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WO2025235801A1 - Anticorps ciblant l'ostéopontine et leurs utilisations pour réduire la résistance de tumeurs solides à une thérapie cellulaire immunitaire - Google Patents

Anticorps ciblant l'ostéopontine et leurs utilisations pour réduire la résistance de tumeurs solides à une thérapie cellulaire immunitaire

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Publication number
WO2025235801A1
WO2025235801A1 PCT/US2025/028476 US2025028476W WO2025235801A1 WO 2025235801 A1 WO2025235801 A1 WO 2025235801A1 US 2025028476 W US2025028476 W US 2025028476W WO 2025235801 A1 WO2025235801 A1 WO 2025235801A1
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seq
cdr1
cdr2
cdr3
car
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Sharareh GHOLAMIN
Christine E. BROWN
John Williams
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California Institute of Technology
City of Hope
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California Institute of Technology
City of Hope
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4214—Receptors for cytokines
    • A61K40/4217—Receptors for interleukins [IL]
    • 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
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • CAR T cell therapy has exhibited notable success in treating certain lymphomas, some forms of leukemia, and multiple myeloma.
  • CAR T cell therapy faces notable limitations in treatment of solid tumors.
  • the suppressive tumor microenvironment (TME) characterized by complex interactions among immune and stromal cells, is gaining recognition in conferring resistance to CAR T cell therapy and there is a need for therapies that reduce resistance to immune cell therapies, e.g., CAR T cell therapy.
  • SUMMARY Described herein, inter alia, are methods for improving the efficacy of CAR T cells by administering antibodies targeted to osteopontin (OPN or SPP1).
  • Glioblastoma stands out as a highly lethal solid tumor, displaying restricted response to conventional as well as emerging treatments.
  • the response to immunotherapies in solid tumors is intricately determined by the dynamic interplay of components within the tumor microenvironment (TME), underscoring an augmented immune response in an inflammatory milieu with activated immune cells.
  • TEE tumor microenvironment
  • the anti-tumor effects of CAR T cell therapies are manifested not only through tumor-directed cytotoxicity, but also through the more pronounced and indirect modulation of the endogenous immune system against malignant cells.
  • an SPP1 antibody can be used together with a CAR targeted to, for example, human IL13Ra2, MMP2, EGFR/EGFRvIII, TAG72, B7H3, CD70, HER2, PSCA, and PSMA.
  • the SPP1 antibody is preferably administered prior to administration of the CAR.
  • the administration of the SSP1 antibody can precede administration of the CAR by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or longer.
  • the SPP1 antibody is administered several times prior to the administration of the CAR and the second or subsequent administration of the SPP1 antibody can be at any point prior to, coincident with, or subsequent to administration of the CAR.
  • Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 are methods of treating cancer (e.g., solid tumors).
  • the cancer being treated for example glioblastoma, can be characterized by infiltration with macrophages with elevated expression of SPP1.
  • the cancer being treated is a glioma.
  • the cancer being treated is a solid tumor.
  • the cancer being treated is a solid tumor that comprises cells that express IL13Ra2, MMP2, EGFR/EGFRvIII, TAG72, B7H3, CD70, HER2, PSCA, and PSMA, including metastases thereof.
  • Methods for treating a human patient suffering from a cancerous solid tumor can comprise: (a) administering to the patient an antibody targeted to osteopontin (SPP-1); and (b) subsequently administering to the human patient a population of immune cells expressing a chimeric antigen receptor (CAR) targeted to a solid tumor antigen.
  • SPP-1 osteopontin
  • CAR chimeric antigen receptor
  • methods of treating a patient suffering from a cancer comprise first administering an SPP1 antibody followed by administering a population of immune cells expressing a CAR (e.g., a IL-13 CAR, CLTX CAR, EGFR CAR, TAG72 CAR, B7H3 CAR, CD70 CAR, HER2 CAR, PSCA CAR, or PSMA CAR); and optionally wherein the SPP1 antibody is administered to the patient before the population of immune cells expressing the CAR.
  • a cancer e.g., a cancer comprising cells with elevated SPP1 expression and/or a cancer comprising cells expressing IL13Ra2, MMP2, EGFR/EGFRvIII, TAG72, B7H3, CD70, HER2, PSCA, and PSMA
  • a CAR e.g., a IL-13 CAR, CLTX CAR, EGFR CAR, TAG72 CAR, B7H3 CAR, CD70 CAR, HER2 CAR, PSCA
  • SPP1 Antibody Described herein, inter alia, are SPP1 antibodies and methods of use. A variety of antibodies targeted to SPP1 are useful in the methods of the invention.
  • US 2021/0188959 describes four different SPP-1 antibodies that bind to mouse OPN: 1) Clone 100G2-2 comprising a VL domain comprising or consisting of: MKLPVRLLVLMFWIPASNSDVVMTQTPLSLPVRLGDQASISCRPSQSIVHGNRKTYLE WYLQKPGQSPKWYKVSNRFSGVPDRFSGSRSGTDFTLKISRVEAADLGVYYCFQGSH VPWTFGGGTKLEIK (SEQ ID NO:61 with the signal sequence (underlined); SEQ ID NO: 62 without the signal sequence) and having CDR1: RPSQSIVHGNRKTYLE (SEQ ID NO: 63); CDR2: KVSNRFS (SEQ ID NO:64); and CDR3: FQGSHVPWT (SEQ ID NO: 65
  • VL domain comprising or consisting of: MSPAQFLILLVLWIRETNGDVVMTQTPLTLSVTIGRPASISCKSSQSLLESDGKTYLNWL LQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFP RTFGGGTKLEIK (SEQ ID NO:71 with the signal sequence (underlined); SEQ ID NO: 72 without the signal sequence) and having CDR1: KSSQSLLESDGKTYLN (SEQ ID NO:73); CDR2 LVSKLDS (SEQ ID NO:74); and CDR3 WQGTHFPRT (SEQ ID NO:75); and a VH domain comprising or consisting of: MGWSCIMLFLAATATGVHSQVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWV KQRPGRGLEWIGRIVPKSGDTKYNEKFKSKATLTVDKPSSTAY
  • Clone 103D6-1 comprising a VL domain comprising or consisting of: MRFSAQLLGLLVLWIPGSTAEIVMTQAAFSKPVTLGTSASISCRSSKSLLQRNGITYLC WFLQRPGQSPQLLIYQMSNLASGVPDRFSCSGSGTEFTLRISRVEAEDVGVYYCAQDLE LPPTFGGGTKLEIR (SEQ ID NO:81 with the signal sequence (underlined); SEQ ID NO: 82 without the signal sequence) and having CDR1 RSSKSLLQRNGITYL (SEQ ID NO:83); CDR2: QMSNLAS (SEQ ID NO:84); and CDR3: AQDLELPPT (SEQ ID NO:85); and a VH domain comprising or consisting of MDSRLNLVFLVLILKGVQCEVQLVESGGGLVKPGGSLKLSCAASGFTFSDYGMHFVRQ APERGLEWVAYINSRSDTIYYVDTVKGRFTISRDNAKNTLFLQMTSL
  • Clone 89G9-1 comprising a VL domain comprising or consisting of: MRFSAQLLGLLVLWIPGSTADIVMTQAAFSNPVTLGTSASISCRSSKSLLHTNGITYLY WFLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLE LPPTFGGGTKLEIK (SEQ ID NO:91 with the signal sequence (underlined); SEQ ID NO: 92 without the signal sequence) and having CDR1: RSSKSLLHTNGITYLY (SEQ ID NO:93); CDR2: QMSNLAS (SEQ ID NO: 94); and CDR3: AQNLELPPT (SEQ ID NO:95); and a VH Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 domain comprising or consisting of: MDSRLNLVFLVLILKGVQCEVQLVESGGGLVKPGGSLKLSCVVSGFTFNNYEMHWVRL
  • the antibody targeted to SPP1 comprises: (a) a light chain domain (LC) comprising a variable light (VL) chain domain comprising of any one of: i. CDR1 comprising or consisting of SEQ ID NO: 63; CDR2 comprising or consisting of SEQ ID NO:64; and CDR3 comprising or consisting of SEQ ID NO: 65; ii. CDR1 comprising or consisting of SEQ ID NO:73; CDR2 comprising or consisting of SEQ ID NO:74; and CDR3 comprising or consisting of SEQ ID NO:75; iii.
  • LC light chain domain
  • VL variable light chain domain
  • CDR1 comprising or consisting of SEQ ID NO:83; CDR2 comprising or consisting of SEQ ID NO:84; and CDR3 comprising or consisting of SEQ ID NO:85; iv. CDR1 comprising or consisting of SEQ ID NO:93; CDR2 comprising or consisting of SEQ ID NO: 94; and CDR3 comprising or consisting of SEQ ID NO:95; v. CDR1 comprising or consisting of SEQ ID NO:P2; CDR2 comprising or consisting of SEQ ID NO:P3; and CDR3 comprising or consisting of SEQ ID NO:P4; vi.
  • CDR1 comprising or consisting of SEQ ID NO:P10; CDR2 comprising or consisting of SEQ ID NO: P11; and CDR3 comprising or consisting of SEQ ID NO: P12); vii. CDR1 comprising or consisting of SEQ ID NO:P18; CDR2 comprising or consisting of SEQ ID NO: P19; and CDR3 comprising or consisting of SEQ ID NO: P20); viii. CDR1 comprising or consisting of SEQ ID NO:P26; CDR2 comprising or consisting of SEQ ID NO: P27; and CDR3 comprising or consisting of SEQ ID NO:P28); Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 ix.
  • CDR1 comprising or consisting of SEQ ID NO:P34; CDR2 comprising or consisting of SEQ ID NO: P35; and CDR3 comprising or consisting of SEQ ID NO: P36); x. CDR1 comprising or consisting of SEQ ID NO:P42; CDR2 comprising or consisting of SEQ ID NO: P43; and CDR3 comprising or consisting of SEQ ID NO:P44); xi. CDR1 comprising or consisting of SEQ ID NO:P50; CDR2 comprising or consisting of SEQ ID NO: P51; and CDR3 comprising or consisting of SEQ ID NO:P52); xii.
  • CDR1 comprising or consisting of SEQ ID NO:P58; CDR2 comprising or consisting of SEQ ID NO: P59; and CDR3 comprising or consisting of SEQ ID NO:P60); xiii. CDR1 comprising or consisting of SEQ ID NO:P66; CDR2 comprising or consisting of SEQ ID NO: P67; and CDR3 comprising or consisting of SEQ ID NO:P68); xiv. CDR1 comprising or consisting of SEQ ID NO:P74; CDR2 comprising or consisting of SEQ ID NO:P75; and CDR3 comprising or consisting of SEQ ID NO:P76); xv.
  • CDR1 comprising or consisting of SEQ ID NO:P82; CDR2 comprising or consisting of SEQ ID NO: P83; and CDR3 comprising or consisting of SEQ ID NO:P84); xvi. CDR1 comprising or consisting of SEQ ID NO:P90; CDR2 comprising or consisting of SEQ ID NO: P91; and CDR3 comprising or consisting of SEQ ID NO:P92); xvii. CDR1 comprising or consisting of SEQ ID NO:P98; CDR2 comprising or consisting of SEQ ID NO: P99; and CDR3 comprising or consisting of SEQ ID NO:P100); xviii.
  • CDR1 comprising or consisting of SEQ ID NO:P106; CDR2 comprising or consisting of SEQ ID NO: P107; and CDR3 comprising or consisting of SEQ ID NO:P108); xix.
  • CDR1 comprising or consisting of SEQ ID NO:P114; CDR2 comprising or consisting of SEQ ID NO: P115; and CDR3 comprising or consisting of SEQ ID NO:P116); Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 xx.
  • CDR1 comprising or consisting of SEQ ID NO:P122; CDR2 comprising or consisting of SEQ ID NO: P123; and CDR3 comprising or consisting of SEQ ID NO: P124); xxi.
  • CDR1 comprising or consisting of SEQ ID NO:P130; CDR2 comprising or consisting of SEQ ID NO: P131; and CDR3 comprising or consisting of SEQ ID NO:P132); xxii. CDR1 comprising or consisting of SEQ ID NO:P138; CDR2 comprising or consisting of SEQ ID NO: P139; and CDR3 comprising or consisting of SEQ ID NO:P140); xxiii. CDR1 comprising or consisting of SEQ ID NO:P146; CDR2 comprising or consisting of SEQ ID NO: P147; and CDR3 comprising or consisting of SEQ ID NO:P148); xxiv.
  • CDR1 comprising or consisting of SEQ ID NO:P154; CDR2 comprising or consisting of SEQ ID NO: P155; and CDR3 comprising or consisting of SEQ ID NO:P156); xxv. CDR1 comprising or consisting of SEQ ID NO:P162; CDR2 comprising or consisting of SEQ ID NO: P163; and CDR3 comprising or consisting of SEQ ID NO:P164); xxvi. CDR1 comprising or consisting of SEQ ID NO:P170; CDR2 comprising or consisting of SEQ ID NO: P171; and CDR3 comprising or consisting of SEQ ID NO:P172); and xxvii.
  • CDR1 comprising or consisting of SEQ ID NO:P178; CDR2 comprising or consisting of SEQ ID NO: P179; and CDR3 comprising or consisting of SEQ ID NO:P180; and (b) a heavy chain domain (HC) comprising a variable heavy (VH) chain domain comprising of any one of: i. CDR1 comprising or consisting of SEQ ID NO:68 or 88; CDR2 comprising or consisting of SEQ ID NO: 69; and CDR3 comprising or consisting of SEQ ID NO:70; ii.
  • CDR1 comprising or consisting of SEQ ID NO:78; CDR2 comprising or consisting of SEQ ID NO:79; and CDR3 comprising or consisting of SEQ ID NO:80; iii. CDR1 comprising or consisting of SEQ ID NO:88; CDR2 comprising or consisting of SEQ ID NO:89; and CDR3 comprising or consisting of SEQ ID NO:90; Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 iv. CDR1 comprising or consisting of SEQ ID NO:98; CDR2 comprising or consisting of SEQ ID NO:99; and CDR3 comprising or consisting of SEQ ID NO: 100; v.
  • CDR1 comprising or consisting of SEQ ID NO:P6; CDR2 comprising or consisting of SEQ ID NO:P7; and CDR3 comprising or consisting of SEQ ID NO:P8; vi. CDR1 comprising or consisting of SEQ ID NO: P14; CDR2 comprising or consisting of SEQ ID NO: P15; and CDR3 comprising or consisting of SEQ ID NO:P16; vii. CDR1 comprising or consisting of SEQ ID NO:P22; CDR2 comprising or consisting of SEQ ID NO: P23; and CDR3 comprising or consisting of SEQ ID NO:P24; viii.
  • CDR1 comprising or consisting of SEQ ID NO:P30; CDR2 comprising or consisting of SEQ ID NO: P31; and CDR3 comprising or consisting of SEQ ID NO:P32; ix.
  • CDR1 comprising or consisting of SEQ ID NO:P38; CDR2 comprising or consisting of SEQ ID NO: P39; and CDR3 comprising or consisting of SEQ ID NO:P40; x.
  • CDR1 comprising or consisting of SEQ ID NO:P46; CDR2 comprising or consisting of SEQ ID NO: P47; and CDR3 comprising or consisting of SEQ ID NO:P48; xi.
  • CDR1 comprising or consisting of SEQ ID NO:P54; CDR2 comprising or consisting of SEQ ID NO: P55, and CDR3 comprising or consisting of SEQ ID NO:P56; xii. CDR1 comprising or consisting of SEQ ID NO:P62; CDR2 comprising or consisting of SEQ ID NO: P63; and CDR3 comprising or consisting of SEQ ID NO:P64; xiii. CDR1 comprising or consisting of SEQ ID NO:P70; CDR2 comprising or consisting of SEQ ID NO: P71; and CDR3 comprising or consisting of SEQ ID NO:P72; xiv.
  • CDR1 comprising or consisting of SEQ ID NO:P78; CDR2 comprising or consisting of SEQ ID NO: P79; and CDR3 comprising or consisting of SEQ ID NO:P80; Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 xv.
  • CDR1 comprising or consisting of SEQ ID NO:P86; CDR2 comprising or consisting of SEQ ID NO: P87; and CDR3 comprising or consisting of SEQ ID NO:P88; xvi.
  • CDR1 comprising or consisting of SEQ ID NO:P94; CDR2 comprising or consisting of SEQ ID NO: P95; and CDR3 comprising or consisting of SEQ ID NO:P96; xvii.
  • CDR1 comprising or consisting of SEQ ID NO:P102; CDR2 comprising or consisting of SEQ ID NO: P103; and CDR3 comprising or consisting of SEQ ID NO:P104; xviii.
  • CDR1 comprising or consisting of SEQ ID NO:P110; CDR2 comprising or consisting of SEQ ID NO: P111; and CDR3 comprising or consisting of SEQ ID NO:P112; xix.
  • CDR1 comprising or consisting of SEQ ID NO:P118; CDR2 comprising or consisting of SEQ ID NO: P119; and CDR3 comprising or consisting of SEQ ID NO: P120; xx. CDR1 comprising or consisting of SEQ ID NO:P126; CDR2 comprising or consisting of SEQ ID NO: P127; and CDR3 comprising or consisting of SEQ ID NO:P128; xxi. CDR1 comprising or consisting of SEQ ID NO:P134; CDR2 comprising or consisting of SEQ ID NO: P135; and CDR3 comprising or consisting of SEQ ID NO:P136; xxii.
  • CDR1 comprising or consisting of SEQ ID NO:P142; CDR2 comprising or consisting of SEQ ID NO: P143; and CDR3 comprising or consisting of SEQ ID NO:P144; xxiii. CDR1 comprising or consisting of SEQ ID NO:P150; CDR2 comprising or consisting of SEQ ID NO: P151; and CDR3 comprising or consisting of SEQ ID NO:P152; xxiv. CDR1 comprising or consisting of SEQ ID NO:P158; CDR2 comprising or consisting of SEQ ID NO: P159; and CDR3 comprising or consisting of SEQ ID NO:P160; xxv.
  • CDR1 comprising or consisting of SEQ ID NO:P166; CDR2 comprising or consisting of SEQ ID NO: P167; and CDR3 comprising or consisting of SEQ ID NO:P168; Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 xxvi.
  • CDR1 comprising or consisting of SEQ ID NO:P174; CDR2 comprising or consisting of SEQ ID NO: P175; and CDR3 comprising or consisting of SEQ ID NO:P176; and xxvii.
  • CDR1 comprising or consisting of SEQ ID NO:P182; CDR2 comprising or consisting of SEQ ID NO: P183; and CDR3 comprising or consisting of SEQ ID NO:P184.
  • a useful SPP1 Ab can also comprise a VL domain comprising or consisting of: DIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVP DRFSSSGSGTDFTLRISRVEAEDVGFYYCAQNLELPWTFGGGSKLEIK (SEQ ID NO:P1 (CDRs underlined)) and having CDR1: KSLLHSNGITY (SEQ ID NO:P2); CDR2: LLIYQMS (SEQ ID NO:P3); and CDR3: AQNLELPWT (SEQ ID NO:P4); and a VH domain comprising or consisting of QVQLQQPGAELVNPGASVRLSCKASGYIFTSYWMHWIKQRPGQGLEWIGEINPTSGRTNYNA PFKNKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARLTGTGFYWGQGTLVTVSA (SEQ ID NO
  • the SPP1 antibody comprises: a variable light chain comprising the amino acid sequence of any one of SEQ ID NOs:61, 62, 71, 72, 81, 82, 91, 92, P1, P9, P17, P25, P33, P41, P49, P57, P65, P73, P81, P89, P97, P105, P113, P121, P129, P137, P145, P153, P161, P169, P177, and variants thereof with 1-5 single amino acid modifications (e.g., 1, 2, 3, 4, or 5 single amino acid substitutions), wherein the modifications (e.g., substitutions) are not in the CDRs; and a variable heavy chain comprises the amino acid sequence of any one of SEQ ID NOs:66, 67, 76, 77, 86, 87, 96, 97, P5, P13, P21, P29 , P37 , P45 , P53 , P61 , P69, P77 , P85
  • the SPP1 Ab comprises comprise a VL domain comprising or consisting of an amino acid sequence of any one of the VL sequences listed in Table 6, or a variant thereof with 1-5 single amino acid modifications (e.g., 1, 2, 3, 4, or 5 single amino acid substitutions), wherein the modifications (e.g., substitutions) are not in the CDRs; and a VH domain comprising Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 or consisting of an amino acid sequence of any one of the VH sequences listed in Table 6, or a variant thereof with 1-5 single amino acid modifications (e.g., 1, 2, 3, 4, or 5 single amino acid substitutions), wherein the modifications (e.g., substitutions) are not in the CDRs.
  • VL domain comprising or consisting of an amino acid sequence of any one of the VL sequences listed in Table 6, or a variant thereof with 1-5 single amino acid modifications (e.g., 1, 2, 3, 4, or 5 single amino acid substitutions), wherein the modifications (
  • a useful SPP1 Ab can also comprise a light chain (LC) domain comprising or consisting of: DIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVP DRFSSSGSGTDFTLRISRVEAEDVGFYYCAQNLELPWTFGGGSKLEIKRADAAPTVSIFPPSSE QLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEY ERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO:213 (CDRs underlined)) and having CDR1 comprising or consisting of SEQ ID NO:P2; CDR2 comprising or consisting of SEQ ID NO:P3; and CDR3 comprising or consisting of (SEQ ID NO:P4); and a heavy chain (HC) domain comprising or consisting of QVQLQQPGAELVNPGASVRLSCKA
  • the SPP1 antibodies described herein bind to human OPN. Additional anti-SPP1 antibodies are described in: Fan, K. et al. A humanized anti-osteopontin antibody protects from Concanavalin A induced-liver injury in mice. European journal of pharmacology 657, 144–151, https://doi.org/10.1016/j.ejphar.2011.01.041 (2011); Hirano, Y., Aziz, M., Yang, W. L., Ochani, M. & Wang, P. Neutralization of Osteopontin Ameliorates Acute Lung Injury Induced by Intestinal Ischemia-Reperfusion.
  • SPP1 antibodies include: Ab214055, Ab166709, Ab214050, Ab236213 and Ab269441 (ThermoFisher); 2) hu1A12 (Absolute Antibody). SPP1 antibodies are also described in US 2023/0203193; WO 2011/021146; and US 11897950.
  • CAR Targeting Solid Tumors e.g., IL-13 CAR
  • IL13R ⁇ 2 targeted CAR Described herein, inter alia, are IL13R ⁇ 2 targeted CAR that can be used conjunction with SPP1 antibody to treat a variety of cancers, including glioblastoma.
  • the IL13R ⁇ 2 targeted CAR include all or a portion of mature human IL13 or a variant thereof as a targeting domain.
  • a CAR that includes a variant IL13 is referred to as a “variant IL13 CAR” or simply an IL13 CAR or IL13R ⁇ 2 CAR.
  • the variant IL13 can include various mutations (e.g., E11Y, E11R, E90L, and R107K) relative to the sequence of human IL13: GPVPPSTA LRELIEELVN ITQNQKAPLC NGSMVWSINL TAGMYCAALE SLINVSGCSA IEKTQRMLSG FCPHKVSAGQ FSSLHVRDTK IEVAQFVKDL LLHLKKLFRE GRFN (SEQ ID NO: 1) Full sequence of wild-type human IL13 (signal sequence underlined) 10 20 30 40 50 MHPLLNPLLL ALGLMALLLT TVIALTCLGG FASPGPVPPS TALRELIEEL 60 70 80 90 100 VNITQNQKAP LCNGSMVWSI NLTAGMYCAA LESLINVSGC SAIEKTQR
  • a useful IL-13 variant for inclusion in a CAR can comprise 107, 108, 109, 110, 111 contiguous amino acids of SEQ ID NO: 1 or the entirety of SEQ ID NO: 1 with 1, 2, 3, 4 or 5 single amino acid changes, provided that there is not an E at position 90 of SEQ ID NO:1.
  • position 90 can be selected from: G, A, L, P, V, I, M, F, Y, W, S, T, C, N, Q, K, R, and H; or can be selected from: G, A, L, P, V, I, M, F, Y, W, S, T, C, N, and Q; or can be selected from: G, A, L, P, V, I, M, F, Y and W; or can be selected from: G, A, L, P, V, I and M; or can be selected from: G, A, L, V, I and M.
  • a useful IL-13 variant for inclusion in a CAR can comprise 107, 108, 109, 110, 111 contiguous amino acids of SEQ ID NO: 1 or the entirety of SEQ ID NO: 1 with 1, 2, 3, 4 or 5 single amino acid changes, provided that there is an L at position 90 of SEQ ID NO:1.
  • position 90 can be selected from: G, A, L, P, V, I, M, F, Y, W, S, T, C, N, Q, K, R, and H; or can be selected from: G, A, L, P, V, I, M, F, Y, W, S, T, C, N, and Q; or can be selected from: G, A, L, P, V, I, M, F, Y and W; or can be selected from: G, A, L, P, V, I and M; or can be selected from: G, A, L, V, I and M.
  • the variant IL13 CAR described herein include a variant IL-13 comprising or consisting of the amino acid sequence (mutations compared to wt IL13 are bold and double underline): GPVPPSTALRYLIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRM LSGFCPHKVSAGQFSSLHVRDTKILVAQFVKDLLLHLKKLFKEGRFN (“YLK”; SEQ ID NO: 2); or comprising or consisting of the amino acid sequence (mutations compared to wt IL13 are bold and double underline): GPVPPSTALRRLIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQR MLSGFCPHKVSAGQFSSLHVRDTKILVAQFVKDLLLHLKKLFKEGRFN (“RLK”; SEQ ID NO: 3); or comprising or consisting of the amino acid sequence (mutations compared to wt IL13 are bold and
  • the CAR comprise a chlorotoxin (CLTX).
  • CLTX can comprise or consist of the amino acid sequence: MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCR (SEQ ID NO:14) or a variant thereof having 1-5 (e.g., 1 or 2) single amino acid modifications (e.g., substitutions) provided that the cysteine residues are not modified.
  • CLTX variants and related toxins such as GaTx2 & GaTx1, toxins from Leiurus quinquestriatus hebraeus, AaCtx, a toxin from Androctonus australis, etc.
  • methods of making and using CLTX CAR and immune cells expressing CLTX CAR are known in the art; e.g., WO 2017/066481.
  • CAR targeted to an antigen expressed on the surface of a cancer cell e.g., IL13Ra2, MMP2, EGFR/EGFRvIII, TAG72, B7H3, CD70, HER2, PSCA, and PSMA
  • a cancer cell e.g., IL13Ra2, MMP2, EGFR/EGFRvIII, TAG72, B7H3, CD70, HER2, PSCA, and PSMA
  • SPP1 antibody e.g., IL13Ra2, MMP2, EGFR/EGFRvIII, TAG72, B7H3, CD70, HER2, PSCA, and PSMA.
  • the IL13R ⁇ 2 targeted CAR can include all or a portion of mature human IL13 or a variant thereof as a targeting domain.
  • the MMP2 targeted CAR can include all or a portion of a cholorotoxin (e.g., SEQ ID NO:14) or a variant thereof as a targeting domain.
  • the CAR can comprise an scFv targeted to a solid tumor (e.g., EGFR/EGFRvIII, B7H3, TAG72, CD70, HER2, PSCA, and PSMA).
  • the targeting domain can comprise or consist of a scFv targeted to HER2 (also called ErbB2).
  • the HER2 scFv comprises or consists of the amino acid sequence of any one of SED ID NO: 205-208 and variants thereof having no more than 1, 2, 3, 4 or 5 single amino acid modifications (e.g., substitutions), wherein the modifications (e.g., substitutions) are not in the CDRs.
  • HER2 targeting sequences e.g., HER2 CDRs, HER2 variable domains (i.e., VL and VH), and HER2 scFvs
  • HER2 targeted CAR e.g., 4D5, rastuzumab, trastuzumab emtansine, trastuzumab deruxtecan, margetuximab, pertuzumab, disitamab vedotin, U-31402, ISU-104, SIB-001, 9F7- F11, EV-20Sap, U-31402, ARX-788, BAT-8001, HL-02, TAA-013, trastuzumab duocarmazine, A-166, AU-101, AU-105, BPX-603, ISB-1302, KN-026, MB-103, MRG-002, zanidatamab, zenocutuzum
  • the targeting domain can comprise or consist of a scFv targeted to an epidermal growth factor receptor (EGFR), a truncated EGFR (EGFRt), a variant of EGFR (e.g., epidermal growth factor receptor variant III; EGFRvIII), or the extracellular binding domain of EGFR.
  • EGFR epidermal growth factor receptor
  • EGFRt truncated EGFR
  • EGFRvIII epidermal growth factor receptor variant III
  • the extracellular binding domain of EGFR e.g., the extracellular binding domain of EGFR.
  • the EGFR scFv targets both wild-type EGFR and EGFRvIII.
  • the EGFR scFv comprises or consists of the amino acid sequence of SED ID NO: 203 or 204, or a variant thereof having no more than 1, 2, 3, 4 or 5 single amino acid modifications (e.g., substitutions), wherein the modifications (e.g., substitutions) are not in the CDRs.
  • EGFR targeting sequences e.g., EGFR CDRs, EGFR variable domains (i.e., VL and VH), and EGFR scFvs
  • EGFR targeted CAR as well as methods of making and using the same, are known in the art; e.g., CDRs and sequences of cetuximab, panitumumab, necitumumab, nepidermin, nimotuzumab, amivantamab, HS-627, amelimumab, depatuxizumab, FmAb-2, GC-1118A, imgatuzumab, matuzumab, MVC-101, SCT-200, QL-1203, tomuzotuximab, zalutumumab, JMT-101, MCLA-158, QL-1105, SYN-004, MCLA-129, WBP- 297, AM-105, BH-2922, BM
  • CD70 targeting sequences e.g., CD70 CDRs, CD70 variable domains (i.e., VL and VH), and CD70 scFvs
  • CD70 targeted CAR as well as methods of making and using the same, are known in the art; e.g., Xiong, Q., et al. (2024)
  • the targeting domain can comprise or consist of a scFv targeted to PSCA.
  • the PSCA scFv comprises or consists of the amino acid sequence of SEQ ID NO: 209 or a variant thereof having 1-5 (e.g., 1, 2, 3, 4, or 5) single amino acid modifications (e.g., substitutions), wherein the modifications are not in the CDRs.
  • PSCA targeting sequences e.g., PSCA CDRs, PSCA variable domains (i.e., VL and VH), and PSCA scFvs
  • PSCA targeted CAR as well as methods of making and using the same, are known in the art; e.g., WO 2017/062628; WO 2022/115421; US 2024/0041921 A1; Morgenroth et al., (2007) Prostate 67(10):1121-1131 (scFv 7F5); Nejatollahi et al., (2013) J Oncology, article ID 839831 (scFv CS-II); and US 2009/0311181 A1.
  • the targeting domain can comprise or consist of a scFv targeted to PSMA.
  • the PSMA scFv comprises or consists of the amino acid sequence of SEQ ID NO: 210 or a variant thereof having 1-5 (e.g., 1, 2, 3, 4, or 5) single amino acid modifications (e.g., substitutions), wherein the modifications are not in the CDRs.
  • PSMA targeting sequences e.g., PSMA CDRs, PSMA variable domains (i.e., VL and VH), and PSMA scFvs
  • PSMA targeted CAR as well as methods of making and using the same, are known in the art; CIK-CAR.PSMA (Formula Pharmaceuticals Inc), P-PSMA-101 (Poseida Therapeutics Inc), UniCAR-T-PSMA (GEMoaB Monoclonals GmbH), Parker et al., Protein Expr Purif 89(2):136- 145 (2013), US 20110268656 (e.g., J591 ScFv); Frigerio et al, European J Cancer 49(9):2223- 2232 (2013) (e.g., scFvD2B); WO 2006125481 (e.g., mAbs 3/A12, 3/E7, and 3/F11 and (scFv A5 and D7).
  • PSMA CDRs PSMA variable domains (
  • the targeting domain can comprise or consist of a scFv targeted to TAG72.
  • the TAG72 scFv comprises or consists of the amino acid sequence of SEQ ID NO: 211, or a variant thereof having 1-5 (e.g., 1, 2, 3, 4, or 5) single amino acid modifications (e.g., substitutions), wherein the modifications are not in the CDRs.
  • Additional TAG-72 targeting sequences e.g., TAG72 CDRs, TAG72 variable domains (i.e., VL and VH), and TAG72 scFvs
  • TAG72 targeted CAR as well as methods of making and using the same, are known in the art; e.g., WO 2020/028721.
  • the targeting domain can comprise or consist of a CD70 binding domain (e.g., a CD70 scFv).
  • the PSCA scFv comprises or consists of the amino acid sequence of SEQ ID NO: 212 or a variant thereof having 1-5 (e.g., 1, 2, 3, 4, or 5) single amino acid modifications Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 (e.g., single amino acid substitutions), wherein the modifications are not in the CDRs.
  • CD70 targeting sequences e.g., CD70 CDRs, CD70 variable domains (i.e., VL and VH), and CD70 scFvs
  • CD70 targeted CAR as well as methods of making and using the same, are known in the art; e.g., Xiong, Q., et al. (2024)
  • the targeting domain can comprise or consist of a scFv targeted to B7H3.
  • the B7H3 scFv can comprise the CDRs of antibody MGA271.
  • Many B7H3 targeting sequences e.g., B7H3 CDRs, B7H3 variable domains (i.e., VL and VH), and B7H3 scFvs
  • B7H3 targeted CAR as well as methods of making and using the same, are known in the art; e.g., antibody MGA271 (Macrogenics); WO 2024/061306; WO 2024/226468; US20250136697A1; US20240245771A1; US20240252639A1
  • the CAR comprise a targeting domain; a spacer comprising a sequence selected from the group consisting of: SEQ ID NOs: 24-34; a transmembrane domain comprising a sequence selected from the group consisting of SEQ ID NOs: 15-23; a costimulatory domain comprising a sequence selected from the group consisting of SEQ
  • the targeting domain comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 1-14 and 203-212.
  • the spacer region can include an amino acid sequence selected from the group consisting of SEQ ID NOs: 24-34 or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions; the transmembrane domain selected from the group consisting of: a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, and a CD3 ⁇ transmembrane domain; the costimulatory domain selected from the group consisting of: a 28 costimulatory domain, a 41-BB costimulatory domain, an OX40 costimulatory domain, and a 2B4 costimulatory domain.
  • the nucleic acid molecule further comprises an interdomain linker consisting of 1 - 5 amino acids between one or more of: the targeting domain (e.g., scFv) and the spacer domain, the spacer domain and the transmembrane domain, the transmembrane domain and the co-stimulatory domain, and/or the costimulatory domain and the CD3 ⁇ signaling domain; the interdomain linker can consist of 1-5 glycine; in some Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 embodiments, the CAR further comprises an interdomain linker consisting of the sequence GGG is located between the costimulatory domain and the CD3 ⁇ signaling domain.
  • the targeting domain e.g., scFv
  • the spacer domain and the transmembrane domain e.g., the spacer domain and the transmembrane domain, the transmembrane domain and the co-stimulatory domain, and/or the costimulatory domain and the CD
  • the CAR comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 101-212 and variants thereof having 1-15 (e.g., 1, 2, 3, 4, 5, or 6) single amino acid modifications (e.g., substitutions); optionally wherein the amino acid modifications (e.g., substitutions) are not in the targeting domain; optionally wherein the amino acid modifications (e.g., substitutions) are not in the CDRs of the scFv.
  • vectors comprising a nucleic acid molecule described herein (e.g., a nucleic acid encoding an SPP1 antibody, a nucleic acid comprising a CAR); populations of immune cells comprising a vector comprising a nucleotide sequence described here; populations of immune cells expressing a CAR described here; populations of human T cells (e.g., a population comprising central memory T cells) and/or of human NK cells transduced by a vector comprising a nucleic acid molecule described herein.
  • a nucleic acid molecule described herein e.g., a nucleic acid encoding an SPP1 antibody, a nucleic acid comprising a CAR
  • populations of immune cells comprising a vector comprising a nucleotide sequence described here
  • populations of immune cells expressing a CAR described here populations of human T cells (e.g., a population comprising central memory T cells) and/or of human NK cells transduced by a vector compris
  • the T cells comprise PBMC, dPBMC (PBMC with depletion of CD14+ and CD25+ cells), Tn/mem (na ⁇ ve and memory T cells, CD62L+ enriched from dPBMC), or Tcm (central memory T cells).
  • PBMC PBMC with depletion of CD14+ and CD25+ cells
  • Tn/mem na ⁇ ve and memory T cells, CD62L+ enriched from dPBMC
  • Tcm central memory T cells. Additional information, including methods of making and using CAR and immune cells expressing CAR, are known in the art; e.g., WO 2017/015490; WO 2017/066481; WO 2017/136829; WO 2018/102761; WO 2023/107593; US 2024/0398913 A1; US 2023/0295296 A1.
  • a population of immune cell can comprise one or more of helper T cells, cytotoxic T cells, memory T cells, na ⁇ ve T cells, regulatory T cells, natural killer T cells, or combinations thereof. In various embodiments: at least 20%, 30%, 40%, or 50% of the CAR T cells are CD8+ T cells.
  • Various T cell subsets, including both alpha beta T cells and gamma delta T cells, can be used.
  • the CAR can be expressed in other immune cells such as NK cells. Where a patient is treated with an immune cell expressing a CAR described herein the cell can be an autologous T cell or an allogenic T cell.
  • the cells used are a cell population that includes both CD4+ and CD8+ central memory T cells (TCM), which are CD62L+, CCR7+, CD45RO+, and CD45RA-, or the cells used are a cell population that includes CD4+ and CD8+ T CM cells, stem central memory T cells and na ⁇ ve T cells (i.e., a population of TCM/SCM/N cells).
  • TCM central memory T cells
  • a population of TCM/SCM/N cells are CD62L+, CCR7+ and include both CD45RA+ and CD45RO+ cells as well as both CD4+ cells and CD8+ cells.
  • a composition comprising CAR T cells or CAR NK cells described herein is administered locally or systemically. In some embodiments, a composition comprising CAR T cells or CAR NK cells described herein is administered by single or repeat dosing.
  • a composition comprising CAR T cells or CAR NK cells described herein is administered to a patient having a glioma (e.g., glioblastoma).
  • a composition comprising CAR T cells or CAR NK cells described herein is administered to a patient having a solid tumor.
  • a composition comprising CAR T cells or CAR NK cells described herein is administered to a patient having a cancer comprising cells expressing SPP1,
  • An amino acid modification refers to an amino acid substitution, insertion, and/or deletion in a protein or peptide sequence.
  • amino acid substitution refers to replacement of an amino acid at a particular position in a parent peptide or protein sequence with another amino acid.
  • a substitution can be made to change an amino acid in the resulting protein in a non-conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping).
  • a conservative change generally leads to less change in the structure and function of the resulting protein.
  • Amino acids with nonpolar R groups Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, Tryptophan, Methionine
  • Amino acids with uncharged polar R groups Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine, Glutamine
  • Amino acids with charged polar R groups negatively charged at pH 6.0: Aspartic acid, Glutamic acid
  • Basic amino acids positively charged at pH 6.0: Lysine, Arginine, Histidine (at pH 6.0).
  • CDR regions are well known to those skilled in the art and have been defined by well-known numbering systems. Generally, the CDRs within the structure of the immunoglobulin variable domain is conserved between species and present in structures called loops.
  • CDRs Kabat Complementarity Determining Regions
  • Chothia refers instead to the location of the structural loops (see, e.g., Chothia and Lesk (1987) J. Mol. Biol.196:901-17).
  • the end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34).
  • the numbering systems including, for example, the Kabat numbering and the ImMunoGeneTics (IMGT) Information System (Lafranc et al., (2003) Dev. Comp. Immunol.27(1):55-77) numbering system, is well known to one skilled in the art.
  • FIG.1 Suppressive tumor microenvironment limits response to CAR T therapy in high- grade glioma
  • a Schematic illustration of the Phase I trial and sample processing.
  • b Numbers of glioblastoma tumors (41 total patient samples) with high (3-4) or low (0-2) immunohistochemistry CD3 scores and samples that did or did not respond to CAR T therapy.
  • c Suppressive tumor microenvironment limits response to CAR T therapy in high- grade glioma
  • Differentially regulated ligand-receptor signaling pathways in immune subclusters of the TME comparing SD/CR vs. PD tumors (top) and CD3-med/high vs, CD3-low tumors (bottom).
  • Significant pathways were selected with a two-sided Wilcoxon p ⁇ 0.01 in the comparison of SD/CR vs. PD, and pathways with an absolute delta of information flow > 0.8 were plotted, with positive values indicating increased signaling in PD or CD3-low tumors.
  • the information flow for a given signaling pathway is defined by the sum of communication probability among all pairs of cell groups in the inferred network. i.
  • FIG.2 Characterization of SPP1 expressing macrophages in non-responders to IL13R2 targeted therapies a. Hazard ratios for survival by expression of macrophage markers CD14, CD68, C1QC, C1QB, C1QA, and SPP1 in a cohort of 41 patients. The HRs and 95% confidence intervals are shown.
  • b. Violin plots displaying the differential expression levels of SPP1 across various macrophage subclusters, comparing samples PD (right) to those with CR/SD (left), and samples with CD3 low (right) to those with CD3 high expression (left).
  • d Forest plot representing the distribution of subclusters among patients from the top and bottom marked areas in FIG 2C.
  • e. Violin plots displaying differential expression of various markers involved in antigen presentation and functionality of macrophages between SPP1 high and low SPP1 macrophages.
  • Pathway enrichment analysis revealing the differential expression of pathways involved in lower antigen presentation, lower phagocytosis, higher ECM synthesis and remodeling, lower inflammatory cytokines in SPP1 high (lower portion) versus SPP1 low (upper portion) expressing macrophages.
  • Pathway enrichment analysis depicting SPP1 high macrophages are lipid associated macrophages.
  • f. Immunofluorescent staining for SPP1, COL1A1, and CD68 shows a higher expression of SPP1 and enrichment of fibroblasts in PD tumors compared to SD/CR tumors. Bars indicate 50 ⁇ m.
  • FIG.3 Congruent pathways drive the suppressive tumor microenvironment in glioblastoma and a syngeneic mouse model
  • a Schematic illustration of the integration analysis of single-cell RNA sequencing data from patient GBM tumor samples and mouse YUMM JAK1/KO tumor samples, CD45+ immune and stromal cells from 41 GBM tumors and 4 YUMM JAK1KO mouse melanomas were integrated to examine regulatory programs shared with the two tumor types.
  • b Schematic illustration of the integration analysis of single-cell RNA sequencing data from patient GBM tumor samples and mouse YUMM JAK1/KO tumor samples, CD45+ immune and stromal cells from 41 GBM tumors and 4 YUMM JAK1KO mouse melanomas were integrated to examine regulatory programs shared with the two tumor types.
  • FIG.4 Priming of YUMM JAK1/KO tumors with SPP-1 targeted monoclonal antibody before delivering mIL13 CAR T cells significantly enhances therapeutic efficacy
  • a Schematic illustration of the experimental design of a study combining anti-SPP1 antibody and mIL13Ra2 CAR T cell therapy against YUMM JAK1/KO tumors.
  • b Tumor volumes comparing combination therapy to mIL13Ra2 CAR T cell therapy alone and anti-SPP1 antibody treatment alone.
  • FIG 5 Depicts the amino acid sequence of CAR having: a variant IL13 targeting domain, an IgG4 (EQ) spacer, CD28 TM domain, 41-BB co-stimulatory domain and a CD3zeta domain (SEQ ID NOs: 101-102).
  • FIG 6 Depicts the amino acid sequence of CAR having: a variant IL13 targeting domain, an IgG4 (EQ) spacer, CD28 TM domain, 41-BB co-stimulatory domain and a CD3zeta domain (SEQ ID NOs: 103-104).
  • FIG 7 Depicts the amino acid sequence of CAR having: a variant IL13 targeting domain, an IgG4 (EQ) spacer, CD28 TM domain, 41-BB co-stimulatory domain and a CD3zeta domain (SEQ ID NOs: 105-106).
  • FIG 8 Depicts the amino acid sequence of CAR having: a variant IL13 targeting domain, an IgG4 (EQ) spacer, CD28 TM domain, 41-BB co-stimulatory domain and a CD3zeta domain (SEQ ID NOs: 107-108).
  • FIG 9 Depicts the amino acid sequence of CAR having: a variant IL13 targeting domain, an IgG4 (EQ) spacer, CD4 TM domain, CD28 co-stimulatory domain and a CD3zeta domain (SEQ ID NOs: 109-110).
  • FIG 10 Depicts the amino acid sequence of CAR having: a variant IL13 targeting domain, an IgG4 (EQ) spacer, CD4 TM domain, CD28 co-stimulatory domain and a CD3zeta domain (SEQ ID NOs: 111-112).
  • FIG 11 Depicts the amino acid sequence of CAR having: a variant IL13 targeting domain, an IgG4 (EQ) spacer, CD4 TM domain, CD28 co-stimulatory domain and a CD3zeta domain (SEQ ID NOs: 113-114).
  • FIG 12 Depicts the amino acid sequence of CAR having: a variant IL13 targeting domain, an IgG4 (EQ) spacer, CD4 TM domain, CD28 co-stimulatory domain and a CD3zeta domain (SEQ ID NOs: 115-116).
  • FIG 13 Depicts the amino acid sequences of CARs having: a variant IL13 targeting domain, an IgG4 (EQ) spacer, CD4 TM domain, 4-1BB co-stimulatory domain and a CD3zeta domain (SEQ ID NOs: 117-120).
  • FIG 14 Depicts the amino acid sequences of CARs having: a variant IL13 targeting domain, an IgG4 (EQ) spacer, CD8 TM domain, 41-BB co-stimulatory domain and a CD3zeta domain (SEQ ID NOs: 121-124).
  • FIG 15 Depicts the amino acid sequence of IL13(C4)-IgG4(L235E,N297Q)-CD28gg-Zeta without a signal sequence (SEQ ID NO: 125).
  • FIG 16 Depicts the amino acid sequence of a CAR having C4 mutein targeting domain. Amino acid sequence of IL13(C4)-IgG4(HL-CH3)-CD4tm-41BB-Zeta (SEQ ID NO: 126).
  • FIG 17 Depicts the amino acid sequence of a CAR having D7 mutein targeting domain.
  • FIG 18 Depicts the amino acid sequence of a CAR having D7 mutein targeting domain. Amino acid sequence of (A) IL13(D7)-IgG4(HL-CH3)-CD4tm-41BB-Zeta (SEQ ID NO: 128).
  • FIG 19 Depicts that amino acid sequence of and IL-13 CAR having a variant IL-13 targeting domain (SEQ ID NO: 129; without the signal peptide, SEQ ID NO:130).
  • FIG 20 Depicts that amino acid sequence of and IL-13 CAR having a variant IL-13 targeting domain (SEQ ID NO: 131; without the signal peptide, SEQ ID NO:132).
  • FIG 21 Depicts that amino acid sequence of and IL-13 CAR having a variant IL-13 targeting domain (SEQ ID NO: 133; without the signal peptide, SEQ ID NO:134).
  • FIG 22 Depicts that amino acid sequence of and IL-13 CAR having a variant IL-13 targeting domain (SEQ ID NO: 135; without the signal peptide, SEQ ID NO:136).
  • FIG 23 Depicts that amino acid sequence of and IL-13 CAR having a variant IL-13 targeting domain (SEQ ID NO: 137; without the signal peptide, SEQ ID NO:138).
  • FIG 24 Depicts that amino acid sequence of and IL-13 CAR having a variant IL-13 targeting domain (SEQ ID NO: 139; without the signal peptide, SEQ ID NO:140).
  • FIG 25 Depicts that amino acid sequence of and IL-13 CAR having a variant IL-13 targeting domain (SEQ ID NO: 141; without the signal peptide, SEQ ID NO:142).
  • FIG 26 Depicts that amino acid sequence of and IL-13 CAR having a variant IL-13 targeting domain (SEQ ID NO: 143; without the signal peptide, SEQ ID NO:144). Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017
  • FIG 27 Depicts that amino acid sequence of and IL-13 CAR having a variant IL-13 targeting domain (SEQ ID NO: 145; without the signal peptide, SEQ ID NO:146)
  • FIG 28 Depicts that amino acid sequence of and IL-13 CAR having a variant IL-13 targeting domain (SEQ ID NO: 147; without the signal peptide, SEQ ID NO:148).
  • FIG.29A-29M Depicts that amino acid sequence of and IL-13 CAR having a variant IL-13 targeting domain.
  • Flow cytometric analysis shows a significantly reduced percentage of CD3+ cells in the blood of mice bearing YUMM JAK1/KO tumors treated with mIL13R ⁇ 2 CAR T cells compared to YUMM WT (** p ⁇ 0.01). Data are mean ⁇ SD.
  • d Flow cytometric analysis showing reduced CAR T cell percentages in the blood of YUMM JAK1/KO tumor-bearing mice compared to YUMM WT (* p ⁇ 0.05). Data are mean ⁇ SD.
  • f Volumes of tumors untreated or treated with mIL13R ⁇ 2 CAR T cells, exhibiting significant response in YUMM WT tumors in comparison to suboptimal response in YUMM JAK1/KO tumors (* p ⁇ 0.05). Data are mean ⁇ SEM.
  • g Clustering of scRNA-seq data of 51,820 CD45+ cells from 6 JAK1/KO (23,848 cells, including 14,831 cells from mIL13R ⁇ 2 CAR T treated and 9,017 cells from untreated control tumors) and 6 WT (27,972 cells, 14,128 treated, 13,844 untreated) tumors, annotated by cluster identity.
  • j The percentage of mIL13R ⁇ 2 CAR T cells identified in scRNA-seq analysis shows a reduction in JAK1/KO YUMM tumors relative to WT YUMM tumors.
  • k Differentially regulated ligand-receptor signaling pathways in a comparison of the TME of CAR treated YUMM JAK1/KO and WT tumors. Significant pathways were selected with a p ⁇ 0.01, and pathways with an absolute delta of >0.8 were selected for plotting.
  • the information flow for a given signaling pathway is defined by the sum of communication Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 probability among all pairs of cell groups in the inferred network.
  • FIG. 30B UMAP dimensionality reduction of GBM/HGG (28,156 cells) and YUMM JAK1/KO (40,324 cells) scRNA-seq data, annotated by cluster identity.
  • FIGS.31A-31B Anti-SPP1 mAb restores SPP1-mediated inhibition on proliferation and activation of CAR T cell.
  • FIG.31A Human IL13R ⁇ 2-CAR T cell stained by CFSE was activated by 2.5 ⁇ g/mL anti-CD3 and incubated with indicated concentration of rhSPP1.
  • FIG. 31B The first IL13R ⁇ 2-CAR T cell stained by CFSE was activated by 2.5 ⁇ g/mL anti-CD3 and incubated with indicated concentration of rhSPP1.
  • Human IL13R ⁇ 2-CAR T cell stained by CFSE was activated by indicated amount of human IL13R ⁇ 2-Fc or 2.5 ⁇ g/mL anti-CD3 and incubated with 10 ⁇ g/mL recombinant human SPP1 or Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 not. Cell proliferation and activation (measured by CD69+41BB+) was detected by flow cytometry.
  • FIG.33 Sequence of anti-mouse/human/rat SPP1 Ab.
  • FIG.34A depicts the amino acid sequence of CLTX-IgG4(L235E, N297Q)-CD28tm-CD28gg- zeta (SEQ ID NO:149; mature sequence without the signal sequence is SEQ ID NO:150).
  • FIG.34B depicts the amino acid sequence of CLTX-IgG4(HL-CH3)-CD28tm -CD28gg-zeta (SEQ ID NO:151; mature sequence without the signal sequence is SEQ ID NO:152).
  • FIG.34C depicts the amino acid sequence of CLTX-CD8h-CD28tm-CD28gg-zeta (SEQ ID NO:153; mature sequence without the signal sequence is SEQ ID NO:154).
  • FIG.34D depicts the amino acid sequence of CLTX-IgG4(hinge)-CD28tm -CD28gg-zeta (SEQ ID NO:155; mature sequence without the signal sequence is SEQ ID NO:156).
  • FIG.34E depicts the amino acid sequence of CLTX-L--CD28tm-CD28gg-zeta (SEQ ID NO:157; mature sequence without the signal sequence is SEQ ID NO:158).
  • FIG.34F depicts the amino acid sequence of CLTX-IgG4(L235E, N297Q)-CD28tm-CD28gg-4- 1BB-zeta (SEQ ID NO:159; mature sequence without the signal sequence is SEQ ID NO:160).
  • FIG.34G depicts the amino acid sequence of CLTX-IgG4(HL-CH3)-CD28tm -CD28gg-4-1BB- zeta (SEQ ID NO:161; mature sequence without the signal sequence is SEQ ID NO:162).
  • FIG.34H depicts the amino acid sequence of CLTX-CD8h-CD28tm-CD28gg-4-1BB-zeta (SEQ ID NO:163; mature sequence without the signal sequence is SEQ ID NO:164).
  • FIG.34I depicts the amino acid sequence of CLTX-IgG4(hinge)-CD28tm-CD28gg-4-1BB-zeta (SEQ ID NO:165; mature sequence without the signal sequence is SEQ ID NO:166).
  • FIG.34J depicts the amino acid sequence of CLTX-L-CD28tm-CD28gg-4-1BB-zeta (SEQ ID NO:167; mature sequence without the signal sequence is SEQ ID NO:168).
  • FIG.34K depicts the amino acid sequence of CLTX-IgG4(L235E, N297Q)-CD28tm-4-1BB-zeta (SEQ ID NO:169; mature sequence without the signal sequence is SEQ ID NO:170).
  • FIG.34L depicts the amino acid sequence of CLTX-IgG4(HL-CH3)-CD4tm-4-1BB-zeta (SEQ ID NO:171; mature sequence without the signal sequence is SEQ ID NO:172).
  • FIG.34M depicts the amino acid sequence of CLTX-CD8h-CD28tm-4-1BB-zeta (SEQ ID NO:173; mature sequence without the signal sequence is SEQ ID NO:174).
  • FIG.34N depicts the amino acid sequence of CLTX-IgG4(hinge)-CD28tm-4-1BB-zeta (SEQ ID NO:175; mature sequence without the signal sequence is SEQ ID NO:176).
  • FIG.34O depicts the amino acid sequence of CLTX-L-CD28tm-4-1BB-zeta (SEQ ID NO:177; mature sequence without the signal sequence is SEQ ID NO:178).
  • FIG.35A depicts the amino acid sequence of HER2 scFv- IgG4(S228P, L235E, N297Q)- CD8tm-41BB-Zeta (SEQ ID NO:179; mature sequence without the signal sequence is SEQ ID NO:180).
  • FIG.35B depicts the amino acid sequence of HER2 scFv- IgG4(S228P, L235E, N297Q)- CD28tm-CD28gg-Zeta (SEQ ID NO:181; mature sequence without the signal sequence is SEQ ID NO:182).
  • FIG.35C depicts the amino acid sequence of HER2 scFv -CD8hinge-CD8tm-41BB-Zeta (SEQ ID NO:183; mature sequence without the signal sequence is SEQ ID NO:184).
  • FIG.35D depicts the amino acid sequence of HER2 scFv - IgG4hinge(S228P)-linker-CD8tm- 41BB-Zeta (SEQ ID NO:185; mature sequence without the signal sequence is SEQ ID NO:186).
  • FIG.35E depicts the amino acid sequence of HER2 scFv - IgG4(S228P)-Linker-IgG4 CH3- CD8tm-41BB-Zeta (SEQ ID NO:187; mature sequence without the signal sequence is SEQ ID NO:188).
  • FIG.35F depicts the amino acid sequence of HER2 scFv - IgG4(S228P)-Linker-IgG4 CH3-CD28tm-CD28gg-Zeta (SEQ ID NO:189; mature sequence without the signal sequence is SEQ ID NO:190).
  • FIG.35G depicts the amino acid sequence of HER2 scFv - Linker-CD28tm-CD28gg-Zeta (SEQ ID NO:191; mature sequence without the signal sequence is SEQ ID NO:192).
  • FIG.35H depicts the amino acid sequence of HER2 scFv - Linker-CD8tm-41BB-Zeta (SEQ ID NO:193; mature sequence without the signal sequence is SEQ ID NO:194).
  • FIG.35I depicts the amino acid sequence of HER2 scFv - CD8h-CD8tm-Zeta (SEQ ID NO:195; mature sequence without the signal sequence is SEQ ID NO:196).
  • SPP1 Antibodies Described herein, inter alia, are antibodies targeted to SPP1 and uses thereof.
  • SPP1 antibodies can comprise a variable light (VL) chain domain (e.g., any one of the VL listed in Table 6) and a variable heavy (VH) chain domain (e.g., any one of the VH listed in Table 6).
  • VL variable light
  • VH variable heavy
  • the SPP1 antibody comprises: a VL domain comprising the amino acid sequence of a VL listed in Table 6 or a variant thereof having 1-5 (e.g., 1, 2, or 3) single amino acid modifications (e.g., substitutions), wherein the modifications are not in the CDRs; and a VH domain comprising the amino acid sequence of a VH listed in Table 6 or a variant thereof having 1-5 (e.g., 1, 2, or 3) single amino acid modifications (e.g., substitutions), wherein the modifications are not in the CDRs.
  • the SPP1 antibody comprises the CDRs of a VL listed in Table 6 and the CDRs of a VH listed in Table 6.
  • SPP1 Antibody Sequences Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 Attorney Docket No.: 40056-0107WO1 / COH Ref:
  • the SPP1 antibody comprises a light chain (LC) comprising a variable light (VL) chain domain comprising the amino acid sequence of any one of SEQ ID NOs: P1, P9, P17, P25, P33, P41, P49, P57, P65, P73, P81, P89, P97, P105, P113, P121, P129, P137, P145, P153, P161, P169, P177, and variants thereof with 1-5 single amino acid modifications (e.g., 1 or 2 single amino acid substitutions), wherein the modifications (e.g., substitutions) are not in the CDRs; and Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 a heavy chain (HC) comprising a variable heavy (VH) chain domain comprising the amino acid sequence of any one of SEQ ID NOs: P5, P13, P21, P29, P37, P45, P53, P61 , P69, P77, P85,
  • HC
  • the SPP1 antibody is targeted to human SPP1 and/or is humanized.
  • a variety of antibodies targeted to SPP1 can be useful in the compositions and methods of the present application. Additional SPP1 targeting sequences and antibodies are known in the art; for example, US 11,897,950; US 2023/0203193; WO 2011/021146; Fan, K. et al. (2011) European journal of pharmacology 657, 144–151; Hirano, Y., et al. (2016) Shock 46, 431–438; Yamamoto, N. et al. (2007) International immunopharmacology 7, 1460–1470; Yamamoto, N. et al. (2003) The Journal of clinical investigation 112, 181–188; Zhang, B.
  • SPP1 antibodies include: Ab214055, Ab166709, Ab214050, Ab236213, Ab269441 (ThermoFisher); and hu1A12 (Absolute Antibody). IB.
  • a chimeric antigen receptor refers to an artificial immune cell receptor that is engineered to recognize and bind to a surface antigen.
  • a T cell that expresses a CAR polypeptide is referred to as a CAR T cell.
  • CAR have the ability to redirect T-cell specificity and reactivity toward a selected target in a non-MHC-restricted manner.
  • the non-MHC-restricted antigen recognition gives CAR T cells the ability to recognize an antigen independent of antigen processing, thereby bypassing a major mechanism of tumor escape.
  • First generation CARs join an antibody-derived scFv to the CD3 ⁇ intracellular signaling domain of the T cell receptor through a spacer region (also called a hinge domain) and a transmembrane domain.
  • Second generation CARs incorporate an additional co-stimulatory domain (e.g., CD28, Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 4-BB, or ICOS) to supply a co-stimulatory signal.
  • Third generation CARs contain two co- stimulatory domains (e.g., a combination of CD27, CD28, 4-1BB, ICOS, or OX40) fused with the TCR CD3 ⁇ chain.
  • a CAR is often fused to a signal peptide at the N-terminus for surface expression.
  • the CAR can be co-expressed with a polypeptide that can serve as marker, for example a truncated EGFR receptor lacking signaling function or a truncated CD19 receptor lacking signaling function.
  • the targeting domain (also called the extracellular binding domain) of useful CAR can be targeted to one or more antigen(s) present on the cell surface of a cancer cell.
  • the CAR comprise an scFv targeted to an antigen expressed on the surface of a cancer cell (e.g., a solid tumor).
  • the CAR include all or a portion of mature human IL13 or a variant thereof as a targeting domain (e.i., IL13R ⁇ 2 targeted CAR).
  • the CAR can comprise a targeting domain (e.g., an IL-13, a variant IL13, a chlorotoxin, a variant chlorotoxin or related toxin, a HER2 scFV, a PSCA scFv, a PSMA scFv, an EGFR scFv, CD70 scFv, etc.).
  • the extracellular binding domain comprises an scFv targeting a molecule expressed on the surface of a cancer cell, such as an antigen expressed on a solid tumor (e.g., HER2, PMSA, etc.).
  • a cancer cell such as an antigen expressed on a solid tumor (e.g., HER2, PMSA, etc.).
  • the extracellular binding domain of useful CAR can be targeted to, for example, IL13R ⁇ 2, MMP2, EGFR, HER2, PSCA, TAG72, PMSA, CD70, B7H3, etc.
  • the extracellular binding domain can comprise or consist of any one of: GPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRM LSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFN (SEQ ID NO: 1); GPVPPSTALRYLIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRM LSGFCPHKVSAGQFSSLHVRDTKILVAQFVKDLLLHLKKLFKEGRFN (“YLK”; SEQ ID NO: 2); Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 GPVPPSTALRRLIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQR MLSGFCPHKVSAGQFSSLHVRDTKILV
  • a IL-13 CAR can comprise or consist of the amino acid sequence of any one of SEQ ID NOs:149-178, or a variant thereof having 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) single amino acid modifications (e.g., substitutions); optionally, wherein the modifications are not in the targeting domain (e.g., the IL13).
  • IL-13 variants and IL-13R ⁇ 2 targeting sequences are known in the art; e.g., WO 2008/146911, WO 2004/087758, WO 2021/154543, WO 2021/183960, WO 2022/212525, US 2023/0265147 A1.
  • Chlorotoxin in the case of a CAR comprising a chlorotoxin (CLTX), variant thereof, or related toxin:
  • the extracellular binding domain can comprise or consist of: Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCR (SEQ ID NO:14) or a variant thereof having 1-5 (e.g., 1 or 2) single amino acid modifications (e.g., substitutions) provided that the cysteine residues are not modified.
  • a CLTX CAR can comprise or consist of the amino acid sequence of any one of SEQ ID NOs:149-178, or a variant thereof having 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) single amino acid modifications (e.g., substitutions); optionally, wherein the modifications are not in the targeting domain (e.g., the CLTX).
  • CLTX variants and related toxins such as GaTx2 & GaTx1, toxins from Leiurus quinquestriatus hebraeus, AaCtx, a toxin from Androctonus australis, etc.
  • sequences of the same as well as methods of making and using CLTX CAR and immune cells expressing CLTX CAR, are known in the art; e.g., WO 2017/066481.
  • Table 4 Examples of CAR Comprising Chlorotoxin Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 *SEQ ID NOs for sequence including signal sequence / SEQ ID NOs for sequence excluding signal sequence.
  • EGFR In the case of CAR comprises an scFv targeted to an epidermal growth factor receptor (EGFR), a truncated EGFR (EGFRt), a variant of EGFR (e.g., epidermal growth factor receptor variant III; EGFRvIII), or the extracellular binding domain of EGFR.
  • an anti-EGFR scFv targets both wild-type EGFR and EGFRvIII.
  • An EGFR scFv can comprise or consist of the amino acid sequence: DILMTQSPLSLPVSLGDQASISCRSSQNIVHNNGITYLEWYLQRPGQSPKLLIYKVSDRFSGVPD RFSGSGSGTDFTLKISRVEAEDLGIYYCFQGSHIPPTFGGGTKLEIKRAAGGGGSGGGGSGGG GSQVQLQQSGSEMARPGASVKLPCKASGDTFTSYWMHWVKQRHGHGPEWIGNIYPGSGGT NYAEKFKNKVTLTVDRSSRTVYMHLSRLTSEDSAVYYCTRSGGPYFFDYWGQGTTLTVSS (SEQ ID NO: 203; CDRs are underlines), or a variant thereof having 1-5 (e.g., 1, 2, 3, 4, or 5) single amino acid modifications (e.g., single amino acid substitutions); optionally, wherein the modifications are not in the CDRs.
  • 1-5 e.g., 1, 2, 3, 4, or 5
  • An EGFR scFv can comprise or consist of the amino acid sequence: EIQLVQSGA EVKKPGESLR ISCKGSGFNI EDYYIHWVRQ MPGKGLEWMG RIDPENDETK YGPIFQGHVT ISADTSINTV YLQWSSLKAS DTAMYYCAFR GGVYWGQGTT VTVSSGGGGS GGGGSGGGGS GGGGSDVVMT QSPDSLAVSL GERATINC KS Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 SQSLLDSDGK TYLNWLQQKP GQPPKRLISL VSKLDSGVPD RFSGSGTD FTLTISSLQA EDVAVYYCWQ GTHFPGTFGG GTKVEIK (SEQ ID NO: 204; CDRs are underlines), or a variant thereof having 1-5 (e.g., 1, 2, 3, 4, or 5) single amino acid modifications (e.g., single amino acid substitutions);
  • An EGFR CAR can comprise or consist of the amino acid sequence: DILMTQSPLSLPVSLGDQASISCRSSQNIVHNNGITYLEWYLQRPGQSPKLLIYKVSDRFSGVPD RFSGSGSGTDFTLKISRVEAEDLGIYYCFQGSHIPPTFGGGTKLEIKRAAGGGGSGGGGSGGG GSQVQLQQSGSEMARPGASVKLPCKASGDTFTSYWMHWVKQRHGHGPEWIGNIYPGSGGT NYAEKFKNKVTLTVDRSSRTVYMHLSRLTSEDSAVYYCTRSGGPYFFDYWGQGTTLTVSSPK SCDKTHTCPPCPDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPG PTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR DPEMGGKPRRKNPQEGLYNEL
  • EGFR targeting sequences e.g., EGFR CDRs, EGFR variable domains (i.e., VL and VH), and EGFR scFvs
  • EGFR targeted CAR as well as methods of making and using the same, are known in the art; e.g., CDRs and sequences of cetuximab, panitumumab, necitumumab, nepidermin, nimotuzumab, amivantamab, HS-627, amelimumab, depatuxizumab, FmAb-2, GC-1118A, imgatuzumab, matuzumab, MVC-101, SCT-200, QL-1203, tomuzotuximab, zalutumumab, JMT-101, MCLA-158, QL-1105, SYN-004, MCLA-129, WBP- 297, AM-105, BH-2922, BM
  • HER2 also called ErbB2
  • the extracellular binding domain can comprise or consist of a HER2 scFv.
  • the HER2 scFv comprises or consists of the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFS Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 GSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGSTSGGGSGGGSGGGGSS EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADS VKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (S
  • the HER2 scFv comprises or consists of the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFS GSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEV QLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVK GRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSS (SEQ ID NO: 206; CDRs are underlined), or a variant thereof having 1-5 (e.g., 1, 2, 3, 4, or 5) single amino acid modifications (e.g., single amino acid substitutions); optionally, wherein the modifications are not in the CDRs.
  • 1-5 e.g., 1, 2, 3, 4, or 5
  • the HER2 scFv comprises or consists of the amino acid sequence: DIQMTQSP SSLSASVGDR VTITCRASQD VNTAVAWYQQ KPGKAPKLLI YSASFLYSGV PSRFSGSRSG TDFTLTISSL QPEDFATYYC QQHYTTPPTF GQGTKVEIKR TGSTSGSGKP GSGEGSEVQL VESGGGLVQP GGSLRLSCAA SGFNIKDTYI HWVRQAPGKG LEWVARIYPT NGYTRYADSV KGRFTISADT SKNTAYLQMN SLRAEDTAVY YCSRWGGDGF YAMDVWGQGT LVTVSS (SEQ ID NO:207) or a variant thereof having 1-5 (e.g., 1, 2, 3, 4, or 5) single amino acid modifications (e.g., single amino acid substitutions); optionally, wherein the modifications are not in the CDRs.
  • 1-5 e.g., 1, 2, 3, 4, or
  • the HER2 scFv comprises or consists of the amino acid sequence: DIQMTQSP SSLSASVGDR VTITCRASQD VNTAVAWYQQ KPGKAPKLLI YSASFLESGV PSRFSGSRSG TDFTLTISSL QPEDFATYYC QQHYTTPPTF GQGTKVEIKR TGSTSGSGKP GSGEGSEVQL VESGGGLVQP GGSLRLSCAA SGFNIKDTYI HWVRQAPGKG LEWVARIYPT NGYTRYADSV KGRFTISADT SKNTAYLQMN SLRAEDTAVY YCSRWGGDGF VAMDVWGQGT LVTVSS (SEQ ID NO:208) or a variant thereof having 1-5 (e.g., 1, 2, 3, 4, or 5) single amino acid modifications (e.g., single amino acid substitutions); optionally, wherein the modifications are not in the CDRs.
  • 1-5 e.g., 1, 2, 3, 4, or 5
  • the HER2 CAR comprises or consists of the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFS GSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGSTSGGGSGGGSGGGGSS EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADS VKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSESKY GPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYK
  • a HER2 CAR can include, from amino to carboxy terminus, an scFv (underlined) and spacer (not underlined) a CD8 transmembrane domain (underlined), a 4-1BB co-stimulatory domain (not underlined), a GGG linker (underlined) and a CD3zeta domain (not underlined).
  • the spacer in this HER2 CAR of SEQ ID NO:180 is IgG4(S228P, L235E, N297Q).
  • a HER2 CAR can comprise or consist of the amino acid sequence of any one of SEQ ID NOs:179-196, or a variant thereof having 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) single amino acid modifications (e.g., substitutions); optionally, wherein the modifications are not in the targeting domain (e.g., the HER2scFv).
  • HER2 targeting sequences e.g., HER2 CDRs, HER2 variable domains (i.e., VL and VH), and HER2 scFvs
  • HER2 targeted CAR e.g., 4D5, rastuzumab, trastuzumab emtansine, trastuzumab deruxtecan, margetuximab, pertuzumab, disitamab vedotin, U-31402, ISU-104, SIB-001, 9F7- F11, EV-20Sap, U-31402, ARX-788, BAT-8001, HL-02, TAA-013, trastuzumab duocarmazine, A-166, AU-101, AU-105, BPX-603, ISB-1302, KN-026, MB-103, MRG-002, zanidatamab, zenocutuzum
  • the scFv can comprise or consist of the amino acid sequence: DIQLTQSPSTLSASVGDRVTITCSASSSVRFIHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGS GSGTDFTLTISSLQPEDFATYYCQQWGSSPFTFGQGTKVEIKGSTSGGGSGGGSGGGGSSEV QLVEYGGGLVQPGGSLRLSCAASGFNIKDYYIHWVRQAPGKGLEWVAWIDPENGDTEFVPKF QGRATMSADTSKNTAYLQMNSLRAEDTAVYYCKTGGFWGQGTLVTVSS (SEQ ID NO:209) or a variant thereof having 1-5 (e.g., 1, 2, 3, 4, or 5) single amino acid modifications (e.g., single amino acid substitutions); optionally, wherein the modifications are not in the CDRs.
  • 1-5 e.g., 1, 2, 3, 4, or 5
  • single amino acid modifications e.g., single amino acid substitutions
  • PSCA targeting sequences e.g., PSCA CDRs, PSCA variable domains (i.e., VL and VH), and PSCA scFvs
  • PSCA targeted CAR as well as methods of making and using the same, are known in the art; e.g., WO 2017/062628; WO 2022/115421; US20240041921A1; Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 Morgenroth et al., (2007) Prostate 67(10):1121-1131 (scFv 7F5); Nejatollahi et al., (2013) J Oncology, article ID 839831 (scFv CS-II); and US 2009/0311181 A1.
  • the extracellular binding domain can comprise or consist of a PSMA scFv.
  • the scFv can comprise or consist of the amino acid sequence of: DIVMTQS HKFMSTSVGD RVSIICKASQ DVGTAVDWYQ QKPGQSPKLL IYWASTRHTG VPDRFTGSGS GTDFTLTITN VQSEDLADYF CQQYNSYPLT FGAGTMLDLK GGGGSGGGGS SGGGSEVQLQ QSGPELVKPG TSVRISCKTS GYTFTEYTIH WVKQSHGKSL EWIGNINPNN GGTTYNQKFE DKATLTVDKS SSTAYMELRS LTSEDSAVYY CAAGWNFDYW GQGTTLTVSS ASSG (SEQ ID NO: 210; CDRs are underlined), or a variant thereof having 1-5 (e.g., 1, 2, 3, 4, or 5) single amino acids
  • PSMA targeting sequences e.g., PSMA CDRs, PSMA variable domains (i.e., VL and VH), and PSMA scFvs
  • PSMA targeted CAR as well as methods of making and using the same, are known in the art; CIK-CAR.PSMA (Formula Pharmaceuticals Inc), P-PSMA-101 (Poseida Therapeutics Inc), UniCAR-T-PSMA (GEMoaB Monoclonals GmbH), Parker et al., Protein Expr Purif 89(2):136-145 (2013), US 20110268656 (e.g., J591 ScFv); Frigerio et al, European J Cancer 49(9):2223-2232 (2013) (e.g., scFvD2B); WO 2006125481 (e.g., mAbs 3/A12, 3/E7, and 3/F11 and (scFv A5 and D7).
  • PSMA CDRs PSMA variable domains (i.e
  • the extracellular binding domain can comprise or consist of a TAG72 scFv.
  • the scFv can comprise or consist of: QVQLVQSGAEVVKPGASVKISCKASGYTFTDHAIHWVKQNPGQRLEWIGYFSPGNDDFKYSQ KFQGKATLTADTSASTAYVELSSLRSEDTAVYFCTRSLNMAYWGQGTLVTVSSGSTSGGGSG GGSGGGGSSDIVMSQSPDSLAVSLGERVTLNCKSSQSVLYSSNSKNYLAWYQQKPGQSPKLL IYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYPLSFGAGTKLELK (SEQ ID NO: 211; CDRs are underlined), or a variant thereof having 1-5 (e.g., 1, 2, 3, 4, or 5) single amino acid modifications (e.g., substitutions); optionally,
  • a Tag72 CAR can comprise or consist of the amino acid sequence QVQLVQSGAEVVKPGASVKISCKASGYTFTDHAIHWVKQNPGQRLEWIGY FSPGNDDFKYSQKFQGKATLTADTSASTAYVELSSLRSEDTAVYFCTRSL NMAYWGQGTLVTVSS GSTS GGGSGGGSGGGGSS DIVMSQSPDSLAVS LGERVTLNCKSSQSVLYSSNSKNYLAWYQQKPGQSPKLLIYWASTRESGV PDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYPLSFGAGTKLELKE SKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTV
  • TAG-72 targeting sequences e.g., TAG72 CDRs, TAG72 variable domains (i.e., VL and VH), and TAG72 scFvs
  • TAG72 targeted CAR e.g., WO 2020/028721.
  • CD70 In the case of CAR targeted to CD70, the extracellular binding domain can comprise or consist of a CD70 binding domain (e.g., a CD70 scFv).
  • the scFv can comprise or consist of the amino acid sequence: QAVVTQEPS LTVSPGGTVT LTCGLKSGSV TSDNFPTWYQ QTPGQAPRLL IYNTNTRHSG VPDRFSGSIL GNKAALTITG AQADDEAEYF CALFISNPSV EFGGGTQLTV LGGGGGSGGG GSGGGGSGGG GSEVQLVESG GGLVQPGGSL RLSCAAS GFT FSVYYMNWVR QAPGKGLEWV SDINNEGGTT YYADSVKGRF TISRDNSKNS LYLQMN SLRA EDTAVYYCAR DAGYSNHVPI FDSWGQGTLV TVSS (SEQ ID NO: 212; CDRs are underlined), or a variant thereof having 1-5 (e.g., 1, 2, 3, 4, or 5) single amino acid modifications (e.g., single amino acid substitutions); optionally, wherein the modifications are not in the CDRs.
  • 1-5 e
  • a CD70 CAR comprises or consist of the amino acid sequence MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAA Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 QCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDP LPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSL CSSDFIRILVIFSGMFLVFTLAGALFLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFP EEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL
  • CD70 targeting sequences e.g., CD70 CDRs, CD70 variable domains (i.e., VL and VH), and CD70 scFvs
  • CD70 targeted CAR e.g., CD70 targeted CAR, as well as methods of making and using the same, are known in the art; e.g., Xiong, Q., et al. (2024)
  • CD70 CDRs CD70 variable domains
  • VL and VH variable domains
  • CD70 scFvs CD70 targeted CAR
  • the extracellular binding domain can comprise or consist of a B7H3 scFv.
  • the scFv can comprise the CDRs of antibody MGA271.
  • B7H3 targeting sequences e.g., B7H3 CDRs, B7H3 variable domains (i.e., VL and VH), and B7H3 scFvs
  • B7H3 targeted CAR as well as methods of making and using the same, are known in the art; e.g., antibody MGA271 (Macrogenics); WO 2024/061306; WO 2024/226468; US20250136697A1; US20240245771A1; US20240252639A1.
  • Commercial and Clinical CAR Additional CAR known in the art can be used in combination with the SPP1 antibodies described herein.
  • the transmembrane domain of the CAR constructs used in the Examples has CD4 transmembrane domain having the sequence: MALIVLGGVAGLLLFIGLGIFF (SEQ ID NO: 18) or a CD28 TM having the sequence MFWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO:17).
  • Other transmembrane domains can be used including those shown below.
  • Table 1 Examples of Transmembrane Domains (c) Spacer Domain
  • the CAR or polypeptide described herein can include a spacer domain located between the targeting domain and the transmembrane domain.
  • the spacer region can function to provide flexibility to the CAR, or domains thereof, or to prevent steric hindrance of the CAR, or domains thereof.
  • a variety of different spacers can be used. Some of them include at least portion of a human Fc region, for example a hinge portion of a human Fc region or a CH3 domain or variants thereof. Table 2 below provides various spacer domains that can be used in the CARs described herein.
  • Spacer Domains include all or part of an immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4) hinge region, i.e., the sequence that falls between the CH1 and CH2 domains of an immunoglobulin, e.g., an IgG4 Fc hinge or a CD8 hinge.
  • Some spacer domains include an immunoglobulin CH3 domain (called CH3 or ⁇ CH2) or both a CH3 domain and a CH2 domain.
  • the immunoglobulin derived sequences can include one or more amino acid modifications, for example, 1, 2, 3, 4 or 5 substitutions, e.g., substitutions that reduce off-target binding.
  • the spacer domain can also comprise an IgG4 hinge region having the sequence ESKYGPPCPSCP (SEQ ID NO: 26) or ESKYGPPCPPCP (SEQ ID NO: 25).
  • the spacer domain can comprise the sequence: ESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL SLSLGK (SEQ ID NO: 31).
  • the spacer has 1, 2, 3, 4, or 5 single amino acid changes (e.g., conservative changes) compared to SEQ ID NO: 31.
  • the IgG4 Fc hinge/linker region that is mutated at two positions (L235E; N297Q) in a manner that reduces binding by Fc receptors (FcRs).
  • Intracellular Signaling Domains Any of the CAR constructs described herein contain one or more intracellular signaling domains (e.g., CD3 ⁇ , and optionally one or more co-stimulatory domains), which are the functional end of the receptor. Following antigen recognition, receptors cluster and a signal is transmitted to the cell.
  • CD3 ⁇ is the cytoplasmic signaling domain of the T cell receptor complex.
  • CD3 ⁇ contains three immunoreceptor tyrosine-based activation motifs (ITAMs), which transmit an activation signal to the T cell after the T cell is engaged with a cognate antigen.
  • ITAMs immunoreceptor tyrosine-based activation motifs
  • CD3 ⁇ provides a primary T cell activation signal but not a fully competent activation signal, which requires a co- stimulatory signal.
  • the CAR polypeptides disclosed herein may further comprise one or more co-stimulatory signaling domains in addition to CD3 ⁇ .
  • the co- stimulatory domain CD28 and/or 4-1BB can be used to transmit a proliferative/survival signal together with the primary signaling mediated by CD3 ⁇ .
  • the co-stimulatory domain(s) are located between the transmembrane domain and the CD3 ⁇ signaling domain. Table 3 includes examples of suitable co-stimulatory domains together with the sequence of the CD3 ⁇ signaling domain.
  • CD3 ⁇ Domain and Examples of Co-stimulatory Domains Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017
  • the CD3 ⁇ signaling domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 98% identical to SEQ ID NO: 35.
  • the CD3 ⁇ signaling domain has 1, 2, 3, 4, or 5 amino acid changes (preferably conservative substitutions) compared to SEQ ID NO: 35.
  • the CD3 ⁇ signaling domain is SEQ ID NO: 35.
  • the co-stimulatory domain is selected from the group consisting of: a co-stimulatory domain depicted in Table 3 or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications, a CD28 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications, a 4-1BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications and an OX40 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications.
  • a 4-1BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications is present in the CAR polypeptides described herein.
  • there are two co-stimulatory domains for example, a CD28 co- stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions) and a 4-1BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions).
  • the 1-5 (e.g., 1 or 2) amino acid modification are substitutions.
  • the co-stimulatory domain is amino terminal to the CD3 ⁇ signaling domain and a short linker consisting of 2 – 10, e.g., 3 amino acids (e.g., GGG) can be positioned between the co-stimulatory domain and the CD3 ⁇ signaling domain.
  • a short linker consisting of 2 – 10, e.g., 3 amino acids (e.g., GGG) can be positioned between the co-stimulatory domain and the CD3 ⁇ signaling domain.
  • the CAR can be produced using a vector in which the CAR open reading frame is followed by a T2A ribosome skip sequence and a truncated EGFR (EGFRt), which lacks the cytoplasmic signaling tail, or a truncated CD19R (also called CD19t).
  • co- expression of EGFRt or CD19t provides an inert, non-immunogenic surface marker that allows for accurate measurement of gene modified cells, and enables positive selection of gene- modified cells, as well as efficient cell tracking of the therapeutic T cells in vivo following adoptive transfer. Efficiently controlling proliferation to avoid cytokine storm and off-target toxicity is an important hurdle for the success of T cell immunotherapy.
  • the EGFRt or the CD19t incorporated in the CAR lentiviral vector can act as suicide gene to ablate the CAR+ T cells in cases of treatment-related toxicity.
  • the CD3 ⁇ signaling domain can be followed by a ribosomal skip sequence (e.g., LEGGGEGRGSLLTCGDVEENPGPR; SEQ ID NO: 45) and a truncated EGFR having a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to: LVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFT HTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSL GLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPE Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 GCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGR GPD
  • the truncated EGFR has 1, 2, 3, 4 of 5 amino acid changes (preferably conservative) compared to SEQ ID NO: 46.
  • the CD3 ⁇ signaling domain can be followed by a ribosomal skip sequence (e.g., LEGGGEGRGSLLTCGDVEENPGPR; SEQ ID NO: 45) and a truncated CD19R (also called CD19t) having a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to: MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLK LSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWN VSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCVPPRDSLNQSLSQD LTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSL
  • the CAR described herein can be produced by any means known in the art, though preferably it is produced using recombinant DNA techniques.
  • Nucleic acids encoding the several regions of the chimeric receptor can be prepared and assembled into a complete coding sequence by standard techniques of molecular cloning known in the art (genomic library screening, overlapping PCR, primer-assisted ligation, site-directed mutagenesis, etc.) as is convenient.
  • the resulting coding region is preferably inserted into an expression vector and used to transform a suitable expression host cell line, preferably a T lymphocyte, and most preferably an autologous T lymphocyte.
  • a suitable expression host cell line preferably a T lymphocyte, and most preferably an autologous T lymphocyte.
  • Various T cell subsets isolated from the patient can be transduced with a vector for CAR or polypeptide expression.
  • Central memory T cells are one useful T cell subset.
  • Central memory T cell can be isolated from peripheral blood mononuclear cells (PBMC) by selecting for CD45RO+/CD62L+ cells, using, for example, the CliniMACS® device to immunomagnetically select cells expressing the desired receptors.
  • the cells enriched for central memory T cells can be activated with anti-CD3/CD28, transduced with, for example, a lentiviral vector that directs the expression of a CAR or as well as a non-immunogenic surface marker for in vivo detection, ablation, and potential ex vivo selection.
  • the activated/genetically modified central memory T Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 cells can be expanded in vitro with IL-2/IL-15 and then cryopreserved. Additional methods of preparing CAR T cells can be found in PCT/US2016/043392. Methods for preparing useful T cell populations are described in, for example, WO 2017/015490 and WO 2018/102761. In some cases, it may be useful to use natural killer (NK) cells, e.g., allogenic NK cells derived from peripheral blood or cord blood. In other cases, NK cells can be derived from human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs).
  • NK natural killer
  • NK cells can be derived from human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs).
  • a composition comprising the iPSC-derived CAR T cells or CAR NK cells.
  • a composition comprising iPSC-derived CAR T cells or CAR NK cells has enhanced therapeutic properties.
  • the iPSC- derived CAR T cells or CAR NK cells demonstrate enhanced functional activity including potent cytokine production, cytotoxicity and cytostatic inhibition of tumor growth, e.g., as activity that reduces the amount of tumor load.
  • the CAR can be transiently expressed in a T cell population by an mRNA encoding the CAR.
  • mRNA can be introduced into the T cells by electroporation (Wiesinger et al.2019 Cancers (Basel) 11:1198).
  • a composition comprising the CAR T cells comprise one or more of helper T cells, cytotoxic T cells, memory T cells, na ⁇ ve T cells, regulatory T cells, natural killer T cells, or combinations thereof. II.
  • aspects of the present disclosure provide methods for treating a subject with cancer (e.g., gliomas, such as glioblastoma, and solid tumors, including metastases thereof) by administering an antibody targeted to human SPP1 and a population of immune cells, e.g., IL13 CAR T cells, HER2 CAR T cells, PSMA CAR T cells, and PSCA CAR T cells.
  • the cancer can include cells that express IL13R ⁇ , MMP2, EGFR/EGFRvIII, TAG72, B7H3, CD70, HER2, PSCA, and PSMA.
  • Subjects The terms “subject” and “patient” are used interchangeably throughout.
  • the subject to be treated by the methods described can be a human subject having a cancer, such as a cancer comprising cells that express any one of IL13R ⁇ 2 MMP2, EGFR/EGFRvIII, TAG72, B7H3, Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 CD70, HER2, PSCA, and PSMA.
  • a subject has a glioma, a, glioblastoma, a solid tumor, and/or a metastasis of any thereof.
  • An effective amount of a therapy can be administered to a subject (e.g., a human) in need of the treatment via any suitable route (e.g., administered locally or systemically to a subject).
  • a suitable route e.g., administered locally or systemically to a subject.
  • suitable modes of administration include injection, infusion, instillation, or ingestion.
  • Injection includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intraventricular, intradermal, intraperitoneal, subcutaneous injection, and infusion.
  • an SPP1 antibody described herein can administered together with a population of immune cells expressing a CAR (e.g., CAR T cells, CAR NK cells, CAR M cells) targeted to, for example, human IL-13R ⁇ 2, MMP2, EGFR/EGFRvIII, TAG72, B7H3, CD70, HER2, PSCA or PSMA.
  • a CAR e.g., CAR T cells, CAR NK cells, CAR M cells
  • the SPP1 antibody is administered prior to administration of the CAR (e.g., CAR T cells).
  • the administration of the SSP1 antibody can precede administration of the CAR by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or longer.
  • the SPP1 antibody is administered several times prior to the administration of the CAR and the second or subsequent administration of the SPP1 antibody can be at any point prior to, coincident with, or subsequent to administration of the CAR (e.g., CAR T cells).
  • the SPP1 antibody can be administered to a subject in need thereof (e.g., suffering from a cancer) two or three times prior to the administration of the CAR and a subsequent administration of the SPP1 antibody can be administered coincident with the administration of the CAR (e.g., population of CAR immune cells, e.g., CAR T cells).
  • An effective amount refers to the amount of each active agent required to confer therapeutic effect on the subject, either alone or in combination with one or more other active agents.
  • Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of treatment, the nature of concurrent therapy, if any, the specific route of administration and like factors.
  • a therapeutic effect on the subject can be determined as the amelioration of one symptom associated with the condition being treated or a reduction of size or number of cancer cells in the subject (e.g., 1%, 2%, 3%, 4%, or 5% decrease in size or number of cancer cells).
  • Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 EXAMPLES The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
  • GBM/HGG tumor samples acquisition and processing Baseline tumor tissue samples were obtained from 41 patients with recurrent/refractory malignant glioma participating in a Phase I study (IRB #13384) on cellular immunotherapy using central memory-enriched IL13R ⁇ 2-targeting Chimeric Antigen Receptor (CAR) T cells. Tumor resection material was collected through the COH Department of Pathology according to the clinical protocol.
  • BioLegend Total Seq-C hashtag antibodies were used to allow sample deconvolution after pooled processing, with barcoded samples sorted at equal proportions into a single collection tube. Samples in the remaining batches were sequenced without pooling.60,000 cells were loaded to a single Gel Bead-in-Emulsion (GEM) reaction onto the Chromium instrument. ScRNA-seq library preparation was performed according to manufacturer protocols and sequenced on Illumina iSeq100 for cell count validation and NovaSeq6000 at the recommended depth. Sequence data were processed using 10x Genomics Cell Ranger V5.0 and Ensemble 98.
  • GEM Gel Bead-in-Emulsion
  • GBM/HGG tumor single-cell data processing and analysis Single-cell sequencing data were analyzed using Seurat v5.
  • CellRanger objects for each batch were imported to create a Seurat object for each of the 37 batches.
  • sample identities were deconvoluted, and multiplets were identified using Demuxlet.
  • Exome sequencing FASTQ reads were chunked to 40M reads, aligned to GRCh38 using BWA, and processed with samtools (v1.10) fixmates and samtools sort (v1.10). Individual chunks were merged, and PCR and optical duplicates were marked with samtools.
  • Genotypes were called with DeepVariant Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 (github.com/google/deepvariant).
  • demultiplexing was done using the HTODemux() function of Seurat.
  • the single-cell data were filtered to retain singlets with >500 unique RNA features detected, >1,000 RNA feature counts, and ⁇ 10% of reads mapping to mitochondrial genes.
  • possible doublets were identified using DoubletFinder v2.0.3.
  • PCs principal components
  • Expression data were preprocessed for the cell-cell communication analysis by identifying over-expressed ligands or receptors in one group and then identifying over-expressed ligand-receptor interactions. Then, gene expression data were projected onto the protein-protein interaction network using the function projectData(). Next, communication probabilities were computed using computeCommunProb(), and the results were filtered to retain instances with a minimum of ten cells in each group.
  • Cell-cell communication was inferred at a pathway level by summarizing the Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 communication probabilities of all ligands-receptor interactions associated with each signaling pathway using computeCommunProbPathway(), and the aggregated communication network was calculated by counting the number of links and summarizing the communication probability using aggregateNet().
  • Cox proportional-hazards model using the coxph() function of the R package survival v 3.5-5 (github.com/therneau/survival).
  • the “mkfastq” command was used to generate FASTQ files and the “count” command was used to generate raw gene-barcode matrices aligned to the 10X Genomics GRCm38 reference genome (mm10).
  • the data from all samples were combined in R (4.0.4) using the Read10X() function from the Seurat package (v4.0.3) and an aggregate Seurat object was generated. Filtering was conducted by retaining cells that had unique molecular identifiers (UMIs) greater than 400, expressed 200 and 9000 genes inclusive, and had mitochondrial content less than 15 percent. No sample batch correction was performed. Data were normalized using the “LogNormalize” method and using a scale factor of 10,000.
  • Clustering was conducted with the FindClusters() function using 20 PCA components and a Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 resolution parameter set to 0.3.
  • Cell cycle analysis was conducted using the CellCycleScoring() in Seurat package with a list of cell cycle markers from Aviv Regev et al’s study. Differentially expressed genes among clusters and treatments were identified with logFC greater than 0.25 (adjusted p ⁇ 0.05) as determined in Wilcoxon rank-sum test from Seurat.
  • the markers for different cell types were retrieved from CellMarker database, which is a manually curated resource of cell markers in human and mouse.
  • the pathway analysis was done using Gene Ontology and canonical pathways in enrichr, with adjusted P ⁇ 0.05 as the cutoff for statistically differential pathways.
  • Cell–cell interactions based on the expression of known ligand–receptor pairs in different cell types were inferred using CellChatDB (v1.0.0).
  • the total counts of interactions and interaction strengths were calculated using the compareInteractions function in CellChat.
  • the differential edge list was passed through CircosDiff (a wrapper around the R package ‘circlize’) and netVisual_chord_gene in CellChat to filter receptor ligand edges and generate Circos plots.
  • Immune cells were isolated using the CD45+ isolation kit (EasySepTM Mouse CD45 Positive Selection Kit, STEMCELL Technologies). A panel of 35 immune markers was used for analysis. Samples were analyzed using the Fluidigm Helios Mass Cytometry System at the UCLA Flow Cytometry core. Manual gating was performed using FlowJo software (version 10.4.2) to identify cells, singlets, and viable CD45+ populations. Data files were analyzed using OMIQ software. Cluster median data were normalized, and a threshold of >0.5 was used to define positive immune markers for cluster identification and annotation. Murine RNA Isolation and RNA-seq analysis For in vitro experiments, melanoma cell lines were seeded of 1.5 ⁇ 105 cells per 6-well plate for treatment.
  • the culture media were replaced with fresh media containing IFN ⁇ (BD Pharmingen, catalog no.554616) or supernatant of mIL13R ⁇ 2 CAR T cells with YUMM2.1 WT tumors.
  • Cells were harvested 8 hours after treatment. The cell pellets were lysed in TRIzol reagent (Invitrogen, catalog no.15596018) and stored at ⁇ 80°C until RNA extraction.
  • RNA-seq libraries were constructed using KAPA mRNA HyperPrep Kit.
  • RNA-seq was performed in the Illumina HiSeq2000 System using 51 bp single-end sequencing. High-quality reads were obtained by trimming the raw reads using fastp (v0.23.3) and by carrying out FastQC (v0.11.9) for the quality control assessment. The clean reads were aligned to the mouse reference genome GRCm38 with HISAT-STRINGTIE analytic pipeline (HISAT2 v2.1.0; Stringtie v1.3.4). The DESeq2 (v.1.26.0) framework was adopted for differential expression analysis.
  • YUMM WT and YUMM JAK1/KO were graciously provided by the Antoni Ribas laboratory, located within the comprehensive cancer center at UCLA. Both the YUMM and MC38 sublines were cultured at 37°C with 5% CO2 in DMEM (Invitrogen), supplemented with 10% FBS, 100 U/mL penicillin, 100 ⁇ g/mL streptomycin, and 0.25 ⁇ g/mL amphotericin B.
  • mice 1 ⁇ 10 6 MC38 WT and JAK1/KO cells were injected into the mice, followed by the same treatment after 8 days.
  • 5 ⁇ 10 6 mIL13R ⁇ 2 CAR T cells were infused via the tail vein on day 8 post-tumor inoculation. Tumor volumes were regularly measured using calipers.
  • 1 ⁇ 10 5 tumor cells were stereotactically implanted intracranially into the right forebrain of mice. Successful engraftment was confirmed by bioluminescence imaging the day before injecting the CAR T cells. Mice were grouped based on bioluminescence intensity.
  • mice received an intracranial administration of Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 1 ⁇ 10 6 mIL13R ⁇ 2 CAR T cells. Tumor progression was monitored using SPECTRAL LagoX and analyzed with Aura software. Survival outcomes were charted using GraphPad Prism Software (v10).
  • the pretreatment group was administered three doses every other day before CAR T cell therapy and received three additional doses afterward.
  • the simultaneous treatment group was given an initial dose of anti-OPN antibody (bioXcell Clone: 100D3) on the day of CAR T cell therapy, followed by five more doses to ensure parity in antibody delivery across groups, all given intraperitoneally (IP).
  • the combination group received three doses of anti-SPP1 antibody (clone MPIIIB10) prior to the CAR T cell therapy on day 8, continuing with five subsequent doses IP. All mice were continually observed by the Center for Comparative Medicine at City of Hope for signs of tumor progression and overall survival, with euthanasia applied in adherence to the American Veterinary Medical Association Guidelines.
  • Biostatistics Statistical significance in the in vivo studies was assessed utilizing either a Student t-test for two groups or a one-way ANOVA with Bonferroni correction for three or more groups. Survival analysis was represented via Kaplan-Meier survival curves, with statistical significance determined by the log-rank (Mantel-Cox) test. All statistical analyses were conducted using GraphPad Prism software (v10). Significance levels were denoted as follows: *, P ⁇ 0.05; **, P ⁇ 0.01; ***, P ⁇ 0.001; ****, P ⁇ 0.0001.
  • YUMM and MC38 sublines expansion Mouse tumor cells expanded in vitro were stained with an unconjugated goat anti-mouse IL13R ⁇ 2 (R&D Systems) followed by secondary donkey anti-goat NL637 (R&D Systems). Live murine CAR T cells were stained with CD3 (eBioscience) and CD19 (BD Biosciences) as a surrogate to detect the CAR.
  • ICAM-1 engineering of the tumor cells The plasmid containing the ICAM-1 gene and the packaging plasmids were provided by VectorBuilder. The lentiviral backbone was used for the construction of the expression vector. The cells were then incubated with the lentivirus following the protocol provided by Vectorbuilder.
  • Neomycin (Geneticin) was used for positive selection of the transduced cells.
  • the overexpression of ICAM-1 was confirmed using flow cytometry analysis.
  • the engineered YUMM JAK1/KO and YUMM-WT as well as MC38 JAK1/KO and MC38 WT were exposed to Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 supernatants derived from CAR T cell mediated cytotoxicity with matched YUMM and MC38 WT tumors. The cells, subsequently, were sent to bulk RNA-sequencing for transcriptional analysis.
  • Murine CAR T cells The murine IL13R ⁇ 2 CAR was engineered within an MSCV retroviral backbone (Addgene), comprising the murine IL13 extracellular domain, murine CD8 hinge, murine CD8 transmembrane domain, and intracellular murine 4-1BB costimulatory and murine CD3 ⁇ signaling domains.
  • MSCV retroviral backbone Additional structural protein
  • a truncated murine CD19 was inserted downstream via a T2A ribosomal skip as a transduction marker.
  • the resulting plasmid was transfected into PlatE cells (courtesy of Dr. Zuoming Sun’s lab) using Fugene (Promega).
  • Murine T cells were isolated from spleens of na ⁇ ve C57BL/6J mice or using the EasySep Mouse T cell Isolation Kit (STEMCELL Technologies) and activated with Dynabead Mouse T-Activator CD3/CD28 beads (Gibco) at a 1:1 ratio. Transduction of T cells occurred on RetroNectin-coated plates (Takara Bio) using retrovirus-containing supernatants (as described above).
  • Target cells were dissociated using trypsin or scraped to remove enzymes, resuspended in assay media, and passed through a 40 ⁇ m cell strainer to eliminate clumps. Cell counts were determined using the Muse Cell Analyzer, and cells were resuspended at the desired concentration. A total of 50 ⁇ L of assay media was added to each well of the E-plate, and background signal was recorded on the Xcelligence instrument, ensuring no bubbles were present. The plate was then removed, and 100 ⁇ L of the target cell suspension was added to each well. Cells were allowed to settle for 45 minutes at room temperature before placing the plate back on the instrument to minimize edge effects and ensure accurate readings.
  • Target cells were cultured for 24 hours at 37°C in a humidified incubator with 5% CO!.
  • effector cells were prepared at a concentration of 1.0 ⁇ 106 cells/mL for each cell line, followed by serial dilutions to achieve the desired effector- to-target (E:T) ratio.
  • the Xcelligence experiment was paused, and 100 ⁇ L of the prepared effector cell suspension was added to each well in a tissue culture hood. Cells were allowed to Attorney Docket No.: 40056-0107WO1 / COH Ref: TEC 24-017 settle for 45 minutes at room temperature before resuming the experiment on the Xcelligence instrument.
  • Immunofluorescence staining was done on 5 ⁇ m-thick sections of FFPE (formalin-fixed paraffin- embedded) specimens placed on positively charged glass slides.
  • the slides were deparaffinized in xylene, rehydrated in an ethanol gradient, and underwent antigen retrieval (10 min, 110-116 o C) in citrate-based antigen unmasking solution (#H-3300, Vector Laboratories) using a pressure cooker. The slides were then washed and incubated with carbohydrate-free blocking solution (#SP- 5040, Vector Laboratories) prior to staining.
  • the slides were then incubated with anti- COL1A1 antibody (E3E1X, Cell Signaling, #66948), anti-SPP1 (#AF1433, R&D) and anti-CD68 (EPR20545, Abcam, #ab213363) overnight at 4 °C, followed by incubation with donkey anti- mouse Alexa Fluor 555 (#A-31570), donkey anti-goat Alexa Fluor 488 (#A-11055), donkey anti- rabbit Alexa Fluor 647 (#A-31573) and Hoechst 33342 (#H3570, Thermofisher Scientific) for 1 hour (RT).
  • the slides were then coverslipped and scanned using a Zeiss LSM880 confocal microscope.
  • Murine tissues were incubated with anti-COL1A1 antibody (E8F4L, Cell Signaling, #72026), anti-SPP1 (#AF808, R&D) and anti-CD68 (FA-11, Invitrogen, #14-0681-82), followed by incubation with donkey anti-rat Alexa Fluor 594 (#A-21209), donkey anti-mouse Alexa Fluor 647 (#A-31571), donkey ant-rabbit Alexa Fluor 647 (#A-31573) and Hoechst 33342 (#H3570).
  • Cell Proliferation and Activation Human IL13Ra2 CAR T cells (25000) were incubated with 1 ⁇ M CFSE at room temperature for 20 min.
  • the CFSE-stained CAR T cells were added in 96-well plate coated with 2.5 ⁇ g/mL anti-CD3 antibody (clone: OKT3).
  • the presence and absence of 10 ⁇ g/mL human recombinant SPP1, as well as the presence and absence of 10 ⁇ g/mL anti-SPP1 antibody (clone: MPIIIB10) was tested, as indicated in figures (e.g., FIGS. 31A-31B).
  • cells were collected and subjected to flow cytometry to measure cell proliferation (measured by CFSE) and activation (measured by CD69 and CD25).
  • Human IL13Ra2 CAR T cells (25000) were incubated with 1 ⁇ M CFSE at room temperature for 20 min. After washes three times with PBS, the CFSE-stained CAR T cells were added in 96-well plate coated with different titrations of human IL13Ra2-Fc (0, 31, 62.5, 125, 250, 500 ⁇ g/mL) or 2.5 ⁇ g/mL anti-CD3 antibody (clone: OKT3) and in the presence and absence of 10 ⁇ g/mL human recombinant SPP1. After 3 days of incubation, cells were collected and subjected to flow cytometry to measure cell proliferation (measured by CFSE) and activation (measured by CD69 and 41BB).
  • RNA sequencing Single-cell RNA sequencing (scRNA-seq) analysis of tumors from 41 glioma patients undergoing IL13R ⁇ 2-targeted CAR T cell therapy. This study identified elevated suppressive SPP-1 signatures in macrophages from patients who were resistant to treatment. Also described is integrative scRNA-seq analysis of glioblastoma tumor samples and an interferon-signaling deficient syngeneic mouse model—both resistant to CAR T therapy that demonstrate the role of congruent suppressive pathways in mediating resistance to CAR T cells.
  • Example 1 Suppressive tumor microenvironment limits response to CAR T therapy in high-grade glioma Recognizing the unique challenges posed by solid tumors to CAR T cell therapy, a series of experiments was performed to identify dominant networks within GBM/HGG associated with patient outcomes to adoptive T cell therapy.
  • FIG. 1A To elucidate the resistance features within gliomas in response to CAR T cells, we performed scRNA-seq on freshly dissociated pretreatment tumors obtained from 41 glioma patients undergoing IL13Ra2-targeted CAR T cell therapy (FIG 1A). Glioma tumors were characterized demographically and stratified based on CD3+ T cell infiltration (CD3 low/CD3 high) and therapeutic outcome to CAR T cells (progressive diseases (PD) in comparison to complete response, partial response or stable disease (CR/PR/SD)) (FIG 1B).
  • CD3+ T cell infiltration CD3 low/CD3 high
  • PD progressive diseases
  • CR/PR/SD partial response or stable disease
  • This dual stratification aimed to uncover shared pathways that could reveal universal features of the TME associated with GBM/HGG response to CAR T cell therapy.
  • FIG.1G The differences in immune and stromal cell type proportions between complete response/stable disease (CR/SD) and progressive disease (PD) and GBM tumors with high (3- 4) and low (0-2) IHC CD3 scores are shown in FIG.1G.
  • CR/SD complete response/stable disease
  • PD progressive disease
  • FIG.1G To identify pretreatment cellular communication networks underlying clinical response to CAR T therapy, we evaluated receptor-ligand interaction probabilities among known cell-cell communication networks by calculating the sum of communication probabilities among all pairs of cell types in the TME subsets as well as the whole tumor. In favorably responding patients, those who achieved SD/CR or had increased intra-tumoral CD3 infiltrates, the pretreatment TME signaling patterns were significantly different from non-responders (FIG 1H).
  • MHC-presentation pathways including both MHCI and MHCII, were up-regulated in responding patients (SD/CR and CD3-med/high; p ⁇ 0.01) when considering the whole tumor (FIGS 1C-1D) as well as the TME subset (FIGS 1E-1H). Consistent with this observation, total lymphocyte proportions (Lymph 1 and Lymph 2) were significantly increased in SD/CR compared to PD (FIGS 1D-1G). These results align with improved patient outcomes following CAR T cell therapy being associated with higher pretreatment intratumoral T cell levels, as assessed by immunohistochemistry, and increased IFN-signaling, as previously reported.
  • SPP1+ myeloid cells specifically clusters M1, M2, M3, M5 and M6, as well as fibroblast cluster F3, show high expression of SPP1 and engage through three dominant cell-cell interaction
  • Example 2 Priming of YUMM JAK1/KO tumors with OPN targeted monoclonal antibody before delivering mIL13 CAR T cells significantly enhances therapeutic efficacy Given the convergence of SPP1 mediated signaling associated with an immuno-suppressive TME, we hypothesized targeting SPP1 alongside CAR T cell therapy may overcome resistance. To test this, experiments were conducted in three preclinical models of solid tumors with known suppressive TMEs and IFN deficiencies. To evaluate the functional impact of SPP1 in promoting solid tumor resistance to CART cell therapy, we employed a combined strategy using an anti-SPP1 antibody alongside CAR T cell therapy targeting YUMM JAK1/KO and MC38 JAK1/KO tumors (FIG.4A).
  • Example 3 Multifunctional SPP1-expressing myeloid cells are associated with therapeutic resistance to CAR T cell therapy in GBM/HGGs.
  • M1, M2, M3, and M5 make up a significant portion of the suppressive myeloid cell population within the tumors and higher SPP1 expression was associated with poor outcomes to CAR therapy, total subcluster abundance was not significantly differentially distributed in outcome comparisons (FIGS 2H-2I). Additionally, the fibroblast subcluster F3 exhibits high SPP1 and APOE expression and is enriched in PD and CD3 low non-responding groups compared to their corresponding counterparts (FIGS 2H-2I). In contrast, M4, M8, and M9 are SPP1-negative myeloid cells lacking suppressive features, such as TREM2, IL1B, and CCL4 (FIGS 2H-2I).
  • M9 is a CD4+ expressing subcluster with upregulation of GZMB potentially representing activated myeloid cells and is found in greater abundance in SD/CR group (FIGS 2H-2I).
  • Immunofluorescent staining of PD tumor samples compared to SD/CR tumor samples confirmed differences in SPP1 expression non-responding patients, consistent with transcriptomic findings (FIGS 2H-2j).
  • SPP1 High myeloid cells displayed upregulation of suppressive interleukin (IL) signaling pathways (FIG.2F), in stark contrast to the inflammatory IL pathways enriched in SPP1 Low myeloid cells. Elevated ligand-receptor interactions, particularly involving FGFR and its ligands, were also observed in SPP1 High myeloid cells (FIG.2F). Furthermore, pathways associated with increased ECM activity and hypoxia, along with decreased phagocytosis and a marked downregulation of antigen processing and presentation pathways were associated with SPP1 High myeloid cells (FIGS.2E & 2F).
  • IL interleukin
  • GBM/HGGs and JAK1/KO tumors showed strong concordance in the high expression of canonical suppressive macrophage and fibroblast markers.
  • These genes include APOE, C1QA, C1QB, C1QC, COL1A1, and SPP1, all expressed in multiple integrated macrophage subclusters, as well as S100A8 and S100A9, which were expressed in neutrophil subclusters.
  • these genes were not as correlated in the comparison of GBM/HGGs and YUMM WT tumors that were more responsive to CAR T cell therapy (FIG.30E).
  • the expression of these conserved signatures across immune components predominantly shapes the suppressive TME, thereby reinforcing its unresponsiveness to CAR T cell therapy.
  • IL13R ⁇ 2-engineered YUMM JAK1/KO and WT tumor cells were injected subcutaneously into immunocompetent mice and treated with murine IL13R ⁇ 2-targeted CAR (mIL13R ⁇ 2 CAR) T cells administered intravenously (IV) (FIG.29A).
  • mIL13R ⁇ 2 CAR murine IL13R ⁇ 2-targeted CAR
  • IV intravenously
  • YUMM JAK1/KO tumors demonstrated significant resistance to IV delivered mIL13R ⁇ 2 CAR T cell therapy as compared to WT YUMM counterparts (FIG.29B).
  • JAK1/KO tumors displayed a significantly reduced number of circulating CD3+ and CAR T cells in the blood (FIGS.29C-29D).
  • SPP1 emerged as one of the top upregulated pathways in YUMM JAK1/KO tumors, while MHCII was notably upregulated in WT tumors (FIG. 29K).
  • Cell-cell communication analysis further demonstrated increased interaction probabilities between fibroblasts and myeloid cells in YUMM JAK1/KO tumors relative to YUMM WT, with SPP1 representing one of the most prominent pathways driving these interactions (FIGS.29K- 29L).
  • both SPP1 and its cognate receptor CD44 were significantly upregulated across all immune-driven populations, including fibroblasts, neutrophils, and myeloid cells, in JAK1/KO tumors compared to YUMM WT (FIGS.3H & 29M).
  • Human IL13R ⁇ 2-CAR T cell stained by CFSE was activated using human IL13R ⁇ 2-Fc or 2.5 ⁇ g/mL anti-CD3 in the presence and absence of 10 ⁇ g/mL recombinant human SPP1 to measure the effect on the T cell proliferation and activation as measured by CD69+41BB+ (FIG. 32).
  • the results demonstrate that SPP1 inhibits IL13Ra2-Fc-mediated CAR T cell activation and proliferation.
  • the IL13R ⁇ 2-CAR T cells were successfully activated when incubated with more than 125 ⁇ g of human recombinant IL13R ⁇ 2-Fc (through CAR signaling).
  • human recombinant SPP1 suppresses CAR T cell proliferation and activation. However, this suppressive effect is not observed when CAR T cells are incubated with 500 ⁇ g of IL13R ⁇ 2-Fc.
  • human IL13R ⁇ 2-CAR T cell stained by CFSE was activated by 2.5 ⁇ g/mL anti-CD3 and incubated with indicated concentration of human recombinant SPP1 (rhSPP1) in FIGS.31A- 31B. Cell proliferation and activation as measured by CD69+CD25+ was detected.
  • Human recombinant SPP1 is able to inhibits the proliferation of IL13R ⁇ 2-CAR T cell (activated through TCR signaling) in a dose-dependent manner (FIG.31A). Similarly, the activation level of IL13R ⁇ 2-CAR T cell, as indicated by the proportion of CD69+CD25+ population, is notably inhibited by human recombinant SPP1 (FIG.31A).
  • an anti-SPP1 mAb was added at the indicated concentration to human IL13R ⁇ 2-CAR T cell stained by CFSE and activated by 2.5 ⁇ g/mL anti-CD3 in the presence and absence of 10 ⁇ g/mL rhSPP1 (FIG.31B). Again, Cell proliferation and activation as measured by CD69+CD25+ was detected. The addition of anti-SPP1 antibody partially reversed SPP1-mediated suppression on CAR T cell proliferation and activation, while the antibody alone has no notable effect on CAR T cell proliferation and activation (FIG.31B).

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Abstract

L'invention concerne des méthodes et des compositions pour traiter des patients atteints d'un cancer par administration d'anticorps ciblant l'ostéopontine (OPN ou SPP1) et une population de cellules immunitaires exprimant un récepteur antigénique chimérique (CAR). L'invention concerne également des méthodes d'amélioration de l'efficacité d'une thérapie cellulaire immunitaire à l'aide de CAR (par exemple, des lymphocytes T CAR) par administration d'anticorps ciblés sur SPP1.
PCT/US2025/028476 2024-05-08 2025-05-08 Anticorps ciblant l'ostéopontine et leurs utilisations pour réduire la résistance de tumeurs solides à une thérapie cellulaire immunitaire Pending WO2025235801A1 (fr)

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