Biacore Affinity Research Articles

Abstract 6365: TRIM21 is a novel endogenous partner of the inhibitory myeloid checkpoint CLEC-1 involved in tumor antigen cross-presentation

Abstract Myeloid cells represent the most abundant immune component of the tumor microenvironment, where they often assume immunosuppressive roles. Therefore, understanding the mechanisms regulating the immunosuppressive activity of myeloid cells is of major interest in oncology. We have identified CLEC-1, a member of the C-type lectin receptor (CLR) family, as a myeloid cell-driven brake for anti-tumor T cell response in different murine models, including mesothelioma, hepatocellular and colorectal carcinomas. However, the molecular mechanism underlying the immunomodulatory role of CLEC-1 remained to be fully elucidated. Here, using both murine and human cells, we identified and characterized a novel endogenous ligand for CLEC-1, shedding new light on CLEC-1 driven inhibitory myeloid checkpoint activity. First, using single cell RNA sequencing, we confirmed that CLEC-1 is expressed by human myeloid cells, especially by conventional type-one dendritic cells and by tumor-associated macrophages. Using an Fc-fusion human CLEC-1 protein (huCLEC-1) as a bait for potential endogenous ligands, we observed both in human and murine cells that huCLEC-1 binds specifically to stressed or dying cells following programmed necrosis, for instance after X-ray or chemotherapy treatments. CLEC-1 interaction with its ligand expressed by necrotic cells was able to activate the NFAT promoter in a CLEC-1-CD3zeta NFAT reporter cell line in vitro; furthermore, CLEC-1 ligand targeting via Fc-CLEC-1 treatment in vivo extended the overall survival of chemotherapy-treated mc38-colorectal cancer bearing mice, altogether demonstrating the functional relevance of CLEC-1 interaction with its ligand. Immunoprecipitation of proteins bound to huCLEC-1 subsequently enabled the identification of TRIM21, an intra-cellular Fc receptor and E3 ubiquitin ligase as a novel interaction partner for CLEC-1. Direct interaction between recombinant TRIM21 and CLEC-1 human proteins was subsequently confirmed by ELISA and Biacore affinity analysis. TRIM21 was indeed detected at the cell surface of necrotic cells following genotoxic treatments in vitro in several human cancer cell lines and, interestingly, high TRIM21 expression was predictive of worse overall survival in patients with hepatocellular carcinoma, pancreatic cancer, or glioma. Mechanistically, we demonstrated that CLEC-1 genetic deletion in mice enhances the capacity of dendritic cells to cross-present tumor antigens, a process known to be regulated by TRIM21 through its E3 ubiquitin ligase activity. Antagonist anti-CLEC-1 or anti-TRIM21 antibodies are therefore being evaluated to further confirm the involvement of the newly identified CLEC-1 interaction with TRIM21 in the regulation of CLEC-1’s function as an inhibitory myeloid checkpoint. Altogether our data highlight the CLEC-1/TRIM21 axis as a new target for cancer immunotherapy. Citation Format: Irene Baccelli, Vanessa Gauttier, Marion Drouin, Caroline Mary, Isabelle Girault, Emmanuelle Wilhelm, Sabrina Pengam, Géraldine Teppaz, Julien Taurelle, Ariane Desselle, Virginie Thepenier, Nicolas Poirier, Elise Chiffoleau. TRIM21 is a novel endogenous partner of the inhibitory myeloid checkpoint CLEC-1 involved in tumor antigen cross-presentation [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 6365.

Effect of baicalin on expression of fibrogenic factors TGF-β1, mmp2 and timp2 in tissue of mice with pulmonary fibrosis

To investigate the effect of baicalin extracted from Qinbai Qingfei Concentrated Pills on the expressions of TGF-β1, mmp2 and timp2 in mice with pulmonary fibrosis induced by bleomycin. The Biacore technique was used to detect the specific binding between Qinbai Qingfei Concentrated Pills and TGF-β1, and the affinity components were enriched, regenerated and recovered by Biacore fishing. Then ultra-performance liquid chromatography and quadrupole time of flight mass spectrometry(UPLC-Q-TOF-MS) were used to determine whether the monomer was baicalin. Biacore was used to verify the affinity kinetics of baicalin, which was validated by pharmacodynamics in vivo. Totally 30 BALB/C mice were randomly divided into three groups: baicalin group, blank group and model group. The blank group was given sodium chloride injection(0.08 mL·kg~(-1)), while the model group and the baicalin group were injected with 4 mg·kg~(-1) bleomycin. The localization of TGF-β1, mmp2 and timp2 protein in the cells and the mRNA expressions of TGF-β1, mmp2 and timp2 were detected by RT-PCR 14 days later. The results of Biacore affinity analysis showed that the peak of binding response between Qinbai Qingfei Concentrated Pills and TGF-β1 protein reached 1 524.0 RU, with specific binding. The affinity constant K_D of baicalin and TGF-β1 was 1.620 06 μmol·L~(-1), which was determined by SPR kinetic analysis, suggesting a stable binding between baicalin and TGF-β1, which verified the results of angulation. The results of immunohistochemistry showed that the deposition of cellulose in baicalin group was significantly less than that in model group, the mRNA expressions of TGF-β1, mmp2 and timp2 were decreased in baicalin solution compared with the model group. Baicalin combined with TGF-β1 could inhibit the expressions of mmp2 and timp2 and delay the progress of pulmonary fibrosis.

Abstract 1544: Cell penetrating single domain antibody (sdAb) SBT-100 binds KRAS and inhibits growth of human cancers with KRAS activating mutations

Abstract Background: Despite nearly fourth years of research, scientists have failed to develop a clinically viable therapy against KRAS, one of the deadliest families of cancer-causing proteins. Mutations in KRAS are prevalent amongst the top three most deadly cancer types in the United States: pancreatic (95%), colorectal (45%), and lung (35%). KRAS has been thought to be undruggable due to: 1) its intracellular location and lack of binding pockets for small molecules; 2) the high (pM) affinity of RAS for GTP precludes direct targeting of the nucleotide binding pocket; 3) high intracellular concentrations of GTP (uM) inhibits competition for the nucleotide-binding pocket by small molecules; and 4) possible toxicity. Mutations of KRAS result in it being perpetually turned on to propagate signal down the MAPK pathway. This results in constant production of P-ERK and plays an important role in malignant development. To overcome these challenges, Singh Biotechnology has developed SBT-100 a first in class & best in class novel sdAb that penetrates the cell membrane to bind KRAS to inhibit its GTPase activity. Methods: Human cancer cell lines were purchased from ATCC. BIAcore affinity studies were conducted by Precision Antibody. KRAS GTPase assay was purchased from Promega. Levels of P-ERK were determined using western blots. In vitro cell growth suppression was tested with MTT assay. Athymic nude mice for xenograft studies were purchased from Envigo. Results: SBT-100 binds KRAS with KD=10-9 and KRAS(G12D) with KD=10-7 as demonstrated by BIAcore affinity assay. Both SBT-100 and SBT-102 significantly inhibit KRAS GTPase activity in vitro and inhibition is comparable to polyclonal antibody to KRAS. Growth of MDA-MB-231 cells with KRAS(G13D) mutation and PANC-1 cells with KRAS(G12D) mutation are significantly decreased in the MTT assay when incubated with SBT-100. Additionally, same cell lines have significantly decreased P-ERK expression when cultured with SBT-100. Xenograft studies demonstrate significant growth suppression of MDA-MB-231 and PANC-1 when treated with SBT-100 in vivo. Conclusion: SBT-100, crosses the cell membrane, binds to KRAS intracellularly and its most common mutant with nanomolar affinity, inhibits KRAS GTPase activity, downregulates P-ERK signaling, and suppresses the growth of cancers cells in vitro and in vivo without showing any toxic effects. Citation Format: Sunanda Singh, Genoveva Murillo, Avani Singh, Samara Singh, Meenakshi Parihar, Anjali Singh, Rajendra Mehta, Ashutosh Parihar. Cell penetrating single domain antibody (sdAb) SBT-100 binds KRAS and inhibits growth of human cancers with KRAS activating mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1544.

A mutated recombinant subunit vaccine protects mice and guinea pigs against botulinum type A intoxication

ABSTRACTBotulinum neurotoxins (BoNTs) are the most potent toxins to mammals. A toxoid vaccine was previously used for prevention of botulinum intoxication; however, this vaccine is no longer available. Currently, no approved botulinum vaccines are available from the Food and Drug Administration (FDA). Recently, a recombinant host cell receptor-binding subunit created for use as a potential vaccine completed phase 2 clinical trials. The current study designed a vaccine candidate against BoNT type A (BoNT/A) using a structural design. Our vaccine candidate was the BoNT/A heavy chain C-terminal region (HCR) that contained the point mutation BA15 (R1269A) within the ganglioside-binding site. A Biacore affinity test showed that the affinity of BA15 for ganglioside GT1b was 100 times lower than that of the HCR. A SNAP25 cleavage assay revealed that immunized sera blocked SNAP25 cleavage of the BoNT/A toxin via BA15. In an in vivo experiment, mice and guinea pigs immunized with BA15 produced neutralizing antibodies that protected against 3,000 LD50 of BoNT/A. In conclusion, the results of both in vitro and in vivo assays showed that our BA15 vaccine candidate was similar to the recombinant host cell receptor-binding subunit vaccine. The inability of BA15to bind ganglioside shows that BA15 is a potential safe vaccine candidate.

Single domain antibody (SBT-100) crosses the blood brain barrier (BBB) and inhibits human glioblastoma by selectively targeting KRAS and P-STAT3.

e13547 Background: Glioblastoma is difficult to treat malignancy that has a high incidence of KRAS mutations ( > 90%) and hyper-expression of P-STAT3 ( > 90%). Most chemotherapeutic agents and large biologics (four chain heterotetrameric IgG) cannot cross the BBB. Therefore Singh Biotechnology’s (SBT) propriety technology has developed a novel targeted therapy and engineered SBT-100 a 15 kD single domain antibody (sdAb) that is bispecific for KRAS and STAT3, which can cross the BBB to target gliablastomas. Methods: Human glioblastoma (U87MG) cell line was used and obtained from ATCC. Biacore affinity assay was used to demonstrate SBT-100 binding to KRAS, KRAS (G12D), and STAT3. The glioblastoma cells were incubated with 0µg/ml to 200µg/ml of SBT-100. MTT assay was performed after 3 days of treatment with SBT-100 and the growth inhibition was calculated. Xenograft (athymic nude mouse) with a well established tumor growing for 6 weeks was injected with SBT-100 intraperitoneally (IP). After 15 minutes the brain of this animal was harvested for immunohistochemical staining. Results: Biacore studies showed SBT-100 binds KRAS with affinity constant of 10-9M, KRAS (G12D) at 10-8M, and STAT3 at 10-8M. MTT assay reveals 62% (p < 0.01) growth inhibition of U87MG within 3 days. A xenograft mouse with a well established tumor ( > 150mm3), when injected IP with 5mg/kg of SBT-100 showed localization in the brain within 15 minutes. Therefore SBT-100 (sdAb) crosses the BBB, and shows intracellular localization in the animal’s neurons and glial cells. Conclusions: SBT-100 significantly inhibits the growth of glioblastoma. KRAS mutations and/or over expression of P-STAT3 in glioblastomas are promising targets for sdAbs like SBT-100, which is bispecific for KRAS and STAT3. SBT-100 crosses the BBB and localizes within the neurons and glial cells of the brain. SBT’s proprietary single domain antibody technology platform used to engineer SBT-100 holds promise for targeting primary brain malignancies, metastatic cancers that go to the brain, and for neurologic disease amenable to targeted therapy. Many chemo-resistant and radiation-resistant cancers use STAT3 as an escape mechanism.

Control of the molecular behavior of SHIP by FcγRIIB and Nck (P1122)

Abstract Leukocyte activity is controlled by balancing activating and inhibitory signals in order to generate effective immune responses while avoiding tissue damage and autoimmunity. This project examines an important regulatory circuit in B cells that involves the lipid phosphatase SHIP. Localization, phosphorylation, and binding to interaction partners are important factors in the regulation of this protein, however knowledge of their inter-relationships is incomplete. Here, we examine the influence of two interaction partners on the molecular behaviour and function of SHIP. First, we examine in detail how the inhibitory receptor FcγRIIB controls SHIP localization dynamics. We have observed that co-engagement of FcγRIIB along with the B cell receptor in A20 cells does not influence the magnitude or kinetics of SHIP-EGFP recruitment to the membrane as assessed by confocal microscopy, however it does alter mobility at the cell periphery as measured by fluorescence recovery after photobleaching. Next, we probe the influence of a novel binding partner, the adaptor protein Nck. We have demonstrated an interaction between SHIP and Nck by both Biacore affinity analysis and pull-down assays. Functional relevance will be addressed through mutagenesis experiments. With these aims we hope to enhance our understanding of an interaction that is already recognized as relevant and investigate the function of a novel interaction that occurs at a previously uncharacterized regulatory site.

Combinatorial Design of an Anticalin Directed against the Extra-Domain B for the Specific Targeting of Oncofetal Fibronectin

The oncofetal isoform of the extracellular matrix protein fibronectin (Fn), which carries the extra-domain B (ED-B) and is exclusively expressed in neovasculature, has gained interest for tumor diagnosis and therapy using engineered antibody fragments. We have employed the human lipocalin 2 (Lcn2) as a small and robust non-immunoglobulin scaffold to select ED-B-specific Anticalins from a new advanced random library using bacterial phage display and ELISA screening against appropriately engineered Fn fragments. As a result, we have isolated and biochemically characterized four different Anticalins that all show low nanomolar affinities for ED-B, right in the range between the monomeric and dimeric forms of the single-chain variable antibody fragment L19 that has been widely applied in this area before. All Anticalins can be readily expressed in Escherichia coli as soluble and strictly monomeric proteins, and they show specific staining of ED-B-positive tumor cells in immunofluorescence microscopy while BIAcore affinity analyses indicate recognition of distinct ED-B epitopes. The crystal structure for one Anticalin, N7A, in complex with the Fn7B8 fragment, was solved at 2.6Å resolution and reveals binding to the gfcc′ sheet and cc′ loop on ED-B. This is the second example of a protein-specific Lcn2-based Anticalin, which illustrates the remarkable plasticity of the calyx-like ligand pocket of lipocalins with their four structurally hypervariable loops supported by a highly conserved β-barrel. The ED-B-specific Anticalins resulting from this study should provide useful reagents in research and biomedical drug development, both for in vivo imaging and for directed cancer therapy.

Selection and characterization of human antibody fragments specific for psoriasin – A cancer associated protein

S100A7 (psoriasin) is a calcium-binding protein that is upregulated in many types of cancer and often associated with poor prognosis. Its role in carcinogenesis has been associated with the stimulation of VEGF and EGF activity. The recent research showed that psoriasin directly interacts with αvβ6 integrin, a protein related to the invasive phenotype of cancer. Moreover, this interaction promotes the αvβ6-dependent invasive activity. The important function of S100A7 in carcinoma development determines a great need for valuable tools enabling its detection, quantification and also activity inhibition. Here, we show the selection of S100A7 specific antibody fragments from the human scFv phage library ETH-2 Gold. We have selected antibody fragments specific for psoriasin, purified them and analyzed by BIAcore affinity measurements. The best clone was subjected to affinity maturation procedure yielding molecule with a subnanomolar affinity towards human S100A7 protein. Selected clone was expressed in a bivalent format and applied for immunostaining analysis, which confirmed the ability of the antigen recognition in physiological conditions. We therefore propose that obtained antibody, that is the first phage display-derived human antibody against psoriasin, can serve as a useful psoriasin binding platform in research, diagnostics and therapy of cancer.

Affibody molecules for in vivo characterization of HER2-positive tumors by near-infrared imaging.

HER2 overexpression has been associated with a poor prognosis and resistance to therapy in breast cancer patients. We are developing molecular probes for in vivo quantitative imaging of HER2 receptors using near-infrared (NIR) optical imaging. The goal is to provide probes that will minimally interfere with the studied system, that is, whose binding does not interfere with the binding of the therapeutic agents and whose effect on the target cells is minimal. We used three different types of HER2-specific Affibody molecules [monomer ZHER2:342, dimer (ZHER2:477)2, and albumin-binding domain-fused-(ZHER2:342)2] as targeting agents and labeled them with Alexa Fluor dyes. Trastuzumab was also conjugated, using commercially available kits, as a standard control. The resulting conjugates were characterized in vitro by toxicity assays, Biacore affinity measurements, flow cytometry, and confocal microscopy. Semiquantitative in vivo NIR optical imaging studies were carried out using mice with s.c. xenografts of HER2-positive tumors. The HER2-specific Affibody molecules were not toxic to HER2-overexpressing cells and their binding to HER2 did interfere with neither binding nor effectives of trastuzumab. The binding affinities and specificities of the Affibody-Alexa Fluor fluorescent conjugates to HER2 were unchanged or minimally affected by the modifications. Pharmacokinetics and biodistribution studies showed the albumin-binding domain-fused-(ZHER2:342)2-Alexa Fluor 750 conjugate to be an optimal probe for optical imaging of HER2 in vivo. Our results suggest that Affibody-Alexa Fluor conjugates may be used as a specific NIR probe for the noninvasive semiquantitative imaging of HER2 expression in vivo.

Increased Affinity and Stability of an Anti-HIV-1 Envelope Immunotoxin by Structure-based Mutagenesis

HIV-infected cells are selectively killed by an immunotoxin in which a truncated form of Pseudomonas exotoxin A is joined to the variable region of a broadly neutralizing antibody (3B3) that recognizes the viral envelope glycoprotein (Env). To improve the efficacy of this molecule, we used three-dimensional structural information and phage selection data to design 23 single and multiple point mutations in the antibody variable region sequences that contact Env. Substituting an aromatic residue for an aspartate in the third complementarity-determining region of V(H) increased the potency of the immunotoxin by approximately 10-fold in a cell-killing assay. Detailed analysis of one such mutant, N31H/Q100eY, revealed both a higher affinity for monomeric and cell surface Env and an increased stability against aggregation compared with the starting immunotoxin. Conversion to a disulfide-linked two-chain format further stabilized the protein. N31H/Q100eY retained the ability to bind to Env from multiple viral isolates, to inhibit Env-mediated cell fusion, and to limit spreading viral infection in peripheral blood mononuclear cells. Such site-directed mutants may increase the utility of immunotoxins for reducing or eradicating persistent HIV-1 infection in humans.

The Cytoplasmic Tail of L-selectin Interacts with Members of the Ezrin-Radixin-Moesin (ERM) Family of Proteins: CELL ACTIVATION-DEPENDENT BINDING OF MOESIN BUT NOT EZRIN

L-selectin regulates the recruitment of naive lymphocytes from the bloodstream to secondary lymphoid organs, mediating their initial capture and subsequent rolling along high endothelial cell surface-expressed ligands in peripheral lymph nodes. In vivo, distribution of L-selectin and cell surface levels determine the tethering efficiency and rolling velocity of leukocytes, respectively. Treatment of naive lymphocytes with phorbol myristate acetate (PMA) induces rapid ectodomain proteolytic down-regulation (shedding) of surface L-selectin via a protein kinase C (PKC)-dependent pathway. In an attempt to isolate proteins that are involved in regulating L-selectin expression, an affinity column was constructed using the 17-amino acid cytoplasmic tail of L-selectin. Affinity purification of extracts from lymphocytes, pre-treated with or without PMA, allowed identification of proteins that interact with the affinity column under one condition but not the other. By using this approach, members of the Ezrin-Radixin-Moesin family of proteins were found to interact specifically with the cytoplasmic tail of L-selectin. Moesin from PMA-stimulated lymphocytes, but not from unstimulated lymphocytes, bound to L-selectin tail. In contrast, ezrin from unstimulated or PMA-stimulated lymphocytes associated with L-selectin tail with equal affinity. Furthermore, the PKC inhibitor Ro 31-8220 significantly reduced the interaction of moesin, but not ezrin, with L-selectin. Alanine mutations of membrane-proximal basic amino acid residues in the cytoplasmic domain of L-selectin identified arginine 357 as a critical residue for both ezrin and moesin interaction. Finally, BIAcore affinity analysis confirmed that N-terminal moesin interacts specifically with L-selectin cytoplasmic tail, with relatively high affinity (K(d) approximately 40 nm). Based on these findings, although moesin and ezrin bind to a similar region of the cytoplasmic tail of L-selectin, moesin binding is dependent on PKC activation, which suggests that ezrin and moesin are regulated differently in lymphocytes.