Cell-based Binding Assay Research Articles

Molecular mechanism of contactin 2 homophilic interaction

Contactin 2 (CNTN2) is a cell adhesion molecule involved in axon guidance, neuronal migration, and fasciculation. The ectodomains of CNTN1-CNTN6 are composed of six Ig domains (Ig1-Ig6) and four FN domains. Here, we show that CNTN2 forms transient homophilic interactions (KD ∼200nM). Cryo-EM structures of full-length CNTN2 and CNTN2_Ig1-Ig6 reveal a T-shaped homodimer formed by intertwined, parallel monomers. Unexpectedly, the horseshoe-shaped Ig1-Ig4 headpieces extend their Ig2-Ig3 tips outwards on either side of the homodimer, while Ig4, Ig5, Ig6, and the FN domains form a central stalk. Cross-linking mass spectrometry and cell-based binding assays confirm the 3D assembly of the CNTN2 homodimer. The interface mediating homodimer formation differs between CNTNs, as do the homophilic versus heterophilic interaction mechanisms. The CNTN family thus encodes a versatile molecular platform that supports a very diverse portfolio of protein interactions and that can be leveraged to strategically guide neural circuit development.

Characterization of Product Related Variants in Therapeutic Monoclonal Antibodies

The characterization of product-related variants in monoclonal antibodies involves identifying and quantifying the size and charge of variants that can impact the activity, efficacy, and safety of the antibodies. These variants represent distinct molecular forms that may arise from processes such as fragmentation, dimerization, aggregation, or post-translational modifications. The characterization of variants typically involves isolating the relevant species using a semi-preparative scale HPLC and analyzing them using various analytical techniques and biological assays. The commonly used analytical techniques include size-exclusion and ion-exchange chromatography, light scattering, mass spectrometry, capillary isoelectric focusing, and capillary electrophoresis-sodium dodecyl sulfate with ultraviolet or laser-induced fluorescence detection, among others. Additionally, functional assessments are performed using cell-based assays and binding assays to assess the biological activities of the variants. Identifying product-related variants through characterization enables the recognition of impurities that compromise the quality and safety of the drug.

Second generation Al18F-labeled D-amino acid peptide for CXCR4 targeted molecular imaging

BackgroundThe C-X-C chemokine receptor type 4 (CXCR4) is overexpressed in many cancers, e.g. multiple myeloma and acute leukemia, yet solely [68Ga]PentixaFor is used for clinical PET imaging. The aim of this study was to develop and assess a second generation Al18F-labeled D-amino acid peptide based on the viral macrophage inflammatory protein II for CXCR4 targeted molecular imaging. MethodsWe designed a library of monomer and multimer constructs and evaluated their binding affinity for human and mouse CXCR4. Based on these results, we selected the best vector molecule for development of an Al18F-labeled ligand, [18F]AlF-NOTA-2xDV1(c11sc12s), which was further evaluated in a cell-based binding assay to assess its binding properties and specificity for CXCR4. Next, pharmacokinetics and tumor uptake of [18F]AlF-NOTA-2xDV1(c11sc12s) were evaluated in naïve mice and mice with xenografts derived from U87.CXCR4 cells. Finally, we performed an imaging study in a non-human primate to assess the in vivo distribution of this novel radioligand in a species closely related to humans. ResultsThe lead ligand AlF-NOTA-2xDV1(c11sc12s) showed six-fold higher affinity for human CXCR4 compared to Ga-Pentixafor. The corresponding radiotracer was obtained in a good radiochemical yield of 40.1 ± 13.5 % (n = 4) and apparent molar activity of 20.4 ± 3.3 MBq/nmol (n = 4) after optimization. In U87.CD4.CXCR4 cell binding assays, the total bound fraction of [18F]AlF-NOTA-(2×)DV1(c11sc12s) was 32.4 ± 1.8 %. This fraction could be reduced by 82.5 % in the presence of 75 μM AMD3100. In naïve mice, [18F]AlF-NOTA-2xDV1(c11sc12s) accumulated in organs expressing mouse CXCR4, e.g. the liver (SUVmean (mean standardized uptake value) 75 min p.i. 11.7 ± 0.6), which was blockable by co-injecting AMD3100 (5 mg/kg). In U87.CXCR4 xenografted tumor mice, the tumor uptake of [18F]AlF-NOTA-2xDV1(c11sc12s) remained low (SUVmean 0.5 ± 0.1), but was reduced by co-administration of AMD3100. Surprisingly, [18F]AlF-NOTA-2xDV1(c11sc12s) exhibited a similar biodistribution in a non-human primate as in mice indicating off-target binding of [18F]AlF-NOTA-2xDV1(c11sc12s) in liver tissue. We confirmed that [18F]AlF-NOTA-2xDV1(c11sc12s) is taken up by hepatocytes using in vitro studies and that the uptake can be blocked with AMD3100 and rifampicin, a potent organic anion-transporting-polypeptide (OATP)1B1 and OATP1B3 inhibitor. ConclusionThe second generation D-peptide AlF-NOTA-2xDV1(c11sc12s) showed high affinity for human CXCR4 and the corresponding radiotracer was produced in good radiochemical yields. However, [18F]AlF-NOTA-2xDV1(c11sc12s) is not specific for CXCR4 and is also a substrate for OATP1B1 and/or OATP1B3, known to mediate hepatic uptake. Therefore, D-amino acid peptides, based on the viral macrophage inflammatory protein II, are not the prefered vector molecule for the development of CXCR4 targeting molecular imaging tools.

The totality of evidence approach in the development of AVT02 (adalimumab), a biosimilar to Humira.

The development of a biosimilar is based on comparative structural, physicochemical, functional and clinical assessments. The sum of these analyses encompasses the 'totality of evidence', which demonstrates no clinically meaningful differences between the biosimilar and the reference product (RP). Once biosimilarity has been established, provided there is suitable scientific justification, clinical data may be extrapolated to other indications of the RP. AVT02 has been developed as a biosimilar to high-concentration, low-volume Humira (adalimumab), an anti-tumour necrosis factor-alpha monoclonal antibody approved for various chronic inflammatory indications. The totality of evidence for AVT02 is described, supporting its approval as an adalimumab biosimilar for all approved indications globally. Analytical similarity assessments using mass spectrometry methods demonstrated identical amino acid sequences for AVT02 and the RP, with high similarity in terms of primary structure, post-translational modifications and higher-order structural attributes. The mechanism of action was assessed by various cell-based potency assays and binding assays, and the results demonstrated that AVT02 is highly similar to the RP. No clinically meaningful differences in terms of purity, potency and safety were observed, and minor differences in a few physiochemical attributes did not impact the in vitro biologic activity and were not considered clinically relevant. Clinical similarity was demonstrated by comparing the pharmacokinetic, efficacy, safety and immunogenicity profiles of AVT02 with those of the RP. Clinical studies supported similar pharmacokinetic and comparable immunogenicity profiles between AVT02 and the RP in healthy participants and participants with moderate-to-severe chronic plaque psoriasis, with no new safety signals detected. The totality of evidence described demonstrates the biosimilarity of AVT02 to the RP, thereby fulfilling the scientific and regulatory requirements for AVT02 as a high-concentration biosimilar for the treatment of chronic plaque psoriasis and all approved indications of the RP.

Addressing Domain Specificity in the Development of a Cell-Based Binding Assay for the Detection of Neutralizing Antibodies Against a CD47xPD-L1 Bispecific Antibody

PF-07257876 is a bispecific antibody being developed for the treatment of certain advanced or metastatic solid tumors. To support clinical development of PF-07257876, neutralizing antibody (NAb) assays were developed as part of a tiered immunogenicity testing approach. Because PF-07257876 targets both CD47 and PD-L1, determination of domain specificity of a NAb response may provide additional insight relating to PK, efficacy, and safety. Due to limitations of functional cell systems, two cell-based binding assays were developed using electrochemiluminescence to detect domain-specific NAb. While both NAb assays utilized a cell-based binding approach and shared certain requirements, such as sensitivity and tolerance to potentially interfering substances, the development of each assay faced unique challenges. Among the hurdles encountered, achieving drug tolerance while preserving domain specificity for CD47 proved particularly challenging. Consequently, a sample pretreatment procedure to isolate NAb from potentially interfering substances was necessary. The sample pretreatment procedure developed was based on a bead-extraction and acid dissociation (BEAD) approach. However, the use of the standard BEAD approach with whole drug to capture NAb resulted in loss of NAb detection under certain circumstances. Specifically, mock samples containing a mixture of NAb positive controls against both binding domains of the bispecific antibody produced false-negative results in the cell-based binding assay. An adaptation made to the standard BEAD approach restored domain-specific NAb detection, while also contributing to an assay sensitivity of 1 µg/mL in the presence of a clinically relevant drug tolerance level of up to 400 µg/mL.Graphical

N-Alkyl Carbamoylimidazoles as Versatile Synthons for the Synthesis of Urea-Based PSMA Inhibitors.

We describe N-alkyl carbamoylimidazoles as readily available and highly versatile synthons for synthesizing urea-based prostate-specific membrane antigen (PSMA) inhibitors. Urea formation proceeded in high yields (>80%) at room temperature under aqueous conditions. All novel compounds were tested for their PSMA inhibitory potency in a cell-based radiometric binding assay. Compound 17 was identified as a novel high-affinity PSMA inhibitor (IC50 = 0.013 μM) suitable for developing an 18F-labeled radioligand for PET imaging of PSMA in prostate cancer.

Abstract 6357: Generation of a mutant SIRPa fusion protein with highly-improved affinity and favorable safety profile

Abstract Background/Purpose: CD47 has been validated to be expressed on various tumor types including acute myeloid leukemia, myelodysplastic syndrome and other hematologic or solid tumors. As primary ‘don’t eat me’ signal, high expression of CD47 on tumor cells interacts with SIRPα negatively regulating the phagocytosis level. However, poor safety profile as well as insufficient combinatory effect remain to be the issues limiting the clinical outcomes of CD47-targeting molecules. Herein, we used rational design strategies to generate SCR9168 with potent affinity improvement, and maintained favorable safety profiles in different animal species, including cynomolgus monkeys. Procedures: Mutagenesis was introduced to the residues critical for CD47/SIRPα binding interface. Affinity was measured by SPR analysis. In vitro activities were determined by biochemical- or cell-based binding, blocking and antibody-dependent cellular phagocytosis (ADCP) assays. The efficacy in vivo was assessed with the OE19 xenograft model. Pharmacokinetics (PK) and safety were monitored in both mice and cynomolgus monkeys. Results: The affinity of SCR9168 achieves 178 pM for human CD47 which is nearly 50-fold increase in comparison with wild-type SIRPα. Enhanced affinity has also been confirmed in binding to OE19 or DLD1 cell lines, as well as in blocking human SIRPα binding to Raji cells. SCR9168 remains strong binding potency to monkey, mouse or rat CD47 which allows the PK and safety assessment using these animal species. The combined effects of SCR9168 and multiple therapeutic antibodies targeting tumor-associated antigens (TAAs) have been evaluated with ADCP assays using human monocyte-derived macrophages. SCR9168 markedly increases the ratio of phagocytic cells combining with antibodies, such as cetuximab. However, due to the inert function of Fc fragment, no phagocytosis of red blood cells or platelets was caused after the incubation in vitro. In addition, SCR9168 treatment in combination with trastuzumab leads to significantly improved suppression of tumor growth in a dose-dependent manner. Moreover, administration of two doses at day 1 and day 11 in cynomolgus monkeys results in no toxicity events related to SCR9168 based on the data from hematological analysis. Conclusion: SCR9168 demonstrates the best-in-class potential among SIRPα mutein molecules. It elicits improved and dose-dependent efficacy in phagocytosis or tumor suppression combining with therapeutic antibodies, such as trastuzumab or cetuximab. Favorable safety profile with no phagocytosis of RBC or platelets in vitro as well as no hematological toxicity observation in cynomolgus monkeys allows broader dose range exploration in early clinical phase. SCR9168 is currently in development stage and IND enabling will be expected in the end of 2023. Citation Format: Yingying Hu, Yayuan Fu, Lei Liu, Wenjing Li, Liting Xue, Zhiyong Yu, Yun Zhang, Meijuan Gao, Yixin Tan, Fudong Wang, Yadan Wu, Jie Li, Zhenzhen Li, Feng Zhou, Wenqing Yang, Zhuoxiao Cao, Renhong Tang. Generation of a mutant SIRPa fusion protein with highly-improved affinity and favorable safety profile [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 6357.

Characterization of Viloxazine Effects on Cortical Serotonin Neurotransmission at Doses Relevant for ADHD Treatment

AbstractIntroductionMost ADHD treatments are thought to be effective due to augmentation of dopamine (DA) and norepinephrine (NE). Our prior preclinical studies found that the ADHD treatment, viloxazine, may augment serotonin (5-HT) in addition to NE and DA; however, it was unclear if these effects occurred at clinically relevant concentrations. To further understand these potential 5-HT effects, we conducted a series of experiments with two objectives: 1) Can we confirm and better elucidate the previously observed serotonergic effects of viloxazine and determine if they occur at clinically relevant concentrations? 2) Are these effects observed in species with close physiology to humans?MethodsObjective 1: The affinity of viloxazine for human isoforms of 5-HT2A, 5-HT2B, 5-HT2C, and 5-HT7 receptors was assessed via cell-based binding assays. Viloxazine agonism of 5-HT2C and antagonism at 5-HT7 was elucidated with IP1, Ca2+, β-arrestin, internalization, and cAMP assays in cells expressing human receptor isoforms. A microdialysis study was conducted in rats to determine the relationship between viloxazine concentrations in the interstitial fluid (ISF) and changes in NE, DA, 5-HT, and their metabolite concentrations in the prefrontal cortex (PFC). Objective 2: A PET imaging study using a 5-HT2A/2C radioligand agonist, [11C]CIMBI-36, is being conducted in non-human primates (NHPs) to evaluate if viloxazine binds these receptors and/or increases 5-HT release.Animal research was approved by animal care and use committees. Animals were cared for according to international standards.ResultsObjective 1: Cell-based assays to measure viloxazine affinity for NET, 5-HT2B, 5-HT2C, and 5-HT7 found Ki values of 0.14, 0.65, 0.84, 1.90 μM respectively. These values were lower than therapeutically relevant rat ISF concentrations (3.5 ± 1.6 μM) approximating pediatric ADHD patients unbound plasma concentrations (2.1-3.3 μM), indicating receptor recruitment. Binding affinity and functional activity assays found viloxazine had negligible activity for 5-HT2A and SERT at therapeutic concentrations. Viloxazine 5-HT2C agonism activated Gq-protein signaling (EC50=1.6 μM, Ca2+ assay), but not β-arrestin or internalization pathways (EC50 values >150 μM). Viloxazine 5-HT7 antagonism decreased Gs-protein signaling (IC50 =6.7 μM). The microdialysis study found that at therapeutically relevant ISF concentrations, 5-HT levels were significantly increased over baseline; no changes were seen in the 5-HIAA metabolite, indicating 5-HT increase is not due to 5-HT reuptake inhibition. Objective 2: PET imaging studies are ongoing.ConclusionsTo date, our experiments to further elucidate the potential 5-HT effects of viloxazine have shown that the previously observed effects of viloxazine on 5-HT receptors and its augmentation of 5-HT in rat PFC occur at clinically relevant concentrations. Further exploration is needed to ascertain if these effects occur in NHPs and are relevant to ADHD.FundingSupernus Pharmaceuticals, Inc.

Sibling rivalry among the ZBTB transcription factor family: homodimers versus heterodimers.

The BTB domain is an oligomerization domain found in over 300 proteins encoded in the human genome. In the family of BTB domain and zinc finger-containing (ZBTB) transcription factors, 49 members share the same protein architecture. The N-terminal BTB domain is structurally conserved among the family members and serves as the dimerization site, whereas the C-terminal zinc finger motifs mediate DNA binding. The available BTB domain structures from this family reveal a natural inclination for homodimerization. In this study, we investigated the potential for heterodimer formation in the cellular environment. We selected five BTB homodimers and four heterodimer structures. We performed cell-based binding assays with fluorescent protein-BTB domain fusions to assess dimer formation. We tested the binding of several BTB pairs, and we were able to confirm the heterodimeric physical interaction between the BTB domains of PATZ1 and PATZ2, previously reported only in an interactome mapping experiment. We also found this pair to be co-expressed in several immune system cell types. Finally, we used the available structures of BTB domain dimers and newly constructed models in extended molecular dynamics simulations (500 ns) to understand the energetic determinants of homo- and heterodimer formation. We conclude that heterodimer formation, although frequently described as less preferred than homodimers, is a possible mechanism to increase the combinatorial specificity of this transcription factor family.

Abstract 5558: Discovery of a novel Claudin 6 X 4-1BB bispecific antibody with potent anti-tumor activity through conditional 4-1BB activation

Abstract Background Claudin 6 (CLDN6) is a tight junction transmembrane protein normally only expressed during embryonic development, but is aberrantly expressed in a variety of tumors, including ovarian cancer, testicular cancer, hepatocellular and lung adenocarcinoma. Here, we generated a CLDN6 X 4-1BB bispecific antibody (BsAb) which could conditionally activate T cells through 4-1BB stimulation upon CLDN6 engagement, positioning it as a potential novel immunotherapy for ovarian cancer and other CLDN6 positive tumors. Methods CLDN6 X 4-1BB BsAb was evaluated for its antigen binding through cell-based binding assay. In vitro function of the antibody was confirmed in a 4-1BB signaling reporter assay and primary human PBMC assay. In vivo efficacy of the antibody was further demonstrated in 4-1BB humanized syngeneic mouse model. Ex vivo analysis of tumor infiltrating and peripheral immune cells was conducted by FACS. Safety parameters such as liver enzymes and pathology upon antibody administration were also assessed. Results CLDN6 X 4-1BB BsAb showed strong binding to several ovarian cancer cell lines with different levels of CLDN6 expression. Functional evaluation of CLDN6X4-1BB BsAb indicated that the activation of 4-1BB signaling was dependent on CLDN6 expression on tumor cells and the activity was stronger than reference 4-1BB monoclonal antibody urelumab. In a humanized 4-1BB mouse model, CLDN6X4-1BB BsAb showed tumor growth inhibition superior to the combination of anti-CLDN6 and anti-4-1BB monospecific antibodies, and the treatment effect was associated with an increase in tumor infiltrating CD45 and CD8 cells as well as CD8/Treg ratio. From the safety perspective, there were no significant changes in liver enzymes or liver histopathology following repeated BsAb administration, suggesting little risks for liver toxicity commonly induced by other 4-1BB agonist antibodies. Conclusions We have successfully generated a novel CLDN6-targeted 4-1BB bispecific antibody which could induce potent 4-1BB stimulation and anti-tumor activity in a CLDN6-dependent manner while minimizing the risk of liver toxicity. Taken together, these data support further development of CLDN6 X 4-1BB bispecific antibody towards IND and clinical trial in 2022. Citation Format: Jian Li, Wenqing Jiang, Yan Liu, Zhengyi Wang, Xi Chen, Taylor B. Guo. Discovery of a novel Claudin 6 X 4-1BB bispecific antibody with potent anti-tumor activity through conditional 4-1BB activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5558.

Abstract 6328: GPR56 as a therapeutic target for the development of antibody-drug conjugates for the treatment of colorectal cancer

Abstract Cancer stem cell (CSC) marker LGR5 is highly expressed in colorectal cancer (CRC) and has been considered as a therapeutic target for the treatment of CRC. Previously, we and others showed that CRC tumors were eliminated by treatment with LGR5-targeted antibody-drug conjugates (ADCs). However, a fraction of tumors eventually recurred, likely due to CSC plasticity or the ability of LGR5-positive CSCs to interconvert into more drug resistant LGR5-negative cancer cells. Through our efforts to identify underlying mechanisms of drug resistance and plasticity and develop therapies to eliminate CRC, we showed that GPR56 expression is increased with loss of LGR5 protein expression. GPR56 is a member of the adhesion G-protein coupled receptor (GPCR) family which mediates diverse functions, including cell adhesion and tumorigenesis. GPR56 is also upregulated in CRC and increased expression levels correlate with poor patient survival. We recently showed GPR56 can promote tumor growth and drug resistance of CRC cells through upregulation of multidrug resistance proteins. These findings identify GPR56 as a potential therapeutic target for the treatment of CRC. Presently, we are focused on the development and characterization of anti-GPR56 ADCs that selectively bind GPR56-high CRC cells to deliver potent cytotoxic drugs and destroy them. We reported the generation of unique anti-GPR56 monoclonal antibodies (mAbs) and are currently developing and optimizing GPR56-targeted ADCs conjugated with different drugs. Cell-based binding assays show that our anti-GPR56 mAb binds with high affinity and specificity to 293T cells overexpressing recombinant GPR56 or GPR56-high CRC cells, with no detectable binding to GPR56-negative cells. Using cytotoxicity assays, we evaluated the in vitro efficacy of anti-GPR56 ADCs against several CRC lines with different levels of GPR56 expression and showed that the ADC is only effective against CRC cells expressing GPR56. Moreover, we evaluated the therapeutic efficacy of our anti-GPR56-ADC against CRC cell line and patient-derived tumor xenograft models in vivo and demonstrated significant inhibition of tumor growth. Altogether, these findings suggest that novel GPR56-targeted ADCs may be a promising therapeutic strategy to overcome resistance for the improved treatment of CRC. Citation Format: Liezl E. Francisco, Treena Chatterjee, Zhengdong Liang, Sheng Zhang, Kendra Carmon. GPR56 as a therapeutic target for the development of antibody-drug conjugates for the treatment of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6328.

Rapid, sensitive and cost-effective determination of immune checkpoint inhibitor activity using a magnetic bead-based binding assay

Immune checkpoint Inhibitors (ICIs) are effective immunno-therapeutic agents for cancer. Rapid and sensitive determination of the blocking activity of ICIs is important for ICIs development and immunological research. Among various immune checkpoint (IC) binding assays, cell-based binding assays are widely regarded, and the functional ELISA is a convenient alternative. However, these methodologies are limited by time-consuming preparation of cell lines stably expressing IC molecules, or long turnaround time with high cost. In this study, two magnetic bead based binding assays were developed to evaluate activity of ICIs, which was determined by a soluble ligand/bead immobilized receptor based binding assay (sL/bR binding assay) that assessed efficacy to block binding of one soluble IC ligand on its cognate receptor immobilized beads, or by a soluble receptor/bead immobilized ligand based binding assay (sR/bL binding assay) that assessed efficacy to block binding of soluble IC receptor on its cognate ligand immobilized beads. Half maximal inhibitory concentration (IC50) values of ICIs were calculated to determine ICIs activity. The sL/bR binding assay accurately determined the activity of two TIGIT blocking antibodies, since the relative blocking activity of two TIGIT antibodies determined by the sL/bR binding assay established in this study and that by the cell based binding assay were almost identical. In contrast, the sR/bL binding assay showed significantly improved sensitivity to determine activity of two PD-1 blocking antibodies than the sL/bR binding assay that was tested in this study and previous reports. Moreover, both amount of the used recombinant protein of ICI receptor/ligand and turnaround time of the two binding assays were more than 10 times less than those of the functional ELISA. These data indicate that the two magnetic bead based binding assays are sensitive, rapid and cost-effective methods to determine blocking activity of ICIs.

Functional Similarity of Proposed Biosimilar ABP 798 with Rituximab

▪BackgroundRegulatory guidelines governing the development of biosimilars require that proposed biosimilars undergo comprehensive structural and functional characterization prior to the initiation of clinical investigation. ABP 798 is being developed as a biosimilar to rituximab. The reference product is approved in the US for treatment of non-Hodgkin's lymphoma, chronic lymphocytic leukemia, severe rheumatoid arthritis, granulomatosis with polyangiitis, and microscopic polyangiitis. Development of biosimilars is challenging as even small variations in structure have the potential to alter the function of the molecule and thus may impact efficacy, safety, and/or bioavailability. Here we compare ABP 798 and rituximab reference product with respect to binding properties (CD20, C1q, FcRn, and Fcγ receptors), antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and induction of apoptosis. The reference product was sourced from the US (rituximab US) and the EU (rituximab EU).MethodsBinding of ABP 798 and rituximab to the CD20 antigen was characterized using a cell-based CD20 binding assay utilizing the CD20 expressing WIL2-S human B-lymphoblastoid cell line. A direct binding ELISA was used to assess the binding of the Fc domain of ABP 798 to C1q. Binding of the Fc moiety of ABP 798 and rituximab to FcγRIa, FcγRIIa, FcγRIIb, and FcγRIIIa (158V and 158F) were evaluated in AlphaLISAÒ competitive binding assays. Binding to FcRn was evaluated by an AlphaScreenÒ competitive binding assay. ADCC activity was evaluated in a functional cell-based assay, with WIL2-S cells used as target cells and NK92-MI cells, stably transfected with human CD16 (FcγRIIIa [158V]), used as effector cells. CDC activity was evaluated in a functional cell-based assay using the WIL2-S cell line and baby rabbit complement. Induction of apoptosis was assessed by measuring activation of caspase 3/7 in SU-DHL-4 cells, a CD20 expressing human B cell lymphoma cell line.ResultsRelative binding (%) as compared to an ABP 798 reference standard was similar between ABP 798 and rituximab (Table 1).The dose response profiles and relative activity for ADCC and CDC were also similar (ADCC relative cytotoxicity: ABP 798, 88%; rituximab US, 85%; rituximab EU, 86%; CDC relative cytotoxicity: ABP 798, 103%; rituximab US, 102%; rituximab EU, 104%). The dose-response profile for induction of caspase 3/7 was similar between ABP 798 and rituximab as well.ConclusionsThe results presented here demonstrate that ABP 798 is similar to rituximab sourced from both US and EU in terms of biological activity across the range of tested binding and functional assays. The functional assays tested the major and minor mechanisms of action related to clinical efficacy across approved rituximab indications and these results provide a foundation for further clinical development of ABP 798. [Display omitted] DisclosuresMcBride:amgen: Employment; amgen: Other: stockholder. Maher:amgen: Employment; amgen: Other: stockholder. Sweet:amgen: Employment; amgen: Other: stockholder. Foltz:amgen: Employment; amgen: Other: stockholder. Kuhns:amgen: Other: stockholder; amgen: Employment.

O-Fucose and Fringe-Modified Recombinant Notch Peptides As Decoy Molecules to Promote Hematopoietic Progenitor Mobilization

Nearly all autologous hematopoietic cell therapy (HCT) procedures are performed using mobilized hematopoietic progenitor cells (HPCs). Successful outcome is dependent on infusing an adequate number of functionally active HPCs. Mobilization strategy of AMD3100, in combination with G-CSF, increases total CD34+ cells mobilized, and often is used for HPC mobilization in myeloma and non-Hodgkin lymphoma patients. Unfortunately, this approach does not always lead to adequate HPC collection in patients who previously have been exposed to extensive cytotoxic therapy. Furthermore, suboptimal number of HPCs mobilized in some patients may result in slower hematopoietic reconstitution and increased risk of complications during the transplant. Hence developing new mobilization strategy is highly desired for patients who are considered poor-mobilizers. Notch is a signaling molecule important for stem cell self-renewal and fate determination in many tissues including the hematopoietic system. An important feature of Notch is its adhesive nature which was first described by cell aggregation assays in Drosophila studies. We previously reported that hematopoietic stem cell and progenitors (HSPCs) with faulty Notch-ligand interaction display increased cell cycling, decreased adhesion to marrow osteoblastic lineage cells, and enhanced egress from the marrow.Since Notch2 is the major Notch isotype expressed on HSPCs, we showed that prior treatment in mice with Notch2 blocking antibody but not Notch1 blocking antibody sensitizes HSPC to the mobilizing stimuli of G-CSF and AMD3100 with a 3~4 - fold increase in mobilization. We showed that transient Notch2 blockade leads to decreased CXCR4 expression by HSPCs with CXCR4 transcription being directly regulated by the Notch transcriptional protein RBPJ. Notch2 blockade also increases cell cycling of HPCs during mobilization temporarily without affecting hematopoietic stem cell (HSC) homeostasis and self-renewal. Moreover, transient Notch2 blockade leads to greater HSPC homing to the marrow that is accompanied with a faster hematopoietic recovery and a competitive repopulating advantage of HSPCs.Herein, to search for alternative agents to Notch blocking antibody, we evaluate the ability of recombinant Notch peptides as decoy molecules to promote disengagement of Notch2-ligand interaction between HSPCs and niche supporting cells. By using OP9 cell-based binding assay and surface plasmon resonance (SPR) analyses, we showed that recombinant Notch111-13, Notch19-13, Notch18-13, Notch211-13, Notch29-13, and Notch28-13 peptide comprising EGF-like repeats 11-13, 8-13, and 9-13, of Notch1 or Notch2, respectively, all bind to Notch ligand Dll4, Dll1 and Jag2. Because EGF-like repeat 12 (EGF 12) has been shown located within the essential ligand binding domain, and the O -fucose attached to the threonine residue of Notch1 EGF12 acts as a surrogate amino acid to make specific and functional contact to residues of Notch ligand, we generated peptides in CHO cells with O -fucose attached to the threonine residue of EGF12 and further extended by Fringe. We found that binding affinities of the glycosylated peptides further increased compared to those of non-glycosylated peptides. Compared to Notch2 peptides, O -fucose and Fringe-modified Notch111-13 peptide shows the highest biding affinity to Notch ligand Dll4 with dissociation constant (Kd) reaching 34 nM. Similar to Notch2 blocking antibody, the O -fucose and Fringe-modified Notch111-13 peptide effectively outcompetes Notch2-ligand binding and thus blocks the adhesion of marrow HSPCs to the primary osteoblasts or OP9 cells expressing Notch ligands. Further, O -fucose and Fringe-modified Notch111-13 peptide can effectively induce HSPCs emigration from a 3D osteoblastic “spheroid niche”, an in vitro model of HSC niche. Compared to Notch2 blocking antibody, O -fucose and Fringe-modified Notch111-13 peptide caused a further decrease of HSPC cells remained in the “spheroid niche” and more HSPC emigration out of the “niche”.In summary, our findings suggest that recombinant Notch decoy peptides may be considered as alternatives to Notch2 blockade to be combined with the mobilizing regimen of AMD3100 and G-CSF for more effective HSPC mobilization. DisclosuresNo relevant conflicts of interest to declare.

Identification of the Antidepressant Function of the Edible Mushroom Pleurotus eryngii.

Pleurotus eryngii produces various functional molecules that mediate physiological functions in humans. Recently, we observed that P. eryngii produces molecules that have antidepressant functions. An ethanol extract of the fruiting body of P. eryngii was obtained, and the extract was purified by XAD-16 resin using an open column system. The ethanol eluate was separated by HPLC, and the fraction with an antidepressant function was identified. Using LC-MS, the molecular structure of the HPLC fraction with antidepressant function was identified as that of tryptamine, a functional molecule that is a tryptophan derivative. The antidepressant effect was identified from the ethanol extract, XAD-16 column eluate, and HPLC fraction by a serotonin receptor binding assay and a cell-based binding assay. Furthermore, a forced swimming test (FST) showed that the mice treated with purified fractions of P. eryngii exhibited decreased immobility time compared with nontreated mice. From these results, we suggest that the extract of P. eryngii has an antidepressant function and that it may be employed as an antidepressant health supplement.

A multimodal molecular imaging approach targeting urokinase plasminogen activator receptor for the diagnosis, resection and surveillance of urothelial cell carcinoma

With a 5-year recurrence rate of 30–78%, urothelial cell carcinoma (UCC) rates amongst the highest of all solid malignancies. Consequently, after transurethral resection, patients are subjugated to life-long endoscopic surveillance. A multimodal near-infrared (NIR) fluorescence-based imaging strategy can improve diagnosis, resection and surveillance, hence increasing quality of life. MethodsExpression of urokinase plasminogen activator receptor (uPAR) and epithelial cell adhesion molecule (EpCAM) are determined on paraffin-embedded human UCC using immunohistochemistry and on UCC cell lines by flow cytometry. MNPR-101, a humanised monoclonal antibody targeting uPAR is conjugated to IRDye800CW and binding is validated in vitro using surface plasmon resonance and cell-based binding assays. In vivo NIR fluorescence and photoacoustic three-dimensional (3D) imaging are performed with subcutaneously growing human UM-UC-3luc2 cells in BALB/c-nude mice. The translational potential is confirmed in a metastasising UM-UC-3luc2 orthotopic mouse model. Infliximab-IRDye800CW and rituximab-IRDye800CW are used as controls. ResultsUCCs show prominent uPAR expression at the tumour-stroma interface and EpCAM on epithelial cells. uPAR and EpCAM are expressed by 6/7 and 4/7 UCC cell lines, respectively. In vitro, MNPR-101-IRDye800CW has a picomolar affinity for domain 2-3 of uPAR. In vivo fluorescence imaging with MNPR-101-IRDye800CW, specifically delineates both subcutaneous and orthotopic tumours with tumour-to-background ratios reaching as high as 6.8, differing significantly from controls (p < 0.0001). Photoacoustic 3D in depth imaging confirms the homogenous distribution of MNPR-101-IRDye800CW through the tumour. ConclusionsMNPR-101-IRDye800CW is suitable for multimodal imaging of UCC, awaiting clinical translation.

Identification of Mutated Peptides in Bladder Cancer From Exomic Sequencing Data Reveals Negative Correlation Between Mutation-Specific Immunoreactivity and Inflammation.

Introduction and ObjectiveNeoantigen-based immunotherapy is one of the breakthroughs in cancer immunotherapy. Benefit from the Cancer Genome Atlas database, we intended to identify mutant peptides with neoantigen property in bladder cancer (BC). Correlations between the immunoreactivity of candidate neoantigens and clinical manifestations were further analyzed.MethodsHLA-A*02:01 restricted mutant (MT) and wildtype (WT) peptides were predicted by using whole exome sequencing data of 412 BC patients in the TCGA database. Binding affinity to HLA-A2 molecules was determined by using T2 cell-based binding assay. The immunoreactivity to WT and MT peptides in HLA-A2+ BC patients was determined by using an ELISPOT assay upon in vitro stimulation with MT and WT peptides individually. Clinical relevance to peptide-specific immunoreactivity was analyzed by Pearson correlation analysis. The disease free survival (DFS) curves were plotted using the Kaplan–Meier method in BC patients with or without mutations and compared using the log-rank test online.ResultsFifty-seven HLA-A*02:01 restricted WT and MT peptides were selected based on predicted high affinity and expression frequency, among which 12 MT peptides from 12 individual genes exhibited strong affinity to HLA-A2 molecules when compared to WT counterparts. MT peptides induced more peptide-specific IFNγ spot forming units (SFUs) than WT counterparts in HLA-A2+ BC patients upon in vitro stimulation. They were negatively correlated to the counts of peripheral leukocytes and platelets. Patients with higher C-reactive protein level exhibited lower immunoreactivity to MT peptides. Combination of MT peptides from 6 genes, including CDKN1AG61V, RHOBP75L, DDB1S25L, AHNAKD4855Y, ANP32AS56L and MKI67H84L covered 47.5% of the patients under investigation. Patients harboring combinational mutations in these genes were associated with a longer DFS according to the cBioportal online analysis.ConclusionTwelve HLA-A*02:01 restricted MT peptides have been identified exhibiting higher binding affinity to HLA-A2 molecules and stronger immunoreactivity than WT counterparts in BC patients. Combination of MT peptides from six genes might be potential as neoantigen candidates in cancer immunotherapy against BC in the future. Inflammatory modulation is inclined to be a strategy to enhance the efficacy of neoantigen-based immunotherapy.

Abstract 5644: Claudin 18.2 X 4-1BB bispecific antibody induced potent tumor inhibition through tumor-specific 4-1BB activation

Abstract Background Claudin 18.2 (CLDN18.2) is a gastric-specific membrane protein. In the healthy tissue, CLDN18.2 expression is restricted to the short-lived differentiated cells of gastric mucosa as a component of tight junction with limited accessibility of antibody treatment. However, it was ectopically expressed at significant levels in a variety of primary lesion and metastases of epithelial tumor entities, including gastric, pancreatic, esophageal, and lung adenocarcinoma cells. Taken together, CLDN18.2 was believed to be an ideal tumor antigen for immunotherapy. Here, we generated and described a bispecific antibody TJ-CD4B which could redirect and activate T cells to CLDN18.2 positive tumor cells by engaging 4-1BB antigen, thus positioned as a potential novel therapy for gastric cancer and other CLDN18.2 positive tumors. Methods TJ-CD4B was evaluated for its antigen binding through protein-based binding and cell based binding assay. The cell-based function of TJ-CD4B was further evaluated in a 4-1BB signaling reporter assay and primary human T cell assay. The in vivo efficacy was validated in 4-1BB humanized C57BL/6 mice transferred with colon cancer cell line MC38 overexpressed with human CLDN18.2. Percentage of tumor infiltrating and peripheral CD8+ T cells was evaluated by FACS analysis. Results TJ-CD4B showed stronger binding capability to CLDN18.2 when compared with benchmark monoclonal antibody against CLDN18.2 (IMAB362). Functional evaluation of TJ-CD4B indicated the activation of 4-1BB signaling was solely dependent on CLDN18.2 expression on the cell. Furthermore, TJ-CD4B showed superior 4-1BB activity than benchmark 4-1BB monoclonal antibody (Urelumab) in the presence of cells expressing a wide range of CLDN18.2 level including CLDN18.2-low cells. Using a humanized 4-1BB mouse model, TJ-CD4B showed strong tumor growth inhibition (TGI) of CLDN18.2 expressing tumor cells. Meanwhile, we also observed TJ-CD4B increased tumor infiltrating lymphocytes while it had no impact on peripheral lymphocytes. Drug developability assessment indicated that TJ-CD4B was a reasonably developable molecule to be taken forward. Conclusions Our data showed that CLDN18.2-targeted 4-1BB bispecific antibody, TJ-CD4B, displayed potent anti-tumor activity only in tumor microenvironment, possibly minimizing the risk of peripheral toxicity. These compelling data supported the further development of TJ-CD4B, which aims to enter clinical phase in early 2021. Citation Format: Wenqing Jiang, Lei Fang, Zhengyi Wang, Taylor B. Guo, Eunyoung Park, Eunsil Sung, Jaeho Jung. Claudin 18.2 X 4-1BB bispecific antibody induced potent tumor inhibition through tumor-specific 4-1BB activation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5644.

Redesigning HVEM Interface for Selective Binding to LIGHT, BTLA, and CD160

Herpes virus entry mediator (HVEM) regulates positive and negative signals for Tcell activation through co-signaling pathways. Dysfunction of the HVEM co-signaling network is associated with multiple pathologies related to autoimmunity, infectious disease, and cancer, making the associated molecules biologically and therapeutically attractive targets. HVEM interacts with three ligands from two different superfamilies using two different binding interfaces. The engagement with ligands CD160 and B- and T-lymphocyte attenuator (BTLA), members of immunoglobulin superfamily, is associated with inhibitory signals, whereas inflammatory responses are regulated through the interaction with LIGHT from the TNF superfamily. We computationally redesigned the HVEM recognition interfaces using a residue-specific pharmacophore approach, ProtLID, to achieve switchable-binding specificity. In subsequent cell-based binding assays the new interfaces, designed with only single or double mutations, exhibited selective binding to only one or two out of the three cognate ligands.

NanoBRET binding assay for histamine H2 receptor ligands using live recombinant HEK293T cells

Fluorescence/luminescence-based techniques play an increasingly important role in the development of test systems for the characterization of future drug candidates, especially in terms of receptor binding in the field of G protein-coupled receptors (GPCRs). In this article, we present the establishment of a homogeneous live cell-based BRET binding assay for the histamine H2 receptor with different fluorescently labeled squaramide-type compounds synthesized in the course of this study. Py-1-labeled ligand 8 (UR-KAT478) was found to be most suitable in BRET saturation binding experiments with respect to receptor affinity (pKd = 7.35) and signal intensity. Real-time kinetic experiments showed a full association of 8 within approximately 30 min and a slow dissociation of the ligand from the receptor. Investigation of reference compounds in BRET-based competition binding with 8 yielded pKi values in agreement with radioligand binding data. This study shows that the BRET binding assay is a versatile test system for the characterization of putative new ligands at the histamine H2 receptor and represents a valuable fluorescence-based alternative to canonical binding assays.