-CD27 antibody significantly increase the long term presence of CD27+ antigen specific memory T cells after vaccination against tumor associated antigen HER2. As consequence, combination of anti-CD27 with HER2 sensitized the immune unresponsive breast cancer toward anti-PD1 antibody. Our study suggests that the vaccination against tumor-associated antigen with mAb targeting CD27 leads to the robust cellular immunity, which is required for successful ICIs against breast cancer. Citation Format: Bin-Jin Hwang, Erika Crosby, Timothy Trotter, Li-Chung Tsao, Tao Wang, Xiao Yang, Herbert Kim Lyerly, Zachary Hartman. Sensitizing breast cancers to immune checkpoint inhibitors through CD27 agonism and vaccination against tumor-associated antigen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 696.

Biochemical separation of Cetuximab-Fab from papain-digested antibody fragments and radiolabeling with 64Cu for potential use in radioimmunotheranostics

Engineered Fab fragments of monoclonal antibodies (mAbs) after radiolabeling with suitable radiometals have the potential to play a key role in personalized radioimmunotheranostics of cancer patients. In this study, we have generated Fab fragment of Cetuximab, a mAb targeting epidermal growth factor receptor (EGFR) expression and purified from the Fc and other fragments by ultrafiltration and affinity chromatography. The Cetuximab-Fab was conjugated with a suitable bifunctional chelator and radiolabeled with no-carrier-added (NCA) 64Cu produced via 64Zn (n, p) 64Cu reaction in a nuclear reactor. The radioimmunoconjugate obtained after size exclusion chromatographic separation possessed >95% radiochemical purity and it retained its integrity over at least three half-lives of the radiometal. Biodistribution studies was performed in fibrosarcoma tumor bearing Swiss mice, which demonstrated the explicit need for purification of the Cetuximab-Fab from Fc fragments. Enhanced and rapid tumor uptake with decent tumor-to-background ratio with prolonged retention was observed when radiolabeled purified Cetuximab-Fab was intravenously administered in animal models. Overall, this preclinical study established the pivotal role of separation science and technology to obtain the radioimmunoconjugate with requisite purity in order to demonstrate optimal pharmacokinetics and maximized treatment efficacy.

SARS-CoV-2 neutralizing antibody epitopes are overlapping and highly mutated which raises the chances of escape variants and requires development of broadly reactive vaccines.

The rapid adaptation of SARS-CoV-2 within the host species and the increased viral transmission triggered the evolution of different SARS-CoV-2 variants. Though numerous monoclonal antibodies (mAbs) have been identified as prophylactic therapy for SARS-CoV-2, the ongoing surge in the number of SARS-CoV-2 infections shows the importance of understanding the mutations in the spike and developing novel vaccine strategies to target all variants. Here, we report the map of experimentally validated 74 SARS-CoV-2 neutralizing mAb binding epitopes of all variants. The majority (87.84%) of the potent neutralizing epitopes are localized to the receptor-binding domain (RBD) and overlap with each other, whereas limited (12.16%) epitopes are found in the N-terminal domain (NTD). Notably, 69 out of 74 mAb targets have at least one mutation at the epitope sites. The potent epitopes found in the RBD show higher mutations (4-10aa) compared to lower or modest neutralizing antibodies, suggesting that these epitopes might co-evolve with the immune pressure. The current study shows the importance of determining the critical mutations at the antibody recognition epitopes, leading to the development of broadly reactive immunogens targeting multiple SARS-CoV-2 variants. Further, vaccines inducing both humoral and cell-mediated immune responses might prevent the escape of SARS-CoV-2 variants from neutralizing antibodies.

Abstract OT1-03-04: Datopotamab deruxtecan (Dato-DXd), a TROP2 antibody-drug conjugate, vs investigators’ choice of chemotherapy in previously-treated, inoperable or metastatic HR+/HER2– breast cancer: TROPION-Breast01

Abstract Background: Chemotherapy is the main treatment in patients with pre-treated endocrine-resistant HR+/HER2– metastatic breast cancer, but has limited efficacy and substantial toxicities. The antibody-drug conjugate Dato-DXd consists of a humanized IgG1 mAb targeting TROP2 attached via a stable cleavable linker to a topoisomerase I (TopI) inhibitor payload. Heavily pre-treated patients with metastatic triple-negative breast cancer in the TROPION-PanTumor01 (NCT03401385) study of Dato-DXd showed a manageable safety profile and highly encouraging objective response rates (ORR by blinded independent central review [BICR]: 34% in all patients; 52% in patients treatment-naïve to TopI inhibitor-based therapies). The metastatic HR+/HER2– breast cancer cohort of TROPION-PanTumor01 has completed enrollment (n=41); data are currently maturing. Trial design: TROPION-Breast01 (NCT05104866) is an ongoing, global, phase 3, open-label, randomized trial evaluating efficacy and safety of Dato-DXd vs investigators’ choice of chemotherapy (ICC) in patients with inoperable or metastatic HR+/HER2– breast cancer. Patients (n≈700) are randomized 1:1 to Dato-DXd 6 mg/kg IV Q3W or ICC (eribulin, capecitabine, vinorelbine, or gemcitabine) until progression. Adults with an ECOG performance status of 0–1, who experienced progression on or are unsuitable for endocrine therapy, and received 1–2 prior lines of standard-of-care chemotherapy in the inoperable or metastatic setting are eligible. Monotherapy treatment with inhibitors of mTOR, PD-[L]1, CDK4/6 and PARP do not count as prior chemotherapy lines. Patients must have ≥1 measurable lesion per RECIST 1.1 and an archival or fresh formalin-fixed and paraffin-embedded tumor sample. Clinically inactive brain metastases are permitted. Dual primary endpoints are progression-free survival (PFS) by BICR, and overall survival. Secondary endpoints include PFS per investigator, ORR, disease control rate, patient-reported outcomes, and Dato-DXd pharmacokinetics and immunogenicity. Exploratory endpoints include TROP2 expression and exposure–efficacy relationship. Patients are stratified by number of prior chemotherapy lines, prior CDK4/6 inhibitor use, and region. At the time of writing 236 patients have been enrolled across 19 countries. Citation Format: Aditya Bardia, Kevin Kalinsky, Junji Tsurutani, Erika Hamilton, Joo Hyuk Sohn, Kyong Hwa Park, Yeon Hee Park, Seock-Ah Im, Keun Seok Lee, Daisy Dastur, Vincent Haddad, Sabrina Khan, Binghe Xu, Barbara Pistilli, Hope Rugo. Datopotamab deruxtecan (Dato-DXd), a TROP2 antibody-drug conjugate, vs investigators’ choice of chemotherapy in previously-treated, inoperable or metastatic HR+/HER2– breast cancer: TROPION-Breast01 [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT1-03-04.

Best Practices in mAb and Soluble Target Assay Selection for Quantitative Modelling and Qualitative Interpretation.

Biologics, especially monoclonal antibodies (mAbs), are an increasingly important part of the drug discovery and development portfolio across the pharmaceutical industry. To enable robust demonstration of pillars 1 and 2 [1] for mAbs, specialised assays are required to measure the complex interactions between mAb and target. This is especially important for the interpretation of soluble target interactions. In some instances, multiple assays with overlapping purposes (e.g., developing both complex and total assays) have been developed. In retrospect, these efforts may have led to excessive time and resources spent in assay development and the generation of data that is contradictory or misleading. Our recommendation is to invest resources early into the development of total assays for both mAb and target. Free target assay data may be inaccurate and report higher levels of free target than are present in the sample at collection due to re-equilibrium during measurement. Total assay formats are inherently less sensitive to the effects of sample preparation, assay conditions, and re-equilibration than free or complex assays. It is acknowledged that pathology/pharmacology is ultimately driven by the free target and knowledge of its dynamics are critical. However, generation of appropriate total target data and using model-based estimation of free target concentrations is a more robust approach than utilisation of direct assay derived estimates. Where free data are utilised, the potential biases should be prospectively considered when developing the assay and utilising the data for quantitative analyses.

Interaction of epitopic missense variants of VEGFA with therapeutic monoclonal antibodies

Vascular Endothelial Growth Factor A (VEGFA) is a glycoprotein that mediates various biological processes, including angiogenesis, vascular permeability, and cellular migration. Aberrant VEGFA signaling is also one of the hallmarks of many types of cancer and has been implicated in various ophthalmological conditions such as diabetic macular edema and age-related macular degeneration. Consequently, a number of therapeutic monoclonal antibodies (mAb) targeting VEGFA have been developed and are widely used to treat these conditions. Bevacizumab (BVZ) and Ranibizumab (RBZ) are two such antibodies that are commercially available and used to treat various cancers and ophthalmological conditions. Nevertheless, a very high rate of non-responsiveness to these mAb treatments has been reported. Therefore, it is important to predict the response to these therapeutic mAb treatments in patients in a personalized approach. This study was aimed at analyzing the impacts of missense variants in the respective VEGFA epitopes for these two therapeutic anti-VEGFA mAbs (BVZ and RBZ) on their interaction with VEGFA through the use of multiple in silico tools. Three missense variants (VEGFAR82W, VEGFAR82Q, and VEGFAG92R) in VEGFA epitopes appear to significantly destabilize VEGFA-BVZ interaction, while only two variants (VEGFAR82W and VEGFAR82Q) affect interaction of VEGFA with RBZ. The VEGFAR82W variant may be pathogenic as well. These missense variants may play roles in the observed heterogeneous response to anti-VEGFA mAb treatments in patients and, therefore, may be used as pharmacogenetic markers for the prediction of responses before administration, and thus for the improvement of therapeutic outcomes.

A novel monoclonal antibody efficiently blocks the infection of duck adenovirus 3 by targeting Fiber-2

Duck adenovirus 3 (DAdV-3), identified as the causative agent of a disease characterized by swelling and hemorrhage of liver and kidney, has caused substantial economic losses to duck industry in China. However, the neutralizing epitopes and the infection mechanism of DAdV-3 have not been extensively elucidated. In this study, a novel monoclonal antibody (mAb) targeting Fiber-2 protein of DAdV-3 was generated and designated as mAb 3E7. Indirect immunofluorescence assay showed that mAb 3E7 specifically reacted with the Fiber-2 in LMH cells transfected with pcDNA3.1-Fiber-2 or infected with DAdV-3. Moreover, mAb 3E7 could immunoprecipitate the Fiber-2 and efficiently inhibit the infection of DAdV-3 in vitro. Further epitope mapping revealed mAb 3E7 recognized the epitope 108LALGDGLE115 in Fiber-2, which was highly conserved among DAdV-3 strains. These findings not only identified a novel neutralizing epitope in Fiber-2, but also paved the way for further elucidating the vital roles of Fiber-2 in the infection and pathogenesis of DAdV-3.

Optimized ACI‐24, an amyloid beta (Abeta) vaccine that safely drives immunity to oligomers and Pyroglutamate Abeta, key pathological species of Alzheimer’s disease (AD)

AbstractBackgroundAmyloid immunotherapy has received recent clinical validation with monoclonal antibodies. Vaccines, such as optimized ACI‐24, are designed to produce a broader, robust antibody response against multiple pathologic forms of Abeta with the added advantage to reduce treatment frequency. Optimized ACI‐24 builds on the initial safety, immunogenicity and pharmacodynamic data of ACI‐24 that has been studied in AD and Down Syndrome‐related AD. Optimized ACI‐24 is formulated with the additional immune cell help via a non‐Abeta, universal T‐helper cell peptide, aiming to insure sustained antibody production, particularly against pathological Abeta species, such as oligomers and truncated pyroglutamate.MethodOptimized ACI‐24 was generated using the SupraAntigen® platform. Mice and non‐human primates were immunized and sera/plasma obtained to measure titers against Abeta1‐42, pyroglutamate (Abeta3‐42) and oligomers. Epitope mapping, immunohistochemistry on different human tissues, including AD brains were performed.ResultImmunization of mice and cynomolgus monkeys with optimized ACI‐24 resulted in potent antigen specific IgG levels. The polyclonal antibody response was observed to mature over time with higher affinity IgGs emerging as well as an increase in titers of IgGs that recognized the highly neurotoxic Abeta species including truncated pyroglutamate and pathological Abeta oligomers. Epitope mapping confirmed the induction of a broader spectrum of antibodies recognizing the N‐terminal containing and the truncated species of pathological Abeta, as compared to the previously clinically tested Abeta vaccines. In addition, target engagement was demonstrated as the immunized monkey sera labeled Abeta plaques in AD patient brain sections while did not show any binding to other human tissues.ConclusionThese preclinical data illustrate that optimized ACI‐24 generates IgGs with a broader N‐terminal Abeta sequence recognition than offered by clinically tested mAbs or Abeta vaccines. This unique binding profile for the pathogenic forms of Abeta, including truncated pyroglutamate and toxic oligomers, is encouraging as it combines the targets of mAbs, such as Aducanumab and Donanemab, recently linked to clinical benefits, and thus, the potential of enhancing plaque reduction. The presented data support the fast progression of optimized ACI‐24 into clinical development as a disease modifying treatment with potential for prevention in AD and Down Syndrome‐related AD.

Human antibody recognition and neutralization mode on the NTD and RBD domains of SARS-CoV-2 spike protein

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). Variants of concern (VOCs) such as Delta and Omicron have developed, which continue to spread the pandemic. It has been reported that these VOCs reduce vaccine efficacy and evade many neutralizing monoclonal antibodies (mAbs) that target the receptor binding domain (RBD) of the glycosylated spike (S) protein, which consists of the S1 and S2 subunits. Therefore, identification of optimal target regions is required to obtain neutralizing antibodies that can counter VOCs. Such regions have not been identified to date. We obtained 2 mAbs, NIBIC-71 and 7G7, using peripheral blood mononuclear cells derived from volunteers who recovered from COVID-19. Both mAbs had neutralizing activity against wild-type SARS-CoV-2 and Delta, but not Omicron. NIBIC-71 binds to the RBD, whereas 7G7 recognizes the N-terminal domain of the S1. In particular, 7G7 inhibited S1/S2 cleavage but not the interaction between the S protein and angiotensin-converting enzyme 2; it suppressed viral entry. Thus, the efficacy of a neutralizing mAb targeting inhibition of S1/2 cleavage was demonstrated. These results suggest that neutralizing mAbs targeting blockade of S1/S2 cleavage are likely to be cross-reactive against various VOCs.

Phase II Trial of Elotuzumab with Pomalidomide and Dexamethasone for Relapsed/Refractory Multiple Myeloma (RRMM) in the Post-Daratumumab Progression Setting

Introduction: Elotuzumab (Elo) is an immunostimulatory (via natural killer [NK] cell activation) monoclonal antibody (mAb) targeting signaling lymphocytic activation molecule F7 (SLAMF7) that has shown activity in combination with the immunomodulatory drugs (IMiDs) lenalidomide (Len) or pomalidomide (Pom) and dexamethasone (Dex), in patients (pts) with RRMM in the ELOQUENT 2 and 3 trials, respectively. However, in ELOQUENT 2, pts were not Len refractory or previously treated with daratumumab (Dara) and in ELOQUENT 3, <5% of patients were previously treated with Dara. The efficacy of Elo-based regimens in Dara-refractory pts needs prospective evaluation to understand optimal sequencing of regimens in RRMM. We report data from a phase 2 trial evaluating the efficacy of Elo-Pom-Dex (EPd) in pts with Dara-refractory RRMM. Methods: This phase 2 trial (NCT03713294) enrolled pts with RRMM who had disease progression and were refractory to Dara. Key eligibility criteria included: measurable disease as per IMWG criteria, at least one prior line of therapy, adequate marrow/organ function (neutrophil count ≥1.0 x 109, platelets ≥50x 109, creatinine clearance ≥30 mL/min), ECOG performance status ≤2, and disease not refractory to Pom. EPd was dosed as per the ELOQUENT 3 study. The trial utilized a two-stage design with an interim analysis to test the null hypothesis that the true overall response rate (ORR) in this patient population is most 20% vs. the alternative hypothesis that it is at least 40%, with 90% power and 9% type I error. Treatment was given until disease progression, intolerance, or patient refusal. Results: 37 pts were enrolled. Median follow-up time was 23.4 months (95% CI: 14.6-37.2). The median age was 70 years (range: 42-84), 12 (32.4%) pts had ISS III disease and 16 (43.2%) pts had high risk cytogenetics. Pts had received a median of 4 (range: 1-9) prior lines of therapy. Prior anti-MM agents included bortezomib (35; 94.6%), Len (34; 91.9%), carfilzomib (9; 24.3%), ixazomib (9; 24.3%), stem cell transplantation (18; 48.6%) and CAR-T cell therapy (1; 2.7%). The majority of pts (33; 89%) were previously exposed to both a proteasome inhibitor and Len. 25 (67.6%) pts got Dara-based therapy as the immediate line of therapy prior to EPd (after a median of 4 prior lines [range 1-7]) whereas 12 (32%) pts got Dara-based therapy ≥1 line prior to EPd (after a median of 6 prior lines [range 2-9]). ORR in all pts was 35.1% (13 pts), with no CRs, 1 (2.7%) VGPR, and 12 (32.4%) PRs. Four pts (10.8%) achieved an MR and 15 (40.5%) achieved SD. The study met its primary endpoint. The ORR in pts exposed to Len was 11/34 (32.4%) compared to 2/3 (66.7%) in patients not exposed to Len. The ORR in pts with Dara as the immediate prior line of therapy was 44% (11/25) compared to 16.7% (2/12) in pts who had received Dara-based therapy ≥1 line prior to EPd (p=0.15). The median PFS for the entire cohort was 3.7 mth (95% CI: 2.9-11.1) (Figure 1A). The median time to best response was 2.9 mth. The median time to next treatment was 8.5 mth (95% CI: 6.8-20.4). Median PFS of pts with Dara as the immediate prior line of therapy was 4.7 mth (95% CI: 3.2-19.6) compared to 2.8 mth (95% CI: 2.0-NA) in patients who had received Dara-based therapy ≥1 line prior to EPd (p=0.03; Figure 1B). The most common grade 3/4 adverse events were neutropenia (57%), anemia (22%), thrombocytopenia (16%), leucopenia (16%), and fatigue (8%). Conclusion: This was a positive study with EPd showing clinically meaningful benefit in pts with Dara-refractory RRMM. The reported PFS of 3.7 mths is less than the 10.3 mths reported in ELOQUENT 3 trial which suggests that Elo-based regimens are less effective in Dara-refractory pts. Interestingly, pts had a significantly superior PFS if they received EPd immediately following progression on Dara, however they were less heavily pretreated comparted to pts who got EPd ≥1 line after Dara failure. Nonetheless, EPd is an effective regimen in the post-Dara setting. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeting C3d in Combination with Daratumumab:a Strategy to Enhance Therapeutic Potential in Multiple Myeloma

The monoclonal antibody (mAb) daratumumab (DARA) targeting CD38 can induce minimal residual disease negative remissions in patients with multiple myeloma (MM). However, majority of patients subsequently relapse or become refractory to treatment (RRMM). Rapid reduction of surface CD38 levels in MM cells following DARA infusion due to trogocytosis by monocytes and granulocytes leads to reduced complement dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC), effector functions that rely on antigen density. High expression of complement regulatory proteins, CD55 and CD59 on MM cell surface can also impede CDC activity. Therefore, modalities to overcome these limitations and improve clinical outcome for patients with RRMM are needed. Daratumumab is a strong activator of complement. While a subset of cells may be lysed by the membrane attack complex, all cells reacted with daratumumab are opsonized by C3d, the terminal component of C3. C3d remains covalently attached at the cell surface. Thus, the newly acquired membrane bound C3d constitutes a target for additional intervention, particularly when the cell surface CD38 is lost or low following treatment with DARA. Using phage display technology, we generated and characterized recombinant rabbit/human chimeric mAbs that selectively bind C3 fragments including C3d but not full length C3. We selected two high affinity anti-C3d mAbs (clones 5S8 and d301 with kD values 3.35 nM and 0.99 nM, respectively) that showed reactivity against both human and mouse C3d. To deposit C3d on myeloma cell lines (MM1-S, MM1-R, RPMI8226, NCI-H929, KMS-12-PE) without activating CDC, the cells were reacted with DARA in C5 depleted normal human serum. Both 5S8 and d301 bound to the C3d opsonized cells. Based on SPR analysis these two mAbs do not compete for C3d binding suggesting that they recognize distinct epitopes. In normal human serum, the anti-C3d mAbs enhanced killing by CDC of DARA-opsonized target cells compared to DARA alone (P<0.0001). The non-targeting IgG1 isotype control mAb trastuzumab (TRA) did not bind C3d or enhance DARA-mediated killing. To test the combination of DARA with anti-C3d mAbs in vivo, we used Balb/c-SCID mice bearing subcutaneous (s. c.) MM1-R tumors. First, using clones 5S8 and d301, we demonstrated in vivo binding of mouse C3d on MM1-R tumor cells harvested 24 h after a single intraperitoneal (i. p.) injection of DARA. Next, we challenged the mice s. c. with MM1-R cells and randomly divided them into the following treatment groups: 20 mg/kg DARA single agent (n=7), or combination treatments of 10 mg/kg of each of two mAbs: DARA + TRA (n=10), DARA + 5S8 (n=13), DARA + d301 (n=13). The mAbs were given via i. p. injections weekly starting on day 7 post tumor challenge and continued for 6 weeks. Palpable tumors were noticed in mice between days 7 and 14, and tumor growth was monitored by caliper measurements twice weekly. Mice were euthanized when tumors reached pre-defined limits. All the mice that received DARA + TRA showed progressive tumor growth with median survival of 38 days and all mice had to be euthanized by day 50. Doubling the dose of DARA to 20mg/kg slightly increased median survival to 43 days (P = 0.12 vs DARA+TRA). Two of 7 mice that received DARA alone (at 20 mg/kg) survived to day 175. Median survival for mice treated with DARA (10mg/kg) plus an anti-C3d mAb was 162 days (P = 0.006 for comparison to DARA 20mg/kg), and 13 of 26 (50%) mice in this cohort survived tumor-free beyond 200 days. There was no statistically significant difference between groups treated with 5S8 or d301 in combination with DARA (P = 0.23). All the surviving mice showed no evidence of tumor, weight loss or other morbidity indicating that they are essentially tumor-free. In summary, these results demonstrate that combination treatment of DARA with anti-C3d mAbs improves efficacy over single agent DARA leading to long-term survival in the MM.1R xenograft model. The combination of C3d targeting mAbs with DARA could improve clinical efficacy and overcome resistance to single agent therapy.

Epitope Mapping of an Anti-Mouse CCR2 Monoclonal Antibody (C2Mab-6) Using Enzyme-Linked Immunosorbent Assay.

CC chemokine receptor type-2 (CCR2) is a member of the G protein-coupled receptors, and is mainly expressed on cell surface of immune cells. CCR2 binds to its ligand, C-C motif chemokine 2 (also named as monocyte chemoattractant protein-1), which involves in the tumor progression by modulating the tumor microenvironment. Therefore, the monoclonal antibody (mAb) targeting CCR2 could be one of the strategies for cancer treatment. In this study, we investigated the critical epitope of C2Mab-6, an anti-mouse CCR2 (mCCR2) mAb developed by N-terminal peptides immunization. We first performed enzyme-linked immunosorbent assay (ELISA) using N-terminal peptides of mCCR2 and demonstrated that C2Mab-6 recognizes 1-19 amino acids of mCCR2. We further performed ELISA using 20 alanine-substituted peptides of mCCR2. C2Mab-6 lost the reaction to the alanine-substituted peptides of D3A, N4A, M6A, P8A, Q9A, and F10A. These results indicate that the binding epitope of C2Mab-6 includes Asp3, Asn4, Met6, Pro8, Gln9, and Phe10 of mCCR2.

Discovery of Aptamers Against Cell Surface Markers Using Ligand-Guided Selection.

Oligonucleotide ligands (DNA, RNA, or XNA), also known as aptamers, are selected against various target molecules using an iterative, evolutionary process called systematic evolution of ligands by exponential enrichment (SELEX). To select aptamers against complex cell surface proteins in their native state, a variant of SELEX termed ligand-guided selection (LIGS) was recently introduced. The significance of LIGS is rooted in its strategy of exploiting the selection step in SELEX to identify highly specific aptamers against known cell surface markers. Thus, in LIGS, a higher-affinity secondary ligand, such as a monoclonal antibody (mAb) to a whole-cell bound to an evolved SELEX library, is introduced to outcompete sequences against the mAb targeting cell surface protein or induce a conformational switch to destabilize the aptamer-surface cell surface protein resulting in elution of the sequences. Here, we describe the detailed method of LIGS utilized in identifying aptamers against T-cell receptor cluster of differentiation three complex (TCR-CD3) expressed in human T-cells and T-cell leukemia.

Preclinical characterization of amubarvimab and romlusevimab, a pair of non-competing neutralizing monoclonal antibody cocktail, against SARS-CoV-2.

Monoclonal antibodies (mAbs) targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein have demonstrated clinical efficacy in preventing or treating coronavirus disease 2019 (COVID-19), resulting in the emergency use authorization (EUA) for several SARS-CoV-2 targeting mAb by regulatory authority. However, the continuous virus evolution requires diverse mAb options to combat variants. Here we describe two fully human mAbs, amubarvimab (BRII-196) and romlusevimab (BRII-198) that bind to non-competing epitopes on the receptor binding domain (RBD) of spike protein and effectively neutralize SARS-CoV-2 variants. A YTE modification was introduced to the fragment crystallizable (Fc) region of both mAbs to prolong serum half-life and reduce effector function. The amubarvimab and romlusevimab combination retained activity against most mutations associated with reduced susceptibility to previously authorized mAbs and against variants containing amino acid substitutions in their epitope regions. Consistently, the combination of amubarvimab and romlusevimab effectively neutralized a wide range of viruses including most variants of concern and interest in vitro. In a Syrian golden hamster model of SARS-CoV-2 infection, animals receiving combination of amubarvimab and romlusevimab either pre- or post-infection demonstrated less weight loss, significantly decreased viral load in the lungs, and reduced lung pathology compared to controls. These preclinical findings support their development as an antibody cocktail therapeutic option against COVID-19 in the clinic.