Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/0005—Vertebrate antigens
  • A61K39/0011—Cancer antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
  • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
  • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • 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
  • C07K16/2818—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 against CD28 or CD152
• • 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
  • C07K16/283—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 against Fc-receptors, e.g. CD16, CD32, CD64
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
  • A61K2039/507—Comprising a combination of two or more separate antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
  • A61K2039/6031—Proteins
  • A61K2039/6056—Antibodies

Definitions

• BACKGROUND While immunotherapy using immune checkpoint inhibitors (ICIs) is a promising treatment for many cancers, its success has been limited in “cold” tumors that lack T-cell infiltration (Liu and Sun, Theranostics.2021; 11(11): 5365–5386).
• SUMMARY The present invention is based, at least in part, on the discovery that Anti-CD16B- antigen conjugates (AACs) binding neutrophils can drive T cells into tumor tissues, turning “cold” tumors into “hot” tumors that respond to treatment with anti-PD1.
• AACs Anti-CD16B- antigen conjugates
• compositions comprising (i) a conjugate comprising a tumor antigen and an antibody comprising an antigen-binding domain that binds to Fc ⁇ RIII, optionally wherein the conjugate is a fusion protein or chemical conjugate, and (ii) an immune checkpoint inhibitor (ICI).
• the tumor antigen is listed in Table A.
• the one or more ICIs are selected from an antibody that binds to PD-1, CD40, PD-L1, Tim3, Lag3, CTLA-4, or T-cell immunoglobulin and ITIM domains (TIGIT).
• the ICI is an antibody that binds PD-1.
• compositions described herein for use in a method of treating a subject who has cancer.
• the subject has a solid tumor.
• the solid tumor does not have infiltrating leukocytes, or is immunologically cold.
• the solid tumor is melanoma, pancreatic cancer, prostrate cancer, colon cancer, glioblastoma, or ovarian cancer.
• methods of treating a subject who has cancer comprising administering to the subject an effective amount of a composition described herein.
• a conjugate comprising an antigen and an antibody comprising an antigen-binding domain that binds to Fc ⁇ RIII, optionally wherein the conjugate is a fusion protein or chemical conjugate, and (ii) an immune checkpoint inhibitor (ICI).
• the tumor antigen is shown in Table A.
• the one or more ICIs are selected from an antibody that binds to PD-1, CD40, PD-L1, Tim3, Lag3, CTLA-4, or T-cell immunoglobulin and ITIM domains (TIGIT).
• the ICI is an antibody that binds PD-1.
• the subject has a solid tumor.
• the tumor does not have infiltrating leukocytes, or is immunologically cold.
• the tumor is melanoma, pancreatic cancer, prostrate cancer, glioblastoma, colon cancer, or ovarian cancer.
• all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting.
• FIGs.1A-B AAC but not anti-PD-1 increases early T cell and Natural Killer (NK) cell accumulation in an established B16F10-Ova melanoma.
• NK Natural Killer
• hFcJR humanized FcJR mice expressing FcJRIIIB (CD16B) and FcJRIIA (CD32A) selectively on neutrophils of mice lacking their endogenous activating FcJRs (Tsuboi et al. Immunity 2008, 28: 833).
• B16F10-Ova murine melanoma cells expressing Ovalbumin (Ova), a model T cell-dependent antigen.
• Ova Ovalbumin
• OT-I na ⁇ ve Ova-specific CD8 + T cells.
• AAC anti-FcJRIIIB conjugated to Ova.
• IgG- Ova non-targeting isotype of anti-FcJRIIIB conjugated to Ova.
• DPD1 anti-PD1.
• Iso P isotype control of anti-PD-1.
• FIGs.2A-B AAC reduces tumor growth independently and significantly enhances the efficacy of anti-PD-1.
• a Schematic for the timeline of indicated treatments.
• hFcJR humanized FcJR mice expressing FcJRIIIB (CD16B) and FcJRIIA (CD32A) selectively on neutrophils of mice lacking their endogenous activating FcJRs (Tsuboi et al. Immunity 2008, 28: 833).
• B16F10-Ova murine melanoma cells expressing Ovalbumin (Ova), a model T cell-dependent antigen.
• Ova Ovalbumin
• OT-I na ⁇ ve Ova-specific CD8 + T cells.
• AAC anti-FcJRIIIB conjugated to Ova.
• IgG-Ova non-targeting isotype of anti- FcJRIIIB conjugated to Ova.
• DPD1 anti-PD1.
• Iso P isotype control of anti-PD-1.
• FIGs.3A-C Anti-PD-1 in combination with AAC increases tumor accumulation of antigen-specific effector T cells, and T SCM and T RM memory T cells.
• Tumors were harvested on day 19 and analyzed by flow cytometry for surface and Attorney Docket No.29618-0401WO1/BWH 2023-020 intracellular T cell markers and the number of cells per mm 3 volume of tumor is given.
• a. Ova-specific CD8 + T cells detected with MHC-I tetramers (Tet + ).
• B16F10 melanoma in mice has been shown to be a "cold" tumor with minimal infiltration with T cells, NK cells and other leukocytes with anti-tumor functions due to an immunosuppressive microenvironment (Urs et al., 2019, available online at biopharma.labcorp.com/industry-solutions/by-therapeutic- area/oncology/preclinical/tumor-spotlights/b16-f10-a-murine-melanoma-model.html).
• Tumors include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.
• adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.
• carcinoma is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas.
• the disease is renal carcinoma or melanoma.
• exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary.
• carcinosarcomas e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues.
• An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.
• the term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.
• Anti-Fc ⁇ RIIIB antibodies are known in the art, as are methods of making them. Anti-Fc ⁇ RIIIB antibodies are commercially available, e.g., from Abbexa Ltd; Abcam; Abeomics; antibodies-online; Aviva Systems Biology; Biogems International, Inc.; BioLegend; Biorbyt; CEDARLANE; Cell Sciences; Creative Diagnostics; Elabscience Biotechnology Inc.; EXBIO Praha, a.s.; GeneTex; Invitrogen Antibodies; LifeSpan BioSciences; MBL International; Miltenyi Biotec; MyBioSource.com; NSJ Bioreagents; OriGene Technologies; Peninsula Laboratories International, Inc.; ProSci, Inc; R&D Systems; Santa Cruz Biotechnology, Inc.; Signalway Antibody LLC; Sino Biological, Inc.; SouthernBiotech; STEMCELL Technologies, Inc.; and United States Biological.
• the antibody is 3G8 (e.g., available from biolegend, Santa Cruz Biotechnology, and others; see, e.g., Perussia and Trinchieri, J Immunol.1984 Mar;132(3):1410-5). These antibodies can be used as-is or modified, e.g., to reduce immunogenicity or alter half-life.
• the antibody also recognizes Fc ⁇ RIIIA (CD16a: a transmembrane isoform of the GPI-linked Fc ⁇ RIIIB, CD16b) that is present on macrophages and NK cells.
• the antibody is a Fc ⁇ RIIIB specific antibody that does not bind to Fc ⁇ RIIIA.
• antibody refers to an immunoglobulin molecules, preferably IgG, as well as modified forms, or antigen-binding fragments, variants, or derivatives thereof.
• the antibody can be polyclonal, monoclonal, recombinant, chimeric, de-immunized or humanized, fully human, non-human, (e.g., murine), or single chain antibody.
• the antibody has effector function and can fix complement.
• xenogeneic antibodies such as human-like antibodies in mice
• xenogeneic antibodies such as human-like antibodies in mice
• Modified forms of antibodies, or antigen-binding fragments, variants, or derivatives thereof described herein can be made from whole precursor or parent antibodies using techniques known in the art. Exemplary techniques are discussed in more detail herein.
• Antibodies, or antigen-binding fragments, variants, or derivatives thereof described herein can be made or manufactured using techniques that are known in the art.
• antibody molecules or fragments thereof are “recombinantly produced,” i.e., are produced using recombinant DNA technology.
• Antibodies, or antigen-binding fragments, variants, or derivatives thereof described herein also include derivatives that are modified, e.g., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from specifically binding to its cognate epitope.
• the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
• antibodies, or antigen-binding fragments, variants, or derivatives thereof described herein will not elicit a deleterious immune response in the animal to be treated, e.g., in a human.
• binding molecules, e.g., antibodies, or antigen-binding fragments thereof described herein are derived from a patient, e.g., a human patient, and are subsequently used in the same species from which they are derived, e.g., human, alleviating or minimizing the occurrence of deleterious immune responses. De-immunization can also be used to decrease the immunogenicity of an antibody.
• the term “de-immunization” includes alteration of an antibody to modify T cell epitopes; see, e.g., international applications WO98/52976 and WO00/34317.
• VH and VL sequences from the starting antibody are analyzed and a human T cell epitope “map” from each V region showing the location of epitopes in relation to complementarity determining regions (CDRs) and other key residues within the sequence.
• CDRs complementarity determining regions
• VH and VL sequences are designed comprising combinations of amino acid substitutions and these sequences are subsequently incorporated into a range of binding polypeptides, e.g., Fc ⁇ RIII-specific antibodies or immunospecific fragments thereof for use in the diagnostic and treatment methods disclosed herein, which are then tested for function.
• binding polypeptides e.g., Fc ⁇ RIII-specific antibodies or immunospecific fragments thereof for use in the diagnostic and treatment methods disclosed herein, which are then tested for function.
• Complete heavy and light chain genes comprising modified V and human C regions are then cloned into expression vectors and the subsequent plasmids introduced into cell lines for the production of whole antibody.
• Attorney Docket No.29618-0401WO1/BWH 2023-020 The antibodies are then compared in appropriate biochemical and biological assays, and the optimal variant is identified.
• a humanized version of the 3G8 antibody is used.
• 3G8 is a well-characterized mouse IgG k1 mAb specific for F ⁇ RIII (CD16).
• a humanized 3G8 antibody will be as generated using standard methods. The most common method, as described for Fc ⁇ RIIA mouse antibody, IV.3 (Chen et al. Ann Rheum Dis 78, 228-237 (2019)), is the grafting of the complementary determining regions (CDR) of the heavy and light chains of the mouse 3G8 antibody on to the closest human germline variable heavy and variable kappa chain genes.
• CDR complementary determining regions
• an antibody polypeptide described herein may comprise, consist essentially of, or consist of a fusion protein.
• fusion proteins are chimeric molecules which comprise, for example, an immunoglobulin Fc ⁇ RIII-binding antibody and at least one heterologous tumor antigen sequence.
• the amino acid sequences may normally exist in separate proteins that are brought together in the fusion polypeptide or they may normally exist in the same protein but are placed in a new arrangement in the fusion polypeptide. Fusion proteins may be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.
• heterologous as applied to a polynucleotide or a polypeptide, means that the polynucleotide or polypeptide is derived from a distinct entity from that of the rest of the entity to which it is being compared.
• a “heterologous polypeptide” to be fused to an antibody, or an antigen-binding fragment, variant, or analog thereof is derived from a non-immunoglobulin polypeptide of the same species, or an immunoglobulin or non-immunoglobulin polypeptide of a different species.
• antibodies, or antigen-binding fragments, variants, or derivatives thereof described herein may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalent and non-covalent conjugations) to polypeptides or other compositions.
• a given antibody may contain many types of modifications.
• Antibodies may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic antibodies may result from posttranslation natural processes or may be made by synthetic methods.
• Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer- RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination; see, e.g
• the antibody-antigen conjugates can be fusion proteins, or can be chemical conjugates assembled using a variety of techniques.
• cross-linking reagents include glutaraldehyde (links molecules to N-terminus of peptides), carbodiimide (EDC) (attaches to C-terminus of peptide); succinimide esters (e.g., MBS, SMCC) (binds free amino group and Cysteine residues); benzidine (BDB) (links to Tyrosine residues), periodate (attaches to carbohydrate groups); isothiocyanate; carbodiimide/activated ester (EDC/NHS) coupling;
• a reactive azide group can be site specifically introduced to the protein surface using enzymatic ligation as a posttranslational modification, which can in turn be conjugated to an alkyne- containing polymer using highly efficient click chemistry.
• conjugates with biotin can be prepared, e.g., by reacting an FC ⁇ RIII- antibody polypeptide with an activated ester of biotin such as the biotin N- hydroxysuccinimide ester.
• the antibody-antigen conjugates described herein can also be fused to other heterologous polypeptides, e.g., to increase the in vivo half-life of the polypeptides.
• PEG can be conjugated to the antibodies described herein to increase their half-life in vivo; see, e.g., Leong et al., Cytokine 16 (2001), 106- 119; Adv. In Drug Deliv. Rev.54 (2002), 531; or Weir et al., Biochem. Soc. Transactions 30 (2002), 512.
• the AACs described herein can synergize with other immunomodulatory pharmaceutical agents.
• the present methods include administering one or more immunotherapy agents, e.g., immune checkpoint inhibitors, e.g., an inhibitor of PD-1 signaling, e.g., an antibody that binds to PD-1, CD40, or PD-L1, or an inhibitor of Tim3 Attorney Docket No.29618-0401WO1/BWH 2023-020 or Lag3, e.g., an antibody that binds to Tim3 or Lag3, or an antibody that binds to CTLA- 4, or an antibody that binds to T-cell immunoglobulin and ITIM domains (TIGIT).
• an anti-PD-1 antibody is used.
• Exemplary anti-PD-1 antibodies that can be used in the methods described herein include those that bind to human PD-1; an exemplary PD-l protein sequence is provided at NCBI Accession No. NP_005009.2. Exemplary antibodies are described in US8008449; US9073994; and US20110271358, including PF-06801591, AMP-224, BGB-A317, BI 754091, JS001, MEDI0680, PDR001, REGN2810, SHR-1210, TSR-042, pembrolizumab, nivolumab, avelumab, pidilizumab, and atezolizumab.
• Exemplary CTLA-4 antibodies that can be used in the methods described herein include those that bind to human CTLA-4; exemplary CTLA-4 protein sequences are provided at NCBI Acc No. NP_005205.2. Exemplary antibodies include those described in Tarhini and Iqbal, Onco Targets Ther.3:15-25 (2010); Storz, Mabs.2016 Jan; 8(1):10– 26; US2009025274; US7605238; US6984720; EP1212422; US5811097; US5855887; US6051227; US6682736; EP1141028; and US7741345; and include ipilimumab, Tremelimumab, and EPR1476.
• Exemplary antibodies are described in US20170058033; WO2016/061142A1; WO2016/007235A1; WO2014/195852A1; and Attorney Docket No.29618-0401WO1/BWH 2023-020 WO2013/079174A1, including BMS-936559 (MDX-1105), FAZ053, KN035, Atezolizumab (Tecentriq, MPDL3280A), Avelumab (Bavencio), and Durvalumab (Imfinzi, MEDI-4736).
• Exemplary anti-Tim3 also known as hepatitis A virus cellular receptor 2 or HAVCR2 antibodies that can be used in the methods described herein include those that bind to human Tim3; exemplary Tim3 sequences are provided at NCBI Accession No. NP_116171.3. Exemplary antibodies are described in WO2016071448; US8552156; and US PGPub. Nos.20180298097; 20180251549; 20180230431; 20180072804; 20180016336; 20170313783; 20170114135; 20160257758; 20160257749; 20150086574; and 20130022623, and include LY3321367, DCB-8, MBG453 and TSR-022.
• Exemplary anti-Lag3 antibodies that can be used in the methods described herein include those that bind to human Lag3; exemplary Lag3 sequences are provided at NCBI Accession No. NP_002277.4. Exemplary antibodies are described in Andrews et al., Immunol Rev.2017 Mar;276(1):80-96; Antoni et al., Am Soc Clin Oncol Educ Book. 2016;35:e450-8; US PGPub. Nos.20180326054; 20180251767; 20180230431; 20170334995; 20170290914; 20170101472; 20170022273; 20160303124, and include BMS-986016.
• Exemplary anti-TIGIT antibodies that can be used in the methods described herein include those that bind to human TIGIT; an exemplary human TIGIT sequence is provided at NCBI Accession No. NP_776160.2.
• Exemplary antibodies include AB154; MK ⁇ 7684; BMS ⁇ 986207; ASP8374; Tiragolumab (MTIG7192A; RG6058); (Etigilimab (OMP ⁇ 313M32)); 313R12. See, e.g., Harjun university and Guillerey, Clin Exp Immunol 2019 Dec 11[Online ahead of print], DOI: 10.1111/cei.13407; 20200062859; and 20200040082.
• compositions and Methods of Administration can include the use of pharmaceutical compositions comprising an anti-Fc ⁇ RIII antibody-antigen complex (AAC) as an active ingredient, and compositions comprising anti-PD1 antibodies as an active ingredient.
• AAC anti-Fc ⁇ RIII antibody-antigen complex
• anti-PD1 antibodies as an active ingredient.
• the AAC and anti-PD1 antibodies are combined in a single composition for co-administration, or are administered in separate compositions (e.g., the AAC is Attorney Docket No.29618-0401WO1/BWH 2023-020 given first and then anti-PD1 at different intervals followed by a repetition of this regimen).
• Pharmaceutical compositions typically include a pharmaceutically acceptable carrier.
• compositions can also include an adjuvant to increase T cell response.
• nanoparticles that enhance T cell response can be included, e.g., as described in Stano et al., Vaccine (2012) 30:7541–6 and Swaminathan et al., Vaccine (2016) 34:110–9.
• an adjuvant comprising poly-ICLC (carboxymethylcellulose, polyinosinic- polycytidylic acid, and poly-L-lysine double-stranded RNA), Imiquimods, CpG oligodeoxynuceotides and formulations (IC31, QB10), AS04 (aluminium salt formulated with 3-O-desacyl-4′-monophosphoryl lipid A (MPL)), AS01 (MPL and the saponin QS- 21), and/or MPLA can also be used.
• ICLC carboxymethylcellulose, polyinosinic- polycytidylic acid, and poly-L-lysine double-stranded RNA
• Imiquimods CpG oligodeoxynuceotides and formulations
• AS04 aluminium salt formulated with 3-O-desacyl-4′-monophosphoryl lipid A (MPL)
• AS01 MPL and the saponin QS- 21
• MPLA
• compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous administration.
• solutions or suspensions used for parenteral, application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as Attorney Docket No.29618-0401WO1/BWH 2023-020 ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
• a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
• antibacterial agents such as benzyl alcohol or methyl parabens
• antioxidants such as ascorbic acid or sodium bisul
• compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
• suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
• the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
• isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
• Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.
• Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
• dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above.
• a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
• the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
• the therapeutic compounds are prepared with carriers that will protect the therapeutic compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
• OT-I mice express a transgenic T cell receptor (TCR) recognizing Ovalbumin (Ova) residues 257–264 (SIINFEKL) in the context of H2K b on CD8 T cells (The Jackson Laboratory #003831).
• TCR transgenic T cell receptor
• Ova Ovalbumin residues 257–264
• OT-I/ ⁇ actin-GFP were obtained by crossing OT-I mice with ⁇ actin-GFP mice. Animals were maintained in a specific pathogen-free facility. All in vivo experiments were conducted with age and sex matched Attorney Docket No.29618-0401WO1/BWH 2023-020 animals. The Brigham and Women’s Hospital Animal Care and Use Committee approved all procedures in this study.
• Reagents Anti-CD16B (3G8) or mouse IgG ⁇ (non-targeting isotype control for the CD16B antibody) (Biolegend) were biochemically conjugated to FITC-Ova (#O23020, Thermofisher) as a custom order (Biolegend) to generate AAC and IgG-Ova, respectively.
• H-2 K b Ova Tetramer (Ova 257–264 ) was from the NIH Tetramer Core Facility, and anti-PD-1 (clone RMP1-14) and Isotype control were from BioXcell.
• B16F10-Ova tumor challenge and treatment B16F10 cells expressing soluble Ova were cultured in vitro in DMEM/high glucose supplemented with 10% fetal calf serum and maintained at sub-confluent density. Mice were anesthetized, shaved and injected in the flank subcutaneously with 1 ⁇ 10 5 tumor cells in 100 ⁇ l HBSS. Tumors were measured with a caliper every 2 days once palpable in any one group (long diameter and short diameter) and tumor volume was calculated using an ellipsoid formula (1/2XDxd 2 ) where “D” and “d” are the longer and shorter diameter respectively.
• the tumors were gently dissociated in FACS buffer (PBS supplemented with 2% FCS and 2 mM EDTA) by shearing the tissue on a 70 ⁇ m nylon cell strainer (FisherBrand) using a 3 ml syringe plunger. Tumors were minced/digested in Collagenase type I and dissociated using gentle MACS Dissociator 1X and resuspended in FACS buffer. Cells were subjected to red blood cell lysis using ACK lysis buffer solution (Lonza Cat 10-548E) for 2 min at room temperature, washed once with PBS and resuspended in FACS buffer.
• FACS buffer PBS supplemented with 2% FCS and 2 mM EDTA
• Fig 1b The observed AAC induced early increase in T cell and NK cells in an established B16F10-Ova melanoma (Fig 1b) predicts that AAC would reduce tumor growth and improve the efficacy of immune checkpoint inhibitors such as anti-PD-1, which rely on pre-existing intratumoral T cells.
• CD16B-CD32A/ ⁇ - /- mice were injected with na ⁇ ve Ova-specific CD8 + T cells, followed at day 6 by AAC or IgG-Ova and anti-PD-1 or isotype antibody at day 9, 12 and 15.
• Melanoma was measured in individual mice with calipers over time and harvested at day 19 after euthanizing animals (Fig 2a).
• FIG. 3a A larger number of Ova-specific CD8 + T cells (Fig 3a) and those expressing markers of effector functions (granzyme B, perforin, IFN ⁇ and TNF ⁇ ) (Fig 3b) were observed in mice treated with a combination of AAC plus anti-PD- 1 compared to those given IgG-Ova control, AAC or anti-PD-1. The same was observed for Stem Cell Memory T cells (T SCM , PD-1 + TCF1 hi ) and Tissue-Resident memory T cells (T RM , CD103 + CD69 + ) (Fig 2b), which are known to associate with anti-tumor immunity.
• T SCM Stem Cell Memory T cells
• PD-1 + TCF1 hi Tissue-Resident memory T cells
• T RM Tissue-Resident memory T cells

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