Journal: Journal for Immunotherapy of Cancer

Article Title: Antibody therapeutics with high affinity for FcγRs exacerbate anaphylaxis via FcγR-mediated capture by tumor-associated myeloid cells

doi: 10.1136/jitc-2025-013316

Figure Lengend Snippet: High-affinity FcγR-binding anti-PD-L1 antibody clone, 10F.9G2, but not low-affinity clones nor deglycosylated 10F.9G2 induced anaphylaxis. (A) Experimental scheme for treatment of CT26 tumor-bearing mice with anti-mouse PD-L1 antibodies. CT26 tumor-bearing mice were treated with antibodies (200 µg/mouse) on days 10, 13 and 17 after inoculation. Body temperature and survival were measured after the third antibody injection. (B) Changes in body temperature of CT26 tumor-bearing mice after the third injection of 10F.9G2 (n=4), Control IgG2b (n=4), or MIH6 (n=3). (C) Survival curve of CT26 tumor-bearing mice after the third injection of antibodies in (B). (D) Concentrations of IgG ADA against 10F.9G2 (n=6), Control IgG2b (n=6), and MIH6 (n=4) in serum evaluated by ELISA. (E) Survival of CT26 tumor-bearing mice after the third injection of 10F.9G2 (n=4), 10F.9G2-CP001 (n=4), or 10F.9G2-CP154 (n=4). (F) Concentrations of IgG ADA against 10F.9G2 (n=4), 10F.9G2-CP001 (n=4), and 10F.9G2-CP154 (n=4) in the serum evaluated by ELISA. (G) Survival of CT26 tumor-bearing mice after the third injection of 10F.9G2 (n=6) or deglycosylated 10F.9G2 (n=6). *p<0.05, using log-rank (Mantel-Cox) tests. (H) Serum concentrations of the IgG ADAs against 10F.9G2 (n=3) and deglycosylated 10F.9G2 (n=3) evaluated by ELISA. (I) Normalized ADA production against 10F.9G2 (n=3) and deglycosylated 10F.9G2 (n=3) in serum. *p<0.05, unpaired t-test with Welch’s correction. Data are represented as mean±SE. ADAs, antidrug antibodies; FcγR, Fcγ receptor; i.v., intravenous; PD-L1, programmed death-ligand 1; s.c., subcutaneous.

Article Snippet: Murine colon carcinoma CT26 (CRL-2638) cells and breast cancer 4T1 (CRL-2539) were obtained from the American Type Culture Collection (Manassas, Virginia, USA).

Techniques: Binding Assay, Clone Assay, Injection, Control, Enzyme-linked Immunosorbent Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Antibody therapeutics with high affinity for FcγRs exacerbate anaphylaxis via FcγR-mediated capture by tumor-associated myeloid cells

doi: 10.1136/jitc-2025-013316

Figure Lengend Snippet: FcγRII/III blockade on tumor-associated myeloid cells reduces 10F.9G2, captures and protects mice from anaphylaxis-induced death. ( A ) Overlaid relative histogram of FcγRII/III expression on the surface of Ly6C hi Ly6G − cells (left), monocyte (middle) and macrophage (right). Gray: unstained sample; Blue: healthy mice; Red: CT26 tumor-bearing mice. ( B ) Percentages of FcγRII/III + Ly6C hi Ly6G − cells, monocyte and macrophage in spleen from healthy mice (n=4) and CT26 tumor-bearing mice (n=4). ***p<0.001, unpaired t-test with Welch’s correction. Data are represented as mean±SE. ( C ) FcγRII/III expression measured by MFI of FcγRII/III-BV510. Blue: healthy mice (n=4); Red: CT26 tumor-bearing mice, (n=4). ***p<0.001, ****p<0.0001, unpaired t-test with Welch’s correction. Data are represented as mean±SE. ( D ) Percentage of 10F.9G2 capturing CD11b + myeloid cells out of total myeloid cells.***p<0.001, Tukey one-way ANOVA. Data are represented as mean±SE. ( E ) Capture ability of different myeloid cells measured by MFI of secondary antibody binding with anti-mouse PD-L1 antibodies after FcγR blocking. Data are represented as mean±SE. ( F ) Experimental schedule for FcγR blocking before 10F.9G2 injection. ( G ) Survival of CT26 tumor-bearing mice after the second injection of 10F.9G2. Phosphate-buffered saline administered 30 min before first administration of 10F.9G2 as control (n=8) and FcγRII/III blocking antibody treatment before first administration of 10F.9G2 (n=8). p=0.09, Using Log-rank (Mantel-Cox) test. ( H ) Concentrations of the IgG ADA against 10F.9G2 with control (n=4) and FcγRII/III blocking (n=4) in the serum evaluated by ELISA. ADA, antidrug antibody; ANOVA, analysis of variance; FcγR, Fcγ receptor; i.v., intravenous; MFI, mean fluorescence intensity; n.s., not significant; PD-L1, programmed death-ligand 1; s.c., subcutaneous.

Article Snippet: Murine colon carcinoma CT26 (CRL-2638) cells and breast cancer 4T1 (CRL-2539) were obtained from the American Type Culture Collection (Manassas, Virginia, USA).

Techniques: Expressing, Binding Assay, Blocking Assay, Injection, Saline, Control, Enzyme-linked Immunosorbent Assay, Fluorescence

Journal: Journal for Immunotherapy of Cancer

Article Title: Antibody therapeutics with high affinity for FcγRs exacerbate anaphylaxis via FcγR-mediated capture by tumor-associated myeloid cells

doi: 10.1136/jitc-2025-013316

Figure Lengend Snippet: 10F.9G2 stimulates monocytic–macrophage lineage cells to function as APCs, initiating T-cell dependent humoral immunity. ( A ) Representative t-SNE plots of splenic myeloid cells from CT26 tumor-bearing mice 24 hours after the second administration of MIH6, 10F.9G2, or deglycosylated 10F.9G2. tSNE analysis was performed using FlowJo software based on CD11b, Ly6C, Ly6G, and F4/80 expression. ( B ) Representative t-SNE plots of MHC-II expression in ( A ). ( C ) Histogram showing cell surface MHC-II expression in spleen Ly6C hi Ly6G − cells (left), monocytes (middle), and macrophages (right) 24 hours after the second administration of MIH6, 10F.9G2, or deglycosylated 10F.9G2. The MFI of MHC-II is shown in the upper-left corner of each picture. ( D–F ) Percentages of MHC-II + Ly6C hi Ly6G − cells ( D ), monocytes ( E ), and macrophages ( F ) in total myeloid cells 24 hours after the second administration of MIH6 (n=3), 10F.9G2 (n=4), or deglycosylated 10F.9G2 (n=4). *p<0.05, **p<0.01, ***p<0.001, using Tukey one-way ANOVA. Data are represented as mean±SE. ( G ) Representative dot plots of activated helper T cells (CD3 + CD4 + CD69 + ) in the spleen 24 hours after the second administration of MIH6, 10F.9G2 or deglycosylated 10F.9G2. ( H ) Percentage of activated helper T cells in splenocytes 24 hours after the second administration of MIH6 (n=3), 10F.9G2 (n=4), or deglycosylated 10F.9G2 (n=4). **p<0.01, ***p<0.001, Tukey one-way ANOVA. Data are represented as mean±SE. ( I ) Concentrations of IgG ADA against 10F.9G2 (n=4) compared with control (n=4) in the serum of BALB/c nu/nu CT26-bearing mice evaluated by ELISA. ( J ) Representative dot plots of IgG ADA-secreting plasma cells (CD138 + B220 − IgG + ) from living splenocytes 1 week after the administration of 10F.9G2 to BALB/c nu/nu CT26-bearing mice. ( K ) Percentage of IgG ADA-secreting plasma cells of splenocytes 1 week after administration of 10F.9G2 to BALB/c nu/nu CT26-bearing mice, using t-test with Welch’s correction. Data are represented as mean±SE. ADA, antidrug antibody; ANOVA, analysis of variance; APC, antigen presenting cell; MFI, mean fluorescence intensity; MHC, major histocompatibility complex; n.s., not significant; t-SNE, t-distributed stochastic neighbor embedding.

Article Snippet: Murine colon carcinoma CT26 (CRL-2638) cells and breast cancer 4T1 (CRL-2539) were obtained from the American Type Culture Collection (Manassas, Virginia, USA).

Techniques: Software, Expressing, Control, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Fluorescence, Immunopeptidomics

Journal: Journal for Immunotherapy of Cancer

Article Title: Antibody therapeutics with high affinity for FcγRs exacerbate anaphylaxis via FcγR-mediated capture by tumor-associated myeloid cells

doi: 10.1136/jitc-2025-013316

Figure Lengend Snippet: Antibody therapeutics with high FcγR affinities induce anaphylaxis via the same mechanism with 10F.9G2 in mouse models. ( A ) Binding affinities for mouse FcγRIII of anti-mouse Gr-1 (left), PD-L1 10F.9G2 (middle), and anti-mouse CD20 SA271G2 (right) evaluated by surface plasmon resonance. ( B ) Changes in the body temperature of CT26 tumor-bearing mice after repeated administration of RB6-8C5 (n=4), 10F.9G2 (n=4), SA271G2 (n=4), and murine IgG2b isotype control (n=4). ( C ) Survival curve of CT26 tumor-bearing mice after the second injection of antibodies in ( B ). ( D ) Concentrations of IgG ADAs against RB6-8C5 (n=4), 10F.9G2 (n=4), SA271G2 (n=4), and murine IgG2b isotype control (n=4) in serum evaluated by ELISA. ( E ) Normalized ADA production against RB6-8C5 (n=3) and SA271G2 (n=3) in serum. ***p<0.001, ****p<0.0001, unpaired t-test with Welch’s correction. Data are represented as mean±SE. ( F ) Immunofluorescence staining of specific markers in spleen sections from CT26 tumor-bearing mice. Representative images show the distribution of B220 (cyan), anti-mouse Gr-1 antibodies (red), and F4/80 (green) in tissue samples. Scale bars=100 µm. ( G ) Hierarchical clustering heatmap of core glycosylation and anaphylactic grouping of antibodies: control rat IgG2b (n=3, no anaphylaxis), 10F.9G2 (n=3, anaphylaxis), SA271G2 (n=3, anaphylaxis), and RB6-8C5 (n=3, anaphylaxis). ( H–J ) Number of Ly6C hi Ly6G − cells ( H ), monocytes ( I ) and macrophages ( J ) in splenocytes. **p<0.01, ***p<0.001, unpaired t-test with Welch’s correction. Data are represented as mean±SE. ( K ) Immunofluorescence staining of specific markers in spleen sections from 4T1 tumor-bearing mice. Representative images show the distribution of B220 (cyan), anti-mouse PD-L1 antibodies (red), and F4/80 (green) in tissue samples. Scale bars=100 µm. ( L ) Changes in body temperature of 4T1 tumor-bearing mice after repeated administration of 10F.9G2 (n=4) and murine IgG2b isotype control (n=4). ( M ) Survival curve of 4T1 tumor-bearing mice after the second injection of antibodies in ( L ). ( N ) Concentrations of IgG ADAs against 10F.9G2 (n=4) and rat IgG2b isotype control (n=4) in serum evaluated by ELISA. ( O ) Illustration of the mechanism by which antibody therapeutics with high FcγR affinities induce IgG ADA production in tumor-bearing mice. ADA, antidrug antibody; FcγR, Fcγ receptor; MHC, major histocompatibility complex; n.s., not significant; PD-L1, programmed death-ligand 1.

Article Snippet: Murine colon carcinoma CT26 (CRL-2638) cells and breast cancer 4T1 (CRL-2539) were obtained from the American Type Culture Collection (Manassas, Virginia, USA).

Techniques: Binding Assay, SPR Assay, Control, Injection, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Glycoproteomics, Immunopeptidomics

Journal: PLoS Pathogens

Article Title: Neutrophil-Derived CCL3 Is Essential for the Rapid Recruitment of Dendritic Cells to the Site of Leishmania major Inoculation in Resistant Mice

doi: 10.1371/journal.ppat.1000755

Figure Lengend Snippet: L. major –i.p. recruited C57BL/6 and BALB/c neutrophils were purified by MACS and incubated with medium, L. major (5∶1 parasite∶neutrophil ratio), IFNγ, or both. (A) Sixteen hours later, neutrophil CCL3, CCL4 and CCL5 mRNA levels were assessed by real-time quantitative RT-PCR, and (B) 24 hours after initiation of culture, chemokine content was measured in cell-free culture supernatants by ELISA. Data are the mean triplicate measurement ± SEM of neutrophil mRNA or chemokine content in the supernatants. *: p<0.05 compared to neutrophils cultured with medium only. (C) Supernatants from inflammatory neutrophils cultured in presence or in absence of L. major were tested for their chemotactic activity towards bone marrow-derived DCs in a transwell cell migration assay. The number of DCs that migrated toward neutrophil supernatant is represented. (D) DC migration was similarly assessed in response to neutrophil supernatants depleted of CCL3. *: p<0.05 as compared to values measured in response to supernatant of neutrophil incubated with medium alone. Data are expressed as mean number ± SEM of DC that migrated towards neutrophil supernatant (n = 3 per group). The results of a representative experiment out of three are shown.

Article Snippet: Female BALB/c and C57BL/6 mice were purchased from Harlan Olac Ltd. (Bicester, UK).

Techniques: Purification, Incubation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, Activity Assay, Derivative Assay, Cell Migration Assay, Migration

Journal: PLoS Pathogens

Article Title: Neutrophil-Derived CCL3 Is Essential for the Rapid Recruitment of Dendritic Cells to the Site of Leishmania major Inoculation in Resistant Mice

doi: 10.1371/journal.ppat.1000755

Figure Lengend Snippet: (A) The number of neutrophils, Langerhans and dermal DCs, and monocyte-derived DCs emigrating from ear explants was measured at 0, 6, 24 and 48 hours post L. major inoculation *: p<0.05 comparing cell number in C57BL/6 versus BALB/c mice. Values from ear explants of six mice per group are expressed as mean values ± SEM . The data are representative of three separate experiments. (B) Gating strategy by flow cytometry of Langerhans and dermal DCs, and monocyte-derived DCs emigrating from L. major -infected C57BL/6 ear explants. For Langerhans and dermal DCs, MHCII and DEC205 positive cells were analyzed on CD11c + gated cells. For monocyte-derived DCs, four colour FACS analysis was performed, CD11b + and LY6C + cells were analyzed on a CCD11c dim and Ly6G − gated cell population. A representative flow cytometry plot is shown.

Article Snippet: Female BALB/c and C57BL/6 mice were purchased from Harlan Olac Ltd. (Bicester, UK).

Techniques: Derivative Assay, Flow Cytometry, Infection

Journal: PLoS Pathogens

Article Title: Neutrophil-Derived CCL3 Is Essential for the Rapid Recruitment of Dendritic Cells to the Site of Leishmania major Inoculation in Resistant Mice

doi: 10.1371/journal.ppat.1000755

Figure Lengend Snippet: C57BL/6 and BALB/c mice were depleted of neutrophils by an injection i.p. of the NIMP-R14 anti-neutrophil mAb, or injected with a control mAb, 6 hours prior to infection i.d. with L. major stationary promastigotes. The number of (A) Langerhans and dermal DCs and (B) monocyte-derived DCs recruited in the ear dermis at 0h (naïve) or 24h following L. major inoculation was quantified and compared in mice depleted or not of neutrophils. Data are given as mean values ± SEM (n = 6 per group) and are representative of three experiments. * : p<0.05 between mice depleted or not of neutrophils.

Article Snippet: Female BALB/c and C57BL/6 mice were purchased from Harlan Olac Ltd. (Bicester, UK).

Techniques: Injection, Control, Infection, Derivative Assay

Journal: PLoS Pathogens

Article Title: Neutrophil-Derived CCL3 Is Essential for the Rapid Recruitment of Dendritic Cells to the Site of Leishmania major Inoculation in Resistant Mice

doi: 10.1371/journal.ppat.1000755

Figure Lengend Snippet: (A) C57BL/6 and BALB/c mice were infected with L. major in the ear dermis. mRNA expression of CCL3, CCL4 and CCL5 at the site of infection was measured 0, 6, 24 and 48 hours post infection by quantitative Real-time PCR and normalized relative to HPRT mRNA levels. Data are represented as the mean ± SEM mRNA transcript levels of individual infected ear (n = 6 per time point). One representative experiment of three is shown. (B) Twenty four hours post-infection, the ear chemokine mRNA level was compared in C57BL/6 mice that were given either the NIMP-R14 neutrophil depleting mAb or a control mAb 6 hours prior to L. major inoculation in the ear. Results are represented as fold increase in mRNA levels relative to levels measured in uninfected mice given a value of 1, and are representative of two experiments.

Article Snippet: Female BALB/c and C57BL/6 mice were purchased from Harlan Olac Ltd. (Bicester, UK).

Techniques: Infection, Expressing, Real-time Polymerase Chain Reaction, Control

Journal: PLoS Pathogens

Article Title: Neutrophil-Derived CCL3 Is Essential for the Rapid Recruitment of Dendritic Cells to the Site of Leishmania major Inoculation in Resistant Mice

doi: 10.1371/journal.ppat.1000755

Figure Lengend Snippet: (A) C57BL/6 and BALB/c mice were injected i.p. with Evasin-1, a chemokine binding protein that neutralizes CCL3. Twenty four hours post L. major inoculation, the number of neutrophils, Langerhans and dermal DCs, and monocyte-derived DCs, spontaneously emigrating out of ear explants was measured and compared to that obtained from ear explants from mice similarly infected but which were not given Evasin-1. * : p<0.05 between mice treated or not with Evasin-1. (B) 24 hours after infection i.d. with L. major , the number of leukocytes emigrating out of ear skin explants of CCL3 −/− mice was compared to that measured in similarly infected C57BL/6 ear explants. The number of neutrophils, Langerhans and dermal DCs, and monocyte-derived DCs is presented as the mean ± SEM (n = 4 for Evasin-1 treated mice, and n = 6 for CCL3 −/− mice) and are representative of three experiments. *: p<0.05 between CCL3 −/− and C57BL/6 mice.

Article Snippet: Female BALB/c and C57BL/6 mice were purchased from Harlan Olac Ltd. (Bicester, UK).

Techniques: Injection, Binding Assay, Derivative Assay, Infection