Fc Fusion Research Articles

Biodistribution of recombinant factor IX, extended half-life recombinant factor IX Fc fusion protein, and glycoPEGylated recombinant factor IX in hemophilia B mice.

Extended half-life recombinant FIX (rFIX) molecules have been generated to reduce the dosing burden and increase the protection of patients with hemophilia B. Clinical pharmacology studies with recombinant factor IX Fc fusion protein (rFIXFc) report a similar initial peak plasma recovery to that of rFIX, but with a larger volume of distribution. Although the pegylation of N9-GP results in a larger plasma recovery, there is a smaller volume of distribution, suggesting less extravasation of the latter drug. In this study, we set out to compare the biodistribution and tissue localization of rFIX, rFIXFc, and glycoPEGylated rFIX in a hemophilia B mouse model. Radiolabeled rFIX, rFIXFc, and rFIX-GP were employed in in vivo single-photon emission computed tomography imaging (SPECT/CT), microautoradiography (MARG), and histology to assess the distribution of FIX reagents over time. Immediately following injection, vascularized tissues demonstrated intense signal irrespective of FIX reagent. rFIX and rFIXFc were retained in joint and muscle areas through 5 half-lives, unlike rFIX-GP (assessed by SPECT). MARG and immunohistochemistry showed FIX agents localized at blood vessels among tissues, including liver, spleen, and kidney. Microautoradiographs, as well as fluorescent-labeled images of knee joint areas, demonstrated retention over time of FIX signal at the trabecular area of bone. Data indicate that rFIXFc is similar to rFIX in that it distributes outside the plasma compartment and is retained in certain tissues over time, while also retained at higher plasma levels. Overall, data suggest that Fc fusion does not impede the extravascular distribution of FIX.

Efficacy of rFIXFc versus N9-GP Prophylaxis in Patients with Hemophilia B: Matching-Adjusted Indirect Comparison of B-LONG and PARADIGM 2 Trials.

For patients with hemophilia B, extended half-life factor IX (FIX) products are available for prophylaxis and for treating bleeds. Different methods are used to extend the half-lives of recombinant FIX Fc fusion protein (rFIXFc) and nonacog beta pegol (N9-GP). This affects their biodistribution and plasma FIX levels, although differences do not always correlate with clinical outcomes. A matching-adjusted indirect comparison (MAIC) of prophylaxis with rFIXFc and N9-GP was performed, based on licensed dosing in the European Union. Combined rFIXFc data from the weekly and individualized interval prophylaxis arms of the B-LONG clinical trial, and N9-GP data from the 40 IU/kg once-weekly prophylaxis arm of PARADIGM 2 were used in a MAIC. Individual patient data for rFIXFc (n=87) were matched to aggregated data for N9-GP (n=29). Estimated annualized bleeding rates (ABRs) for rFIXFc were recalculated using a Poisson regression model with adjustment for over-dispersion, and compared with ABRs reported for N9-GP, using incidence rate ratios (IRRs) with 95% confidence interval (CI). There was no evidence of significant differences in estimated ABRs between prophylaxis with rFIXFc and N9-GP. Analysis of pooled rFIXFc weekly and interval-adjusted dosing compared with N9-GP 40 IU/kg once weekly produced estimated ABRs of 2.59 versus 2.51 (IRR 1.03; 95% CI 0.56-1.89), as well as 1.34 versus 1.22 (IRR 1.10; 95% CI 0.42-2.91) and 1.13 versus 1.29 (IRR 0.88; 95% CI 0.47-1.63) for overall, spontaneous, and traumatic bleeding events, respectively. The study did not reveal any significant differences in the efficacy of rFIXFc and N9-GP prophylaxis. Given differences in trough levels (rFIXFc dosing was targeted to achieve a trough 1-3 IU/dL above baseline versus a reported estimated N9-GP mean trough of 27.3 IU/dL), interpreting plasma FIX levels as potential surrogate efficacy markers requires consideration of compound-specific pharmacokinetic profiles.

Post hoc longitudinal assessment of the efficacy and safety of recombinant factor IX Fc fusion protein in hemophilia B.

Long-term efficacy and safety of the extended half-life recombinant factor IX Fc fusion protein (rFIXFc) has been established among previously treated patients with severe hemophilia B in 2 phase 3 trials (B-LONG [#NCT01027364] and Kids B-LONG [#NCT01440946]) and a long-term extension study (B-YOND [#NCT01425723]). In this study, we report post hoc analyses of pooled longitudinal data for up to 6.5 years for rFIXFc prophylaxis. In the B-LONG study, subjects ≥12 years received weekly dose-adjusted prophylaxis (WP; starting dose, 50 IU/kg), individualized interval-adjusted prophylaxis (IP;initially, 100 IU/kg every 10days), or on-demand dosing. In the Kids B-LONG study, subjects <12 years received 50 to 60 IU/kg every 7days, adjusted as needed. In the B-YOND study, subjects received WP (20-100 IU/kg every 7days), IP (100 IU/kg every 8-16days), modified prophylaxis, or on-demand dosing; switching between treatment groups was permitted. A total of 123 subjects from B-LONG and 30 from Kids B-LONG study were included, of whom 93 and 27, respectively, enrolled in the B-YOND study. The median cumulative duration of treatment was 3.63 years (range, 0.003-6.48 years) in B-LONG/B-YOND and 2.88 years (range, 0.30-4.80 years) in Kids B-LONG/B-YOND group. Annualized bleed rates (ABRs) remained low, annualized factor consumption remained stable, and adherence remained high throughout treatment. Low ABRs were also maintained in subjects with dosing intervals ≥14days or with target joints at baseline. Complete resolution of evaluable target joints and no recurrence in 90.2% of baseline target joints during follow-up were observed. rFIXFc prophylaxis was associated with sustained clinical benefits, including long-term bleed prevention and target joint resolution, for severe hemophilia B.

Determination of HER2 binding domain in antigen-antibody complexes based on chemical crosslinking mass spectrometry

Chemical crosslinking (XL) of non-covalent antigen-antibody complexes followed by mass spectrometric identification (MS) of inter-protein crosslinks can provide spatial constraints between relevant residues, which are valuable structural information associated with the molecular binding interface. To highlight the potential of XL/MS in the biopharmaceutical industry, we herein developed and validated an XL/MS workflow that employed a zero-length linker, 1,1′‑carbonyldiimidazole (CDI), and a widely used medium-length linker, disuccinimidyl sulfoxide (DSSO), for fast, accurate determination of antigen domains targeted by therapeutic antibodies. To avoid false identification, system suitability samples and negative samples were designed for all experiments, and all tandem mass spectra were manually examined. To validate the proposed XL/MS workflow, two complexes involving human epidermal growth factor receptor 2 Fc fusion protein (HER2Fc) with known crystal structures, including HER2Fc-pertuzumab and HER2Fc-trastuzumab, have been subjected to CDI and DSSO crosslinking. Crosslinks established by CDI and DSSO between HER2Fc and pertuzumab accurately revealed their interaction interface. CDI crosslinking contributes more than DSSO because of its short spacer arm and high reactivity towards hydroxyl groups, demonstrating its capacity in protein interaction analysis. The correct binding domain cannot be revealed solely based on DSSO in the HER2Fc-trastuzumab complex, because domain proximity revealed by this 7-atom spacer linker cannot be directly translated as binding interfaces. As the first successful XL/MS application in early-stage therapeutic antibody discovery, we analyzed the molecular binding interface between HER2Fc and H-mab, an innovant drug candidate whose paratopes have not been studied yet. We predict that H-mab probably targets HER2 Domain I. The proposed XL/MS workflow can serve as an accurate, fast, and low-cost method to study the interaction between antibodies and large multi-domain antigens. SignificanceThis article described a fast, low-consumption approach based on chemical crosslinking mass spectrometry (XL/MS) using two linkers for binding domain determination in multidomain antigen-antibody complexes. Our results highlighted the higher importance of zero-length crosslinks established by CDI than 7-atom DSSO crosslinks, as residue proximity revealed by zero-length crosslinks is closely related to epitope-paratope interaction surfaces. Furthermore, the higher reactivity of CDI towards hydroxyl groups broadens the ranges of possible crosslinks, despite the necessity of delicate operation in CDI crosslinking. We suggest that all established CDI and DSSO crosslinks should be comprehensively considered for correct binding domain analysis because predictions solely based on DSSO might be ambiguous. We have determined the binding interface in the HER2-H-mab using CDI and DSSO, which is the first successful application of XL/MS in real-world early-stage biopharmaceutical development.

Dependency of Tamoxifen Sensitive and Resistant ER+ Breast Cancer Cells on Semaphorin 3C (SEMA3C) for Growth.

Estrogen receptor positive (ER+) breast cancer (BCa) accounts for the highest proportion of breast cancer-related deaths. While endocrine therapy is highly effective for this subpopulation, endocrine resistance remains a major challenge and the identification of novel targets is urgently needed. Previously, we have shown that Semaphorin 3C (SEMA3C) is an autocrine growth factor that drives the growth and treatment resistance of various cancers, but its role in breast cancer progression and endocrine resistance is poorly understood. Here, we report that SEMA3C plays a role in maintaining the growth of ER+ BCa cells and is a novel, tractable therapeutic target for the treatment of ER+ BCa patients. Analyses of publicly available clinical datasets indicate that ER+ BCa patients express significantly higher levels of SEMA3C mRNA than other subtypes. Furthermore, SEMA3C mRNA expression was positively correlated with ESR1 mRNA expression. ER+ BCa cell lines (MCF7 and T47D) expressed higher levels of SEMA3C mRNA and protein than a normal mammary epithelial MCF10A cell line. ER siRNA knockdown was suppressed, while dose-dependent beta-estradiol treatment induced SEMA3C expression in both MCF7 and T47D cells, suggesting that SEMA3C is an ER-regulated gene. The stimulation of ER+ BCa cells with recombinant SEMA3C activated MAPK and AKT signaling in a dose-dependent manner. Conversely, SEMA3C silencing inhibited Estrogen Receptor (ER) expression, MAPK and AKT signaling pathways while simultaneously inducing apoptosis, as monitored by flow cytometry and Western blot analyses. SEMA3C silencing significantly inhibited the growth of ER+ BCa cells, implicating a growth dependency of ER+ BCa cells on SEMA3C. Moreover, the analysis of tamoxifen resistant (TamR) cell models (TamC3 and TamR3) showed that SEMA3C levels remain high despite treatment with tamoxifen. Tamoxifen-resistant cells remained dependent on SEMA3C for growth and survival. Treatment with B1SP Fc fusion protein, a SEMA3C pathway inhibitor, attenuated SEMA3C-induced signaling and growth across a panel of tamoxifen sensitive and resistant ER+ breast cancer cells. Furthermore, SEMA3C silencing and B1SP treatment were associated with decreased EGFR signaling in TamR cells. Here, our study implicates SEMA3C in a functional role in ER+ breast cancer signaling and growth that suggests ER+ BCa patients may benefit from SEMA3C-targeted therapy.

Real-World Effectiveness of rFIXFc Prophylaxis in Patients with HaemophiliaB Switched from Standard Half-Life Therapy in Three European Countries.

The current study describes real-world clinical outcomes and factor usage among patients with haemophiliaB switching from standard half-life factorIX (SHL FIX) treatment to recombinant factorIX Fc fusion protein (rFIXFc) prophylaxis in European treatment centres. This non-interventional, retrospective, multicentre chart review evaluated medical records from adult and paediatric patients with haemophiliaB in Denmark, Germany and the UK. Patients had documented SHL FIX treatment, on-demand or prophylaxis, for ≥ 6months before starting rFIXFc prophylaxis, and subsequent data for ≥ 6months afterwards (up to 24months). Primary endpoints included annualised bleeding rates (ABRs), prophylactic factor consumption and injection frequency. Data from 30 patients (24/30 [80.0%] with severe disease) showed overall mean (standard deviation, SD) ABRs of 4.7 (6.3) on SHL FIX treatment and 1.7 (2.3) after switching to rFIXFc prophylaxis. The reduction in mean (SD) ABRs was greater when switching from SHL FIX on-demand treatment (n = 6), with a decrease from 10.5 (9.9) to 2.6 (4.5), than when switching from SHL FIX prophylaxis (n = 24), with a decrease from 3.3 (4.3) to 1.5 (1.4). Among prior SHL FIX prophylaxis patients, switching to rFIXFc prophylaxis increased the proportion of those with zero bleeds from 21.7% to 45.8% during the 6months before and after switching, respectively. In the total population, five of six target joints (83.3%) present when patients started rFIXFc prophylaxis subsequently resolved. In patients switching from SHL FIX prophylaxis to rFIXFc prophylaxis, mean (SD) weekly injection frequency was reduced by 1.0 (0.7) and mean (SD) factor consumption was reduced by 27.7 (49.6) IU/kg/week. This study demonstrates the effectiveness of rFIXFc prophylaxis in real-world clinical practice. Improvements in both clinical effectiveness and factor usage associated with rFIXFc prophylaxis may potentially reduce patient burden and improve quality of life.

#4024 PHASE 1 STUDY OF THE SAFETY, TOLERABILITY, PHARMACOKINETICS, AND PHARMACODYNAMICS OF POVETACICEPT FOR AUTOIMMUNE GLOMERULONEPHRITIDES

Abstract Background and Aims B cell activating factor (BAFF) of the tumor necrosis factor (TNF) family and a proliferation-inducing ligand (APRIL), cytokines which bind and signal through BAFF-R, transmembrane activator and CAML interactor (TACI), and/or B cell maturation antigen (BCMA) on B cells, play overlapping and non-redundant roles in B cell development, proliferation, function, and survival. Therapeutic agents targeting BAFF and/or APRIL have demonstrated promising clinical potential in autoantibody-related glomerulonephritides (GN) such as lupus nephritis (LN), immunoglobulin (Ig) A nephropathy (IgAN), membranous nephropathy, and other B cell-related diseases such as systemic lupus erythematosus (SLE); however, there is still need for more safe and efficacious therapies. Povetacicept (ALPN-303) is an Fc fusion protein of an engineered TACI variant TNFRSF domain (vTD) with enhanced affinity for APRIL and BAFF which mediates more potent inhibitory activity than wild type (WT) TACI-Fc or BAFF- or APRIL-specific antibodies. In preclinical studies, povetacicept demonstrated enhanced pharmacokinetic (PK) and immunomodulatory properties vs. WT TACI-Fc, which may translate to lower and/or less frequent doses in humans. Povetacicept also suppressed autoantibodies, renal IgG deposition, and nephritis in mouse models. Povetacicept may therefore significantly improve clinical outcomes in autoantibody-mediated GNs and other B cell-related diseases. Method In this first-in-human study (NCT05034484), 66 healthy adult volunteers were randomized 4:2 into single ascending dose cohorts of intravenous (IV) or subcutaneous (SC) povetacicept or placebo. Participants were followed to assess safety and PK, circulating Ig, galactose-deficient IgA1 (Gd-IgA1), and circulating leukocyte populations. Results Povetacicept has been well tolerated in all cohorts evaluated as single IV or SC doses of up to 960 mg. Overall, it exhibits dose-related PK and expected pharmacodynamic (PD) effects, including dose-related reductions in serum IgA, IgM, IgG, and Gd-IgA1 (Figure 1), and in circulating antibody-secreting cells (ASC; plasmablasts and plasma cells) (Figure 2). In the same setting, these PD effects appear greater than those reported for WT TACI-Fc molecules and appear to be saturated at doses ≥80 mg. Coverage of free APRIL was maintained for 2-3 weeks with 80 mg and ≥4 weeks with 240 mg, respectively. The most frequent adverse event has been mild headache. To date, there have been no imbalances of infections between placebo and povetacicept groups, no treatment-related reactions other than mild injection site pain, and no adverse trends in safety laboratories. Conclusion To date, povetacicept has demonstrated acceptable safety and tolerability as single IV or SC doses, exhibiting dose-dependent PK and PD that appear to differentiate favorably vs WT TACI-Fc. Based on the magnitude and duration of the observed PD effects, dose regimens of 80-240 mg SC every 4 weeks are anticipated for use in future studies. Overall, the study findings support future clinical development of povetacicept in multiple autoantibody-related GN, as well as other B cell- and/or autoantibody-related diseases such as SLE and autoantibody-associated cytopenias.

CD24-Fc suppression of immune related adverse events in a therapeutic cancer vaccine model of murine neuroblastoma.

The combination of Myc-suppressed whole tumor cells with checkpoint inhibitors targeting CTLA-4 and PD-L1 generates a potent therapeutic cancer vaccine in a mouse neuroblastoma model. As immunotherapies translate from pre-clinical to clinical trials, the potential immune-related adverse events (irAEs) associated with induction of potent immunity must be addressed. The CD24-Siglec 10/G interaction is an innate checkpoint that abrogates inflammatory responses to molecules released by damaged cells, but its role in cancer immunology is not well defined. We investigate irAEs of an effective whole cell neuroblastoma vaccine and subsequently the effect of CD24-Fc, a CD24 and Fc fusion protein, on both the vaccine efficacy and induced irAEs in a mouse neuroblastoma model. To test whether the whole tumor cell vaccination leads to autoimmune responses in other organ systems we harvested lung, heart, kidney and colon from naïve mice (n=3), unvaccinated tumor only mice (n=3), and vaccinated mice with CD24 Fc (n=12) or human IgG-Fc control (n=12) after tumor inoculation and vaccination therapy at day 30. The Immune cell infiltrates and immunogenic pathway signatures in different organ systems were investigated using NanoString Autoimmune Profiling arrays. Nanostring RNA transcript results were validated with immunohistochemistry staining. The whole tumor cell vaccine combined with immune checkpoint therapy triggers occult organ specific immune cell infiltrates, primarily in cardiac tissue and to a lesser extent in the renal and lung tissue, but not in the colon. CD24-Fc administration with vaccination partially impedes anti-tumor immunity but delaying CD24-Fc administration after initial vaccination reverses this effect. CD24-Fc treatment also ameliorates the autoimmune response induced by effective tumor vaccination in the heart. This study illustrates that the combination of Myc suppressed whole tumor cell vaccination with checkpoint inhibitors is an effective therapy, but occult immune infiltrates are induced in several organ systems in a mouse neuroblastoma model. The systemic administration of CD24-Fc suppresses autoimmune tissue responses, but appropriate timing of administration is critical for maintaining efficacy of the therapeutic vaccine.

Co-transient expression of PSA-Fc and PAP-Fc fusion protein in plant as prostate cancer vaccine candidates and immune responses in mice.

PAP-FcK and PSA-FcK prostate cancer antigenic proteins transiently co-expressed in plant induce their specific humoral immune responses in mice. Prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) have been considered as immunotherapeutic antigens for prostate cancer. The use of a single antigenic agent is unlikely to be effective in eliciting immunotherapeutic responses due to the heterogeneous and multifocal nature of prostate cancer. Thus, multiple antigens have been combined to enhance their anti-cancer effects. In the current study, PSA and PAP were fused to the crystallizable region (Fc region) of immunoglobulin G1 and tagged with KDEL, the endoplasmic reticulum (ER) retention signal motif, to generate PSA-FcK and PAP-FcK, respectively, and were transiently co-expressed in Nicotiana benthamiana. Western blot analysis confirmed the co-expression of PSA-FcK and PAP-FcK (PSA-FcK + PAP-FcK) with a 1:3 ratios in the co-infiltrated plants. PSA-FcK, PAP-FcK, and PSA-FcK + PAP-FcK proteins were successfully purified from N. benthamiana by protein A affinity chromatography. ELISA showed that anti-PAP and anti-PSA antibodies successfully detected PAP-FcK and PSA-FcK, respectively, and both detected PSA-FcK + PAP-FcK. Surface plasmon resonance (SPR) analysis confirmed the binding affinity of the plant-derived Fc fusion proteins to FcγRI/CD64. Furthermore, we also confirmed that mice injected with PSA-FcK + PAP-FcK produced both PSA- and PAP-specific IgGs, demonstrating their immunogenicity. This study suggested that the transient plant expression system can be applied to produce the dual-antigen Fc fusion protein (PSA-FcK + PAP-FcK) for prostate cancer immunotherapy.

Phase 1b study of batiraxcept (AVB-S6-500, BT) plus gemcitabine (G) and nab-paclitaxel (NP) as first-line treatment (1L) for advanced pancreatic adenocarcinoma (PDAC).

e16258 Background: PDAC is a deadly aggressive cancer characterized by up to 80% tumor stroma enriched by macrophages, fibroblasts, and collagen deposition that influences resistance to therapy. Tyrosine kinase receptor AXL and ligand expression of growth arrest specific protein 6 (GAS6) produce tumor associated macrophages and fibroblasts and correlate with poor prognosis. GAS6 blockade partially reverses epithelial-to-mesenchymal transition of tumor and supports NK cell activation, inhibiting metastasis. As a novel decoy FC fusion protein, batiraxcept (BT) binds to GAS6 and demonstrates highly potent and specific AXL inhibition. Preclinical studies showed a relationship between AXL signaling and response to DNA damage in PDAC models. Methods: This dose escalation study tested BT 15 mg/kg on day 1 and 15 in combination with G 1000 mg/m2 and NP 125 mg/m2 days 1, 8, 15 of a 28-day cycle. Primary endpoints are safety and objective response rate (ORR) using RECIST v1.1; key secondary endpoints are progression free survival (PFS) and overall survival (OS); pharmacokinetic data were collected to determine exposure-PFS response (E-R) relationship. Results: As of January 17, 2023, 21 patients (pts) received BT/G/NP of which 10 (48%) were male and 3 (14%) received prior neoadjuvant/adjuvant therapy. BT-related AEs &gt; 10% pts included fatigue (n=7, 33%), diarrhea (n=4, 19%), infusion related reaction and neutropenia (n=3, 14% each). Grade ≥ 3 BT-related AE occurred in 6 pts (29%). BT administration reduced GAS6 to non-quantifiable levels until treatment ended. 18 pts (86%) discontinued treatment, primarily due to toxicity (n=2) and progressive disease (n=13). Median (m) BT/G/NP treatment duration was 13.1 weeks (range 0.1─74.7). ORR is 29%; mPFS and mOS were 5.4 mo (2.1, 5.6) and 12.3 mo (4.11─NE) respectively. BT efficacy results are described by a minimally efficacious concentration (MEC) of 14.5 mg/L identified by E-R modeling. Conclusions: BT/G/NP combination was well tolerated, and the BT safety profile is consistent with prior trials. Patients with BT trough &gt; MEC had longer PFS and OS. In addition, in these patients OS appears longer than historical data of 8.5 months. Due to increased stroma seen in PDAC, a higher BT MEC may be required to maximize efficacy. Higher BT dose levels in combination with G/NP are under study. Clinical trial information: NCT04983407 . [Table: see text]

A phase Ib trial of the VEGF fusion protein HB002.1T plus chemotherapy in patients with advanced solid tumors.

2530 Background: HB002.1T is a recombinant human vascular endothelial growth factor receptor (VEGF)-IgG Fc fusion protein, which functions as a decoy receptor to bind VEGF-A, VEGF-B and placental growth factor (PlGF). In phase Ia, HB002.1T monotherapy showed encouraging anti-tumor activity in gallbladder cancer. Methods: This study aimed to evaluate efficacy and safety of HB002.1T plus chemotherapy (mainly based on platinum, paclitaxel, gemcitabine, etc.) in advanced solid tumors. The tumor assessment was evaluated by investigator according to RECIST v1.1. The study included part 1 and part 2, part 1 study was dose escalation (12mg/kg, 16mg/kg Q3W) and dose expansion conducted in patients (pts) with advanced solid tumors, part 2 study was dose expansion (16mg/kgQ3W) in specified tumors (cohort 1: ovarian and cervical, cohort 2: pancreatic, gallbladder and bile duct cancer). Results: As of Dec 20,2022,68 (42F/26M) advanced solid tumor pts were enrolled in the study with 32 pts in part 1 and 36 pts in part 2. 33.3% pts failed at least 3 lines of therapy before enrollment. 63(92.6%) pts had stage IV disease. Median age was 58. It is to be note that 12 efficacy assessment evaluablepts with ovarian cancer (1 with 12mg/kg in part 1,11with 16mg/kg in part 2) were all stage IV, which included 3 complete response (CR),6 partial responses (PR) and 3 stable disease (SD). Median progression free survival (PFS) was 7.3 months and 6-month PFS rate was 58.3%. The ORR and DCR were 75%, 100% respectively. The most common treatment related adverse events (TRAEs) included neutropenia (9/12, 75.0%) and leukocytopenia (8/12, 66.7%). 8 (8/12, 66.7%) pts had grade ≥ 3 TRAEs, which neutropenia (4/12, 33.3%) and leukocytopenia (3/12, 25.0%) were commonly observed. Part 1: In 28 pts whose tumor assessment were available, including 1 gastric cancer patient achieved CR, 11 pts achieved PR (2 gastric, 2 ovarian, 2 cervical, 1 maxillary, 1 lung, 1 parotid, 1 nasopharynx, 1 pancreatic) and 15 achieved SD. Part 2: 32 pts were available for tumor assessment, including 4 achieved CR (3 ovarian, 1 cervical), 10 achieved PR (4 ovarian, 2 cervical, 3 bileduct, 1 pancreatic), and 16 achieved SD. Overall, in Part 1 and 2, TRAEs occurred in 64 (64/68, 94.1%) pts. The most common TRAEs were leukocytopenia (28/68, 41.2%), neutropenia (26/68, 38.2%) and proteinuria (25/68, 36.8%). 34 (34/68, 50.0%) pts had grade ≥ 3 TRAEs, which neutropenia (15/68, 22.1%) and leukocytopenia (11/68, 16.2%) were commonly observed. TRAE leading to discontinuation occurred in 3 pts. No death was reported related with HB002.1T. Conclusions: The Pts were well tolerated to 12mg/kg and 16mg/kg HB002.1T dose combined with chemotherapy, and HB002.1T showed acceptable safety profile. As well as, promising anti-tumor activity was observed in various tumors, especially in advanced ovarian cancer. More data is being accumulated, and confirmatory clinical trials are being prepared. Clinical trial information: NCT04802980 .

Adenovirotherapy delivering cross-hybrid IgGA Fc engineering PD-L1 inhibitors for enhanced cancer immunotherapy

Adenovirotherapy delivering cross-hybrid IgGA Fc engineering PD-L1 inhibitors for enhanced cancer immunotherapy

Non-Redundant Roles of T Cell Costimulation Pathways in Inflammatory Arthritis Revealed by Dual Blockade of ICOS and CD28 with Acazicolcept (ALPN-101).

CD28 and inducible T cell costimulator (ICOS) appear to have nonredundant roles in T cell activation and adaptive immunity. We undertook this study to characterize in vitro and in vivo the therapeutic potential of acazicolcept (ALPN-101), an Fc fusion protein of a human variant ICOS ligand (ICOSL) domain designed to inhibit both CD28 and ICOS costimulation, in inflammatory arthritis. Acazicolcept was compared in vitro with inhibitors of either the CD28 or ICOS pathways (abatacept and belatacept [CTLA-4Ig], prezalumab [anti-ICOSL monoclonal antibody]) in receptor binding and signaling assays, and in a collagen-induced arthritis (CIA) model. Acazicolcept was also compared in cytokine and gene expression assays of peripheral blood mononuclear cells (PBMCs) from healthy donors or rheumatoid arthritis (RA) or psoriatic arthritis (PsA) patients stimulated with artificial antigen-presenting cells (APCs) expressing CD28 and ICOS ligands*. Acazicolcept bound CD28 and ICOS, prevented ligand binding, and inhibited human T cell functional interactions, matching or exceeding the activity of CD28 or ICOS costimulatory single-pathway inhibitors tested individually or in combination. Acazicolcept administration significantly reduced disease in the CIA model and more potently than abatacept. Acazicolcept also inhibited proinflammatory cytokine production from stimulated PBMCs in cocultures with artificial APCs and demonstrated unique effects on gene expression distinct from those induced by abatacept, prezalumab, or a combination of both. Both CD28 and ICOS signaling play critical roles in inflammatory arthritis. Therapeutic agents such as acazicolcept that coinhibit both ICOS and CD28 signaling may mitigate inflammation and/or disease progression in RA and PsA more effectively than inhibitors of either pathway alone.

TGFβ4 alleviates the phenotype of Charcot-Marie-Tooth disease type 1A.

The duplication of the peripheral myelin protein 22 (PMP22) gene causes a demyelinating type of neuropathy, commonly known as Charcot-Marie-Tooth disease type 1A (CMT1A). Development of effective drugs for CMT1A still remains as an unmet medical need. In the present study, we assessed the role of the transforming growth factor beta 4 (TGFβ4)/Nodal axis in the pathogenesis of CMT1A. First, we identified PMP22 overexpression-induced Nodal expression in Schwann cells, which might be one of the downstream effectors in CMT1A. Administration of Nodal protein at the developmental stage of peripheral nerves induced the demyelinating phenotype in vivo. Second, we further isolated TGFβ4 as an antagonist that could abolish Nodal-induced demyelination. Finally, we developed a recombinant TGFβ4-fragment crystallizable (Fc) fusion protein, CX201, and demonstrated that its application had promyelinating efficacy in Schwann cells. CX201 administration improved the demyelinating phenotypes of CMT1A mouse models at both pre-symptomatic and post-symptomatic stages. These results suggest that the TGFβ4/Nodal axis plays a crucial role in the pathogenesis of CMT1A and might be a potential therapeutic target for CMT1A.

Enhancement of immunogenicity and neutralizing responses against SARS-CoV-2 spike protein using the Fc fusion fragment.

Vaccination has played an important role in protecting against death and the severity of COVID-19. The recombinant protein vaccine platform has a long track record of safety and efficacy. Here, we fused the SARS-CoV-2 spike S1 subunit to the Fc region of IgG and investigated immunogenicity, reactivity to human vaccinated sera, and neutralizing activity as a candidate protein vaccine. We evaluated the immunogenicity of CHO-expressed S1-Fc fusion protein and tag-free S1 protein in rabbits via the production of S1-specific polyclonal antibodies. We subsequently compared the neutralizing activities of sera from immunized rabbits and human-vaccinated individuals using a surrogate Virus Neutralization Test (sVNT). The results indicate that S1-specific polyclonal antibodies were induced in all groups; however, antibody levels were higher in rabbits immunized with S1-Fc fusion protein than tag-free S1 protein. Moreover, the reactivity of human vaccinated sera against S1-Fc fusion protein was significantly higher than tag-free S1 protein. Lastly, the results of the virus-neutralizing activity revealed that vaccination with S1-Fc fusion protein induced the highest level of neutralizing antibody response against SARS-CoV-2. Our results demonstrate that the S1 protein accompanied by the Fc fragment significantly enhances the immunogenicity and neutralizing responses against SARS-CoV-2. It is hoped that this platform can be used for human vaccination.

Eftrenonacog Alfa: A Review in Haemophilia B.

Eftrenonacog alfa (Alprolix®) is an extended half-life recombinant factor IX (rFIX)-Fc fusion protein (hereafter referred to as rFIXFc). Administered as an intravenous bolus, it is approved for prophylactic use and the treatment of bleeding in patients with haemophilia B in various countries worldwide, including those of the EU, as well as the USA. In multinational, phase III trials, rFIXFc was effective for the prophylaxis, perioperative management or on-demand treatment of bleeding in male patients with severe haemophilia B regardless of age and irrespective of whether or not they had been previously treated with FIX replacement products. Prophylactic efficacy was maintained over the longer term (up to 5years) in previously treated patients. rFIXFc effectiveness in the real-world setting is supported by results of prospective studies, as well as the outcomes of several retrospective trials. rFIXFc was well tolerated in clinical trials in previously treated and untreated children, adolescents and/or adults with severe haemophilia B. Thus, rFIXFc continues to represent a useful treatment option among the haemophilia B patient population.

Vaccination with recombinant Lactococcus lactis expressing HA1-IgY Fc fusion protein provides protective mucosal immunity against H9N2 avian influenza virus in chickens

BackgroundH9N2 virus is mainly transmitted through the respiratory mucosal pathway, so mucosal immunity is considered to play a good role in controlling avian influenza infection. It is commonly accepted that no adequate mucosal immunity is achieved by inactivated vaccines, which was widely used to prevent and control avian influenza virus infection. Thus, an improved vaccine to induce both mucosal immunity and systemic immunity is urgently required to control H9N2 avian influenza outbreaks in poultry farms.MethodsIn this study, we constructed a novel Lactococcus lactis (L. lactis) strain expressing a recombinant fusion protein consisting of the HA1 proteins derived from an endemic H9N2 virus strain and chicken IgY Fc fragment. We evaluated the immunogenicity and protective efficacy of this recombinant L. lactis HA1-Fc strain.ResultsOur data demonstrated that chickens immunized with L. lactis HA1-Fc strain showed significantly increased levels of serum antibodies, mucosal secretory IgA, T cell-mediated immune responses, and lymphocyte proliferation. Furthermore, following challenge with H9N2 avian influenza virus, chickens immunized with L. lactis HA1-Fc strain showed reduced the weight loss, relieved clinical symptoms, and decreased the viral titers and the pathological damage in the lung. Moreover, oropharyngeal and cloacal shedding of the H9N2 influenza virus was detected in chicken immunized with L. lactis HA1-Fc after infection, the results showed the titer was low and reduced quickly to reach undetectable levels at 7 days after infection.ConclusionOur data showed that the recombinant L. lactis HA1-Fc strain could induce protective mucosal and systemic immunity, and this study provides a theoretical basis for improving immune responses to prevent and control H9N2 virus infection.

Complex PK-PD of an engineered IL-15/IL-15Rα–Fc fusion protein in cynomolgus monkeys: QSP modeling of lymphocyte dynamics

XmAb24306 is a lymphoproliferative interleukin (IL)-15/IL-15 receptor α (IL-15Rα) Fc-fusion protein currently under clinical investigation as an immunotherapeutic agent for cancer treatment. XmAb24306 contains mutations in IL-15 that attenuate its affinity to the heterodimeric IL-15 receptor βγ (IL-15R). We observe substantially prolonged pharmacokinetics (PK) (half-life ∼ 2.5 to 4.5 days) in single- and repeat-dose cynomolgus monkey (cyno) studies compared to wild-type IL-15 (half-life ∼ 1 hour), leading to increased exposure and enhanced and durable expansion of NK cells, CD8+ T cells and CD4–CD8– (double negative [DN]) T cells. Drug clearance varied with dose level and time post-dose, and PK exposure decreased upon repeated dosing, which we attribute to increased target-mediated drug disposition (TMDD) resulting from drug-induced lymphocyte expansion (i.e., pharmacodynamic (PD)-enhanced TMDD). We developed a quantitative systems pharmacology (QSP) model to quantify the complex PKPD behaviors due to the interactions of XmAb24306 with multiple cell types (CD8+, CD4+, DN T cells, and NK cells) in the peripheral blood (PB) and lymphoid tissues. The model, which includes nonspecific drug clearance, binding to and TMDD by IL15R differentially expressed on lymphocyte subsets, and resultant lymphocyte margination/migration out of PB, expansion in lymphoid tissues, and redistribution to the blood, successfully describes the systemic PK and lymphocyte kinetics observed in the cyno studies. Results suggest that after 3 doses of every-two-week (Q2W) doses up to 70 days, the relative contributions of each elimination pathway to XmAb24306 clearance are: DN T cells > NK cells > CD8+ T cells > nonspecific clearance > CD4+ T cells. Modeling suggests that observed cellular expansion in blood results from the influx of cells expanded by the drug in lymphoid tissues. The model is used to predict lymphoid tissue expansion and to simulate PK-PD for different dose regimens. Thus, the model provides insight into the mechanisms underlying the observed PK-PD behavior of an engineered cytokine and can serve as a framework for the rapid integration and analysis of data that emerges from ongoing clinical studies in cancer patients as single-agent or given in combination.

Plant-derived PAP proteins fused to immunoglobulin A and M Fc domains induce anti-prostate cancer immune response in mice

In this study, recombinant Fc-fused Prostate acid phosphatase (PAP) proteins were produced in transgenic plants. PAP was fused to immunoglobulin (Ig) A and M Fc domain (PAP-IgA Fc and PAP-IgM Fc), which were tagged to the ER retention sequence KDEL to generate PAP-IgA FcK and PAP-IgM FcK. Agrobacterium-meditated transformation was performed to produce transgenic tobacco plants expressing four recombinant proteins. Genomic PCR and RT-PCR analyses confirmed the transgene insertion and mRNA transcription of PAP-IgA Fc, PAP-IgM Fc, PAP-IgA FcK, and PAP-IgM FcK in tobacco plant leaves. Western blot confirmed the expression of PAP-IgA Fc, PAP-IgM Fc, PAP-IgA FcK, and PAP-IgM FcK proteins. SEC-HPLC and Bio-TEM analyses were performed to confirm the size and shape of the plant-derived recombinant PAP-Fc fusion proteins. In mice experiments, the plant-derived IgA and IgM Fc fused proteins induced production of total IgGs including IgG1 against PAP. This result suggests that IgA and IgM Fc fusion can be applied to produce recombinant PAP proteins as a prostate cancer vaccine in plant expression system.

Abstract LB333: Cytokine affinity tuning using the AlphaSeq platform to generate targeted immuno-oncology therapeutics

Abstract Here, we demonstrate a novel approach for generating affinity detuned cytokines that can be fused to antibodies for selective immune cell activation to improve anti-tumor responses and mitigate systemic toxicity. Although cytokine therapies have demonstrated curative effects in some cancer patients, clinical use remains limited with current toxicity profiles accompanying systemic administration. Next generation cytokine approaches include conditional signaling focused to sites of interest, including the tumor microenvironment or specific immune cell populations. In this presentation, we share a novel approach for generating detuned cytokine therapeutic candidates using the AlphaSeq platform, which involves the repurposing of yeast agglutination and mating to quantitatively measure protein-protein interactions at a library-on-library scale. We show, for the first time, how AlphaSeq can be used to measure interactions between cytokines and their receptors as well as generate engineered cytokines with a broad range of affinities. A saturated mutational library was created for a cytokine of interest and subsequently screened against a second library consisting of axis receptor chains, their species orthologs and off-target receptors. Cytokine variants with lower affinity than parental 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. Finally, detuned cytokine candidates were fused to antibodies recognizing surface antigens of interest to demonstrate cell population-specific signaling. Our results show the AlphaSeq platform can accurately quantitate thousands of cytokine variant affinities simultaneously against multiple relevant receptors which translates to signaling potency in primary human cells. When engineered as antibody fusion proteins, candidate immunocytokines are identified that only induce signaling in intended cell populations. AlphaSeq’s rapid, comprehensive affinity determination can be utilized to develop a portfolio of clinically relevant therapeutic immunocytokines. Citation Format: Ryan Swanson, Davis Goodnight, Jeff Adamo, Kyle Minch, Emily Engelhart, Randolph Lopez, David Younger. Cytokine affinity tuning using the AlphaSeq platform to generate targeted immuno-oncology therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB333.