Extracellular Loop Research Articles

The Plasmodium vivax MSP1P-19 is involved in binding of reticulocytes through interactions with the membrane proteins band3 and CD71

The parasite Plasmodium vivax preferentially invades human reticulocytes. Its merozoite surface protein 1 paralog (PvMSP1P), particularly the 19-kDa C-terminal region (PvMSP1P-19), has been shown to bind to reticulocytes and this binding can be inhibited by antisera obtained by PvMSP1P-19 immunization. The molecular mechanism of interactions between PvMSP1P-19 and reticulocytes during P. vivax invasion, however, remains unclear. In this study, we analyzed the ability of MSP1P-19 to bind to different concentrations of reticulocytes and confirmed its reticulocyte preference. LC-MS analysis was used to identify two potential reticulocyte receptors, band3 and CD71, that interact with MSP1P-19. Both PvMSP1P-19 and its sister taxon Plasmodium cynomolgi MSP1P-19 (PcMSP1P-19) were found to bind to the extracellular loop (loop 5) of band3, where the interaction of MSP1P-19 with band3 was chymotrypsin-sensitive. Antibodies against band3-P5, CD71, and MSP1P-19 reduced the binding activity of PvMSP1P-19 and PcMSP1P-19 to reticulocytes, while MSP1P-19 proteins inhibited P. falciparum invasion in vitro in a concentration-dependent manner. To sum up, identification and characterization of the reticulocyte receptor is important for understanding the binding of reticulocytes by MSP1P-19.

Cryo-EM structures of adenosine receptor A3AR bound to selective agonists

The adenosine A3 receptor (A3AR), a key member of the G protein-coupled receptor family, is a promising therapeutic target for inflammatory and cancerous conditions. The selective A3AR agonists, CF101 and CF102, are clinically significant, yet their recognition mechanisms remained elusive. Here we report the cryogenic electron microscopy structures of the full-length human A3AR bound to CF101 and CF102 with heterotrimeric Gi protein in complex at 3.3-3.2 Å resolution. These agonists reside in the orthosteric pocket, forming conserved interactions via their adenine moieties, while their 3-iodobenzyl groups exhibit distinct orientations. Functional assays reveal the critical role of extracellular loop 3 in A3AR’s ligand selectivity and receptor activation. Key mutations, including His3.37, Ser5.42, and Ser6.52, in a unique sub-pocket of A3AR, significantly impact receptor activation. Comparative analysis with the inactive A2AAR structure highlights a conserved receptor activation mechanism. Our findings provide comprehensive insights into the molecular recognition and signaling of A3AR, paving the way for designing subtype-selective adenosine receptor ligands.

Cryo-EM Structure of the Human Amylin 1 Receptor in Complex with CGRP and Gs Protein.

Inhibition of calcitonin gene-related peptide (CGRP) or its cognate CGRP receptor (CGRPR) has arisen as a major breakthrough in the treatment of migraine. However, a second CGRP-responsive receptor exists, the amylin (Amy) 1 receptor (AMY1R), yet its involvement in the pathology of migraine is poorly understood. AMY1R and CGRPR are heterodimers consisting of receptor activity-modifying protein 1 (RAMP1) with the calcitonin receptor (CTR) and the calcitonin receptor-like receptor (CLR), respectively. Here, we present the structure of AMY1R in complex with CGRP and Gs protein and compare it with the reported structures of the AMY1R complex with rat amylin (rAmy) and the CGRPR in complex with CGRP. Despite similar protein backbones observed within the receptors and the N- and C-termini of the two peptides bound to the AMY1R complexes, they have distinct organization in the peptide midregions (the bypass motif) that is correlated with differences in the dynamics of the respective receptor extracellular domains. Moreover, divergent conformations of extracellular loop (ECL) 3, intracellular loop (ICL) 2, and ICL3 within the CTR and CLR protomers are evident when comparing the CGRP bound to the CGRPR and AMY1R, which influences the binding mode of CGRP. However, the conserved interactions made by the C-terminus of CGRP to the CGRPR and AMY1R are likely to account for cross-reactivity of nonpeptide CGRPR antagonists observed at AMY1R, which also extends to other clinically used CGRPR blockers, including antibodies.

Synthetic Peptides with Genetic‐Codon‐Tailored Affinity for Assembling Tetraspanin CD81 at Cell Interfaces and Inhibiting Cancer Metastasis

AbstractProbing biomolecular interactions at cellular interfaces is crucial for understanding and interfering with life processes. Although affinity binders with site specificity for membrane proteins are unparalleled molecular tools, a high demand remains for novel multi‐functional ligands. In this study, a synthetic peptide (APQQ) with tight and specific binding to the untargeted extracellular loop of CD81 evolved from a genetically encoded peptide pool. With tailored affinity, APQQ flexibly accesses, site‐specifically binds, and forms a complex with CD81, enabling in‐situ tracking of the dynamics and activity of this protein in living cells, which has rarely been explored because of the lack of ligands. Furthermore, APQQ triggers the relocalization of CD81 from diffuse to densely clustered at cell junctions and modulates the interplay of membrane proteins at cellular interfaces. Motivated by these, efficient suppression of cancer cell migration, and inhibition of breast cancer metastasis were achieved in vivo.

Synthetic Peptides with Genetic-Codon-Tailored Affinity for Assembling Tetraspanin CD81 at Cell Interfaces and Inhibiting Cancer Metastasis.

Probing biomolecular interactions at cellular interfaces is crucial for understanding and interfering with life processes. Although affinity binders with site specificity for membrane proteins are unparalleled molecular tools, a high demand remains for novel multi-functional ligands. In this study, a synthetic peptide (APQQ) with tight and specific binding to the untargeted extracellular loop of CD81 evolved from a genetically encoded peptide pool. With tailored affinity, APQQ flexibly accesses, site-specifically binds, and forms a complex with CD81, enabling in-situ tracking of the dynamics and activity of this protein in living cells, which has rarely been explored because of the lack of ligands. Furthermore, APQQ triggers the relocalization of CD81 from diffuse to densely clustered at cell junctions and modulates the interplay of membrane proteins at cellular interfaces. Motivated by these, efficient suppression of cancer cell migration, and inhibition of breast cancer metastasis were achieved in vivo.

Mechano-sensitivity of β2-adrenoceptors enhances constitutive activation of cAMP generation that is inhibited by inverse agonists

The concept of agonist-independent signalling that can be attenuated by inverse agonists is a fundamental element of the cubic ternary complex model of G protein-coupled receptor (GPCR) activation. This model shows how a GPCR can exist in two conformational states in the absence of ligands; an inactive R state and an active R* state that differ in their affinities for agonists, inverse agonists, and G-protein alpha subunits. The proportion of R* receptors that exist in the absence of agonists determines the level of constitutive receptor activity. In this study we demonstrate that mechanical stimulation can induce β2-adrenoceptor agonist-independent Gs-mediated cAMP signalling that is sensitive to inhibition by inverse agonists such as ICI-118551 and propranolol. The size of the mechano-sensitive response is dependent on the cell surface receptor expression level in HEK293G cells, is still observed in a ligand-binding deficient D113A mutant β2-adrenoceptor and can be attenuated by site-directed mutagenesis of the extracellular N-glycosylation sites on the N-terminus and second extracellular loop of the β2-adrenoceptor. Similar mechano-sensitive agonist-independent responses are observed in HEK293G cells overexpressing the A2A-adenosine receptor. These data provide new insights into how agonist-independent constitutive receptor activity can be enhanced by mechanical stimulation and regulated by inverse agonists.

Computational Insights into the Interaction of the Conserved Cysteine-Noose Domain of the Human Respiratory Syncytial Virus G Protein with the Canonical Fractalkine Binding site of Transmembrane Receptor CX3CR1 Isoforms.

The human Respiratory Syncytial Virus (hRSV) stands as one of the most common causes of acute respiratory diseases. The infectivity of this virus is intricately linked to its membrane proteins, notably the attachment glycoprotein (G protein). The latter plays a key role in facilitating the attachment of hRSV to respiratory tract epithelial cells, thereby initiating the infection process. The present study aimed to characterize the interaction of the conserved cysteine-noose domain of hRSV G protein (cndG) with the transmembrane CX3C motif chemokine receptor 1 (CX3CR1) isoforms using computational tools of molecular modeling, docking, molecular dynamics simulations, and binding free energy calculations. From MD simulations of the molecular system embedded in the POPC lipid bilayer, we showed a stable interaction of cndG with the canonical fractalkine binding site in the N-terminal cavity of the CX3CR1 isoforms and identified that residues in the extracellular loop 2 (ECL2) region and Glu279 of this receptor are pivotal for the stabilization of CX3CR1/cndG binding, corroborating what was reported for the interaction of the chemokine fractalkine with CX3CR1 and its structure homolog US28. Therefore, the results presented here contribute by revealing key structural points for the CX3CR1/G interaction, allowing us to better understand the biology of hRSV from its attachment process and to develop new strategies to combat it.

A Conserved Intramolecular Ion-Pair Plays a Critical but Divergent Role in Regulation of Dimerization and Transport Function among the Monoamine Transporters.

The monoamine transporters, including the serotonin transporter (SERT), dopamine transporter (DAT), and norepinephrine transporter (NET), are the therapeutic targets for the treatment of many neuropsychiatric disorders. Despite significant progress in characterizing the structures and transport mechanisms of these transporters, the regulation of their transport functions through dimerization or oligomerization remains to be understood. In the present study, we identified a conserved intramolecular ion-pair at the third extracellular loop (EL3) connecting TM5 and TM6 that plays a critical but divergent role in the modulation of dimerization and transport functions among the monoamine transporters. The disruption of the ion-pair interactions by mutations induced a significant spontaneous cross-linking of a cysteine mutant of SERT and an increase in cell surface expression but with an impaired specific transport activity. On the other hand, similar mutations of the corresponding ion-pair residues in both DAT and NET resulted in an opposite effect on their oxidation-induced dimerization, cell surface expression, and transport function. Reversible biotinylation experiments indicated that the ion-pair mutations slowed down the internalization of SERT but stimulated the internalization of DAT. In addition, cysteine accessibility measurements for monitoring SERT conformational changes indicated that substitution of the ion-pair residues resulted in profound effects on the rate constants for cysteine modification in both the extracellular and cytoplasmatic substrate permeation pathways. Furthermore, molecular dynamics simulations showed that the ion-pair mutations increased the interfacial interactions in a SERT dimer but decreased it in a DAT dimer. Taken together, we propose that the transport function is modulated by the equilibrium between monomers and dimers on the cell surface, which is regulated by a potential compensatory mechanism but with different molecular solutions among the monoamine transporters. The present study provided new insights into the structural elements regulating the transport function of the monoamine transporters through their dimerization.

Imidacloprid effects on acetylcholinesterase and nicotinic acetylcholine receptor in Apis mellifera. Experimental and molecular modeling approaches

Although the neonicotinoid insecticides have good selectivity towards insects rather than vertebrates, they have severe effects on honeybee production and pollination activities. Therefore, the effects of imidacloprid (IMI), the most used neonicotinoid, on the two main bioreceptors, acetylcholinesterase (AChE) and nicotinic acetylcholine receptor alpha subunit (nAChRα1) of honeybees were examined to identify their roles in honeybee toxicity and possible binding sites which assist in selecting and designing neonicotinoids. In vivo, IMI showed a high inhibitory effect on AChE (IC50 5.63 mg/L); however, the effect was much lower in vitro experiment (IC50 719 mg/L). This result induced us to examine the IMI effect on AChE gene expression which revealed that the AChE-2 gene expression was severely affected by IMI explaining the observed high enzyme inhibition. In addition, although toxicity increased by increasing exposure to IMI (LC50 2.9 mg/L after 4h and 0.75 mg/L after 48h), AChE was not elevated (IC50 5.63 and 5.52 mg/L respectively). Besides, Despite resuming most enzyme activity (77% during 2 h and 84.14% after 4 h), a high mortality level was observed with LC50 2.9 mg/L. These results reinforced that the observed high toxicity is a multifactor process. Accordingly, Molecular modeling and docking of IMI into honeybee AChE and nAChRα1were also performed to examine their possible interactions and identify the important binding sites. Results models indicated that the first two binding sites in AChE were found in the esteratic subunit in the active site explaining the observed in vitro inhibition. In nAChRα1, four of the highest five free energy binding sites are located in the large TM3-TM4 loop and one in the extracellular loops. Consequently, the present work revealed that IMI toxicity is attributed to various factors including direct interaction with both AChE and nAChRα1 as well as downregulating AChE-2 gene expression.

Structural basis of ligand specificity and channel activation in an insect gustatory receptor

Gustatory receptors (GRs) are critical for insect chemosensation and are potential targets for controlling pests and disease vectors, making their structural investigation a vital step toward such applications. We present structures of Bombyx mori Gr9 (BmGr9), a fructose-gated cation channel, in agonist-free and fructose-bound states. BmGr9 forms a tetramer similar to distantly related insect odorant receptors (ORs). Upon fructose binding, BmGr9's channel gate opens through helix S7b movements. In contrast to ORs, BmGr9's ligand-binding pocket, shaped by a kinked helix S4 and a shorter extracellular S3-S4 loop, is larger and solvent accessible in both agonist-free and fructose-bound states. Also, unlike ORs, fructose binding by BmGr9 involves helix S5 and a pocket lined with aromatic and polar residues. Structure-based sequence alignments reveal distinct patterns of ligand-binding pocket residue conservation in GR subfamilies associated with different ligand classes. These data provide insight into the molecular basis of GR ligand specificity and function.

Structural insights into the mechanism of protein transport by the Type 9 Secretion System translocon.

Secretion systems are protein export machines that enable bacteria to exploit their environment through the release of protein effectors. The Type 9 Secretion System (T9SS) is responsible for protein export across the outer membrane (OM) of bacteria of the phylum Bacteroidota. Here we trap the T9SS of Flavobacterium johnsoniae in the process of substrate transport by disrupting the T9SS motor complex. Cryo-EM analysis of purified substrate-bound T9SS translocons reveals an extended translocon structure in which the previously described translocon core is augmented by a periplasmic structure incorporating the proteins SprE, PorD and a homologue of the canonical periplasmic chaperone Skp. Substrate proteins bind to the extracellular loops of a carrier protein within the translocon pore. As transport intermediates accumulate on the translocon when energetic input is removed, we deduce that release of the substrate-carrier protein complex from the translocon is the energy-requiring step in T9SS transport.

Simultaneous Recording of Remote Domain Dynamics in Membrane Proteins Using the Double-Labeled DXB/DXT Technique.

Protein dynamics play important roles in biological functions, which accompany allosteric structure changes. Diffracted X-ray blinking (DXB) uses monochromatic X-rays and nanocrystal probes. The intramolecular motion of target proteins is analyzed from the intensity changes in detector signals at the diffraction rings. In contrast, diffracted X-ray tracking (DXT) elucidates molecular dynamics by analyzing the trajectories of Laue spots. In this study, we have developed a dual-labeling technique for DXB and DXT, allowing the simultaneous observation of motions at different domains in proteins. We identified zinc oxide (ZnO) crystals as promising candidates for the second labeling probes due to their excellent diffraction patterns, high chemical stability, and favorable binding properties with proteins. The diffraction spots from the ZnO crystals are sufficiently separated from those of gold, enabling independent motion analysis at different domains. Dual-labeling DXB was employed for the motion analysis of the 5-HT2A receptor in living cells. Simultaneous motion recording of the N-terminus and the second extracellular loop demonstrated ligand-induced motion suppression at both domains. The dual-labeling DXT technique demonstrated a capsaicin-induced peak shift in the two-dimensional motion maps at the N-terminus of the TRPV1 protein, but the peak shift was not obvious in the C-terminus. The capsaicin-induced motion modulation was recovered by the addition of the competitive inhibitor AMG9810.

Abstract 3189: A novel monoclonal antibody targeting TM4SF4 enhances antitumor activity through regulation of cellular levels of immune checkpoint ligands and antibody dependent cellular cytotoxicity

Abstract Transmembrane 4 super family member 4(TM4SF4) protein has been shown to be involved in EMT-associated stemness through autocrine of insulin-like growth factor 1(IGF1) and osteopontin(OPN) in non-small cell lung cancer(NSCLC) cells. However, its potential as a therapeutic target has not been evaluated. In this study, anti-human TM4SF4 monoclonal antibodies(anti-hTM4SF4 mAbs) were prepared in mouse as part of a strategy for developing anticancer drug using humanized antibody. A synthetic peptide(15 mer, 126-140th AA) derived from the extracellular loop(ECL) 2 of human TM4SF4 exposed on outer cell surface was used as an immunization antigen. Among the selected anti-hTM4SF4 mAbs, ECL-2B7 and ECL-12A8 showed the best affinity for the antigen(Kd), approximately 2.66 nM and 8.34 nM, as determined by surface plasmon resonance analysis. Using mouse xenograft of NSCLC cell line, it was confirmed that ECL-2B7 was superior to ECL-12A8 in producing humanized antibody. ECL-2B7 inhibited EMT-associated stemness of TM4SF4 positive cancer cells by blocking cellular signaling events such as IGF1Rβ and CD44 and their downstream signals, PI3K/AKT/GSK3β/NF-κB and JAK2/STAT3 pathways. ECL-2B7 also enhanced antitumor activity by downregulating cellular and exosomal level of PD-L1 and B7-H4, immune-checkpoint ligands that elicit immunosuppression of T cell, and by mediating antibody-dependent cellular cytotoxicity. Treatment with OPN- and IGF1-neutralizing antibodies suppressed intracellular PD-L1 and B7-H4 level. It means that the regulation of PD-L1 and B7-H4 levels by TM4SF4 is closely related to the autocrine action of OPN and IGF1 induced by TM4SF4. Using patient-derived xenograft model, it was confirmed that the ECL-2B7 has excellent antitumor activity that effectively inhibits tumor progress. These results strongly suggest that it is necessary to construct humanized antibody based on ECL-2B7 to confirm its potential as a novel anticancer drug. Citation Format: Rae Kwon Kim, Chang-Kyu Heo, Yeon-jee Kahm, Uhee Jung, Byung-Chul Shin, Won-Yong Kim, Bum-Jin Kim, Sung-Chul Kim, Chun-jeih Ryu, Eun-Wie Cho, In-Gyu Kim. A novel monoclonal antibody targeting TM4SF4 enhances antitumor activity through regulation of cellular levels of immune checkpoint ligands and antibody dependent cellular cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3189.

Abstract 6706: An anti-claudin 18.2/CD3 bispecific antibody for the treatment of claudin 18.2 positive gastric cancer

Abstract The claudin18.2 (CLDN18.2) protein shows four transmembrane domains (TMDs) with two extracellular loops (ECL), ECL-1 and ECL-2. It is a highly selective biomarker with limited expression in normal tissues and often exhibits abnormal expression during the occurrence and development of various primary malignant tumors. The specific expression pattern supports the notion that CLDN18.2 is a distinctive molecule for targeted therapy in various cancers, particularly gastric and pancreatic cancers. Various drugs targeting CLDN18.2, including monoclonal antibodies, ADCs, CAR-T, and bispecific antibodies, have also entered clinical trials. As of this writing, there are currently no commercially available products worldwide that specifically target CLDN18.2. However, Zolbetuximab (IMAB362) has successfully reached the Phase III end point, indicating positive clinical outcomes and establishing CLDN18.2 as a promising cellular and antibody therapeutic option. Here, we describe LNF2007, an anti-CLDN 18.2/CD3 bispecific antibody that can redirect CD3+ effector T cells to CLDN18.2+ tumor sites, resulting in the activation of T cells and subsequent killing of target cells. The design of LNF2007 focuses on selecting a relatively weak and reasonable affinity to CD3, accompanied by the suppression of Fc-mediated effector functions. This aim is to minimize off-target toxicity and maximize therapeutic efficacy. In vitro data demonstrate that LNF2007 binds more strongly to CLDN18.2+cells than to CD3+ cells. LNF2007 exhibited good dose-dependent activation and cytotoxicity against engineered cell lines BXPC3-hCLDN18.2-Luc and CHOK1-hCLDN18.2-Luc, which are positive for CLDN18.2 expression .In the in vivo pharmacodynamics assay, LNF2007 also demonstrated superior efficacy in the humanized NUGC4-CLDN18.2 model and BxPC3-CLDN18.2 model. Furthermore, a 4-week repeated dose (up to 1 mg/kg) toxicity study in cynomolgus monkeys showed that LNF2007 was safe and well tolerated. In vitro cytokine release was lower in the LNF2007 group than that in the AMG 910 and AZD5863 (two another anti-claudin18.2/CD3 bispecific antibody) groups. In conclusion, the novel anti-CLDN18.2 × anti-CD3 bispecific antibody LNF2007 demonstrates similar efficacy to AMG 910 and AZD5863 and is safer. LNF2007 provides a promising therapy for claudin 18.2-positive cancer patients.Taishan Industry Leading Talents Project of Shandong Province was a sponsor of this research. Citation Format: Lili Zhao, Guimin Zhang, Zhong Liu, Jinhua Xu, Lizhi Zhao, Guangyan Li, Bin Li, Zhongsong Zhu, Wei Peng, Yuqiang Zhu, Zhaowei Chen, Zhenyu Li. An anti-claudin 18.2/CD3 bispecific antibody for the treatment of claudin 18.2 positive gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6706.

Abstract 3122: Preclinical development of a novel camptothecin-based antibody-drug conjugate targeting solid tumors expressing Claudin-6

Abstract Claudin-6 is a member of the claudin family and consists of four transmembrane helices, two extracellular loops, and an amino- and carboxyl-terminal tail with a PDZ-binding motif in the cytoplasm. It is involved in the formation of tight junctions and is expressed in developing human epithelial structures during early-to-mid gestation while expression in iadult tissues is mostly absent. Claudin-6 expression is upregulated in ovarian cancer, testicular cancer, endometrial cancer, non-small cell lung cancer (NSCLC) and gastric cancer. Here, the generation and preclinical characterization of a novel Claudin-6 specific antibody-drug conjugate (ADC) is described, composed of a humanized IgG1 directed against human Claudin-6 site-specifically conjugated a novel payload containing a valine-alanine cleavable linker and a camptothecin warhead with a drug to antibody ratio of 6. FcγR-mediated effector function of the ADC was abrogated via mutations in the Fc portion of the antibody. The purpose of this study was to evaluate the expression of Claudin-6 in different tumour indications and characterise the in vitro and in vivo anti-tumour activity in human cancer cell lines and patient-derived xenograft (PDX) models. In addition, the tolerability of the ADC was evaluated in a repeat-dose toxicity study cynomolgus monkeys. Cell binding studies using Fabs showed high affinity binding to Claudin-6, while affinity for Claudin-9 was 16-fold lower with no binding to Claudin-3 and Claudin-4. Cross-reactivity with cynomolgus monkey and mouse Claudin-6 was also demonstrated. Fcgamma receptor binding studies confirmed the strongly reduced binding of the antibody to FcγI, FcγI,IIa, FcγIIb and FcγIIa, consistent with the introduction of mutations in the Fc domain of the antibody. In vitro, the Claudin-6 ADC showed potent and specific cytotoxicity against various Claudin-6-expressing solid cancer cell lines, including OVCAR3, PA-1, OV-90, FU-97, BeWo, NTERA-2 and NUGC3 in comparison to a non-binding control ADC conjugated to the same payload, and it also showed potent bystander killing. In vivo, a single dose intravenous (IV) of the Claudin-6 ADC demonstrated potent anti-tumour activity in multiple solid tumour xenograft models, including OVCAR3 and PA-1. Furthermore, administration of the Claudin-6 ADC to cynomolgus monkeys was well tolerated up to 40 mg/kg IV when given on Day 1 and 22. Cell membrane expression of Claudin-6 was confirmed by immunohistochemistry (IHC) in ovarian cancer (70%), testicular cancer (98%), endometrial cancer (30%), NSCLC (28%) and gastric cancer (24%), confirming expression of Claudin-6 in indications with high unmet need. In summary, this novel Claudin-6 ADC demonstrated potent, specific anti-tumour activity in Claudin-6 expressing cancer-derived models and was well tolerated in cynomolgus monkey, supporting future clinical development of this ADC. Citation Format: Nicola Tsang, Nicolas Veillard, Elizabeth Horsley, Karin Havenith, Narinder Janghra, Kristina Zaitseva, Cecile Oblette, Ian Kirby, Paul W. Hogg, Francesca Zammarchi, Lolke de Haan, Patrick H. van Berkel. Preclinical development of a novel camptothecin-based antibody-drug conjugate targeting solid tumors expressing Claudin-6 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3122.

Abstract 3138: Discovery of AT65474, a highly selective anti-CLDN6 ADC with a proprietary payload

Abstract CLDN6 is a membrane-bound oncofetal protein and a promising target for cancers such as ovarian and testicular cancer. CLDN6 shares high sequence homology with other CLDN family members which are expressed in normal tissues, making a high degree of selectivity required for CLDN6-targeting therapies. In particular, CLDN6 is closest to CLDN9 in sequence with a difference of only three residues in the extracellular loops, making it challenging to generate a highly selective antibody. We report the development of a humanized anti-CLDN6 antibody with high affinity and high selectivity for targeted cells which displays > 800-fold higher binding affinity for CLDN6 over CLDN9. The antibody was refined for use as an ADC and incorporates AxcynCYSTM technology for site-specific conjugation which can reproducibly achieve high DAR4 ratios of greater than 97% by HIC analysis. AT65474 is an ADC candidate that incorporates a proprietary payload derived from an FDA approved drug that was optimized in-house for increased potency and improved PK properties to limit systemic exposure. In vitro, the payload displays broad sub-nanomolar activity across a wide range of cancer cell lines including those resistant to paclitaxel, DM1, MMAE and DXd. The payload is conjugated via an enzyme-cleavable linker optimized for solubility, stability, and high drug to antibody ratios. We provide evidence characterizing AT65474 as a highly potent ADC with high DAR4 homogeneity and strong in vivo activity at low doses. Compared with a benchmark ADC in clinical development, AT65474 shows stronger binding, higher internalization, superior in vitro cytotoxicity, and stronger bystander killing effect. When evaluated in PA-1 and OV90 xenograft models of ovarian cancer, AT65474 displays significantly higher efficacy over the benchmark ADC. Furthermore, we show that AT65474 displays an ADCC effect that contributes to tumor suppression in the OV90 model. AT65474 was shown to be well tolerated in all xenograft mouse models and a pre-tox study in non-human primates. The combined data support further development of AT65474.​ Citation Format: Chen Zhong, You Xue Wu, Xi Yun Zhang, Yin Wen Cheng, Qian Wang, Ping Du, Shao Jun Liu, Bin Zou. Discovery of AT65474, a highly selective anti-CLDN6 ADC with a proprietary payload [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3138.

Abstract 1032: Development of a novel companion diagnostic immunohistochemistry antibody for claudin 18.2-targeted therapies

Abstract Background: CLDN18.2, the isoform 2 of CLDN18, is physiologically confined to gastric mucosa tight junctions, the epitopes of which would be exposed on the cancer cell surface upon malignant transformation and is highly expressed in a significant proportion of cancers, e.g., gastric and pancreatic adenocarcinomas, which makes it a promising drug target for cancer therapy. Currently, multiple drug modalities, such as monoclonal antibodies, ADCs, and CARTs targeting CLDN18.2 are being evaluated in clinical trials. A substantial correlation exists between the expression of CLDN18.2 and the efficacy of the these therapies. However, to date, no companion diagnostic (CDx) antibodies that are specific to CLDN18.2 have been approved. Therefore, it is important to promptly develop an anti-CLDN18.2 CDx antibody with a high specificity, sensitivity, and suitability for immunohistochemistry (IHC). In this study, we present the discovery and validation of a novel, highly sensitive IHC antibody that selectively identifies CLDN18.2. Methods: 3 CLDN18.2 specific peptide fragments from extracellular domain of human CLDN18.2 were conjugated with KLH. Antibodies were discovered by immunizing BALB/c and SJL mice with these peptides-KLH conjugates. Screening and selection process was relied on positive ELISA results against the immunogen, as well as FACS and Acumen Explorer Microplate staining of CLDN18.2-experssing cells. Hit antibodies were evaluated for IHC on formalin-fixed paraffin-embedded CHOK1 cells expressing full-length CLDN18.2 or CLDN18.1. The antibodies were further validated on human gastric cancer tissue samples to establish the analytical sensitivity and specificity of the IHC assay. Results: The mAb clone 43F11 exhibited binding to a fusion polypeptide comprising the first extracellular loop of human CLDN18.2 (aa32-53) at a half maximal effective concentration (EC50) of 0.212 nM. 43F11 showed positive cell surface IHC staining on CLDN18.2-experssing cells after fixation but demonstrated no staining on CLDN18.1-experssing cells. The IHC test method was established by utilizing clinical tumor tissues and PDX samples that had predetermined expression levels of CLDN18.2. The 43F11 antibody accurately identified the expression level of CLDN18.2 in the IHC assay, with a minimum concentration of 0.25 μg/mL. When compared to the commercial CLDN18.2 IHC antibody EPR19202, 43F11 demonstrated greater sensitivity, allowing for positive staining on cancer tissues that have significantly lower levels of CLDN18.2 expression. Conclusion: 43F11 exhibits specific recognition of human CLDN18.2 in IHC assays, while showing no reactivity towards CLDN18.1. It demonstrated superior sensitivity compared to the benchmark antibody. These data suggest that 43F11 has the capacity to serve as an effective diagnostic tool for identifying patients who positively express CLDN18.2. Citation Format: Jishun Chen, Suya Bai, Yu Bai, Zaoshun Hu, Peng Chen, Jay Mei, Bo Shan, Bing Hou. Development of a novel companion diagnostic immunohistochemistry antibody for claudin 18.2-targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1032.

Abstract 4153: PET/CT imaging of pancreatic ductal adenocarcinoma with a claudin-4 binding peptide in preclinical mouse model

Abstract There is a critical need to develop new imaging and therapeutic strategies for pancreatic cancer. Current positron emission tomography (PET) imaging assessments of pancreatic ductal adenocarcinoma (PDAC) rely on 18F-FDG as a radiopharmaceutical. To increase the sensitivity of PDAC detection, we identified Claudin-4 (CLDN4), a tight junction transmembrane protein, as a PDAC biomarker through spatial transcriptomic and proteomic analyses. CLDN4 expression has previously been reported to be enhanced in 99% of primary and 100% of metastatic pancreatic cancer tissue samples and in 10 of 11 precancerous pancreatic intraepithelial neoplasia (PanIN) specimens. A small fragment of naturally occurring Clostridium perfringens enterotoxin (CPE) was previously reported to bind to the extracellular loop domain of CLDN4 protein (PDB:7kp4) with nanomolar (nM) affinity (Kd). A CLDN4 binding peptide (C4BP) was developed as a radioligand by modifying the n-terminus of C4BP which does not impact receptor binding. Here, we designed and synthesized a DOTA-PEG1-C4BP ligand on a rink amide resin using a microwave-assisted peptide synthesizer. Radiolabeling of DOTA-PEG1-C4BP (2 nmol) with 64CuCl2 (70 MBq) conferred 64Cu-C4BP (28 MBq/nmol) with 80% non-decay corrected yield and >99% radiochemical purity. PET/CT imaging of 64Cu-C4BP (~0.1 μg/mouse) performed in an inducible PDAC (KrasLSL-G12D/+;Rosa26LSL-tdTomato/LSL-tdTomato(KT);Ptf1aCreER;Trp53fl/fl) model at 84 day after tamoxifen injection resulted in ~25% injected activity per cubic centimeter (IA/cc) accumulation in metastases and at ~18% IA/cc retention in pancreatic tumors within 30 minutes of tail vein injection with minimal background accumulation. Ex vivo PET imaging confirmed the localized accumulation of 64Cu-C4BP in transgenic mice within the colon, pancreas, and liver. Blocking of 64Cu-C4BP accumulation with the cold compound in a xenograft model (NOD/SCID) bearing Capan-1 PDAC tumors demonstrated the specificity of 64Cu-C4BP binding. Here we report that CLDN4 is a promising target and 64Cu-C4BP is a promising ligand for the detection of pancreatic cancer. Citation Format: Jai Woong Seo, James Wang, Aris J. Kare, Spencer K. Tumbale, Bo Wu, Marina N. Raie, Mallesh Pandrala, Katherine W. Ferrara. PET/CT imaging of pancreatic ductal adenocarcinoma with a claudin-4 binding peptide in preclinical mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4153.

Abstract 5981: Treatment of cholangiocarcinoma using humanized monoclonal antibodies targeting claudin-1

Abstract Introduction: Cholangiocarcinoma (CCA) is a highly aggressive adenocarcinoma of the hepatobiliary system showing an alarming rise in incidence and mortality with unsatisfactory treatment options. Claudin-1 (CLDN1) is a transmembrane protein expressed in tight junctions, but exposed at the cell surface on cancer epithelial cells. Using highly specific monoclonal antibodies (mAbs) targeting the extracellular loop 1 of exposed CLDN1 with an excellent safety profile (Roehlen, Saviano et al. Science Transl Med 2022), we aimed to investigate the role of CLDN1 as therapeutic target for CCA. Methods: Integrative CCA patient CLDN1 expression analyses, spatial transcriptomics and mouse models were used to evaluate the role of CLDN1 as an oncogenic driver for CCA. Proof-of-concept studies were performed in cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse as well as patient CCA organoid models using humanized CLDN1 mAbs. Results: Integrative expression analyses of CLDN1 in CCA patient tissues revealed robust CLDN1 upregulation across several cohorts and patients with well-characterized driver mutations. scRNASeq and spatial transcriptomics of patient CCA showed that CLDN1 expression in cancer cells is associated with stemness, oncogenic signaling and EMT. Gain-of-function studies using an orthotopic HDTVi and syngeneic mouse model revealed a decrease in survival and an enhanced tumor growth, unraveling a functional role of CLDN1 as an oncogenic driver in CCA. Targeting exposed CLDN1 using highly CLDN1-specific mAb demonstrated a robust anti-tumoral effect across intra- and extra-hepatic CCA mouse models, with a significant inhibition of metastatic disease including models with medium or low CLDN1 expression. Functional studies in patient-derived CCA organoids demonstrated that CLDN1 mAb decreased cellular viability and altered cancer cell plasticity and fate. Mechanistically, CLDN1 mAb treatment suppressed gene expression of pathways mediating proliferation, stemness and EMT by inhibition of Notch1, SRC-FAK, and Hippo-YAP signaling. Conclusion: These results demonstrate that CLDN1 is a CCA driver and therapeutic target. The proof-of-concept studies in patient-derived models pave the way for the clinical development of CLDN1 mAbs to improve the outcomes of patients with advanced CCA. Citation Format: Zeina Nehme, Marion Muller, Emilie Crouchet, Frank Juehling, Julien Moehlin, Romain Désert, Jade Brochon, Fabio Del Zompo, Natascha Roehlen, Christine Thumann, Patrick Pessaux, Emanuele Felli, Aïna Venkatasamy, Patrice Marchand, Mihaela Alina Onea, Roberto Iacone, Markus Meyer, Alberto Toso, Nabeel Bardeesy, Lipika Goyal, Vikas Prakash Ranvir, Mirian Fernández-Vaquero, Mathias Heikenwälder, Tessa Ostyn, Tania Roskams, Patrice Laquerriere, Catherine Schuster, Laurent Mailly, Thomas F. Baumert. Treatment of cholangiocarcinoma using humanized monoclonal antibodies targeting claudin-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5981.

Abstract 2622: TUB-040, a novel Napi2b-targeting ADC built with ethynylphosphonamidate conjugation chemistry, demonstrates high and long-lasting anti-tumor efficacy via Topoisomerase-I inhibition and excellent tolerability predictive of a wide therapeutic window in humans

Abstract TUB-040 is a novel Antibody-Drug Conjugate (ADC) targeting Napi2b, a highly overexpressed target in ovarian cancer and lung adenocarcinoma. Napi2b is a member of the solute-carrier transporter family with eight transmembrane domains and an antibody-accessible extracellular loop, that regulates sodium-dependent phosphate homeostasis. Ethynylphosphonamidate conjugation chemistry was used to build TUB-040 with a homogenous DAR (drug-antibody-ratio) of 8 and the Topoisomerase I inhibitor Exatecan as payload, which is connected to a humanized, Fc-silenced IgG1 antibody targeting Napi2b using a cleavable linker system. In vitro, TUB-040 is characterized by sub-nanomolar affinity and specific binding to Napi2b, efficient target-mediated internalization and high cytotoxicity towards Napi2b-expressing ovarian and lung cancer cell lines as well as bystander activity against co-cultured target-negative cells. TUB-040 induces DNA damage and apoptosis in a dose-dependent fashion as well as markers of immunogenic cell death. In addition, no unspecific uptake and cytotoxicity in healthy, target-negative human cells could be detected, reducing the risk of target-independent toxicities. Pharmacokinetic analysis showed that - in contrast to maleimide-conjugated ADCs - TUB-040 is highly stable - and does not lose or transfer its linker-payload to serum proteins during blood circulation, thus enabling most efficient and continued delivery of Exatecan to the tumor. Across a variety of cell line derived xenograft (CDX) and patient derived xenograft (PDX) models of ovarian and non-small cell lung cancer, including models with low target expression, single dose administration of TUB-040 results in long-lasting tumor growth inhibition with high complete remission rates in vivo. The minimally effective dose (MED) leading to complete remission is 1 mg/kg. Preliminary repeat-dose toxicological assessment of TUB-040 in the pharmacologically relevant species cynomolgus monkey demonstrated that TUB-040 is well tolerated with no signs of lung toxicity and thrombocytopenia. Allometric scaling and PK/PD modelling predicts a human serum half-life greater 10 days and a therapeutic index in humans up to 55. Based on these results, TUB-040, designed with differentiating technology for optimal efficacy and tolerability, is ready for testing in clinical trials. Citation Format: Saskia Schmitt, Isabelle Mai, Paul Machui, Sarah Herterich, Danila Hauswald, Philipp Ochtrop, Philipp Cyprys, Izabela Kozlowska, Annabel Kitowski, Marcus Gerlach, Olivier Marcq, Pamela A. Trail, Dominik Schumacher, Marc-André Kasper, Günter Fingerle-Rowson, Björn Hock, Jonas Helma-Smets, Annette M. Vogl. TUB-040, a novel Napi2b-targeting ADC built with ethynylphosphonamidate conjugation chemistry, demonstrates high and long-lasting anti-tumor efficacy via Topoisomerase-I inhibition and excellent tolerability predictive of a wide therapeutic window in humans [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2622.