EP4611807A1 - Rééquilibrage du système immunitaire par déplétion de cellules souches hématopoïétiques orientées production de myéloïdes - Google Patents

Rééquilibrage du système immunitaire par déplétion de cellules souches hématopoïétiques orientées production de myéloïdes

Info

Publication number
EP4611807A1
EP4611807A1 EP23886580.2A EP23886580A EP4611807A1 EP 4611807 A1 EP4611807 A1 EP 4611807A1 EP 23886580 A EP23886580 A EP 23886580A EP 4611807 A1 EP4611807 A1 EP 4611807A1
Authority
EP
European Patent Office
Prior art keywords
cells
hsc
hscs
mice
antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23886580.2A
Other languages
German (de)
English (en)
Inventor
Jason B. ROSS
Lara MYERS
Joseph NOH
Kim J. HASENKRUG
Irving L. Weissman
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Leland Stanford Junior University
US Department of Health and Human Services
Original Assignee
Leland Stanford Junior University
US Department of Health and Human Services
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Leland Stanford Junior University, US Department of Health and Human Services filed Critical Leland Stanford Junior University
Publication of EP4611807A1 publication Critical patent/EP4611807A1/fr
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • HSC Hematopoietic stem cells
  • Aging is associated with a transition to mainly myeloid biased HSC.
  • myeloid biased HSC reduce the number of naive lymphoid cells in aged individuals, leading to poor T and B cell responses to new pathogens, including microbes such as SARS-CoV-2, influenza, HIV, etc. and vaccine responses.
  • myeloid biased HSC contribute to a chronic inflammatory milieu in the aged (known as inflammaging) that is associated with numerous age-related pathologies.
  • myeloid biased HSC can transform to cause human hematopoietic diseases such as MPN (myeloproliferative neoplasms), MDS (myelodysplastic syndrome), chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), and clonal hematopoiesis of indeterminate potential (CHIP).
  • MPN myeloproliferative neoplasms
  • MDS myelodysplastic syndrome
  • CML chronic myelogenous leukemia
  • AML acute myelogenous leukemia
  • CHIP clonal hematopoiesis of indeterminate potential
  • B cells and T cells Animals, including humans, respond well to the microbes in their local geography, first by eliciting an innate immune response predominated by cells of the myeloid lineage (such as macrophages, neutrophils and granulocytes), and secondly by eliciting a much more specific adaptive response by lymphocytes (B cells and T cells).
  • B cells and T cells expresses a distinct receptor capable of recognizing a specific antigen from a pathogen.
  • those cells with specificity expand into both effector cells to contain the pathogen, and into long-lived memory cells that can respond much faster and more potently if the pathogen is re-encountered.
  • HSC myeloid progenitors
  • CMP common myeloid progenitors
  • CLP common lymphocyte progenitors
  • SARS-CoV-2 novel pathogens
  • myeloid biased HSC are pro-inflammatory, producing or eliciting inflammatory cytokines such as TNF ⁇ , IL1, IL6, etc.
  • cytokines such as TNF ⁇ , IL1, IL6, etc.
  • unbalanced inflammatory responses in the elderly cause much more morbidity and mortality due to inflamed and fibrotic lungs.
  • the predominance of myeloid biased HSCs in the elderly is a two-edged sword in the battle with novel pathogens, resulting not only in a poor adaptive immune response, but also in a detrimental inflammatory response.
  • compositions and methods are provided for rebalancing the immune system of a mammalian subject, including without limitation an aged mammal, by selective depletion of myeloid-biased hematopoietic stem cells (my-HSC).
  • my-HSC myeloid-biased hematopoietic stem cells
  • bal-HSC balanced hematopoietic stem cells
  • the result of this selective depletion can be a relative enhancement of circulating na ⁇ ve lymphocyte populations, and decreased myeloid cell populations and exhausted T cell populations.
  • the rebalanced immune system has an improved capacity to respond to novel infections, including vaccinations, and has reduced inflammaging properties.
  • Conditions that can be treated with the methods include, for example, clonal hematopoiesis of indeterminate potential (CHIP), myeloproliferative neoplasms (MPN), myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), including pre-malignant AML, atherosclerosis, inflammatory and fibrotic conditions, pathogenic infections, e.g. influenza, Covid-19, etc., inadequate response to vaccination, prevention or treatment of liquid and solid cancers, immune recovery after cytotoxic agents, and the like.
  • CHIP indeterminate potential
  • MDN myeloproliferative neoplasms
  • MDS myelodysplastic syndrome
  • AML acute myeloid leukemia
  • pathogenic infections e.g. influenza, Covid-19, etc.
  • Human HSC can be phenotyped by their expression of cell surface markers, and on the basis of this expression that can be categorized as my-HSC or bal-HSC.
  • my-HSC or bal-HSC all human HSC are positive for expression of CD34, CD90, and CD117.
  • the disclosure herein identifies cell surface markers that are differentially expressed on human and mouse my-HSC relative to bal-HSC, which markers are used in the selective depletion of my-HSC.
  • the markers may be referred to herein as “my-HSC selective markers”.
  • the my-HSC selective markers comprise one or more of CD304, TIE2, ESAM, CD9, CD105, CD166, CD150 (Slamf1), CD61 (Itgb3), CD41 (Itga2b), CD62p, and NEO1.
  • the my-HSC selective markers comprise one or more of CD150 (Slamf1), CD61 (Itgb3), CD41 (Itga2b), CD62p, and NEO1.
  • Human my-HSC selective markers include, for example, CD150, NEO1 and CD62p.
  • the methods comprise contacting a population of cells, e.g. cells in bone marrow, comprising HSC with an effective dose of one or more agents that specifically bind to a my-HSC selective marker, which may be referred to as a my-HSC selective agent.
  • a cocktail of binding agents is used, which bind to a plurality of my-HSC selective markers.
  • the my-HSC selective marker is CD150.
  • the my-HSC selective marker is CD62p.
  • the my-HSC selective marker is NEO1.
  • methods of selective immunodepletion comprise administering an effective dose of an agent specific for CD117 in combination with the my-HSC selective agent(s).
  • methods of selective immunodepletion comprise administering an effective dose of an agent that blocks CD47 interaction with SIRP ⁇ , in combination with the my-HSC selective agent(s). In some embodiment, methods of selective immunodepletion comprise administering an effective dose of an agent specific for CD117, and an agent that blocks CD47 interaction with SIRP ⁇ , in combination with the my-HSC selective agent(s).
  • a cocktail of antibodies is administered, comprising an antibody specific for CD47, an antibody specific for a my-HSC marker, for example one or more of anti- CD150, anti-CD62p, anti-NEO1, and an antibody specific for CD117.
  • a cocktail of agents is administered, comprising an antibody specific for SIRP ⁇ , an antibody specific for a my-HSC marker, for example one or more of anti-CD150, anti-CD62p, anti- NEO1, and an antibody specific for CD117.
  • a cocktail of agent is administered, comprising a soluble SIRP ⁇ polypeptide, an antibody specific for a my-HSC marker, for example one or more of anti-CD150, anti-CD62p, anti-NEO1, and an antibody specific for CD117.
  • one or all of the agents is an antibody.
  • the antibody is a humanized monoclonal antibody.
  • An antibody may comprise an Fc region sequence.
  • a single dose of the antibody is administered in vivo.
  • the dose of antibody is delivered by intravenous infusion.
  • the effective dose of the antibody may be up to about 50 mg/kg, up to about 25 mg/kg, up to about 10 mg/kg; up to about 5 mg/kg; up to about 1 mg/kg; up to about 0.1 mg/kg.
  • an antibody dose is from about 0.1 mg/kg to about 25 mg/kg, from about 0.5 mg/kg to about 15 mg/kg, from about 1 to about 5 mg/kg.
  • the antibody is optionally conjugated to a cytotoxic agent. [013]
  • the subject being treated is an aged, or elderly, mammal.
  • the rate of aging is species specific, where a human may be aged at about 50 years; and a rodent at about 2 years.
  • a natural progressive decline in body systems starts in early adulthood, but it becomes most evident several decades later.
  • One arbitrary way to define elderly more precisely in humans is to say that it begins at conventional retirement age, around about 60, around about 65 years of age.
  • Another definition sets parameters for aging coincident with the loss of reproductive ability, which is around about age 45, more usually around about 50 in humans, but will, however, vary with the individual.
  • an individual diagnosed with CHIP, or a myelodysplastic condition e.g.
  • bal-HSC balanced hematopoietic stem cells
  • the method of selective depletion may provide for an enrichment of bal-HSC to my- HSC of at least 1.5-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 7.5-fold, at least 10-fold, at least 15-fold, or more.
  • the ratio of the number of lymphoid progenitors in bone marrow e.g.
  • common lymphoid progenitors to the number of myeloid progenitors, e.g. common myeloid progenitors, may be increased at least 1.5-fold, at least 2- fold, at least 3-fold, at least 5-fold, at least 7.5-fold, at least 10-fold, at least 15-fold, or more.
  • the number of circulating na ⁇ ve T cells relative to the total circulating lymphocyte population may be increased at least 2-fold, at least 3-fold, at least 5-fold, at least 7.5-fold, at least 10- fold, at least 15-fold, or more.
  • the level of circulating “age-associated B cells” (ABC), and/or exhausted T cells relative to the total circulating lymphocyte population may be decreased at least 1.5-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 7.5-fold, at least 10-fold, at least 15-fold, or more.
  • the basal circulating level of ‘inflammaging’ markers e.g. IL-1a, CXCL5, IL1RL1, IL-23, IL-1b, CXCL2, IL-31, IL-5, GM-CSF, may be decreased at least at least 1.5-fold, 2-fold, at least 3-fold, at least 5-fold, at least 7.5-fold, at least 10-fold, at least 15-fold, or more upon treatment with the methods of the invention.
  • the balance of immune cells e.g. the relative number of one or more of na ⁇ ve T cells, exhausted T cells, ABC, myeloid progenitors and lymphoid progenitors is determined before my-HSC-selective depletion.
  • the balance of immune cells e.g. the relative number of one or more of na ⁇ ve T cells, exhausted T cells, ABC, myeloid progenitors and lymphoid progenitors is determined before my-HSC-selective depletion, where an improvement in the desired balance of lymphoid to myeloid cells is associated with successful selective depletion.
  • the method of selective depletion may provide for an improved immune response, e.g.
  • an antigen-specific CD8+ T cell response can be increased at least 1.5-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 7.5-fold, at least 10-fold, at least 15-fold, or more in a rebalanced individual.
  • An antigen-specific antibody response can be increased at least 1.5-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 7.5-fold, at least 10-fold, at least 15-fold, or more in a rebalanced individual.
  • the severity of infection or tumor burden may be reduced, e.g. a decrease in hospitalization, infected cells, mortality, tumor burden, metastases, cancer relapse and the like.
  • BRIEF DESCRIPTION OF THE FIGURES [018] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings.
  • the patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures. [019] FIGS. 1A-1L.
  • a Schematic to identify genes encoding candidate myeloid-biased HSC cell- surface antigens (left) and validate their presence on my-HSCs vs. bal-HSCs (right).
  • b Heatmap depicting expression of candidate markers across independent datasets (left), with ranked mean log2 fold-change (Old HSC vs. Young HSC; My-HSC vs. Bal-HSC) of each candidate (right). Datasets include comparison of Old vs. Young HSCs (a, b, c, d, e, f, g, h) and My-HSCs vs.
  • Bal-HSC (i, j, k).
  • c Comparison of percent-positive for each candidate marker on my-HSCs vs. bal-HSCs (left); plot of my-HSC fold-enrichment for each marker, calculated as: (% marker positive of my-HSC)/(% marker positive of bal-HSC) (right).
  • d Plot of (my-HSC)/(bal-HSC) Fold-Change (log2) of RNA expression (y-axis) vs. Cell-surface Protein expression (x-axis) for 12 candidate markers.
  • g–i Relative cell-surface expression of CD150 (g), NEO1 (h), and CD62p (i), on total HSC (my-HSC & bal-HSC), my-HSC, bal-HSC, MPPa, MPPb, MPPc, CMP (CMP&GMP), MkP, and CLP populations.
  • Flow-cytometry median fluorescent intensity (MFI) values for antibodies to each marker were obtained for each population and normalized from 0–1 based on the lowest to highest expression.
  • FIGS.2A-2K Antibody-mediated depletion of myeloid-biased hematopoietic stem cells in vivo.
  • a Schematic of strategy to restore balanced lineage output from HSCs by depleting my-HSCs using antibody-conditioning.
  • b Schematic to deplete my-HSCs by targeting my- HSC specific antigens (CD150, CD62p, or NEO1), in combination with antibodies to CD47 and to cKIT (left), followed by phenotypic analysis (right).
  • c–e Percentage of total HSCs that are my-HSCs (left) in mice receiving: anti-CD150 (c), anti-CD62p (d), or anti-NEO1 (e), optimized antibody-conditioning protocols, which include anti-CD47 and anti-cKIT.
  • HSC HSC
  • My my- HSCs
  • Bal bal-HSCs
  • MPPs MPPs
  • CLP lymphoid and myeloid progenitors
  • CMP&GMP, MkP myeloid progenitors
  • Total HSCs (KLS FLT3 – CD34 – CD150 + ) were FACS-sorted from aged control mice (Aged, A) or aged mice with my-HSC depletion (Aged+Conditioning, A+C) and underwent bulk RNA-sequencing. My-HSC depletion was performed with anti-NEO1+anti-CD62p+anti-cKIT/CD47 and cells were collected at day 9 post-treatment.
  • FPKM of top 200 differentially expressed genes between A HSCs vs.
  • A+C HSCs ranked by p-value.
  • GSEA applied to differentially expressed genes of A HSCs vs.
  • FIGS. 3A-3J Depletion of my-HSCs in aged mice restores features of a youthful immune system. a, Schematic of time-course experiment to determine the impact of antibody- conditioning on aged mice.
  • Young-adult mice (Y) were compared to aged mice (A), with or without antibody conditioning with anti-NEO1 optimized protocol (A+C), at approximately 1-, 8-, or 16-weeks post-treatment.
  • Values are relative to the mean value for aged control mice at each time-point and log2- transformed. Values for aged mice receiving antibody-conditioning are in closed filled circles; values for control aged mice are in open unfilled circles.
  • FIGS.4A-4E Antibody-conditioning enhances functional immunity to infection in aged mice.
  • spleen Percentage of CD8+ T cells in the spleen that are FV antigen-specific (Dextramer + CD44 + ) in aged-matched (20-26 months) mice without (A), or with antibody-conditioning (A+C), 10-14 days after intravenous (i.v.) vaccination with live- attenuated virus (left).
  • My-HSC depletion was conducted 2 months prior to vaccination with anti-NEO1 v2 conditioning protocol.
  • Units are in mg and log10-transformed. Graph bars depict median.
  • e Percent of CD8+ T cells in the spleen that are FV antigen-specific (Dextramer + CD44 + ) in vaccinated aged mice without (A), or with antibody conditioning (A+C), 14 days after infection with FV (left).
  • mouse ages are at time of analysis: Y, young-adult (3-6 months) mice; A, aged (21-22 months) mice; A+C, aged (21-22 months) mice receiving antibody-conditioning.
  • FIGS.5A-5J Mouse myeloid-biased HSC markers are enriched in aged human HSCs.
  • a Heatmap depicting RNA expression of candidate human my-HSC antigens in independent datasets of human Old vs. Young HSCs (a, b, c, d).
  • b Relative RNA expression of CD62p (Selp), CD41 (Itga2b), CD150 (Slamf1), and NEO1 (Neo1) in human HSCs isolated from young (ages 20-31), middle & old (ages 42-85) donors.
  • c–e Correlation of relative RNA expression of CD62p (c), CD41 (d), and CD150 (e) in human HSCs compared to donor age.
  • f Representative flow-cytometry of CD34 + -enriched donor bone-marrow to identify human HSCs (Lin – CD34 + CD38 – CD45RA – CD90 + ). For b–e, values are relative to mean of young samples.
  • h–i Histograms for flow-cytometry staining of HSCs with antibodies to CD304, CD150, TIE2, CD62p, ESAM, CD9, CD47, CD105, CD166; black line represents FMO control (h), with percent of HSCs positive for each marker (i).
  • j Model to rejuvenate aged immune systems by depleting myeloid-biased hematopoietic stem cells.
  • FIGS.6A-6O Expression of candidate my-HSC markers in hematopoietic progenitors, mature cells, and non-hematopoietic tissues.
  • a-l Expression of my-HSC candidate markers, Slamf1 (CD150) (a), Neo1 (NEO1) (b), Itga2b (CD41) (c), Selp (CD62p) (d), Cd38 (CD38) (e), Itgb3 (CD61) (f), Itgav (CD51) (g), Procr (CD201) (h), Tie2 (i), Esam (j), Eng (CD105) (k), Cd9 (CD9) (l), in hematopoietic stem and progenitor cells (HSPCs) in normal mouse bone marrow (top panels), and in young versus old bone marrow (bottom panels). Data from a–l obtained from Gene Expression Commons.
  • Tabula Muris n, GSE132040
  • Kadoki o, GSE87633
  • FIGS. 7A-7K Gating strategy for total HSCs, my-HSCs, bal-HSCs, and HPCs.
  • Flow-cytometry median fluorescent intensity (MFI) values for each marker were obtained for each population and normalized from 0–1 based on the lowest to highest expression.
  • MFI values for each marker were obtained for each population and normalized from 0–100 based on the lowest to highest expression.
  • k Comparison of percent-positive of my-HSCs vs.
  • FIGS. 8A-8L Identification of non-masking anti-CD150 antibodies.
  • FIGS.9A-9S Antibody-mediated depletion of my-HSCs in vivo.
  • Total HSCs e.g., my-HSCs + bal-HSCs
  • f Percentage of total HSCs that are my-HSCs in mice receiving anti-CD47 alone
  • n 5 mice.
  • j–n Frequency as a percentage of live cells for CLPs (j), IL7Ra + cells (k), CMPs&GMPs (l), MkPs (m), and MEPs (n), after CD150, CD62p, or NEO1 antibody- conditioning protocols.
  • bone-marrow was cKIT-enriched prior to FACS analysis.
  • total bone-marrow (non cKIT-enriched) was examined.
  • p-values were obtained by ordinary one-way ANOVA followed by one-tailed Dunnett’s multiple comparisons test with non-treated as control (a–d), or by unpaired parametric one-tailed t-test (i-k, o–p), or by unpaired parametric two-tailed t-test (e–h, l–n).
  • p-values and R values calculated with one-tailed Pearson correlation coefficient (q–s).
  • CD150 v1 is rat IgG2b anti- CD150 protocol
  • CD150 v2 is rat IgG2a anti-CD150 protocol
  • NEO1 v2 is protocol including mouse IgG2a secondary antibody
  • ⁇ anti-; ns, not significant.
  • FIGS.10A-10Q Optimization of NEO1 depletion protocol in vitro and in vivo.
  • a–f Anti- NEO1 antibody saturation curve (a) determined from in vitro antibody concentration dilution series (b–f).
  • g Schematic of in vivo saturation experiments with anti-NEO1 antibody; used in panels h–k.
  • l Schematic illustrating paradigm for double-antibody strategy to target NEO1, whereby mouse monoclonal anti-goat IgG2a or IgG2b antibodies are administered 24 hours after goat anti-NEO1.
  • FIGS.11A-11N My-HSC depletion restores features of a youthful immune system.
  • b–d Volcano plots of statistical significance (y-axis, -log10p) vs. fold-change (x-axis, log2) for Aged / Young (b), Aged / Aged+Conditioning (c), or Aged / (Young & Aged+Conditioning) (d), mice comparison.
  • My-HSC depletion increases na ⁇ ve T cells and B cells in aged mice.
  • h–j Frequency relative to aged mice of T cell (CD4 & CD8) subsets in Young (Y), Aged (A), and Aged+Conditioning (A+C) mice 8-weeks after antibody treatment (h).
  • CM central memory
  • Mouse ages are at time of antibody-conditioning: Y, young-adult (3-6 months) mice; A, aged (18-24 months) mice; A+C, aged (18-24 months) mice receiving antibody-conditioning.
  • FIGS.13A-13L Flow-cytometry gating strategy for T cells, B cells, and myeloid cells.
  • a–c Gating strategy to identify: (b) na ⁇ ve (CD44 – CD62L + ), central memory (CD44 + CD62L + ), and effector memory (CD44 + CD62L-) T cells (combined CD4 & CD8), or (c) CD4 T cells that are PD1 + CD62L – or PD1 – CD62L + , in the blood.
  • d–f Gating strategy to identify: (e) mature B cells (CD19 + B220 + IgM + IgD + ), or (f) Aged B Cells ABCs (CD19 + IgM + CD93-CD43- CD21/CD35- CD23-), in the blood.
  • FIGS. 14A-14J Antibody-conditioning enhances functional immunity to infection.
  • a Schematic of infectious disease model to determine the impact of antibody-conditioning on functional immunity of aged mice.
  • mice Young-adult mice (Y) were compared to aged mice (A), with or without antibody conditioning with anti-NEO1 optimized protocol (A+C). Mice were vaccinated, or were not vaccinated, at Week-8 post-antibody conditioning, infected at Week- 14, and analyzed at Week-16.
  • Gating strategy to identify Ter119 + cells Te119 + CD19-CD3- CD45 +/lo
  • antigen-infected cells Ag34 + Ter119 +
  • Total number of Ter119 + cells per mouse spleen was evaluated in young-adult (Y), aged (A), or aged+conditioning mice (A+C) that were Na ⁇ ve, Infected, or Vaccinated & Infected with FV. Representative flow-cytometry histogram plots for Ter119 expression, gated on all single cells. Each row represents an independent mouse.
  • NEO1 v2 protocol is NEO1 v1 protocol (anti-NEO1+ anti- CD47+anti-cKIT) + mouse IgG2a secondary antibody.
  • Y young-adult mice; A, aged mice; A+C, aged mice receiving antibody-conditioning; Inf., FV infected without vaccination; Vacc. & Inf., FV infected with vaccination, Vacc.
  • b Relative mRNA expression of CD62p (Selp), CD41 (Itga2b), CD61 (Itgb3), and NEO1 (Neo1) in human HSCs isolated from young (age 18-30) or old (age 65-75) donors.
  • c–e Correlation of relative mRNA expression of CD62p (c), CD41 (d), and CD61 (e) in human HSCs as compared to donor age. For a–e, values are relative to mean of young samples.
  • f Heatmap depicting expression of candidate markers across independent datasets comparing human: HMGA2 + vs. HMGA2 – CD34 + cells (e), MPN (f) or MDS (g) vs.
  • FIGS. 16A-16M Mouse my-HSC antigens mark subsets of human HSCs.
  • FMO fluorescence-minus-one
  • h Illustration depicting human Hematopoietic Stem and Progenitor Cell (HSPC) Tree Analysis (h), with colors for each cell population corresponding to gating scheme in (a).
  • i–l Relative expression of CD62p (i), CD150 (j), ESAM (k), and CD166 (l), on human HSCs, MPPs, LMPPs, CMPs & MEPs, and GMPs.
  • m Percentage of positive HSCs and normalized MFI for each marker in HSCs and HSPCs for CD90, CD62p, TIE2, CD304, CD150, ESAM, CD166, CD105, CD47, and CD9.
  • MFI median fluorescent intensity
  • HSC Hematopoietic stem cells
  • HSC therefore refers to multipotent cells capable of differentiating into all the cell types of the hematopoietic system, including, but not limited to, granulocytes, monocytes, erythrocytes, megakaryocytes, lymphocytes, dendritic cells; and self-renewal activity, i.e. the ability to divide and generate at least one daughter cell with the identical (e.g., self-renewing) characteristics of the parent cell.
  • Human HSC are, for example, CD34 + ; CD90 (thy-1) + ; CD59 + ; CD110 (c-mpl) + ; c-kit (CD-117) + .
  • a human HSC cell may be characterized or selected by the phenotype, for example, Lin-CD34 + CD38 – CD90 + CD45RA – .
  • Mouse HSC are, for example, CD90 (thy-1) lo ; Sca1 + ; c-kit (CD-117) + .
  • a mouse HSC cell may be characterized or selected by the phenotype, for example, Lin – cKIT + Sca1 + Flk2 – CD34 – CD150 + .
  • a “lin” or lineage panel may comprise one or more of the markers CD3, CD4, CD8, CD19, CD20, CD56, CD11b, CD14, and CD15.
  • CLP common lymphocyte progenitor
  • CMP common myeloid progenitor
  • my-HSC and bal-HSC A myeloid-biased HSC generates differentiated progeny with a greater proportion of myeloid progenitors, relative to a balanced HSC.
  • my-HSCs can be defined by the ratio between lymphoid and myeloid cells in blood that are derived from the my- HSC.
  • Balanced HSCs give rise to a blood population that is from about 10% to about 20% myeloid cells, with the remainder lymphocytes.
  • the mean lymphoid- to-myeloid cell ratio in the blood can be around 3.0 ⁇ 3.0.
  • My-HSCs generate a mean lymphoid- to-myeloid cell ratio in the blood of less than about 3 but greater than 0.
  • My-HSC generate myeloid and lymphoid progeny, but with an altered bias toward myeloid cells.
  • human my-HSC can be distinguished from bal-HSC by cell surface markers, including without limitation CD304, TIE2, ESAM, CD9, CD105, CD166, CD150 (Slamf1), CD61 (Itgb3), CD41 (Itga2b), CD62p, and NEO1, where these markers are expressed at higher levels on the my-HSC relative to the bal- HSC.
  • the marker expression can be increased at least 1.5-fold, 2-fold, 3-fold, 5-fold, 10-fold or more on my-HSC relative to bal-HSC.
  • my-HSC selective markers comprise one or more of CD150 (Slamf1), CD61 (Itgb3), CD41 (Itga2b), CD62p, and NEO1.
  • my-selective markers are CD150, CD62p and NEO1.
  • Markers of mouse my-HSC include, for example, CD150, CD62p, NEO1, CD38, CD51 (Itgav), CD201 (Procr), CD202b (Tie2), ESAM (Esam), CD105 (Eng), and CD9.
  • Myeloid progenitor cells are examples of mouse my-HSC.
  • Myeloid progenitor cells comprise one or more of: common myeloid progenitor cells (CMP); and the committed myeloid progenitors: erythroid/megakaryocytic progenitor (MEP), granulocyte/monocyte progenitors (GMP); and megakaryocyte progenitor (MKP).
  • CMP common myeloid progenitor cells
  • MKP megakaryocyte progenitor
  • the cells are CD34 positive, and CD38 positive.
  • the CMP is also characterized as IL-3R ⁇ lo CD45RA-.
  • the CMP are Sca-1 negative, (Ly-6E and Ly-6A), c-kit hi , and Fc ⁇ R lo .
  • CLP Common lymphoid progenitors, express low levels of c-kit (CD117) on their cell surface. Antibodies that specifically bind c-kit in humans, mice, rats, etc. are known in the art. Alternatively, the c-kit ligand, steel factor (Slf) may be used to identify cells expressing c-kit.
  • the CLP cells express high levels of the IL-7 receptor alpha chain (CDw127).
  • Murine CLPs express low levels of Sca-1 (Ly-6E and Ly-6A, see van de Rijn (1989) Proc Natl Acad Sci 86:4634-4638). Human CLPs express low levels of CD34. Human CLP cells are also characterized as CD38 positive and CD10 positive.
  • the CLP subset also has the phenotype of lacking expression of lineage specific markers, exemplified by B220, CD4, CD8, CD3, Gr-1 and Mac-1.
  • the CLP cells are characterized as lacking expression of Thy-1, a marker that is characteristic of hematopoietic stem cells.
  • the phenotype of the CLP may be further characterized as Mel-14-, CD43 lo , HSA lo , CD45 + and common cytokine receptor ⁇ chain positive.
  • Aged As used herein, the term aged refers to the effects or the characteristics of increasing age, particularly with respect to the bias of hematopoietic stem cells towards cells of the myeloid lineage.
  • the rate of aging is species specific, where a human may be aged at about 50 years; and a rodent at about 2 years.
  • a natural progressive decline in body systems starts in early adulthood, but it becomes most evident several decades later.
  • One arbitrary way to define old age more precisely in humans is to say that it begins at conventional retirement age, around about 60, around about 65 years of age.
  • Another definition sets parameters for aging coincident with the loss of reproductive ability, which is around about age 45, more usually around about 50 in humans, but may, however, vary with the individual.
  • individuals may suffer from a similar phenotype due to inflammation, genetic causes, and the like.
  • antibody also includes antigen binding forms of antibodies, including fragments with antigen- binding capability (e.g., Fab', F(ab')2, Fab, Fv and rIgG. The term also refers to recombinant single chain Fv fragments (scFv).
  • the term antibody also includes bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies. The term “entire” antibody is used to refer to an antibody comprising both variable regions and constant regions, i.e. an Fc region. [053] Selection of antibodies for stem cell depletion may be based on a variety of criteria, including selectivity, affinity, cytotoxicity, etc.
  • the specified antibodies bind to a particular protein sequences at least two times the background and more typically more than 10 to 100 times background.
  • antibodies of the present invention bind antigens on the surface of target cells in the presence of effector cells (such as natural killer cells or macrophages). Fc receptors on effector cells recognize bound antibodies.
  • An antibody immunologically reactive with a particular antigen can be generated by recombinant methods such as selection of libraries of recombinant antibodies in phage or similar vectors, or by immunizing an animal with the antigen or with DNA encoding the antigen.
  • Methods of preparing polyclonal antibodies are known to the skilled artisan.
  • the antibodies may, alternatively, be monoclonal antibodies.
  • Monoclonal antibodies may be prepared using hybridoma methods. In a hybridoma method, an appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.
  • antibody also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries.
  • a "humanized antibody” is an immunoglobulin molecule that contains minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • Antibodies of interest may be tested for their ability to induce ADCC (antibody- dependent cellular cytotoxicity).
  • Antibody-associated ADCC activity can be monitored and quantified through detection of either the release of label or lactate dehydrogenase from the lysed cells, or detection of reduced target cell viability (e.g. annexin assay).
  • Assays for apoptosis may be performed by terminal deoxynucleotidyl transferase-mediated digoxigenin- 11-dUTP nick end labeling (TUNEL) assay (Lazebnik et al., Nature: 371, 346 (1994).
  • Agents of interest that bind to CD62p include antibodies specific for human CD62p.
  • Such antibodies are known in the art and commercially available, for example and without limitation inclacumab; Crizanlizumab; HuEP5C7; Clone REA389; clone AK-6; clone Psel.KO.2.12; etc.
  • Agents of interest that bind to NEO1 include antibodies specific for human NEO1.
  • CD117 is a receptor tyrosine kinase type III, which binds to stem cell factor (a substance that causes certain types of cells to grow), also known as "steel factor” or "c-kit ligand".
  • CD117 is an important cell surface marker used to identify certain types of hematopoietic (blood) progenitors in the bone marrow.
  • HSC Hematopoietic stem cells
  • MPP multipotent progenitors
  • CMP common myeloid progenitors
  • anti-CD47 agent or “agent that interferes with the binding between CD47 and SIRP ⁇ ” refers to any agent that reduces the binding of CD47 (e.g., on a target cell) to SIRP ⁇ (e.g., on a phagocytic cell).
  • Non-limiting examples of suitable anti-CD47 reagents include high affinity SIRP ⁇ polypeptides, anti-SIRP ⁇ antibodies, and anti-CD47 antibodies or antibody fragments.
  • a suitable anti-CD47 agent e.g. an anti-CD47 antibody, a SIRP ⁇ reagent, etc. specifically binds CD47 to reduce the binding of CD47 to SIRP ⁇ .
  • Anti-human CD47 antibodies suitable for clinical use include, without limitation, magrolimab (hu5F9-G4, see U.S.
  • Soluble SIRP ⁇ agents include, for example, Evorpacept (ALX148), and CV1-Fc (see, for example, Weiskopf et al. (2013) Science 341 (6141): 88–91). Such antibodies may comprise an Fc region sequence.
  • an anti-CD47 agent is a “high affinity SIRP ⁇ reagent”, which includes SIRP ⁇ -derived polypeptides and analogs thereof (e.g., CV1-hIgG4, and CV1 monomer, ALX148).
  • High affinity SIRP ⁇ reagents are described in international application PCT/US13/21937, which is hereby specifically incorporated by reference.
  • High affinity SIRP ⁇ reagents are variants of the native SIRP ⁇ protein. The amino acid changes that provide for increased affinity are localized in the d1 domain, and thus high affinity SIRP ⁇ reagents comprise a d1 domain of human SIRP ⁇ , with at least one amino acid change relative to the wild-type sequence within the d1 domain.
  • a high affinity SIRP ⁇ reagent is soluble, where the polypeptide lacks the SIRP ⁇ transmembrane domain and comprises at least one amino acid change relative to the wild-type SIRP ⁇ sequence, and wherein the amino acid change increases the affinity of the SIRP ⁇ polypeptide binding to CD47, for example by decreasing the off-rate by at least 10-fold, at least 20-fold, at least 50- fold, at least 100-fold, at least 500-fold, or more.
  • a SIRP ⁇ reagent is a fusion protein, e.g., fused in frame with a second polypeptide.
  • the second polypeptide is capable of increasing the size of the fusion protein, e.g., so that the fusion protein will not be cleared from the circulation rapidly.
  • the second polypeptide is part or whole of an immunoglobulin Fc region. The Fc region aids in phagocytosis by providing an “eat me” signal, which enhances the block of the “don’t eat me” signal provided by the high affinity SIRP ⁇ reagent.
  • the second polypeptide is any suitable polypeptide that is substantially similar to Fc, e.g., providing increased size, multimerization domains, and/or additional binding or interaction with Ig molecules.
  • a subject anti-CD47 agent is an antibody that specifically binds SIRP ⁇ (i.e., an anti-SIRP ⁇ antibody) and reduces the interaction between CD47 on one cell (e.g., an infected cell) and SIRP ⁇ on another cell (e.g., a phagocytic cell).
  • SIRP ⁇ i.e., an anti-SIRP ⁇ antibody
  • Suitable anti-SIRP ⁇ antibodies can bind SIRP ⁇ without activating or stimulating signaling through SIRP ⁇ because activation of SIRP ⁇ would inhibit phagocytosis. Instead, suitable anti-SIRP ⁇ antibodies facilitate the preferential phagocytosis of inflicted cells over normal cells.
  • Antibodies of interest include humanized antibodies, or caninized, felinized, equinized, bovinized, porcinized, etc., antibodies, and variants thereof.
  • Anti-SIRP ⁇ antibodies in clinical and preclinical trials for human use include, for example, CC-95251; BYON4228; SIRP ⁇ -targeting antibody BR105; BI 770371 and BI- 765063/OSE172 (Boehringer Ingelheim); and GS-189 (FSI-189) (Gilead Sciences).
  • a "patient” for the purposes of the present invention includes both humans and other animals, particularly mammals, including pet and laboratory animals, e.g. mice, rats, rabbits, etc.
  • Those in need of treatment include those already affected (e.g., those with cancer, those with an infection, etc.) as well as those in which prevention is desired (e.g., those with increased susceptibility to cancer, those with an increased likelihood of infection, those suspected of having cancer, those suspected of harboring an infection, etc.).
  • Selective Depletion [073] Methods of selective depletion of my-HSC provide for an improved balance in the levels of myeloid versus lymphoid cells in a subject after depletion.
  • the recipient is conditioned with the administration of an effective dose of conditioning agents, e.g. an antibody, specific for a my-HSC selective marker, or a combination of my-HSC selective agents.
  • common lymphoid progenitors to the number of myeloid progenitors, e.g. common myeloid progenitors, may be increased at least 1.5-fold,at least 2-fold, at least 3-fold, at least 5-fold, at least 7.5-fold, at least 10-fold, at least 15-fold, or more.
  • the number of circulating na ⁇ ve T cells relative to the total circulating lymphocyte population may be increased at least 1.5-fold,at least 2-fold, at least 3-fold, at least 5-fold, at least 7.5-fold, at least 10-fold, at least 15-fold, or more.
  • the level of circulating “age-associated B cells” (ABC), and/or exhausted T cells relative to the total circulating lymphocyte population may be decreased at least 1.5-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 7.5-fold, at least 10-fold, at least 15-fold, or more.
  • the effective dose of a my-HSC selective agent e.g.
  • the agent is a CV1 (high affinity SIRP ⁇ ) monomer or CV1 microbody dimer. In other embodiments the agent is an anti-CD47 antibody. In other embodiments the agent is an anti-SIRP ⁇ antibody.
  • the depleting agents can be administered daily, twice daily, every other day, every third day, etc. for a period of time sufficient to affect the desired selective depletion, which may be at least about 1 day, up to about 2 days, up to about 3, 4, 5, 6, 7, 8 or more days. In some embodiments from 4-7 days is sufficient. In some embodiments a single dose is administered. In other embodiments a plurality of doses is administered, e.g.2, 3, 4, 5 or more.
  • the agents may be formulated together or separately, but are administered concomitantly.
  • Concomitant and “concomitantly” as used herein refer to the administration of at least two agents, or at least three agents, or more to a patient either simultaneously or within a time period during which the effects of the first administered agent are still operative in the patient.
  • the concomitant administration of the second agent can occur one to two days after the first, preferably within one to seven days, after the administration of the first agent.
  • compositions containing depleting agents e.g. antibodies, soluble SIRP ⁇ , etc. can be administered for therapeutic treatment.
  • Compositions are administered to a patient in an amount sufficient to selectively deplete my-HSC, as described above.
  • compositions may be administered depending on the dosage and frequency as required and tolerated by the patient.
  • the particular dose required for a treatment will depend upon the medical condition and history of the mammal, as well as other factors such as age, weight, gender, administration route, efficiency, etc.
  • the pharmaceutical compositions are in a water-soluble form, such as being present as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts.
  • “Pharmaceutically acceptable acid addition salt” refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like
  • organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid,
  • the pharmaceutical compositions may also include one or more of the following: carrier proteins such as serum albumin; buffers; fillers such as microcrystalline cellulose, lactose, corn and other starches; binding agents; sweeteners and other flavoring agents; coloring agents; and polyethylene glycol.
  • carrier proteins such as serum albumin
  • buffers such as buffers
  • fillers such as microcrystalline cellulose, lactose, corn and other starches
  • binding agents such as microcrystalline cellulose, lactose, corn and other starches
  • sweeteners and other flavoring agents such as microcrystalline cellulose, lactose, corn and other starches
  • binding agents such as microcrystalline cellulose, lactose, corn and other starches
  • sweeteners and other flavoring agents such as microcrystalline cellulose, lactose, corn and other starches
  • binding agents such as microcrystalline cellulose, lactose, corn and other starches
  • sweeteners and other flavoring agents such as microcrystalline cellulose, lactose, corn and other starches
  • binding agents such as
  • compositions for administration will commonly comprise an antibody or other agent dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier.
  • a pharmaceutically acceptable carrier preferably an aqueous carrier.
  • aqueous carriers can be used, e.g., buffered saline and the like. These solutions are sterile and generally free of undesirable matter.
  • These compositions may be sterilized by conventional, well known sterilization techniques.
  • compositions are administered to a patient in an amount sufficient to substantially deplete targeted myHSC, as described above.
  • An amount adequate to accomplish this is defined as a "therapeutically effective dose.”
  • Single or multiple administrations of the compositions may be administered depending on the dosage and frequency as required and tolerated by the patient.
  • the particular dose required for a treatment will depend upon the medical condition and history of the mammal, as well as other factors such as age, weight, gender, administration route, efficiency, etc.
  • USES [087] The methods disclosed herein provide for a rebalancing of immune systems, generally to increase the production of lymphoid cells relative to myeloid cells. An imbalance is associated with aging and the elderly.
  • myeloid-biased HSCs are pro-inflammatory, producing or eliciting inflammatory cytokines (TNF- ⁇ , IL-1, IL-6, etc.), in response to microbes or endogenous antigens.
  • the disclosure provides compositions and methods for use in a therapeutic method of rebalancing the immune system in a human subject in need thereof. These methods bring the body from a pathological state back into its normal, healthy state, or prevent a pathological state. In some embodiments, the disclosure provides compositions and methods for use in a therapeutic method of improved response to infection and/or vaccination.
  • the disclosure provides compositions and methods for use in a therapeutic method in reducing inflammation, e.g. inflammation associate with infection. In some embodiments, the disclosure provides compositions and methods for use in in a therapeutic method improving surveillance of cancer cells. In some embodiments, the disclosure provides compositions and methods for use in in a therapeutic method that reduces the population of myeloid cells that suppress tumor immunity. [091] In some embodiments, the disclosure provides compositions and methods for rebalancing the immune system of individuals suffering or at risk of a hematologic malignancy.
  • leukemias examples include leukemias, lymphomas, and myelomas, including but not limited to acute biphenotypic leukemia, acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), acute promyelocytic leukemia (APL), biphenotypic acute leukemia (BAL) blastic plasmacytoid dendritic cell neoplasm, chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), chronic lymphocytic leukemia (CLL) (called small lymphocytic lymphoma (SLL) when leukemic cells are absent), acute monocytic leukemia (AMOL), Hodgkin's lymphomas, Non-Hodgkin's lymphomas (e.g.
  • CLL chronic lymphocytic leukemia
  • DLBCL diffuse large B-cell lymphoma
  • FL Follicular lymphoma
  • MCL Mantle cell lymphoma
  • MZL Marginal zone lymphoma
  • BL Hairy cell leukemia
  • PTLD Post-transplant lymphoproliferative disorder
  • Waldenstrom's macroglobulinemia/lymphoplasmacytic lymphoma hepatosplenic-T cell lymphoma, and cutaneous T cell lymphoma (including Sezary's syndrome)
  • multiple myeloma myelodysplastic syndrome
  • myeloproliferative neoplasms myeloplasms.
  • the subject methods find utility in treatment of leukemias, e.g. acute biphenotypic leukemia, acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), acute promyelocytic leukemia, chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia, chronic lymphocytic leukemia (CLL), acute monocytic leukemia (AMOL).
  • leukemias e.g. acute biphenotypic leukemia, acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), acute promyelocytic leukemia, chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia, chronic lymphocytic leukemia (CLL), acute monocytic leukemia (AMOL).
  • leukemias e.g. acute biphenotypic leukemia, acute myelogenous leukemia (
  • Individuals selected for treatment may include, for example, individuals diagnosed with CHIP, pre-malignant AML patients or MDS patients, na ⁇ ve AML patients who are ineligible for standard induction chemotherapy or allogeneic hematopoietic cell transplant due to age and/or co-morbidities; previously untreated intermediate and high risk myelodysplastic syndrome (MDS) patients; and MDS patients who are relapsed and/or refractory to frontline hypomethylating agents.
  • MDS myelodysplastic syndrome
  • Myelodysplastic syndromes are characterized by ineffective and dysplastic hematopoiesis and include the following: Refractory anemia: Anemia with reticulocytopenia; normal or hypercellular marrow with erythroid hyperplasia, and dyserythropoiesis; blasts ⁇ 5% of nucleated marrow cells; Refractory anemia with ringed sideroblasts: Same as refractory anemia with reticulocytopenia, except that ringed sideroblasts are > 15% of nucleated marrow cells; Refractory cytopenia with multilineage dysplasia: Cytopenia not restricted to red cells; prominent dysplasia of white cell precursors and megakaryocytes; Refractory cytopenia with multilineage dysplasia and ringed sideroblasts: With ringed sideroblasts that are > 15% of nucleated marrow cells; Refractory anemia with excess blasts (RAEB): Cytopenia of ⁇ 2 cell lines with
  • the presence of CHIP can be determined by methods known in the art, for example by analyzing a patient sample(s) comprising hematopoietic cells.
  • the cells can be isolated from a bone marrow, blood or blood-derived sample.
  • a plurality of cells in the sample(s) are analyzed for the presence of clonality, usually by high throughput sequencing of polynucleotides isolated from the cell, for example whole exome sequencing, targeted sequencing of frequently mutated genes, etc.
  • the number of cells analyzed may be at least 10 2 , at least 10 3 , at least 10 4 , at least 10 5 or more.
  • the sequencing can be performed on bulk blood cells, e.g. PBLs, or on selected cell populations, e.g.
  • the presence of CHIP can be defined by the presence of somatic mutations, where the most frequently mutated genes include, for example, DNMT3A, TET2, ASXL1, SF3B1, and GNB1.
  • the variant allele fraction (VAF) can be determined, i.e. as the fraction of alleles present in the plurality of cells that comprise a specific somatic mutation.
  • An individual is determined to be a CHIP carrier if the VAF is >0.08, >0.09, >0.1, >0.125, >0.15, >0.175, >0.2 or more.
  • a cut-off of a VAF >0.2 is used to define an individual as having CHIP.
  • kits comprising an effective dose of the one or more agents disclosed herein for selective depletion of my-HSC.
  • Experimental Example 1 Rejuvenating the immune system by depleting myeloid-biased hematopoietic stem cells
  • Aging of the hematopoietic system is characterized by decreased lymphopoiesis and adaptive immunity, and increased inflammation and myeloid pathologies. Age-related changes in the function of hematopoietic stem cells (HSCs), which generate all blood cells throughout life, are thought to underlie these phenomena.
  • HSCs with balanced output of lymphoid and myeloid cells predominate over HSCs with myeloid-biased output, thereby promoting the lymphopoiesis required for adaptive immune responses, while limiting the production of pro-inflammatory myeloid cells.
  • aging is associated with increased proportions of myeloid-biased HSCs resulting in decreased lymphopoiesis and increased myeloid cell-driven inflammation. Whether these age-related changes to HSCs can be reversed to restore youthful immune function is unclear.
  • my-HSCs myeloid-biased HSCs
  • CLPs common lymphocyte progenitors
  • B cells na ⁇ ve T cells
  • the targeted my-HSC antigens are also enriched in aged human HSCs, nominating them as therapeutic targets to rejuvenate the immune system in humans.
  • antibody-mediated depletion of my-HSCs in aged mice improves vaccination responses and enhances vaccine-induced protection from viral infection.
  • HSC hematopoietic stem cell
  • a single hematopoietic stem cell can generate all blood cells and self-renew to maintain the stem cell pool throughout life.
  • HSCs demonstrate functional heterogeneity and can differ in their contribution to the lymphoid and myeloid cell lineages.
  • At least two HSC subsets exist: (i) balanced HSCs (bal-HSC) that provide balanced production of lymphoid and myeloid cells, and (ii) myeloid-biased HSCs (my-HSC) that are biased towards predominant production of myeloid cells.
  • bal-HSCs The frequency of my-HSCs relative to bal-HSCs increases with age. This age-related shift from bal-HSCs to my-HSCs decreases lymphopoiesis and increases myelopoiesis, thereby contributing to numerous pathologies of the elderly, including reduced adaptive immunity, ‘inflammaging’, and several myeloid-related diseases.
  • CD150 emerged from this analysis, along with several markers associated with myeloid-biased HSCs: CD41, CD61, CD62p, and NEO1.
  • CD41, CD61, CD62p, and NEO1 markers associated with myeloid-biased HSCs: CD41, CD61, CD62p, and NEO1.
  • CD41, CD61, CD62p, and NEO1 markers associated with myeloid-biased HSCs: CD41, CD61, CD62p, and NEO1.
  • the ideal target antigen to deplete my-HSCs would be highly expressed on the cell- surface of my-HSCs relative to bal-HSCs.
  • the fold-enrichment was calculated for my-HSCs based on the proportion of my-HSCs (CD150 High HSCs) relative to bal-HSCs (CD150 Low HSCs) that were marker-positive.
  • Antibodies to NEO1 and CD41 resulted in a significantly increased frequency of staining of my-HSCs (Fig.1c, 1f), consistent with NEO1 and CD41 marking HSCs with myeloid bias.
  • CD62p led to the greatest enrichment for my-HSCs (Fig. 1c–f).
  • the most highly enriched cell-surface proteins on my-HSCs relative to bal-HSCs were CD41, CD62p, and NEO1 (Fig. 1c–d).
  • HSCs hematopoietic progenitor cells
  • CMPs lineage-restricted common myeloid progenitors
  • CLPs common lymphoid progenitors
  • GSEA Gene Set Enrichment Analysis
  • CLPs common lymphoid progenitors
  • CMPs & GMPs, MkPs, and MEPs myeloid progenitors
  • the protocols targeting CD62p or NEO1 decreased the frequency of myeloid progenitors (Fig.2g–h and FIG.9l–m) and increased the ratio of lymphoid progenitors (CLPs) to myeloid progenitors (CMPs & GMPs) by up to 4-fold (FIG. 9o).
  • CLPs lymphoid progenitors
  • CMPs & GMPs myeloid progenitors
  • Antibody conditioning increases na ⁇ ve T cells and B cells in aged mice.
  • a critical deficit of aged immune systems is the reduced generation of T and B lymphocytes capable of recognizing novel antigens. Given that depletion of my-HSCs in aged mice increased lymphocyte progenitors, we sought to determine if these changes were sufficient to increase na ⁇ ve T and B cells.
  • mice We evaluated mice after 8-weeks, since the generation of new T and B cells from HSCs peaks between 7-11 weeks. Although we did not observe significant differences in thymus weight (FIG. 11l), treated mice contained all the thymic progenitor subsets associated with thymus function (FIG. 11m–n). After approximately 8-weeks, aged mice receiving antibody-conditioning demonstrated a significant increase in the frequency (Fig.3e) and absolute number (FIG.12a) of circulating na ⁇ ve T cells (CD4+ or CD8+, CD44- CD62L + cells) compared to age-matched controls.
  • Antibody treatment did not significantly impact the total number of circulating CD45 + leukocytes (FIG.12e). Overall, these results demonstrated that antibody-mediated depletion of my-HSCs selectively increased both na ⁇ ve T cells and mature B cells in aged mice. [0116] Antibody conditioning decreases T and B cells with age-related cellular phenotypes. In addition to their decreased frequency and production in aged animals, lymphocytes undergo age-related accumulation of markers of exhaustion and/or inflammation that are thought to contribute to immune decline.
  • CD4 T cells with an exhausted phenotype (PD1 + CD62L – ) increase relative to those with a non-exhausted phenotype (PD1 – CD62L + ), which we confirmed in our experimental cohort (Fig.3g).
  • antibody- conditioning decreased exhausted T cells relative to non-exhausted T cells (Fig. 3g).
  • Aged mice also accumulate a distinct population of ‘age-associated B cells’ (ABCs) correlated with reduced humoral immunity.
  • Our control cohort of aged mice had an increased frequency of ABCs (CD19 + IgM + CD93-CD43- CD21/CD35-CD23-) relative to young-adult mice, which was significantly decreased after antibody conditioning (Fig. 3h).
  • Antibody conditioning decreases systemic pro-inflammatory markers.
  • aging is also associated with increased levels of circulating pro- inflammatory mediators, referred to as ‘inflammaging’, which has been linked to HSC dysfunction and myeloid bias.
  • inflammaging circulating pro-inflammatory mediators
  • the most elevated proteins in aged animals relative to young-adult mice were the pro-inflammatory factors IL-1 ⁇ and CXCL5 (FIG. 11b), which were also the most decreased proteins in aged mice receiving antibody-conditioning (Fig.3i– j and FIG. 11c–d).
  • Antibody-conditioning also decreased numerous additional pro- inflammatory mediators in aged mice, including IL-1 ⁇ , CXCL2 (MIP-2), and IL-23 (Fig.3j and FIG.11c, 6e).
  • my-HSC depletion decreased the levels of circulating pro-inflammatory mediators several months after treatment.
  • Depletion of my-HSC enhances functional immunity to viral infection in aged mice.
  • mice were vaccinated intravenously (i.v.) with live-attenuated virus approximately 8-weeks after receiving anti-NEO1 antibody-conditioning.
  • the spleens were harvested 10-14 days later at the peak of CD8+ T cell response.
  • Aged mice receiving my-HSC depletion demonstrated an increase in virus-specific CD8+ T cell responses (dextramer+) following vaccination as compared to old mice (Fig.4b), demonstrating that my-HSC depletion improved the response to vaccination.
  • aged mice were vaccinated approximately 8-weeks after receiving anti-NEO1 antibody-conditioning and then infected with FV 6-weeks after vaccination (FIG.14a).
  • Spleen cells were examined at two-weeks post-infection, the peak of virus replication. Controls included na ⁇ ve mice, non-depleted aged mice, and unvaccinated mice. We verified that anti- NEO1 antibody conditioning decreased my-HSC by both frequency and absolute number under these conditions and time-points (Fig. 4a and FIG. 14c–h). Control young-adult mice inoculated with FV had approximately three-fold larger spleens than na ⁇ ve mice (Fig.4c) and a per spleen median of 7,000 infectious centers (Fig.4d), a measure of live, infectious virus. Vaccination of young-adult mice prevented splenomegaly (Fig.4c) and significantly reduced infectious centers (Fig.4d).
  • mice Aged-infected mice fared much worse than young-adult mice in all parameters associated with infection: their median increase in spleen weights was ten-fold (Fig. 4c) and their median infectious centers per spleen was 4 million (Fig. 4d), which was more than 500-fold the median in infected young-adult mice. Vaccination of aged mice resulted in a slight but not statistically significant decrease in splenomegaly (Fig.4c), and only 1 out of 8 mice was able to control infection (Fig. 4d and FIG. 14). In contrast, mice that had been conditioned by my-HSC-depletion 2 months prior to vaccination demonstrated significantly reduced splenomegaly (Fig.
  • Mouse myeloid-biased HSC antigen targets are enriched in aged human HSCs.
  • the age-associated expansion of HSCs with myeloid bias occurs in both mouse and humans.
  • antibody-mediated depletion of my-HSCs reverses several features of age-related immune decline in mice, we investigated if the my-HSC antigenic targets used in our conditioning protocol might be applicable to humans.
  • human homologs to mouse my-HSC genes were expressed by aged human HSCs.
  • mice my-HSC genes including CD62p (Selp), CD150 (Slamf1), and CD41 (Itga2b), were significantly increased in aged human HSCs from independent datasets (Fig.5a–b and FIG.60a–b) and were also correlated with age across adulthood (Fig.5c–e and FIG.60c–e).
  • genes for multiple mouse my-HSC antigens were enriched in HSCs isolated from pathologies related to aging of the human hematopoietic system, including aberrant and pre- malignant human HSCs (FIG.60f).
  • the most enriched gene across all datasets – in both mouse and human – was CD62p.
  • Etiology of hematopoietic stem cell clonal heterogeneity Evolution of the vertebrate immune system occurred in the context of populations of individuals that were geographically limited. Immune responses to pathogens are predominated first by an innate response by cells of the myeloid lineage (macrophages, neutrophils, and granulocytes), and second by eliciting a more specific adaptive response by cells of the lymphoid lineage (B cells and T cells). Each of the millions of na ⁇ ve B cells and T cells expresses a distinct receptor capable of recognizing a specific antigen from a pathogen, one receptor specificity for each antigen.
  • T and B stem/memory cells that can respond much faster and more potently if the pathogen is re-encountered.
  • machine-mediated transportation i.e., trains, planes, and cars – individuals were likely to be exposed to the majority of pathogens in their local geography by the time of reproductive age. Since T and B memory/stem cells can survive the lifetime of the individual, they should be sufficient to provide adaptive immune memory to all local microbial pathogens. Thus, the generation of new T and B lymphocytes in later life was likely no longer advantageous. In contrast, the production of short-lived myeloid cells would remain important for acute innate responses, even in later life.
  • Myeloid- biased HSCs are pro-inflammatory, producing or eliciting inflammatory cytokines, which provide a much more serious response to microbes or endogenous antigens.
  • myeloid-biased HSCs in the elderly is a double-edged sword in the battle with novel pathogens, resulting not only in a poor adaptive immune response, but also in detrimental inflammatory responses.
  • Molecular regulators of HSC function in age and disease.
  • the molecular mechanisms that regulate the expansion of myeloid-biased HSCs with age are not fully known. There are at least two models that may explain the expansion of myeloid-biased HSCs with age: (i) changes to the clonal competition between distinct subtypes of HSCs over time, or (ii) epigenetic changes to the functional properties of stem cells over time. This study is not intended to distinguish between these two models.
  • mice were C57BL/6 or (C57BL/10 ⁇ A.BY)F1 (H-2 b/b , Fv1 b , Rfv3 r/s ) and between 8-weeks to 120-weeks old.
  • Mouse ages were defined as follows: mature young-adult (3 to 6 months; 12 to 24 weeks), middle-aged (10 to 14 months; 40 to 56 weeks), and aged (18 to >24 months; 72 to >96 weeks).
  • mature young-adult 3 to 6 months
  • aged (18 to >24 months) mice were compared.
  • mice 6-12 months were used (e.g., between mature young-adult and middle-aged).
  • mice For routine antibody validation experiments, mature young-adult (3 to 6 months) mice were used. Mice were routinely monitored, and abnormal or sick mice were excluded from further analysis. Mice were bred and maintained at Stanford University’s Research Animal Facility or at the Rocky Mountain Laboratories. All animal experiments were performed according to guidelines established by the Administrative Panel on Laboratory Animal Care of Stanford University or on an Animal Study Proposal approved by the Animal Care and Use Committee of the Rocky Mountain Laboratories (RML 2018-058, RML 2021-046) and carried out by certified staff in an Association for Assessment and Accreditation of Laboratory Animal Care International- accredited facility according to the institution’s guidelines for animal use, the basic principles in the NIH Guide for the Care and Use of Laboratory Animals, the Animal Welfare Act, and the United States Department of Agriculture and the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals.
  • Bone Marrow Cell Isolation Mice were euthanized and bone marrow was harvested following one of two methods. The unilateral or bilateral femurs, tibias, and pelvises were dissected, cleaned, and collected in a mortar bowl containing PBS supplemented with 2% FBS (FACS-buffer) and 1mg/mL DNAse-I (LS002007; Worthington). Bones were crushed, and the resulting cell suspension was passed through a 40 ⁇ m filter.
  • FBS FACS-buffer
  • 1mg/mL DNAse-I LS002007; Worthington
  • the femurs and tibias were dissected, cleaned, and cut at the joints and the bone marrow was flushed using an inserted 25-gauge needle and phosphate-buffered balanced salt solution (PBBS) with cells passed through a 100 ⁇ m filter. Cells were collected by centrifugation and washed with FACS- buffer multiple times. Red blood cells were depleted by ACK-lysis or by cKIT-enrichment. For ACK-lysis, cells were resuspended in 1mL ACK Lysing Buffer (A1049201; ThermoFisher) and incubated for 10 minutes at room-temperature.
  • PBBS phosphate-buffered balanced salt solution
  • cKIT-enrichment cells were Fc-blocked by incubation with 1mg/mL rat IgG (ab37361; abcam) for 30 minutes on ice, followed by the addition of anti-cKIT APC-eFluor780 (47-1171-82; ThermoFisher) for 30 minutes. Cells were collected by centrifugation and resuspended in FACS-buffer containing 10uL anti-APC MicroBeads (130-090-855; Miltenyi Biotec) and incubated for 20 minutes on ice.
  • Flow cytometry Flow cytometry was performed on a FACS Aria II (BD Biosciences) or FACS Symphony (BD Biosciences). For absolute cell counts, cells were counted prior to flow- cytometry, or a known volume of Precision Count Beads TM (424902; BioLegend) was added to a known volume of cells, and calculations were performed according to manufacturer’s instructions. For all experiments with Precision Count Beads TM , the stock concentration was assumed to be 1x10 6 particles/mL, based on manufacturer’s documentation.
  • HSPC stain anti-FLT3 APC (ThermoFisher; 17-1351-82) or PerCP-eFluor710 (eBioscience; 46-1351-82), goat anti- mouse NEO1 (R&D; AF1079), anti-CD150 PE-Cy7 (BioLegend; 115914; clone TC15- 12F12.2), anti-IL7Ra PE-Cy5 (ThermoFisher; 15-1271-82 or BioLegend; 135016) or APC (BioLegend; 135012), anti-CD16/32 BV510 (BioLegend; 101308), anti-cKit APC-eFluor780 (ThermoFischer; 47-1171-82), anti-mouse Lineage Cocktail (includes anti-CD3, anti-Ly-6G/C, anti-CD11b, anti-CD45R,
  • anti-CD150 clone mShad150 PE eBioscience; 12-1502-80
  • PE-Cy7 eBioscience; 25-1502-82
  • anti-CD150 clone 9D1 PE eBioscience; 12-1501-80
  • anti-CD150 clone Q38-480 PE BD; 562651
  • anti- CD62p PE BioLegend; 148308
  • anti-Ly6D PE eBioscience; 12-5974-80
  • anti-CD51 PE (12-0512-81; ThermoFisher
  • anti-CD61 PE 561910; BD
  • anti-CD31 PE 561073; BD
  • anti- CD38 PE (12-0381-81; ThermoFisher
  • anti-CD47 clone MIAP301 PE (127507; BioLegend
  • anti-CD47 clone MIAP410 PE LS-C810701-25; LSBio
  • anti-CD62p PE 148305; BioLegend
  • anti-ALCAM PE (12-1661-82; ThermoFisher
  • anti-CD9 PE (124805; BioLegend
  • anti-ESAM PE 136203, BioLegend
  • anti-TIE2 PE (124007; BioLegend
  • anti-CD201 PE 141503; BioLegend
  • anti-cKIT clone ACK2 PE (135105; BioLegend).
  • the absolute numbers of cells was quantified in total bone marrow (non-cKIT enriched).
  • HSCs and HSC subsets e.g., my-HSCs, bal-HSCs, etc.
  • the absolute numbers of cells was quantified in total bone marrow (non-cKIT enriched), or the percentage of HSC/HSC subsets per KLS (Lin – cKIT + Sca1 + ) cells was calculated in the cKIT-enriched fraction and multiplied by the total number of KLS cells quantified in a paired sample of total bone marrow (non-cKIT enriched).
  • Intracellular staining was performed as described. For erythroid cell analysis, spleen cells were first incubated for 30 min with mAb 34, a mouse IgG2b specific for the FV glycoGag protein expressed on infected cells, then stained with anti-mouse IgG2b FITC (BD; 553395) and anti-Ter119 PE-Cy7 (Invitrogen; 25-5921-82). Cells from uninfected controls were used for gating strategy. For non-fixed cells, to determine viability, cells were incubated in buffer containing SYTOX Red Dead Cell Stain (Life Technologies) or SYTOX Blue Dead Cell Stain (ThermoFisher; S34857).
  • anti-CD150 antibody clone 4 (mShad150) does not block anti- CD150 clone 2 (Q38)
  • bone-marrow cells were incubated with saturating concentrations (200ug/mL) of unlabeled anti-CD150 clone mShad150 and then stained with PE-conjugated anti-CD150 clone Q38; PE-Cy7 conjugated anti-CD150 clone mShad150 was used as a control.
  • mouse IgG2a (SB115d; SouthernBiotech) and IgG2b (SB115h; SouthernBiotech) anti-goat antibodies do not block donkey anti-goat IgG AF488 (abcam; ab150129)
  • bone-marrow HSPC stained cells were incubated with saturating concentrations (100ug/mL) of unlabeled mouse IgG2a (6158-01; SouthernBiotech) or IgG2b (6157-01; SouthernBiotech) anti-goat antibodies and then stained with donkey anti-goat AF488.
  • Antibody staining was performed in FACS-buffer solution (PBS with 2% FBS and DNAse-I) at a 1:1 ratio to Brilliant Stain Buffer (563794; BD Biosciences). Non-specific binding was blocked with FcR Blocking Reagent (130-059-901; Miltenyi Biotec) for 5 minutes on ice, followed by the addition of the following antibodies: anti-lineage panel PE-Cy5 (anti-CD3, anti- CD4, anti-CD8, anti-CD11b, anti-CD14, anti-CD19, anti-CD20, anti-CD56, anti-CD235a), anti- CD34 APC-Cy7 (343514; Biolegend), anti-CD45RA BV-785 (304139; Biolegend), anti-CD38 APC (555462; BD), anti-CD90 FITC (328107; Biolegend), and one of anti-human PE: anti- CD62P clone AK4 (304905; Biolegend), anti-CD62P clone
  • mice received injections of antibodies resuspended in PBS intraperitonially, unless otherwise specified.
  • Control animals received an equivalent volume of PBS or an equivalent amount of isotype control antibodies: mouse IgG1 (clone MOPC-21, Bio X Cell), rat IgG2b (clone LTF-2, Bio X Cell), or rat IgG2a (clone RTK2758, BioLegend).
  • isotype control antibodies demonstrated no impact on phenotype, PBS was used as a control in many experiments to minimize costs, as described.
  • NEO1 For NEO1, 30 ⁇ g, 90 ⁇ g, or 200 ⁇ g goat anti-NEO1 (polyclonal cat# AF1079, R&D) was administered on Day -9 for NEO1 v1 protocol, and when indicated, 150 ⁇ g mouse IgG2a (SB115d; SouthernBiotech) or IgG2b (SB115h; SouthernBiotech) anti-goat was administered 24-hours later on Day -8, for NEO1 v2 protocol.
  • mouse IgG1 anti-CD47 (clone MIAP410, Bio X Cell) was administered on Day -11 (100 ⁇ g) and on Days -9 to Day -5 (500 ⁇ g daily), as previously described.
  • rat anti-cKIT (clone ACK2, Bio X Cell) was injected retro-orbitally on Day -9 (30 ⁇ g, 50 ⁇ g, or 100 ⁇ g), and mice were administered 400 ⁇ g of diphenhydramine at least 30 min prior to administration, as previously described. Mice were euthanized for bone-marrow analysis on Day 0 (e.g., approximately 1-week), at approximately 8-10 weeks, or at approximately 14-16 weeks. [0140] Blood Cell Isolation and Plasma Immunoassays.
  • mouse peripheral blood was collected in EDTA tubes after removal of cells through centrifugation at 500 RCF for 10 min, whereupon plasma was transferred to a clean tube and centrifuged for an additional 10 min at 13,000 RCF, while the red blood cells were depleted with ACK-lysis, followed by a PBS wash, and then stained for flow cytometry as described above.
  • For absolute cell counts per mL the volume of blood obtained per animal was recorded, and a known volume of Precision Count Beads TM (424902; BioLegend) was added to a known volume of cells, and calculations were performed according to manufacturer’s instructions assuming a Precision Count Beads TM stock concentration of 1x10 6 particles/mL.
  • Plasma was frozen at -80C until processing by the Stanford Human Immune Monitoring Center (HIMC), as described. Samples were run in technical triplicate using the 48- Plex Mouse ProcartaPlexPanel TM (EPX480-20834-901; ThermoFisher Scientific) or the Mouse Acute Phase Magnetic Bead Panel 2 (MAP2MAG-76K; Millipore Sigma). MFI average value were compared after removal of statistical outliers using the extreme studentized deviate (ESD) Grubbs statistical test ( ⁇ 0.0001). For comparison of estimated concentrations, values below the limit of detection were assigned the value equal to this lower limit. [0141] Friend Virus Mouse Model. Ethics and biosafety statement.
  • the Friend retrovirus (FV) stock used in these experiments was FV-NB, a lactate dehydrogenase virus (LDV)-free complex containing NB- tropic Friend murine leukemia helper virus (F-MuLV) and polycythemia-inducing spleen focus- forming virus (SFFV) generated as a spleen cell homogenate from infected BALB/C mice.
  • the live attenuated vaccine was an NB-tropic F-MuLV helper stock, which replicates poorly without SFFV-induced proliferation, generated as a supernatant from infected Mus dunni cells.
  • mice of (C57BL/10 x A.BY)F1 background were vaccinated by 0.1 ml intravenous (i.v.) injection of 10 5 focus-forming units (FFU) of virus in phosphate-buffered, balanced salt solution (PBBS).
  • PBBS phosphate-buffered, balanced salt solution
  • mice were injected i.v. with 0.2 ml PBBS containing 20,000 spleen focus- forming units of FV-NB complex.
  • Infectious centers assay Titrations of single cell spleen suspensions were plated onto susceptible Mus dunni cells and allowed to incubate in vitro for 2 days at 37 o C and 5% CO2.
  • Antigen-expressing cells in vivo To quantify Ag34+ expressing cells in vivo, Ag34 expression was determined by mAb 34 antibody staining by flow-cytometry. Cells from uninfected controls were used to define the background level of staining. A positive vs. negative threshold was set equal to the highest level of background staining observed in non- infected animals, and only samples with values higher than this threshold were considered positive.
  • HSCs Beerman (a, GSE43729), Bersenev (b, GSE39553), Flach (c, GSE48893), Maryanovich (d, GSE109546), Norddahl (e, GSE27686), Wahlestedt (f, GSE44923), Renders (g, GSE128050), Sun (h, GSE47819).
  • the following datasets were used to compare mouse myeloid-biased HSCs vs. balanced HSCs: Gulati (i, GSE130504), Montecino-Rodriguez (j, GSE112769) Sanjuan-Pla (k, E-MEXP-3935). The following datasets were used to compare human old vs.
  • HSCs Pang (a, GSE32719), Adelman (b, GSE104406), Nilsson (c, GSE69408), Hennrich (d, GSE115348).
  • RNA-sequencing of FACS-purified mouse HSCs was processed and analyzed with GREIN or GEO2R.
  • Murine progenitors, mature cells, and tissues To determine gene expression of mouse progenitors and mature cells, processed data was obtained directly from Gulati on 23 hematopoietic phenotypes based on 64 microarray expression profiles extracted by the Gene Expression Commons. Gene expression data from bulk mouse tissues was obtained from: Tabula Muris (GSE132040) and (Kadoki, GSE87633). Data was processed with Phantasus (v1.19.3).
  • RNA-sequencing of FACS-purified mouse HSCs was processed and analyzed with GREIN or GEO2R.
  • RNA-sequencing of purified mouse HSCs approximately 1,000 total HSCs (KLS FLT3 – CD34 – CD150 + ) were FACS-sorted from aged control mice or aged mice that received antibody-conditioning 9 days earlier and immediately added to lysis buffer.
  • Libraries were prepared using Takara SMART-Seq v4 Ultra low Input RNA kit and sequencing was performed with NovaSeq with approximately 20 million paired reads per sample by MedGenome Inc. Differential gene expression was performed using DESeq2 with fold change shrinkage. Heatmaps were generated using Phantasus (v1.21.5) with FPKM values as input and Limma to define differentially expressed genes.
  • GSEA was conducted on genes ranked by DESeq2 test statistic using WEB-based GEne SeT AnaLysis Toolkit (WebGestalt 2019) with default parameters using a custom list of curated gene-signatures.
  • the following datasets were used to obtain gene-signatures Young vs. Old HSCs: Svendsen (i), Kuribayashi (ii), Maryanovich (iii, GSE109546), Norddahl (iv, GSE27686), Montecino-Rodriguez (v, GSE112769), Wahlestedt (vi, GSE44923), Mann (vii, GSE100428), Renders (viii, GSE128050).
  • a clonogenic common myeloid progenitor that gives rise to all myeloid lineages Nature 404, 193-197.
  • a clonogenic common myeloid progenitor that gives rise to all myeloid lineages Nature 404, 193-197.
  • Beerman I., Bhattacharya, D., Zandi, S., Sigvardsson, M., Weissman, I.L., Bryder, D., and Rossi, D.J. (2010). Functionally distinct hematopoietic stem cells modulate hematopoietic lineage potential during aging by a mechanism of clonal expansion. Proc Natl Acad Sci U S A 107, 5465-5470.10.1073/pnas.1000834107. [0154] Beerman, I., Bock, C., Brian, Zachary, Gu, H., Meissner, A., and Derrick (2013).
  • Jaiswal, S., and Ebert, B.L. 2019. Clonal hematopoiesis in human aging and disease. Science 366.10.1126/science.aan4673.
  • CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis. Cell 138, 271-285. 10.1016/j.cell.2009.05.046.
  • HMGA2 promotes long-term engraftment and myeloerythroid differentiation of human hematopoietic stem and progenitor cells. Blood Adv 3, 681-691. 10.1182/bloodadvances.2018023986.
  • Lymphoid-Biased Hematopoietic Stem Cells are Maintained with Age and Efficiently Generate Lymphoid Progeny. Stem Cell Reports 12, 584-596. 10.1016/j.stemcr.2019.01.016.
  • Hematopoietic stem cell self-renewal versus differentiation. Wiley Interdiscip Rev Syst Biol Med 2, 640-653.10.1002/wsbm.86. [0219] Siegel, R.L., Miller, K.D., Fuchs, H.E., and Jemal, A. (2022). Cancer statistics, 2022. CA Cancer J Clin 72, 7-33.10.3322/caac.21708. [0220] Spangrude, G.J., Heimfeld, S., and Weissman, I.L. (1988). Purification and characterization of mouse hematopoietic stem cells. Science 241, 58-62. 10.1126/science.2898810.
  • Micro-environmental sensing by bone marrow stroma identifies IL-6 and TGF ⁇ 1 as regulators of hematopoietic ageing. Nat Commun 11, 4075.10.1038/s41467-020- 17942-7. [0227] Wahlestedt, M., Norddahl, G.L., Sten, G., Ugale, A., Frisk, M.A., Mattsson, R., Deierborg, T., Sigvardsson, M., and Bryder, D. (2013). An epigenetic component of hematopoietic stem cell aging amenable to reprogramming into a young state. Blood 121, 4257-4264.10.1182/blood- 2012-11-469080.
  • Stem cells units of development, units of regeneration, and units in evolution. Cell 100, 157-168.10.1016/s0092-8674(00)81692-x.
  • Weissman, I.L. (2015a) Stem cells are units of natural selection for tissue formation, for germline development, and in cancer development. Proceedings of the National Academy of Sciences 112, 8922-8928.10.1073/pnas.1505464112.
  • Weissman, I.L. (2015b) Stem cells are units of natural selection for tissue formation, for germline development, and in cancer development. Proc Natl Acad Sci U S A 112, 8922- 8928.10.1073/pnas.1505464112.
  • Circulating hematopoietic stem and progenitor cells are myeloid-biased in cancer patients.
  • Clonal analysis unveils self-renewing lineage-restricted progenitors generated directly from hematopoietic stem cells.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Engineering & Computer Science (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La présente invention concerne un procédé cliniquement applicable pour rééquilibrer la production de cellules hématopoïétiques chez un mammifère, y compris, sans s'y limiter, chez un mammifère âgé, par déplétion sélective de cellules souches hématopoïétiques orientées production de myéloïdes (my-HSC). Cette déplétion sélective permet une production accrue de cellules lymphoïdes par rapport aux cellules myéloïdes.
EP23886580.2A 2022-11-04 2023-10-30 Rééquilibrage du système immunitaire par déplétion de cellules souches hématopoïétiques orientées production de myéloïdes Pending EP4611807A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202263422765P 2022-11-04 2022-11-04
PCT/US2023/036280 WO2024097131A1 (fr) 2022-11-04 2023-10-30 Rééquilibrage du système immunitaire par déplétion de cellules souches hématopoïétiques orientées production de myéloïdes

Publications (1)

Publication Number Publication Date
EP4611807A1 true EP4611807A1 (fr) 2025-09-10

Family

ID=90931289

Family Applications (1)

Application Number Title Priority Date Filing Date
EP23886580.2A Pending EP4611807A1 (fr) 2022-11-04 2023-10-30 Rééquilibrage du système immunitaire par déplétion de cellules souches hématopoïétiques orientées production de myéloïdes

Country Status (2)

Country Link
EP (1) EP4611807A1 (fr)
WO (1) WO2024097131A1 (fr)

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2003245752A1 (en) * 2002-06-28 2004-01-19 Bio Transplant, Inc. Process for promoting graft acceptance by depletion of hematopoietic stem cells
US11066648B2 (en) * 2016-08-29 2021-07-20 Hackensack University Medical Center Compositions and methods for reprogramming adult cells through the stemness of a platelet rich fraction of blood containing platelet-like cells in humans
LT3535392T (lt) * 2016-11-02 2024-04-25 Universität Basel Imunologiškai atpažįstami ląstelių paviršiaus variantai, skirti naudoti ląstelių terapijoje
US20250049887A1 (en) * 2021-12-09 2025-02-13 Fred Hutchinson Cancer Center Methods and compositions for modulating the myeloid arm of the immune system

Also Published As

Publication number Publication date
WO2024097131A1 (fr) 2024-05-10

Similar Documents

Publication Publication Date Title
US20220265711A1 (en) Engineered immune cells comprising a recognition molecule
JP7447388B2 (ja) 感染性疾患の治療のための共受容体システム
US20230048244A1 (en) Anti-tcr antibody molecules and uses thereof
US11906519B2 (en) Methods for identifying LILRB-blocking antibodies
JP7628155B2 (ja) Ｉｌ－１ｒａｐを標的とするｃａｒ－ｔ細胞及びその使用
JP2017535283A (ja) 抗ｃｌｄｎキメラ抗原受容体および使用方法
JP2018506981A (ja) 抗ｄｌｌ３キメラ抗原受容体および使用方法
FI75364C (fi) Foerfarande foer framstaellning av en monoklonal antikropp foer maenskliga cytotoxiska och suppressor-t-celler medelst en ny hybridcellinje.
JP7650351B2 (ja) 非古典的ｈｌａ－ｉとネオアンチゲンとを含む複合体を標的とする抗体、およびその使用方法
EP3439699A2 (fr) Procédé d'élimination de cellules souches hématopoïétique/progéniteurs hématopoïétiques (csh/ph) chez un patient à l'aide d'anticorps bispécifiques
CN114828861A (zh) 表达多特异性免疫细胞接合器的溶瘤病毒
JP2006528627A (ja) アロ反応性ナチュラルキラー細胞を使用する治療用抗体の有効性を増加するための方法および組成物
Graves et al. Development and characterization of a canine-specific anti-CD94 (KLRD-1) monoclonal antibody
JP2024095755A (ja) 白血病幹細胞を根絶することによる急性骨髄性白血病の治療のための方法および医薬組成物
WO2024097131A1 (fr) Rééquilibrage du système immunitaire par déplétion de cellules souches hématopoïétiques orientées production de myéloïdes
US20030055223A1 (en) Monoclonal antibodies to antigens expressed by hematopoietic facilitatory cells
ES2875550T3 (es) Anticuerpos monoclonales que reconocen específicamente el antígeno DLA-DR de canino y sus usos
WO2026025030A1 (fr) Rééquilibrage du système immunitaire par déplétion de cellules souches hématopoïétiques orientées myéloïde exprimant ccr5
EP3997128A1 (fr) Procédé d'élimination de cellules souches hématopoïétique/progéniteurs hématopoïétiques (csh/ph) chez un patient à l'aide d'anticorps bispécifiques
US20250241953A1 (en) Tdt-specific chimeric receptors and methods of their use
Sierro-Martínez Translational Advances in CAR-T Therapy: from clinical validation of an academic CAR-T cell to post-infusion biomarker discovery
Bergua et al. First-in-class anti-human CD45RC antibody targets CD45RChigh T and B cells to mitigate pathogenic immune responses
Grain et al. Evaluation of multi-antigen targeting ADCC strategies in pediatric BCP-ALL
Scroggins Generation and characterization of regulatory dendritic cells for the amelioration of acute graft versus host disease
Morabito et al. Expression of myelomonocytic antigens on chronic lymphocytic leukemia

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20250508

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC ME MK MT NL NO PL PT RO RS SE SI SK SM TR

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)