Fc Fusion Protein Research Articles

Comparison of the Immunogenicity of the LZ901 Vaccine and HZ/su Vaccine in a Mouse Model

Herpes zoster (HZ) is an infectious disease caused by the reactivation of varicella zoster virus (VZV), with 68% of cases occurring in adults over 50 years of age. HZ/su (Shingrix®) was approved by the Food and Drug Administration in 2017 for the prevention of HZ in individuals ≥ 50 years of age and showed very good protection from HZ. However, due to the use of the adjuvant AS01B, adverse reactions caused by Shingrix are a concern. Aluminum hydroxide is the most commonly used adjuvant and is widely used in a variety of vaccines. We developed a recombinant zoster vaccine (code: LZ901) consisting of a tetramer of VZV glycoprotein E (gE) and a human Fc fusion protein expressed in CHO cells, an immune complex-like molecule that can be adsorbed with an aluminum hydroxide adjuvant. We compared the immunogenicity of LZ901 with that of HZ/su in BALB/c mice. The results showed that LZ901 induced levels of gE-specific IgG antibodies comparable to those induced by HZ/su, and the results of FAMA titers further demonstrated their similar neutralizing antibody abilities. Most importantly, LZ901 induced higher levels of cell-mediated immunity (CMI) (which plays a decisive role in the efficacy of zoster vaccines) than HZ/su in BALB/c mice. The numbers of cytokine-producing T cells in LZ901-vaccinated mice were significantly greater than those in v-vaccinated mice, and the proportions of CD4+ and CD8+ T cells producing at least two types of cytokines in LZ901-vaccinated mice were significantly greater than those in HZ/su-vaccinated mice.

Combining CD38 antibody with CD47 blockade is a promising strategy for treating hematologic malignancies expressing CD38.

CD38 and CD47 are expressed in many hematologic malignancies, including multiple myeloma (MM), B-cell non-Hodgkin lymphoma (NHL), B-cell acute lymphoblastic leukemia (ALL), and B-cell chronic lymphocytic leukemia (CLL). Here, we evaluated the antitumor activities of CD38/CD47 bispecific antibodies (BsAbs). Five suitable anti-CD38 antibodies for co-targeting CD47 and CD38 BsAb were developed using a 2 + 2 "mAb-trap" platform. The activity characteristics of the CD38/CD47 BsAbs were evaluated using in vitro and in vivo systems. Using hybridoma screening technology, we obtained nine suitable anti-CD38 antibodies. All anti-CD38 antibodies bind to CD38+ tumor cells and kill tumor cells via antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Five anti-CD38 antibodies (4A8, 12C10, 26B4, 35G5, and 65A7) were selected for designing CD38/CD47 BsAbs (IMM5605) using a "mAb-trap" platform. BsAbs had higher affinity and binding activity to the CD38 target than those to the CD47 target, decreasing the potential on-target potential and off-tumor effects. The CD38/CD47 BsAbs did not bind to RBCs and did not induce RBC agglutination; thus, BsAbs had much lower blood toxicity. The CD38/CD47 BsAbs had a greater ability to block the CD47/SIRPα signal in CD38+/CD47+ tumor cells than IMM01 (SIRPα Fc fusion protein). Through Fc domain engineering, CD38/CD47 BsAbs were shown to kill tumors more effectively by inducing ADCC and ADCP. IMM5605-26B4 had the strongest inhibitory effect on cellular CD38 enzymatic activity. IMM5605-12C10 had the strongest ability to directly induce the apoptosis of tumor cells. The anti-CD38 antibody 26B4 combined with the SIRPα-Fc fusion proteins showed strong antitumor effects, which were better than any of the mono-therapeutic agents used alone in the NCI-H929 cell xenograft model. The CD38/CD47 BsAbs exhibited strong antitumor effects; specifically, IMM5605-12C10 efficiently eradicated all established tumors in all mice. A panel of BsAbs targeting CD38 and CD47 developed based on the "mAb-tarp" platform showed potent tumor-killing ability in vitro and in vivo. As BsAbs had lower affinity for binding to CD47, higher affinity for binding to CD38, no affinity for binding to RBCs, and did not induce RBC agglutination, we concluded that CD38/CD47 BsAbs are safe and have a satisfactory tolerability profile.

The promising feeder-free allogenic NK cell therapy: Clinical outcomes of GIC-102 monotherapy (GIC-102101) and synergistic potential with GI-101 (CD80-IgG4 Fc-IL2v) in advanced solid tumors and hematologic malignancies.

e14526 Background: The allogeneic Natural Killer (NK) cell therapy derived from healthy donors may overcome the short lifespan and dysfunctional anti-tumor activity of current autologous approach. This limitation often restricts treatment efficacy and necessitating high dosages and frequent infusions. GIC-102, non-engineered / feeder-free, highly potent allogeneic NK cell derived from healthy donors, is a promising alternative. Here, we present the clinical outcomes from the Phase 1 study of GIC-102, along with nonclinical evidence demonstrating outstanding pharmacokinetics (PK) and pharmacodynamics (PD) when used in combination with a novel bi-specific Fc fusion protein, GI-101 (CD80-IgG4 Fc-IL2v/NCT04977453/KEYNOTE-B59). Methods: GIC-102101, a first-in-human study, evaluated GIC-102 monotherapy at doses of 1x109 QW and 3x109 QW in patients with advanced solid tumors or hematologic malignancies. A lympho-conditioning strategy using cyclophosphamide (300 mg/m2) and fludarabine (30 mg/m2) was implemented every two cycles before GIC-102 treatment. The study employed a conventional 3+3 dose escalation scheme. The primary endpoint was to determine the safety and tolerability of GIC-102, with tumor response assessed as secondary endpoints. Additionally, the PK/PD profile of GIC-102 in combination with GI-101 was evaluated in a mouse model. Results: As of December 31, 2023, six patients have been treated with GIC-102 monotherapy including two patients with Non-Hodgkin lymphoma (NHL), and four patients with metastatic colorectal cancer (CRC). The median number of prior treatments for patients with CRC and NHL was 4 and 6, respectively. No dose limiting toxicities (DLTs) were observed and maximum tolerated dose was not reached. Treatment-related adverse events (TRAEs) occurred in 5 patients (83%), with the majority being associated with the lympho-conditioning procedure; hyponatremia, neutropenia, lymphopenia and anemia. The best overall response was complete response (CR) in 1/6 patient (NHL) and stable disease (SD) in 4/6 patients (3 CRC, 1 NHL). Treatment for 1 CR (NHL) and 2 SD (CRC) is ongoing (for NHL, 215+ days/for CRC, 160+ days and 153+ days). The in-vivo study of GIC-102 in combination with GI-101 showed significantly enhanced anti-tumor activity and in-vivo NK cell proliferation lasting up to 4 weeks. Conclusions: The GIC-102, feeder-free allogeneic NK cell, was well tolerated without DLTs and demonstrated early promise in efficacy in patients for advanced solid tumors and hematologic malignancy. Further clinical investigation of GIC-102 in combination with GI-101, bi-specific Fc fusion protein, is currently ongoing. Clinical trial information: NCT05880043 .

A phase I clinical study to evaluate the safety, tolerability, immunogenicity, pharmacokinetics and preliminary efficacy of SCB-219M in patients with chemotherapy-induced thrombocytopenia (CIT): An interim analysis.

e15015 Background: CIT is a serious, chemotherapy-associated complication observed in a wide range of cancer patients with various types of cancer. Incidence of CIT can occur in about 15-25% of patients undergoing standard chemotherapy regiments with platinum, taxane and/or gemcitabine-based regimen. CIT can lead to chemotherapy dose delay or dose reduction, and potentially bleeding events. Currently, there is no FDA-approved therapy for CIT. SCB-219M, a thrombopoietin receptor agonist (TPO-RA) mimetic bispecific Fc-fusion protein produced from CHO cells can efficiently raise PLT count under normal and pathological conditions in laboratory animals. Methods: This is a single-arm, multi-center, open-label, dose escalation Phase Ia clinical study to evaluate the safety, tolerability, and immunogenicity, PK profile and preliminary efficacy of a single ascending dose of SCB-219M administered one day after a chemotherapy in prior CIT patients with malignant solid tumors and lymphoma. The study design of accelerated titration combined with "3 + 3" is used, and the number of subjects planned to enroll is 12 to be assigned to 4 cohorts with 2, 6, 10 and 15 mg/kg subcu administration of SCB-219M, respectively. Enrollment criteria include episode of CIT experienced (<75 × 109/L in PLT count) in prior cycle of chemotherapies and PLT count has recovered to >75 × 109/L prior to initiation of this study . The chemotherapy regimen allowed include either mono or combination chemotherapy (including targeted therapy and/or immunotherapy). The observation period of dose-limiting toxicities (DLTs) is 21 days. Chemotherapy is planned at each study site. There will be no change in the planned dose or frequency of chemotherapy in the study. Results: As of Dec 20, 2023, 9 prior CIT patients including 3 at 15 mg/kg were enrolled. SCB-219M was well tolerated. There were no SAEs or AESIs (Adverse Event of Special Interest) observed. DLT was not reached. A favorable PK profile, in particular in serum availability (AUC) and half-life (T1/2) was obtained. All 9 prior CIT patients in the study maintained PLT counts >75 x 109/L from 1-week following chemotherapies with a single dose of SCB-219M, and durable responses experienced through at least 3 weeks. Conclusions: In this interim analysis, drug safety, PK profile and durable efficacy are expected to support SCB-219M dosing interval of ≥2-weeks or a single dosing regimen within a chemotherapy cycle. Accordingly, a Phase Ib trial evaluating repeated dosing of SCB-219M is planned to initiate in 2024. Clinical trial information: NCT05426369 .

#1342 Updated results from the RUBY-3 study of povetacicept, an enhanced dual BAFF/APRIL antagonist, in autoantibody-associated glomerulonephritis

Abstract Background and Aims Inhibition of BAFF and/or APRIL has shown promise in IgA nephropathy (IgAN), systemic lupus erythematosus (SLE), lupus nephritis (LN), and primary membranous nephropathy (pMN), and has the potential to exert a disease-modifying effect. Povetacicept (ALPN-303) is an Fc fusion protein of a variant TACI domain engineered for more potent dual BAFF/APRIL inhibition than wild-type TACI or anti-BAFF or anti-APRIL antibodies. In a previous report, povetacicept 80 mg every 4 weeks was initially well tolerated and demonstrated promising, clinically meaningful reductions in urine protein to creatinine ratio (UPCR) and Gd-IgA1 in participants with IgAN (Tumlin J, et al. Poster TH-PO1125 presented at ASN 2023). Updated data are reported here. Method RUBY-3 is an open-label, multiple ascending dose, phase 1b/2a study of povetacicept 80 or 240 mg administered subcutaneously once every 4 weeks. Eligible participants are aged ≥18 years with biopsy-confirmed IgAN, LN, or pMN and on maximally tolerated ACE inhibitor/ARB therapy, with well-controlled blood pressure, and disease-specific immunosuppressive therapy where applicable. The primary objective is safety; secondary objectives include pharmacokinetics, pharmacodynamics, immunogenicity, biomarkers, and efficacy. Results As of 1 Dec 2023, 12 participants with IgAN have enrolled and received povetacicept 80 mg, with 7 (58%) having received ≥24 weeks of treatment. Povetacicept continues to be well tolerated, with the majority of treatment-emergent adverse events being of low grade. There have been no incidences of severe hypogammaglobulinemia (IgG <3 g/L) or severe infections. Povetacicept 80 mg was associated with a UPCR reduction of 52.6% at 24 weeks (n = 7). Reductions in Gd-IgA1, stable renal function, and pharmacodynamically expected decreases in immunoglobulin levels were also observed. Additional results from the 80 mg cohort with longer duration of follow-up, as well as initial results from the 240 mg dosing cohort, are planned to be presented. Conclusion Povetacicept remains well tolerated with multiple dosing and continues to demonstrate very promising activity in IgAN, strongly supporting further study in IgAN as well as other glomerulonephritis and autoantibody-associated diseases.

Development of nanoparticle vaccines utilizing designed Fc-binding homo-oligomers and RBD-Fc of SARS-CoV-2

The Fc-fused receptor binding domain (RBD-Fc) vaccine for SARS-CoV-2 has garnered significant attention for its capacity to provide effective and specific immune protection. However, its immunogenicity is limited, highlighting the need for improvement in clinical application. Nanoparticle delivery has been shown to be an effective method for enhancing antigen immunogenicity. In this study, we developed bivalent nanoparticle recombinant protein vaccines by assembling the RBD-Fc of SARS-CoV-2 and Fc-binding homo-oligomers o42.1 and i52.3 into octahedral and icosahedral nanoparticles. The formation of RBD-Fc nanoparticles was confirmed through structural characterization and cell binding experiments. Compared to RBD-Fc dimers, the nanoparticle vaccines induced more potent neutralizing antibodies (nAb) and stronger cellular immune responses. Therefore, using bivalent nanoparticle vaccines based on RBD-Fc presents a promising vaccination strategy against SARS-CoV-2 and offers a universal approach for enhancing the immunogenicity of Fc fusion protein vaccines.

Comprehensive analysis and characterization of glycan pairing in therapeutic antibodies and Fc-containing biotherapeutics: Addressing current limitations and implications for N-glycan impact

N-glycosylation of the Fc part is a (critical) quality attribute of therapeutic antibodies and Fc-containing biotherapeutics, that impacts their stability, immunogenicity, pharmacokinetics, and effector functions. Current glycosylation analysis methods focus on the absolute amounts of glycans, neglecting the apparent glycan distribution over the entirety of proteins. The combination of the two Fc N-glycans, herein referred to as glyco-pair, therefore remains unknown, which is a major drawback for N-glycan impact assessment. This study presents a comprehensive workflow for the analysis and characterization of Fc N-glycan pairing in biotherapeutics, addressing the limitations of current glycosylation analysis methods. The applicability of the method across various biotherapeutic proteins including antibodies, bispecific antibody formats, and a Fc-Fusion protein is demonstrated, and the impact of method conditions on glycan pairing analysis is highlighted. Moreover, the influence of the molecular format, Fc backbone, production process, and cell line on glycan pairing pattern was investigated. The results underscore the significance of comprehensive glycan pairing analysis to accurately assess the impact of N-glycans on important product quality attributes of therapeutic antibodies and Fc-containing biotherapeutics.

IL-15/IL-15Rα-Fc fusion protein XmAb24306 potentiates activity of CD3 bispecific antibodies through enhancing T cell expansion.

Insufficient quantity of functional T cells is a likely factor limiting clinical activity of T cell bispecific antibodies, especially in solid tumor indications. We hypothesized that XmAb24306 (efbalropendekin alfa), a lymphoproliferative interleukin (IL)-15/IL-15 receptor α (IL-15Rα) Fc-fusion protein, may potentiate the activity of T cell dependent (TDB) antibodies. Activation of human peripheral T cells by cevostamab, an anti-FcRH5/CD3 TDB, or anti-HER2/CD3 TDB resulted in upregulation of IL-2/15Rβ (CD122) receptor subunit in nearly all CD8+ and majority of CD4+ T cells, suggesting that TDB treatment may sensitize T cells to the IL-15. XmAb24306 enhanced T cell bispecific antibody induced CD8+ and CD4+ T cell proliferation and expansion. In vitro combination of XmAb24306 with cevostamab or anti-HER2/CD3 TDB resulted in significant enhancement of tumor cell killing, which was reversed when T cell numbers were normalized, suggesting that T cell expansion is the main mechanism for the observed benefit. Pre-treatment of immune competent mice with a mouse-reactive surrogate of XmAb24306 (mIL-15-Fc) resulted in significant increase of T cells in blood, spleen and in tumors and converted transient anti-HER2/CD3 TDB responses to complete durable responses. In summary, our results support the hypothesis where the number of tumor infiltrating T cells is rate limiting for the activity of solid tumor targeting TDBs. Upregulation of CD122 by TDB treatment and the observed synergy with XmAb24306 and T cell bispecific antibodies supports clinical evaluation of this novel immunotherapy combination.

Broadly Reactive Nanobody Targeting the H3 Hemagglutinin of the Influenza A Virus.

Monoclonal antibodies and recombinant antibody fragments are a very promising therapeutic tool to combat infectious diseases. Due to their unique paratope structure, nanobodies (VHHs) hold several advantages over conventional monoclonal antibodies, especially in relation to viral infections. Influenza A viruses (IAVs) remain a major threat to public health. The hemagglutinin (HA) protein is the main protective and immunodominant antigen of IAVs. In this study, three broadly reactive nanobodies (D9.2, E12.2, and D4.2) to H3N2 influenza strains were isolated and Fc-fusion proteins (VHH-Fcs) were obtained and characterized in vitro. This modification improved the nanobodies' binding activity and allowed for their interaction with a wider range of strains. The D9.2-Fc antibody showed a 100% protection rate against mortality in vivo in a mouse lethal model. Furthermore, we demonstrated that the observed protection has to do with Fc-FcγR interactions. These results indicate that D9.2-Fc can serve as an effective antiviral agent against the H3N2 influenza infection.

Effect of 4-phenylbutyrate addition timing on titer of Fc-fusion protein in Chinese hamster ovary cell cultures

Effect of 4-phenylbutyrate addition timing on titer of Fc-fusion protein in Chinese hamster ovary cell cultures

Impact of fed-batch process intensification on the productivity and product quality of two CHO cell lines expressing unique novel molecular format proteins.

While monospecific antibodies have long been the foundational offering of protein therapeutics, recent advancements in antibody engineering have allowed for the development of far more complex antibody structures. Novel molecular format (NMF) proteins, such as bispecific antibodies (BsAbs), are structures capable of multispecific binding, allowing for expanded therapeutic functionality. As demand for NMF proteins continues to rise, biomanufacturers face the challenge of increasing bioreactor process productivity while simultaneously maintaining consistent product quality. This challenge is exacerbated when producing structurally complex proteins with asymmetric modalities, as seen in NMFs. In this study, the impact of a high inoculation density (HID) fed-batch process on the productivity and product quality attributes of two CHO cell lines expressing unique NMFs, a monospecific antibody with an Fc-fusion protein and a bispecific antibody, compared to low inoculation density (LID) platform fed-batch processes was evaluated. It was observed that an intensified platform fed-batch process increased product concentrations by 33 and 109% for the two uniquely structured complex proteins in a shorter culture duration while maintaining similar product quality attributes to traditional fed-batch processes.

Cost-Effectiveness of Recombinant FactorIX Fc Prophylaxis and Recombinant FactorIX On-Demand Treatment in Patients with HaemophiliaB Without Inhibitors.

Recombinant factor IX (rFIX) and recombinant FIX Fc fusion protein (rFIXFc) are standard half-life and extended half-life FIX replacement therapies, respectively, and represent established treatment options indicated for adults and children with haemophiliaB. These FIX replacement therapies can be administered as prophylaxis (to prevent bleeding) or 'on-demand' (to stop bleeding). This analysis aimed to estimate the cost-effectiveness of once-weekly prophylaxis with rFIXFc versus on-demand treatment with rFIX in patients with haemophiliaB without inhibitors in the Italian healthcare setting. A Markov model was developed to assess a hypothetical cohort of adolescent or adult male patients (≥ 12years) with haemophiliaB (FIX level of ≤ 2IU/dL) without inhibitors. Model inputs were derived from the pivotal phase3 clinical studies for rFIXFc and rFIX, published literature and assumptions when published data were unavailable. The model employed a lifelong time horizon with 6-monthly transitions between health states, and it estimated total costs, total quality-adjusted life years (QALYs), number of bleeds, number of surgeries and incremental cost-effectiveness ratio. rFIXFc prophylaxis was associated with lower total costs per patient (€5,308,625 versus €6,564,510) and greater total QALYs per patient (15.936 versus 11.943) compared with rFIX on-demand; rFIXFc prophylaxis was therefore the dominant treatment strategy. The model also demonstrated that rFIXFc prophylaxis was associated with fewer incremental bleeds (- 682.29) and surgeries (- 0.39) compared with rFIX on-demand. rFIXFc prophylaxis provides improved health outcomes and lower costs, and represents a cost-effective treatment option compared with rFIX on-demand for adolescent and adult male patients with haemophiliaB. This comparative assessment of cost-effectiveness should help to inform both clinicians and healthcare policy makers when making treatment decisions for patients with haemophiliaB.

Quantification of Biopharmaceutically Relevant Nonionic Surfactant Excipients Using Benchtop qNMR.

Nonionic surfactant excipients (NISEs) are commonly added to biologics formulations to mitigate the effects of stress incurred by the active biotherapeutic during manufacturing, transport, and storage. During manufacturing, NISEs are added by dilution of a stock solution directly into a protein formulation, and their accurate addition is critical in maintaining the quality and integrity of the drug product and thus ensuring patient safety. This is especially true for the common NISEs, polysorbates 20 and 80 (PS20 and PS80, respectively) and poloxamer 188 (P188). With the increasing diversity of biologic modalities within modern pharmaceutical pipelines, there is thus a critical need to develop and deploy convenient and user-accessible analytical techniques that can rapidly and reliably quantify these NISEs under biopharmaceutically relevant conditions. We thus pursued 60 MHz benchtop quantitative NMR (qNMR) as a nondestructive and user-friendly analytical technique for the quantification of PS20, PS80, and P188 under such conditions. We demonstrated the ability of benchtop qNMR (1) to quantify simulated PS20, PS80, and P188 stock solutions representative of those used during the drug substance (DS) formulation step in biomanufacturing and (2) to quantify these NISEs at and below their target concentrations (≤0.025% w/v) directly in biologics formulations containing histidine, sucrose, and one of three biotherapeutic modalities (monoclonal antibody, antibody-drug conjugate, and Fc-fusion protein). Our results demonstrate that benchtop qNMR offers a fit-for-purpose, reliable, user-friendly, and green analytical route by which NISE of interest to the biopharmaceutical industry may be readily and reliably quantified. We conclude that benchtop qNMR has the potential to be applied to other excipient formulation components in the presence of various biological modalities as well as the potential for routine integration within analytical and QC laboratories across pharmaceutical development and manufacturing sites.

Covid-19 Prevention and Treatment by Targeting Fc-fusion Proteins: An Experience to Fight Emerging Diseases.

The coronavirus disease 2019 (Covid-19) pandemic has been considered a major threat to human health. Effective therapeutic approaches are urgently required. Spike protein and the Angiotensin-converting enzyme 2 (ACE2) receptors have critical roles in SARS-CoV-2 infection. As a result, these two proteins are considered potential targets for the development of a wide variety of biotherapeutics and vaccines for controlling Covid-19. The fusion proteins have desirable medicinal properties, including high serum half-life, stability, and solubility in the body. Moreover, other Fc-fusion proteins used to treat other diseases have no known side effects. These Fc-fusion proteins are valuable biopharmaceuticals and have been proposed as therapeutic candidates for the treatment and prevention of Covid-19 owing to their potential therapeutic benefits.

Kinetic modeling of the plasma pharmacokinetic profiles of ADAMTS13 fragment and its Fc-fusion counterpart in mice.

Introduction: Fusion of the fragment crystallizable (Fc) to protein therapeutics is commonly used to extend the circulation time by enhancing neonatal Fc-receptor (FcRn)-mediated endosomal recycling and slowing renal clearance. This study applied kinetic modeling to gain insights into the cellular processing contributing to the observed pharmacokinetic (PK) differences between the novel recombinant ADAMTS13 fragment (MDTCS) and its Fc-fusion protein (MDTCS-Fc). Methods: For MDTCS and MDTCS-Fc, their plasma PK profiles were obtained at two dose levels following intravenous administration of the respective proteins to mice. The plasma PK profiles of MDTCS were fitted to a kinetic model with three unknown protein-dependent parameters representing the fraction recycled (FR) and the rate constants for endocytosis (kup, for the uptake into the endosomes) and for the transfer from the plasma to the interstitial fluid (kpi). For MDTCS-Fc, the model was modified to include an additional parameter for binding to FcRn. Parameter optimization was done using the Cluster Gauss-Newton Method (CGNM), an algorithm that identifies multiple sets of approximate solutions ("accepted" parameter sets) to nonlinear least-squares problems. Results: As expected, the kinetic modeling results yielded the FR of MDTCS-Fc to be 2.8-fold greater than that of MDTCS (0.8497 and 0.3061, respectively). In addition, MDTCS-Fc was predicted to undergo endocytosis (the uptake into the endosomes) at a slower rate than MDTCS. Sensitivity analyses identified the association rate constant (kon) between MDTCS-Fc and FcRn as a potentially important factor influencing the plasma half-life in vivo. Discussion: Our analyses suggested that Fc fusion to MDTCS leads to changes in not only the FR but also the uptake into the endosomes, impacting the systemic plasma PK profiles. These findings may be used to develop recombinant protein therapeutics with extended circulation time.

Abstract 2124: Validation of the intelligent glycotransferase-dependent conjugation (iGDC) platform: A groundbreaking approach for next-generation HER2-targeting ADC GQ1012 with enhanced druggability, potency, and safety

Abstract Background: Antibody glycan remodeling has emerged as a promising site-specific conjugation technology to develop ADCs, while most reported cases suffer from complicated CMC process and scalability challenges. We report herein an innovative, streamlined one-enzyme, one-step glycan remodeling technology, i.e. intelligent Glycotransferase-Dependent Conjugation (iGDC), that enables the site-specific conjugation of various biologic modalities including monoclonal antibody, bispecific antibody and Fc fusion proteins. Employing immobilized enzymes and a continuous-flow process, iGDC efficiently tethers payloads to the N-297 glycan site without requiring antibody engineering. In contrast to alternative conjugation techniques, iGDC offers superior versatility, product quality, process efficiency, scalability, and cost-effectiveness. Validation of the iGDC platform is exemplified by GQ1012, a HER2-targeting ADC featuring the potent Topoisomerase I inhibitor, TopoIx. Preclinical studies demonstrated GQ1012's outstanding anti-tumor efficacy in diverse in vitro and in vivo models, displaying robust synergistic effects with anti-PD-1 therapy and excellent tolerability in pre-tox studies involving monkeys with no signs of lung diseases, surpassing those of Enhertu. Results: GQ1012, developed through iGDC, utilized a highly hydrophilic, stable branch linker to conjugate TopoIx to an anti-HER2 antibody, achieving high conjugation efficiency confirmed by DAR analysis. The ADC maintained comparable antigen binding affinity and internalization compared to the parental antibody, exhibiting potent cytotoxicity against HER2-positive cancer cells. Despite glycan remodeling with iGDC leading to the removal of ADCC function, GQ1012 demonstrated no off-target cytotoxicity in HER2-negative cells and displayed a robust bystander killing ability in co-culture assays. In various in vivo models, GQ1012 demonstrated similar or superior antitumor efficacy compared to Enhertu, showcasing strong synergy with anti-PD-1 therapy. Stability studies confirmed GQ1012 maintains a highly stable linker, minimizing DAR reduction. Preliminary toxicity studies in monkeys following repeated intravenous infusions of GQ1012 showcased excellent safety and pharmacokinetics at specified doses and the potential for an extended therapeutic window compared to Enhertu. Conclusion: The validation of the iGDC platform through GQ1012 highlights its adaptability and efficiency in producing highly homogeneous next-generation ADCs without requiring antibody engineering. GQ1012 presents promising potential as a next generation HER2 ADC with an expanded therapeutic window compared to Enhertu, supported by extensive in vitro, in vivo assessments, and preliminary toxicity studies. Citation Format: Meijun Xiong, Zengyan Mu, Meiling Xu, Chong Liu, Bo Dai, Junhao Wang, Xinju Gao, Lili Shi, Paul H. Song, Gang Qin. Validation of the intelligent glycotransferase-dependent conjugation (iGDC) platform: A groundbreaking approach for next-generation HER2-targeting ADC GQ1012 with enhanced druggability, potency, and safety [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 2124.

The effects of Fc fusion protein glucagon-like peptide-1 and glucagon dual receptor agonist with different receptor selectivity in vivo studies

BackgroundGlucagon-like peptide-1 (GLP-1)/glucagon (GCG) dual receptor agonists with different receptor selectivity are under investigation and have shown significant improvement in both weight loss and glycemic control, but the optimal potency ratio between the two receptors to balance efficacy and safety remains unclear. Experimental approachWe designed and constructed several dual receptor agonists with different receptor potency ratios using Fc fusion protein technology. The long-term effects of the candidates on body weight and metabolic dysfunction-associated steatotic liver disease (MASLD) were evaluated in diet-induced obese (DIO) model mice, high-fat diet (HFD)-ob/ob mice and AMLN diet-induced MASLD mice. Repeat dose toxicity assays were performed to investigate the safety profile of the candidate (HEC-C070) in Sprague Dawley (SD) rats. Key resultsThe high GCG receptor (GCGR) selectivity of HEC-C046 makes it more prominent than other compounds for weight loss and most MASLD parameters but may lead to safety concerns. The weight change of HEC-C052 with the lowest GCG agonism was inferior to that of selective GLP-1 receptor agonist (GLP-1RA) semaglutide in DIO model mice. The GLP-1R selectivity of HEC-C070 with moderate GCG agonism has a significant effect on weight loss and liver function in obese mice, and its lowest observed adverse effect level (LOAEL) was 30 nmol/kg in the repeat dose toxicity study. ConclusionWe compared the potential of the Fc fusion protein GLP-1/GCG dual receptor agonists with different receptor selectivity to provide the setting for future GLP-1/GCG dual receptor agonists to treat obesity and MASLD.

Abstract 2477: Selective IL-21 and IFN-alpha immunocytokines engineered using the AlphaSeq platform

Abstract Although cytokine therapies have demonstrated curative effects in some cancer patients, clinical use remains limited due to undesired toxicity profiles accompanying systemic administration. Next generation cytokine approaches include conditional signaling focused on sites of interest, such as the tumor microenvironment or specific immune cell populations. Here, we share a novel approach for generating detuned cytokine therapeutic candidates using the AlphaSeq platform, which involves the re-engineering of yeast agglutination and mating to quantitatively measure protein-protein interactions at a library-on-library scale. Engineering interferon-alpha (IFNα) and interleukin-21 (IL-21), we show how AlphaSeq measures cytokine-receptor interactions and identifies cytokine variants with a broad range of affinities. A saturated mutational library was created for IFNα and IL-21and subsequently screened against a second library consisting of human IFNAR2 or human IL-21R, species orthologs and off-target receptors. AlphaSeq enabled identification of hundreds of detuned IFNα and IL-21 variants against both human and mouse receptors in parallel assays. Cytokine variants with lower affinity than parental IFNα or IL-21 were recombinantly expressed as Fc fusion proteins to orthogonally measure affinity with biolayer interferometry and characterize potency with an in vitro human PBMC phosflow assay, which showed strong correlation with AlphaSeq affinity measurements. Finally, detuned cytokine candidates were fused to anti-CD8 and other localizing antibodies to demonstrate cell population-specific signaling. Candidate molecules showed 1000-fold or greater potency in the targeted cell population than non-targeted populations. Our results show the AlphaSeq platform can accurately quantitate thousands of cytokine variant affinities simultaneously against multiple relevant receptors, enabling the selection of candidate immunocytokine antibody fusion proteins with targeted cell biased signaling. AlphaSeq’s rapid, comprehensive affinity determination is being used to develop clinically relevant IFNα and IL-21 therapeutic immunocytokines with accompanying preclinical data. Citation Format: Charles Lin, Colleen Shikany, Leah Homad, Jessica Fint, Ruchi Bansal, Davis Goodnight, Jeff Adamo, Danielle Van Citters, Ryan Swanson, Randolph Lopez. Selective IL-21 and IFN-alpha immunocytokines engineered using the AlphaSeq platform [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 2477.

Development of an Engineered Single-Domain Antibody for Targeting MET in Non-Small Cell Lung Cancer.

The Mesenchymal Epithelial Transition (MET) receptor tyrosine kinase is upregulated or mutated in 5% of non-small-cell lung cancer (NSCLC) patients and overexpressed in multiple other cancers. We sought to develop a novel single-domain camelid antibody with high affinity for MET that could be used to deliver conjugated payloads to MET expressing cancers. From a naïve camelid variable-heavy-heavy (VHH) domain phage display library, we identified a VHH clone termed 1E7 that displayed high affinity for human MET and was cross-reactive with MET across multiple species. When expressed as a bivalent human Fc fusion protein, 1E7-Fc was found to selectively bind to EBC-1 (MET amplified) and UW-Lung 21 (MET exon 14 mutated) cell lines by flow cytometry and immunofluorescence imaging. Next, we investigated the ability of [89Zr]Zr-1E7-Fc to detect MET expression in vivo by PET/CT imaging. [89Zr]Zr-1E7-Fc demonstrated rapid localization and high tumor uptake in both xenografts with a %ID/g of 6.4 and 5.8 for EBC-1 and UW-Lung 21 at 24 h, respectively. At the 24 h time point, clearance from secondary and nontarget tissues was also observed. Altogether, our data suggest that 1E7-Fc represents a platform technology that can be employed to potentially both image and treat MET-altered NSCLC.

Identification of structural origins of complex charge heterogeneity in therapeutic ACE2Fc fusion protein facilitated by free-flow isoelectric focusing

Fc Fusion protein represents a versatile molecular platform with considerable potential as protein therapeutics of which the charge heterogeneity should be well characterized according to regulatory guidelines. Angiotensin-converting enzyme 2 Fc fusion protein (ACE2Fc) has been investigated as a potential neutralizing agent to various coronaviruses, including the lingering SARS-CoV-2, as this coronavirus must bind to ACE2 to allow for its entry into host cells. ACE2Fc, an investigational new drug developed by Henlius (Shanghai China), has passed the Phase I clinical trial, but its huge amount of charge isoforms and complicated charge heterogeneity posed a challenge to charge variant investigation in pharmaceutical development. We employed offline free-flow isoelectric focusing (FF-IEF) fractionation, followed by detailed characterization of enriched ACE2Fc fractions, to unveil the structural origins of charge heterogeneity in ACE2Fc expressed by recombinant CHO cells. We adopted a well-tuned 3-component separation medium for ACE2Fc fractionation, the highest allowable voltage to maximize the FF-IEF separation window and a mild Protein A elution method for preservation of protein structural integrity. Through peptide mapping and other characterizations, we revealed that the intricate profiles of ACE2Fc charge heterogeneity are mainly caused by highly sialylated multi-antenna N-glycosylation. In addition, based on fraction characterization and in silico glycoprotein model analysis, we discovered that the large acidic glycans at N36, N73, and N305 of ACE2Fc were able to decrease the binding activity towards Spike (S) protein of SARS-CoV-2. Our study exemplifies the value of FF-IEF in highly complex fusion protein characterization and revealed a quantitative sialylation-activity relationship in ACE2Fc.