human fcrn homozygous transgenic mice  (Jackson Laboratory)

Journal: bioRxiv

Article Title: Engineering HIV antibodies with enhanced breadth and potency of neutralization through multistate affinity maturation

doi: 10.1101/2025.10.22.684031

Figure Lengend Snippet: A ) Polyreactivity of N49P7 variants with CHO cell membrane proteins (CHO-SMP), insulin, and ssDNA, measured by ELISA in duplicate. Adalimumab (non-polyreactive) and 4E10 (highly polyreactive) were included as antibody controls. B ) Reactivity of N49P7 variants with HEp-2 cells, assessed by indirect immunofluorescence. Controls included adalimumab (non-reactive) and polyclonal highly reactive antibodies (homogenous and centromere positive controls). The experiment was performed in duplicate. C ) Analytical size-exclusion chromatography retention time (SEC t R ) of N49P7 variants on a mAb column (30 cm TSKgel SuperSW mAb HR). Adalimumab and ipilimumab (favorable biophysical properties), and bococizumab (aggregation-prone) were included as antibody controls. The experiment was performed in duplicate. D ) Thermal stability of N49P7 variants measured by differential scanning fluorimetry (DSF) in triplicate. Melting temperature (T m ) values were calculated from the peaks of the melt curves, derived from the first derivatives of fluorescence intensity versus temperature. Adalimumab (a stable antibody) served as a control. E ) Solubility of VRC01 and N49P7 variants in 8% PEG. The horizontal dotted line marks OD 350 =0.03, indicating the onset of visible precipitation. Antibodies with OD 350 >0.03 are predicted to be insoluble at high concentrations (>50 mg/mL). Adalimumab (solution-stable) and bococizumab (aggregation-prone) were used as antibody controls. The experiment was performed in duplicate. F ) Hydrophobic interaction chromatography retention time (HIC t R ) of N49P7 variants on a butyl column (10 cm TSKgel Butyl-NPR). Adalimumab and ipilimumab (favorable biophysical properties), and 4E10 (hydrophobic) were included as antibody controls. The experiment was performed in duplicate. G ) Pharmacokinetics (PK) and plasma half-life (t 1/2 ) of N49P7 variants with the LS mutations in hFcRn transgenic mice. Mean ± SD values for 4-7 mice per group are shown.

Article Snippet: PK analysis and t 1/2 determination were performed in homozygous human FcRn transgenic mice (FcRn−/− hFcRn+/+, Jackson Laboratory), which lack the murine FcRn α-chain (Fcgrt tm1Dcr ) and instead express the human FcRn α-chain.

Techniques: Membrane, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Size-exclusion Chromatography, Derivative Assay, Fluorescence, Control, Solubility, Hydrophobic Interaction Chromatography, Drug discovery, Clinical Proteomics, Transgenic Assay

Journal: Nature Communications

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

doi: 10.1038/s41467-024-46321-9

Figure Lengend Snippet: a Illustration outlining the in vivo lung localization experiment in Tg32 mice. b Amount of NIP IgG1 WT and REW in BALF 23 days post i.v. administration of 5 mg/kg of the antibody variants in IVIg (500 mg/kg) pre-loaded mice. Shown as mean ± s.e.m ( n = 4 animals (WT) and n = 5 animals (REW). c BALF/plasma ratio of NIP IgG1 WT and REW, 23 days post i.v. administration. Shown as mean ± s.d ( n = 4 animals per group). d Illustration outlining i.n. delivery experiment in Tg32 mice. e Plasma concentration of NIP IgG1 WT and REW 24 h post i.n. delivery (2.23 mg/kg) in Tg32 mice. Shown as mean ± s.d. ( n = 4 animals (WT) or n = 6 animals (REW)). f Illustration outlining FcRn mediated transcytosis experiments in a transwell system. g Apical to basolateral (A→B) and basolateral to apical (B→A) directed transport of NIP IgG1 WT, REW and IHH across polarized human T84 cells, shown as mean ± s.d ( n = 8 independent monolayers from 2 independent experiments) (two data points was excluded from REW A→B and one data point excluded for REW (B→A) due to disrupted cell monolayers). h Apical to basolateral (A→B) and basolateral to apical (B→A) directed transport of NIP IgG1 WT, REW and IHH across MDCK-hFcRn cells, show as mean ± s.d ( n = 6 independent cellular monolayers from 2 independent experiments) (one data point were excluded from REW A→B and REW (B→A) due to disrupted cell monolayers). i Illustration outlining mucosal vaccine and challenge experiment in Tg32 mice. j Percent survival of Tg32 mice i.n. vaccinated with HA WT and REW monovalent Fc fusions or NaCl control following challenge with a lethal dose of H1N1 virus. k Illustration showing the ex vivo human placental perfusion model used to measure maternal-to-fetal transport of anti-NIP IgG1 WT and REW. l Ex vivo human placental perfusion model showing maternal/fetal (FM) transport ratio of NIP IgG1 WT and REW, shown as mean ± s.d. ( n = 4 placentas per group). b , c , e , g , h Unpaired two-tailed t -test, ( l ) Two-tailed Wilcoxon t -test. Source data are provided as a Source Data file. ( a , d , f , I , k ) were created using BioRender.com.

Article Snippet: Homozygous human FcRn transgenic mice (B6.Cg-Fcgrttm1Dcr Tg(FcGRT)32Dcr/DcrJ) (Tg32) (The Jackson Laboratory) were injected i.v. with 10 mg/kg anti-NIP IgG1 WT, REW, and RGY variants (3 mice per group, age 8–10 weeks, 2 female and 1 male per group) followed by terminal bleeding and plasma collection 24 h post-administration.

Techniques: In Vivo, Clinical Proteomics, Concentration Assay, Control, Virus, Ex Vivo, Two Tailed Test

Journal: Nature Communications

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

doi: 10.1038/s41467-024-46321-9

Figure Lengend Snippet: a Illustration of ELISA setup and binding of anti-NIP IgG1 WT, REW and low-fucose (2FF) WT and REW to ( b ) FcγRIIIa-V158 and ( c ) FcγRIIIa-F158, shown as mean ± s.d of duplicates. d Overview of anti-CD20 IgG1 mAb2 variants and B cell lines used to measure ADCC activity. e The 51 Cr release assay used to measure ADCC activity of MNCs against target cell lines in presence of the antibodies. f – h MNC mediated ADCC of WSU-NHL (low CD20), Carnaval (low CD20) and SU-DHL-4 (high CD20) lymphoma cell lines by the WT mAb2 compared with the Fc-engineered versions; REW, REW-2FF and PGLALA, shown as mean±s.e.m. from 5 replicates performed in parallel. i Experimental outline and ( j ) plasma clearance of anti-NIP IgG1 WT and REW-2FF in IVIg pre-loaded (500 mg/kg) Tg32 mice, shown as mean±s.d of percent antibody remaining in plasma over time ( n = 5 animals per group). k Illustration showing REW-engineered IgG subclasses and a recombinant form of Fc-fusion etanercept. l Cellular FcRn mediated rescue from intracellular degradation of WT and REW engineered IgG subclasses and Fc-fusion etanercept in HERA, shown as mean±s.d of triplicates from three independent experiments. m Plasma clearance of anti-NIP IgG2 WT and REW following i.v. administration (5 mg/kg) in IVIg pre-loaded (500 mg/kg) Tg32 mice, shown as mean ± s.d of percent antibody remaining in plasma over time ( n = 4 animals per group). n Plasma clearance of recombinant etanercept WT and REW following i.v. administration (5 mg/kg) in Tg32 mice, shown as mean ± s.d of percent antibody remaining in plasma over time ( n = 5 animals per group). o ELISA showing binding of human C1q to titrated amounts of antigen captured anti-NIP IgG2 WT and REW, shown as mean±s.d of duplicates. p CDC activity of mAb2 anti-CD20 IgG2 WT and REW against WSU-NHL (low CD20) using the 51 Cr release assay, shown as mean±s.d of duplicates. q ELISA showing binding of human C1q to titrated amounts of antigen captured anti-NIP IgG4 WT and REW, shown as mean±s.d of duplicates. r , s ELISAs showing relative binding of FcyRIIIa-V158 or FcyRIIIa-F158 to antigen captured anti-NIP IgG2 and IgG4 WT and REW, shown as relative binding compared to anti-NIP IgG1-WT, mean ± s.d of duplicates. f , g , h , j , m , n RM Two-way ANOVA with Šídák’s multiple comparison test. l Unpaired two-tailed t -test. Source data are provided as a Source Data file. a , d , e , I , k were created using BioRender.com.

Article Snippet: Homozygous human FcRn transgenic mice (B6.Cg-Fcgrttm1Dcr Tg(FcGRT)32Dcr/DcrJ) (Tg32) (The Jackson Laboratory) were injected i.v. with 10 mg/kg anti-NIP IgG1 WT, REW, and RGY variants (3 mice per group, age 8–10 weeks, 2 female and 1 male per group) followed by terminal bleeding and plasma collection 24 h post-administration.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Activity Assay, Release Assay, Clinical Proteomics, Recombinant, Comparison, Two Tailed Test

Journal: mAbs

Article Title: Efficient production of bispecific antibody by FAST-Ig TM and its application to NXT007 for the treatment of hemophilia A

doi: 10.1080/19420862.2023.2222441

Figure Lengend Snippet: Pharmacokinetic profiles of the selected FAST-Ig variants Comparable pharmacokinetics of NXT007 with different FAST-Ig mutations were confirmed in human FcRn homozyhous transgenic mice strain, B6.Cg-Fcgrttm1Dcr Tg(FCGRT)32Dcr. One mg/kg dose of each BsAb was administered by single intravenous injection via tail vein. Individual data points were collected from three mice in each group and are expressed as mean ± SD.

Article Snippet: One mg/kg doses of each bispecific antibody were administered to human FcRn homozygous transgenic mice, line #32 (B6.Cg-Fcgrt < tm1Dcr > Tg(FCGRT)32Dcr/DcrJ, Jackson Laboratories) by single intravenous injection via the tail vein ( n = 3 for each group).

Techniques: Drug discovery, Transgenic Assay, Injection

Journal: mAbs

Article Title: Efficient production of bispecific antibody by FAST-Ig TM and its application to NXT007 for the treatment of hemophilia A

doi: 10.1080/19420862.2023.2222441

Figure Lengend Snippet: AUC parameters of NXT007 with various FAST-Ig mutations and its parent from Day 0 to Day 7 in human FcRn transgenic mice. AUC profiles comparable to that of the parent were observed in tested FAST-Ig variants. A 1 mg/kg dose of each BsAb was administered by single intravenous injection via tail vein. Data were collected from three mice in each group and are expressed as mean ± SD. The BsAb yields of all antibodies used in this study were confirmed >95% by CIEX analysis.

Article Snippet: One mg/kg doses of each bispecific antibody were administered to human FcRn homozygous transgenic mice, line #32 (B6.Cg-Fcgrt < tm1Dcr > Tg(FCGRT)32Dcr/DcrJ, Jackson Laboratories) by single intravenous injection via the tail vein ( n = 3 for each group).

Techniques: Transgenic Assay, Injection, Variant Assay

Journal: mAbs

Article Title: Efficient production of bispecific antibody by FAST-Ig TM and its application to NXT007 for the treatment of hemophilia A

doi: 10.1080/19420862.2023.2222441

Figure Lengend Snippet: Comparison of stability after low pH exposure and pairing potency between dKiH v14 and ART-S3 (A-C) Acid-induced denature of NXT007 with different Fc mutations, dKiH v14 (a) and ART-S3 (b), was compared by SEC analysis at pH 6.0 (indicated as initial), pH 3.6, and pH 3.2 for 3 hours, followed by neutralization with or without incubation for 24 hours at room temperature (RT). (c) Y axis indicates the extent of denature of antibody as the whole area ratio (arbitrary unit) of ANS fluorescence at 510 nm divided by UV detection at 280 nm. (d) Stability of the human FcRn (hFcrn) binding of NXT007 with dKiH v14 and ART-S3 mutations was compared by SPR analysis. The hFcrn binding ability was calculated as relative binding response (Resonance Unit) per antibody capture amount. Samples were same as in (a-c). (e, f) CIEX chromatography analysis with an optimized CIEX method (pH and NaCl concentration gradient, described in materials and methods) for NXT007 with dKiH v14 (e) or ART-S3 (f). The same chromatograms with reference mispairs are shown in Figure S9. Antibodies were expressed at the optimized plasmid mass ratio H1:L1:H2:L2 = 1.3:2:1:2 in Expi293F and purified by protein A.

Article Snippet: One mg/kg doses of each bispecific antibody were administered to human FcRn homozygous transgenic mice, line #32 (B6.Cg-Fcgrt < tm1Dcr > Tg(FCGRT)32Dcr/DcrJ, Jackson Laboratories) by single intravenous injection via the tail vein ( n = 3 for each group).

Techniques: Comparison, Neutralization, Incubation, Fluorescence, Binding Assay, Chromatography, Concentration Assay, Plasmid Preparation, Purification