Human FcRn Research Articles

Bioengineered Nanomedicines Targeting the Intestinal Fc Receptor Achieve the Improved Glucoregulatory Effect of Semaglutide in a Type 2 Diabetic Mice Model.

The oral administration of the glucagon-like peptide-1 analogue, semaglutide, remains a hurdle due to its limited bioavailability. Herein, neonatal Fc receptor (FcRn)-targeted nanoparticles (NPs) were designed to enhance the oral delivery of semaglutide. The nanocarriers were covalently linked to the FcRn-binding peptide FcBP or the affibody molecule ZFcRn that specifically binds to the human FcRn (hFcRn) in a pH-dependent manner. These FcRn-targeted ligands were selected over the endogenous ligands of the receptor (albumin and IgG) due to their smaller size and simpler structure, which could facilitate the transport of functionalized NPs through the tissues. The capacity of FcRn-targeted semaglutide-NPs in controlling the blood glucose levels was evaluated in an hFcRn transgenic mice model, where type 2 diabetes mellitus (T2DM) was induced via intraperitoneal injection of nicotinamide followed by streptozotocin. The encapsulation of semaglutide into FcRn-targeted NPs was translated in an improved glucoregulatory effect in T2DM-induced mice when compared to the oral free semaglutide or nontargeted NP groups, after daily oral administrations for 7 days. Notably, a similar glucose-lowering response was observed between both FcRn-targeted NPs and the subcutaneous semaglutide groups. An increase in insulin pancreatic content and a recovery in β cell mass were visualized in the mice treated with FcRn-targeted semaglutide-NPs. The biodistribution of fluorescently labeled NPs through the gastrointestinal tract demonstrated that the nanosystems targeting the hFcRn are retained longer in the ileum and colorectum, where the expression of FcRn is more prevalent, than nontargeted NPs. Therefore, FcRn-targeted nanocarriers proved to be an effective platform for improving the pharmacological effect of semaglutide in a T2DM-induced mice model.

Neonatal Fc receptor is a functional receptor for classical human astrovirus.

Human astrovirus (HAstV) is a global cause of gastroenteritis in infants, the elderly, and the immunocompromised. However, the molecular mechanisms that control its susceptibility are not fully understood, as the functional receptor used by the virus has yet to be identified. Here, a genome-wide CRISPR-Cas9 library screen in Caco2 cells revealed that the neonatal Fc receptor (FcRn) can function as a receptor for classical HAstV (Mamastrovirus genotype 1). Deletion of FCGRT or B2M, which encode subunits of FcRn, rendered Caco2 cells and intestinal organoid cells resistant to HAstV infection. We also showed that human FcRn expression renders non-susceptible cells permissive to viral infection and that FcRn binds directly to the HAstV spike protein. Therefore, our findings provide insight into the entry mechanism of HAstV into susceptible cells. We anticipate that this information can be used to develop new therapies targeting human astroviruses, providing new strategies to treat this global health issue.

Engineering of pH-dependent antigen binding properties for toxin-targeting IgG1 antibodies using light-chain shuffling

Immunoglobulin G (IgG) antibodies that bind their cognate antigen in a pH-dependent manner (acid-switched antibodies) can release their bound antigen for degradation in the acidic environment of endosomes, while the IgGs are rescued by the neonatal Fc receptor (FcRn). Thus, such IgGs can neutralize multiple antigens over time and therefore be used at lower doses than their non-pH-responsive counterparts. Here, we show that light-chain shuffling combined with phage display technology can be used to discover IgG1 antibodies with increased pH-dependent antigen binding properties, using the snake venom toxins, myotoxin II and α-cobratoxin, as examples. We reveal differences in how the selected IgG1s engage their antigens and human FcRn and show how these differences translate into distinct cellular handling properties related to their pH-dependent antigen binding phenotypes and Fc-engineering for improved FcRn binding. Our study showcases the complexity of engineering pH-dependent antigen binding IgG1s and demonstrates the effects on cellular antibody-antigen recycling.

Prediction of human serum concentration–time profiles of therapeutic monoclonal antibodies using common marmosets (Callithrix jacchus): initial assessment with canakinumab, adalimumab, and bevacizumab

Cynomolgus monkeys and human FcRn transgenic mice are generally used for pharmacokinetic predictions of therapeutic monoclonal antibodies (mAbs). In the present study, the application of the common marmoset, a small nonhuman primate, as a potential animal model for prediction was evaluated for the first time. Canakinumab, adalimumab, and bevacizumab, which exhibited linear pharmacokinetics in humans, were selected as the model compounds. Marmoset pharmacokinetic data were reportedly available only for canakinumab, and those for adalimumab and bevacizumab were acquired in-house. Four pharmacokinetic parameters for a two-compartment model (i.e. clearance and volume of distribution in the central and peripheral compartments) in marmosets were extrapolated to the values in humans with allometric scaling using the average exponents of the three mAbs. As a result, the observed human serum concentration–time curves of the three mAbs following intravenous administration and those of canakinumab and adalimumab following subcutaneous injections (with an assumed absorption rate constant and bioavailability) were reasonably predicted. Although further prediction studies using a sufficient number of other mAbs are necessary to evaluate the versatility of this model, the findings indicate that marmosets can be an alternative to preceding animals for human pharmacokinetic predictions of therapeutic mAbs.

Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation

The Spike of SARS-CoV-2 recognizes a transmembrane protease, angiotensin-converting enzyme 2 (ACE2), on host cells to initiate infection. Soluble derivatives of ACE2, in which Spike affinity is enhanced and the protein is fused to Fc of an immunoglobulin, are potent decoy receptors that reduce disease in animal models of COVID-19. Mutations were introduced into an ACE2 decoy receptor, including adding custom N-glycosylation sites and a cavity-filling substitution together with Fc modifications, which increased the decoy's catalytic activity and provided small to moderate enhancements of pharmacokinetics following intravenous and subcutaneous administration in humanized FcRn mice. Most prominently, sialylation of native glycans increases exposures by orders of magnitude, and the optimized decoy is therapeutically efficacious in a mouse COVID-19 model. Ultimately, an engineered and highly sialylated decoy receptor produced using methods suitable for manufacture of representative drug substance has high exposure with a 5- to 9-day half-life. Finally, peptide epitopes at mutated sites in the decoys generally have low binding to common HLA class II alleles and the predicted immunogenicity risk is low. Overall, glycosylation is a critical molecular attribute of ACE2 decoy receptors and modifications that combine tighter blocking of Spike with enhanced pharmacokinetics elevate this class of molecules as viable drug candidates.

Monoclonal Antibodies Engineered with Fc Region Mutations to Extend Protection against Fentanyl Toxicity.

Fentanyl and other synthetic opioids are the leading cause of drug-related deaths in the United States. mAbs that selectively target fentanyl and fentanyl analogues offer a promising strategy for treating both opioid-related overdoses and opioid use disorders. To increase the duration of efficacy of a candidate mAb against fentanyl, we selected three sets of mutations in the Fc region of an IgG1 anti-fentanyl mAb (HY6-F9DF215, HY6-F9DHS, HY6-F9YTE) to increase binding to the neonatal Fc receptor (FcRn). The mAb mutants were compared against unmodified (wild-type [WT], HY6-F9WT) anti-fentanyl mAb for fentanyl binding, thermal stability, and FcRn affinity invitro, and for efficacy against fentanyl and mAb half-life invivo in mice. Biolayer interferometry showed a >10-fold increase in the affinity for recombinant FcRn of the three mutant mAbs compared with HY6-F9WT. During an acute fentanyl challenge in mice, all FcRn-mutated mAbs provided equal protection against fentanyl-induced effects, and all mAbs reduced brain fentanyl levels compared with the saline group. Serum persistence of the mutant mAbs was tested in Tg276 transgenic mice expressing human FcRn. After administration of 40 mg/kg HY6-F9WT, HY6-F9DF215, HY6-F9DHS, and HY6-F9YTE, the mAbs showed half-lives of 6.3, 26.4, 14.7, and 6.9 d, respectively. These data suggest that modification of mAbs against fentanyl to bind to FcRn with higher affinity can increase their half-life relative to WT mAbs while maintaining efficacy against the toxic effects of fentanyl, further supporting their potential role as a therapeutic treatment option for opioid use disorder and overdose.

Cellular Neonatal Fc Receptor Recycling Efficiencies can Differentiate Target-Independent Clearance Mechanisms of Monoclonal Antibodies

In vivo clearance mechanisms of therapeutic monoclonal antibodies (mAbs) encompass both target-mediated and target-independent processes. Two distinct determinants of overall mAb clearance largely separate of target-mediated influences are non-specific cellular endocytosis and subsequent pH-dependent mAb recycling mediated by the neonatal Fc receptor (FcRn), where inter-mAb variability in the efficiency of both processes is observed. Here, we implemented a functional cell-based FcRn recycling assay via Madin-Darby canine kidney type II cells stably co-transfected with human FcRn and its light chain β2-microglobulin. Next, a series of pH-dependent internalization studies using a model antibody demonstrated proper function of the human FcRn complex. We then applied our cellular assays to assess the contribution of both FcRn and non-specific interactions in the cellular turnover for a panel of 8 clinically relevant mAbs exhibiting variable human pharmacokinetic behavior. Our results demonstrate that the interplay of non-specific endocytosis rates, pH-dependent non-specific interactions, and engagement with FcRn all contribute to the overall recycling efficiency of therapeutic monoclonal antibodies. The predictive capacity of our assay approach was highlighted by successful identification of all mAbs within our panel possessing clearance in humans greater than 5 mL/day/kg. These results demonstrate that a combination of cell-based in vitro assays can properly resolve individual mechanisms underlying the overall in vivo recycling efficiency and non-target mediated clearance of therapeutic mAbs.

GenSci120, a humanized PD-1 agonistic monoclonal antibody, enhances the binding of both PD-L1 and PD-L2 to PD-1, and mitigates the symptoms of GVHD in a mouse model

Abstract Background: Autoimmune diseases impose tremendous health and economic burden to the individuals and families they affect. The fundamental mechanism of autoimmune diseases is a break in immune tolerance and homeostasis. PD-1 plays an important role in maintaining immune tolerance by antagonizing CD28/TCR signaling in autoreactive T cells. Stimulating the PD-1 co-inhibitory pathway by an agonistic antibody would suppress pathogenic immune responses and restore immune homeostasis. Methods: A PD-1 agonistic monoclonal antibody, GenSci120, was generated by Beacon single plasma cell cloning and murine antibody humanization technologies. In vitro T cell inhibition, ADCC and ligand blocking activities of GenSci120 were characterized. In vivo efficacy in a mouse GVHD model and PK in the human FcRn transgenic mice were evaluated. Results: GenSci120 exhibited potent T cell inhibition activity in a luciferase reporter assay and a primary T cell proliferation assay. GenSci120 also showed ADCC activity in an LDH release assay. Remarkably, GenSci120 enhanced the binding of both PD-L1 and PD-L2 to PD-1 in a flow cytometry assay. In a mouse model, GenSci120 significantly alleviated the symptoms of GVHD. Furthermore, GenSci120 demonstrated longer half-life than competitors’ antibodies in the human FcRn transgenic mice. Conclusion: These data suggest the potential of GenSci120 as a novel therapy to treat autoimmune disorders and support the evaluation of this molecule in clinical studies.

Targeting CEACAM5-positive solid tumors using NILK-2401, a novel CEACAM5xCD47 κλ bispecific antibody.

Blocking the CD47 "don't eat me"-signal on tumor cells with monoclonal antibodies or fusion proteins has shown limited clinical activity in hematologic malignancies and solid tumors thus far. Main side effects are associated with non-tumor targeted binding to CD47 particularly on blood cells. We present here the generation and preclinical development of NILK-2401, a CEACAM5×CD47 bispecific antibody (BsAb) composed of a common heavy chain and two different light chains, one kappa and one lambda, determining specificity (so-called κλ body format). NILK-2401 is a fully human BsAb binding the CEACAM5 N-terminal domain on tumor cells by its lambda light chain arm with an affinity of ≈4 nM and CD47 with its kappa chain arm with an intendedly low affinity of ≈500 nM to enabling tumor-specific blockade of the CD47-SIRPα interaction. For increased activity, NILK-2401 features a functional IgG1 Fc-part. NILK-2401 eliminates CEACAM5-positive tumor cell lines (3/3 colorectal, 2/2 gastric, 2/2 lung) with EC50 for antibody-dependent cellular phagocytosis and antibody-dependent cellular cytotoxicity ranging from 0.38 to 25.84 nM and 0.04 to 0.25 nM, respectively. NILK-2401 binds neither CD47-positive/CEACAM5-negative cell lines nor primary epithelial cells. No erythrophagocytosis or platelet activation is observed. Quantification of the pre-existing NILK-2401-reactive T-cell repertoire in the blood of 14 healthy donors with diverse HLA molecules shows a low immunogenic potential. In vivo, NILK-2401 significantly delayed tumor growth in a NOD-SCID colon cancer model and a syngeneic mouse model using human CD47/human SIRPα transgenic mice and prolonged survival. In cynomolgus monkeys, single doses of 0.5 and 20 mg/kg were well tolerated; PK linked to anti-CD47 and Fc-binding seemed to be more than dose-proportional for Cmax and AUC0-inf. Data were validated in human FcRn TG32 mice. Combination of a CEACAM5-targeting T-cell engager (NILK-2301) with NILK-2401 can either boost NILK-2301 activity (Emax) up to 2.5-fold or allows reaching equal NILK-2301 activity at >600-fold (LS174T) to >3,000-fold (MKN-45) lower doses. NILK-2401 combines promising preclinical activity with limited potential side effects due to the tumor-targeted blockade of CD47 and low immunogenicity and is planned to enter clinical testing.

Abstract 7174: Pharmacokinetics and immunogenicity of anti-PD-1 antibody in humanized FcRn mouse models compared to immunocompetent mouse models

Abstract Introduction: The development and use of therapeutic antibodies in cancer therapy has increased significantly in recent years, led by the success of approved immune checkpoint blockade antibodies targeting PD-1 and PD-L1 axis. However, obtaining more clinically relevant pharmacokinetics (PK), safety and efficacy data for antibodies is still challenging in preclinical phase. Humanized FcRn (hFcRn) transgenic mouse model has a more faithful catabolism compared to WT mice, providing more accurate and predictable PK data of innovative antibody-based therapeutics at discovery stage[1]. In this study, we sought to investigate pharmacokinetics and immunogenicity characteristics of anti-PD-1 antibody in hFcRn transgenic mice and provide guidance for its differentiation of prediction value for drug PK and safety by comparing to WT mice. Methods: Non-tumor bearing hFcRn mice and wild type C57BL/6 mice were divided into 2 groups, 5 mice were enrolled into each group. Mice were administered anti-PD-1(Nivolumab) at 10 mg/kg single dose after grouping by intravenous (IV) injection. Blood samples at 12 timepoints (15min, 6h, 1, 2, 4, 7, 10, 14, 21, 28, 35, 42 days) post-dosing were collected for PK and Anti-Drug Antibody (ADA) analysis by MSD and ELISA. Results: Nivolumab clearance in hFcRn mice followed a typical linear profile with rapid distribution phase and slow elimination phase (t1/2 = 350 to 400 h). While the PK profile in C57BL/6 mice displayed non-linear clearance and very short elimination phase (t1/2 = 45 to 50 h). This data demonstrated that hFcRn mice have better correlated half-life with human (Nivolumab single dose by IV with 10mg/kg, t1/2 = 595 h) [2]. ADA analysis revealed that it was produced starting from Day 10 in wild type C57BL/6 mice and the frequency of ADA induced by Nivolumab in wild type mice (2/5) was higher than hFcRn mice (0/5). The variation of Nivolumab level was correlated with ADA production in wild type mice. This suggested hFcRn shows lower immunogenicity than wild type mice for human IgG drug and can potentially improve negative effect of ADA interference on PK evaluation. Conclusion: The result of this study demonstrated that the PK data in hFcRn mouse model was more predictive of human PK profile compared to wild type C57BL/6 mice. This advances innovation in antibody-based therapeutics and translational applications, with improved PK, safety and efficacy evaluation for preclinical human IgG drug development in cancer therapy.

Abstract 2668: CBO421: A novel drug Fc-conjugate to prevent tumor immune evasion via the CD73/adenosine pathway

Abstract Background CD73 (5’-nucleotidase or NT5E) is a cell surface enzyme responsible for the rate limiting step in adenosine production; the conversion of adenosine monophosphate (AMP) into adenosine. Adenosine contributes to immune evasion by solid tumors by concentrating immunosuppressive adenosine in the tumor microenvironment. CBO421, a Drug Fc-Conjugate (DFC), comprises a novel small molecule CD73 inhibitor stably conjugated to an immune-silent human IgG1 Fc, combining the strengths of small molecule inhibitors and CD73-targeting monoclonal antibodies (mAbs) for potential best-in-class efficacy. Methods CBO421’s binding affinity to human CD73 and Fc receptors was assessed by surface plasmon resonance. Antibody-dependent cellular cytotoxicity (ADCC) was evaluated using a commercial kit. Immunophenotyping and binding to cancer cells was measured by flow cytometry. Inhibition of soluble and membrane-bound CD73 on human PBMCs and mouse cancer cells was determined by measuring free phosphate levels. Functional activity was measured using PBMC rescue assays. In vivo efficacy of CBO421 monotherapy was evaluated in a syngeneic mouse model. Results CBO421 exhibited a binding affinity to human CD73 (KD = 0.8 nM) that was comparable to or greater to the affinity observed with small molecule inhibitors (AB680, OP-5244) and CD73-targeting mAbs (oleclumab, mupadolimab biosimilars). As expected, human FcRn binding of CBO421 was comparable to the unconjugated Fc and the full-length wild-type IgG1 control mAb. CBO421 binding to human Fc gamma receptors and ADCC activity was significantly reduced or abolished compared to the control mAb, such that it poses a minimal risk for undesired autoimmune activity. CBO421 potently inhibited soluble CD73 and CD73 expressed on human MDA-MB-231 and mouse EMT6 cancer cells. Immunophenotyping revealed that CD73 expression was primarily observed on CD8+ T and B cells from human PBMCs. In functional PBMC rescue assays, CBO421 demonstrated potent reactivation of AMP- or ATP-suppressed CD4+ or CD8+ T cells at levels comparable to small molecule inhibitors and greater than anti-CD73 mAb comparators. CBO421 monotherapy demonstrated a dose-dependent reduction in tumor volume in a syngeneic mouse model using MC38, a colorectal cancer cell line. Conclusions CBO421 exhibited strong binding and potent inhibition of both soluble and membrane-bound CD73, differentiating it from mAb CD73 inhibitors. The proprietary immune-silent Fc of CBO421 did not affect FcRn binding but substantially reduced Fc gamma receptor binding, leading to the elimination of ADCC activity to prevent undesired immune cell depletion in host tissues. CBO421 demonstrated high potency in functional cell-based assays and robust antitumor efficacy in a syngeneic mouse model. Currently, CBO421 is being advanced as a clinical development candidate for the treatment of solid cancers. Citation Format: Amanda Almaguer, Doug Zuill, Simon Döhrmann, James Levin, Nicholas Dedeic, Elizabeth Abelovski, Joanne Fortier, Qiping Zhao, Maria Hernandez, Karin Amundson, Madison Moniz, Hongyuan Chen, Dhanya Panickar, Thanh Lam, Tom Brady, Allen Borchardt, Grayson Hough, Jeffrey B. Locke, Jason N. Cole, Leslie W. Tari. CBO421: A novel drug Fc-conjugate to prevent tumor immune evasion via the CD73/adenosine pathway [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 2668.

Abstract 5975: Characterization and pharmacokinetics of ligand traps targeting the human epidermal growth factor receptor (HER) family

Abstract Antibodies targeting HER receptors have led to successful therapeutics for multiple human malignancies. Targeting the family of HER ligands provides an alternative strategy to inhibit signaling and subsequent proliferation of cancer cells. We have engineered ligand traps that bind the growth factors activating the HER family and have characterized a series of molecules (EN2000-Fc) using Surface Plasmon Resonance (SPR). Five distinct molecules were further evaluated for improved in vivo pharmacokinetics in mice that express human neonatal Fc receptor (FcRn). Mutations in the IgG Fc known to extend the in-vivo half-life of monoclonal antibodies were incorporated into the parental EN2000-Fc to generate 3 more distinct pan HER ligand traps. In addition, an EN2000-Fc variant with a full-length hinge was engineered, which is 5-amino-acid longer N-terminally than the parental molecule. These five molecules were synthesized (> 300 mg of each), purified (>98%), and characterized for in vitro and in vivo testing. Two variants were compared by evaluating the ligand binding affinity to 11 HER ligands using SPR. Both molecules showed comparable affinity against all ligands tested, confirming reproducibility of the pan HER ligand trap engineering technology. Among the ligands, HB-EGF had the highest affinity. High binding affinities to EGF, NRG1, NRG2, betacellulin and TGFα were also measured. The serum pharmacokinetics of the different ligand traps were compared in 6- to 8-week-old female C57BL/6 mice engineered to express human FcRn. Ligand traps were administered by single tail vein injection, and serum samples collected over a 28-day interval were analyzed by ELISA. Ligand traps with engineered Fc-mutations had 3- to 4-fold greater systemic exposure (area-under-the-curve) compared to ligand traps with the parental sequence, demonstrating improved in vivo characteristics of selected engineered fusion proteins. These results provide useful information for product candidate selection and for further preclinical development of a ligand trap targeting HER family receptors. Citation Format: Daniel C. Maneval, Cindy Yang, Cynthia Trevisiol, H. Michael Shepard, Pei Jin. Characterization and pharmacokinetics of ligand traps targeting the human epidermal growth factor receptor (HER) family [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 5975.

A Potent Neutralizing Monoclonal Antibody to Human Growth Hormone Suppresses Insulin-Like Growth Factor-1 in Female Rats.

Acromegaly and gigantism are disorders caused by hypersecretion of growth hormone (GH), usually from pituitary adenomas. Although somatostatin analogues (SSA), dopamine agonists, and GH receptor antagonists are important therapeutic agents, all of these have issues with their effectiveness, safety, and/or convenience of use. To overcome these, we developed a GH-specific potent neutralizing a mouse monoclonal antibody (mAb) named 13H02. 13H02 selectively bound both to human and monkey GH with high affinity, and strongly inhibited the biological activity of GH in the Nb2 rat lymphoma cell proliferation assay. In hypophysectomized/GH-supplemented rats, a single subcutaneous administration of 13H02 significantly and dose-dependently lowered the serum insulin-like growth factor-1 levels. To pursue the therapeutic potential of this antibody for acromegaly and gigantism, we humanized 13H02 to reduce its immunogenicity and applied a single amino acid mutation in the Fc region to extend its serum half-life. The resulting antibody, Hu-13H02m, also showed GH-specific neutralizing activity, similar to the parental 13H02, and showed improved binding affinity to human FcRn.

Human IgG Fc-engineering for enhanced plasma half-life, mucosal distribution and killing of cancer cells and bacteria

Monoclonal IgG antibodies constitute the fastest growing class of therapeutics. Thus, there is an intense interest to design more potent antibody formats, where long plasma half-life is a commercially competitive differentiator affecting dosing, frequency of administration and thereby potentially patient compliance. Here, we report on an Fc-engineered variant with three amino acid substitutions Q311R/M428E/N434W (REW), that enhances plasma half-life and mucosal distribution, as well as allows for needle-free delivery across respiratory epithelial barriers in human FcRn transgenic mice. In addition, the Fc-engineered variant improves on-target complement-mediated killing of cancer cells as well as both gram-positive and gram-negative bacteria. Hence, this versatile Fc technology should be broadly applicable in antibody design aiming for long-acting prophylactic or therapeutic interventions.

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.

P765 A Novel Monoclonal Antibody Drug Candidate SPY001 Targeting Integrin ɑ4β7 for the Treatment of IBD Demonstrates Prolonged Half-Life in Non-Human Primates

Abstract Background Background: Antagonism of the interaction between the cellular adhesion integrin ɑ4β7 and endothelial ligand mucosal addressin cell-adhesion molecule-1 (MAdCAM-1) has proven to be relatively safe and effective in the treatment for Crohn’s disease (CD) and ulcerative colitis (UC). Additional benefit may be gained from an ɑ4β7 antagonist given via the subcutaneous (SC) route at extended intervals (eg, every 8 to 12 weeks) during maintenance therapy. Methods The humanized monoclonal IgG1 antibody SPY001 binds to the same ɑ4β7 epitope as vedolizumab and includes a YTE modification within the Fc region to increase its serum half-life via increased affinity for the neonatal Fc receptor (FcRn), thereby favoring recycling into the circulation over degradation. Half-life extension was evaluated via pharmacokinetic analysis in Tg276 transgenic mice (hemizygous for human FcRn) following a single intravenous bolus dose of SPY001 (10 mg/kg, 5 mg/kg, 1 mg/kg) and in cynomolgus monkeys following a single bolus dose of SPY001 (150 mg/kg), given by either intravenous (IV) and subcutaneous (SC) administration. PK simulations in humans were based on allometric scaling of SPY001 clearance observed in cynomolgus monkey and conducted in MATLAB. Results In Tg276 mice, the half-life of SPY001 was 10-11 days across all dose cohorts compared to approximately 3 days for vedolizumab. In cynomolgus monkeys, the half-life of SPY001 was 19-23 days following a single IV or SC infusion, a significant increase over the reported half-life of 10 days for vedolizumab in cynomolgus monkeys. Based on allometric scaling of the clearance of SPY001 observed in this study, predictive simulations of SPY001 PK in humans suggested that Q8W-Q12W SC dosing at 300 mg would be able to achieve a C6-wks, induction ≥ 35 mg/mL and Ctrough, ss ≥ 6 mg/mL for simulated patients covering a large range of body weights and albumin concentrations. Conclusion SPY001 is a novel humanized monoclonal IgG1 with an extended half-life over that of vedolizumab in Tg276 mice and cynomolgus monkeys. It offers the 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.

Development and characterization of NILK-2301, a novel CEACAM5xCD3 κλ bispecific antibody for immunotherapy of CEACAM5-expressing cancers

BackgroundT-cell retargeting to eliminate CEACAM5-expressing cancer cells via CEACAM5xCD3 bispecific antibodies (BsAbs) showed limited clinical activity so far, mostly due to insufficient T-cell activation, dose-limiting toxicities, and formation of anti-drug antibodies (ADA).MethodsWe present here the generation and preclinical development of NILK-2301, a BsAb composed of a common heavy chain and two different light chains, one kappa and one lambda, determining specificity (so-called κλ body format).ResultsNILK-2301 binds CD3ɛ on T-cells with its lambda light chain arm with an affinity of ≈100 nM, and the CEACAM5 A2 domain on tumor cells by its kappa light chain arm with an affinity of ≈5 nM. FcγR-binding is abrogated by the “LALAPA” mutation (Leu234Ala, Leu235Ala, Pro329Ala). NILK-2301 induced T-cell activation, proliferation, cytokine release, and T-cell dependent cellular cytotoxicity of CEACAM5-positive tumor cell lines (5/5 colorectal, 2/2 gastric, 2/2 lung), e.g., SK-CO-1 (Emax = 89%), MKN-45 (Emax = 84%), and H2122 (Emax = 97%), with EC50 ranging from 0.02 to 0.14 nM. NILK-2301 binds neither to CEACAM5-negative or primary colon epithelial cells nor to other CEACAM family members. NILK-2301 alone or in combination with checkpoint inhibition showed activity in organotypic tumor tissue slices and colorectal cancer organoid models. In vivo, NILK-2301 at 10 mg/kg significantly delayed tumor progression in colon- and a pancreatic adenocarcinoma model. Single-dose pharmacokinetics (PK) and tolerability in cynomolgus monkeys at 0.5 or 10 mg/kg intravenously or 20 mg subcutaneously showed dose-proportional PK, bioavailability ≈100%, and a projected half-life in humans of 13.1 days. NILK-2301 was well-tolerated. Data were confirmed in human FcRn TG32 mice.ConclusionsIn summary, NILK-2301 combines promising preclinical activity and safety with lower probability of ADA-generation due to its format compared to other molecules and is scheduled to enter clinical testing at the end of 2023.

Cellular and In Vivo Preclinical Pharmacodynamics and Pharmacology of Nipocalimab, an Anti-FcRn Blocking Therapeutic Antibody (P10-5.011)

<h3>Objective:</h3> To characterize the mechanism of action, pharmacodynamics and pharmacology of nipocalimab in preclinical models. <h3>Background:</h3> Nipocalimab is a fully human antibody being evaluated in over 10 autoantibody diseases including MG and CIDP. Nipocalimab binds with high affinity to the neonatal Fc receptor (FcRn) inhibiting IgG recycling and inducing autoantibody destruction while permitting key immune functions including IgG production. Here, the molecular, cellular and in vivo pharmacology of nipocalimab are shown to be well aligned with the pharmacodynamic and pharmacological effects observed in MG patients. <h3>Design/Methods:</h3> Nipocalimab in vitro receptor occupancy (RO) was assessed in a fluorescence-activated cell sorting assay in human aortic endothelial cells. In vivo, RO in circulating monocytes and total serum IgG were evaluated in human FcRn transgenic mice (Tg32 strain) or cynomolgus monkeys after i.v. infusions of up to 100 or 300 mg/kg nipocalimab, respectively. <h3>Results:</h3> Nipocalimab exposure at picomolar concentrations results in full RO within 30 minutes and inhibits IgG recycling in endothelial cells. In vivo, full RO was established within 2 hours in mice and 4 hours in monkeys and maintained at picomolar concentrations. The onset of IgG lowering due to inhibition of IgG recycling was detected within 1–2 days and was maintained during the period of full FcRn saturation. Duration of RO in peripheral blood monocytes in both preclinical species was dose-dependent, as was the duration of IgG clearance, suggesting a relationship between RO and maintenance of an increased IgG clearance rate. Nipocalimab in vitro FcRn RO and IgG recycling correlated well with in vivo RO and IgG lowering. <h3>Conclusions:</h3> Nipocalimab is a potent FcRn blocker inducing rapid and sustained lowering of IgG and IgG autoantibodies in preclinical studies. These results are consistent with findings from the phase 1 healthy volunteer (NCT02828046) and phase 2 VIVACITY study in gMG patients (NCT03772587). <b>Disclosure:</b> Dr. Ling has received personal compensation for serving as an employee of Janssen Pharmaceuticals (Johnson&amp;Johnson). Dr. Ling has stock in Janssen Pharmaceuticals (Johnson&amp;Johnson). Dr. Ling has received intellectual property interests from a discovery or technology relating to health care. Dr. Ling has received intellectual property interests from a discovery or technology relating to health care. Dr. Choudhury has stock in Janssen Pharmaceuticals. Mr. Tyler has received personal compensation for serving as an employee of AVROBIO, INC . Mr. Tyler has received personal compensation for serving as an employee of Orna Therapeutics. Mr. Tyler has received personal compensation for serving as an employee of Genevant Sciences. Dr. Avery has received personal compensation for serving as an employee of Lyndra Therapeutics. Dr. Avery has received personal compensation for serving as an employee of Xilio Therapeutics. Dr. Avery has received personal compensation for serving as an employee of Kisbee Therapeutics. Dr. Avery has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Momenta Pharmaceutical. Dr. Avery has received stock or an ownership interest from Life Biosciences.

Abstract 2658: ZW251, a novel glypican-3-targeting antibody drug conjugate bearing a topoisomerase 1 inhibitor payload

Abstract Background: Glypican-3 (GPC3) is a cell-surface oncofetal glycoprotein frequently expressed in hepatocellular carcinoma (HCC) with minimal presence in normal adult tissues. ZW251 is an antibody-drug conjugate (ADC) targeting human GPC3, composed of a humanized IgG1 antibody conjugated to a novel camptothecin-based topoisomerase 1 inhibitor, ZD06519, via a maleimide anchor and a glycyl glycyl phenylalanyl glycine (GGFG)-aminomethyl (AM) cleavable linker. Materials and Methods: Extensive functional characterization was performed to assess the mechanism of action and therapeutic potential of the ZW251 ADC. Antibody binding to human and cynomolgus monkey GPC3 was assessed by surface plasmon resonance and flow cytometry. A screen of off-target binding and target specificity was conducted using a membrane proteome array. ZW251 antibody internalization in GPC3-expressing tumor cell lines was assessed by flow cytometry. In vitro ADC cytotoxicity against tumor monolayers and spheroids was assessed in a panel of HCC cell lines. Tumor cell co-culture assays were also performed to assess bystander-mediated cell killing by ZW251. The pharmacokinetic (PK) profile of the ZW251 antibody was assessed in Tg32 mice expressing human FcRn. Anti-tumor activity of ZW251 was investigated in a large panel of cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse models representing a range of GPC3 expression. Results: The ZW251 antibody backbone demonstrated nanomolar binding affinity to both human and cynomolgus monkey GPC3, and strong binding to target-expressing cancer cell lines. Rapid internalization of ZW251 antibody was observed in GPC3-expressing HCC cell lines. ZW251 exhibited potent and target-specific cytotoxicity in a panel of HCC cells cultured either in monolayer or as 3D spheroids. ZW251 showed effective bystander-mediated killing of GPC3 negative cancer cells when in co-culture with GPC3 positive cancer cells. The ZW251 antibody demonstrated a favorable PK profile in Tg32 mice. A single administration of ZW251 resulted in robust tumor growth inhibition of a large panel of CDX and PDX models representing a range of GPC3-expression. Overall, these results support the potential of ZW251 as a novel therapeutic agent against GPC3-bearing cancers. Citation Format: Laurence Madera, Andrea Hernández Rojas, Raffaele Colombo, Alex Wu, Chayne L. Piscitelli, Dunja Urosev, Allysha Bissessur, Chi Wing Cheng, Renee Duan, Catrina Kim, Kevin Yin, Vincent Fung, Kaylee Wu, Winnie Cheung, Diego A. Alonzo, Mark E. Petersen, Stuart D. Barnscher, Jamie R. Rich. ZW251, a novel glypican-3-targeting antibody drug conjugate bearing a topoisomerase 1 inhibitor payload [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 2658.

Evidence for the Role of a Second Fc-Binding Receptor in Placental IgG Transfer in Nonhuman Primates.

Transplacental transfer of maternal antibodies provides the fetus and newborn with passive protection against infectious diseases. While the role of the highly conserved neonatal Fc receptor (FcRn) in transfer of IgG in mammals is undisputed, recent reports have suggested that a second receptor may contribute to transport in humans. We report poor transfer efficiency of plant-expressed recombinant HIV-specific antibodies, including engineered variants with high FcRn affinity, following subcutaneous infusion into rhesus macaques close to parturition. Unexpectedly, unlike those derived from mammalian tissue culture, plant-derived antibodies were essentially unable to cross macaque placentas. This defect was associated with poor Fcγ receptor binding and altered Fc glycans and was not recapitulated in mice. These results suggest that maternal-fetal transfer of IgG across the three-layer primate placenta may require a second receptor and suggest a means of providing maternal antibody treatments during pregnancy while avoiding fetal harm. IMPORTANCE This study compared the ability of several human HIV envelope-directed monoclonal antibodies produced in plants with the same antibodies produced in mammalian cells for their ability to cross monkey and mouse placentas. We found that the two types of antibodies have comparable transfer efficiencies in mice, but they are differentially transferred across macaque placentas, consistent with a two-receptor IgG transport model in primates. Importantly, plant-produced monoclonal antibodies have excellent binding characteristics for human FcRn receptors, permitting desirable pharmacokinetics in humans. The lack of efficient transfer across the primate placenta suggests that therapeutic plant-based antibody treatments against autoimmune diseases and cancer could be provided to the mother while avoiding transfer and preventing harm to the fetus.