Human IgG1 Fc Research Articles

Single-domain antibodies reveal unique borrelicidal epitopes on the Lyme disease vaccine antigen, outer surface protein A (OspA).

Camelid-derived, single-domain antibodies (VHHs) have proven to be extremely powerful tools in defining the antigenic landscape of immunologically heterogeneous surface proteins. In this report, we generated a phage-displayed VHH library directed against the candidate Lyme disease vaccine antigen, outer surface protein A (OspA). Two alpacas were immunized with recombinant OspA serotype 1 from Borrelia burgdorferi sensu stricto strain B31, in combination with the canine vaccine RECOMBITEK Lyme containing lipidated OspA. The phage library was subjected to two rounds of affinity enrichment ("panning") against recombinant OspA, yielding 21 unique VHHs within two epitope bins, as determined through competition enzyme linked immunosorbent assays (ELISAs) with a panel of OspA-specific human monoclonal antibodies. Epitope refinement was conducted by hydrogen exchange-mass spectrometry. Six of the monovalent VHHs were expressed as human IgG1-Fc fusion proteins and shown to have functional properties associated with protective human monoclonal antibodies, including B. burgdorferi agglutination, outer membrane damage, and complement-dependent borreliacidal activity. The VHHs displayed unique reactivity profiles with the seven OspA serotypes associated with B. burgdorferi genospecies in the United States and Europe consistent with there being unique epitopes across OspA serotypes that should be considered when designing and evaluating multivalent Lyme disease vaccines.

An antigen-specific immunotherapeutic, AKS-107, deletes insulin-specific B cells and prevents murine autoimmune diabetes.

The antigen-presenting cell function of insulin-reactive B cells promotes type 1 diabetes (T1D) in non-obese diabetic (NOD) mice by stimulating pathogenic T cells leading to destruction of insulin-producing β-cells of pancreatic islets. To target insulin-reactive B cells, AKS-107, a human IgG1 Fc molecule fused with human insulin A and B chains, was engineered to retain conformational insulin epitopes that bound mouse and human B cell receptors but prevented binding to the insulin metabolic receptor. AKS-107 Fc-mediated deletion of insulin-reactive B cells was demonstrated via ex vivo and in vivo experiments with insulin-reactive B cell receptor transgenic mouse strains, VH125Tg/NOD and Tg125(H+L)/NOD. As an additional immune tolerance feature, the Y16A mutation of the insulin B(9-23) dominant T cell epitope was engineered into AKS-107 to suppress activation of insulin-specific T cells. In mice and non-human primates, AKS-107 was well-tolerated, non-immunogenic, did not cause hypoglycemia even at high doses, and showed an expectedly protracted pharmacokinetic profile. AKS-107 reproducibly prevented spontaneous diabetes from developing in NOD and VH125Tg/NOD mice that persisted for months after cessation of treatment, demonstrating durable immune tolerance. These preclinical outcomes position AKS-107 for clinical development in T1D prevention settings.

Novel bispecific nanobody mitigates experimental intestinal inflammation in mice by targeting TNF-α and IL-23p19 bioactivities.

Inflammatory bowel diseases (IBDs) pose significant challenges in terms of treatment non-response, necessitating the development of novel therapeutic approaches. Although biological medicines that target TNF-α (tumour necrosis factor-α) have shown clinical success in some IBD patients, a substantial proportion still fails to respond. We designed bispecific nanobodies (BsNbs) with the ability tosimultaneously target human macrophage-expressed membrane TNF-α (hmTNF-α) and IL-23. Additionally, we fused the constant region of human IgG1 Fc (hIgG1 Fc) to BsNb to create BsNb-Fc. Our study encompassed in vitro and in vivo characterization of BsNb and BsNb-Fc. BsNb-Fc exhibited an improved serum half-life, targeting capability and effector function than BsNb. It's demonstrated that BsNb-Fc exhibited superior anti-inflammatory effects compared to the anti-TNF-α mAb (infliximab, IFX) combined with anti-IL-12/IL-23p40 mAb (ustekinumab, UST) by Transwell co-culture assays. Notably, in murine models of acute colitis brought on by 2,4,6-trinitrobenzene sulfonic acid（TNBS) and dextran sulphate sodium (DSS), BsNb-Fc effectively alleviated colitis severity. Additionally, BsNb-Fc outperformed the IFX&UST combination in TNBS-induced colitis, significantly reducing colon inflammation in mice with colitis produced by TNBS and DSS. These findings highlight an enhanced efficacy and improved biostability of BsNb-Fc, suggesting its potential as a promising therapeutic option for IBD patients with insufficient response to TNF-α inhibition. A bispecific nanobody (BsNb) was created to target TNF-α and IL-23p19, exhibiting high affinity and remarkable stability. BsNb-Fc inhibited the release of cytokines in CD4+T cells during co-culture experiments. BsNb-Fc effectively alleviated colitis severity in mouse model with acute colitis induced by DSS or TNBS, outperforming the IFX&UST combination.

Activin A levels are raised during human tuberculosis and blockade of the activin signaling axis influences murine responses to M. tuberculosis infection.

Tuberculosis remains the leading cause of death by a bacterial pathogen. The etiologic agent of tuberculosis, Mycobacterium tuberculosis, can remain dormant in the infected host for years before causing disease. Significant effort has been made to identify biomarkers that can discriminate between latently infected and actively diseased individuals. We found that serum levels of the cytokine activin A were associated with increased lung pathology and could discriminate between active tuberculosis and tuberculin skin-test-positive healthy controls. Activin A signals through the ActRIIB receptor, which can be blocked by administration of the ligand trap ActRIIB-Fc, a soluble activin type IIB receptor fused to human IgG1 Fc. In a murine model of tuberculosis, we found that ActRIIB-Fc treatment reduced mycobacterial loads. Strikingly, ActRIIB-Fc treatment significantly increased the number of tissue-resident memory T cells. These results suggest a role for ActRIIB signaling pathways in host responses to Mycobacterium tuberculosis and activin A as a biomarker of ongoing disease.

Evaluation of RNase therapy in systemic lupus erythematosus: a randomised phase 2a clinical trial of RSLV-132

BackgroundCirculating, extracellular RNA is the primary trigger of type I interferon in systemic lupus erythematosus (SLE), and interferon is known to play a central pathogenic role in the disease. RSLV-132...

A neutralizing single-domain antibody that targets the trimer interface of the human metapneumovirus fusion protein.

Human metapneumovirus (hMPV) is an important respiratory pathogen for which no licensed antivirals or vaccines exist. Single-domain antibodies represent promising antiviral biologics that can be easily produced and formatted. We describe the isolation and detailed characterization of two hMPV-neutralizing single-domain antibodies that are directed against the fusion protein F. One of these single-domain antibodies broadly neutralizes hMPV A and B strains, can prevent proteolytic maturation of F, and binds to an epitope in the F trimer interface. This suggests that hMPV pre-F undergoes trimer opening or "breathing" on infectious virions, exposing a vulnerable site for neutralizing antibodies. Finally, we show that this single-domain antibody, fused to a human IgG1 Fc, can protect cotton rats against hMPV replication, an important finding for potential future clinical applications.

VISTA Is the Predominant 2nd Generation of ICI (Immune Check-Point Inhibitor) in Ph(-) myeloproliferative Neoplasm

VISTA Is the Predominant 2nd Generation of ICI (Immune Check-Point Inhibitor) in Ph(-) myeloproliferative Neoplasm

295. Humanized Mice and Convalescent Humans Yield an Antibody Panel against the Highly Immunogenic Decorin Binding Protein A (DbpA) of the Lyme Disease Spirochete, Borrelia Burgdorferi

Abstract Background Lyme disease is the most commonly diagnosed tick-borne infection, affecting more than 300000 people in the United States annually. The disease is principally caused by the transmission of the spirochete bacteria, by the black-legged (deer) ticks. With a renewed call for interest in a Lyme disease vaccine, antibodies relevance continues due to their efficacy, safety and versatile nature. Humanized mice and humans recovering from the disease have successfully been used to generate therapeutic antibodies against Ebola and SARS-CoV-2 diseases. Methods Our study used humanized mice and convalescent patients to generate recombinant human antibodies that recognize decorin-binding protein A (DbpA), a lipoprotein that is proposed to function as spirochete adhesin. The mAbs expressed as human IgG-Fc fragments were characterized for reactivity with recombinant DbpA, native DbpA on the surface of live Borrelia burgdorferi, borreliacidal activity in the presence of human complement and, finally subject to epitope mapping using an array of methodologies. Results The predominant lineage of humanized mice antibodies utilized VH5-51, VH3-20, VH3-23 and VH3-53 all paired with VK1-33 families. Other common families included VH4-31 paired with VK1-39, VH4-4 and VH4-8 paired with VK-9. The human derived antibody utilized VH3-30 paired with VK1-33. We observed an overlap in the repertoire of kappa chains between humanized mice and human-derived mAbs. Conclusion Combining the humanized mice and human platforms enabled the identification of antibodies with a high binding and specificity to DbpA, which have potential prophylactic and therapeutic properties. Disclosures All Authors: No reported disclosures

EDIII-Fc induces protective immune responses against the Zika virus in mice and rhesus macaque.

Zika virus can infect the fetus through the placental barrier, causing ZIKV congenital syndrome and even miscarriage, which can cause great harm to pregnant women and infants. Currently, there is no vaccine and drug available to combat the Zika virus. In this study, we designed a fusion protein named EDIII-Fc, including the EDIII region of Zika E protein and human IgG Fc fragment, and obtained 293T cells that stably secreted EDIII-Fc protein using the lentiviral expression system. Mice were immunized with the EDIII-Fc protein, and it was observed that viral replication was significantly inhibited in the immunized mice compared to non-immunized mice. In rhesus macaques, we found that EDIII-Fc effectively induce the secretion of neutralizing antibodies and T cell immunity. These experimental data provide valid data for further use of Zika virus E protein to prepare an effective, safe, affordable Zika vaccine.

A specific nanobody-based affinity chromatography resin as a platform for small ubiquitin-related modifier fusion protein purification

As an excellent fusion tag for expressing heterologous proteins, yeast SUMO (small ubiquitin-related modifier) has unique advantages such as improving solubility, promoting stability, and reducing degradation, but it lacks a simple and rapid purification method. Camelid single-domain antibodies (VHHs or nanobodies) show great promise as an efficient tool in analytical application. In this study, VHHs against SUMO protein were isolated for the first time using biopanning of an immune camelid nanobody library. Among these nanobodies, VS2 demonstrated a high expression level (1.12 g L − 1), and a high affinity for SUMO (2.26 nM). Meanwhile, VHHs were coupled to agarose resins by cysteine at the C-terminal to form affinity chromatography resins. The VS2 resin showed excellent specificity and a dynamic binding capacity for SUMO, SUMO-DsbA (disulfide oxidoreductase) and SUMO-SAM (S-adenosylmethionine synthetase) were 2.41 mg/mL resin, 7.57 mg/mL resin and 16.23 mg/mL resin, respectively. Furthermore, the VS2 resin enabled one-step purification of SUMO-fusions [SUMO-Fc (human IgG1-Fc fragment), SUMO-IGF1 (human insulin-like growth factor 1), SUMO-FGF21 (human fibroblast growth factor 21), SUMO-G-CSF (human Granulocyte colony-stimulating factor), SUMO-PDGF (human platelet-derived growth factor) and SUMO-PAS200 (conformationally disordered polypeptide chains with expanded hydrodynamic volume comprising the small residues Pro, Ala-and Ser)], and maintained binding capacity and selectivity over 25 purification cycles, each including 15 min of cleaning-in-place with 0.1 M NaOH. This study demonstrated that the VS2 resin was a useful tool at the laboratory scale for one-step purification of various SUMO fusions from complex mixtures.

Time to Achieve Platelet Count Response after Intravenous Efgartigimod in Adults with Primary Immune Thrombocytopenia: A Phase 3, Multicenter, Double-Blinded, Placebo-Controlled, Randomized Clinical Trial (ADVANCE IV)

Background In primary immune thrombocytopenia (ITP), immunoglobulin G (IgG) anti-platelet autoantibodies contribute to platelet clearance and impair platelet production. IgG homeostasis is regulated by the neonatal Fc receptor (FcRn), which binds to IgG in a pH-dependent manner and recycles bound IgG. Efgartigimod (EFG), a human IgG1 Fc fragment, is a natural ligand of FcRn engineered to bind competitively to FcRn with high affinity, preventing recycling of endogenous IgG, thereby reducing IgG levels, including pathogenic IgG autoantibody levels. Methods Intravenous (IV) EFG was evaluated in ADVANCE IV, a phase 3, multicenter, randomized, double-blinded, placebo (PBO)-controlled trial (NCT04188379) in adults with persistent or chronic ITP (Broome CM, et al. ASH 2022). Participants who received 2 prior or 1 prior and 1 concurrent ITP therapy with an average of 2 platelet (PLT) counts of <30×10 9/L during screening were randomized 2:1 to receive EFG IV 10 mg/kg or PBO IV for 24 weeks. Concurrent oral corticosteroids, immunosuppressants, dapsone, danazol, fostamatinib, and oral thrombopoietin receptor agonists were permitted but required to remain at the entry dosage and frequency. Participants received weekly dosing of either EFG or PBO (weeks 1-4) followed by response-dependent weekly or every-other-week dosing (weeks 5-16) and then maintained their dosing regimen from weeks 17-24. PLT counts were measured weekly starting at day 7 after treatment initiation. The extent of disease control, defined as cumulative weeks with PLT counts of ≥50×10 9/L in the chronic ITP population, was assessed as a key secondary endpoint. This analysis evaluates trends in time to response in ADVANCE IV. The time to achieve 2 consecutive PLT counts of ≥50×10 9/L was evaluated as a secondary endpoint. Time to 2 consecutive PLT counts of ≥100×10 9/L and time to single counts of ≥50×10 9/L and 100×10 9/L were analyzed as defined a priori. Post hoc Kaplan-Meier (KM) analyses, including time to a single PLT of ≥30×10 9/L and time to the International Working Group (IWG) response, were also performed. Change from baseline in mean PLT counts over time was evaluated based on a mixed model for repeated measurements. Results One hundred thirty-one participants (118 chronic ITP, 13 persistent ITP) were randomized (86 EFG, 45 PBO). Least-squares mean estimate changes from baseline in PLT counts were significantly different in EFG group compared with PBO group starting at day 7 (22×10 9/L vs -1×10 9/L; P=0.0293) and remained significant at most time points throughout the study. 38.4% of EFG-treated participants compared with 11.1% of PBO treated participants achieved a PLT count of ≥30×10 9/L in 7 days. The EFG group showed a higher proportion of PLT count responders ≥50×10 9/L on ≥4 occasions until week 12 compared with PBO (34.9% vs 4.4%, respectively). Based on KM analysis, 31.8% of participants receiving EFG and 2.2% of those receiving PBO achieved 2 consecutive PLT counts of ≥50×10 9/L by day 42 (Figure). 41.5% of participants receiving EFG vs 11.4% of those receiving PBO achieved the first PLT count ≥50×10 9/L by day 42. 22.5% of participants receiving EFG vs 2.2% of those receiving PBO achieved the first PLT count ≥100×10 9/L by day 42 (Table). The cumulative number of weeks in disease control was significantly higher in the EFG group compared with PBO (mean [SD]: 6.1 [7.66] vs. 1.5 [3.23]; P=0.0009) (Broome CM, et al. ASH 2022).KM estimates for IWG response by day 42 in the overall (chronic and persistent ITP) population were 29.7% in participants receiving EFG vs 4.6% in those receiving PBO. Overall, EFG was well tolerated; most adverse events were mild or moderate in severity. Conclusions In ADVANCE IV, EFG demonstrated a rapid PLT count increase, as early as the first time point measured (7 days after EFG treatment). Irrespective of the specific measure of response utilized, these data demonstrate that at all time points evaluated more participants in the EFG group achieved response compared with the PBO group. In addition to this early response, significantly more sustained responses were observed in the EFG group compared with the PBO group. The response rate, regardless of definition, in participants who received EFG continued to increase over time suggesting that some participants may have delayed responses to EFG. These data further demonstrate early and sustained responses with EFG in ITP.

IL1RAP-Specific T Cell Engager (TCE) Antibody Efficiently Depletes Acute Myeloid Leukemia (AML) Leukemic Stem Cells (LSCs)

LSCs are self-renewal primitive cells that initiate and maintain AML. Because they are highly therapy resistant, they are often responsible for treatment failures. Due to the lack of a specific antigen (Ag) and their quiescent nature, LSCs have been challenging to target and eliminate. IL1RAP is highly expressed on most human AML cell lines and primary blasts, including LSC-enriched CD34+CD38- cells, but not on normal hematopoietic stem cells (HSCs). To leverage this observation, we generated over 50 unique mouse IL1RAP monoclonal (m) antibodies (Abs), grouped them based on sequence, and selected representative members from the clades to generate murine-human chimeric mAbs. One of the mAbs, termed #24, had the strongest antibody-dependent cell-mediated cytotoxicity (ADCC) properties and the greatest in vivo anti-AML activity. Thus, it was advanced to generate a bispecific (Bs) anti-IL1RAP/CD3 TCE Ab termed BiF002, which incorporates a human IgG1 Fc mutated to lack FcR binding. Incubation of BIF002 with healthy donor T cells and AML cell lines or primary AML blasts induced IL1RAP dependent T cell activation, and Ab-dependent T cell lysis of leukemic cells in a dose-, time- and effector to target (E: T) ratio-dependent manner. The IC 50 against MOLM13 and THP-1 was subnanomolar (0.047nM, 95%CI:0.036-0.060nM and 0.025nM, 95%CI:0.14-0.40nM, respectively), at E:T ratio 5:1 at 48 hours using resting T cells. The IC 50 of IL1RAP pos AML bulk and CD34+ blasts (n=6) was also subnanomolar (0.45nM; 95%CI: 0.28-1.96nM). Co-cultures of normal CD34+ cells with BIF002 and T cells did not activate T cells or eliminate the normal CD34+ cells. To test the antileukemic activity of BIF002 in vivo, we engrafted luciferase (luc)-expressing MOLM13 cells into NSG-SGM3 (NSGS) mice. On day (D) 3, in vitro expanded healthy donors' T cells (3x10 6/mouse, weekly), with either BIF002 or a control IgG (10µg,) were administered intravenously (IV) every 3 days x 4-weeks. Mice treated with T cells + BIF002 exhibited a 100-fold greater tumor reduction on D33 than other groups, as measured by bioluminescence imaging (BLI), and had a significantly longer overall survival (OS; median 54.5 D) compared to IgG (27 D, p=0.0045), BIF002 alone (36 D, p=0.0049), or T cells + IgG (33 D, p=0.0062) treated controls. In initial dose finding studies using a similar schema, lower BIF002 doses (0.1 and 1 µg) were tested. Mice treated with T cells and 1 µg of BIF002 had a 100-fold tumor reduction on D29 and longer OS (median 57 D) compared to T cells + 0.1µg of BIF002 (31 D, p=0.0018) or T cells + vehicle (34 D, p=0.0019). We also engrafted luc-expressing primary blasts from a relapsed/refractory complex karyotype AML patient into NSGS mice that treated for 3 weeks using the above described schema (10µg). In this experiment, we used as control BIF026, a bispecific Ab (BsAb) carrying point mutations in both Fab arms to abrogate target binding to IL1RAP and CD3. Mice treated with T cells + BIF002 exhibited a >1000-fold reduction in disease burden and longer OS than BIF026 or BIF002 alone or T-cells+ BIF026-treated mice (median 46 D vs 26, 25 or 30 D, respectively, all comparisons, p=0.0003). In a second patient-derived xenograft (PDX) model, blasts from a relapsed complex karyotype AML patient were engrafted into NSG mice. Treatment was initiated on D10, with T cells given weekly along with 10µg of BIF002 or BIF026 for 3 weeks. After 70 days, all mice treated with T cells + BIF002 were alive, with no detectable blasts in peripheral blood or bone marrow (BM), while all vehicle (median 38 D, p=0.0002) and T cells + BIF026 (38 D, p=0.0001) treated controls had died of leukemia. To determine if T cells + BIF002 eradicated LSCs, we randomly selected 3 female mice from each group on D 34 and transplanted BM mononuclear cells (MNC) into secondary NSGS recipients (n=7). At D 90, no recipients of BM from donors treated with T cells + BIF002 had detectable blasts and remained alive, while all vehicle (median 26 D, p=0.0004) and T cells + BIF026 (median 26 D, p=0.0002) treated controls succumbed to AML. Importantly, no obvious hematopoietic or non-hematologic side effects were observed after BIF002 administration alone or with activated T cells in normal and leukemic mice. In summary, we provide evidence of the subnanomolar potency and efficacy of BIF002, a novel anti-IL1RAP/CD3 TCE, in eliminating AML blasts, including LSCs. BIF002 is currently undergoing IND-enabling studies.

Production of recombinant human long-acting IL-18 binding protein: inhibitory effect on ulcerative colitis in mice.

Interleukin-18 binding protein (IL-18BP) is a natural IL-18 inhibitor in vivo, which can effectively neutralize IL-18 and inhibit the inflammatory signaling pathway induced by IL-18, thus playing an anti-inflammatory role. Traditional production methods primarily rely on eukaryotic animal cell expression systems, which often entail complex processes, lower yields, and increase production costs. In this study, we present a novel approach for expressing IL-18BP fusion protein using the Escherichia coli (E. coli) system. The N-terminal segment of IL-18BP was fused with the small ubiquitin-related modifier (SUMO) tag, enabling soluble expression, while the C-terminal segment was fused with the human IgG1 Fc fragment to prolong its in vivo lifespan. Through screening, we obtained a high-expression engineering strain from a single colony and developed optimized protocols for fermentation and purification of the recombinant SUMO-IL-18BP-Fc protein. The SUMO tag was subsequently cleaved using SUMO protease, and the purified recombinant human IL-18BP-Fc (rhIL-18BP-Fc) exhibited a purity exceeding 90% with a yield of 1 g per liter of bacterial solution. The biological activities and underlying mechanisms of rhIL-18BP-Fc were evaluated using cell lines and a mouse model. Our results demonstrated that rhIL-18BP-Fc effectively inhibited IL-18-stimulated IFN-γ production in KG-1a cells in vitro and ameliorated DSS-induced ulcerative colitis in mice. In conclusion, we successfully employed the SUMO fusion system to achieve high-level production, soluble expression, and prolonged activity of rhIL-18BP-Fc in E. coli. These findings lay the groundwork for future large-scale industrial production and pharmaceutical development of rhIL-18BP-Fc protein. KEY POINTS: • Effective expression, fermentation, and purification of bioactive rhIL-18BP-Fc protein in E. coli. • The rhIL-18BP-Fc protein has a great potential for the therapy of ulcerative colitis by inhibiting the expression of inflammatory factors.

Efficacy and safety of the neonatal Fc receptor inhibitor efgartigimod in adults with primary immune thrombocytopenia (ADVANCE IV): a multicentre, randomised, placebo-controlled, phase 3 trial

Primary immune thrombocytopenia is an autoimmune disorder mediated partly by platelet autoantibodies, resulting in thrombocytopenia, bleeding, and constitutional symptoms. Efgartigimod, a first-in-class novel human IgG1 Fc fragment, binds the neonatal Fc receptor with high affinity and thus reduces serum IgG concentrations, including autoantibodies. The objective of this study was to evaluate the efficacy and safety of efgartigimod in adults with persistent and chronic primary immune thrombocytopenia. This phase 3, multicentre, randomised, double-blinded, placebo-controlled, 24-week study evaluated the efficacy and safety of intravenous efgartigimod in adults aged 18 years or older with chronic or persistent primary immune thrombocytopenia who had an average platelet count of less than 30 000, had responded to at least one previous immune thrombocytopenia therapy, and were on a concurrent therapy at baseline or had received at least a second previous immune thrombocytopenia therapy. The study took place in 71 participating sites from Asia, Europe, and North America. Patients were randomly assigned 2:1 to receive either efgartigimod (10 mg/kg) or placebo intravenously for the first 4 weeks, after which the dosing schedule could be altered to once per week or every other week depending on the patients' platelet count. The primary endpoint, evaluated in the chronic population, was sustained platelet count response (≥50 × 109 for at least 4 of the last 6 weeks). This study is registered with ClinicalTrials.gov (NCT04188379) and is completed. A total of 205 patients were screened from Dec 9, 2019, to Feb 3, 2022, and 131 (86 in the efgartigimod group; 45 in the placebo group) were randomly assigned. These patients represented a population with long-term disease who had a mean time since diagnosis of 10·6 years and 67% (88/131) of whom had received at least three previous immune thrombocytopenia treatments. 22% (17/78) of patients with chronic immune thrombocytopenia receiving efgartigimod reached the primary endpoint compared with 5% (2/40) of those receiving placebo (p=0·032; adjusted difference in response, 16% [95% CI 2·6-26·4]). The median number of weeks of disease control in patients with chronic immune thrombocytopenia was 2·0 (IQR 0·0-11·0) for efgartigimod versus 0·0 (0·0-1·0) for placebo (p=0·0009). Efgartigimod was well tolerated; most adverse events were mild to moderate in severity. The most common adverse events of interest in both groups were headache (16% in efgartigimod and 13% in placebo), haematuria (16% in efgartigimod and 16% in placebo), and petechiae (15% in efgartigimod and 27% in placebo). Efgartigimod significantly increased sustained platelet count responses compared with placebo in patients with chronic immune thrombocytopenia, including those who had received multiple previous immune thrombocytopenia therapies. Upon completion of the ADVANCE IV study, patients could enroll in the ongoing open-label extension. Subcutaneous efgartigimod is currently being evaluated in patients with immune thrombocytopenia in the ADVANCE SC+ trial. argenx.

Preclinical assessment of a novel human antibody VH domain targeting mesothelin as an antibody-drug conjugate

Mesothelin (MSLN) has been a validated tumor-associated antigen target for several solid tumors for over a decade, making it an attractive option for therapeutic interventions. Novel antibodies with high affinity and better therapeutic properties are needed. In the current study, we have isolated and characterized a novel heavy chain variable (VH) domain 3C9 from a large-size human immunoglobulin VH domain library. 3C9 exhibited high affinity (KD [dissociation constant] <3nM) and binding specificity in a membrane proteome array (MPA). In a mouse xenograft model, 3C9 fused to human IgG1 Fc was detected at tumor sites as early as 8h post-infusion and remained at the site for over 10days. Furthermore, 3C9 fused to a human Fc domain drug conjugate effectively inhibited MSLN-positive tumor growth in a mouse xenograft model. The X-ray crystal structure of full-length MSLN in complex with 3C9 reveals interaction of the 3C9 domains with two distinctive residue patches on the MSLN surface. This newly discovered VH antibody domain has a high potential as a therapeutic candidate for MSLN-expressing cancers.

Efficacy and safety of tripterygium wilfordii Hook F plus TNF inhibitor for active rheumatoid arthritis: A multicentre, randomized, double-blind, triple-dummy controlled trial

An investigator-initiated, multicentre, randomized, double-blind, triple-dummy, controlled trial was conducted at 14 tertiary rheumatology centers in China to evaluate the efficacy and safety of Tripterygium wilfordii Hook F (TwHF) with recombinant human TNF receptor IgGFc fusion protein (rhTNFR-Fc) in active Rheumatoid Arthritis (RA). Primary endpoint was the proportion of patients achieved a 50% improvement of American College of Rheumatology criteria (ACR50) in TwHF+rhTNFR-Fc vs. methotrexate (MTX) group at week 12. ACR50 was achieved in 57.1% (72/126), 41.3% (52/126), 23.0% (29/126), and 26.2% (33/126) patients receiving TwHF+rhTNFR-Fc, MTX + rhTNFR-Fc, TwHF and MTX monotherapy, respectively, at week 12 (TwHF+rhTNFR-Fc vs. other three groups, all p < 0.05). No statistical difference in serious adverse events or adverse events leading to discontinuation of study across all groups was documented. TwHF+rhTNFR-Fc was superior to MTX for active RA, and was more effective than MTX + rhTNFR-Fc on ACR50, with a similar safety profile.Trial registration:ClinicalTrials.govNCT03589833.

A NOVEL MSLN×4-1BB BISPECIFIC ANTIBODY FOR SOLID TUMOR

Abstract Background Mesothelin (MSLN) is a 70 KD glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein that is rarely expressed in normal tissues but overexpressed in a variety of cancers, including mesothelioma, ovarian cancer, pancreatic cancer and breast cancer et.al. 4-1BB is a member of the tumor necrosis factor receptor superfamily that functions as a co-stimulatory molecule. Agonistic antibodies bind to 4-1BB, triggering a signaling cascade leading to T-cell activation and expansion of cytotoxic CD8+ T lymphocytes. Here, we developed two bispecific antibodies (bsAbs) targeting both MSLN and 4-1BB with an intact Fc fragment from human IgG1 or IgG4, named HK013-G1 and HK013-G4 respectively. We suspected that HK013-G1 can simultaneously exert the cytotoxic effect of CD8+T cells and NK cells on tumor cells expressing MSLN to achieve better antitumor efficacy. Methods Both HK013-G1 and HK013-G4 were constructed by fused a single-chain variable fragment (scFv) targeting hu4-1BB to the C terminus of an anti-MSLA nanobody. And their affinity was optimized to making it highly effective in tumor localization. Next, we tested the killing ability of bsAbs-mediated PBMC or NK92 against tumor cells with different expression levels of MSLN in vitro. And the IFN-γ secretion was detected when CD8+T cells co-cultured with MSLN+ or MSLN- cells in the presence of antibodies. Also, the 4-1BB agonist activity of bsAbs was measured in a luciferase report gene assay. To confirm the safety of HK013-G1, non-specific activation of 4-1BB signal mediated by Fc receptor and CRS was evaluated in vitro. Finally, we compared the antitumor activity of two bispecific antibodies in both MC38/hMSLN and CT26/hMSLN tumor model and hepatotoxicity as well as cardiotoxicity was evaluated. Results Affinity-optimized HK013-G1 has an order of magnitude greater affinity for MSLN(KD≈10−9M) than 4-1BB (KD≈10−8M). HK013-G1 induced stronger PBMC against tumor cells than MOARb009 while HK013-G4 does not. Also, HK013-G1 could only mediate the killing of NK92 on MSLN-positive tumor cells. In co-cultured assay, HK013-G1 had superior ability to stimulate CD8+T cell secretion of IFN-γ than urelumab in the presence of MSLN. In luciferase reporter assay, the bsAbs-induced 4-1BB activation is dependent on expression level of MSLN. In addition, HK013-G1 was shown no stronger ability to inducing non-specific activation of 4-1BB signal mediated by Fc receptor and CRS in vitro. Compared with HK013-G4, HK013-G1 showed a more significant anti-tumor effect in both MC38/hMSLN and CT26/hMSLN tumor model. And, HK013-G4 showed significant hepatotoxicity in mice while HK013-G1 not. Moreover, HK013-G1 can protect mice against tumor re-challenge. Conclusions HK013-G1, an MSLN×4-1BB bsAb with human IgG1 Fc fragment, prevents tumor development by killing tumor cells directly via effector functions mediated by NK and cytotoxic T cells. More importantly, HK013-G1 showed no stronger toxic side effects both in vitro and in vivo. These results show that HK013-G1 has the potential to develop into a new clinical therapy for cancer types with MSLN expression.

CHO-produced RBD-Fc subunit vaccines with alternative adjuvants generate immune responses against SARS-CoV-2.

Subunit vaccines feature critical advantages over other vaccine platforms such as stability, price, and minimal adverse effects. To maximize immunological protection of subunit vaccines, adjuvants are considered as main components that are formulated within the subunit vaccine. They can modulate adverse effects and enhance immune outcomes. However, the most suitable formulation providing the best immunological outcomes and safety are still under investigation. In this report, we combined recombinant RBD with human IgG1 Fc to create an RBD dimer. This fusion protein was expressed in CHO and formulated with alternative adjuvants with different immune activation including Montanide ISA51, Poly (I:C), and MPLA/Quil-A® as potential vaccine candidate formulations. Using the murine model, a potent induction of anti-RBD IgG antibodies in immunized mice sera were observed. IgG subclass analyses (IgG1/IgG2a) illustrated that all adjuvanted formulations could stimulate both Th1 and Th2-type immune responses in particular Poly (I:C) and MPLA/Quil-A®, eliciting greater balance. In addition, Montanide ISA51-formulated RBD-Fc vaccination provided a promising level of neutralizing antibodies against live wild-type SARS-CoV-2 in vitro followed by Poly (I:C) and MPLA/Quil-A®, respectively. Also, mice sera from adjuvanted formulations could strongly inhibit RBD:ACE2 interaction. This study offers immunogenicity profiles, forecasted safety based on Vaccine-associated enhanced disease (VAED) caused by Th1-skewed immunity, and neutralizing antibody analysis of candidates of RBD-Fc-based subunit vaccine formulations to obtain an alternative subunit vaccine formulation against SARS-CoV-2.

811-P: Initiation of Once-Weekly (QW) Basal Insulin Fc (BIF) in Three Patient Populations in Phase 2 Studies Using a One-Time Loading Dose (LD) Minimizes Transient Hyperglycemia and Is Not Associated with Excess Hypoglycemia Risk vs. Degludec

BIF is a fusion protein combining a novel single chain insulin variant with a human IgG Fc domain. The safety population from 3 randomized, open-label, phase 2 treat-to-target studies included 942 people with T2D previously treated with basal insulin (T2D-Switch) or new to insulin therapy (T2D-Naïve), or with T1D on multiple daily injections (T1D). QW BIF was compared to once daily (OD) DEG for 26 or 32 wks. Given BIF's slow accumulation and extended PK profile, initiation of or transition from prior OD basal insulin to BIF was managed by “boosting” initial exposure with a one-time ~ 3-fold LD strategy to achieve therapeutic concentration more quickly. In the absence of a definitive mg to unit conversion factor, determination of the starting QW dose differed by study and was based on PK/PD modelling and individual patient characteristics. Fasting glucose and patient reported hypoglycemia &amp;lt;70 mg/dL were compared during the first 6 wks of treatment and showed no clinically significant differences between treatments (Table). T2D-Switch and T1D studies included CGM and data during first 6 wks of the study were reviewed; no severe hypoglycemia was reported during this time. These data suggest that the BIF LD strategy is safe and effective in reducing transient hyperglycemia, especially in T2D. Disclosure J.M.Bue-valleskey: Employee; Eli Lilly and Company. C.M.Kazda: Employee; Eli Lilly and Company. Q.Zhang: Employee; Eli Lilly and Company, Stock/Shareholder; Eli Lilly and Company. A.Haupt: Employee; Lilly, Stock/Shareholder; Lilly. D.Dahl: Research Support; Lilly, Novo Nordisk, Eli Lilly and Company, Boehringer Ingelheim Inc., Amgen Inc. J.P.Frias: Advisory Panel; Becton, Dickinson and Company, Pfizer Inc., Sanofi, Consultant; Akero Therapeutics, Inc., 89bio, Inc., Aimmune, Boehringer Ingelheim Inc., Eli Lilly and Company, Carmot Therapeutics, Inc., Echosens, Merck &amp; Co., Inc., Metacrine, Inc., Novo Nordisk, Pfizer Inc., Sanofi, Employee; Ionis Pharmaceuticals, Research Support; Akero Therapeutics, Inc., 89bio, Inc., Altimmune, Axcella Health Inc., Boehringer Ingelheim Inc., Eli Lilly and Company, Intercept Pharmaceuticals, Inc., Carmot Therapeutics, Inc., Janssen Pharmaceuticals, Inc., Madrigal Pharmaceuticals, Inc., Merck &amp; Co., Inc., Metacrine, Inc., Novo Nordisk, Oramed Pharmaceuticals, Novartis, Pfizer Inc., Sanofi, Speaker's Bureau; Eli Lilly and Company, Sanofi. Funding Eli Lilly and Company

806-P: Once Weekly (QW) Basal Insulin Fc (BIF) and Daily Degludec (DEG) Achieve Comparable Glycemic Control in Three Patient Populations Studied during Phase 2—Clinical Insights from CGM Assessments

BIF is a fusion protein combining a novel single chain insulin variant with a human IgG Fc domain. Three randomized, open-label, treat-to-target phase 2 studies of 26 or 32 week duration assessed 912 people with T2D previously treated with basal insulin (T2D-Switch) or new to insulin therapy (T2D-Naïve), or T1D on multiple daily injections (T1D). Studies assessed QW BIF vs once daily DEG. Key results were previously disclosed and met the primary endpoint of non-inferiority in HbA1c change from baseline (non-inferiority margin=0.4%). Two studies had CGM during entire study and T2D-Naïve had blinded, intermittent CGM using 14-day sessions. Patients had baseline HbA1c of 8.1, 8.0, and 7.5% for T2D-Switch, T2D-Naïve, and T1D respectively. Figure shows average hourly mean glucose at endpoint and time in range (inset). Glucose profiles appeared comparable between treatments with 24-hour average glucose for BIF vs DEG in T2D-Naïve: 137 vs 127 mg/dL p=.042 and T1D: 175 vs 169 p=.075 (T2D-Switch not available). Average duration of hypoglycemia, &amp;lt;70mg/dL (min) per patient for BIF vs DEG: T2D-Switch 31 vs 35 p=NS, T2D-Naïve 51 vs 91 p&amp;lt;.001, and T1D 35 vs 35 p=NS. QW BIF achieved similar glycemic control vs DEG as seen in several CGM derived measures providing useful and reassuring insights into the use of QW BIF in these patient populations. Disclosure C.M.Kazda: Employee; Eli Lilly and Company. J.M.Bue-valleskey: Employee; Eli Lilly and Company. Q.Zhang: Employee; Eli Lilly and Company, Stock/Shareholder; Eli Lilly and Company. A.Haupt: Employee; Lilly, Stock/Shareholder; Lilly. J.P.Frias: Advisory Panel; Becton, Dickinson and Company, Pfizer Inc., Sanofi, Consultant; Akero Therapeutics, Inc., 89bio, Inc., Aimmune, Boehringer Ingelheim Inc., Eli Lilly and Company, Carmot Therapeutics, Inc., Echosens, Merck &amp; Co., Inc., Metacrine, Inc., Novo Nordisk, Pfizer Inc., Sanofi, Employee; Ionis Pharmaceuticals, Research Support; Akero Therapeutics, Inc., 89bio, Inc., Altimmune, Axcella Health Inc., Boehringer Ingelheim Inc., Eli Lilly and Company, Intercept Pharmaceuticals, Inc., Carmot Therapeutics, Inc., Janssen Pharmaceuticals, Inc., Madrigal Pharmaceuticals, Inc., Merck &amp; Co., Inc., Metacrine, Inc., Novo Nordisk, Oramed Pharmaceuticals, Novartis, Pfizer Inc., Sanofi, Speaker's Bureau; Eli Lilly and Company, Sanofi. D.Dahl: Research Support; Lilly, Novo Nordisk, Eli Lilly and Company, Boehringer Ingelheim Inc., Amgen Inc. Funding Eli Lilly and Company