Antigen-presenting neutrophil-derived dendritic cells and methods of use thereof

Abstract

Methods and compositions for use in generating or promoting an immune response against cancer or an infectious disease, comprising promoting the differentiation of neutrophils into dendritic cells using a combination of GMCSF and (i) an immune complex comprising an antigen and an antibody comprising an Fc region that binds to FcγRIIA or FcγRIIIB, (ii) a conjugate comprising an antigen and an anti-FcγRIIIB antibody, or (iii) an anti-FcγRIIIB antibody.

Description

[Technical field]

[0001] Claiming priority This application claims the benefit of U.S. Provisional Patent Application No. 62 / 795,498, filed Jan. 22, 2019, the entire contents of which are incorporated herein by reference.

[0002] Federally funded research or development This invention was made with Government support under Grant No. HL065095 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] Described herein are methods and compositions for use in generating or promoting an immune response against cancer or infectious disease, comprising promoting the differentiation of neutrophils into dendritic cells using a combination of GMCSF and (i) an immune complex comprising an antigen and an antibody against the antigen that comprises an Fc region that binds to FcγRIIA or FcγRIIIB, (ii) a conjugate comprising an antigen and an anti-FcγRIIIB antibody, or (iii) an anti-FcγRIIIB antibody. [Background technology]

[0004] Dendritic cells (DCs) are essential in lymphoid organs for presenting antigens to naive T cells. DC uptake of extracellular proteins leads to presentation of peptides on MHCII, whereas peptides generated from cytoplasmic proteins are loaded onto MHC1 and mediate expression of antigens on naive CD4 T cells, respectively. + Helper T cells and CD8 + DCs also cross-present antigens, where extracellular antigens taken up in vesicles reach the cytosol and the MHCI pathway, and are expressed by CD8 + Prime T cells, a property thought to be exclusive to DCs and exploited in cell-based immunotherapy for treating cancer and infectious diseases 1、2、3 . Summary of the Invention [Means for solving the problem]

[0005] The present invention is based, at least in part, on the discovery that FcγR-mediated antigen uptake by neutrophils leads to their differentiation into migratory dendritic-like cells that activate naive CD4+ and CD8+ T cells.

[0006] Neutrophils, specialized in microbial clearance, can acquire the function of antigen-presenting cells after cytokine stimulation. Here, we show that uptake of antigen-containing immune complexes converts neutrophils into cells with dendritic cell-like characteristics (neutrophil-derived DCs, NDDCs) that activate naive CD4+ and CD8+ T cells and induce delayed-type hypersensitivity responses and tumor immunity in vivo. Neutrophils from humanized mice show that both FcγRIIIB and FcγRIIA support the generation of NDDCs. Intravenously delivered antigen-conjugated antibodies against mouse neutrophil FcγRIIIB generate NDDCs in the blood and spleen, support the proliferation and cytolytic function of CD8 T cells, and reduce tumor growth in the presence of GM-CSF. Similarly, antigen-conjugated anti-FcγRIIIB converts human neutrophils into NDDCs. Thus, FcγRIIIB-mediated antigen delivery can be exploited to elicit effective adaptive immunity against tumors and pathogens.

[0007] Thus, provided herein is an in vitro method for generating a population of dendritic cells (DCs), comprising the steps of: providing a population of neutrophils comprising neutrophils expressing one or both of FcγRIIA and / or FcγRIIIB; contacting the neutrophils in culture with granulocyte-macrophage colony-stimulating factor (GM-CSF) and an immune complex (IC) comprising an antigen and an antibody, wherein the antibody comprises an antigen-binding portion that binds the antigen and an Fc region that binds FcγRIIA and / or FcγRIIIB; and maintaining the population of neutrophils in culture in the presence of GM-CSF and IC under conditions and for a time sufficient for the neutrophils to differentiate into DCs.

[0008] In some embodiments, the antigen comprises a tag and the antibody binds to the tag portion of the antigen.

[0009] In some embodiments, the antigen is a tumor antigen. These cells can be used, for example, in a method for treating cancer. Also provided herein is a method for treating a subject with cancer, comprising administering to the subject an effective amount of the cells produced by this method.

[0010] In some embodiments, the antigen is derived from a pathogen. These cells can be used, for example, in a method of treating an infectious disease, where the antigen is derived from a pathogen that the subject is infected with. Also provided herein is a method of treating a subject infected with a pathogen, comprising administering to the subject an effective amount of these generated cells, where the antigen is derived from a pathogen that the subject is infected with.

[0011] Also provided herein is a conjugate (e.g., a fusion protein or a chemical conjugate) comprising (i) an antigen and (ii) an antibody comprising an antigen-binding domain that binds to FcγRIII. In some embodiments, the antigen is a tumor antigen or an antigen derived from a pathogen.

[0012] Further provided herein is an in vitro method for generating a population of dendritic cells (DCs), the method comprising the steps of: providing a population of neutrophils, comprising neutrophils expressing one or both of FcγRIIA and / or FcγRIIIB; contacting the population of neutrophils in culture with a conjugate described herein and, optionally, with granulocyte-macrophage colony-stimulating factor (GM-CSF); and maintaining the population of neutrophils in culture in the presence of the conjugate and GM-CSF under conditions and for a time sufficient for the neutrophils to differentiate into DCs.

[0013] In some embodiments, antigen is tumor antigen.These DCs can be used, for example, in the method of treating cancer.Also provided herein is a method of treating a subject with cancer, comprising administering to the subject an effective amount of the DCs produced by these methods.

[0014] In some embodiments, the antigen is derived from a pathogen. These DCs can be used in, for example, a method for treating infectious diseases, where the antigen is derived from a pathogen that the subject is infected with. Also provided is a method for treating a subject infected with a pathogen, comprising administering an effective amount of these DCs to the subject, where the antigen is derived from a pathogen that the subject is infected with.

[0015] Further provided herein is a method for treating a subject, comprising administering to the subject an effective amount of a conjugate described herein comprising (i) an antigen and (ii) an antibody comprising an antigen-binding domain that binds to FcγRIII, and optionally granulocyte-macrophage colony-stimulating factor (GM-CSF). In some embodiments, the subject has cancer and the antigen is a tumor antigen. In some embodiments, the subject has an infection caused by a pathogen and the antigen is derived from the pathogen.

[0016] Also provided herein is a method for generating a population of dendritic cells (DCs), comprising the steps of providing, e.g., from a subject, a population of neutrophils that express one or both of FcγRIIA and / or FcγRIIIB; contacting the neutrophils in culture with an antibody that comprises an antigen-binding portion that binds an antigen and an Fc region that binds FcγRIIA and / or FcγRIIIB; and maintaining the population of neutrophils in culture under conditions, and for a time sufficient for the neutrophils to differentiate into DCs.

[0017] In some embodiments, the population of neutrophils is maintained in the presence of GM-CSF and / or ICs.

[0018] Also provided herein are populations of dendritic cells, eg, generated by the methods described herein, for use in methods of treating a subject, preferably a subject from which a population of neutrophils has been obtained.

[0019] In some embodiments, a population of neutrophils comprising neutrophils expressing one or both of FcγRIIA and / or FcγRIIIB is obtained from a subject, and the method further comprises administering differentiated dendritic cells to the subject from which the population of neutrophils was obtained.

[0020] In some embodiments, the subject has a myeloid tumor or an infection, eg, a bacterial, fungal, or viral infection.

[0021] Also provided herein is a method for treating a subject with myeloid tumor or infectious disease, such as bacterial, fungal or viral infection.The method comprises administering to the subject an effective amount of an antibody comprising an antigen-binding domain that binds to FcγRIII.In some embodiments, the subject has a myeloid tumor selected from the group consisting of acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) or myeloproliferative neoplasm (MPN).

[0022] The term "antigen" includes any structure capable of inducing an immune response in an organism, either by itself or when linked to a suitable carrier molecule or cell. Antigens according to the present invention therefore include small compounds that act as haptens as well as whole cells, such as tumor cells, and parts thereof, such as polypeptides, oligopeptides derived therefrom, lipids, such as glycolipids, polysaccharides and nucleic acids.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention belongs.Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used.Materials, methods, and examples are illustrative only and are not intended to be limiting.All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein in their entirety by reference.In case of conflict, the present specification, including definitions, shall control.

[0024] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. [Brief description of the drawings]

[0025] [Figure 1A-B]Engagement of FcγR by immune complexes promotes the differentiation of neutrophils into dendritic cells. A-B) Mature neutrophils isolated from wild-type mice were incubated for 2 h with the indicated model immune complexes (ICs) composed of OVA-anti-OVA, Nip-OVA-anti-Nip or BSA-anti-BSA or with individual components, washed and cultured with GM-CSF (A) or treated with ICs generated by SLE patient's or normal (NH) serum, or IgG from anti-RNP positive SLE serum and RNP (SLE-IgG+RNP) or normal serum and IgG from RNP (NHS-IgG+RNP) and cultured in medium without GM-CSF (B). After 3 days, cells were harvested, assessed for viability and analyzed for markers of neutrophils (Ly6G) and DC (CD11c, MHCII) by flow cytometry. This population was analyzed for additional DC markers, CD80, CD86 and CCR7. Data are means ± sem. P values ​​were calculated by one-way ANOVA and Dunnett's multiple comparison test in A) and multiple t-test in B). *P<0.05 and **P<0.005. C-D) Blood samples from normal human controls (N) and SLE patients were analyzed for the indicated neutrophil (CD10, CD15) and DC (CD11c, MHCII) markers in lineage-negative (Lin-; CD3-CD19-CD56-) cells (C), SLE samples were further evaluated for CD80, CCR7 and dendritic subset markers (Clec9A, CD141) (D) and correlated with SLE Disease Activity Index (SLEDAI) scores. Statistical significance was determined by non-parametric tests using Mann-Whitney analysis and correlations were analyzed by Spearman test (*P<0.05). E) Mice with a knock-in of tdTomato at the Ly6G locus were subjected to anti-GBM nephritis. On day 14, the indicated tissues were isolated from naive mice (control) and mice subjected to anti-GBM nephritis and CD45+, Lin- and tdTomato positive cells were assessed for DC markers (CD11c+MHCII+).This population was further analyzed for DC markers (middle panel) and DC subset markers (lower panel) were further evaluated in mice subjected to anti-GBM nephritis. Student's t-test for unpaired comparisons using Dambon-Ferrone was used to compare the mean percentage of tdTomato+CD11c+MHCII+, CD86+ or CCR7+ events in control versus anti-GBM nephritis animals. *P<0.05 and **P<0.005. F-G) Neutrophils from wild-type, γ- / -, FcγRIIA (2A) / γ- / - and FcγRIIIB (3B) / γ- / - mice were incubated with SLE IC (F) or FcgRIIIB antibody-antigen conjugate (3G8-OVA) (G) and analyzed as in A). Multiple t-tests were used to determine significant differences between OVA and OVA-IC or between normal (N) and SLE (S) sera (**P<0.005). [Figure 1C] (As stated above.) [Figure 1D] (As stated above.) [Figure 1E] (As stated above.) [Figure 1F-G] (As stated above.) [Figure 2A]Antigen uptake by neutrophil FcγRIIA or FcγRIIIB generates DCs that activate CD4 and CD8 cells. A) Isolated WT and γ- / - neutrophils were incubated with FITC-OVA or FITC-OVA-IC for the indicated times. FITC positive signal is a measure of FITC-OVA uptake. Mean % of positive cells ± sem is given. n=5 per group. Unpaired Student's t-test was used to compare the mean percentage of Ly6G+FITC+ events between WT and γ- / - at various time points (**P<0.005). B-C) ​​NDDCs were generated with GM-CSF alone (-), OVA or OVA-IC. After 3 days of culture, adherent cells were incubated with OT-I, CFSE-labeled CD8 T cells that recognize the OVA257-264 peptide in the context of H2Kb-MHC class I. As positive controls, NDDCs generated with GM-CSF were pulse-labeled with OVA peptide SIINFEK (B), or OT-II (C), CD4 T cells that recognize the OVA323-339 peptide in the context of I-Ab-MHC class II. CFSE dye dilution represents distinct generations of proliferating cells. Graphs show mean % of cell division ± sem. One-way ANOVA with Dunnett's multiple comparisons was used to compare mean percentages of GM-CSF(-), OVA and OVA-IC in different genotypes as indicated. *P<0.05 and **P<0.005. D) Neutrophils isolated from MyD88 / TRIF- / - mice were treated as in B-C and OTI and OTII proliferation was assessed (n=2). [Figure 2B] (As stated above.) [Figure 2C] (As stated above.) [Figure 2D] (As stated above.) [Figure 3A-B]Neutrophil-derived DCs are migratory and promote antigen-dependent delayed-type hypersensitivity responses and B16F10 tumor immunity. OVA-IC- or OVA-fed neutrophils from wild-type (A, C-E) or β-actin-RFP (B) mice were cultured ex vivo to generate NDDCs. A) NDDCs were CFSE-labeled and injected iv on day -3. On day 0, their presence in blood, spleen, liver and lungs was determined by flow cytometry. B) On days -7 and 0, mice were injected with OVA-IC β-actin-RFP+ NDDCs in the footpad and intravital microscopy was performed on popliteal lymph nodes. The seconds of dwell time, the number of OT-I cells in the field of view (FOV) in the presence or absence of NDDCs and the number of OT-I cells interacting with RFP+-NDDCs were assessed. Data were averaged from 3 fields per mouse for n=3 mice (*P<0.05, **P<0.005). Representative images of cluster formation of OT-I-GFP CD8+ T cells around NDDCs and random distribution of CD8+ T cells in NDDC-free areas are shown. Scale bar: 15uM. C) NDDCs generated with OVA or OVA-IC were injected iv on day -7. On day 0, soluble OVA (+) or PBS (-) was injected into the right or left footpad, respectively. On day 1, footpad swelling in both footpads was assessed and paws were harvested for histological analysis. Inflammation scores are shown in the right panel. Mean percentages of footpad thickness and inflammation scores were compared using unpaired Student's t-test. Significant differences were assessed by t-test (*P<0.05 and **P<0.005). D) NDDCs (without OVA) or NDDCs loaded with OVA or OVA-IC from WT or MyD88-TRIF- / - were injected iv on day -7. B16F10-OVA cells were implanted sc on day 0 and tumor volumes were measured over time. Numbers of mice per group are in parentheses. One-way ANOVA was used for data comparison of tumor volumes of OVA-IC versus all other groups (*P<0.05 and **P<0.01).At harvest, OVA peptide-specific CD8 T cells in the spleen (top) and draining lymph nodes (bottom) of the OVA (left) and OVA-IC (right) groups were quantified by FAC using OVA-peptide tetramer complexes. Mean ± sem Unpaired Student's t-test was used to compare OVA to OVA-IC samples. **P<0.01. E) Recipient WT mice were injected iv with NDDCs loaded with OVA-IC as in D) and sc with B16F10-OVA and subjected to treatment with anti-CD4 or anti-CD8 depleting antibodies or isotype control as indicated in the timeline. Tumor growth was assessed over time. Number of mice per group is in parentheses. Tumor volumes were compared between anti-CD8 and anti-CD4 or isotype using one-way ANOVA (*P<0.05 and **P<0.01). CD8 and CD4 T cell depletion was confirmed by FAC analysis of blood samples (right panel). Unpaired Student's t-test was performed and compared with isotype (**P<0.01). [Figure 3C] (As stated above.) [Figure 3D] (As stated above.) [Figure 3E] (As stated above.) [Figure 4A]Immunogenic DCs are generated by engagement of FcγRIIIB on blood neutrophils with antibody-conjugated antigen. Wild-type, γ- / -, FcγRIIA(2A) / γ- / -, FcγRIIIB(3B) / γ- / - and FcγRIIIB+FcγRIIA(2A3B) / γ- / - mice were used. A) Mice were given iv with anti-FcγRIIIB 3G8 antibody conjugated to FITC-OVA (3G8-OVA) and uptake of FITC-OVA (upper panel) as well as markers of neutrophils and DCs (middle and lower panels) were assessed in blood and spleen at the indicated times. Representative FAC profiles in blood are shown. One-way ANOVA was performed to compare mean ± sem between γ- / - and 3B / γ- / - or 2A3B / γ- / - (**P<0.005). B) Mice were given an iv injection of 3G8-OVA on day 3 and CFSE-labeled OT-I CD8 T cells on day 0. On day 3, spleens were harvested and CD8 proliferation was assessed by CFSE dilution. Representative histograms of CD8 dividing cells (bars) and starting CD8 population (Con, light grey) in spleens are shown. ANOVA and Dunnett's correction were used to determine significant differences between γ- / - and 2A3B / γ- / - or 3B / γ- / - mice (**P<0.005). C) Mice were given an iv injection of 3G8-OVA on day 10 and target wild-type splenocytes, highly and lowly CFSE-labeled, pulsed and unpulsed with OVA-peptide, respectively, on day 0. Spleens were harvested 16 hours later and % of dead target cells were assessed. Means ± sem were assessed and compared for statistical significance using ANOVA and Dunnett's correction. WT was compared with 2A3B / γ- / -, 3B / γ- / -, 2A / γ- / - and γ- / -. **P<0.005. D-F) Mice were given daily ip injections of GM-CSF from day -10 to day -5 and iv injections of 3G8-OVA or FITC-OVA on day -10. On day 0, B16F10-OVA cells were implanted subcutaneously. D) Tumor growth was assessed. Numbers of mice per group are in parentheses, scale bar = 5 mm, statistical analysis of tumor growth rates is described in additional Fig. S3.**p<0.005 between 2A3B / γ- / -+3G8-OVA and other groups. E) At harvest, spleens were analyzed for CD8 effector T cells (CD62lo CD44hi) specific for OVA-peptide using MHCI tetramers, CD4 and Treg cells. For significant differences of 2A3B / γ- / - versus γ- / - and WT, one-way ANOVA with Dunnett's multiple comparisons was used. *P<0.05. F) GM-CSF-pretreated human whole blood was incubated with 3G8-OVA or FITC-isotype control and %FITC-positive neutrophils were determined at the indicated times. FcγRIIIB internalization was assessed using anti-FcγRIIIB antibody REA(589), which binds to a distinct epitope from 3G8. Antibody IV.3 was used to assess FcγRIIA levels. A multiple t-test was used to assess the significance of differences between isotype and 3G8-OVA at the indicated time points (**P<0.005). G) Blood treated as in F) was subjected to Hetasep treatment and RBC lysis to isolate leukocytes. Cells were cultured for 2 days and analyzed for neutrophil (CD10, CD15) and DC (CD11c, MHCII) markers. This population was further investigated for co-stimulatory molecules, CD80, CD86, chemokine receptor CCR7 and DC subset markers (XCR1, Clec9A, CD141). Comparisons of significance between isotype and 3G8-OVA treated blood samples were performed by multiple t-test (*P<0.05 and **P<0.005). [Figure 4A-2] (As stated above.) [Figure 4B-C] (As stated above.) [Figure 4D] (As stated above.) [Figure 4E-F] (As stated above.) [Figure 4G] (As stated above.) [Figure 5A]A) Gating strategy for evaluating the purity of mouse neutrophils isolated from mouse bone marrow. Cells were stained and evaluated for CD11b, Ly6G, MHCII, CD11c and CD115. B) Gating strategy for expression of DC markers CD11c and MHCII, costimulatory molecules CD80 and CD86 and migration receptor CCR7 on NDDCs generated from neutrophils treated with model IC or their individual components. A representative strategy is shown for NDDCs generated with OVA-IC. Single cells were gated using FSC-H and FSC-A. All live cells negative for lineage markers (CD3, NK1.1, CD19) and negative for fixable viability dye were further gated for CD11c, MHCII and Ly6G. Positive cells were then checked for expression of CD80, CD86 and CCR7. C) NDDCs were generated using the indicated model IC or individual components. After 3 days, cells were analyzed for XCR1, CD8a and CD103 (DC1 subset) or Siglec-H (plasmacytoid DC marker) on CD11c+MHCII+Ly6G+ cells. Data are comparisons between the mean ± sem of OVA-IC or OVA against anti-OVA for all model ICs based on one-way ANOVA and Dunnett's multiple comparison test. D) Gating strategy for expression of DC markers, costimulatory molecules and CCR7 on neutrophils cultured with ICs generated with normal serum (NHS), SLE patient serum or RNP and IgG from normal or anti-RNP positive SLE serum. A representative strategy for NDDCs generated with SLE serum is shown. E) Subset analysis of XCR1, CD8a and CD103 (DC1 subset) on NDDCs generated from ICs generated with NHS, SLE patient serum or RNP and IgG from normal or anti-RNP positive SLE serum. F) Gating strategy for blood samples from human controls and SLE patients analyzed for CD80, CCR7 and DC subset markers. G) WT and MyD88- / -TRIF- / - derived NDDCs were analyzed for expression of CD11c, MHCII and Ly6G (upper panel).Positive cells were further evaluated for CD86, CD80 (middle panel) and CCR7 (lower panel) using Student's t-test for unpaired comparisons according to Dambon-Ferrone (**P<0.005). [Figure 5B-C] (As stated above.) [Figure 5D-E] (As stated above.) [Figure 5F] (As stated above.) [Figure 5G] (As stated above.) [Figure 6] Neutrophils isolated from WT and β2-microglobulin-deficient (- / -) mice were treated with GM-CSF alone (-), OVA, or OVA-IC and cultured for 3 days. Adherent NDDCs were cocultured with CFSE-labeled OT-I or OT-II T cells. As a positive control for OT-I proliferation, GM-CSF-generated NDDCs were pulsed with the OVA peptide SIINFEKL. Percent proliferation was measured by CFSE dye dilution. [Figure 7A]A) Anti-FcγRIIIB 3G8 antibody conjugated to FITC-OVA (3G8-OVA) was given iv to mice. After 3.5 hours, blood was isolated and internalization of FITC-OVA, FcγRIIIB (CD16) and FcγRIIA (CD32) was assessed. The anti-CD16 antibody, REA 589, binds to a distinct epitope from 3G8. Gating strategies for FITC-OVA, FcγRIIIB and FcγRIIA are shown. B) Gating strategy for determining basal levels of FcγRIIIB and FcγRIIA expression in CD115+ and Ly6Chi cells in humanized transgenic mice expressing FcγRIIIB+FcγRIIA. Means ± sem for FcγRIIIB and FcγRIIA are given for the CD115+Ly6Chi population (n=4). C) Gating strategy to assess the presence of 3G8-OVA positive events of CD11c+MHCII+NDDC in spleens of 2A3B / γ- / - and γ- / - mice 72 hours after iv injection of 3G8-OVA conjugate. D) Regression analysis was used to compare the slope curve coefficients (tumor growth rate) between different groups. Regression analysis with Prism was used to test whether the slopes and intercepts were different. One-way ANOVA and Dunnett's multiple comparisons were used for global comparisons. Trend lines for tumor volumes of all groups were generated using Excel and are shown. Tumor growth in 2A3B / γ- / -+3G8-OVA was compared to WT+FITC-OVA, γ- / -+3G8-OVA and 3B / γ- / - (**P<0.005). E) Table of T cell subset analysis of spleens harvested from mice injected with B16F10-OVA. Spleens were analyzed for CD8 effector T cells (CD62lo CD44hi) specific for OVA peptide using MHCI-tetramer and markers for CD8 effector, CD4 and Treg cells. Results for T cell activation markers (CD25;CD69), proliferation markers (Ki67), transcription factors (t-bet) and PD1 are also shown. **P<0.05 for 2A3B / γ- / -+3G8-OVA compared to other groups. [Figure 7B] (As stated above.) [Figure 7C-D](As stated above.) [Figure 7E] (As stated above.) [Figure 8] FcγR-mediated antigen-antibody uptake leads to the differentiation of neutrophils into antigen-loaded dendritic cells that promote antigen-specific T cell responses. Neutrophils incubated ex vivo with antigen-antibody immune complexes (ICs) enhance antigen uptake (1). iv-injected anti-FcγRIIIB antigen (protein or peptide) conjugates (FcγRIIIB vaccines) selectively bind antigen and induce antigen internalization by neutrophils (2). Engagement of FcγRs by methods 1) or 2) delivers signals that promote the differentiation of neutrophils into dendritic cells and efficient loading of antigen on MHC1 and MHCII. iv-injected NDDCs (1) or in vivo-generated NDDCs (2) migrate to the spleen. Here, they present antigen to naive CD4+ and CD8+ T cells and migrate from the spleen to generate antigen-specific effector T cells that promote helper T cell responses and cytolytic responses, respectively. [Figure 9A-B] A) Schematic of NDDC vaccine with anti-FcγRIIIB antibody conjugated to tumor antigen. B) Schematic of method using only anti-FcγRIIIB antibody (no exogenous antigen) targeting neutrophils. C) One experiment shows quantification of ELISPOT assay detecting IFN production by activated T cells. This experiment is representative of one of two experiments on an independent normal donor and patient 1 (diagnosed with acute myeloid leukemia) and patient 2 (diagnosed with myelodysplastic syndrome). D) Representative images are shown for the indicated samples incubated with isotype control or vaccine. [Figure 9C-D] (As stated above.) DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] In humans, 60-70% of circulating white blood cells are neutrophils; these are immune effector cells that destroy pathogens and are thought to be terminally differentiated forms with a limited life span.4 However, neutrophils cultured with the survival factor GM-CSF and cytokines were able to acquire DC phenotypic markers and functional characteristics. 5 , detected in inflammatory lesions in mice and human patients 5、6 The potency of neutrophil-derived DCs in effector immunity, potentially the identification of molecular pathways that enable neutrophil-derived DCs to migrate and cross-present, and strategies to exploit these cells as best as possible for therapeutic benefit remain largely unexplored.

[0027] Low-affinity FcγRs present on myeloid cells bind antigen-antibody immune complexes (ICs) and mediate several immune functions. 7、8 In mice, FcγRIII and FcγRIV require a γ chain containing immunoreceptor tyrosine-based activation motif (ITAM) for cell surface expression and signal transduction, and mice with γ chain deficiency (γ - / - ) protects against tissue injury in several autoimmune disease models. However, the FcγR repertoires of mice and non-human primates differ from those of humans. 7、8 Humans express a low-affinity ITAM-containing single polypeptide, FcγRIIA, on neutrophils, monocytes, eosinophils, DCs, and platelets, whereas glycosylphosphatidylinositol (GPI)-linked FcγRIIIB is expressed almost exclusively on neutrophils. γ-chain associated FcγRIIIA is expressed on natural killer cells and monocytes / macrophages. 9 FcγRIIA promotes the cytotoxic function of neutrophils. 10 , as is its mouse counterpart in DC. 12 , enhancing maturation and cross-presentation 11 Transgenic expression of FcγRIIA in mice increases susceptibility to autoimmune-mediated injury and drives antitumor vaccine responses. 11、12 , which may promote anaphylaxis 9 Little is known about the function of FcγRIIIB, which cooperates with FcγRIIA to promote the function of some neutrophils. 10, alone, can support IC-induced neutrophil accumulation 13 Furthermore, like FcγRIIA, FcγRIIIB can induce plasma membrane invagination of ICs and support internalization of ICs deposited within the vessel wall. 14 FcγRIIB is a conserved inhibitory receptor in humans and mice that is expressed primarily on B cells, macrophages, and DCs. 8 It is also detected on neutrophils, but primarily in individuals with a rare FcγRIIB.4 promoter haplotype. 15 .

[0028] Without wishing to be bound by theory, as shown herein, FcγR-mediated antigen uptake of blood neutrophils delivers signals that simultaneously drive antigen uptake and differentiation of neutrophils into a mature DC-like phenotype, thus ensuring a large number of antigen-loaded DCs that accumulate in the spleen and promote T cell activation that is dependent on MHCI and MHCII (see schematic in FIG. 8). The presence of NDDCs in models of antibody-mediated renal inflammation and in SLE patients with high circulating levels of immune complexes suggests that immune complexes can promote NDDC generation in vivo. Coupling of FcγR-mediated antigen-antibody uptake with differentiation allows for the uptake of peptides derived from opsonized non-self antigens, as occurs during secondary exposure to antigen, by CD8 + Because presentation to cytotoxic cells is preferentially favored 11、12, autoimmune responses can be avoided. Engagement of FcγRIIIB alone is sufficient to induce differentiation of neutrophils into migratory, cross-presenting DCs. This identifies a novel role for this enigmatic neutrophil-specific GPI-linked receptor in linking innate and adaptive immunity in response to blood-borne antigens and shows that differentiation induced by ICs does not require ITAM signaling. However, despite comparable ability to stimulate T cell proliferation in vitro (Figure 2B-C), mice expressing both FcγRIIA and FcγRIIIB had superior CTL proliferation and target cell killing (Figure 4B-C). In classical DCs, simultaneous delivery of TLR ligands and / or additional costimulatory signals is required for sustained T cell responses. 1 The generation of FcγR-dependent NDDCs occurred in the absence of adjuvants and instead required GM-CSF. Clinically, ex vivo generation of DCs from abundant blood neutrophils could circumvent the current need for apheresis in the generation of autologous DCs for cancer immunotherapy. 1 A monoclonal antibody that is an antitumor antibody known to exhibit both ADCC and FcγR-mediated vaccine effects. 11 Incubation of neutrophils with FcγRIIIB-opsonized tumor cells can also promote antigen sampling of opsonized tumor cells and simultaneously drive neutrophils toward DC differentiation. Intravenous immunization with FcγRIIIB antibody-antigen conjugates can be a robust platform to both generate large amounts of DC in situ and deliver antigens for optimal T cell activation and cross-presentation. Of note, although pro- and anti-tumorigenic functions of neutrophils in cancer have been described, 32By engaging FcγR, a well-known opsonic receptor in host defense, the tumorigenic potential of neutrophils may be exploited. Collectively, this study identifies FcγRIIIB-mediated antigen delivery as a new pathway for both generating DCs and delivering antigens to compartments favorable for cross-presentation. This process, optionally combined with GM-CSF treatment, can be exploited to generate immunogenic DCs to fight cancer and infectious diseases.

[0029] Thus, methods of inducing antigen-specific immunity (e.g., treating cancer or infectious diseases) are provided herein. These methods deliver antigens to neutrophils, which induce their differentiation into antigen-presenting DCs that activate CD4+ helper T cells and CD8+ cytotoxic T cells by antigen uptake. The methods of delivery are 1) antigen-antibody immune complexes that engage human-specific FcγRIIA and GPI-linked FcgRIIIB or mouse counterparts on neutrophils to generate NDDCs in vitro, which are then administered to patients, and 2) anti-FcγRIIIB antibodies conjugated to antigens (vaccines) that engage activating human FcγRIIIB, which is selectively expressed on the surface of human neutrophils and known to generate NDDCs in vivo. In addition to inducing differentiation of neutrophils into DCs, the above method of antigen delivery resulted in superior antigen presentation over that of soluble antigens, consistent with published findings in classical DCs where antigen presentation present in immune complexes exceeds that of soluble antigens (Wen et al., EMBO Mol Med 8, 1120-1133 (2016)).

[0030] Thus, two methods for treating subjects are described herein.Either method 1 or 2 can be used to treat patients with cancer or infectious disease, and potentially be used as a preventive vaccine to reduce the risk of cancer or infectious disease, and increase antigen-specific immunity.

[0031] As used in this context, "treating" cancer means improving at least one symptom of cancer. Administration of a therapeutically effective amount of a compound described herein for the treatment of cancer may result in reduced or stabilized tumor burden, reduced or stabilized tumor size, reduced or stabilized tumor growth rate, reduced or stabilized tumor serum markers, and reduced or stabilized risk of metastasis. The subject may be, for example, a mammal, for example, a human or veterinary subject. Although humans are used as examples herein, other mammals may also be treated using the methods of the present invention, using species-appropriate antibodies and other reagents.

[0032] The method generally includes identifying a subject having a tumor, e.g., cancer. As used herein, the term "cancer" refers to cells having an abnormal state or condition characterized by the capacity for autonomous growth, i.e., rapidly proliferating cell growth. Hyperproliferative and neoplastic disease states may be classified as pathological, i.e., characterizing or constituting a disease state, or non-pathological, i.e., deviating from normal but not associated with a disease state. Generally, cancer will be associated with the presence of one or more tumors, i.e., abnormal cell populations. The term "tumor" is meant to include all types of cancerous growth or oncogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasiveness. "Pathological hyperproliferative" cells arise in disease states characterized by malignant tumor growth. Generally, the methods described herein may be practiced in subjects having solid tumors or hematopoietic tumors, which are malignant tumors of cells of the immune system. Methods for identifying or diagnosing subjects having cancer are known in the art.

[0033] Tumors include malignancies of various organ systems, such as lung, breast, thyroid, lymphatic system, gastrointestinal, and genitourinary tract, as well as adenocarcinomas, including malignancies such as most colon cancers, renal cell carcinoma, prostate cancer and / or testicular cancer, non-small cell carcinoma of the lung, cancer of the small intestine, and cancer of the esophagus. The term "cancer" is recognized in the art and refers to malignancies of epithelial or endocrine tissues, including respiratory system cancer, digestive system cancer, genitourinary system cancer, testicular cancer, breast cancer, prostate cancer, endocrine system cancer, and melanoma. In some embodiments, the disease is renal carcinoma or melanoma. Exemplary cancers include those formed from tissues of the cervix, lung, prostate, breast, head and neck, colon, and ovary. The term also includes carcinosarcoma, including malignancies composed of, for example, carcinoma and sarcoma tissue. "Adenocarcinoma" refers to cancers derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term "sarcoma" is art-recognized and refers to malignant tumors of mesenchymal derivation.

[0034] In some embodiments, the cancers evaluated or treated by the methods described herein include epithelial cancers, such as lung cancer (e.g., non-small cell lung cancer (NSCLC)), breast cancer, colorectal cancer, renal cancer, head and neck cancer, prostate cancer, or ovarian cancer. Epithelial malignancies are cancers that affect epithelial tissues.

[0035] Additional examples of cancers that are potential targets for NDDC therapy include hematological malignancies.As used herein, the term "hematological tumors" includes cancers that arise against precursor cells of bone marrow, such as acute leukemia, myeloproliferative neoplasms, and myelodysplastic syndromes; cancers that arise from peripheral lymphoid tissue, such as non-Hodgkin's lymphoma, Hodgkin's lymphoma, and leukemia or mature T and B cells (e.g., chronic lymphocytic leukemia); or cancers that arise from plasma cells or plasma cell precursors (e.g., multiple myeloma and lymphoplasmocytic lymphoma).

[0036] In some embodiments, the cancer to be evaluated or treated by the methods described herein includes a myeloid tumor, an immune system malignancy, such as acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), or myeloproliferative neoplasm (MPN).

[0037] Method 1 - Cell therapy The first method involves the use of neutrophils exposed to antigen-antibody immune complexes that bind to FcγRIIA and / or FcγRIIIB in the presence of GMCSF and promote the differentiation of neutrophils into dendritic cells (DCs). These DCs, referred to herein as NDDCs, are expected to be more effective at promoting the proliferation of CD4+ and CD8+ T cells than DCs generated by GM-CSF and soluble antigens. Because neutrophils represent 55-70% of circulating white blood cells in humans, a greater number of neutrophils can be recovered in a single blood draw. The method delivers antigen in vitro in immune complexes that engage activating FcγRs, a specific feature of classical DCs, both to promote differentiation and to facilitate antigen presentation to CD4 T cells and cross-presentation to CD8 cells.

[0038] Generally, the method includes preparing a population of neutrophils. Preferably, the neutrophils are autologous, but HLA-matched xenogeneic neutrophils may also be used. Methods for obtaining neutrophils from a sample of peripheral blood are known in the art and may include commercially available human neutrophil enrichment kits (EASYSEP). In some embodiments, the population of neutrophils is enriched for cells expressing FcγRIIA and / or FcγRIIIB, for example, using FACS, microfluidic devices, or other cell sorting methods.

[0039] Neutrophils are incubated in the presence of GMCSF (e.g., 5-15 ng / ml, e.g., 8-12 ng / ml, e.g., 10 ng / ml GMCSF) and antigen-antibody immune complexes. These immune complexes include tumor-associated antigens (e.g., cancer-testis (CT) antigens, proteins normally expressed only in human germline cells but absent in a subset of malignant tumors), e.g., CT antigens listed in Table A, or neoantigens resulting from tumor-specific mutations. Neoantigens can result from any genomic mutation that alters the protein sequence. See, e.g., Hu, Ott, Wu "Towards personalized, tumor-specific, therapeutic vaccines for cancer." Nat Rev Immunol. 2018 Mar;18(3):168-182;Vigneron et al., Cancer Immun. 2013; 13: 15;Hutchison and Pritchard, Mamm Genome. 2018; 29(11): 714-730;Teku and Vihinen, Sci Rep. 2018; 8: 12735;Renkvist et al., Cancer Immunology and Immunotherapy 2001; 50:3-15.

[0040] [Table 1-1]

[0041] [Table 1-2]

[0042] If neoantigens are used, preferably neoantigens expressed in an individual subject are identified and used as described, for example, in Hu et al., Nat Rev Immunol. 2018 Mar;18(3):168-182; Ott et al., Nature. 2017 Jul 13;547(7662):217-221; Carreno et al., Science. 2015 348(6236): 803-808; and Linette and Carreno, Trends Mol Med. 2017 Oct;23(10):869-871.

[0043] In some embodiments, the tumor antigen binds to an antibody that recognizes the antigen (through the antigen-binding domain) and binds to FcγRIIA and / or FcγRIIIB (through the Fc domain), resulting in clustering of receptors on the cell surface, followed by internalization of the FcγR and the bound antibody-antigen complex. In some embodiments, the antibody is an IgG, preferably an IgG isolated from the individual subject to be treated. In some embodiments, to purify the IgG, a serum sample is incubated with protein G high performance agarose beads; other methods for purifying IgG from subjects may be used. Alternatively, IgG from other individuals may be used, for example, commercially available IgG. Recombinant IgG may be used. For human subjects, preferably, human or humanized IgG is used. In some embodiments, the antibody is selected or optimized to bind to FcγRIIIB and / or FcγRIIA. The Fab region recognizes the antigen, while the Fc region binds to the FcγR. The affinity to Fc is affected by variants of FcγRIIA and FcγRIIIB, as well as the subclass of IgG. There are four subclasses of human IgG (IgG1-4). Binding affinity of IgG to FcγRIIA and / or FcγRIIIB is highest for IgG1 and IgG3 (Bruhns et al., Blood 113, 3716-3725 (2009); Ivan and Colovai, Hum Immunol 67, 479-491 (2006); Bruggeman et al., J Immunol 199, 204-211 (2017)). Lower complement activation potential favors FcγR binding, making IgG1 preferred over IgG3. Most antitumor therapeutic antibodies are also IgG1. FcγR binding is influenced by N-linked glycosylation at Asn297 of the Fc domain. The core N-linked glycoform can be modified with galactose, fucose and / or sialic acid to generate up to 500 different glycoforms (Jefferis, Nat Rev Drug Discov 8, 226-234 (2009)).Hypofucosylation favors IgG1 interaction with FcγRIIIB, but has minimal effect on FcγRIIA (Bruhns et al., Blood 113, 3716-3725 (2009);Bruggeman et al., J Immunol 199, 204-211 (2017);Jefferis, Nat Rev Drug Discov 8, 226-234 (2009);Mizushima et al., Genes Cells 16, 1071-1080 (2011);Ferrara et al., Proc Natl Acad Sci USA 108, 12669-12674 (2011);Subedi and Barb, MAbs 8, 1512-1524 (2016);Peipp et al., Blood 112, 2390-2399 (2008)). Thus, hypofucosylated IgG1 is generated to engage neutrophils with FcγRIIA and FcγRIIIB. See, e.g., Derer et al., MAbs. 2014 Mar 1; 6(2): 409-421;Zhang et al., J Biol Chem. 2016 Dec 30; 291(53): 27134-27146;Derer et al., Blood 2013 122:3466;Bruhns et al., Blood 2009 113:3716-3725.

[0044] In some embodiments, the antibody is an antibody that recognizes and directly binds to an antigen. For example, existing or new therapeutic antibodies to extracellular or intracellular antigens of tumors (e.g., HER2 / neu for breast cancer, many epithelial tumors such as non-small cell lung cancer, EGFR for colorectal cancer (see, for example, Corraliza-Gorjon et al., 2017 Front Immunol. Doi.org / 10.3389 / fimmu. 2017.01804), or PRL-3, a phosphatase associated with externalizing intracellular cancer (Trenevska et al., 2017 Front Immunol. Doi.org / 10.3389 / fimmu.2017.01001)); alternatively, the antigen can be produced as a fusion protein with a tag that the antibody binds to. For example, in some embodiments, antigen-antibody complexes are generated by methods in which the antigen is generated with a "tag" (e.g., KLH currently used in patients, Wimmers et al., Scientific Reports 7, Article number: 43486 (2017), or polyhistidine, FLAG, Softag1 and Softag3, and Streptag II (see, e.g., Arnau et al., Protein Expression and Purification 48 (2006) 1-13)) or a human peptide sequence to which a humanized antibody binds. For example, a humanized mouse anti-KLH monoclonal antibody (Abcam, Sino Biologicals, Rockland Immunochemicals) can be used. In this way, antibodies would not need to be adapted for each antigen. The antigen / antibody immune complexes are incubated with neutrophils to produce NDDC-presenting antigens.

[0045] Neutrophils can also be incubated in the presence of tumor cells opsonized with anti-tumor antibodies, resulting in uptake of debris and antigens by neutrophils and their subsequent differentiation into antigen-presenting DCs.

[0046] Neutrophils may also be incubated with anti-FcgRIIIB (3G8)-antigen conjugates (described in method 2) and FcgRIIA (IV3)-antigen conjugates, where IV3 specifically recognizes FcgRIIA. Another embodiment is a bispecific antibody that contains antigen binding sites for FcγRIIIB and FcγRIIA within one IgG1 molecule, and thus will engage and cluster with both receptors simultaneously (Sedykh et al., 2018. Bispecific antibodies: design, therapy, perspectives Drug Des Devel Ther. 12: 195-208).

[0047] The neutrophils are incubated in the presence of immune complexes and GMCSF long enough for the cells to mature into dendritic type cells (referred to herein as NDDCs, as described above), for example, 2-5 days.

[0048] The NDDCs may be maintained in culture for an additional period of time, if desired, to allow for proliferation. The cells may then be washed, optionally purified and / or concentrated, and administered to a subject, for example, intravenously. The harvested NDDCs may be frozen prior to use.

[0049] As shown herein, intravenous delivery of in vitro generated NDDCs with antigen-antibody complexes made with the "model" antigens ovalbumin (OVA) and anti-OVA results in their accumulation in the spleen. Immunized mice showed immunity to B16F10-OVA melanoma that typically associates with antigen-specific CD8 T cells in the tumor-draining lymph nodes. This immunity was reversed when CD8 T cells were depleted from the mice. Furthermore, immunized mice showed delayed-type hypersensitivity responses to the antigen, suggesting that NDDCs stimulate CD4 T cell function.

[0050] These features are superior to the two current technologies utilized for antigen delivery:

[0051] The methods of the invention offer advantages over currently used techniques that rely on blood apheresis to isolate autologous monocytes and then generate antigen-loaded DCs in vitro by culturing the cells with cytokines and antigens, and then reintroduce the cells into the patient (see, e.g., Van Willigen et al., Frontiers in Immunol. 2018 Oct 1;9:2265; Tanyi et al., Sci Transl Med. 2018 Apr 11;10(436)). Unlike blood monocytes (2%), which represent only a small population of peripheral blood cells, neutrophils represent 55-70% of peripheral blood leukocytes, allowing large numbers of these cells to be isolated from a single blood draw and differentiated into NDDCs, eliminating the need for blood apheresis. Presentation of antigen in immune complexes that engage FcγRIIA and FcγRIIIB also promotes antigen cross-presentation and activation of CD8 T cells, which is minimal when soluble antigen is given alone.

[0052] Method 2 - Antigen-conjugated anti-FcγRIIIB antibody Also provided herein are methods in which constructs comprising anti-FcγRIIIB antibodies conjugated to tumor antigens are used to prepare cells for use in cell therapy methods in vitro / ex vivo or to stimulate an immune response to an antigen in vivo.

[0053] As shown herein, incubation of neutrophils expressing human FcγRIIIB or FcγRIIIB+FcγRIIA with anti-FcγRIIIB-OVA conjugate (vaccine) resulted in antigen uptake and neutrophil transdifferentiation into NDDCs. Neutrophils lacking FcγRIIIB did not respond to this treatment. Human neutrophils incubated with FcγRIIIB antibody conjugated to antigen (vaccine) internalize antigen and differentiate into NDDCs.

[0054] Thus, in some embodiments, the conjugates can be incubated with human neutrophils in the presence of GMCSF, e.g., as described above in Method 1, to drive differentiation into NDDCs and then administered to a subject in need thereof.

[0055] In vivo experiments, intravenous delivery of anti-FcγRIIIB-OVA antigen conjugates resulted in antigen uptake and generation of NDDCs in the blood and accumulated in the spleen. Immunized mice developed antigen-specific cytolytic CD8 T cells as assessed in in vivo CD8 T cell proliferation and cytotoxic T lymphocyte (CTL) assays, and developed antigen (i.e., OVA)-specific CD8 T cells in the B16F10-OVA melanoma model. Mice treated with five consecutive injections of anti-FcgRIIIB-OVA antigen and GM-CSF developed antigen (i.e., OVA)-specific CD8+ T cells in the B16F10-OVA melanoma model, and showed a significant reduction in tumor growth.

[0056] Thus, in some embodiments, the conjugate is delivered intravenously to a subject, optionally in addition to GMCSF. These methods can be used to deliver antigens intravenously and promote the differentiation of neutrophils into NDDCs in vivo, which in turn generate antigen-specific cytotoxic T cells. The in vivo generation of NDDCs has the advantage of allowing neutrophils to take up additional antigens by phagocytosis and trogocytosis of tumor cells as they differentiate into NDDCs.

[0057] The methods of the present invention provide advantages over currently used techniques that rely on the injection of antigen-conjugated antibodies to classical DC receptors and require the inclusion of adjuvants such as toll-like receptor agonists for efficacy (Macri et al., Clin Transl Immunology. 2016 Mar 18;5(3):e66; these adjuvants are not required to use the methods of the present invention). The delivery route to access the relevant classical DC subsets also remains a challenge. The DC receptors targeted in currently used techniques are often present on other cell types, which increases the possibility of undesirable side effects, whereas the approach described herein specifically targets FcγRIIIB, which is almost exclusively expressed on neutrophils. Furthermore, as mentioned above, targeting neutrophils to DCs is advantageous since neutrophils represent a large proportion of circulating leukocytes, which increases the number of antigen-presenting cells that can be generated not only in vitro but also in vivo.

[0058] The method of the present invention uses a construct comprising anti-FcγRIIIB conjugated to a tumor antigen (see, for example, FIG. 9A). The tumor antigen is described above. Anti-FcγRIIIB is known in the art, as are the methods of making them. Anti-FcγRIIIB antibodies are commercially available from, for example, Abbexa Ltd; Abcam; Abeomics; antibodies-online; Aviva Systems Biology; Biogems International, Inc.; BioLegend; Bioorbyt; CEDARLANE; Cell Sciences; Creative Diagnostics; Elabscience Biotechnology Inc.; EXBIO Praha, as; GeneTex; Invitrogen Antibodies; LifeSpan BioSciences; MBL International; Miltenyi Biotec; MyBioSource.com; NSJ Bioreagents; OriGene Technologies; Peninsula Laboratories International, Inc.; ProSci, Inc; R&D Systems; Santa Cruz Biotechnology, Inc.; Signalway Antibody LLC; Sino Biological, Inc.; SouthernBiotech; STEMCELL Technologies, Inc.; and United States Biological. In some embodiments, the antibody is 3G8 (e.g., available from biolegend, Santa Cruz Biotechnology, etc.; see, e.g., Perussia and Trinchieri, J Immunol. 1984 Mar;132(3):1410-5). These antibodies may be used as is or may be modified, e.g., to reduce immunogenicity or alter half-life.

[0059] In some embodiments, the antibody also recognizes FcγRIIIA (CD16a: the GPI-linked transmembrane isoform of FcγRIIIB, CD16b) present on macrophages and NK cells. In some embodiments, the antibody is an FcγRIIIB-specific antibody that does not bind to FcγRIIIA.

[0060] Method 3 - Anti-FcγRIIIB antibody (not conjugated to antigen) In some embodiments, anti-FcγRIIIB may be used alone (without the associated conjugated antigen, see e.g., Figure 9B) for conditions in which neutrophils are the source of antigen, including, but not limited to, myeloid tumors and infectious diseases in which neutrophils acquire antigen following uptake of pathogens or infected cellular debris or following infection of the neutrophils themselves by pathogens.

[0061] Myeloid tumors A special cancer treatment problem is found in myeloid neoplasms (including acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), and myeloproliferative neoplasms (MPN)), common tumors of the elderly derived from progenitor cells of the innate immune system that harbor driver and passenger mutations that give rise to neoantigens (Lindsley and Ebert, 2013). These tumors usually relapse after treatment with current chemotherapy regimens, and patients are often too old and / or too sick to tolerate allogeneic stem cell transplantation (Webster and Pratz, 2018).

[0062] Several immunotherapeutic strategies have been developed for these tumors, particularly AML, involving their ex vivo manipulation to convert tumor cells into antigen-presenting cells. These methods are still being evaluated in preclinical studies and clinical trials, and variously involve the conversion of tumor cells in GMP laboratories with cDNA encoding antigen-presenting and costimulatory molecules (Shi et al., 2018); the fusion of tumor cells with autologous dendritic cells expressing these molecules (Rosenblatt et al., 2016); or the loading of autologous dendritic cells with tumor neo-antigens (see, e.g., (Van Acker et al., 2019)), each of which is then reinfused into the patient. In some embodiments, the methods described herein use FcγRIIIB antibodies (without exogenous antigens) that convert neutrophils originating from the mutated clone into NDDCs. This may be done in vitro using neutrophils from the subject as described above in method 1, or in vivo, which would also eliminate the need for ex vivo manipulation and the need to isolate large numbers of autologous dendritic cells (which may be a challenge due to the low abundance of neutrophils and thus require blood apheresis). NDDCs induced by FcγRIIIB antibodies loaded with endogenous neoantigens on MHCI will stimulate the activation of autologous T cells and kill malignant myeloid cells, including the leukocyte stem cells at the root of these neoplasms. This strategy does not require ex vivo manipulation of tumor cells or any knowledge of tumor neoantigens. This is a strategy in which cancer cells are induced to mount an immune response against themselves, which as such represents a substantial advance over currently available methods.

[0063] infectious disease Neutrophils may be a source of immunogenic "foreign" antigens in infectious diseases, either by uptake of pathogens (e.g., bacteria, fungi, and viruses) or infected cellular material, or by direct infection with certain organisms, such as intracellular bacteria (Rungelrath et al., 2020), herpes viruses (Larochelle et al., 1998; Saez-Lopez et al., 2005), and influenza A viruses (Cassidy et al., 1988; Hufford et al., 2012; Wang et al., 2008; Zhao et al., 2008). Virus-infected neutrophils have been shown to stimulate CD8 effector T cells to produce IFNg but not the cytotoxic response essential for antitumor immunity (Hufford et al., 2012). Moreover, neutrophils were not shown to prime naive CD8 T cells, an essential property restricted to classical antigen-presenting cells such as DCs and required for the initiation of subsequent immune responses (Hufford et al., 2012). The observed ability to present peptides in MHCII for recognition by CD8 effector T cells and subsequent killing of target cells is a common property of most nucleated cells that have evolved to protect the host against pathogens. Furthermore, virus-infected neutrophils do not express MHCII, which is necessary for the activation of naive and CD4 effector T cells that support CD8 T cell responses and other immune functions and thus have limited antigen-presenting function. These methods include the use of FcγRIIIB (without exogenous antigen) to convert directly infected or pathogen-engulfed neutrophils into NDDCs that promote the activation of naive CD4 and CD8 T cells and promote the cytolytic function of CD8 T cells in vivo, as shown for anti-FcγRIIIB-antigen conjugates (Figure 4C). This can be done in vitro using neutrophils from the subject, as described above in method 1, or in vivo by administration of anti-FcγRIIIB antibodies. As described above, this does not require any knowledge of the antigen from the immunogenic pathogen.

[0064] Antibody The term "antibody" as used herein refers to an immunoglobulin molecule, preferably IgG, and modified forms, or antigen-binding fragments, variants, or derivatives thereof. The antibody may be a polyclonal antibody, a monoclonal antibody, a recombinant antibody, a chimeric antibody, a deimmunized or humanized antibody, a fully human antibody, a non-human antibody, (e.g., murine), or a single chain antibody. In some embodiments, the antibody has effector function and may fix complement. Methods for making antibodies and fragments thereof are known in the art and are described, for example, in Harlow et. Al., editors, Antibodies: A Laboratory Manual (1988); Goding, Monoclonal Antibodies: Principles and Practice, (NY Academic Press 1983); Howard and Kaser, Making and Using Antibodies: A Practical Handbook (CRC Press; 1st edition, Dec 13, 2006); Kontermann and Dubel, Antibody Engineering Volume 1 (Springer Protocols) (Springer; 2nd ed., May 21, 2010); Lo, Antibody Engineering: Methods and Protocols (Methods in Molecular Biology) (Humana Press; Nov 10, 2010); and Dubel, Handbook of Therapeutic Antibodies: Technologies, Emerging Developments and Approved Therapeutics, (Wiley-VCH; 1st edition September 7, Please refer to the following link: http: / / www.nature.com / news2010 / 11023 / If the antibody is obtained by phage display techniques, surface plasmon resonance can be used as in the BIAcore system to increase the availability of phage antibodies that bind to the same epitope as any one of the antibodies described herein (Schier, Human Antibodies Hybridomas 7 (1996), 97-105;Malmborg, J. Immunol. Methods 183 (1995), 7-13). The production of chimeric antibodies is described, for example, in WO 89 / 09622. Methods for the production of humanized antibodies are described, for example, in EP 0239400 and WO 90 / 07861. A further source of antibodies to be utilized according to the present invention are so-called xenogenous antibodies. The general principles for the production of xenogenous antibodies, such as human-like antibodies, in mice are described, for example, in International Publication Nos. WO 91 / 10741, WO 94 / 02602, WO 96 / 34096 and WO 96 / 33735.

[0065] Modified forms of the antibodies described herein, or their antigen-binding fragments, variants, or derivatives, can be made from whole precursors or parent antibodies using techniques known in the art. Exemplary techniques are discussed in more detail herein. The antibodies described herein, or their antigen-binding fragments, variants, or derivatives, can be made or manufactured using techniques known in the art. In certain embodiments, the antibody molecule or its fragment is "recombinantly produced", i.e., produced using recombinant DNA technology. Exemplary techniques for making antibody molecules or their fragments are discussed in more detail elsewhere herein.

[0066] The antibodies, or antigen-binding fragments, variants, or derivatives thereof described herein also include derivatives that are modified, e.g., by the covalent attachment of any type of molecule to the antibody, such that the covalent attachment does not prevent the antibody from specifically binding to its cognate epitope. For example, and without limitation, antibody derivatives include antibodies modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins. Any of a number of chemical modifications may be performed by known techniques, including specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. Additionally, derivatives may contain one or more non-classical amino acids.

[0067] In certain embodiments, the antibodies, or antigen-binding fragments, variants, or derivatives thereof described herein do not elicit a deleterious immune response in the treated animal, e.g., human. In certain embodiments, binding molecules, e.g., antibodies, or antigen-binding fragments thereof described herein, are derived from a patient, e.g., a human patient, and then used in the same species, e.g., human, from which they were derived, to mitigate or minimize the occurrence of a deleterious immune response.

[0068] Deimmunization can also be used to reduce the immunogenicity of antibodies. As used herein, the term "deimmunization" includes antibody modification to modify T cell epitopes, see, for example, International Publication Nos. WO98 / 52976 and WO00 / 34317. For example, the VH and VL sequences from the starting antibody are analyzed, and a "map" of human T cell epitopes from each V region is generated to indicate the location of the epitopes in relation to the complementarity determining regions (CDRs) and other important residues in the sequence. Individual T cell epitopes from the T cell epitope map are analyzed to identify alternative amino acid substitutions that have a low risk of altering the activity of the final antibody. A range of alternative VH and VL sequences that contain combinations of amino acid substitutions are presented, and these sequences are then incorporated into a range of binding polypeptides, such as antibodies specific for FcγRIII or immunospecific fragments thereof that are useful in the diagnostic and treatment methods disclosed herein, and then tested for function. Typically, between 12 and 24 variant antibodies are generated and tested.The complete heavy and light chains, including modified V regions and human C regions, are then cloned into expression vectors and then plasmids that are introduced into cell lines for the production of whole antibodies.The antibodies are then compared in appropriate biochemical and biological assays to identify the best variants.

[0069] In some embodiments, a humanized version of the 3G8 antibody is used. 3G8 is a well-characterized mouse IgG k1 mAb specific for FcγRIII (CD16). Humanized 3G8 antibodies will be generated using standard methods. The most common method described for FcγRIIA mouse antibody IV.3 (Chen et al. Ann Rheum Dis 78, 228-237 (2019)) is the grafting of the heavy and light chain complementarity determining regions (CDRs) of the mouse 3G8 antibody to the closest human germline variable heavy and variable kappa chain genes. Because CDR grafting can result in loss of affinity to the epitope (Safdari et al., Biotechnol Genet Eng Rev 29, 175-186 (2013)), variants with improved binding will be generated by amino acid substitutions in the CDR-grafted domains. These substitutions may follow those identified for a humanized bispecific EGFRxCD16 antibody (Asano et al., FEBS J 279, 223-233 (2012)), using 3G8 as one of the antibody sources.

[0070] Methods for conjugating antigens to antibodies are also known in the art.For example, the antibody polypeptides described herein may comprise, consist essentially of, or consist of fusion proteins.These fusion proteins are, for example, chimeric molecules that comprise the sequence of an immunoglobulin FcγRIII-binding antibody and at least one heterologous tumor antigen.The amino acid sequences may normally be present in separate proteins, which are brought together in the fusion polypeptide, or they may normally be present in the same protein, but are in a position such that they are newly arranged in the fusion polypeptide.Fusion proteins may be created, for example, by chemical synthesis, or by creating and translating a polynucleotide that encodes peptide regions in the desired relationship.

[0071] The term "heterologous," when applied to a polynucleotide or polypeptide, means that the polynucleotide or polypeptide is derived from a separate entity from that of the rest of the entity to which it is compared. For example, as used herein, a "heterologous polypeptide" fused to an antibody, or antigen-binding fragment, variant, or analog thereof, is derived from a non-immunoglobulin polypeptide of the same species, or an immunoglobulin or non-immunoglobulin polypeptide of a different species.

[0072] As discussed in more detail elsewhere herein, the antibodies, or antigen-binding fragments, variants, or derivatives thereof described herein may further be recombinantly fused at the N- or C-terminus to heterologous polypeptides or chemically conjugated (including covalent and non-covalent conjugation) to polypeptides or other compositions.

[0073] Also, a given antibody may contain many types of modifications. Antibodies may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic antibodies may result from post-translation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins, e.g., arginylation, and ubiquitination; see, e.g., Proteins - Structure and Molecular Properties, TE Creighton, WH Freeman and Company, New York 2nd Ed., (1993);Posttranslational Covalent Modification of Proteins, BC See Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol. 182 (1990), 626-646; Rattan et al., Ann. NY Acad. Sci. 663 (1992), 48-62).

[0074] Those skilled in the art will recognize that conjugates can be assembled using a variety of techniques, depending on the agent selected to be conjugated. For example, conjugates containing biotin can be prepared, for example, by reacting an FcγRIII-antibody polypeptide with an activated ester of biotin, such as the biotin N-hydroxysuccinimide ester.Techniques for conjugating various moieties to antibodies, or antigen-binding fragments, variants, or derivatives thereof, are well known and are described, for example, in Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985);Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), Marcel Dekker, Inc., pp. 623-53 (1987);Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), Academic Press pp. 303-16 (1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev. 62 (1982), 119-158.For example, antibodies can be linked to antigens via free amino, carboxylic acid, or sulfhydryl groups using common coupling methods. Some commonly used cross-linking reagents include glutaraldehyde (linking molecules to the N-terminus of peptides), carbodiimide (EDC) (attaching to the C-terminus of peptides); succinimide esters (e.g., MBS, SMCC) (attaching to free amino and cysteine ​​residues); benzidine (BDB) (attaching to tyrosine residues), periodate (attaching to carbohydrate groups); isothiocyanates; carbodiimide / activated ester (EDC / NHS) coupling; reactive azide groups can be sites that are specifically introduced onto protein surfaces as post-translational modifications using enzymatic ligation, which can then be conjugated to alkyne-containing polymers using highly efficient quick chemical reactions. See, for example, Paeth et al., Methods Enzymol. 2017;590:193-224.

[0075] The antibody described herein may be fused to heterologous polypeptide to increase the in vivo half-life of the polypeptide or to be used in immunoassays using methods known in the art.For example, in some embodiments, PEG may be conjugated to the antibody described herein to increase the in vivo half-life;See, for example, Leong et al., Cytokine 16 (2001), 106-119;Adv. In Drug Deliv. Rev. 54 (2002), 531;or Weir et al., Biochem. Soc. Transactions 30 (2002), 512.

[0076] Treating and reducing the risk of infectious diseases In some embodiments of the methods and compositions described herein, instead of tumor antigens, pathogen-associated antigens are used.Pathogens can be, for example, viral, bacterial, fungal, or parasitic pathogens; antigens include viruses and their parts, as well as any prokaryotic and eukaryotic organisms, such as bacteria. For example, the pathogen may be or be associated with E. faecium, S. aureus, K. pneumoniae, A. baumanii, P. aeruginosa, and Enterobacter species, human papillomavirus, Salmonella typhi (typhoid), Vibrio cholera, Yersinia pestis, pulmonary tuberculosis, malaria / Plasmodium falciparum, dengue fever, influenza virus, lyssavirus, meningitis virus, Neisseria meningitidis type B, Chagas' disease, trypanosomiasis, leishmaniasis, babesiosis, or Lyme disease. Methods for identifying peptides for use in targeting these pathogens are known in the art.See, for example, Verma et al., Vaccine. 2018 Jun 27;36(28):4014-4022 (typhoid);Vakli et al., Comput Biol Chem. 2018 Feb;72:16-25 (leishmaniasis);Cockburn and Seder, Nat Immunol. 2018 Nov;19(11):1199-1211 (malaria);Earla et al., J Anc Dis Prev Rem 2014, Vol 2(1): R1-001;and Delany et al., EMBO Mol Med. 2014 Jun;6(6):708-720.Specific examples include the Tat protein, gag p24 / p41 and nef for HIV / AIDs (Steinman, Proc (Bayl Univ Med Cent) 21, 3-8 (2008)); the circumsporozoite of Plasmodium falciparum for malaria (Steinman, Proc (Bayl Univ Med Cent) 21, 3-8 (2008)); the LcrV of Yersinia Pestis for pneumonia (Steinman, Proc (Bayl Univ Med Cent) 21, 3-8 (2008)); the LACK (Leishmania-activated C kinase) antigen of Leishmania major for leishmaniasis (Steinman, Proc (Bayl Univ Med Cent) 21, 3-8 (2008)); the streptococcus pneumoniae (Streptococcus pneumoniae) for leishmaniasis ... pneumoniae capsular serotype antigen for Salmonella pneumoniae (Reglinski et al., NPJ Vaccines 3, 53 (2018)); Vi capsular polysaccharide for Salmonella typhi (Li et al., NPJ Vaccines 3, 1 (2018)); ZIKV nonstructural 1 (NS1) protein for Zika virus (Brault et al., Sci Rep 7, 14769 (2017)); and major outer membrane protein (MOMP) for Chlamydia trachomatis serotype E, a cause of infectious blindness (ocular trachoma) (Pal et al., Infect Immun 73, 8153-8160 (2005)).

[0077] The methods and compositions can be used to treat a subject with an infection or to reduce the risk of or the severity of an acquired infection.

[0078] Pharmaceutical Compositions and Methods of Administration The methods described herein can involve the use of a pharmaceutical composition that comprises an anti-FcγRIII antibody as an active ingredient.

[0079] Pharmaceutical compositions typically include a pharma- ceutically acceptable carrier. As used herein, the term "pharma- ceutically acceptable carrier" includes saline, solvent, dispersion medium, coating, antimicrobial and antifungal agents, isotonic agents, etc., compatible with pharmaceutical administration. Supplementary active compounds may be introduced into the composition, for example, GMCSF may be included in FcgRIII antibody-antigen conjugate immunization. The composition may include an adjuvant to increase T cell responses. For example, nanoparticles that enhance T cell responses (e.g., as described in Stano et al., Vaccine (2012) 30:7541-6 and Swaminathan et al., Vaccine (2016) 34:110-9) may be included. See also Panagioti et al., Front. Immunol., 16 February 2018; doi.org / 10.3389 / fimmu.2018.00276. Alternatively or additionally, adjuvants including poly-ICLC (carboxymethylcellulose, polyinosinic-polycytidylic acid, and poly-L-lysine double-stranded RNA), imiquimod, CpG oligodeoxynucleotides and formulations (IC31, QB10), AS04 (aluminum salts formulated with 3-O-desacyl-4'-monophosphoryl lipid A (MPL), AS01 (MPL and saponin QS-21), and / or MPLA may be used. See, e.g., Coffman et al., Immunity. 2010 Oct 29; 33(4): 492-503;Martins et al., EBioMedicine 3:67-78, 2016;Del Giudice, Seminars in Immunology, 2018, See doi.org / 10.1016 / j.smim.2018.05.001; and Aurisicchio et al., Journal of Experimental & Clinical Cancer Research 2018; 37:86.

[0080] A pharmaceutical composition is typically formulated to be compatible with its intended route of administration, examples of which include parenteral, e.g., intravenous, administration.

[0081] Methods for formulating suitable pharmaceutical compositions are known in the art, see, for example, Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions used for parenteral application may contain the following components: water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for adjusting isotonicity such as sodium chloride or dextrose. pH may be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0082] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents, such as sugars, polyalcohols such as mannitol and sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable composition can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin, in the composition.

[0083] Sterile injectable solution can be prepared by incorporating the active compound in the required amount in a suitable solvent with one or combination of the above-listed components as required, and then sterilizing by filtration.Generally, dispersion is prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and other components required from the above-listed ones.For the preparation of sterile powder for sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying, which obtains the powder of active ingredient and any additional desired components from the solution that has been previously sterilized and filtered.

[0084] In some embodiments, the therapeutic compound is prepared with a carrier that will protect the therapeutic compound against rapid elimination from the body, for example, a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polybutyric acid may be used. Such formulations may be prepared using standard techniques or obtained commercially, for example, from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (containing liposomes targeted to selected cells with monoclonal antibodies against cellular antigens) may also be used as pharma- ceutically acceptable carriers. These may be prepared according to methods known in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0085] The pharmaceutical compositions may be included in a container, pack, or dispenser together with instructions for administration. EXAMPLES

[0086] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0087] material and method The following materials and methods were used in the examples below.

[0088] mouse All mice were on a C57Bl / 6 background and included the following: wild type mice, FcR γ - / - (Takai et al., Cell 76, 519-529 (1994)), which expresses human FcγRs - / - Mouse (FcγRIIA / γ - / - , FcγRIIIB / γ - / - and FcγRIIA+FcγRIIIB / γ- / - )(Tsuboi et al., Immunity 28, 833-846 (2008)), β2m - / - (Koller et al., Science 248, 1227-1230 (1990)) (The Jackson Laboratory number 002087), MyD88 / TRIF - / - (Yamamoto et al., Science 301, 640-643 (2003)), "catch-up" mice expressing tdTomato under the Ly6G promoter (Hasenberg et al., Nat Methods 12, 445-452 (2015)), OT-I (expressing a transgenic T cell receptor recognizing ovalbumin residues 257-264 (SIINFEKL; SEQ ID NO:1)) in the context of H2Kb (The Jackson Laboratory, no. 003831), OT-I / beta-actin-GFP (obtained by crossing OT-I mice with beta-actin-GFP mice) and beta-actin-RFP (The Jackson Laboratory, no. 005884). Animals were maintained in a special pathogen-free facility. All in vivo experiments were performed in age- and sex-matched animals. The Brigham and Women's Hospital Animal Care and Use Committee approved all procedures in this study.

[0089] Human volunteers and SLE patients and sera For the study on SLE patients, peripheral blood samples were obtained from healthy controls and patients with SLE who fulfilled the 1997 ACR classification criteria. The study was approved by the IRB of the Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran (IRE-2297), and all participants signed informed consent. Healthy volunteers with no family history of autoimmune diseases were recruited as controls. The clinical and demographic characteristics of the study participants are summarized in Table 1. Human SLE serum samples were obtained from the Lupus Clinic of the Beth Israel Deaconess Medical Center and Instituto Nacional de Ciencias Medicas y de la Nutricion Salvador Zubiran. For studies on normal human neutrophils, and for collection of normal serum, blood samples were obtained from healthy volunteers in accordance with protocols for the protection of human subjects approved by the Brigham and Women's Hospital Institutional Review Board (P001694 / PHS). All volunteer controls gave written informed consent.

[0090] [Table 2] Abbreviations: SLE, systemic lupus erythematosus; HC, healthy controls; SEM, standard error of the mean; SLEDAI, systemic lupus erythematosus disease activity index; dsDNA, double-stranded DNA antibodies; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein.

[0091] reagent Mouse GM-CSF (no. 315-03), human GM-CSF (no. 300-03), mouse G-CSF (no. AF-250-05) were from Peprotech. BSA (no. A-7970), OVA (no. A5503), anti-BSA rabbit antibody (no. B7276) and anti-OVA rabbit antibody (no. C6534) were from Sigma Aldrich. FITC-OVA (no. O23020) was from Thermofisher. NIP-OVA (11 NIPs per OVA) and anti-NIP human IgG1 (chimeric antibody with mouse lambda light chain and human heavy chain) were kindly provided by Richard Blumberg (Brigham and Women's Hosp, Boston). Anti-FcγRIIIB (3G8) (Biolegend) conjugated to FITC-OVA (no. O23020, Thermofisher) at a ratio of 2 FITC-OVA:1 IgG was produced by Biolegend as described below.

[0092] Accutase cell detachment solution (number 07920) was from STEMCELL Technologies. FACS antibodies used, including catalog number, clone and dilution, are listed in Tables 2.1 and 2.2. For depletion experiments, anti-mouse CD4 (GK1.5, number BE0003), anti-mouse CD8 (2.43, number BE0061) and rat IgG2b isotype control (LTF-2, number BE0090) were obtained from BioCell.

[0093] [Table 3-1]

[0094] [Table 3-2]

[0095] [Table 4]

[0096] [Table 5]

[0097] [Table 6]

[0098] Conjugation Method Used by Biolegend FITC-OVA conjugation to 3G8 is achieved by click chemistry. Lyophilized powder of FITC-OVA (Thermofisher, Cat. No. O23020) was dissolved in phosphate buffer (pH 7.0) and activated with an azide-containing reagent, Azido-PEG4-NHS Ester (Click Chemistry Tools). Azide-activated FITC-OVA is purified by G-25 Sephadex resin. 3G8 antibody is activated by DTT and then activated by DBCO-PEG4-Maleimide (Click Chemistry Tools). DBCO-modified 3G8 antibody is incubated with azide-activated FITC-OVA at a molar ratio of 1:3 (antibody:dye). The conjugation is quenched with sodium azide and loaded onto a Superdex 200 column to separate free antibody from free FITC-OVA. Fractions containing 1:1 and 1:2 (Ab:FITC-OVA) were pooled for a final ratio of approximately 1.4-1.5.

[0099] Model IC Generation To generate insoluble immune complexes, a solution containing 2 mg / ml anti-OVA, anti-NIP-OVA or anti-BSA antibody was added to 0.2 mg / ml OVA, FITC-OVA, NIP-OVA or BSA or 0.1 mg / ml OVA, FITC-OVA, NIP-OVA or BSA in PBS in a 1:1 ratio and incubated overnight at 37° C. to generate ICs designated IC solution 1 and IC solution 2, respectively. Excess antibody in the mixture results in the appearance of a precipitate (insoluble immune complexes).

[0100] Serum, isolation of IgG and formation of immune complexes with RNP Peripheral blood was collected into BD Vacutainer™ Venous Blood Collection Tubes SST. The tubes were centrifuged at 2500 / g for 30 min. Serum was aliquoted and frozen at -80°C. To purify IgG, serum samples were incubated with Protein G high performance agarose beads (Genesee Scientific, no. 20-538). Antibodies were eluted with 0.1 M Glycine (pH 2.7) and samples were immediately neutralized with neutralization buffer (1 M Tris-HCl, pH 9). Samples were then dialyzed overnight against PBS and concentrated using a 100 kDa centrifugal filter (Millipore, UFC910024). To generate SLE-IgG / RNP immune complexes, a solution containing 2mg / ml of purified SLE IgG or normal human IgG was mixed with an equal volume of 0.3mg / ml Sm / RNP complex (No. SRC-3000, Immunovision) in PBS (Ca, Mg free) and incubated at 37C for 4 hours. 20ul / ml of immune complex solution was added to neutrophils to generate human or mouse dendritic cells. RNPs were used at 3ug / ml. SLE-IgG or normal human IgG alone were used at 20ug / ml.

[0101] Isolation, treatment and culture of bone marrow-derived murine neutrophils Neutrophil isolation: Mice were euthanized and femurs and tibias were flushed with ice-cold RPMI 1640 using a 25-gauge needle attached to a 20 cc syringe, and cell clumps were dispersed by passing the cell suspension through a 1 ml pipette tip several times. Harvested cells were filtered through a 70 um strainer into a 50 ml Falcon tube, pelleted, and diluted at 1 x 10 in PBS containing 2% FBS and 1 mM EDTA. 8 The cells were resuspended at 1000x cells / ml. Neutrophils were isolated by negative selection using EasySep Mouse Neutrophil Enrichment Kit 19762 (STEMCELL Technologies). Aliquots of isolated cells were taken to determine neutrophil count and viability by trypan blue dye exclusion. Viability was also determined by FACS using the fluorescent viability dye Fixable Viability Dye eFluor 780 (ThermoFisher). Purity was assessed by flow cytometry (see Figure 6).

[0102] Conditions for generating NDDCs in vitro: Isolated mouse neutrophils were resuspended in RPMI medium (RPMI with L-glutamine and 25 mM HEPES supplemented with 10% fetal bovine serum and penicillin / streptomycin (50 U / ml penicillin and 50 μg / ml streptomycin)). 2 × 10 cells per well were cultured. 6Cells were plated in 2 ml of RPMI medium in 6-well plates and incubated at 37° C. in 5% CO2. Additionally, neutrophils were cultured with one or more of the following: GM-CSF (10 ng / ml), SLE patient IgG or normal human IgG (20 ug / ml) with 10 ng / ml of GM-CSF, or with SLE patient serum or normal human serum (50 ul / ml) alone (without GM-CSF). For treatment with IC, the solution containing IC prepared as described in "Generation of model immune complexes" was mixed thoroughly before use, and cells were incubated with 10 μl / ml of IC solution 1 and 10 μl / ml of IC solution 2 (IC containing OVA, FITC-OVA, NIP-OVA or BSA). For treatment with antigen alone, soluble OVA, FITC-OVA, NIP-OVA or BSA was used at 1.5 μg / ml, an amount equivalent to that contained in their respective IC. For antibody treatment alone, cells were incubated with 20ug / ml anti-OVA, anti-BSA or anti-NIP-OVA. Neutrophils were incubated in medium in Falcon tubes at 37C with 5% CO2 with gentle rocking. Cells were pelleted at 300g, washed with PBS and cultured in RPMI medium supplemented with 10ng / ml GM-CSF. After 3 days of culture, non-adherent and adherent cells were harvested using Accutase (STEMCELL Technologies), washed with PBS and resuspended in FACS buffer for flow cytometry analysis as described. For in vivo injections, only adherent cells harvested with Accutase were used.

[0103] Human neutrophil treatment and in vitro culture conditions Peripheral blood was collected from healthy human volunteers into tubes containing trisodium citrate, citric acid and dextrose (Vacutainer ACD solution A, BD). Autologous serum used for cell culture was obtained by collecting blood into BD Vacutainer™ Venous Blood Collection Tubes SST, centrifuging at 2500×g for 30 min and collecting the supernatant. To generate NDDCs in vitro, blood was supplemented with GM-CSF (10 ng / ml) for 30 min at 37° C., followed by addition of FITC-IgG isotype control or FcγRIIIB (3G8)-FITC-OVA conjugate for 2 h. Blood was then incubated with Hetasep (STEMCELL Technologies) according to the manufacturer's protocol to deplete red blood cells and enrich for white blood cells. The leukocyte-rich plasma layer was washed with PBS, and the remaining red blood cells were lysed using ammonium chloride solution on ice for 10 min, washed with PBS, and resuspended in RPMI containing L-glutamine and 25 mM HEPES, supplemented with 2.5% autologous serum and penicillin / streptomycin (50 U / ml penicillin and 50 μg / ml streptomycin). 6 Cells were plated in 6-well plates in 2 ml of medium supplemented with 10 ng / ml GM-CSF or 50 μl / ml serum from healthy donors or SLE patients. After 48 hours, cells were harvested using Accutase and dendritic cell differentiation was assessed by flow cytometry as described.

[0104] Flow cytometry Flow cytometry was performed on either a FACSCanto II or an LSRFortessa-12-color analyzer. Data were exported using FCS (Flow Cytometry Standard Format) 3.0 data files and analyzed using FlowJo (Mac version 10.5). Compensation controls were created for each fluorochrome. BD multicolor compensation beads were stained as compensation controls for FITC, Pe, Pe / Cy7, PerCP Cy5.5, APC, APC / Cy7, BV421 and BV605. In all experiments, cells were stained with Fixable Viability Dye eFluor 780 (ThermoFisher) to gate out dead cells. A forward gate and a side scatter gate were used to distinguish doublets and debris (FSC-A, FSC-H, SSC-A × SSC-H). Matched isotypes were used as controls and negative gating was based on the FMO (fluorescence minus one) strategy. Only viable cells were included in the study.

[0105] For surface staining, single cell suspensions in FACS buffer (PBS supplemented with 2% FCS and 2 mM EDTA) were incubated with BD Fc Blockade for mouse or TruStain FcX for human for 20 min at 4 C. Samples were incubated with the indicated fluorochrome-conjugated antibodies for 30 min at 4 C, washed with PBS and fixed with 1% paraformaldehyde.

[0106] Flow cytometric analysis of mouse bone marrow-derived neutrophils and NDDCs For phenotypic and functional analysis of bone marrow-derived neutrophils and NDDCs, antibodies against the following cell surface markers were used: CD11c, MHC-II, Ly6G, CD80, CD86 and CCR7 (see Table 2.1 for details). Within the lineage-negative surviving population (CD3, NK1.1 and CD19), CD11c + and MHC-II + Events were gated and analyzed for Ly6G expression. + MHC-II +and Ly6G + Populations were further analyzed for expression of the costimulatory molecules CD80, CD86 and the migratory marker CCR7. CD11c gate was based on FM controls, CD80, CD86 and CCR7 were based on isotype and FM controls. Numbers shown are expressed as % CD11c + , MHC-II + and Ly6G + The subset DC markers XCR1, CD103 and CD8a were detected by CD11c + , MHC-II + and Ly6G + The populations were further analyzed. For the analysis of uptake, FITC and Ly6G gates were set based on isotype controls. Anti-Ly6G was linked to the APC fluorochrome. In the OT-I and OT-II experiments, anti-CD3, anti-CD4 and anti-CD8 antibodies were used. CD3 + , CD4 + or CD8 + Singlets from 1000 to 10000 were gated for further analysis. Histograms are from one representative donor and show the population of CFSE+ events (solid histograms) and were set based on isotype controls. Clear histograms show the population of CFSE+ events (solid histograms) and were set based on isotype controls. + Staining on day 0 of the event is shown.

[0107] For intracellular staining, fixed cells were stained for surface markers and permeabilized with BD perm / wash buffer (BD Biosciences). Cells were then stained with the following antibodies: anti-Foxp3, anti-T-bet, and anti-Ki-67. Cells were washed again with permeabilization buffer and analyzed by flow cytometry.

[0108] Flow cytometric analysis of human blood neutrophils and dendritic cells To evaluate cellular markers in circulating leukocytes in whole blood of SLE patients and matched normal controls, 100 μL of blood was incubated with antibodies against the following surface markers: CD10, CD15, CD11c, MHC-II, CD80, CCR7 (see Table 2.2). Within the lineage-negative viable population (CD3, CD19 and CD56), CD11c + and MHC-II + Events were gated and analyzed for expression of CD10 and CD15. + MHC-II + , CD10 + The CD15+ and CD15+ populations were further analyzed for CD80 and CCR7 markers. DC subset markers Clec9a, XCR1 and CD141 were also assessed. Red blood cells were lysed using lysis buffer (BD Pharm Lyse) and samples were analyzed by flow cytometry in an LSR Fortessa hemocytometer (BD bioscience).

[0109] Analysis of FITC-OVA, FITC-OVA / anti-OVA and anti-FcγRIII-FITC-OVA uptake in vitro. For mouse neutrophil uptake experiments, cells from the indicated genotypes were cultured with FITC-OVA or FITC-OVA-anti-OVA model immune complexes generated as detailed above. At the indicated times, cells were harvested, stained with anti-Ly6G, and subjected to FAC analysis.

[0110] For human neutrophil uptake measurements, blood was incubated with FITC-IgG isotype control or anti-FcγRIIIB (3G8)-FITC-OVA antibody conjugate (8.5 to 12.5 μg / ml) for the indicated times at 37° C. with gentle agitation on a rocking table. Samples were then treated with ammonium chloride solution (STEMCELL Technologies #07800) for 10 min on ice, washed with PBS and subjected to FACS analysis with the indicated antibodies: anti-CD10, anti-CD15, anti-CD16 (REA-589), anti-CD16 (3G8) and anti-CD32 (IV3).

[0111] Immunization with NDDC or FcγRIIIB-FITC-OVA Neutrophils isolated from WT or MyD88 / TRIF- / mice were incubated with PBS (vehicle), OVA or OVA-anti-OVA IC for 2 h and cultured in RPMI medium (RPMI 1640 with L-glutamine and 25 mM HEPES, supplemented with 10% fetal bovine serum and penicillin / streptomycin (50 U / ml penicillin and 50 μg / ml streptomycin)) containing 10 ng / ml GM-CSF. After 3 days, non-adherent cells were gently removed and remaining adherent cells were detached with Accutase. Seven days before inoculation of B16F10-OVA cells, 1 × 10 6 10 cells were injected intravenously into mice anesthetized with isoflurane. For immunization studies, mice were injected iv with 100 μl of PBS solution containing 3G8(anti-FcγRIIIB)-FITC-OVA (30 μg) or FITC-OVA (12 μg) 6 h after intraperitoneal injection of 5 μg of GM-CSF. Four further injections of GM-CSF were given daily. Ten days after injection of 3G8-OVA conjugate or FITC-OVA, 2 × 10 cells were injected intravenously into mice anesthetized with isoflurane. 5 Mice were challenged with B16F10-OVA cells via subcutaneous injection.

[0112] Analysis of blood and spleen for detection of Ag uptake and neutrophil-DC markers. γ- / -, 3B γ- / - and 2A3B γ- / - mice were injected retro-orbitally with 30 μg of 3G8 (anti-FcγRIIIB)-FITC-OVA. Blood samples were taken at the indicated times, subjected to red blood cell lysis using ACK lysis buffer, washed with PBS and analyzed by FACS using antibodies against the following markers: Ly6G, CD11c, CD11b, MHC-II, CD80 and CCR7 (Figure 4A); Ly6C, CD115, CD16 (REA-589) and CD32 (Figure S3). Spleens were harvested 3 days later and processed as described in the organ harvest section.

[0113] In vitro and in vivo OTI and OTII proliferation assays: In vitro: T cells from ovalbumin-specific transgenic OT-I and OT-II mice were isolated from spleens and lymph nodes by positive selection. For enrichment of CD8+ T cells, microbeads (Miltenyi) conjugated to anti-mouse CD8a (Ly-2) or CD4 + For T cell enrichment, cells were enriched using microbeads (Miltenyi) conjugated to anti-mouse CD4 (L3T4). The final preparation contained 85-90% CD8 + or CD4 + T cells were included. 0.5 × 10 6 Bone marrow neutrophils were loaded with OVA, OVA-IC or vehicle control as previously described, seeded in 96-well microplates in RPMI medium (10% FCS) supplemented with GM-CSF, and cultured for 3 days to generate NDDCs. As a positive control, NDDCs were pretreated with 1 μg / ml OVA 24 h prior to the addition of T cells. 257~264 The pulse stimulation was performed with .

[0114] Cells were labeled with CFSE and proliferation was assessed. For this, OT-I and OT-II cells were cultured in PBS (10 7 NDDCs were resuspended in 100 mM NaCl (cells / ml) and incubated with 5 μM CellTrace Violet for 20 min at 37°C. The reaction was stopped with 10% FCS, cells were washed twice with cold PBS and resuspended in RPMI supplemented with 10% FCS, L-glutamine and pen / strep. NDDCs were cultured at 2 × 10 5 After 3 days of culture with OT-I or 5 days of culture with OT-II, cells were washed with PBS and stained for analysis by flow cytometry.

[0115] In vivo: Enriched OT-I and OT-II cells were labeled with CellTrace Violet dye as described above for the in vitro assay. Cells were resuspended in PBS and transferred (3×10) to recipient mice that received an iv injection of 30 ug of FcγRIIIB-FITC-OVA conjugate on day −10. 6 Four days after priming, mice were euthanized and cells from the spleen were harvested and stained for flow cytometry analysis.

[0116] CTL assay On day -7, recipient mice were injected with 30ug FcγRIIIB-FITC-OVA. On day 0, spleens were harvested from WT naïve mice and half of the cells were resuspended in 1μg / ml OVA ("unpulsed") in RPMI supplemented with 10% FCS. 257~264 Target spleen cells were generated by incubating the spleen cells with half of the spleen free of CellTrace Violet (SIINFEKL Analytical Biotechnology Services, Boston) for 1 h at 37 C. Cells were washed and resuspended in PBS, and pulsed and unpulsed cells were labeled with 6 μM CellTrace Violet (Thermofisher, C34557) (pulsed) or 1 μM CellTrace Violet (unpulsed), respectively, for 20 min at 37 C. Cells were washed and resuspended in PBS, and 1 × 10 6 1 x 10 pulse-stimulated cells and 6 Unpulsed cells were injected iv into the indicated recipient mice. 16 hours later, spleens from the recipient mice were harvested and assessed for loss of pulsed targets by flow cytometry. Cell-specific lysis was calculated using the following equation: 6 The actual values ​​were determined as follows: ratio = irrelevant percentage:epitope-specific percentage (unpulsed peak:pulsed peak), percent specific lysis = [1-(untransfected control ratio / experimental ratio)] x 100.

[0117] B16F10-OVA tumor challenge, organ harvest and flow cytometric analysis B16F10 cells expressing soluble OVA were cultured in vitro in DMEM / high glucose supplemented with 10% fetal bovine serum and maintained at subconfluent density. Mice were anesthetized by intraperitoneal injection of a ketamine / xylazine cocktail (90 mg / kg ketamine, 10 mg / kg xylazine), shaved, and cultured at 2 × 10 cells in 100 μl of HBSS. 5 Tumor cells were injected subcutaneously in the flank. Tumors were measured (longest and shortest diameters) with calipers in any one of the groups where they were palpable, once every 1-2 days, and tumor volumes were calculated based on the elliptical equation (1 / 2×D×d), where D and d are the longest and shortest diameters, respectively. 2 ) was used to calculate the mean sigma-based ...

[0118] Briefly, on ice, all excess fat was removed. Organs were gently dissociated in FACS buffer (PBS supplemented with 2% FCS and 2 mM EDTA) by shearing the tissue on a 70 μm nylon cell strainer (FisherBrand) using a 3 ml syringe plunger. Cells from draining and non-draining lymph nodes and tumors were washed with PBS and stained with antibodies for analysis by flow cytometry. Cells from spleens were subjected to red blood cell lysis using ACK lysis buffer solution (Lonza Cat 10-548E) for 2 min at room temperature, washed once with PBS, and resuspended in FACS buffer. Blood was collected from the retroorbital plexus of mice anesthetized with isoflurane using microhematocrit capillary tubes into EDTA tubes (5 mM final). Samples were subjected to red blood cell lysis using ACK lysis buffer for 10 min at room temperature, washed with PBS, and resuspended in FACS buffer. By flow cytometry analysis, cells were analyzed for expression of CD4, CD8, CD44, CD62L, HLA-DR tet (H-2 Kb OVA Tetramer (OVA257-264)), CD69, CD25, PD-1, Foxp3, Ki-67, and T-bet.

[0119] Delayed-type hypersensitivity (DTH) response: Neutrophils given OVA-IC or OVA were cultured in the presence of GM-CSF. After 3 days, non-adherent cells were discarded and adherent cells were harvested using Accutase and cultured at 1 × 10 6NNDCs were injected iv into wild-type recipient mice. Seven days after immunization, 50 μg of soluble OVA in 100 μl of PBS was injected into the right hind footpad, and an equal volume of PBS was injected into the left hind footpad to serve as control. Paw swelling was measured using a digital caliper on day 0 before OVA injection and 24 hours after OVA challenge. The magnitude of the DTH response was calculated as the thickness before and after OVA challenge. For histological analysis of the DTH response, paws were harvested, fixed in neutrally buffered 10% formalin solution (Sigma, HT-50-14-120 ml), and processed for H&E staining using standard histopathology methods. Inflammation scores were determined in a blinded manner.

[0120] Anti-GBM nephrotoxic glomerulonephritis Twelve-week-old mice were pre-immunized subcutaneously with 0.05 mg of rabbit IgG (Thermofisher #31235) in incomplete Freund's adjuvant (Thermo) and nonviable dried Mycobacterium tuberculosis H37Ra (Difco, Michigan) on day -3 and given an intravenous injection of 100 μL of nephrotoxic serum (NTS) on day 0. Control animals received an injection of PBS on day -3 and an intravenous injection of PBS on day 0. Spot urine samples were collected at the indicated time points and mice were euthanized on day 14 for histological and FACS analysis. Kidneys were harvested using Collagenase Type II / Hyaluronidase (StemCell Technologies) and draining and non-draining lymph nodes and spleens were harvested as described above for the B16F10 tumor model. Td-tomato+ cells were gated and analyzed for expression of CD11c and MHC-II, and positive cells were assessed for expression of CD80 and CD86. Subset DC marker analysis included CD8a, XCR1, and CD103.

[0121] Intravital microscopy of popliteal lymph nodes Approximately 5 × 10 cells were generated using OVA-IC as described for the DTH model. 5NDDCs were resuspended in 25 μl of PBS and injected into the right hind footpad of wild-type C57Bl / 6 mice on day 0. (10–10.5) × 10 in 200 μl of PBS 6 OT-1 β-actin-GFP cells were injected i.v. 30 min prior to NDDC injection. Three days after NDDC injection, mice were anesthetized with an intraperitoneal injection of a mixture of ketamine (50 mg / kg) and xylazine (10 mg / kg). The carotid artery of anesthetized mice was cannulated to allow intravenous delivery of Qtracker 655 Vascular dye and further anesthetics if required. The right popliteal lymph node was prepared by microsurgery and positioned on a custom-made microscope stage for intravital microscopy. During microsurgery, care was taken to preserve blood vessels and afferent lymphatics. The exposed lymph node was submerged in normal saline and covered with a glass slide with a thermocouple placed next to the lymph node to monitor the local temperature, which was maintained at 37°C. Two-photon intravital imaging was performed using an upright microscope (Prairie Technologies) and a water-immersion 20× objective (numerical aperture of 0.95). A MaiTai Ti:sapphire laser (Spectra-Physics) was tuned to 870 nm and 900 nm for two-photon excitation and second harmonic generation. Images of xy sections (512 × 512 pixels) were acquired every 1.5–2 s for 5 min (analysis) or 10 min (video) with electronic zoom varying from 1× to 3×. Radioactive light and second harmonic signals were directed through 450 / 80-nm, 525 / 50-nm and 630 / 120-nm bandpass filters and detected on a non-descanned detector. Files were saved as multiple TIF images and imported into FIJI (Schindelin et al., Nat Methods 9, 676-682 (2012)) for analysis or Imaris software (Bitplane) for export as videos.

[0122] statistics Statistical analysis was performed using Graphpad prism 8 (LaJolla, CA), STATA 13 (StataCorp. 2013. College Station, TX) and JMP10 software (SAS Institute, Inc, USA). All data included in this study are expressed as mean ± SEM. One-way ANOVA with Dunnett's multiple comparisons was used for analysis of groups. When comparing only two groups, a two-tailed Student's t test with Bonferroni correction was used. Statistical significance of differences for SLE patients and SLEDAI scores was determined by non-parametric tests using Mann-Whitney analysis. Correlations were analyzed using the Spearman test. * P < 0.05 and ** P<0.005 was considered significant.

[0123] In the statistical analysis of tumor growth in Figure 4D, the change in volume over time from days 7 to 14 was assessed in each animal, controlling for repeated measurements on the same animal by use of a mixed-effects regression model with group and random subject effects. In this model, separate intercepts and slopes were considered for animals in each of the four groups. An additional model considered the possible nonlinear relationship of volume over time in each animal by including a quadratic effect in time. In the evaluation, this effect was centered on the mean time of 12.5 days. Models were fitted using maximum likelihood, and likelihood ratio tests were used to test whether the slopes differed across groups and whether the inclusion of the quadratic term improved model fit. Models were fitted using the Mixed routine in STATA version 13. Models adding quadratic terms were not significantly better than models assuming linearity over time, with separate slopes for each group.

[0124] [Example 1] Fc.GAMMA.RIIIB-targeted antigen uptake converts neutrophils into immunogenic dendritic-like cells As shown herein, purified mouse bone marrow-derived neutrophils incubated with immune complexes (ICs) in the presence of GM-CSF (Figure 5A) exhibited a significantly higher frequency of DC-like phenotypes (CD11c, CD11p, CD11g, CD11p-CD11p-CD11p-CD11p) than neutrophils cultured with GM-CSF alone (Figures 1A and 5B), while retaining expression of the neutrophil marker Ly6G. + ICs also enhanced maturation, which was shown to mediate the expression of costimulatory molecules (CD80, CD86) and CCR7 (required for optimal T cell responses and migration of DCs to secondary lymphoid organs, respectively). 16 ) (Figure 1A). These cells are specialized in cross-presenting exogenous antigens to CD8 T cells. 2 Surface markers of the so-called DC1 subset, XCR1, CD103 and CD8α, were detected in DCs generated in the presence of GM-CSF alone and were further enhanced by ICs (Figure 5C). Siglec-A, a marker of plasmacytoid DCs, was not observed in any of the groups (Figure 5C). Mouse neutrophils were cultured in mice with systemic lupus erythematosus (SLE) (Figure 1B, Figure 5D), an autoimmune disease with abundant circulating ICs. 17 Acquisition of DC-like characteristics was also enhanced when incubated with ICs present in serum from patients with lupus, and ICs generated by IgG and RNP isolated from serum of anti-ribonucleoprotein (RNP) seropositive SLE patients. No differentiation was observed with normal serum or IgG plus RNP isolated from anti-RNP negative normal individuals (Figure 1B, Figure 5E). Consistent with these findings, neutrophil-derived DCs (NDDCs) expressing CD11c, MHC-II and neutrophil markers CD10 and CD15 were increased in the blood of lupus patients compared to normal volunteers. The frequency of NDDCs that expressed the costimulatory molecules CD80, CCR7 and the DC subset marker Clec9A, but not CD141, correlated with the lupus clinical score (Figure 1C-D, Figure 5F). Anti-glomerular basal membrane (anti-GBM)-mediated glomerulonephritis, a surrogate for lupus nephritis 13 Neutrophil reporter mice infected with HIV (Figure 1E) 18NDDC was increased in kidney tissue, draining lymph nodes, and spleen compared to naive animals.

[0125] FcR γ-chain deficient mice (γ - / - )-derived neutrophils failed to differentiate in response to SLE serum, demonstrating the requirement for activating FcγR in this process (Figure 1F). Notably, differentiation into NDDCs was mediated by selective expression of human FcγRIIA (FcγRIIA / γ - / - ) or FcγRIIIB (FcγRIIIB / γ - / - ) expressing γ - / - mouse 13 This was observed in neutrophils isolated from mice (Figure 1F), suggesting that either of the two human activating FcγRs supports the conversion of neutrophils into DCs. Surprisingly, NDDCs remained viable for at least 9 days in culture (Figure 1F). - / - Mouse (FcγRIIIB / γ - / - Anti-FcγRIIIB-OVA treatment of neutrophils isolated from mice converted them into NDDCs. - / - Gamma expressing mouse or human FcγRIIA - / - Mouse (FcγRIIA / γ - / - Similar treated neutrophils from mice (Fig. 1G) did not show transformation. Adjuvants play an important role in DC maturation. 2 Sera from SLE patients contain abundant autoantibodies complexed with Toll-like receptor (TLR) ligands, including RNP and DNA. 17 , canonical and non-canonical TLR signaling 19 WT lacking Myd88 / TRIF - / - Neutrophils from mice showed comparable NDDC differentiation after incubation with SLE ICs ( Fig. 5G ).

[0126] Classical DNA activation of naive CD8 T cells requires a fraction of the concentration of antigen in IC compared to soluble antigen 2、20、21This correlates with trafficking of antigens to degradative lysosomes, distinct from early or late endosomes, rather than the amount of antigen internalized. 2、22、23 Neutrophil uptake of OVA in IC was enhanced 2-fold compared to soluble OVA after 2 h of incubation, and this was dependent on FcγR (Fig. 2A). NDDCs derived from neutrophils fed with OVA-IC showed a strong ability to cross-prime OVA-specific CD8 T cells, whereas NDDCs derived from neutrophils fed with soluble OVA failed to do so (Fig. 2B). Neutrophils from mice lacking β2 microglobulin, which requires MHCI expression on the surface, were unable to activate CD8 T cells (Fig. 6). NDDCs derived from neutrophils fed with OVA-IC also strongly activated naive CD4 T cells, and these were more effective than NDDCs derived from neutrophils fed with soluble OVA (Fig. 2C). γ - / - DCs differentiated from neutrophils lacked OVA-IC-dependent activation of CD4 and CD8 T cells, whereas those directly loaded with OVA peptide showed normal CD8 T cell proliferation. - / - Neutrophil-derived NDDCs supported the activation of both T cell subsets (Figure 2B-C). T cell proliferation was significantly enhanced by OVA-IC-loaded WT and MyD88 / TRIF - / - Similar to that in DCs of neutrophil origin, TLRs were not required (Fig. 2D).

[0127] Both capture of blood-borne antigens by splenic DCs and migration of antigen-loaded DCs from tissues to lymph nodes are important for the initiation of T cell-mediated immune responses 16 As described in melanoma patients who were injected intravenously with autologous DCs. 24 Intravenous injection of NDDCs generated ex vivo from OVA-IC resulted in the accumulation of neutrophils in the spleen and lungs (Figure 3A). Furthermore, local injection of NDDCs generated with OVA-IC into the footpad of mice resulted in their accumulation in the popliteal draining LNs, where they exhibited active dendritic outgrowth and CD8+ The NDDCs generated by OVA-IC showed prolonged contact with T cells (Figure 3B). Thus, NDDCs generated by OVA-IC demonstrated the ability to accumulate in secondary lymphoid organs where they activated T cell function. Indeed, NDDCs generated by OVA-IC induced a CD4 Th1-driven recall response to antigen and a subsequent neutrophil-rich inflammatory response. 26 A delayed-type hypersensitivity (DTH) model that has been reported to rely on 25 Namely, 7 days after receiving soluble OVA subcutaneously in the footpad, mice receiving OVA-IC-generated NDDCs iv developed tissue swelling and infiltration of immune cells. Notably, this exceeded the response observed in mice given OVA-generated NDDCs (Figure 3C). OVA-IC-generated NDDCs were also effective in eliciting CD8 T cell responses, which are important for tumor immunity. Following subcutaneous implantation of OVA-expressing B16F10 melanoma cells, 27 Mice prophylactically immunized with NDDCs generated by OVA developed large tumors with slightly reduced volume compared to mice not immunized with OVA (Fig. 3D). In contrast, tumor growth was completely inhibited in mice fed NDDCs generated by OVA-IC (Fig. 3D). The latter antitumor response was confirmed by the expression of OVA-specific CD8 + The tumor growth in immunized mice was associated with the accumulation of CD8 and not CD4 T cells, as assessed by immunodepletion antibodies (Fig. 3D).

[0128] Antibody conjugated to antigens targeting classical endocytic receptors in monocyte-derived DCs can induce antigen-specific T cell responses and immunity in mice when combined with TLRs 1We hypothesized that by targeting FcγRIIIB in blood neutrophils, DC differentiation signals would be delivered specifically to neutrophils, both to generate a large number of antigen-presenting cells and to deliver antigens via FcγR, which promotes antigen cross-presentation. FITC-OVA conjugated to FcγRIIIB antibody, 3G8 (3G8-OVA), a murine IgG1 with a predicted half-life of 6 to 8 days, was used. 28 A single intravenous injection of 3G8-OVA resulted in selective labeling of neutrophils in the blood (Figure 4A). Analysis of blood within 3.5 h of 3G8-OVA injection revealed neutrophil conjugates and internalization of FcγRIIIB, as assessed by evaluating surface levels of the receptor, while FcγRIIA remained on the surface (Figure 7A). Within 24 h, Ly6G expressed CD11c and MHCII, costimulatory markers, and CCR7. + Cells were detected in the blood, whereas 3G8-OVA labeling and DC differentiation markers were not detected in similarly treated γ - / - In humanized transgenic mice, FcγRIIIB and FcγRIIA expressed downstream of the MRP8 promoter were not observed in the 3G8-OVA conjugates (Figure 4A). The 3G8-OVA conjugate also did not produce NDDCs in the spleen (assessed 72 hours after iv injection) (Figure 4A). In humanized transgenic mice, FcγRIIIB and FcγRIIA expressed downstream of the MRP8 promoter were not observed in the 3G8-OVA conjugates (Figure 4A). + Also CD115 + Monocytes 13 However, 3G8-OVA-positive CD11c + More than 98% of MHCII+ cells expressed Ly6G, suggesting that they were primarily NDDCs (Fig. 7C). These results demonstrated that splenic NDDCs promoted OVA-specific CD8 T cell proliferation (Fig. 4B) and antigen-specific target cell killing in vivo. 29 (Figure 4C). We next tested the ability of 3G8-OVA to promote antitumor immunity. Pre-immunization with 3G8-OVA alone had no effect on the growth of subcutaneously implanted B16F10-OVA melanoma cells, but it did generate OVA-specific CD8 T cells in the draining lymph nodes and spleen. The antitumorogenic potential of ex vivo generated OVA-IC-loaded neutrophils cultured with GM-CSF (Figure 3D), and the success of a GM-CSF-based vaccine, were demonstrated. 30 With this in mind, we treated mice iv with recombinant GM-CSF for five consecutive days starting on the day of immunization with 3G8-OVA. We found that GM-CSF induced B16F10-OVA tumor responses to the 3G8-OVA conjugate (Figure 4D, Figure 7D), which was consistent with wild-type mice immunized with OVA alone or γ-cells immunized with 3G8-OVA. - / - Compared to mice, 3G8-OVA correlated with the generation of OVA-specific CD8 effector T cells, an increased ratio of CD8 to Treg in the spleen (Figure 4E, Figure 7E) and enhanced CD8 T cell infiltration in tumors. These findings with 3G8-OVA may have relevance in humans, as peripheral blood human neutrophils treated with this conjugate rapidly internalized OVA and targeted FcγRIIIB. FcγRIIA internalization was also observed over time (Figure 4A) and may be important for optimal generation of NDDCs as discussed in the following section. This differs from the lack of FcγRIIA internalization in mice 3 hours after 3G8-OVA administration (Figure 7A). FcγRIIA internalization in vivo may occur over a longer period of time. Within 2 days of culture, neutrophils given 3G8-OVA had significantly higher expression of CD11c, MHCII, costimulatory molecules and CCR7 compared to neutrophils treated with isotype control (Figure 4G). Human DC subsets with antigen cross-presenting capacity express XCR1, CD141 and Clec9A 31 XCR1 was increased in neutrophils given 3G8-OVA versus isotype control, whereas CD141 expression was higher but comparable between the two groups, and the slight increase in Clec9A was not statistically significant (Figure 4H).

[0129] [Example 2] Anti-FcγRIIIB antibodies alone convert neutrophils into immunogenic dendritic-like cells Malignancies of the innate immune system, such as in myeloid neoplasms, which are cancers of innate immune cells that carry driver mutations and subsequently express neoantigens. To determine whether neutrophils from these patients can be converted into NDDCs that present antigens to autologous T cells, we treated neutrophils isolated from patients with two different types of myeloid neoplasms with anti-FcgRIIIB-OVA conjugate or isotype control. Of note, OVA is inappropriate in this experiment; this conjugate was used because sufficient amounts of antibody alone were not immediately available. Neutrophils were isolated from normal human volunteers (normal), patient 1 (diagnosed with acute myeloid leukemia) or patient 2 (diagnosed with myelodysplastic syndrome, driving mutations as determined by rapid heme panel), and autologous peripheral blood mononuclear cells (PBMCs) were frozen. Isolated neutrophils were incubated with medium alone, anti-FcγRIIIB-OVA conjugate (vaccine) or non-FcγRIIIB binding antibody isotype control (isotype) in the presence of GM-CSF. Two days later, PBMCs were thawed, T cells were isolated and autologous T cells were incubated with cognate cultures of neutrophils / NDDCs at a ratio of 4:1. Cocultures were performed in triplicate. IFNγ ELISPOT assays were performed 18 hours after coculture to detect IFN production by activated T cells. Each spot represented an individual cell secreting cytokine. NDDCs were generated in these patients as assessed by expression of DC markers as outlined in Figure 1. NDDCs were incubated with autologous T cells from the same patients (predicted to have T cell reactivity against tumor neo-antigens despite their low abundance). Successful antigen presentation to T cells by NDDCs leads to T cell activation and subsequent production of IFNγ as assessed in an Elispot assay, which allows quantification of the frequency of individual cells secreting cytokines. We observed significant production of IFNγ in samples with anti-FcgRIIIB-OVA-generated nDCs and samples with T cells in two patients (Figures 9C-9D).No IFNγ was observed in neutrophils incubated with nDCs or T cells alone or with an isotype antibody control. Furthermore, no responses were observed in neutrophils from two normal human volunteers treated with anti-FcgRIIIB (Figures 9C-9D) and from two additional patients screened.

[0130] References

[0131] [Table 7-1]

[0132] [Table 7-2]

[0133] [Table 7-3]

[0134] [Table 7-4]

[0135] [Table 7-5]

[0136] Other embodiments While the present invention has been described in conjunction with its detailed description, it should be understood that the description is intended to be illustrative and not limiting of the scope of the invention as defined in the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. The inventions described in the original claims of this application are listed below. [Invention 1] 1. A method for generating a population of dendritic cells (DCs), comprising: providing a population of neutrophils comprising neutrophils expressing one or both of FcγRIIA and / or FcγRIIIB; contacting the neutrophils in culture with granulocyte-macrophage colony-stimulating factor (GM-CSF) and an immune complex (IC) comprising an antigen and an antibody, wherein the antibody comprises an antigen-binding portion that binds to the antigen and an Fc region that binds to FcγRIIA and / or FcγRIIIB; maintaining said population of neutrophils in culture in the presence of GM-CSF and IC under conditions and for a time sufficient for the neutrophils to differentiate into DCs. The method comprising: [Invention 2] The method according to claim 1, wherein the antigen comprises a tag and an antibody binds to the tag portion of the antigen. [Invention 3] 3. The method according to claim 1 or 2, wherein the antigen is a tumor antigen. [Invention 4] 4. A method of treating a subject having cancer, comprising administering to said subject an effective amount of the cells produced by the method of invention 3. [Invention 5] 4. A cell produced by the method according to claim 3 for use in a method of treating cancer. [Invention 6] 3. The method according to claim 1 or 2, wherein the antigen is derived from a pathogen. [Invention 7] A method for treating a subject infected with a pathogen, comprising the step of administering to the subject an effective amount of the cells produced by the method of invention 6, wherein the antigen is derived from the pathogen with which the subject is infected. [Invention 8] 7. A cell produced by the method of invention 6 for use in a method of treating an infection by a pathogen, wherein the antigen is derived from the pathogen with which the subject is infected. [Invention 9] A conjugate comprising an antigen and an antibody comprising an antigen-binding domain that binds to FcγRIII, optionally a fusion protein or a chemical conjugate. [Invention 10] 10. The conjugate according to claim 9, wherein the antigen is a tumor antigen or an antigen derived from a pathogen. [Invention 11] 1. An in vitro method for generating a population of dendritic cells (DCs), comprising: providing a population of neutrophils comprising neutrophils expressing one or both of FcγRIIA and / or FcγRIIIB; contacting said population of neutrophils in culture with a conjugate according to invention 9 or 10 and granulocyte-macrophage colony-stimulating factor (GM-CSF); maintaining said population of neutrophils in culture in the presence of said conjugate and GM-CS under conditions and for a time sufficient for the neutrophils to differentiate into DCs. The method comprising: [Invention 12] 12. The method according to claim 11, wherein the antigen is a tumor antigen. [Invention 13] 13. A method for treating a subject having cancer, comprising the step of administering to said subject an effective amount of DCs generated by the method according to claim 12. [Invention 14] A population of dendritic cells generated by the method according to invention 12 for use in a method of treating cancer. [Invention 15] 12. The method of claim 11, wherein the antigen is derived from a pathogen. [Invention 16] A method for treating a subject infected with a pathogen, comprising the step of administering to the subject an effective amount of DCs generated by the method of invention 15, wherein the antigen is derived from the pathogen with which the subject is infected. [Invention 17] 16. A population of dendritic cells generated by the method of invention 15 for use in a method of treating an infection by a pathogen, wherein the antigen is derived from the pathogen with which the subject is infected. [Invention 18] A method for treating a subject, comprising the step of administering to the subject an effective amount of a conjugate according to invention 9 or 10 and granulocyte-macrophage colony-stimulating factor (GM-CSF). [Invention 19] 20. The method of claim 18, wherein the subject has cancer and the antigen is a tumor antigen. [Invention 20] 19. The method of claim 18, wherein the subject has an infection with a pathogen and the antigen is derived from the pathogen. [Invention 21] 1. A method for generating a population of dendritic cells (DCs), comprising: providing a population of neutrophils comprising neutrophils expressing one or both of FcγRIIA and / or FcγRIIIB; contacting the neutrophils in culture with an antibody comprising an antigen-binding portion that binds an antigen and an Fc region that binds FcγRIIA and / or FcγRIIB; maintaining said population of neutrophils in culture under conditions, and for a time sufficient for the neutrophils to differentiate into DCs. The method comprising: [Invention 22] 22. The method of claim 21, wherein said population of neutrophils is maintained in the presence of GM-CSF and / or IC. [Invention 23] 22. A population of dendritic cells generated by the method according to claim 21. [Invention 24] 22. A population of dendritic cells produced by the method according to invention 21 for use in a method of treating a subject, preferably a subject from which said population of neutrophils has been obtained. [Invention 25] 25. The population of dendritic cells for use according to invention 24, wherein the subject has a myeloid tumor or an infectious disease. [Invention 26] 25. The population of dendritic cells for use according to invention 24, wherein the infection is a bacterial, fungal or viral infection. [Invention 27] The method of invention 21, wherein the population of neutrophils comprising neutrophils expressing one or both of FcγRIIA and / or FcγRIIIB is obtained from a subject, and further comprising a step of administering differentiated dendritic cells to the subject from which the population of neutrophils was obtained. [Invention 28] 28. The method of claim 27, wherein the subject has a myeloid tumor or an infectious disease. [Invention 29] A method for treating a subject having a myeloid tumor, comprising administering to the subject an effective amount of an antibody comprising an antigen-binding domain that binds to FcγRIII. [Invention 30] 30. The method of claim 29, wherein the subject has a myeloid neoplasm selected from the group consisting of acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), or myeloproliferative neoplasm (MPN). [Invention 31] A method for treating a subject having an infectious disease, comprising administering to the subject an effective amount of an antibody comprising an antigen-binding domain that binds to FcγRIII. [Invention 32] 32. The method of claim 31, wherein the infection is a bacterial, fungal, or viral infection.

Claims

1. 1. A method for generating a population of dendritic cells, comprising: Providing a population of neutrophils comprising neutrophils expressing one or both of FcγRIIA and / or FcγRIIIB; contacting the neutrophils in culture with granulocyte-macrophage colony-stimulating factor (GM-CSF) and an immune complex (IC) comprising an antigen and an antibody, wherein the antibody comprises an antigen-binding portion that binds the antigen and an Fc region that binds FcγRIIA and / or FcγRIIIB; maintaining said population of neutrophils in culture in the presence of GM-CSF and IC under conditions and for a time sufficient for the neutrophils to differentiate into dendritic cells. The method comprising:

2. The method of claim 1 , wherein the antigen comprises a tag and an antibody binds to the tag portion of the antigen.

3. The method of claim 1 or 2, wherein the antigen is a tumor antigen.

4. 4. A pharmaceutical composition for treating a subject having cancer, comprising the dendritic cells produced by the method of claim 3.

5. 4. A dendritic cell produced by the method of claim 3 for use in a method of treating cancer.

6. The method of claim 1 or 2, wherein the antigen is derived from a pathogen.

7. 10. A pharmaceutical composition for treating a subject infected with a pathogen, comprising the dendritic cells produced by the method of claim 6, wherein the antigen is derived from the pathogen with which the subject is infected.

8. 10. A dendritic cell produced by the method of claim 6 for use in a method of treating an infection by a pathogen, wherein the antigen is derived from the pathogen with which a subject is infected.

9. 1. An in vitro method for generating a population of dendritic cells, comprising: Providing a population of neutrophils comprising neutrophils expressing one or both of FcγRIIA and / or FcγRIIIB; said population of neutrophils in culture, a conjugate which is an antibody comprising an antigen and an antigen-binding domain which binds to FcγRIIA and / or FcγRIIIB, said conjugate being a fusion protein or a chemical conjugate; and Granulocyte-macrophage colony-stimulating factor (GM-CSF) and contacting the maintaining said population of neutrophils in culture in the presence of said conjugate and GM-CSF under conditions and for a time sufficient for the neutrophils to differentiate into dendritic cells. The method comprising:

10. The method of claim 9 , wherein the antigen is a tumor antigen.

11. 11. A pharmaceutical composition for treating a subject having cancer, comprising dendritic cells produced by the method of claim 10.

12. A population of dendritic cells produced by the method of claim 10 for use in a method of treating cancer.

13. The method of claim 9 , wherein the antigen is derived from a pathogen.

14. 14. A pharmaceutical composition for treating a subject infected with a pathogen, comprising the dendritic cells produced by the method of claim 13, wherein the antigen is derived from the pathogen with which the subject is infected.

15. 14. A population of dendritic cells produced by the method of claim 13 for use in a method of treating an infection by a pathogen, wherein the antigen is derived from the pathogen with which a subject is infected.

16. 1. A method for generating a population of dendritic cells, comprising: Providing a population of neutrophils comprising neutrophils expressing one or both of FcγRIIA and / or FcγRIIIB; contacting the neutrophils in culture with an antibody comprising an antigen-binding portion that binds an antigen and an Fc region that binds FcγRIIA and / or FcγRIIB, and granulocyte-macrophage colony-stimulating factor (GM-CSF); maintaining said population of neutrophils in culture in the presence of GM-CSF and said antibody under conditions and for a time sufficient for the neutrophils to differentiate into dendritic cells. The method comprising:

17. A population of dendritic cells generated by the method of claim 16.

18. A population of dendritic cells produced by the method of claim 16 for use in a method of treating a subject from which said population of neutrophils was obtained.

19. The population of dendritic cells for use according to claim 18, wherein the subject has a myeloid tumor or an infectious disease.

20. 20. The population of dendritic cells for use according to claim 19, wherein the infection is a bacterial, fungal, or viral infection.

21. 1. A pharmaceutical composition for use in a method of treating a subject, comprising: The pharmaceutical composition comprises a population of dendritic cells generated by the method of claim 16, 17. The method of claim 16, wherein the population of neutrophils comprising neutrophils expressing one or both of FcγRIIA and / or FcγRIIIB is obtained from the subject; The method includes administering the population of dendritic cells to the subject. The pharmaceutical composition.

22. 22. The pharmaceutical composition of claim 21, wherein the subject has a myeloid tumor or an infection.

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