Fc Protein Research Articles

Engineered IgG Fc-conjugation prolongs the half-life of florfenicol and alleviates pneumonia in mice

Small molecule drugs often exhibit short half-lives, requiring frequent administrations to maintain therapeutic concentrations over an extended period. To address this issue, the fragment crystallizable (Fc) region of IgG, known to prolong the half-life of antibodies via its interaction with the Fc neonatal receptor, was harnessed as a carrier protein to extend the half-life of a small molecule drug, florfenicol. Florfenicol, was chemically coupled to a recombinant Fc protein expressed using the eukaryotic expression system in HEK293 cells. The Fc-florfenicol conjugate exhibited a substantially prolonged half-life of from 3.8 to 9.1 hours compared to unconjugated florfenicol and demonstrated excellent therapeutic properties in treating pneumonia in a mouse model. Our results, combined with the literature analysis on Fc-small molecule conjugates, show that Fc can substantially enhance the drug’s half-life and suggest the potential for its use as a carrier in novel delivery systems.

Chemically Programmed Fc Protein as Antibody Mimetic to Targeting Carbonic Anhydrase IX

Chemically Programmed Fc Protein as Antibody Mimetic to Targeting Carbonic Anhydrase IX

XTX301, a Tumor-Activated Interleukin-12 Has the Potential to Widen the Therapeutic Index of IL12 Treatment for Solid Tumors as Evidenced by Preclinical Studies.

IL12 is a proinflammatory cytokine, that has shown promising antitumor activity in humans by promoting the recruitment and activation of immune cells in tumors. However, the systemic administration of IL12 has been accompanied by considerable toxicity, prompting interest in researching alternatives to drive preferential IL12 bioactivity in the tumor. Here, we have generated XTX301, a tumor-activated IL12 linked to the human Fc protein via a protease cleavable linker that is pharmacologically inactivated by an IL12 receptor subunit beta 2 masking domain. In vitro characterization demonstrates multiple matrix metalloproteases, as well as human primary tumors cultured as cell suspensions, can effectively activate XTX301. Intravenous administration of a mouse surrogate mXTX301 demonstrated significant tumor growth inhibition (TGI) in inflamed and non-inflamed mouse models without causing systemic toxicities. The superiority of mXTX301 in mediating TGI compared with non-activatable control molecules and the greater percentage of active mXTX301 in tumors versus other organs further confirms activation by the tumor microenvironment-associated proteases in vivo. Pharmacodynamic characterization shows tumor selective increases in inflammation and upregulation of immune-related genes involved in IFNγ cell signaling, antigen processing, presentation, and adaptive immune response. XTX301 was tolerated following four repeat doses up to 2.0 mg/kg in a nonhuman primate study; XTX301 exposures were substantially higher than those at the minimally efficacious dose in mice. Thus, XTX301 has the potential to achieve potent antitumor activity while widening the therapeutic index of IL12 treatment and is currently being evaluated in a phase I clinical trial.

Treatment of Bleeding Episodes with Efanesoctocog Alfa in Children with Severe Hemophilia A in the XTEND-Kids Phase 3 Study

Introduction Efanesoctocog alfa (formerly BIVV001) is a new class of high sustained factor VIII (FVIII) replacement therapy, composed of a single recombinant FVIII protein fused to dimeric Fc protein, 2 XTEN polypeptides, and the D'D3 domain of von Willebrand factor (VWF), designed to overcome the endogenous VWF-imposed half-life ceiling. The XTEND-Kids (NCT04759131) study demonstrated once-weekly efanesoctocog alfa was well tolerated and provided effective treatment of and protection from bleeds in previously treated patients <12 years old with severe hemophilia A. Here, we provide a detailed report on efanesoctocog alfa as a treatment for bleeds during XTEND-Kids. Methods Previously treated patients <12 years old (≥150 exposure days [EDs] for patients 6 to <12 years of age and ≥50 EDs for patients <6 years old) with severe hemophilia A (<1 IU/dL [<1%] endogenous FVIII activity or a documented genotype associated with severe hemophilia) received efanesoctocog alfa 50 IU/kg once weekly for 52 weeks. In line with the study protocol, bleeds were treated with a single dose of efanesoctocog alfa (50 IU/kg). Additional doses (30 or 50 IU/kg, every 2-3 days) could be considered if needed. For minor/moderate bleeds occurring within 2-3 days of a recent prophylactic dose, a 30 IU/kg dose could also be used. The number and location of bleeds, as well as dose and number of efanesoctocog alfa injections required to resolve them were recorded. Patient assessment of the response to the first injection for treating a bleed was evaluated using the 4-point International Society on Thrombosis and Haemostasis (ISTH) scale (excellent, good, moderate, and none). Results Of 74 patients in XTEND-Kids, 73 were included in these post hoc analyses, including 38 subjects <6 years of age. One subject in the 6 to <12 group was excluded from these analyses as he did not receive weekly prophylaxis treatment as specified in the protocol, instead receiving intense consolidation treatment (2-3 injections per week for ~4 months) following 2 traumatic self-reported hip bleeds (however, no signs of bleeding or joint damage were observed by magnetic resonance imaging during the consolidation treatment period). The mean (SD) total number of EDs was 52.3 (6.83) for the overall population. The mean (95% confidence interval [CI]) estimated annualized bleed rate (ABR) was 0.61 (0.42-0.90). The mean (95% CI) annualized spontaneous, traumatic, and joint bleed rates were 0.16 (0.08-0.31), 0.40 (0.24-0.65), and 0.30 (0.16-0.57), respectively. Eighty-six percent of participants (n=63) had ≤1 bleed, including 64% (n=47) with no overall bleeds; 88% (n=64) had no spontaneous bleeds and 84% (n=61) had no joint bleeds. There were 43 bleeds treated with efanesoctocog alfa, of which 11 were reported as spontaneous (26%), 28 as traumatic (65%), and 4 (9%) were of unknown etiology (Table). The most common locations for bleeds were the joints (n=21, 49%) and skin/mucosa (n=15, 35%). The rate of bleeding events per week throughout the study remained consistently low (Figure). The mean (SD) duration between a treated bleed and nearest injection taken prior to that bleed was 4.7 (1.95) days, with a mean of 4.5 (1.56) days for a spontaneous bleed and 4.8 (2.12) for traumatic bleeds. A single injection was sufficient to resolve 95% of bleeds, and no bleed required more than 2 injections for resolution. The median (interquartile range) total dose required to resolve a bleed was 52.6 (50.0-55.8) IU/kg. Among first injections for bleeding episodes with an evaluation of response by participants (n=37), the vast majority (n=36, 97%) were rated as excellent or good. Conclusions A single dose of efanesoctocog alfa 50 IU/kg resolved 95% of bleeds in children <12 years of age, regardless of type or location. The hemostatic efficacy of efanesoctocog alfa was rated as excellent or good for nearly all evaluated first injections (97%). Once-weekly efanesoctocog alfa provided efficient prophylaxis with no bleeds in 64% of participants, and consistently low ABRs among all types and locations of bleeds. Efanesoctocog alfa provided effective treatment and prevention of bleeds in previously treated children <12 years of age with severe hemophilia A.

Production of codon-optimized Factor C fragment from Tachypleus gigas in the Pichia pastoris GS115 expression system for endotoxin detection

BackgroundFactor C (FC) is widely used as a standard material for endotoxin testing. It functions as a zymogenic serine protease and serve as a biosensor that detects lipopolysaccharides. Prior investigations involving molecular docking and molecular dynamics simulations of FC demonstrated an interaction between the C-type lectin domain (CLECT) and the ligand lipopolysaccharide (lipid A). In this study, our aim was to assess the stability of the interaction between fragment FC and the lipid A ligand using protein modeling approaches, molecular docking, molecular dynamics simulation, and gene construction into the pPIC9K expression vector. Methods and resultsThe FC structure was modelled by online tools. In this case, both molecular docking and MD simulations were applied to identify the interaction between protein and ligand (lipid A) including its complex stability. The FC structure model using three modeling websites has varied values, according to a Ramachandran plot study. When compared to other models, AlphaFold server modeling produced the best Ramachandran findings, with residues in the most advantageous area at 88.3%, followed by ERRAT values at 89.83% and 3D Verify at 71.93%. From the docking simulation of FC fragments with three ligands including diphosphoryl lipid A, FC-Core lipid A, and Kdo2 lipid A can be an activator of FC protein by binding to receptor regions to form ligand-receptor complexes. MD simulations were performed on all three complexes to assess their stability in water solvents showing that all complexes were stable during the simulation. The optimization of recombinant protein expression in Pichia pastoris was conducted by assessing the OD value and protease activity. Induction was carried out using 1% (v/v) methanol in BMMY media at 30°C for 72 h. ConclusionsProtein fragments of Factor C has been proven to detect endotoxins and serve as a potential biomarker. Molecular docking simulation and MD simulation were employed to study the complex formation of protein fragments FC with ligands. The expression of FC fragments was successfully achieved through heterologous expression. We propose optimizing the expression of FC fragments by inducing them with 1% methanol at 30°C and incubating them for 72 h. These optimized conditions are well-suited for upscaling the production of recombinant FC fragments using a bioreactor.

Characterization and validation of a middle-down hydrophobic interaction chromatography method to monitor methionine oxidation in IgG1

Post-translational modifications (PTMs) of therapeutic monoclonal antibodies (mAbs) can impact the efficacy of a drug. Methionine oxidation can alter the overall hydrophobicity of an antibody, thereby inducing conformational changes and affecting its biological activity. To ensure high quality, safety, and efficacy of mAbs, routine monitoring of PTMs such as methionine (Met) oxidation is essential. Met oxidation in the fragment crystallizable (Fc) region of immunoglobulin-G1 (IgG1) is a potential critical quality attribute because it impacts not only the interaction with the neonatal Fc receptor and protein A but also the half-life of mAbs in serum circulation. Although bottom-up mass spectrometry provides high site specificity, it may have limited application in quality control workflows, and its complicated sample preparation could result in procedure-induced oxidation. In this study, we describe the development and characterization of a rapid and robust middle-down hydrophobic interaction chromatography method for monitoring Met oxidation in the Fc region of IgG1. Additionally, we assessed a comprehensive method validation package and demonstrated the specificity, linearity, precision, and accuracy of the new method within a range of 3.8–37.7%. The relative quantitative data provided by this method may be used in a regulated workflow to support process and formulation development as well as in the later stages of drug development and batch release and stability studies.

Binding to Platelets Differs Among Recombinant Factor VIII Products in Vitro

Introduction: Platelets are activated in response to injury and play a key role in blood clotting. In primary hemostasis, platelets adhere to exposed subendothelial matrix proteins (e.g., collagen) and subsequent downstream signaling pathways induce granule secretion and platelet aggregation. In secondary hemostasis, procoagulant platelets in the periphery activate clotting factors, leading to a more stable fibrin clot. Binding of factor VIII (FVIII) to procoagulant platelets ensures efficient production and localization of thrombin at the site of injury. However, it is less clear how platelet-FVIII interactions spatiotemporally control the initial thrombus formation and pave the way for successful tissue repair. In patients with hemophilia A, replacement FVIII therapy is used to treat and prevent bleeds. A number of recombinant FVIII (rFVIII) products are indicated for use in patients with hemophilia A. Simoctocog alfa (Nuwiq®) is a chemically unmodified rFVIII with no fusion protein produced in a human cell line. Damoctocog alfa pegol (Jivi®) and rurioctocog alfa pegol (Adynovate®) are pegylated rFVIII products, while efmoroctocog alfa (Eloctate®) consists of FVIII fused with a Fc protein. It is not clear whether such modifications impact the platelet binding potential of FVIII. This study aims to examine platelet binding of different rFVIII products in vitro. Methods: Platelets were isolated from healthy donors, activated using thrombin and collagen-related peptide (CRP-XL) and incubated with individual rFVIII concentrates. After 30 min, the reaction was stopped by dilution. Binding was quantified by flow cytometry using one of two approaches: rFVIII immunostaining with a fluorescent (Alexa Fluor 647; AF647) anti-FVIII antibody; or using rFVIII molecules that had been pre-labeled with AF647. For comparison among products, experiments were performed at equimolar concentrations (17.6 nM) or equal activities of rFVIII concentrates. Values were normalized to simoctocog alfa. Statistical analyses were performed using one-way ANOVA with Tukey post-hoc testing. Results: Platelets were obtained from 10 healthy donors. At equimolar concentrations, efmoroctocog alfa and damoctocog alfa pegol bound significantly (p < 0.001) less than simoctocog alfa to stimulated platelets, as visualized via immunostaining (n = 10). Binding to platelets was significantly lower with damoctocog alfa pegol than efmoroctocog alfa (p < 0.001). Similar results were obtained from experiments with pre-labelled rFVIII (n = 6) or when using equal activities of rFVIII concentrates. In experiments with platelets from 2 healthy donors, rurioctocog alfa pegol exhibited lower platelet binding compared with simoctocog alfa, when tested using the immunostaining method and equimolar concentrations of rFVIII. Conclusions: Simoctocog alfa showed increased binding to procoagulant platelets compared with other rFVIII concentrates tested. Further studies, including additional functional platelet assays, will be performed using samples from a larger number of healthy donors and from patients with hemophilia A. The results of this study may provide new insights into the function and efficacy of different rFVIII products in patients with hemophilia A.

A First-in-Human Phase I Dose Escalation Study of IMM01, Sirpα Fc Protein in Patients with Relapsed or Refractory Lymphoma

A First-in-Human Phase I Dose Escalation Study of IMM01, Sirpα Fc Protein in Patients with Relapsed or Refractory Lymphoma

Blockade of interleukin-33 activities by recombinant interleukin-33 Trap Fc protein would be a novel therapeutic strategy in allergic asthma

The majority of autoimmune and allergic diseases are associated with abnormal expression of interleukin (IL)-33, a member of the IL-1 family of cytokines, that function dually as a proinflammatory cytokine and a transcriptional factor. We created an IL-33 inhibitor called "IL-33 Trap Fc" constructed by fusion of an Fc fragment of human immunoglobulin G1 and two distinct extracellular part receptors involved in interacting with IL-33, IL-1 receptors accessory protein, and IL-33 receptor. IL-33 Trap Fc was expressed by two systems, mammalian HEK293 cells and Pichia pastoris yeast. We found that these recombinant proteins were expressed as a glycoprotein and perhaps in dimeric form. IL-33 Trap Fc from HEK293 and P. pastoris suppressed the activity of IL-33 in vitro culture conditions. The glycosylation of IL-33 Trap expressed by P. pastoris yeast was more intensive and heterogeneous than the counterpart protein expressed from HEK293 cells. That is maybe one reason leading to a substantial decrease in the activity of IL-33 Trap Fc from P. pastoris compared with that from HEK293 cells. We also demonstrated that IL-33 Trap Fc expressed from HEK293 cells had therapeutic effects in ovalbumin-induced asthma mouse model. These data collectively suggested that IL-33 Trap Fc potently blocks IL-33 in vitro and in vivo, which may be a novel therapeutic strategy for IL-33-mediated allergic diseases.

In vivo therapeutic effects of small molecule-drug conjugates enhanced by Fc grafting

Small molecule-drug conjugate (SMDC) shows great potential as a new class of targeted chemotherapeutic agents to tackle cancer. However, its in vivo therapeutic effect is compromised by its poor pharmacokinetic parameters. Herein we describe an approach that enables the precise conjugation of SMDC on N-terminus of the Fc protein to produce a SMDC-Fc bioconjugate (Fc1070) with superior specificity, affinity and potency to tumor cells. In vivo, Fc1070 exhibited an antibody-like pharmacokinetic profile with a long circulation half-life (t1/2 = 79 h) and pro-liver clearance pathway, that is distinct from the parent SMDC (t1/2 = 0.5 h and renal clearance). Intravenous injection of Fc1070 can eliminate the tumor with a single dosing of 7 mg/kg. Coupled with a predefined ligand toolbox, this approach allows the fast generation of other SMDC bioconjugates on demand, thus extending the format easily to other tumor targets. This may provide a general approach for the development of SMDC with enhanced therapeutic properties.

De novo Fc-based receptor dimerizers differentially modulate PlexinB1 function

Signaling by single-pass transmembrane receptors often involves a formation of ligand-induced receptor dimers with particular conformation, and bivalent receptor binders can modulate receptor functions by inducing different receptor dimer conformations, although such agents are difficult to design. Here, we describe the generation of both antagonistic and agonistic receptor dimerizers toward PlexinB1 (PlxnB1), a receptor for semaphorin 4D (Sema4D), by grafting two different PlxnB1-binding peptides onto the human immunoglobulin G1 (IgG1) Fc protein. The function-modulating activity of a peptide Fc was strongly dependent on the type of the peptide as well as the grafting site, with the best variants showing activity at an nM concentration range. Structural analysis of each peptide-PlxnB1 complex revealed that the agonistic Fc dimerizes PlxnB1 in a face-to-face fashion similar to that induced by Sema4D, whereas antagonistic Fc would induce signaling-incompetent PlxnB1 dimer conformation, enforcing the idea that plexin activation is primarily controlled by the receptor orientation within the dimer.

In Situ Tumor Vaccine Expressing Anti-CD47 Antibody Enhances Antitumor Immunity.

In situ tumor vaccine is a potential cancer therapy due to their advantages in induction of antitumor immune responses. Oncolytic virotherapy utilizes natural or engineered oncolytic viruses to kill tumors selectively, representing a promising in situ tumor vaccine for cancer immunotherapy. In addition to direct oncolysis, oncolytic viruses elicit potent and durable antitumor immune responses by induction of immunogenic cell death of tumors. Membrane protein CD47 overexpressed on tumor cells engages in “don’t eat me” signal that prevents macrophages from engulfing tumor cells. CD47-targeting agents have been tested via preclinical and clinical trials. As potential tumor vaccine vectors, oncolytic viruses can be engineered to express anti-CD47 antibodies to induce potentiated tumor killing. Therefore, we developed an adenovirus-based tumor vaccine loaded with a CD47-targeting nanobody fused with the IgG2a Fc protein. B16-F10 melanoma, A20 lymphoma, and 4T1 breast cancer models in immunocompetent mice were established to evaluated in vivo antitumor efficacy of in situ tumor vaccination. The tumor vaccine armed with a nanobody against CD47 induced durable suppression of the tumor and long-term survival of tumor-bearing mice, and also elevated the number of tumor-infiltrating immune cells with an activated immunophenotype, suggesting that it could remodel the tumor immune microenvironment. Systemic antitumor effects and immune memory were also observed in immunocompetent mice following in situ vaccination with the anti-CD47 tumor vaccines; tumorigenesis was completely inhibited in these mice after tumor re-challenge. The recombinant anti-CD47 tumor vaccine has an effectual antitumor activity and may be a promising antitumor agent.

Thermostability enhancement of l-glutamate oxidase from Streptomyces sp. NT1 by full consensus protein design

Thermostable l-glutamate oxidases (LGOXs) are desirable for use in l-glutamate (L-Glu) assay kits, enzymatic synthesis of α-ketoglutarate and for biosensor development. However, protein engineering efforts to improve thermostability often lead to a decrease in enzymatic activity. In this report, we aimed to enhance the thermostability (melting temperature, Tm) of a mesophilic LGOX from Streptomyces sp. NT1 (LGOXNT1) without a reduction in activity by a sequence-based protein design approach, termed full consensus (Fc) protein design. Among the 690 amino acids of LGOXNT1, 104 amino acids were substituted by the Fc protein design. The mutant gene was artificially synthesized and expressed in Escherichia coli BL21(DE3) cells. The Tm of the purified, recombinant LGOX mutant (FcLGOX) was determined to be ∼72°C, which is an increase on the Tm of 65°C for LGOXNT1 and the highest among known LGOXs. Importantly, purified FcLGOX showed no loss of specific activity or substrate specificity after a 30-min incubation at 70°C. Our findings provide a new approach to improve the thermostability of enzymes.

Increasing Transfection Efficiency of Lipoplexes by Modulating Complexation Solution for Transient Gene Expression

Transient gene expression is a suitable tool for the production of biopharmaceutical candidates in the early stage of development and provides a simple and rapid alternative to the generation of stable cell line. In this study, an efficient transient gene expression methodology using DC-Chol/DOPE cationic liposomes and pDNA in Chinese hamster ovary suspension cells was established through screening of diverse lipoplex formation conditions. We modulated properties of both the liposome formation and pDNA solution, together called complexation solutions. Protein expression and cellular cytotoxicity were evaluated following transfection over the cell cultivation period to select the optimal complexation solution. Changes in hydrodynamic size, polydispersity index, and ζ potential of the liposomes and lipoplexes were analyzed depending on the various pH ranges of the complexation solutions using dynamic light scattering. The transfer of lipoplexes to the cytosol and their conformation were traced using fluorescence analysis until the early period of transfection. As a result, up to 1785 mg/L and 191 mg/L of human Fc protein and immunoglobulin G (bevacizumab), respectively, were successfully produced using acidic liposome formation and alkaline pDNA solutions. We expect that this lipoplex formation in acidic and alkaline complexation solutions could be an effective methodology for a promising gene delivery strategy.

An Oncolytic Adenovirus Armed with a Nanobody Against CD47 Reprograms Tumor Immune Microenvironment and Drives Durable Antitumor Immunity

Background: Oncolytic virotherapy utilizes natural or engineered viruses to kill tumors selectively, representing a promising cancer immunotherapy. In addition to direct oncolysis, oncolytic viruses (OVs) elicit potent and durable antitumor immune responses by induction of immunogenic cell death of tumors. Membrane protein CD47 overexpressed on tumor cells engages in don't eat me signal that prevents macrophages from engulfing tumor cells. CD47-targeting agents have been tested via preclinical and clinical trials. As ideal gene delivery vectors in cancer therapy, OVs can be engineered to express anti-CD47 antibodies to induce potentiated tumor killing. Importantly, OVs can intratumorally deliver anti-CD47 antibodies without causing uncontrolled off-tumor toxicity. Methods: We developed a recombinant oncolytic adenovirus loaded with a CD47-targeting nanobody fused with the IgG2a Fc protein. An oncolytic adenovirus without transgene of interest was used as control. B16-F10 melanoma, A20 lymphoma, and 4T1 breast cancer mouse models were established to evaluated in vivo antitumor efficacy of oncolytic adenoviruses. Findings: The recombinant oncolytic adenovirus armed with a nanobody against CD47 induced durable suppression of the tumor and long-term survival of tumor-bearing mice. Additionally, the recombinant oncolytic adenovirus elevated the number of tumor-infiltrating immune cells with an activated immunophenotype, suggesting that it could remodel the tumor immune microenvironment. Systemic antitumor effects and immune memory were also observed in mice treated with the anti-CD47 oncolytic adenovirus; tumorigenesis was completely inhibited in these mice after tumor re-challenge. Interpretation: The recombinant anti-CD47 oncolytic adenovirus has an effectual antitumor activity and may be a promising antitumor agent. Funding Statement: This work was supported by the National Science and Technology Major Projects of New Drugs (2018ZX09201018-013), the National Science and Technology Major Project for Infectious Diseases Control (2017ZX10203206-004), the National Natural Science Foundation of China (81101728), Sichuan Regional Innovation Cooperation Project (20QYCX0100), the Innovation Spark Project of Sichuan University (2018SCUH0084). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: All animal experiments were approved by the Institutional Animal Care and Use Committee of Sichuan University, Chengdu, China.

Inhibition of Interleukin-21 prolongs the survival through the promotion of wound healing after myocardial infarction

Ly6Clow macrophages promote scar formation and prevent early infarct expansion after myocardial infarction (MI). Although CD4+ T cells influence the regulation of Ly6Clow macrophages after MI, the mechanism remains largely unknown. Based on the hypothesis that some molecule(s) secreted by CD4+ T cells act on Ly6Clow macrophages, we searched for candidate molecules by focusing on cytokine receptors expressed on Ly6Clow macrophages. Comparing the transcriptome between Ly6Chigh macrophages and Ly6Clow macrophages harvested from the infarcted heart, we found that Ly6Clow macrophages highly expressed the receptor for interleukin (IL)-21, a pleiotropic cytokine which is produced by several types of CD4+ T cells, compared with Ly6Chigh macrophages. Indeed, CD4+ T cells harvested from the infarcted heart produce IL-21 upon stimulation. Importantly, the survival rate and cardiac function after MI were significantly improved in IL-21-deficient (il21−/−) mice compared with those in wild-type (WT) mice. Transcriptome analysis of infarcted heart tissue from WT mice and il21−/− mice at 5 days after MI demonstrated that inflammation is persistent in WT mice compared with il21−/− mice. Consistent with the transcriptome analysis, the number of neutrophils and matrix metalloproteinase (MMP)-9 expression were significantly decreased, whereas the number of Ly6Clow macrophages and MMP-12 expression were significantly increased in il21−/− mice. In addition, collagen deposition and the number of myofibroblasts in the infarcted area were significantly increased in il21−/− mice. Consistently, IL-21 enhanced the apoptosis of Ly6Clow macrophages. Finally, administration of neutralizing IL-21 receptor Fc protein increased the number of Ly6Clow macrophages in the infarcted heart and improved the survival and cardiac function after MI. Thus, IL-21 decreases the survival after MI, possibly through the delay of wound healing by inducing the apoptosis of Ly6Clow macrophages.

Cancer immunotherapy by NC410, a LAIR-2 Fc protein blocking human LAIR-collagen interaction.

Collagens are a primary component of the extracellular matrix and are functional ligands for the inhibitory immune receptor leukocyte-associated immunoglobulin-like receptor (LAIR)-1. LAIR-2 is a secreted protein that can act as a decoy receptor by binding collagen with higher affinity than LAIR-1. We propose that collagens promote immune evasion by interacting with LAIR-1 expressed on immune cells, and that LAIR-2 releases LAIR-1-mediated immune suppression. Analysis of public human datasets shows that collagens, LAIR-1 and LAIR-2 have unique and overlapping associations with survival in certain tumors. We designed a dimeric LAIR-2 with a functional IgG1 Fc tail, NC410, and showed that NC410 increases human T cell expansion and effector function in vivo in a mouse xenogeneic-graft versus-host disease model. In humanized mouse tumor models, NC410 reduces tumor growth that is dependent on T cells. Immunohistochemical analysis of human tumors shows that NC410 binds to collagen-rich areas where LAIR-1+ immune cells are localized. Our findings show that NC410 might be a novel strategy for cancer immunotherapy for immune-excluded tumors.

Preliminary results of a first-in-human phase I dtudy of IMM01, SIRPα Fc protein in patients with relapsed or refractory lymphoma.

2550 Background: IMM01 is a recombinant human signal regulatory protein α (SIRPα) IgG 1 fusion protein that exerts dual-mechanism antitumor activity via engagement of activating tumor cell phagocytosis and stimulating T-cell anti-tumor responses by binding CD47 on tumor cell membrane. IMM01 displays promising preclinical characteristics regarding its receptor occupancy/tumor exposure/efficacy relationship. Unlike anti-CD47 monoclonal antibodies, IMM01 shows unique property of weak human erythrocyte conjugation so as avoiding severe hemolysis. Methods: Monotherapy of IMM01 was conducted in 14 enrolled subjects with relapsed or refractory lymphoma who had failed standard therapies. Dose escalation was performed in routine design of accelerated single-patient followed by standard 3+3 to establish the preliminary data of safety as well as determination of a recommended expansion dosage. Each cycle contains 4 dosing weekly followed by a week rest. The tumor responses were evaluated based on Lugano Classification 2014. IMM01 pharmacokinetics (PK) and pharmacodynamics (PD) analyses were performed. Results: As of February 08, 2021, a total of 14 patients (median age 49 y; median prior therapy 3) were enrolled in 6 escalated dose cohorts (0.003 mg/kg, 0.01 mg/kg, 0.05 mg/kg, 0.15 mg/kg, 0.5 mg/kg and 1.0 mg/kg). The common tumor types were follicular lymphoma, Hodgkin lymphoma, diffuse large B-cell lymphoma. No DLTs were observed up to 1.0 mg/kg. One SAE (grade 2 increased amylase and grade 3 increased lipase) was reported, which induced by disease progression on pancreas. The most common treatment related adverse events were thrombocytopenia (54%), neutrophil count decreased (36%), Pyrexia (36%) and Anaemia (27%). There were grade 1 or 2 except for one patient experienced a grade 3 platelet count decreased (lower baseline at 70×109/L). Transient platelet count decrease after 2 hours and return to baseline at 24 to 48 hours post first infusion. In 12 evaluated patients, one patient with FL had a CR and maintained a 26-week response at the dose of 0.01 mg/kg. One patient with HL who had failed PD-1 inhibitor was confirmed PR at 27 weeks and continues the therapy, and one patient with MZL maintained SD for 12 weeks at the dose of 0.15 mg/kg. One patient with HL failed PD-1 inhibitor and one patient with FL maintained a shrunk SD for 12 weeks at the dose of 0.5 mg/kg. One patient with AITL was evaluated as a shrunk SD after 5 doses treatment at the dose of 1.0 mg/kg. Terminal half-life of IMM01 range from 53.8 hours to 73.3 hours. The AUC and Cmax of IMM01 show nonlinear increases in the dose range of 0.05 mg/kg to 0.5 mg/kg. Conclusions: Preliminary data from the present phase 1 study of IMM01, a SIRPα IgG 1 fusion protein, demonstrate that IMM01 has an excellent preliminary safety, tolerability and promising anti-tumor activity up to doses of 1.0 mg/kg. Clinical trial information: ChiCTR1900024904.

AB0282 SAFETY, TOLERABILITY AND SELECTIVE EXPANSION OF REGULATORY T CELLS BY A SINGLE DOSE OF THE NOVEL IL-2 MUTEIN PT101 IN A PHASE 1 STUDY IN HEALTHY VOLUNTEERS

Background:Activation and expansion of regulatory T cells (Tregs) has been proposed as a strategy to treat autoimmunity. When administered in low doses, IL-2 expands and activates Tregs leading to clinical response in several autoimmune diseases. However, the narrow therapeutic window of IL-2 results in loss of selectivity for Tregs and concurrent activation of conventional T cells (Tconv) and NK cells, limiting its clinical utility. This loss of selectivity may negate the clinical benefit of Treg activation and lead to dose-limiting side effects. PT101 is a novel engineered variant of IL-2 fused to an Fc protein backbone which in preclinical studies selectively activates Tregs without expanding Tconv or NK cells. PT101 is in clinical development for the treatment of patients with autoimmune diseases.Objectives:To assess the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of PT101 after a single dose in healthy human volunteers.Methods:We conducted a randomized, double-blind, single-ascending dose trial of PT101 or placebo (3:1 allocation). Five dose levels from 1 mg to 10 mg were administered by subcutaneous injection. Adverse events, physical examination findings, and clinical laboratory results were assessed for 29 days. Serum PT101 levels and antidrug antibody were assessed. Changes in mononuclear cell populations were measured in peripheral blood by flow cytometry.Results:56 subjects were administered PT101 or placebo. All subjects completed the study. There were no deaths, serious adverse events, dose limiting toxicities, or clinically significant changes in vital sign, ECG, or laboratory results. All adverse events were Grade 1 or 2 and self-limited. Injection site reactions were the most common adverse event. Transient increases in eosinophil counts were observed in some subjects, consistent with the known class effect of IL-2. Peak levels of PT101 occurred 11.0 to 14.6 hours after administration, and declined with a mean half-life of 20.4 to 28.3 hours, demonstrating linear exposure through the dose range. No anti-drug antibodies were induced. PT101 caused dose-related expansion of Tregs that plateaued at doses between 3.5 and 10 mg. Mean maximum expansion above baseline was 3.6-fold for total Tregs and 72.5-fold for the CD25bright subset of Tregs. Maximal expansion was observed by Day 8-10 with a return toward baseline by Day 29. Over 80% of subjects achieved a 2-fold or greater expansion of total Tregs (Table 1). No significant expansion of Tconv or NK cells was observed at any dose level.Table 1.Percent Total Treg RespondersFold Change Total TregsPlacebo(n=14)1 mg(n=6)3.5 mg(n=12)5 mg(n=12)7.5 mg(n=6)10 mg(n=6)≥ 2X7%33%83%83%100%100%≥ 3X0%0%58%75%33%50%≥ 4X0%0%24%42%33%17%Conclusion:PT101 was safe and well tolerated after a single dose in healthy volunteers. Marked expansion of both total Treg and CD25bright Treg cells was observed. High selectivity for Tregs was observed with no significant expansion of pro-inflammatory Tconv and NK cells even at the highest dose studied. These results support the therapeutic potential of PT101 in planned multiple dose studies in systemic lupus erythematosus, ulcerative colitis, and other autoimmune diseases.

Induction of ADAM10 by Radiation Therapy Drives Fibrosis, Resistance, and Epithelial-to-Mesenchyal Transition in Pancreatic Cancer.

Stromal fibrosis activates prosurvival and proepithelial-to-mesenchymal transition (EMT) pathways in pancreatic ductal adenocarcinoma (PDAC). In patient tumors treated with neoadjuvant stereotactic body radiation therapy (SBRT), we found upregulation of fibrosis, extracellular matrix (ECM), and EMT gene signatures, which can drive therapeutic resistance and tumor invasion. Molecular, functional, and translational analysis identified two cell-surface proteins, a disintegrin and metalloprotease 10 (ADAM10) and ephrinB2, as drivers of fibrosis and tumor progression after radiation therapy (RT). RT resulted in increased ADAM10 expression in tumor cells, leading to cleavage of ephrinB2, which was also detected in plasma. Pharmacologic or genetic targeting of ADAM10 decreased RT-induced fibrosis and tissue tension, tumor cell migration, and invasion, sensitizing orthotopic tumors to radiation killing and prolonging mouse survival. Inhibition of ADAM10 and genetic ablation of ephrinB2 in fibroblasts reduced the metastatic potential of tumor cells after RT. Stimulation of tumor cells with ephrinB2 FC protein reversed the reduction in tumor cell invasion with ADAM10 ablation. These findings represent a model of PDAC adaptation that explains resistance and metastasis after RT and identifies a targetable pathway to enhance RT efficacy. SIGNIFICANCE: Targeting a previously unidentified adaptive resistance mechanism to radiation therapy in PDAC tumors in combination with radiation therapy could increase survival of the 40% of PDAC patients with locally advanced disease.See related commentary by Garcia Garcia et al., p. 3158 GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/12/3255/F1.large.jpg.