Targeting AIRE-expressing macrophages for tumor immunotherapy

Abstract

Described herein are methods of identifying a population of Aire-expressing tumor-associate macrophages (ATAMs) and methods of targeting ATAMS for the treatment of cancer.

Description

TARGETING AIRE-EXPRESSING MACROPHAGES FOR TUMOR IMMUNOTHERAPYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority benefit of U.S. Provisional Application No. 63 / 582,458, filed September 13, 2023, which is incorporated by reference for all purposes.BACKGROUND OF THE INVENTION

[0002] Immunotherapy has revolutionized the treatment of cancer, but many malignancies remain resistant to these interventions. While extensive work has focused on effector and regulatory lymphocyte populations in tumors and their roles in the tumor microenvironment, less attention has been paid to the myeloid antigen-presenting populations that shape these immune responses.

[0003] Tolerogenic populations of antigen-presenting cells outside of the thymus characterized by expression of the autoimmune regulator Aire, which play critical roles in immune tolerance and homeostasis, have been previously defined (Gardner, et al. Science 321, 843-847, 2008; Gardner, et al. Immunity 39, 560-572, 2013; Wang, et al. Science immunology 6, eabl5053, 2021). It was reported that such extrathymic Aire-expressing cells (eTACs) are important for the maintenance of maternal-fetal immune tolerance, and may govern commensal-specific peripheral regulatory T cell development (Gardner et al., 2013, supra, Gillis-Buck, et al., Science immunology 6, ,abfl96, 2021; Sun, et al. Immunological reviews 308, 93-104, 2022; Akagbosu, et al. Nature 610, 752-760, 2022; Kedmi, et al. Nature 610, 737-743, 2022).BRIEF SUMMARY

[0004] The following provides a brief summary of certain aspects disclosure, but does not constitute a comprehensive summary of all aspects.

[0005] In one aspect, the disclosure features a method of inhibiting growth of a tumor, the method comprising: administering a multispecific antibody, e.g., a bispecific antibody, that selectively targets a population of Aire-expressing tumor-associated macrophages (ATAMs),wherein the multispecific antibody comprises at least a first and a second binding domain, wherein the first binding domain specifically binds to a first ATAM-specific cell surface marker expressed by an ATAM present in microenvironment of the tumor and the second binding domain specifically binds a second ATAM-specific cell surface marker different from the from the first ATAM-specific cell surface marker, wherein the multispecific antibody is linked to a cytotoxic reagent and / or at least one of the binding domains blocks binding of the target ATAM-specific cell surface marker to a soluble ligand or ligand present on the surface of another cell to block activity. In some embodiments, the multispecific antibody is administered intravenously. In some instances, the multispecific antibody is administered locally to the tumor. In some embodiments, the first cell surface marker is CD81 and the second cell surface marker is selected from the group consisting of CD63, CD72, TMEM119, TMEM171 Psd3, Timp2, Mertk, Pf4, Syngrl, Tnfsfl3b, Itga9, Ctla2b, Tmeml l9. 112ra, Adgrel, Pmepal, Tmeml71, Lhfpl2, and Slamf6. In some embodiments, the tumor is a melanoma, a breast cancer tumor, or a colorectal cancer tumor.

[0006] In a further aspect, the disclosure features a method of inhibiting growth of a tumor, the method comprising locally administering to the tumor a first binding moiety that specifically binds to a first ATAM-specific cell surface marker expressed by an Aire- expressing tumor associated macrophage (ATAM) present in a microenvironment of the tumor, wherein the first binding moiety is an antibody, aptamer, or ligand, and is linked to a cytotoxic agent and / or blocks activity of the cell surface marker, wherein the first binding moiety is selected from the group consisting of CD63, CD72, TMEM119, TMEM171, Psd3, Timp2, Mertk, Pf4, Syngrl, Tnfsfl3b, Itga9, Ctla2b, Tmeml l9. 112ra, Adgrel, Pmepal, Tmeml71, Lhfpl2, and Slamf6. In some embodiments, the method further comprises locally administering to the tumor a second binding moiety that specifically binds to a second ATAM-specific cell surface marker expressed by the ATAM present in the tumor microenvironment, wherein (i) the second ATAM-specific cell surface marker is different from the first ATAM-specific cell surface marker and is selected from the group consisting of CD63, CD72, TMEM119, TMEM171, Psd3, Timp2, Mertk, Pf4, Syngrl, Tnfsfl3b, Itga9, Ctla2b, Tmeml l9. 112ra, Adgrel, Pmepal, Tmeml71, Lhfpl2, and Slamf6; (ii) the second binding moiety is linked to a cytotoxic agent and / or blocks activity of the cell surface marker; and (iii) the second binding moiety is an antibody, aptamer, or ligand. In some embodiments, the first binding moiety binds to CD81. In some embodiments, the tumor is a melanoma, a breast cancer tumor, or a colorectal cancer tumor.

[0007] In another aspect, the disclosure features a method of inhibiting growth of a tumor, the method comprising locally administering to the tumor a first binding moiety that specifically binds to a first ATAM-specific secreted marker selected from a group consisting of Clqa, Ccl2, Clqc, Ccl7, Pf4, Olfml3, Clqb, Grn, Selenop, Ccl4, and Apoe expressed by an Aire-expressing tumor associated macrophage (ATAM) present in a microenvironment of the tumor, wherein the first binding moiety is an antibody, aptamer, or ligand, and blocks binding of the first ATAM-specific marker to a binding partner, thereby blocking activity. In some embodiments, the method further comprises locally administering to the tumor a second binding moiety that specifically binds to a second ATAM-specific secreted marker expressed by the ATAM present in the tumor microenvironment, wherein (i) the second ATAM- specific cell surface marker is different from the first ATAM-specific cell surface marker and is selected from a group consisting of Clqa, Ccl2, Clqc, Ccl7, Pf4, 01fml3, Clqb, Grn, Selenop, Ccl4, and Apoe; and (iii) the second binding moiety is an antibody, aptamer, or ligand that blocks binding of the second ATAM-specific marker to a binding partner, thereby blocking activity. In some embodiments, the tumor is a melanoma, a breast cancer tumor, or a colorectal cancer tumor.

[0008] In some embodiments, a method of the invention as described herein, e.g., as described in the preceding three paragraphs, further comprises administering a monoclonal antibody that selectively binds to receptor activator of nuclear factor-i<B ligand (RANKL). In some embodiments, anti-RANK-L monoclonal antibody is administered systemically. In some instances, the anti-RANK-L monoclonal antibody is administered locally to the tumor. In some embodiments, the anti-RANK-L monoclonal antibody is denosumab. In some embodiments, the anti-RANK-L monoclonal antibody is administered at the same time as the binding agent that binds to the cell surface marker expressed by the ATAM or is administered within one week. In some embodiments, the tumor is a melanoma, a breast cancer tumor, an ovarian tumor or a colorectal cancer tumor.

[0009] In a further aspect, the disclosure features a method of inhibiting growth of a tumor, the method comprising administering to a subject having a tumor with ATAMS present in the tumor microenvironment (i) an anti-CD81 antibody linked to a cytotoxic agent, and (ii) administering an anti-RANK-L monoclonal antibody, e.g., denosumab. In some embodiments, the anti-CD81 linked to the cyotoxic agent and the anti-RANK-L monoclonal antibody are administered intravenously. In some embodiments, the anti-CD81 linked to the cyotoxic agent and the anti-RANK-L monoclonal antibody are administered directly to thetumor, e.g., injected. In some embodiments, the tumor is a melanoma, a breast cancer tumor, an ovarian tumor or a colorectal cancer tumor.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1 shows illustrative RNAs that are differentially expressed in AT AMS.

[0011] FIG. 2A-C: Tumors contain Aire-expressing (Aire+) hematopoietic populations. (A) Flow data form MC38 tumors implanted into WT or Aire-driven GFP (Adig) mice. (B) Immunofluorescent staining of MC38 tumors from WT and Adig mice. (C) Proportion of AireGFP tumor-associated populations classified by flow cytometry as Macrophages (Macs; CD64+, F4 / 80+, Ly-6c-) Monocyte / macrophages (MoMac; CDl lc-, CDl lb+, CD64+, F4 / 80+, Ly-6c+), Monocytes (Monos; CDl lc-, CDl lb+, CD64-, F4 / 80int, Ly-6c+) or dendritic cells (DCs; CD1 lb-, CD1 lc+, CD64-, F4 / 80-). N = 6.

[0012] FIG. 3 A-G: Ablation of extrathymic Aire-expressing cell profoundly inhibits tumor growth in the presence and absence of checkpoint blockade. (A) Experimental design for AireDTR / MC38 model. Treatment was initiated once tumors reached >180mm3, at which point they are resistant to conventional checkpoint blockade. (B) Tumor growth curves for AireDTR+ and AireDTR- mice treated as indicated in (A). (C) Tumor growth curves for WT congenic cohorts (N=6 each group) lethally irradiated and reconstituted with AireDTR+ or AireDTR- bone marrow, validated for reconstitution at 6 weeks, implanted with MC38 tumors as above, and monitored for tumor growth. (D) Experimental design for AireDTR / MC38 model with checkpoint blockade. (E) Tumor growth curves for AireDTR and WT mice, all receiving DT and anti-PD-1 as per design in (D). (F) Tumor growth curves for previously thymectomized cohort of Aire DTR+ and AireDTR- mice treated as in (D). (G) CD206-DTR+ and WT control mice implanted with MC38 tumors and DT-treated using identical implantation and treatment strategy as above for AireDTR system. 3B, 3C, 3E, and 3F: upper line, DTR', lower line, DTR+; 3G: upper line, DTR+, lower line, DTR'.

[0013] FIG. 4: CyTOF analysis of tumor associated ATAM populations across three tumor models - MC38 (colon), B16 (melanoma), AT3 (breast). Leiden clustering and uMAP projection of cell populations captured (left) with feature plot of Aire-GFP intensity per cell (middle) and heatmap with two-dimensional hierarchical clustering of top markers differentially overexpressed on AIRE-GFP+ cells in each tumor. ATAM subset highlighted by arrow (right panel).

[0014] FIG. 5A-B: Preliminary scRNAseq of AireGFP+ and AireGFP- CD45+ populations from MC38 tumors implanted subcutaneously into B6 AireGFP (Adig) mice. (A) Leiden clustering of all cells, sorted on live CD45+ cells, either AireGFP+ (right panel, orange) or GFP- cells (right panel, blue). (B) Feature plots of indicated genes showing scaled expression per cell in populations as sorted from A.

[0015] FIG. 6A-C: Aire-expressing immune cells are detected in human cancers and resemble their murine counterparts. Publicly available human single-cell RNA sequencing tumor immune atlas data (from website pubmed.ncbi.nlm.nih.gov / 34548323 / ) was processed and annotated using an approach mirroring the original publication method (A); and queried for Aire gene expression (B). (C) Aire-expressing immune cells were queried across multiple cancer types and show up across a range of cancers, and are notably enriched in ovarian cancers. The majority of Aire expression in human cancers was observed in M2-like tolerogenic and proliferating TAMS.

[0016] FIG. 7: Impact of ablation of Aire-expressing cells on tumor growth in large B 16 melanoma tumors, which are normally highly resistant to immunotherapy, without checkpoint blockade. Tumor growth curves from AireDTR+ and AireDTR- control mice implanted with B16F10 tumors; ablation with DT initiated when tumor reached 180-200 mm3. Upper line, DTR', lower line, DTR+

[0017] FIG. 8: AT AM ablation-mediated tumor growth suppression required CD8+ T cell activity. Tumor growth curves from AireDTR+ and AireDTR- control mice implanted with MC38 tumors; anti-CD8 antibody depletion and ablation with DT initiated when tumors reached 180-200 mm3. N=6 per group. Lower line, DTR', upper line, DTR+

[0018] FIG. 9A-C: Validation of Aire-driven LSL-DTR-eGFP (deleTAC) conditional deletion model. (A) Schematic of Aire-driven LDL-DTR-eGFP (deleTAC) mouse model. (B) Specificity of targeting for DTR-eGFP in deleTAC X FoxNl-Cre or RORgt-Cre mouse line showing highly selective ablation of Aire-expressing cell subsets dependent on the Cre recombinase used.

[0012] FIG. 10A-E. RANK signaling drives Aire expression in AT AMs, and interfering with this signaling is protective against tumor growth. (A) Feature plot as shown in FIG. 5 of RANK (Tnfrsfl la) expressing in AT AMs. (B) Loss of Aire reporter expression in AT AMs in mice conditionally deficient in RANK signaling in macrophages (LysM-Cre+ x RANK flox / flox) when compared to WT (LysM-Cre- x RANK flox / flox) littermates. (C) Tumorgrowth curves of MC38 tumors implanted into WT (LysM-Cre- x RANK flox / flox) and macrophage-specific conditionally RANK-deficient mice (LysM-Cre+ x RANK flox / flox). (D) Antibody blockade of RANK signaling is synergistic with anti-PDl blockade in MC38 tumors; tumor growth curves by treatment group. (E) Final MC38 tumor sizes in MC38 mice treated with isotype, anti-PDl, anti -RANK, and combination anti-RANK / anti-PDl therapy; IOC: Upper line, Cre', lower line, Cre+; 10D: Upper line, iso; second line from top, aRANKL; third line from top, aPDl; bottom line, combo.

[0019] FIG. 11 A-F. A, Experimental schematic of bone marrow chimera (BMC) generation and tumor injection. B. UMAP of tumor-infiltrating myeloid cells (CD45+CD1 lb+) in Aire- KO BMC mice implanted with MC38 tumors analyzed on day 17 via spectral flow cytometry. C. Log density ratio representing relative abundance of myeloid populations in Aire-KO vs Aire WT mice. D. Relative abundance of myeloid clusters that are significantly changing in Aire-KO BMC mice compared to Aire WT BMC controls. *p<0.05, **p<0.01, ***p<0.001. E. Experimental schematic for LAKO (Vavl-Cre x Aire fl / fl) mice or WT mice implanted with MC38 treated with anti-PD-1 or isotype control. F. Tumor growth curves for LAKO or littermate controls all treated with anti-PD-1. *p<0.05, **p<0.01, ***p<0.001 on day 18 (endpoint).DETAILED DESCRIPTION OF THE INVENTIONDEFINITIONS

[0020] As used in herein, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” can include two or more cells and reference to “an antibody” can include a combination of two or more such molecules, and the like.

[0021] Genes and proteins encodes by the genes are defined by symbol and nomenclature for the human gene as assigned by the Human Genome Organization (HUGO) Gene Nomenclature Committee.

[0022] The term “Aire” as used herein refers to the protein Autoimmune Regulator, which is a transcription factor that play a role in promoting self-tolerance in the thymus. Human Aire protein sequence information is available under UniProt ID 043918 (see, also NCBI reference sequence NP 000374.1. The UniProt entry describes four isoform sequences produced by alternative splicing. 043918-1 is assigned as the canonical Aire sequence. Sequence information for the mRNA can be found under NCBI Reference SequenceNM 000383.4. Mouse Aire protein sequence information is available under UniProt ID Q9Z0E3. Aire sequences from other mammals are also available in the UniProt database.

[0023] An “Aire-expressing tumor associated macrophage” or “ATAM” refers to a macrophage that is found in the tumor microenvironment that play a role in tumor immune evasion. “AT AMs” express Aire as well as additional surface markers, such as CD81, CD64, F4 / 80, Trem2, Clq, and other cell surface markers as detailed herein that distinguishes the cell from other macrophages in the tumor microenvironment.

[0024] In the context of the present invention, an “ATAM-specific marker” refers to a protein that is preferentially expressed in ATAMS compared to other macrophage populations. In typical embodiments, an RNA encoding an ATAM-specific marker is expressed at a level at least two-fold, or at least five-fold higher in a population of ATAMs compared to other macrophages; and is expressed in at least 60% of the cells of the population of ATAMs.

[0025] In the present application, the term “eTAC” generally refers to an extrathymic Aire- expressing cell, which have been shown in various murine models to induce immune tolerance. ATAMs are subsets of eTACs.

[0026] As used herein, the term "antibody" means an isolated or recombinant binding agent that comprises the necessary variable region sequences to specifically bind an antigenic epitope. Therefore, an “antibody” as used herein is any form of antibody of any class or subclass or fragment thereof that exhibits the desired biological activity, e.g., binding a specific target antigen. Thus, it is used in the broadest sense and includes, but is not limited to, a monoclonal antibody (including full-length monoclonal antibodies), human antibodies, chimeric antibodies, single domain antibodies, such as nanobodies, diabodies, camelid- derived antibodies, monovalent antibodies, bivalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments including, but not limited to scFv, Fab, and the like so long as they exhibit the desired biological activity.

[0027] A “bispecific” antibody as used herein refers to an antibody comprising two binding domains, each of which binds to a different epitope, which can be on the same protein, but are often on different proteins. A “multispecific” antibody refers to an antibody that has binding specificities for two or more different epitopes. Bispecific or multispecific antibodies can be derived from full-length antibodies or antibody fragments (e.g., F(ab’)2 bispecific antibodies.

[0028] "Antibody fragments" comprise a portion of an intact antibody, for example, the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific or multivalent antibodies formed from antibody fragments. A "Fab" fragment contains a variable and constant domain of the light chain and a variable domain and the first constant domain (CHI) of the heavy chain. A F(ab')2 fragment has a pair of Fab fragments that are generally covalently linked near their carboxy termini by hinge cysteines. Other chemical couplings of antibody fragments are also known. An "Fv" is a minimal antibody fragment that contains a complete antigen-recognition and binding site and is a dimer of one heavy- and one light-chain variable region domain.

[0029] As used herein, “V-region” refers to an antibody variable region domain comprising the segments of Framework 1, CDR1, Framework 2, CDR2, and Framework 3, including CDR3 and Framework 4.\

[0030] The amino acid sequences of the CDRs and framework regions can be determined using various well-known definitions in the art, e.g., Kabat, Chothia, international ImMunoGeneTics database (IMGT), and AbM (see, e.g., Johnson et al., supra; Chothia & Lesk, 1987, Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. Biol.196, 901-917; Chothia C. et al., 1989, Conformations of immunoglobulin hypervariable regions. Nature 342, 877-883; Chothia C. et al., 1992, structural repertoire of the human VH segments J. Mol. Biol.227, 799-817; Al-Lazikani et al., J .Mol. Biol 1997, 273(4)). Definitions of CDRs are also described in the following: Ruiz et al., IMGT, the international ImMunoGeneTics database. Nucleic Acids Res., 28, 219-221 (2000); and Lefranc, M.-P. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. Jan l;29(l):207-9 (2001); MacCallum et al, Antibody-antigen interactions: Contact analysis and binding site topography, J. Mol. Biol., 262 (5), 732-745 (1996); and Martin et al, Proc. Natl Acad. Sci. USA, 86, 9268-9272 (1989); Martin, et al, Methods Enzymol., 203, 121-153, (1991); Pedersen et al, Immunomethods, 1, 126, (1992); and Rees et al, In Sternberg M.J.E. (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141-1721996). Reference to CDRs as determined by Kabat numbering are based, for example, on Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institute of Health, Bethesda, MD (1991)). Chothia CDRs are determined as defined by Chothia (see, e.g., Chothia and Lesk J. Mol. Biol.196:901-917 (1987)).

[0031] “Epitope" or "antigenic determinant" as used in the present disclosure in the context of antibody binding refers to a site on an antigen to which an antibody binds. Epitopes can be formed from contiguous amino acids and / or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol.66, Glenn E. Morris, Ed (1996). Binding of an antibody to an epitope can be influenced by other environmental factors, such as s the presence of calcium ions.

[0032] The term “bispecific antibody” as used herein, refers to an antibody that binds to two or more different epitopes. In some embodiments, a bispecific antibody binds to epitopes for two different target antigens.

[0033] As used herein, the term “specifically binds” to a target protein, refers to a binding reaction whereby the antibody binds to the target with greater affinity, greater avidity, and / or greater duration than it binds to a different target. In some embodiments, a target-binding protein has at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20- fold, 25-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, or greater affinity for the target compared to an unrelated target when assayed under the same binding affinity assay conditions. The term “specific binding,” “specifically binds to," or “is specific for" a particular target, as used herein, can be exhibited, for example, by a molecule (e.g., an antibody) having an equilibrium dissociation constant KD for the target of, e.g., 10-2 M or smaller, e.g., 10-3 M, 10-4 M, 10-5 M, 10-6 M, 10-7 M, 10-8 M, 10-9 M, 10-10 M, 10-11 M, or 10-12 M. In some embodiments, an antibody has a KD of less than 100 nM or less than 10 nM.

[0034] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0035] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.

[0036] As used herein, "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, and complements thereof. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses degenerate codon substitutions that encode the same amino acid (for protein encoding sequences), complementary sequences, splice variants, and the like as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed- base and / or deoxyinosine residues. The term “nucleic acid” can refer to a gene, cDNA, mRNA, shRNA, siRNA, oligonucleotide, polynucleotide, and the like.

[0037] The term “treat” and “treatment” refer to both therapeutic treatment and prophylactic or preventive measures, wherein the object is to prevent or slow down an undesired physiological change or disorder. For purpose of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. In other embodiments the terms “treat”, “treatment” and “treating” refer to the inhibition of the progression of a proliferative disorder, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g., stabilization of a physical parameter, or both. In other embodiments the terms “treat”, “treatment” and “treating” refer to the reduction or stabilization of tumor size or cancerous cell count.

[0038] As used herein, the term “subject” refers to a mammal, e.g., preferably a human. Mammals include, but are not limited to, humans and domestic and farm animals, such as monkeys (e.g., a cynomolgus monkey), mice, dogs, cats, horses, and cows, etc.

[0039] As used herein, the term “pharmaceutically acceptable carrier” refers to an excipient or diluent in a pharmaceutical composition. The pharmaceutically acceptable carrier must be compatible with the other ingredients of the formulation and not deleterious to the recipient. In the present invention, the pharmaceutically acceptable carrier must provide adequate pharmaceutical stability to the active ingredient. The nature of the carrier differs with the mode of administration. For example, for intravenous administration, an aqueous solution carrier is generally used; for oral administration, a solid carrier is preferred.Introduction

[0040] The inventors have identified an Aire-expressing population of macrophages (ATAMS) in the tumor microenvironment that play a role in tumor immune evasion. The inventors demonstrate that ablation of this population reduces tumor growth and extends overall survival and thus present a therapeutic target for the treatment of cancer.

[0041] ATAMS have patterns of macrophage cell surface markers and expression patterns, e.g., RNA expression profiles, that collectively distinguish these cells from other macrophages and other extrathymic Aire-expressing cells. This marker profile allows for selective targeting of ATAM populations to inhibit tumor growth by using binding agents, e.g., linked to a cytotoxic moiety, that specifically bind to ATAMs and / or disrupt ATAM signaling pathways to reduce or eliminate the population from tumors.ATAM expression profiles

[0042] ATAMs express canonical macrophage markers, including F480, CD64, and CD1 lb, and CD11c', but also have a distinct expression profile of marker mRNAs that are expressed at greater levels in ATAM populations in the tumor microenvironment compared to non-Aire-expressing macrophage populations. Such marker mRNAs include RNAs that encode proteins containing a domain that is expressed on the cell surface and RNAs that encode secreted proteins. CD81, Psd3, Timp2, CD63, Mertk, Syngrl, Tnfsfl3b, Itga9, Ctla2b, Tmeml l9, 112ra, CD72, Adgrel, Pmepal, Tmeml71, Lhfpl2, and Slamf6 proteins include domains expressed on the cell surface. Clqa, Cell 2, Clqc, Ccl7, Pf4, Olfml3, Clqb, Grn, Selenop, Ccl4, and Apoe are secreted proteins.

[0043] In some embodiments, at least 60% of the macrophages in ATAM populations in the tumor microenvironment, or at least 80% of the macrophages, express at least five or more of these markers, or at least 10 or more, at least 15 or more, or at least 20 or more of themarkers, at a level that is at least two-fold higher than in non-Aire-expressing macrophage populations.Therapeutic agents that target ATAMS

[0044] In some embodiments, ATAMS can be targeted using a binding agent (also referred to herein as a binding moiety) such as an antibody or aptamer, that selectively binds to a marker that is overexpressed in ATAMS compared to other macrophages, e.g., CD81 or another marker selected from the group consisting of Psd3, Timp2, CD63, Mertk, Syngrl, Tnfsfl3b, Itga9, Ctla2b, Tmeml l9, 112ra, CD72, Adgrel, Pmepal, Tmeml71, Lhfpl2, and Slamf6. In some embodiments, the binding agent is an antibody. For example, the antibody is a multispecific antibody, such as a bispecific antibody comprising two binding domains each of which targets a different cell surface marker that are characteristic of Aire-expressing macrophages. In some embodiments, such antibodies can be linked to a cytotoxic agent. In other embodiments, the antibody may block activity of the marker protein. In other instance, two or more antibodies, each of which target an ATAM-specific marker can be administered to a subject to inhibit tumor growth. In some embodiments, the one or more binding agents can be delivered administered locally to a tumor.Bispecific and multispecific antibodies

[0045] Generally, bispecific antibodies may be produced in a variety of molecular formats as reviewed by Brinkmann et al., Mabs 9: 182-212, 2017. See also, Wang et al, Antibodies (Basel) 8:43, 2019; Liu et al., Front. Immunol 8:38, 2017. Bispecific or multispecific antibody-based molecules may also be produced by chemical conjugation or coupling of individual full length IgGs or coupling of fragments of IgGs to form multispecific and multivalent antibody derivatives. Examples are chemically coupled Fab fragments, IgG- dimers, etc. In some embodiments, multispecific molecules may also be produced by combining recombinant and chemical methods.

[0046] In some embodiments, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion is typically to an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions.

[0047] Techniques for making bispecific or multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairshaving different specificities (see Milstein and Cuello, Nature 305: 537 (1983)) and “knob-in hole” engineering (see, e.g., U.S. Patent No. 5,731,168, and Atwell et ah, J. Mol. Biol. 270:26 (1997)). Nonlimiting exemplary knob-in-hole substitutions include T366W (knob) and T366S / L368A / Y407V (hole). In some embodiments, the knob-in-hole substitutions are in IgGl constant domains

[0048] Multispecific or bispecific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules. See, e.g., WO 2009 / 089004; Dillon et ah, Mabs 9(2): 213-230 (2017).

[0049] In some embodiments, a bispecific antibody comprises both knob-in-hole substitutions and electrostatic substitutions. See, e.g., WO 2016 / 172485.

[0050] Various additional molecular formats for multispecific antibodies are known in the art (see e.g., Spiess et al., Mol Immunol 67 (2015) 95-106). Multispecific antibodies may also be made by cross-linking two or more antibodies or fragments (see, e.g., US Patent No. 4,676,980, and Brennan et al., Science , 229: 81 (1985)); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J Immunol ., 148(5): 1547-1553 (1992) and WO 2011 / 034605); using the common light chain technology for circumventing the light chain mis-pairing problem (see, e.g., WO 98 / 50431); using “diabody” technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA , 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol ., 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al. J. Immunol. 147: 60 (1991).

[0051] Engineered antibodies with three or more antigen binding sites, including for example, “Octopus antibodies”, or DVD-Ig may also be generated (see, e.g., WO 2001 / 77342 and WO 2008 / 024715). Other examples of multispecific antibodies with three or more antigen binding sites can be found in WO 2010 / 115589, WO 2010 / 112193, WO 2010 / 136172, WO 2010 / 145792, and WO 2013 / 026831.

[0052] Multispecific antibodies may also be provided in an asymmetric form with a domain crossover in one or more binding arms of the same antigen specificity, i.e. by exchanging the VH / VL domains (see e.g., WO 2009 / 080252 and WO 2015 / 150447), the CHI / CL domains (see e.g., WO 2009 / 080253) or the complete Fab arms (see e.g., WO 2009 / 080251, WO 2016 / 016299, also see Schaefer et al, PNAS, 108 (2011) 1187-1191, and Klein at al., MAbs 8 (2016) 1010-20). In one aspect, the multispecific antibody comprises a cross-Fab fragment.The term “cross-Fab fragment” or “xFab fragment” or “crossover Fab fragment” refers to a Fab fragment, wherein either the variable regions or the constant regions of the heavy and light chain are exchanged. A cross-Fab fragment comprises a polypeptide chain composed of the light chain variable region (VL) and the heavy chain constant region 1 (CHI), and a polypeptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL). Asymmetrical Fab arms can also be engineered by introducing charged or non-charged amino acid mutations into domain interfaces to direct correct Fab pairing. See e.g., WO 2016 / 172485.

[0053] Bispecific and multispecific antibodies of the present disclosure are thus produced using methodology apparent to those of skill in the art.Antibody Conjugates

[0054] In some embodiments, an antibody, e.g., a multispecific antibody such as a bispecific antibody, is conjugated to a cytotoxic agent to provide an antibody-drug conjugate (ADS) for treatment. ADCs are well known in the art (e.g., Polakis (2016) Pharmacol. Rev., 68:3-19). The cytotoxic agent can be directly conjugated to an antibody or fragment thereof or conjugated via a linker.

[0055] Examples of cytotoxic agents include, without limitation: antimetabolites, alkylating agents, DNA minor groove binders, DNA intercalators, DNA crosslinkers, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, anti-mitotic agents, anthracyclines, cytoskeletal disruptors, taxanes, epothilones, toxins (e.g., diphtheria, exotoxin, ricin (e.g., ricin A), abrin, Pseudomonas exotoxin, saporin, etc.), topoisomerase I inhibitors, topoisomerase II inhibitors, nucleotide analogs, nucleotide precursor analogs, antibiotics (e.g., peptide antibiotics), platinum-based agents, maytansinoids (e.g., maitansine), auristatins (e.g., monomethyl auri statin E (MMAE)), doxorubicins, duocarmycins (e.g., thienoindoles), irinotecans, and pyrrolobenzodiazepines (PDB) or pyrrolobenzodiazepine dimers (see, e.g., Carlson (2012) Biotechnol. Healthc., 9(4): 28-31; WO 2005 / 040170).

[0056] In some embodiments, In some embodiments, the cytotoxic agent is a radioactive isotopes (e.g.,211At,131I,125I,90Y, 186RE,188Re,153Sm,212Bi,32P,212PB and other radioistoptes); a chemotherapeutic agents or drug such as, methotrexate, adriamicin, or vinca alkaloids (vincristine, vinblastine, or etoposide), doxorubicin, melphalan, mitomycin C,chlorambucil, daunorubicin or other intercalating agents); or a growth inhibitory agent such as an enzyme or fragment thereof such as a nucleolytic enzyme; and the like.Non-antibody binding agents

[0057] In some embodiments, a binding agent that targets an ATAM is an aptamer. Aptamers interact with their targets by recognizing a specific three-dimensional structure. In some instances, the aptamer is a peptide aptamers. Peptide aptamers are composed of a short variable peptide loop attached at both ends to a protein scaffold such as the bacterial protein thioredoxin-A. A peptide aptamer specific to a target of interest may be selected using any method known by the skilled person such as the yeast two-hybrid system or phage display. Peptide aptamers may be produced by chemical synthesis or recombinantly produced.

[0058] In some instances, the aptamer is a nucleic acid aptamer. Nucleic acid aptamers are a class of small nucleic acid ligands that are composed of RNA or single-stranded DNA oligonucleotides folded into a three-dimensional structure that have high specificity and affinity for their targets. For example, Systematic Evolution of Ligands by Exponential enrichment (SELEX) technology can be used to obtain aptamers specific to a particular molecular target. Nucleic acid aptamers can be produced by as chemical synthesis or in vitro transcription for RNA aptamers. Nucleic acid aptamers include DNA aptamers, RNA aptamers, XNA aptamers (nucleic acid aptamer comprising xeno nucleotides) and L-RNA aptamers.

[0059] In some embodiments, an aptamer is directly or indirectly joined, e.g., by chemical conjugation, to a cytotoxic moiety. In other instance, an aptamer may block activity of the target protein to which it binds.

[0060] In other embodiments, the binding agent is a ligand that binds to a target ATAM marker. Such a ligand can be linked to a cytotoxic moiety and / or may be an inhibitory ligand that prevents activity of the ATAM marker.Administration with RANK-RANK-L signaling disruptors

[0061] In some embodiments, ATAM-targeted binding moieties as described herein are administered in conjunction with an agent that disrupts RANK and RANK ligand (RANKL) signaling. Thus, for an example, tumor-infiltrating macrophages that express Aire may be treated with ATAM-targeted binding moieties as described here to inhibitor proliferation, e.g, by cell killing of AT AMs and a RANK -RANKL signaling disruptor such as denosumabcan be administered to disrupt induction of Aire, thereby preventing or reducing generation of ATAMs. An example of such as agent is the monoclonal antibody denosumab (see, e.g., US Patent No. 11, 192,952), which is a RANKL inhibitor that is FDA- approved for the treatment of osteoporosis and various bone-loss disorders including bone problems that arise in individuals treated for cancer.

[0062] A RANK-RANKL disruptor, e.g., denosumab, can be administered before, after, or concurrently, with an ATAM-targeting binding moiety as described herein. In some embodiments, an ATAM-targeting binding moiety as described herein can be administered to a subject having a tumor followed by one or more administrations of denosumab.

[0063] In some embodiments, a tumor is analyzed to determine whether ATAMs are present. Such analysis can be performed using a variety of techniques. Such techniques include protein-detection techniques, such as flow cytometery or imaging to identify macrophages that express AIRE, and / or other ATAM-specific polypeptides encoded by marker RNAs as described herein; as well techniques that assess RNA expression, such as RNA sequencing, quantitative RT-PCR, and the like, to identify macrophage populations that have marker profiles that are characteristic of ATAMs compared to other Aire-expressing eTACs and other non-Aire-expressing macrophage populations.Pharmaceutical compositions

[0064] Pharmaceutical compositions comprising a binding agent, e.g., an antibody such as a multispecific, e.g., bispecific, antibody as described herein can be prepared in accordance with methods well known and routinely practiced in the art. The binding agent is provided in a solution suitable for administration to the patient, such as a sterile isotonic aqueous solution for injection. The agent is dissolved or suspended at a suitable concentration in an acceptable carrier. In some embodiments the carrier is aqueous, e.g., water, saline, phosphate buffered saline, and the like. The compositions may contain auxiliary pharmaceutical substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, and the like. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Applicable methods for formulating the antibodies and determining appropriate dosing and scheduling can be found, for example, in Remington: The Science and Practice of Pharmacy, 21stEd., University of the Sciences in Philadelphia, Eds., Lippincott Williams & Wilkins (2005).

[0065] The pharmaceutical compositions are administered to a patient in an amount sufficient to cure or at least partially arrest the disease or symptoms of the disease and its complications. An amount adequate to accomplish this is defined as a "therapeutically effective dose." A therapeutically effective dose is determined by monitoring a patient’s response to therapy. Typical benchmarks indicative of a therapeutically effective dose include amelioration of symptoms of the cancer in the patient. Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's health, including other factors such as age, weight, gender, administration route, etc. Single or multiple administrations of the binding agent may be administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the methods provide a sufficient quantity of binding agent to effectively treat the patient.

[0066] The binding agent can be administered by any suitable means, including, for example, parenteral, intrapulmonary, and intranasal administration, as well as local administration, such as intratumor administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, a binding agent is locally administered to a tumor, e.g., directly injected into the tumor as a bolus.

[0067] The dose of antibody or binding agent is chosen in order to provide effective therapy for the patient. For example, the dose of an antibody can be in the range of less than 0.01 mg / kg body weight to about 25 mg / kg body weight or in the range 1 mg - 2 g per patient. Preferably the dose is in the range 0.1 - 10 mg / kg or approximately 50 mg - 1000 mg / patient. The dose may be repeated at an appropriate frequency which may be in the range once per day to once every three months, or every six months, depending on the pharmacokinetics of the agent. For example, the dose of an antibody may be in the range once per day to once every three months, or every six months, depending on the pharmacokinetics of the antibody, including half-life of the antibody in the circulation, and the pharmacodynamic response (e.g., the duration of the therapeutic effect of the agent, e.g., antibody). In some embodiments, the in vivo half-life is between about 7 and about 25 days and antibody dosing is repeated between once per week and once every 3 months or once every 6 months. In other embodiments, the antibody is administered approximately once per month.

[0068] The dosage of the pharmaceutical compositions depends on factors including the route of administration, the disease to be treated, and physical characteristics, e.g., age, weight, general health, of the subject. In some embodiments, the amount of active ingredient (ATAM targeting binding agent) contained within a single dose may be an amount that effectively prevents, delays, or treats the disease. The dosage may be adapted by the physician in accordance with conventional factors such as the extent of the disease and different parameters of the subject.

[0069] Any tumor that comprised populations of ATAMs can be treated ATAM-binding agents as described herein to inhibit or eliminate ATAM populations, thereby inhibiting tumor growth. Cancers treated in accordance with the invention include, but are not limited to, colorectal cancer, breast cancer, melanoma, lung cancer, esophageal cancer, stomach cancer, kidney cancer, ovarian cancer, uterine cancer, liver cancer, bladder cancer, head and neck cancer, or testicular cancer.

[0070] In some embodiments, the method further comprises administering an additional therapeutic agent, e.g., a chemotherapeutic agent, radiation; or an immunotherapy. In some embodiments, the therapeutic agent can be a monoclonal antibody that targets tumor cells. Although in some embodiments, a checkpoint inhibitor may be also administered, in other embodiments, treatment of the tumor by targeting ATAMs as described herein omits treatment with a checkpoint inhibitor, e.g., an antibody that blocks PD-1 ligand binding to PD-1.

[0071] In another aspect, the disclosure provides agents to inhibitor AIRE expression in AIRE-expressing macrophages in the tumor microenvironment. In some instances, the inhibitor is an inhibitory RNA, e.g., siRNA, shRNA, dsRNA or miRNA, for reducing the expression level of AIRE. Alternatively, gene editing machinery, such as a CRISPR-CAS, meganuclease, TALEN, or zinc finger system, maybe delivered to a tumor to inhibit expression of AIRE in tumor-associated macrophages, e.g., using viral or nanoparticle delivery. In some embodiments, such an AIRE-targeting agent is administered in conjunction with administration of a checkpoint inhibitor, e.g., an antibody that blocks PD-1 ligand binding to PD-1. Such antibodies include, for example, pembrolizumab, nivolumab, cemiplimab, dostarlimab, durvalumab, atezolizumb, and avelumab. In other embodiments, a checkpoint inhibitor may inhibit CTLA-4, such an anti-CTLA-4-blocking antibody such as ipilimumab. In some embodiments, the checkpoint inhibitor may disrupt the interaction of aB-7 receptor and its ligand. In some embodiments, the checkpoint inhibitor inhibits ICOS, PDL2, IDO1, IDO2, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, GITR, HAVCR2, LAG3, KIR, LAIR1, LIGHT, MARCO, OX-40, SLAM, , 2B4, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137, CD160, CD39, VISTA, TIGIT, CGEN-15049, 2B4, CHK 1, CHK2, or A2aR. The checkpoint inhibitor may be delivered concurrently with the AIRE- targeting agent, or sequentially.Technical section / illustrative techniques, supporting data

[0072] Aire-expressing antigen-presenting cells have previously been shown to play important roles in central immune tolerance in the thymus, while eTACs, their extrathymic counterparts, have recently been described to play important roles in immune tolerance and homeostasis. Whether tumors recruit or induce such immune-regulatory myeloid populations for the purposes of immune evasion is unknown.

[0073] In this section, we describe analyses leading to the identification of new subsets of eTACS in the tumor microenvironment that are primarily composed of tumor-associated macrophages and monocytes (TAMs). These Aire-expressing (Aire+) tumor-associated macrophages (ATAMs) have not been previously identified, but were detected by both flow cytometry and mass cytometry (CyTOF) in multiple murine tumor models, including MC38, B16F10 and AT3 (FIGS. 2 and 4). ATAMs share lineage markers with their non-Aire- expressing (Aire') counterparts, but have unique phenotypic features including unique functional marker and transcriptional profiles.

[0074] To define the transcriptional changes that accompany acquisition of Aire expression in TAM populations, we sorted GFP+ and GFP- TAMs in biological triplicate from MC38 tumors implanted into Aire-GFP reporter mice and wildtype controls (N=6 each). TAMs were sorted based on live, CD45+ CD64+ F4 / 80+ CD11c- from Aire-GFP reporter mice as either GFP+ or GFP-, with thresholds as set from the wildtype control cohort. Single-cell RNA-sequencing was performed using the 10X Genomics workflow. Our initial analysis revealed clusters significantly enriched in ATAMs and some surface markers that are more highly expressed by these subsets (FIG. 5). See, also FIG. 1.

[0075] We also accessed a large publicly available human single cell tumor immune atlas (Nieto, et al., Genome Res. 31 : 1013-1926, 2021). The dataset was queried for Aire expression across a variety of human cancer types. Our preliminary data confirm thepresence of Aire-expressing myeloid cells in diverse human tumors, with enrichment in the tolerogenic macrophage compartment, consistent with our mouse models of cancer (FIG. 6).

[0076] Although ATAMs share some phenotypic characteristics typically associated with the M2 macrophage phenotypes, including expression of CD206, Trem2, and Argl, these known markers did not completely overlap with the ATAM phenotype. For example, use of a CD206-DTR mouse (below) could not phenocopy the effects seen in the Aire-DTR mouse, and has no effect on tumor growth kinetics in this model (FIG. 3E).

[0077] Further, while substantial prior work identified immune-modulatory TAM populations (Pittet, et al., Nature reviews. Clinical oncology 19, 402-421, 2022), ablation of these populations of cells had only modest effects on tumor growth and checkpoint-resistance in a number of models; for example, as noted above, ablation of CD206+TAM populations has no impact on the growth kinetics of large MC38 tumors treated with an immune checkpoint inhibitor. This is in stark contrast to the observations described below for ablation of Aire-expressing cells.

[0078] The data from the ablation analyses demonstrated that ablation of Aire-expressing cells using the Aire-DTR system is sufficient to restrict tumor growth in the MC38 model in the setting of immune checkpoint blockade (ICB) with anti-PDl, even after tumors have reached a size at which they become otherwise checkpoint-resistant (FIG. 3). In fact, the data indicate that checkpoint blockade with anti-PDl may be dispensable for the phenotype, as ablation of ATAMs alone significantly arrested tumor growth (FIG. 7A). These observations are indicative that the loss of ATAMs alone may have a profound effect on tumor growth and tumor immune evasion in this model system.

[0079] Given that MC38 is a relatively checkpoint-sensitive tumor model, we also employed the melanoma model B16F10 to define whether this mechanism of immune evasion via ATAMs is shared across a range of tumor models and tissue types. Indeed, ablation of ATAMS in large (180-200mm3) B16F10 melanoma tumors implanted subcutaneously, again in the absence of checkpoint blockade or other immune therapy, also a significantly reduced tumor growth (FIG. 7B). Given the resistance of the Bl 6F 10 model to other immunotherapy modalities, including anti-PDl therapy, we were surprised at the magnitude of this effect.

[0080] Macrophages have been shown to exert pro-tumor effects by promoting T-cell dysfunction (Pittet et al, supra, Zhou et al. Frontier in Oncol. 10:288, 2020). To test if CD8+T cells are responsible for the reduction of tumor growth in Aire-DTR mice, we depleted CD8+ T cells in MC38-bearing Aire-DTR mice. Aire-DTR or WT mice received either anti- CD8 depleting antibody or isotype control via IP injection beginning the day before tumor inoculation, and continued every 3 days until euthanasia (N=10 per condition). Once tumors reached 180 mm3DT treatment began as previously described, and mice were euthanized 7 days after the first DT dose. At day 7, thymi were harvested and stained to validate Aire ablation, while tumors were harvested to validate depletion of CD8+ T cells. The results indicated a strong CD8-dependence of the eTAC-ablation phenotype on tumor growth in MC38 tumors (FIG. 8).

[0081] The methods described in this section further demonstrated that an engineered transgenic reporter system provides the ability to identify previously uncharacterized populations of Aire-expressing immune cells in the tumor microenvironment. The transgenic Aire-specific deletion systems can be employed to identify roles for these populations in tumor immune evasion in a number of model systems. The system is depicted in FIG. 9. Using this system, we demonstrated that crossing the deleTAC transgene onto a thymic epithelial lineage-specific FoxNl-Cre leads to highly specific labeling of mTECs, but not eTACs.

[0082] Furthermore, we demonstrated that RANK signaling drives Aire expression in AT AMs, and interfering with this signaling is protective against tumor growth (FIG. 10A-D).

[0083] In addition, we performed analyses demonstrating that peripheral Aire promotes formation of ATAMs and loss of peripheral Aire slows tumor growth in combination with ICB therapy (FIG. 11 A-F). FIG. 11 A-D shows that in a mixed bone marrow chimera setting, loss of Aire in hematopoietic cells leads to alterations in tumor immune populations, shifting toward immature mono / mac cells that may be more pro-inflammatory, and away from the immunoregulatory / suppressive Trem2+CD81+population (showing a requirement for Aire itself in the differentiation of this population). FIG. 11E-F show a functional experiment demonstrating that conditional deletion of the Aire gene selectively from macrophages is synergistic with checkpoint blockade in MC38 tumors. These data demonstrate for the first time a cell-intrinsic role for Aire itself in the development of these immunoregulatory populations in the tumor microenvironment, and indicate that therapies targeting Aire or genes in the Aire pathway may be of therapeutic value, particularly in the context of ICB.

[0084] All patents, patent applications, publications, and accession numbers cited in this specification are herein incorporated by reference for the subject matter for which they are cited to the same extent as if each independent patent application, or publication was specifically and individually indicated to be incorporated by reference. The disclosed embodiments are presented for purposes of illustration and not limitation. While the invention has been described with reference to the described embodiments thereof, it will be appreciated by those of skill in the art that modifications can be made to the structure and elements of the invention without departing from the spirit and scope of the invention as a whole.

Claims

WHAT IS CLAIMED IS:

1. A method of inhibiting growth of a tumor, the method comprising: administering a multispecific antibody that selectively targets a population of Aire-expressing tumor-associated macrophages (ATAMs), wherein the multispecific antibody comprises at least a first and a second binding domain, wherein the first binding domain specifically binds to a first ATAM-specific cell surface marker expressed by an ATAM present in microenvironment of the tumor and the second binding domain specifically binds a second ATAM-specific cell surface marker different from the from the first ATAM-specific cell surface marker, wherein the multispecific antibody is linked to a cytotoxic reagent and / or at least one of the binding domains blocks binding of the target ATAM-specific cell surface marker to a soluble ligand or ligand present on the surface of another cell to block activity.

2. The method of claim 1, wherein the multispecific antibody is administered intravenously.

3. The method of claim 1, wherein the multispecific antibody is administered locally to the tumor.

4. The method of any one of claims 1-3, wherein the first cell surface marker is CD81 and the second cell surface marker is selected from the group consisting of CD63, CD72, TMEM119, TMEM171, Psd3, Timp2, Mertk, Pf4, Syngrl, Tnfsfl3b, Itga9, Ctla2b, Tmeml l9. 112ra, Adgrel, Pmepal, Tmeml71, Lhfpl2, and Slamf6.

5. The method of any one of claims 1-4, wherein the multispecific antibody is a bispecific .antibody.

6. A method of inhibiting growth of a tumor, the method comprising locally administering to the tumor a first binding moiety that specifically binds to a first ATAM-specific cell surface marker expressed by an Aire-expressing tumor associated macrophage (ATAM) present in a microenvironment of the tumor, wherein the first binding moiety is an antibody, aptamer, or ligand, and is linked to a cytotoxic agent and / or blocks activity of the cell surface marker, wherein the first binding moiety is selected from the group consisting of CD63, CD72, CD81, TMEM119, TMEM171, Psd3, Timp2, Mertk, Pf4, Syngrl, Tnfsfl3b, Itga9, Ctla2b, Tmeml l9. 112ra, Adgrel, Pmepal, Tmeml71, Lhfpl2, and Slamf6.

7. The method of claim 6, further comprising locally administering to the tumor a second binding moiety that specifically binds to a second ATAM-specific cell surface marker expressed by the ATAM present in the tumor microenvironment, wherein (i) the second ATAM-specific cell surface marker is different from the first ATAM-specific cell surface marker and is selected from the group consisting of CD63, CD72, CTMEM119, TMEM171, Psd3, Timp2, Mertk, Pf4, Syngrl, Tnfsfl3b, Itga9, Ctla2b, Tmeml l9. 112ra, Adgrel, Pmepal, Tmeml71, Lhfpl2, and Slamf6; (ii) the second binding moiety is linked to a cytotoxic agent and / or blocks activity of the cell surface marker; and (iii) the second binding moiety is an antibody, aptamer, or ligand.

8. The method of claim 6 or 7, wherein the first binding moiety binds to CD81.

9. A method of inhibiting growth of a tumor, the method comprising locally administering to the tumor a first binding moiety that specifically binds to a first ATAM-specific secreted marker selected from a group consisting of Clqa, Ccl2, Clqc, Ccl7, Pf4, 01fml3, Clqb, Grn, Selenop, Ccl4, and Apoe expressed by an Aire-expressing tumor associated macrophage (ATAM) present in a microenvironment of the tumor, wherein the first binding moiety is an antibody, aptamer, or ligand, and blocks binding of the first ATAM-specific marker to a binding partner, thereby blocking activity.

10. The method of claim 6, further comprising locally administering to the AIrREtumor a second binding moiety that specifically binds to a second ATAM-specific secreted marker expressed by the ATAM present in the tumor microenvironment, wherein (i) the second ATAM-specific cell surface marker is different from the first ATAM-specific cell surface marker and is selected from a group consisting of Clqa, Ccl2, Clqc, Ccl7, Pf4, Olfml3, Clqb, Gm, Selenop, Ccl4, and Apoe; and (iii) the second binding moiety is an antibody, aptamer, or ligand that blocks binding of the second ATAM-specific marker to a binding partner, thereby blocking activity.

11. The method of claim any one of claims 1-10, further comprising administering a monoclonal antibody that selectively binds to receptor activator of nuclear factor -KB ligand (RANKL).

12. The method of claim 11, wherein the anti-RANK-L monoclonal antibody is administered systemically.

13. The method of claim 11, wherein the anti-RANK-L monoclonal antibody is administered locally to the tumor.

14. The method of any one of claims 11-13, where the anti-RANK-L monoclonal antibody is denosumab.

15. The method of any one of claims 11-14, wherein the anti-RANK-L monoclonal antibody is administered at the same time as the binding agent that binds to the cell surface marker expressed by the ATAM or is administered within one week.

16. The method of any one of claims 1-15, wherein the tumor is a melanoma, a breast cancer tumor, an ovarian tumor, or a colorectal cancer tumor.

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