Subnanomolar Affinity Research Articles

Abstract 2479: Discovery of agonist CD28-specific single domain antibodies from alpaca

Abstract Upon immune activation, in addition to the first signal driven by the TCR-peptide/MHC interactions, the costimulatory signals are indispensable to optimal T-cell activation, the absence of which will trigger naive T cells to an anergic state. The CD28-CD80/CD86 pathway is widely known as the most classical and critical costimulatory signal for the initial activation of T cells. Bispecific T-cell engagers targeting CD3e can activate cytotoxic T-cells and have shown remarkable tumor regressions in clinical trials. However, the absence of a costimulatory signal through CD28 results in insufficient T-cell activation and early exhaustion. Combination of both CD3e and CD28 targeting antibodies offers attractive approach to optimally boost T-cell activity. Development of superagonistic CD28-targeting antibody has been paused after TeGenero‘s TGN1412 Phase 1 trial in 2006 due to life-threatening cytokine release syndrome. Unlike superagonist TGN1412, which activates T cells and induce cytokine storm, tumor targeting antigen and cross-linking dependent CD28 bispecific antibody is supposed to display little or no toxicity. In the present study, we explored Alpaca immunized phage library approach to generate cross-linking dependent agonist CD28 single domain antibody for bispecific antibody development. Alpacas were immunized subsequently with recombinant human CD28 extracellular domain and 293F cells stably expressing human CD28. Serum samples after immunizations showed decent immune response against CD28. At the end of Boost 2 and 3, peripheral blood was collected and phage display libraries were constructed using VHH specific primers. Phage display libraries were panned against recombinant CD28 ECD protein and CHOK1 cells overexpressing CD28. After one round of panning, both protein and cell panning tracks showed robust enrichment in output titer and repeating sequences. Next, 600 clones were randomly picked for screening by the binding to U266B1 cells endogenously expressing CD28. Of the 326 clones showed positive binding to U266B1, 79 unique sequences were identified. Secondary screening by ELISA and FACS using periplasmic extract confirmed 69 unique sequences that bound to both CD28 protein and cells expressing CD28. Seventeen unique sequences were selected for VHH-Fc production. All purified VHH-Fc showed sub-nanomolar affinity by ELISA and bind to primary T cells by flow cytometry. Three VHH-Fc induced IL-2 production in a primary T cells activation assay only when cross-linked and in the presence of CD3 co-stimulation and are suitable for further bispecific T cell engager development. Citation Format: Yunyun Chen, Yannan Ding, Xiaoyan Liu, Qianqian Zhu, Wenfang Xin, Hu Liu, Teddy Yang, Yikai Qiu. Discovery of agonist CD28-specific single domain antibodies from alpaca [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 2479.

Abstract 2623: Enabling 5T4 for targeted cancer therapy: TUB-030, a novel ADC built with ethynylphosphonamidate conjugation chemistry, shows long-lasting anti-tumor activity via Topoisomerase-I inhibition with an optimized therapeutic index

Abstract TUB-030 is a novel Antibody-Drug Conjugate (ADC) targeting the oncofetal 5T4 antigen, a glycoprotein, that is functionally involved in regulation of the Wnt signalling pathway and promoting cellular motility and adhesion. Immunohistochemical analysis identified 5T4 overexpressed in many types of cancers, including bladder, lung, breast, stomach, esophagus, head and neck, colon, and ovarian. Analysis in fresh frozen healthy human tissue with the therapeutic mAb candidate of TUB-030 revealed limited cross reactivity. Ethynylphosphonamidate conjugation chemistry was used to build TUB-030 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 5T4 using a cleavable linker system. Overall, TUB-030 contrasts previous ADC designs targeting 5T4, implementing several layers of differentiation in terms of antibody, payload and conjugation technology. In vitro, TUB-030 is characterized by sub-nanomolar affinity to 5T4, efficient target-mediated internalization and strong cytotoxicity towards cancer cells from different tumor indications with a broad range of 5T4 expression levels. Differently to previous 5T4-targeting ADCs, TUB-030 has efficient bystander activity against co-cultured target-negative cells, which facilitates targeting of tumors with heterogenous 5T4 expression. Pharmacokinetic analysis showed that TUB-030 is highly stable - and does not lose or transfer its linker-payload to serum proteins during blood circulation and antibody-like clearance profile contrasting maleimide-conjugated ADCs and enabling most efficient and continued delivery of Exatecan to the tumor site. Single-dose treatment with TUB-030 results in long-lasting tumor regression with high complete remission rates across a variety of both cell-line derived xenograft (CDX) and patient-derived xenograft (PDX) cancer models of multiple tumor indications in vivo, including models with low target expression. Preliminary repeat-dose toxicological assessment of TUB-030 in a pharmacologically relevant non-human primate species demonstrated that TUB-030 is well tolerated. The preclinical results underline that TUB-030 with novel ADC technology has the potential to enable 5T4 as a therapeutic target and provide treatment options in numerous solid cancer indications with high unmet medical need supporting further development towards the clinic. 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. Enabling 5T4 for targeted cancer therapy: TUB-030, a novel ADC built with ethynylphosphonamidate conjugation chemistry, shows long-lasting anti-tumor activity via Topoisomerase-I inhibition with an optimized therapeutic index [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 2623.

Abstract 1341: SM2235: A bispecific EGFR x CD16A innate immune cell engager for enhanced treatment of EGFR-expressing solid tumors, regardless of RAS mutation status

Abstract Background: Monoclonal antibodies (mAbs) targeting the epidermal growth factor receptor (EGFR) are frontline therapies for numerous EGFR-dependent epithelial tumors. However, their efficacy faces challenges due to on-target skin toxicity and intrinsic or acquired resistance. Within the tumor microenvironment, innate immune cells, including macrophages and natural killer (NK) cells, play a significant role but are hindered by the lack of specific cancer cell targeting and a suppressive microenvironment. To enhance innate immune cell cytotoxicity, we have developed SM2235, a humanized tetravalent bispecific VHH antibody. SM2235 partially blocks EGFR and EGF interaction while conditionally activating innate immune cells through CD16A upon engagement with cancer cells. Methods: SM2235 was engineered by combining a partially blocking EGFR single-domain antibody with a high-affinity CD16A-activating single-domain antibody on an inactivated human IgG1 Fc backbone. The binding affinity and specificity were confirmed using bio-layer interferometry and ELISA with recombinant proteins, followed by flow cytometry with EGFR+ human cancer cell lines and CD16A+ primary NK cells. SM2235's efficacy in blocking EGFR-EGF interaction and downstream signaling was assessed through competitive ELISA and proliferation assays in both EGFR-dependent and independent cancer cell lines. Its ability to induce target-dependent NK cell and macrophage activation was evaluated through activation markers as well as cytotoxicity and phagocytosis assays. In vivo efficacy of SM2235 against EGFR+ human tumors was measured using xenograft assays in NSG mice, covering cetuximab-sensitive and resistant cancer cell lines. Preliminary pharmacokinetics and safety studies were conducted in cynomolgus monkeys. Results: SM2235 exhibits sub-nanomolar affinity for EGFR+ human cancer cell lines and CD16A+ primary human NK cells. It partially inhibits EGFR-dependent cancer cell proliferation. Compared to cetuximab, SM2235 significantly enhances target-dependent CD16A-mediated NK cell and macrophage activation and cytotoxicity against EGFR+ cancer cells in ADCC and ADCP assays in vitro. SM2235 effectively inhibits EGFR+ xenograft tumors in vivo, irrespective of KRAS mutation status. In cynomolgus monkeys, SM2235 demonstrates excellent tolerance with a 20-hour serum half-life of approximately 20 hours and no dose-dependent skin toxicity. Conclusions: Preclinical characterization of SM2235 shows promising in vivo efficacy against EGFR+ human cancers, regardless of KRAS mutation, with a favorable safety profile marked by minimal skin toxicity. Citation Format: Huiqin Geng, Wenjing Song, Xiaodan Liu, Shihao Lu, Hong Zhou, Simin Yang, Siyao Xie, Yi Wei, Xing Zhang, Sheila Zhou, Yanbin Liang. SM2235: A bispecific EGFR x CD16A innate immune cell engager for enhanced treatment of EGFR-expressing solid tumors, regardless of RAS mutation status [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1341.

Stabilization of F-actin by Salmonella effector SipA resembles the structural effects of inorganic phosphate and phalloidin

Entry of Salmonella into host enterocytes relies on its pathogenicity island 1 effector SipA. We found that SipA binds to F-actin in a 1:2 stoichiometry with sub-nanomolar affinity. A cryo-EM reconstruction revealed that SipA's globular core binds at the groove between actin strands, whereas the extended C-terminal arm penetrates deeply into the inter-strand space, stabilizing F-actin from within. The unusually strong binding of SipA is achieved by a combination of fast association via the core and very slow dissociation dictated by the arm. Similar to Pi, BeF3, and phalloidin, SipA potently inhibited actin depolymerization by actin depolymerizing factor (ADF)/cofilin, which correlated with increased filament stiffness, supporting the hypothesis that F-actin's mechanical properties contribute to the recognition of its nucleotide state by protein partners. The remarkably strong binding to F-actin maximizes the toxin's effects at the injection site while minimizing global influence on the cytoskeleton and preventing pathogen detection by the host cell.

Preclinical Characterization of DPI-4452: A 68Ga/177Lu Theranostic Ligand for Carbonic Anhydrase IX.

The membrane protein carbonic anhydrase IX (CAIX) is highly expressed in many hypoxic or von Hippel-Lindau tumor suppressor-mutated tumor types. Its restricted expression in healthy tissues makes CAIX an attractive diagnostic and therapeutic target. DPI-4452 is a CAIX-targeting cyclic peptide with a DOTA cage, allowing radionuclide chelation for theranostic purposes. Here, we report CAIX expression in multiple tumor types and provide invitro and invivo evaluations of 68Ga-labeled DPI-4452 ([68Ga]Ga-DPI-4452) and 177Lu-labeled DPI-4452 ([177Lu]Lu-DPI-4452). Methods: CAIX expression was assessed by immunohistochemistry with a panel of tumor and healthy tissues. The molecular interactions of complexed and uncomplexed DPI-4452 with CAIX were assessed by surface plasmon resonance and cell-binding assays. In vivo characterization of radiolabeled and nonradiolabeled DPI-4452 was performed in HT-29 colorectal cancer (CRC) and SK-RC-52 clear cell renal cell carcinoma (ccRCC) human xenograft mouse models and in healthy beagle dogs. Results: Overexpression of CAIX was shown in several tumor types, including ccRCC, CRC, and pancreatic ductal adenocarcinoma. DPI-4452 specifically and selectively bound CAIX with subnanomolar affinity. In cell-binding assays, DPI-4452 displayed comparably high affinities for human and canine CAIX but a much lower affinity for murine CAIX, demonstrating that the dog is a relevant species for biodistribution studies. DPI-4452 was rapidly eliminated from the systemic circulation of beagle dogs. The highest uptake of [68Ga]Ga-DPI-4452 and [177Lu]Lu-DPI-4452 was observed in the small intestine and stomach, 2 organs known to express CAIX. Uptake in other organs (e.g., kidneys) was remarkably low. In HT-29 and SK-RC-52 xenograft mouse models, both [68Ga]Ga-DPI-4452 and [177Lu]Lu-DPI-4452 showed tumor-selective uptake; in addition, [177Lu]Lu-DPI-4452 significantly reduced tumor growth. These results demonstrated the theranostic potential of DPI-4452. Conclusion: DPI-4452 selectively targets CAIX. [68Ga]Ga-DPI-4452 and [177Lu]Lu-DPI-4452 localized to tumors and were well tolerated in mice. [177Lu]Lu-DPI-4452 demonstrated strong tumor growth inhibition in 2 xenograft mouse models. Thus, the 2 agents potentially provide a theranostic approach for selecting and treating patients with CAIX-expressing tumors such as ccRCC, CRC, and pancreatic ductal adenocarcinoma.

Illuminating Neuropeptide Y Y4 Receptor Binding: Fluorescent Cyclic Peptides with Subnanomolar Binding Affinity as Novel Molecular Tools.

The neuropeptide Y (NPY) Y4 receptor (Y4R), a member of the family of NPY receptors, is physiologically activated by the linear 36-amino acid peptide pancreatic polypeptide (PP). The Y4R is involved in the regulation of various biological processes, most importantly pancreatic secretion, gastrointestinal motility, and regulation of food intake. So far, Y4R binding affinities have been mostly studied in radiochemical binding assays. Except for a few fluorescently labeled PP derivatives, fluorescence-tagged Y4R ligands with high affinity have not been reported. Here, we introduce differently fluorescence-labeled (Sulfo-Cy5, Cy3B, Py-1, Py-5) Y4R ligands derived from recently reported cyclic hexapeptides showing picomolar Y4R binding affinity. With pKi values of 9.22-9.71 (radioligand competition binding assay), all fluorescent ligands (16-19) showed excellent Y4R affinity. Y4R saturation binding, binding kinetics, and competition binding with reference ligands were studied using different fluorescence-based methods: flow cytometry (Sulfo-Cy5, Cy3B, and Py-1 label), fluorescence anisotropy (Cy3B label), and NanoBRET (Cy3B label) binding assays. These experiments confirmed the high binding affinity to Y4R (equilibrium pKd: 9.02-9.9) and proved the applicability of the probes for fluorescence-based Y4R competition binding studies and imaging techniques such as single-receptor molecule tracking.

Pharmacologic Characterization of Substituted Nitazenes at μ, κ, and Δ Opioid Receptors Suggests High Potential for Toxicity.

The benzimidazole opioids (substituted nitazenes) are highly potent μ opiod receptor (MOR) agonists with heroin- or fentanyl-like effects. These compounds have caused hospitalizations and fatal overdoses. We characterized the in vitro pharmacology and structure-activity relationships of 19 nitazenes with substitutions at three positions of the benzimidazole core. Affinities were assessed using agonist radioligand binding assays at human μ, κ, and Δ opioid receptors (MOR, KOR, and DOR, respectively) heterologously expressed in CHO cells. Notably, for MOR binding, nine substituted nitazenes had significantly higher affinities than fentanyl including N-pyrrolidino etonitazene, N-pyrrilidino isonitazene, and N-desethyl isotonitazene; 13 had subnanomolar affinities. Only metodesnitazene and flunitazene had significantly lower affinities than fentanyl. Affinities for the substituted nitazenes at KOR and DOR relative to MOR were 46- to 2580-fold and 180- to 1280-fold lower, respectively. Functional activities were assessed using [35S]GTPγS binding assays. Four nitazenes had subnanomolar potencies at MOR: N-pyrrolidino etonitazene, N-pyrrilidino isonitazene, N-pyrrilidino protonitazene and N-desethyl isotonitazene. Ten substituted nitazenes had significantly higher potencies than fentanyl. All tested nitazenes were full MOR agonists. Potencies at KOR and DOR relative to MOR were 7.3- to 7920-fold and 24- to 9400-fold lower, respectively. Thus, many of these compounds are high affinity/high potency MOR agonists with elevated potential to elicit toxicity and overdose at low doses. SIGNIFICANCE STATEMENT: Substituted nitazenes are a growing public health threat. Although the 19 nitazenes tested vary in their opioid receptor pharmacology, a number are very high affinity, high potency, and high efficacy compounds- higher than fentanyl. Their pharmacology suggests high potential for harm.

IL-33-binding HpARI family homologues with divergent effects in suppressing or enhancing type 2 immune responses.

HpARI is an immunomodulatory protein secreted by the intestinal nematode Heligmosomoides polygyrus bakeri, which binds and blocks IL-33. Here, we find that the H. polygyrus bakeri genome contains three HpARI family members and that these have different effects on IL-33-dependent responses in vitro and in vivo, with HpARI1+2 suppressing and HpARI3 amplifying these responses. All HpARIs have sub-nanomolar affinity for mouse IL-33; however, HpARI3 does not block IL-33-ST2 interactions. Instead, HpARI3 stabilizes IL-33, increasing the half-life of the cytokine and amplifying responses to it in vivo. Together, these data show that H. polygyrus bakeri secretes a family of HpARI proteins with both overlapping and distinct functions, comprising a complex immunomodulatory arsenal of host-targeted proteins.

P911 Development and Characterization of SPY002, a Novel Extended Half-life Monoclonal Antibody Drug Candidate Targeting TL1A for the Treatment of IBD

Abstract Background Tumor necrosis factor (TNF)-like ligand 1A (TL1A) mediates a broad spectrum of pro-inflammatory and fibrotic effects and is implicated in the pathogenesis of several immunologic disorders. Blockade of the interaction of TL1A with its cognate receptor, DR3, has been shown to ameliorate disease activity patients with Crohn’s disease (CD) and ulcerative colitis (UC). SPY002 is a novel, extended half-life fully human IgG1 monoclonal antibody (mAb) that binds soluble TL1A with high affinity and specificity, and potently inhibits TL1A-mediated signaling. Methods SPY002 was evaluated in multiple in vitro and ex vivo assays compared to other clinical anti-TL1A mAbs (PRA023/MK-7240, RVT-3101, and TEV-48574). Binding affinity to soluble TL1A was determined by surface plasmon resonance (SPR) and binding to membrane-bound TL1A was confirmed by flow cytometry (FACS). Competitive blockade of TL1A binding to cell-surface DR3 was determined by FACS, and competition against the decoy receptor, DcR3, was evaluated by ELISA. The binding epitope of SPY002 was determined using cryogenic electron microscopy. TF-1 cell apoptosis and a primary human whole blood-based assay measuring IFNg secretion were used to assess functional blockade of exogenously added TL1A. Half-life extension was measured via pharmacokinetic analysis in both Tg276 transgenic mice (hemizygous for human FcRn) and cynomolgus monkeys given a single bolus of SPY002 by intravenous and/or subcutaneous administration. Results SPY002 binds specifically to monomeric and trimeric TL1A and not to related TNF super family proteins TNF, FasL, TRAIL, or LIGHT. SPY002 demonstrates sub-nanomolar affinity for soluble TL1A and binds to TL1A over-expressed on the surface of human embryonic kidney (HEK) cells. SPY002 potently inhibits both TL1A-induced apoptosis of TF-1 cells and TL1A-induced secretion of IFNg in human whole blood, with IC50 values comparable to those of RVT-3101 and TEV-48574, and a substantially lower IC50 than that of PRA023/MK-7240. The half-life of SPY002 is significantly extended in Tg276 mice compared to RVT-3101. In cynomolgus monkeys, the half-life of SPY002 is at least 18 days, compared to an observed half-life of 9-12 days for RVT-3101 and PRA023/MK-7240. Conclusion SPY002 exhibits high selectivity and affinity for TL1A, demonstrates effective blockade of the TL1A interaction with DR3, and potently inhibits downstream cellular signaling. With an extended half-life in NHP, SPY002 demonstrates therapeutic potential for effective and safe treatment of CD and UC with the advantage of infrequent SC dosing. Further preclinical and clinical studies are warranted to demonstrate this potential.

Peptide recognition by a synthetic receptor at subnanomolar concentrations.

This paper describes the discovery and characterization of a dipeptide sequence, Lys-Phe, that binds to the synthetic receptor cucurbit[8]uril (Q8) in neutral aqueous solution with subnanomolar affinity when located at the N-terminus. The thermodynamic and structural basis for the binding of Q8 to a series of four pentapeptides was characterized by isothermal titration calorimetry, NMR spectroscopy, and X-ray crystallography. Submicromolar binding affinity was observed for the peptides Phe-Lys-Gly-Gly-Tyr (FKGGY, 0.3 μM) and Tyr-Leu-Gly-Gly-Gly (YLGGG, 0.2 μM), whereas the corresponding sequence isomers Lys-Phe-Gly-Gly-Tyr (KFGGY, 0.3 nM) and Leu-Tyr-Gly-Gly-Gly (LYGGG, 1.2 nM) bound to Q8 with 1000-fold and 170-fold increases in affinity, respectively. To our knowledge, these are the highest affinities reported between a synthetic receptor and an unmodified peptide. The high-resolution crystal structures of the Q8·Tyr-Leu-Gly-Gly-Gly and Q8·Leu-Tyr-Gly-Gly-Gly complexes have enabled a detailed analysis of the structural determinants for molecular recognition. The high affinity, sequence-selectivity, minimal size of the target binding site, reversibility in the presence of a competitive guest, compatibility with aqueous media, and low toxicity of Q8 should aid in the development of applications involving low concentrations of target polypeptides.

Abstract PR017: Discovery and clinical evaluation of a potent and selective A2A/2B dual receptor antagonist

Abstract Elevated adenosine levels present in the tumor microenvironment (TME) produce a net immunosuppressive effect through inhibition of T cell function by two mechanisms of action: Direct T cell effects via A2A receptor agonism and indirect T cell effects via agonism of A2A and A2B receptors on myeloid cells. Our team sought to identify a small molecule dual antagonist of the A2A and A2B receptors with the ability to decrease the immunosuppressive effects of adenosine in the TME and restore anti-tumor immune response. Drawing on our prior experience in the design of A2A receptor antagonists for the potential treatment of Parkinson’s disease, we developed a molecule with sub-nanomolar and single-digit nanomolar affinities for the A2A and A2B receptors respectively and greater than 100-fold selectivity over the related A1 and A3 receptors. A single dose assessment of this molecule in human subjects demonstrated the ability to achieve >99% target engagement (TE) at the A2A receptor and >90% TE at the A2B receptor at trough concentration. This presentation will, for the first time, describe the discovery and early clinical evaluation of this molecule, including disclosure of the structure, human pharmacokinetics, safety, and tolerability. Citation Format: Duane DeMong, Sheila Ranganath, Jared Cumming, Matthew Larsen, Yonglian Zhang, Christopher Plummer, Amjad Ali, Anthony Palmieri, Evan Barry, Pierre Daublain, Pranav Gupta, Manash Chatterjee, Vincent Giranda, Jeremy Presland, Sebastian Schneider, Paul Ciaccio, Daniel Tatosian, Aaron Sather, Ben Turnbull, Steven Silverman, Harry Chobanian, Harini Krishnamurthy, Richard Wnek, Roshi Afshar, Stephen Crowley, Alita Miller, Mark Ayers, Alan Whitehead, Marlene Hinton, Derek Chiang, Robert Orr, Jill Chrencik. Discovery and clinical evaluation of a potent and selective A2A/2B dual receptor antagonist [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr PR017.

Discovery of a Tritiated Radioligand with High Affinity and Selectivity for the Histamine H3 Receptor.

Radioligands used previously for histamine H3 receptor (H3R) are accompanied by a number of disadvantages. In this study, we report the synthesis of the new H3R radioligand [3H]UR-MN259 ([3H]11) with high (radio)chemical purity and stability. The radioligand exhibits sub-nanomolar affinity for the target receptor (pKi (H3R) = 9.56) and displays an outstanding selectivity profile within the histamine receptor family (>100,000-fold selective). [3H]UR-MN259 is ideally suitable for the characterization of H3R ligands in competition binding and shows one-site binding to the H3R in saturation binding experiments. The radiotracer shows fast association to the receptor (τassoc = 6.11 min), as well as full dissociation from the receptor (τdissoc = 14.48 min) in kinetic binding studies. The distinguished profile of [3H]UR-MN259 makes it a highly promising pharmacological tool to further investigate the role of the H3R in the CNS.

Next generation fibroblast activation protein (FAP) targeting PET tracers - The tetrazine ligation allows an easy and convenient way to 18F-labeled (4-quinolinoyl)glycyl-2-cyanopyrrolidines

Small-molecular fibroblast activation protein inhibitor (FAPI)-based tracer have been shown to be promising Positron Emission Tomography (PET) 68Ga-labeled radiopharmaceuticals to image a variety of tumors including pancreatic, breast, and colorectal cancers, among others. In this study, we developed a novel 18F-labeled FAPI derivative. [18F]6 was labeled using a synthon approach based on the tetrazine ligation. It showed subnanomolar affinity for the FAP protein and a good selectivity profile against known off-target proteases. Small animal PET studies revealed high tumor uptake and good target-to-background ratios. [18F]6 was excreted via the liver. Overall, [18F]6 showed promising characteristics to be used as a PET tracer and could serve as a lead for further development of halogen-based theranostic FAPI radiopharmaceuticals.

Trivalent nanobody-based ligands mediate powerful activation of GPVI, CLEC-2, and PEAR1 in human platelets whereas FcγRIIA requires a tetravalent ligand

BackgroundClustering of the receptors glycoprotein receptor VI (GPVI), C-type lectin-like receptor 2 (CLEC-2), low-affinity immunoglobulin γ Fc region receptor II-a (FcγRIIA), and platelet endothelial aggregation receptor 1 (PEAR1) leads to powerful activation of platelets through phosphorylation of tyrosine in their cytosolic tails and initiation of downstream signaling cascades. GPVI, CLEC-2, and FcγRIIA signal through YxxL motifs that activate Syk. PEAR1 signals through a YxxM motif that activates phosphoinositide 3-kinase. Current ligands for these receptors have an undefined valency and show significant batch variation and, for some, uncertain specificity. ObjectivesWe have raised nanobodies against each of these receptors and multimerized them to identify the minimum number of epitopes to achieve robust activation of human platelets. MethodsDivalent and trivalent nanobodies were generated using a flexible glycine-serine linker. Tetravalent nanobodies utilize a mouse Fc domain (IgG2a, which does not bind to FcγRIIA) to dimerize the divalent nanobody. Ligand affinity measurements were determined by surface plasmon resonance. Platelet aggregation, adenosine triphosphate secretion, and protein phosphorylation were analyzed using standardized methods. ResultsMultimerization of the nanobodies led to a stepwise increase in affinity with divalent and higher-order nanobody oligomers having sub-nanomolar affinity. The trivalent nanobodies to GPVI, CLEC-2, and PEAR1 stimulated powerful and robust platelet aggregation, secretion, and protein phosphorylation at low nanomolar concentrations. A tetravalent nanobody was required to activate FcγRIIA with the concentration-response relationship showing a greater variability and reduced sensitivity compared with the other nanobody-based ligands, despite a sub-nanomolar binding affinity. ConclusionThe multivalent nanobodies represent a series of standardized, potent agonists for platelet glycoprotein receptors. They have applications as research tools and in clinical assays.

FRI545 Durability Of Treatment Response With VRDN-001, A Full Antagonist Antibody To IGF-1 Receptor, In Patients With Thyroid Eye Disease (TED): Phase 1/2 Clinical Study

Abstract Disclosure: R. Douglas: Consulting Fee; Self; Horizon Therapeutics, Viridian Therapeutics. Research Investigator; Self; Viridian Therapeutics Inc., Horizon Therapeutics. S. Ugradar: Consulting Fee; Self; Viridian Therapeutics Inc. K. Cockerham: Advisory Board Member; Self; Viridian Therapeutics Inc., Horizon Therapeutics. Research Investigator; Self; Viridian Therapeutics Inc. Speaker; Self; Horizon Therapeutics. R.E. Turbin: Advisory Board Member; Self; Horizon Therapeutics. Research Investigator; Self; Viridian Therapeutics Inc. Stock Owner; Self; Viridian Therapeutics Inc. R. Tang: Consulting Fee; Self; Viridian Therapeutics Inc. Research Investigator; Self; Viridian Therapeutics Inc., Horizon Therapeutics. N. Nijhawan: Research Investigator; Self; Viridian Therapeutics Inc. D.J. O’Shaughnessy: Employee; Self; Viridian Therapeutics Inc. R.M. Summerfelt: Employee; Self; Viridian Therapeutics Inc. A. She: Employee; Self; Viridian Therapeutics Inc. B. Katz: Employee; Self; Viridian Therapeutics Inc. Introduction: Clinical and preclinical evidence demonstrate IGF-1 receptor (IGF-1R) antagonism reduces TED-related inflammation and proptosis. VRDN-001, a subnanomolar affinity, full antagonist antibody to IGF-1R, is being evaluated in a phase 1/2 double-masked RCT (NCT05176639) at 3, 10, or 20 mg/kg administered intravenously. Results from the first cohort of TED patients (10 mg/kg) are presented here. Methods: Adults with active moderate-to-severe TED and clinical activity score (CAS) ≥4 were randomized to 2 infusions 3 weeks apart of either 10 mg/kg VRDN-001 or placebo (3:1). Safety, tolerability, and efficacy through 12 weeks were assessed. Endpoints included overall responder rate (% of patients with ≥2 mm reduction in proptosis and ≥2 point reduction in CAS), proptosis responder rate (% of patients with ≥2 mm reduction), change from baseline in proptosis and CAS, proportion of patients with CAS decrease to 0 or 1, and diplopia resolution. Results: Baseline characteristics were similar between VRDN-001 (n=6) and placebo (n=2). After 2 infusions, at 6 weeks, the overall responder rate was 83% (5/6; VRDN-001) vs. 0% (0/2; placebo). Proptosis responder rate was 83% (5/6; VRDN-001) vs. 50% (1/2; placebo). Mean proptosis decreased by 2.4 mm (VRDN-001) vs. 1.0 mm (placebo). MRI analysis of proptosis, available for 4 of 6 drug-treated patients, confirmed proptosis improvement in each; MRI analysis showed slight worsening of proptosis in both placebo patients. CAS decreased to 0 or 1 for 83% (5/6; VRDN-001) vs. 0% (0/2; placebo), with mean decreases of 4.3 (VRDN-001) and 1.5 (placebo). In patients presenting with diplopia, complete resolution occurred for 75% (3/4; VRDN-001) vs. 0% (0/1; placebo). Durability of response in the 5 VRDN-001 treatment responders at 6 weeks persisted through 12 weeks: 80% (4/5) maintained overall responder status and 80% (4/5) maintained proptosis responder status, with mean reduction in proptosis at week 12 of 2.2 mm for all 6 drug-treated patients; MRI analysis confirmed the maintained response for all 4 of the drug-treated patients for whom scans were available. Mean CAS reduction remained consistent (4.2 at Week 12 vs. 4.3 at Week 6) and 80% (4/5) maintained a CAS of 0 or 1. All 4 patients with baseline diplopia had diplopia resolution at Week 12. VRDN-001 was well-tolerated through 12 weeks. AEs were mostly mild, with no severe or serious AEs reported. Conclusions: Two infusions of 10 mg/kg VRDN-001 were well tolerated in this cohort of TED patients, and the rapid, clinically meaningful improvement across all efficacy measures by 6 weeks was sustained through 12 weeks. Further, these results were achieved with a lower dose and fewer treatments than in prior RCTs of other anti-IGF-1R antibodies. Results from the additional 3 mg/kg and 20 mg/kg cohorts may extend these findings and define potential VRDN-001 treatment regimens. Presentation: Friday, June 16, 2023

“Redirecting an anti-IL-1β antibody to bind a new, unrelated and computationally predicted epitope on hIL-17A”

Antibody engineering technology is at the forefront of therapeutic antibody development. The primary goal for engineering a therapeutic antibody is the generation of an antibody with a desired specificity, affinity, function, and developability profile. Mature antibodies are considered antigen specific, which may preclude their use as a starting point for antibody engineering. Here, we explore the plasticity of mature antibodies by engineering novel specificity and function to a pre-selected antibody template. Using a small, focused library, we engineered AAL160, an anti-IL-1β antibody, to bind the unrelated antigen IL-17A, with the introduction of seven mutations. The final redesigned antibody, 11.003, retains favorable biophysical properties, binds IL-17A with sub-nanomolar affinity, inhibits IL-17A binding to its cognate receptor and is functional in a cell-based assay. The epitope of the engineered antibody can be computationally predicted based on the sequence of the template antibody, as is confirmed by the crystal structure of the 11.003/IL-17A complex. The structures of the 11.003/IL-17A and the AAL160/IL-1β complexes highlight the contribution of germline residues to the paratopes of both the template and re-designed antibody. This case study suggests that the inherent plasticity of antibodies allows for re-engineering of mature antibodies to new targets, while maintaining desirable developability profiles.

Widespread monoclonal IgE antibody convergence to an immunodominant, proanaphylactic Ara h 2 epitope in peanut allergy

Despite their central role in peanut allergy, human monoclonal IgE antibodies have eluded characterization. We sought to define the sequences, affinities, clonality, and functional properties of human monoclonal IgE antibodies in peanut allergy. We applied our single-cell RNA sequencing-based SEQ SIFTER discovery platform to samples from allergic individuals who varied by age, sex, ethnicity, and geographic location in order to understand commonalities in the human IgE response to peanut allergens. Select antibodies were then recombinantly expressed and characterized for their allergen and epitope specificity, affinity, and functional properties. We found striking convergent evolution of IgE monoclonal antibodies (mAbs) from several clonal families comprising both memory B cells and plasmablasts. These antibodies bound with subnanomolar affinity to the immunodominant peanut allergen Ara h 2, specifically a linear, repetitive motif. Further characterization of these mAbs revealed their ability to single-handedly cause affinity-dependent degranulation of human mast cells and systemic anaphylaxis on peanut allergen challenge in humanized mice. Finally, we demonstrated that these mAbs, reengineered as IgGs, inhibit significant, but variable, amounts of Ara h 2- and peanut-mediated degranulation of mast cells sensitized with allergic plasma. Convergent evolution of IgE mAbs in peanut allergy is a common phenomenon that can reveal immunodominant epitopes on major allergenic proteins. Understanding the functional properties of these molecules is key to developing therapeutics, such as competitive IgG inhibitors, that are able to stoichiometrically outcompete endogenous IgE for allergen and thereby prevent allergic cascade in cases of accidental allergen exposure.

Development and evaluation of nanobody tracers for noninvasive nuclear imaging of the immune-checkpoint TIGIT.

T cell Ig and ITIM domain receptor (TIGIT) is a next-generation immune checkpoint predominantly expressed on activated T cells and NK cells, exhibiting an unfavorable prognostic association with various malignancies. Despite the emergence of multiple TIGIT-blocking agents entering clinical trials, only a fraction of patients responded positively to anti-TIGIT therapy. Consequently, an urgent demand arises for noninvasive techniques to quantify and monitor TIGIT expression, facilitating patient stratification and enhancing therapeutic outcomes. Small antigen binding moieties such as nanobodies, are promising candidates for such tracer development. We generated a panel of anti-human or anti-mouse TIGIT nanobodies from immunized llamas. In addition, we designed a single-chain variable fragment derived from the clinically tested monoclonal antibody Vibostolimab targeting TIGIT, and assessed its performance alongside the nanobodies. In vitro characterization studies were performed, including binding ability and affinity to cell expressed or recombinant TIGIT. After Technetium-99m labeling, the nanobodies and the single-chain variable fragment were evaluated in vivo for their ability to detect TIGIT expression using SPECT/CT imaging, followed by ex vivo biodistribution analysis. Nine nanobodies were selected for binding to recombinant and cell expressed TIGIT with low sub-nanomolar affinities and are thermostable. A six-fold higher uptake in TIGIT-overexpressing tumor was demonstrated one hour post- injection with Technetium-99m labeled nanobodies compared to an irrelevant control nanobody. Though the single-chain variable fragment exhibited superior binding to TIGIT-expressing peripheral blood mononuclear cells in vitro, its in vivo behavior yielded lower tumor-to-background ratios at one hour post- injection, indicating that nanobodies are better suited for in vivo imaging than the single-chain variable fragment. Despite the good affinity, high specificity and on-target uptake in mice in this setting, imaging of TIGIT expression on tumor- infiltrating lymphocytes within MC38 tumors remained elusive. This is likely due to the low expression levels of TIGIT in this model. The excellent affinity, high specificity and rapid on-target uptake in mice bearing TIGIT- overexpressing tumors showed the promising diagnostic potential of nanobodies to noninvasively image high TIGIT expression within the tumor. These findings hold promise for clinical translation to aid patient selection and improve therapy response.

Development of Radiopharmaceuticals for NPY Receptor-5 (Y5) Nuclear Imaging in Tumors by Synthesis of Specific Agonists and Investigation of Their Binding Mode.

The neuropeptide-Y (NPY) family acts through four G protein-coupled receptor subtypes in humans, namely, Y1, Y2, Y4, and Y5. A growing body of evidence suggest the involvement of the NPY system in several cancers, notably the Y5 subtype, thus acting as a relevant target for the development of radiopharmaceuticals for imaging or targeted radionuclide therapy (TRT). Here, the [cPP(1-7),NPY(19-23),Ala31,Aib32,Gln34]hPP scaffold, further referred to as sY5ago, was modified with a DOTA chelator and radiolabeled with 68Ga and 111In and investigated in vitro and in vivo using the MCF-7 model. For in vivo studies, MCF-7 cells were orthotopically implanted in female nude mice and imaging with small animal positron emission tomography/computed tomography (μPET/CT) was performed. At the end of imaging, the mice were sacrificed. A scrambled version of sY5ago, which was also modified with a DOTA chelator, served as a negative control (DOTA-[Nle]sY5ago_scrambled). sY5ago and DOTA-sY5ago showed subnanomolar affinity toward the Y5 (0.9 ± 0.1 and 0.8 ± 0.1 nM, respectively) and a single binding site at the Y5 was identified. [68Ga]Ga-DOTA-sY5ago and [111In]In-DOTA-sY5ago were hydrophilic and showed high specific internalization (1.61 ± 0.75%/106 cells at 1 h) and moderate efflux (55% of total binding externalized at 45 min). On μPET/CT images, most of the signal was depicted in the kidneys and the liver. MCF-7 tumors were clearly visualized. On biodistribution studies, [68Ga]Ga-DOTA-sY5ago was eliminated by the kidneys (∼60 %ID/g). The kidney uptake is Y5-mediated. A specific uptake was also noted in the liver (5.09 ± 1.15 %ID/g vs 1.13 ± 0.21 %ID/g for [68Ga]Ga-DOTA-[Nle]sY5ago_scrambled, p < 0.05), the lungs (1.03 ± 0.34 %ID/g vs 0.20 %ID/g, p < 0.05), and the spleen (0.85 ± 0.09%ID/g vs 0.16 ± 0.16%ID/g, p < 0.05). In MCF-7 tumors, [68Ga]Ga-DOTA-sY5ago showed 12-fold higher uptake than [68Ga]Ga-DOTA-[Nle]sY5ago_scrambled (3.43 ± 2.32 vs 0.27 ± 0.15 %ID/g, respectively, p = 0.0008) at 1 h post-injection. Finally, a proof-of-principle tissular micro-imaging study on a human primary cancer sample showed weak binding of [111In]In-DOTA-sY5ago in prostatic intra-neoplasia and high binding in the ISUP1 lesion while normal prostate was free of signal.

Interactions of the Kv1.1 Channel with Peptide Pore Blockers: A Fluorescent Analysis on Mammalian Cells.

The voltage-gated potassium channel Kv1.1, which is abundant in the CNS and peripheral nervous system, controls neuronal excitability and neuromuscular transmission and mediates a number of physiological functions in non-excitable cells. The development of some diseases is accompanied by changes in the expression level and/or activity of the channels in particular types of cells. To meet the requirements of studies related to the expression and localization of the Kv1.1 channels, we report on the subnanomolar affinity of hongotoxin 1 N-terminally labeled with Atto 488 fluorophore (A-HgTx) for the Kv1.1 channel and its applicability for fluorescent imaging of the channel in living cells. Taking into consideration the pharmacological potential of the Kv1.1 channel, a fluorescence-based analytical system was developed for the study of peptide ligands that block the ion conductivity of Kv1.1 and are potentially able to correct abnormal activity of the channel. The system is based on analysis of the competitive binding of the studied compounds and A-HgTx to the mKate2-tagged human Kv1.1 (S369T) channel, expressed in the plasma membrane of Neuro2a cells. The system was validated by measuring the affinities of the known Kv1.1-channel peptide blockers, such as agitoxin 2, kaliotoxin 1, hongotoxin 1, and margatoxin. Peptide pore blocker Ce1, from the venom of the scorpion Centruroides elegans, was shown to possess a nanomolar affinity for the Kv1.1 channel. It is reported that interactions of the Kv1.1 channel with the studied peptide blockers are not affected by the transition of the channel from the closed to open state. The conclusion is made that the structural rearrangements accompanying the channel transition into the open state do not change the conformation of the P-loop (including the selectivity filter) involved in the formation of the binding site of the peptide pore blockers.