WO2023215746A2 - Anti-alppl2 antibodies and chimeric antigen receptors, and compositions and methods of use - Google Patents

Anti-alppl2 antibodies and chimeric antigen receptors, and compositions and methods of use Download PDF

Info

Publication number
WO2023215746A2
WO2023215746A2 PCT/US2023/066496 US2023066496W WO2023215746A2 WO 2023215746 A2 WO2023215746 A2 WO 2023215746A2 US 2023066496 W US2023066496 W US 2023066496W WO 2023215746 A2 WO2023215746 A2 WO 2023215746A2
Authority
WO
WIPO (PCT)
Prior art keywords
seq
cdr
human
cell
add sequence
Prior art date
Application number
PCT/US2023/066496
Other languages
French (fr)
Other versions
WO2023215746A3 (en
WO2023215746A9 (en
Inventor
Anthony B. COOPER
Colleen COOPER
Rebecca ARGYROS
Original Assignee
Javelin Oncology, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Javelin Oncology, Inc. filed Critical Javelin Oncology, Inc.
Publication of WO2023215746A2 publication Critical patent/WO2023215746A2/en
Publication of WO2023215746A3 publication Critical patent/WO2023215746A3/en
Publication of WO2023215746A9 publication Critical patent/WO2023215746A9/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464454—Enzymes
    • A61K39/464463—Phosphatases
    • 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
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03001—Alkaline phosphatase (3.1.3.1)

Definitions

  • the present disclosure relates to anti-ALPPL2 agents such as anti-ALPPL2 antibodies (Abs), antigen-binding Ab fragments, multi-specific Abs and antigen-binding Ab fragments, antibody-drug conjugates (ADCs), and chimeric antigen receptors (CARs).
  • the disclosure also relates to polynucleotides and vectors encoding, cells and pharmaceutical compositions comprising such anti-ALPPL2 agents and methods for expanding such cells.
  • the present disclosure further relates to methods of treating a subject using such anti-ALPPL2 agents and compositions, and to methods of treating, preventing, or diagnosing a disease such as cancer and methods of stimulating an immune response.
  • the invention also relates to methods of producing such an anti- ALPPL2 agent or composition.
  • Immunotherapy is a growing field, allowing for treatment of a range of diseases that did not have an effective treatment option before.
  • Many examples of immunotherapy have been employed in oncology, including antibody therapies targeting CD20 (Non-Hodgkin's lymphoma), HER2 (HER2 -positive breast cancer), and immune checkpoints such as PD-1, PD-L1, and CTLA-4 (various cancers).
  • Immunotherapies are also being developed, tested, and/or marketed for non-cancer disease indications, such as autoimmune diseases (Wraith D.C. etal, Front Immunol. 2017 Nov 28;8:1668. doi: 10.3389/fimmu.2017.01668. eCollection 2017).
  • Chimeric antigen receptor (CAR)-based cell therapy is a type of immunotherapy whereby immune cells, such as T cells, NK cells, or macrophages, are genetically modified to express a receptor that allows recognition of a specific antigen. Upon antigen recognition, the cells ere activated via signaling domains, converting them Into potent cdl killers and/or immune stimulators.
  • an anti-CD19 CAR T cdl product received FDA approval for B cell lymphoma, illustrating the potential of this therapeutic approach in cancer, e-g, hematological cancer [Leyftnan Y, et aL, Cancer OB Int 2018 Nov 14;18:182.
  • Another potential challenge of CAR-based therapy is die presence of soluble firms of a taiget protein such as those in the serum (Garda-Guerrero E et at. Front Immunol.2020; 11: 1128.). This may occur, fir example, if die taiget protein on the cell surface becomes cleaved or shed, or if the taiget cell releases exosomes containing the taiget protein. These taiget proteins outside the cell can act as a decoy fir CAR and potentially limit the effectiveness of CAR-based cell therapy.
  • Alkaline phosphatase, placenta-like 2 (ALPPL2) also referred to as “ALPPL”, “alkaline phosphatase, germ cell (APGC)”, or “germ cell alkaline phosphatase (GCAF)”
  • ALPPL alkaline phosphatase, germ cell
  • GCAF germ cell alkaline phosphatase
  • AP placenta-like 2
  • GPI glycosyl phosphatidyl Inositol
  • ALPPL2 In healthy humans, the protein expression of ALPPL2 is primarily in the placenta (bttps://www.proteinatiaMrg/ENSG00000163286-ALPPL2/tiMue) and ALPPL2 regulates naive pluripotency (Bi Y. etaL, OU Rep. 2020 Mar 17;30(ll):3917-3931.e5. doi: 10.1016/j.cdrep2020.02.090.). APPL2 expression in the body may be altered by exposure to and/or intake of an exogenous substance (Nisio A. D. etaL, Chemosphere. 2020 Mar;242: 125208.
  • ALPPL2 is proposed as a potential biomarker that may be used in liquid biopsy-based diagnosis for pancreatic cancer (Sb In HS. et aL, Mol Ther Methods CHn Dev. 2019 Sep 12;15:204-210. doL* 10.1016/j.omtro.2019.08.016. eCollection 2019 Dec 13.).
  • ADC anti-ALPPL2 antibody drug conjugate
  • the present disclosure provides anti-ALPPL2 agents.
  • the anti-ALPPL2 anentls an antibody (Ab) or antigen-binding Ab fragment
  • the Ab or antigen-binding Ab fragment may bind to ALPPL2 and comprise: (a) a heavy chain (HC) variable region (VH); and (b) a light chain (LC) variable region (VL).
  • the VH may comprise: a HC complementarity determining region (CDR) 1 (also referred to as CDR-H1); a HC CDR 2 (also referred to as CDR-H2); and a HC CDR 3 (also referred to as CDR-H3).
  • the VL may comprise: a VL CDR 1 (CDR-L1); and a VL CDR 2 (CDR-L2); a VL CDR 3 (CDR-L3).
  • the CDR-H1, CDR-H2, and CDR-H3 may be the CDR-H1, CDR-H2, and CDR-H3, respectively, comprised in the VH of any anti-ALPPL2 antibodies described herein such as but not limited to 8735, 8506, 8416, 8380, 8001, 185, h3H2, hlE8, hlB2, 3H2, 1E8, or 182; and the CDR-L1, CDR-L2, and CDR-L3 maybe the CDR-L1, CDR-L2, and CDR-L3, respectively, comprised in the VL of any anti-ALPPL2 antibodies described herein such as butnot limited to 8735, 8506, 8416, 8380, 8001, 185, h3H2, hlE8, hlB2, 3H2, 1E8, or 182.
  • the VH may comprise the CDR-H1, CDR-H2, and CDR- H3 sequences contained in SEQ ID NO: 751
  • the VL may comprise the CDR-L1, CDR-L2, and CDR-L3 sequences contained in SEQ ID NO: 755.
  • the VH may comprise the CDR-H1, CDR-H2, and CDR- H3 sequences contained in SEQ ID NO: 741
  • the VL may comprise the CDR-L1, CDR-L2, and CDR-L3 sequences contained in SEQ ID NO: 745.
  • the VH may comprise the CDR-H1, CDR-H2, and CDR- H3 sequences contained in SEQ ID NO: 731
  • the VL may comprise the CDR-L1, CDR-L2, and CDR-L3 sequences contained in SEQ ID NO: 735.
  • the VH may comprise the CDR-H1, CDR-H2, and CDR- H3 sequences contained in SEQ ID NO: 721 and/or the VL may comprise the CDR-L1, CDR-L2, and CDR-L3 sequences contained in SEQ ID NO: 725.
  • the VH may comprise the CDR-H1, CDR-H2, and CDR- H3 sequences contained in SEQ ID NO: 711
  • the VL may comprise the CDR-L1, CDR-L2, and CDR-L3 sequences contained in SEQ ID NO: 715.
  • the VH may comprise the CDR-H1, CDR-H2, and CDR- H3 sequences contained in SEQ ID NO: 701 and/or the VL may comprise the CDR-L1, CDR-L2, and CDR-L3 sequences contained in SEQ ID NO: 705 or encoded in SEQ ID NO: 805.
  • the VH may comprise the CDR-H1, CDR-H2, and CDR- H3 sequences contained in SEQ ID NO: 331 or encoded in SEQ ID NO: 431, and/or the VL may comprise the CDR-L1, CDR-L2, and CDR-L3 sequences contained In SEQ ID NO: 335 or encoded in SEQ ID NO: 435.
  • die VH may comprise the CDR-H1, CDR-H2, and CORNS sequences contained in SEQ ID NO: 321 or encoded in SEQ ID NO: 421, and/or the VL may comprise the CDR-L1, CDR-L2, and CDR-L3 sequences contained In SEQ ID NO: 325 or encoded in SEQ ID NO: 425.
  • die VH may comprise the CDR-H1, CDR-H2, and CDR- H3 sequences contained in SEQ ID NO: 311 or encoded in SEQ ID NO: 411
  • the VL may comprise the CDR-L1, CDR-L2, and CDR-L3 sequences contained In SEQ ID NO: 315 or encoded in SEQ ID NO: 415.
  • die VH may comprise the CDR-H1, CDR-H2, and CORNS sequences contained in SEQ ID NO: 131 or encoded in SEQ ID NO: 231, and/or the VL may comprise the CDR-L1, CDR-L2, and CDR-L3 sequences contained In SEQ ID NO: 135 or encoded in SEQ ID NO: 235.
  • die VH may comprise the CDR-H1, CDR-H2, and CORNS sequences contained in SEQ ID NO: 121 or encoded in SEQ ID NO: 221, and/or the VL may comprise the CDR-L1, CDR-L2, and CDR-L3 sequences contained In SEQ ID NO: 125 or encoded in SEQ ID NO: 225.
  • die VH may comprise the CDR-H1, CDR-H2, and CORNS sequences contained in SEQ ID NO: 111 or encoded in SEQ ID NO: 211
  • the VL may comprise the CDR-L1, CDR-L2, and CDR-L3 sequences contained In SEQ ID NO: 115 or encoded in SEQ ID NO: 215.
  • the CDR-H1, CDR-H2, and CDR-H3 may be the CDR-H1, CDR-H2, and CDR-H3, respectively, of any anti-ALPPL2 antibodies described herein such as but not limited to B735, B506, B416, B380, B001, 1B5, h3H2, hlE8, hlB2, 3H2, 1E8, or 1B2; and/or the CDR-L1, CDR-L2, and CDR-L3 may be the CDR-L1, CDR-L2, and CORLS, respectively, of any anti-ALPPL2 antibodies described herein such as but not limited to B735, B506, B416, B380, B001, IBS, h3H2, hlE8, hlB2, 3H2, 1E8, or 1B2.
  • the CDR-H1, CDR-H2, and CDR-H3 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 752, 753, and 754, respectively; and/or the CDR-L1, CDR-L2, and CDR-L3 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 756, 757, and 758, respectively.
  • the CDR-H1, CDR-H2, and CDR-H3 may comprise or consist of the amino acid sequence set forth In SEQ ID NOs: 742, 743, and 744, respectively; and/or the CDR-L1, CDR-L2, and CDR-L3 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 746, 747, and 748, respectively.
  • the CDR-H1, CDR-H2, and CDR-H3 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 732, 733, and 734, respectively; and/or the CDR-L1, CDR-L2, and CDR-L3 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 736, 737, and 738, respectively.
  • the CDR-H1, CDR-H2, and CDR-H3 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 722, 723, and 724, respectively; and/or the CDR-L1, CDR-L2, and CDR-L3 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 726, 727, and 728, respectively.
  • the CDR-H1, CDR-H2, and CDR-H3 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 712, 713, and 714, respectively; and/or the CDR-L1, CDR-L2, and CDR-L3 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 716, 717, and 718, respectively.
  • the CDR-H1, CDR-H2, and CDR-H3 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 702, 703, and 704, respectively; and/or the CDR-L1, CDR-L2, and CDR-L3 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 706, 707, and 708, respectively or may be encoded by SEQ ID NOs: 806, 807, and 808, respectively.
  • the CDR-H1, CDR-H2, and CDR-H3 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 332, 333, and 334, respectively or may be encoded by SEQ ID NOs: 432, 433, and 434, respectively; and/or the CDR-L1, CDR-L2, and CDR-L3 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 336, 337, and 338, respectively or may be encoded by SEQ ID NOs: 436, 437, and 438, respectively.
  • the CDR-H1, CDR-H2, and CDR-H3 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 322, 323, and 324, respectively or may be encoded by SEQ ID NOs: 422, 423, and 424, respectively; and/or the CDR-L1, CDR-L2, and CDR-L3 may comprise or consist of the amino add sequence set forth in SEQ ID NOs: 326, 327, and 328, respectively or may be encoded by SEQ ID NOs: 426, 427, and 428, respectively.
  • the CDR-H1, CDR-H2, and CDR-H3 may comprise or consist of the amino add sequence set forth in SEQ ID NOs: 312, 313, and 314, respectively or may be encoded by SEQ ID NOs: 412, 413, and 414, respectively; and/or the CDR-L1, CDR-L2, and CDR-L3 may comprise or consist of the amino add sequence set forth in SEQ ID NOs: 316, 317, and 318, respectively or may be encoded by SEQ ID NOs: 416, 417, and 418, respectively.
  • the CDR-H1, CDR-H2, and CDR-H3 may comprise or consist of the amino add sequence set forth in SEQ ID NOs: 132, 133, and 134, respectively or may be encoded by SEQ ID NOs: 232, 233, and 234, respectively; and/or the CDR-L1, CDR-L2, and CDR-L3 may comprise or consist of the amino add sequence set forth in SEQ ID NOs: 136, 137, and 138, respectively or may be encoded by SEQ ID NOs: 236, 237, and 238, respectively.
  • the CDR-H1, CDR-H2, and CDR-H3 may comprise or consist of the amino add sequence set forth in SEQ ID NOs: 122, 123, and 124, respectively or may be encoded by SEQ ID NOs: 222, 223, and 224, respectively; and/or the CDR-L1, CDR-L2, and CDR-L3 may comprise or consist of the amino acid sequence set forth In SEQ ID NOs: 126, 127, and 128, respectively or may be encoded by SEQ ID NOs: 226, 227, and 228, respectively.
  • the CDR-H1, CDR-H2, and CDR-H3 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 112, 113, and 114, respectively or may be encoded by SEQ ID NOs: 212, 213, and 214, respectively; and/or the CDR-L1, CDR-L2, and CDR-L3 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 116, 117, and 118, respectively or may be encoded by SEQ ID NOs: 216, 217, and 218, respectively.
  • the % sequence identity of the VH amino acid sequence to a germline-encoded human VH sequence, optionally to the VH encoded by the human VH2-5 gene segment allele 8, may be about 80% or higher, about 85% or higher, about 90% or higher, about 93% or higher, about 95% or higher, about 96% or higher, about 97% or higher, about 98% or higher, about 98% or higher, about 99% or higher, or about 100%.
  • the % sequence identity of the VL amino acid sequence to a germline-encoded human VL, optionally to the VL encoded by the human IgKl-16 gene segment allele 1 may be about 80% or higher, about 85% or higher, about 86% or higher, about 90% or higher, about 93% or higher, about 95% or higher, about 96% or higher, about 97% or higher, about 98% or higher, about 98% or higher, or about 99% or higher, or about 100%,
  • the VH may comprise a human-like VH framework which may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human VH framework, optionally to any one of the human VH frameworks comprised in any one of the VH polypeptides of SEQ ID NO: 751, 741, 731, 721, 711, 701, 311, 321 and 331.
  • the VL may comprise a human-like VL framework which may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human VL framework, optionally to any one of the human VL frameworks comprised in any one of the VL polypeptides of SEQ ID NO: 755, 745, 735, 725, 715, 315, 325 or 335.
  • the VH and/or VL may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH and/or VH of any anti-ALPPL2 antibodies described herein such as but not limited to 8735, 8506, 8416, 8380, 8001, 185, h3H2, hlE8, hlB2, 3H2, 1E8, or 1B2 or may be encoded by a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH-encodlng sequence and/or VL-encodlng sequence of any anti-ALPPL2 antibodies described herein such as but not limited to 8735
  • the VH may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% Identical to SEQ ID NO: 751; and/or the VL may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 755.
  • the VH may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 741; and/or the VL may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 745.
  • the VH may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% Identical to SEQ ID NO: 731; and/or the VL may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 735.
  • the VH may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 721; and/or the VL may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 725.
  • the VH may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 711; and/or the VL may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 715.
  • the VH may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 701; and/or the VL may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 705 or may be encoded by a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 805.
  • the VH may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% Identical to SEQ ID NO: 111, or may be encoded by a nucleic add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 211; and/or the VL may comprise an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115, or may be encoded by a nucleic acid sequence at least 80%, at least 85%, at least 90%,
  • the VH may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 121, or may be encoded by a nucleic add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% Identical to SEQ ID NO: 221; and/or the VL may comprise an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 125, or may be encoded by a nucleic add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 9
  • the VH may comprise an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 131, or may be encoded by a nucleic add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 231; and/or the VL may comprise an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 135, or may be encoded by a nucleic add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at
  • the VH may comprise an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 311, or may be encoded by a nucleic add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 411; and/or the VL may comprise an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 315, or may be encoded by a nucleic add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at
  • die VH may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 321, or may be encoded by a nucleic add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 421; and/or the VL may comprise an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 325, or may be encoded by a nucleic add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at
  • die VH may comprise an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 331, or may be encoded by a nucleic add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 431; and/or the VL may comprise an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 335, or may be encoded by a nucleic add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at
  • the Ab or antigen-binding Ab fragment may be or may comprise or consist of any one or more of, for example, but not limited to, a monoclonal Ab, a monospedfic Ab, a bispedfic Ab, a multispedfic Ab, a humanized Ab, a tetrameric Ab, a tetravalent Ab, a single chain Ab, a domain-spedfic Ab, a domain-deleted Ab, an scFc fusion protein, a chimeric Ab, a synthetic Ab, a recombinant Ab, a hybrid Ab, a mutated Ab, CDR-grafted Ab, a fragment antigen-binding (Fab), an F(ab')2, an Fab' fragment; a variable fragment (Fv), a single-chain Fv (scFv), an Fd fragment, a diabody, or a minibody.
  • a monoclonal Ab a monospedfic Ab, a bispedfic Ab, a multispedfic
  • the antibody or antigen-binding Ab fragment may comprise or consists of a scFv comprising or consisting of a VH. a VL, and a linker which joins the VH and VL, and the VH and VL may be any of the VH and VL polypeptides described above or herein.
  • the antibody or antigen-binding Ab fragment may comprise such a scFv, wherein the linker comprises the amino add sequence of SEQ ID NO: 168, the amino add sequence encoded by SEQ ID NO: 268 or 68, the amino add sequence of SEQ ID NO: 159, or the amino acid sequence encoded by SEQ ID NO: 259.
  • the antibody or antigen-binding Ab fragment may comprise a scFv, which comprises an amino add sequence (i) which is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 760, 759, 750, 749, 740, 739, 730, 729, 720, 719, 319, 320, 329, 330, 339, 340, 341, or 342; or (ii) encoded by a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 419, 19, 420, 20, 429, 29, 430, 30, 439, 39, 440, 40, 441,
  • the Ab or antigen-binding Ab fragment may comprise two or more binding specificities.
  • the first specificity may be to an epitope in ALPPL2.
  • the second specificity may to another epitope in ALPPL2.
  • the second specificity may be to an epitope in a second antigen other than ALPPL2.
  • the second antigen may be for example, butnot limited to, NKG2D, 4-1BB, or Fc receptor (FcR).
  • the Ab or antigen-binding Ab fragment may comprise a human or human-like CHI, CH2, and/or CH3 domain(s).
  • the human-like CHI, CH2, and/or CH3 domain(s) may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human CHI, CH2, and/or CH3 domain(s), respectively.
  • the human or human-like CHI, CH2, and/or CH3 domain(s) is/are individually derived from the CHI, CH2, and/or CH3 domain(s), respectively, of a human IgM, a human IgD, a human IgG, a human IgE, or a human IgA optionally of a human IgGl, a human IgG2, a human IgG3, or a human IgG4.
  • the Ab or antigen-binding Ab fragment may comprise a human or human-like fragment crystallizable (Fc) region.
  • the human or human-like Fc region may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human Fc region.
  • the Fc region may be or may be derived from the Fc region of a human IgM, a human IgD, a human IgG, a human IgE, or a human IgA optionally of a human IgGl, a human IgG2, a human IgG3, or a human IgG4
  • the human-like Fc region may bind to an Fc receptor (FcR).
  • the FcR may be for example, but not limited to, Fc gamma receptor (FcgR), FcgRl, FcgRllA, FcgRlIBl, FcgRllB2, FcgRIllA, FcgRlllB, Fc epsilon receptor (FceR), FceRI, FceRII, Fc alpha receptor (FcaR), FcaRI, Fc alpha/mu receptor (Fca/mR), and neonatal Fc receptor (FcRn).
  • FcgR Fc gamma receptor
  • FcgRl Fc gamma receptor
  • FcgRl FcgRl
  • FcgRllA FcgRlIBl
  • FcgRllB2 FcgRIllA
  • FcgRlllB Fc epsilon receptor
  • FceR FceRI, FceRII, Fc alpha receptor (FcaR), FcaRI, Fc
  • the Ab or antigen-binding Ab fragment may bind to ALPPL2 with a dissociation constant (KD) of about 10 nM or smaller, about 5 nM or smaller, about 2 nM or smaller, about 1 nM or smaller, about 0.8 nM or smaller, about 0.6 nM or smaller, about 0.4 nM or smaller, about 0.3 nM or smaller, about 0.2 nM or smaller, about 0.1 nM or smaller, about 0.05 nM or smaller, about 0.02 nM or smaller, or about 0.01 nM or smaller.
  • KD dissociation constant
  • the Ab or antigen-binding Ab fragment may bind to ALPPL2 with a dissociation constant (KD) of between about 10 nM and about 0.1 nM, between about 5 nM and about 0.2 nM, between about 10 nM and about 0.01 nM, between about 5 nM and about 0.02 nM, between about 2 nM and about 0.05 nM, between about 2 nM and about 0.1 nM, or between about 1 nM and about 0.3 nM.
  • KD dissociation constant
  • the Ab or antigen-binding Ab fragment may bind to ALPPL2 with a dissociation constant (KD) of about 1.6 nM, about 0.8 nM, about 0.4 nM, about 0.3 nM, or about 0.2 nM.
  • KD dissociation constant
  • die KD may be measured via enzyme-linked immunosorbent assay (ELISA) or Biolayer Interferometry (BLI).
  • ELISA enzyme-linked immunosorbent assay
  • BLI Biolayer Interferometry
  • the Ab or antigen-binding Ab fragment may bind to ALPPL2 with a half maximal effective concentration (ECso) of about 1 pg/mL or smaller, about 0.5 pg/mL or smaller, about 0.2 pg/mL or smaller, about 0.1 pg/mL or smaller, about 0.05 pg/mL or smaller, about 0.02 pg/mL or smaller, about 0.01 pg/mL or smaller, about 0.005 pg/mL or smaller, about 0.002 pg/mL or smaller, about 0.001 pg/mL or smaller.
  • ECso half maximal effective concentration
  • the Ab or antigen-binding Ab fragment may bind to ALPPL2 with an ECso between about 1 pg/mL and about 0.001 pg/mL, between about 0.5 pg/mL and about 0.002 pg/mL, between about 0.2 pg/mL and about 0.005 pg/mL, between about 0.1 pg/mL and about 0.01 pg/mL, between about 0.05 pg/mL and about 0.02 pg/mL
  • the Ab or antigen-binding Ab fragment may bind to ALPPL2 with an ECso of about 0.05 pg/mL or about 0.02 pg/mL.
  • the ECso may be measured via ELISA
  • the Ab or antigen-binding Ab fragment may bind to ALPPL2 -expressing cells, optionally ALPPL2-expressing human cancer cells, with an ECso of about 100 nM or smaller, about 50 nM or smaller, about 20 nM or smaller, about 10 nM or smaller, about 5 nM or smaller, about 2 nM or smaller, about 1 nM or smaller, about 0.5 nM or smaller, about 0.2 nM or smaller, about 0.1 nM or smaller, or about 0.05 nM or smaller.
  • the Ab or antigen-binding Ab fragment may bind to ALPPL2 -expressing cells, optionally ALPPL2-expressing human cancer cells, with an ECso of between about 100 nM and about 0.05 nM, between about 50 nM and about 0.1 nM, or between about 20 nM and about 0.2 nM.
  • the Ab or antigen-binding Ab fragment may bind to ALPPL2 -expressing cells, optionally ALPPL2-expressing human cancer cells, with an ECso of about 15 nM, about 10 nM, about 5 nM, about 1 nM, about 0.5 nM, or about 0.2 nM.
  • the ECso may be measured using flowcytometry, further optionally based on mean fluorescence intensity (MFI).
  • MFI mean fluorescence intensity
  • the anti-ALPPL2 agent of the present disclosure may be an antibody-drug conjugate (ADC).
  • ADC may comprise: (a) any Ab or antigen-binding Ab fragment described above; and (b) a drug conjugated to the Ab or antigen-binding Ab fragment
  • the drug may be for example, but not limited to, an anticancer drug, an anti-proliferative drug, a cytotoxic drug, an anti-angiogenic drug, an apoptotic drug, an immunostimulatory drug, an anti-microbial drug, an antibiotic drug, an antiviral drug, an anti-inflammatory drug, an ALPPL2 inhibitor, an ALPPL2 signaling inhibitor, an enzyme, a hormone, a toxin, a radioisotope, a compound, a small molecule, a small molecule inhibitor, a protein, a peptide, a vector, a plasmid, a viral replicon, a viral particle, a nano
  • ADC antibody-drug conjugate
  • the ADC may comprise a drug selected from the group consisting of doxorubicin, daunorubicin, cucurbitadn, chaetocin, chaetoglobosin, chlamydocin, calicheamicin, nemorubidn, cryptophyscin, mensacardn, ansamitodn, mitomycin C, geldanamydn, mechercharmycin, rebeccamycin, safracin, okilactomydn, ollgomycin, actinomycin, sandramydn, hypothemycln, polyketomydn, hydroxyellipticine, thiocolchicine, methotrexate, triptoHde, taltobulin, lactacystin, dolastatin, auristatin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), telomestatin, tubastatin
  • a drug selected from the
  • the anti-ALPPL2 agent of the present disclosure is a chimeric antigen receptor (CAR).
  • the CAR may comprise: (a) an antigen-binding domain that binds to ALPPL2, (b) a transmembrane (TM) domain, and (c) an intracellular signaling (ICS) domain.
  • TM transmembrane
  • ICS intracellular signaling
  • such a CAR may further comprise (d) a hinge that joins said antigen-binding domain and said TM domain, and (e) one or more costimulatory (CS) domains.
  • the antigen-binding domain may be, comprise, or consist of any of the Abs or antigen-binding Ab fragments described above or herein.
  • the antigen-binding domain may be, comprise, or consist of any of the scFvs described above.
  • the antigen-binding domain may comprise or consist of an amino acid sequence which is (i) at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 760, 759, 750, 749, 740, 739, 730, 729, 720, 719, 319, 320, 329, 330, 339, 340, 341, or 342 or (ii) encoded by a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% Identical to SEQ ID NOs: 419, 19, 420, 20, 429, 29, 430, 30, 439, 39, 440, 40, 441, or 442.
  • the antigen-binding domain may comprise (i) the amino acid sequence of SEQ ID NOs: 339, 340, 341, or 341 or (ii) the amino acid sequence encoded by SEQ ID NOs: 439, 39, 440, 40, 441, or 442.
  • the antigen-binding domain may compete for binding to ALPPL2 with a scFv comprising an amino acid sequence which is (i) at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 760, 759, 750, 749, 740, 739, 730, 729, 720, 719, 319, 320, 329, 330, 339, or 340, or (ii) encoded by a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 419, 19, 420, 20, 429, 29, 430, 30, 439, 39, 440, 40, 441, or
  • the TM domain may be derived from the TM region, or a membrane-spanning portion thereof, of for example, but not limited to, CD28, CD3e, CD4, CDS, CD8, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD 154, TCRa, TCRb, or CD3z.
  • the TM domain may be derived from the TM region of CD28, or a membrane-spanning portion thereof.
  • the TM domain may optionally comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to the amino acid sequence: (i) set forth in SEQ ID NO: 161, or (ii) encoded by SEQ ID NO: 261.
  • the ICS domain may be derived from a cytoplasmic signaling sequence, or a functional fragment thereof, of for example, but not limited to, CD3z, a lymphocyte receptor chain, a TCR/CD3 complex protein, an Fc receptor (FcR) subunit, an IL-2 receptor subunit, FcRg, FcRb, CD3g, CD3d, CD3e, CDS, CD22, CD66d, CD79a, CD79b, CD278 (ICOS), FceRI, DAP10, or DAP12.
  • a cytoplasmic signaling sequence or a functional fragment thereof, of for example, but not limited to, CD3z, a lymphocyte receptor chain, a TCR/CD3 complex protein, an Fc receptor (FcR) subunit, an IL-2 receptor subunit, FcRg, FcRb, CD3g, CD3d, CD3e, CDS, CD22, CD66d, CD79a, CD79b, CD278 (ICOS), FceRI,
  • the ICS domain may be derived from a cytoplasmic signaling sequence of CD3z, or a functional fragment thereof.
  • the ICS domain may optionally comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% Identical to the amino add sequence: (I) set forth in SEQ ID NO: 162, or (ii) encoded by SEQ ID NO: 262.
  • the hinge may be derived from CD28.
  • the hinge may optionally comprise an amino sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% Identical to the amino add sequence: (1) set forth in SEQ ID NO: 163, or (ii) encoded by SEQ ID NO: 263.
  • At least one of the one or more CS domains may be derived from a cytoplasmic signaling sequence, or functional fragment thereof, of for example, but not limited to, CD28, DAP10, 4- IBB (CD137), CD2, CD4, CDS, CD7, CD8a, CD8b, CD 11a, CDllb, CDllc, CDlld, CD18, CD19, CD27, CD29, CD30, CD40, CD49d, CD49f, CD69, CD84, CD96 (Tactile), CD100 (SEMA4D), CD103, 0X40 (CD134), SLAM (SLAMF1, CD150, IPO-3), CD160 (BY55), SELPLG (CD162), DNAM1 (CD226), Ly9 (CD229), SLAMF4 (CD244, 2B4), ICOS (CD278), B7-H3, BAFFR, BTLA, BLAME (SLAMF8), CEACAM1, CDS, CRTAM,
  • the CS domain may be derived from a cytoplasmic signaling sequence of CD28, 4-1BB, or DAP10, or functional fragment thereof.
  • the CS domain may optionally comprise an amino sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to the amino add sequence: (1) set forth in SEQ ID NO: 164, (ii) encoded by SEQ ID NO: 264, (iii) set forth in SEQ ID NO: 165, (iv) encoded by SEQ ID NO: 265, (v) set for the in SEQ ID NO: 166, or (vi) encoded by SEQ ID NO: 266.1n certain embodiments, the CAR may comprise an amino add sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to the amino acid sequence of (i-1) B735scFvHL-CD28H- CD28TM-CD28CS-
  • die CAR may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to the amino acid sequence of (i) hlB2scFvHL-CD28H-CD28TM-CD28CS- CDSzICS (SEQ ID NO: 351), (ii) hlB2scFvHL-CD28H-CD28TM-41BBCS-CD3zICS (SEQ ID NO: 352), (ill) hlB2scFvHL-CD28H-CD28TM-DAP10CS-CD3zICS (SEQ ID NO: 353), (iv) hlB2scFvLH-CD28H-CD28TM-CD28CS-CD3zICS (SEQ ID NO: 354), (v) hlB2scFvLH- CD28H-CD28TM-41BBCS-CD3zICS (SEQ ID NO: 355),
  • the CAR may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% Identical to the amino acid sequence encoded by any one of SEQ ID NOs: 451-456, 461- 466, 471-476, 51-56, 61-66, and 71-76.
  • the CAR may further comprise a cytotoxic drug conjugated to the antigen-binding domain.
  • the present invention also relates to isolated polynucleotides encoding any of the anti-ALPPL2 agents described above and to combinations of polynucleotides which as a combination encode any of the anti-ALPPL2 agents described above.
  • the isolated polynucleotide or the combination of isolated polynucleotides may encode an antibody (Ab) or antigen-binding Ab fragment; which may comprise a VH and a VL as described above.
  • a combination of isolated polynucleotides encodes an antibody (Ab) or antigen-binding Ab fragment; for example, one polynucleotide may encode a VH and another polynucleotide may encode a VL, or one polynucleotide may encode a heavy chain and another polynucleotide may encode a light chain.
  • the CDR-H1, CDR-H2, and CDR-H3-encoding nucleic acid sequences may comprise or consist of the CDR-H1, CDR-H2, and CDR-H3-encoding nucleic acid sequences for any of the anti-ALPPL2 antibodies described herein, such as butnot limited to B735, B506, B416, B380, B001, 1B5, h3H2, hlE8, hlB2, 3H2, 1E8, or 1B2.
  • the CDR-L1, CDR-L2, and CDR-L3-encoding nucleic acid sequences may comprise or consist of the CDR-L1, CDR-L2, and CDR-L3-encoding nucleic acid sequences for any of the anti-ALPPL2 antibodies described herein, such as butnot limited to B735, B506, B416, B380, B001, IBS, h3H2, hlEB, hlB2, 3H2, 1E8, or 1B2.
  • the CDR-L1, CDR-L2, and CDR-L3-encoding nucleic acid sequences may comprise or consist of SEQ ID NOs: 806, 807, and 808, respectively.
  • the CDR-H1, CDR-H2, and CDR-H3-encoding nucleic acid sequences may comprise or consist of SEQ ID NOs: 432, 433, and 434, respectively; and the CDR-L1, CDR-L2, and CDR-L3-encoding nucleic acid sequences may comprise or consist of SEQ ID NOs: 436, 437, and 438, respectively.
  • the CDR-H1, CDR-H2, and CDR-H3-encoding nucleic acid sequences may comprise or consist of SEQ ID NOs: 422, 423, and 424, respectively; and the CDR-L1, CDR-L2, and CDR-L3-encodlng nucleic acid sequences may comprise or consist of SEQ ID NOs: 426, 427, and 428, respectively.
  • the CDR-H1, CDR-H2, and CDR-H3-encoding nucleic acid sequences may comprise or consist of SEQ ID NOs: 412, 413, and 414, respectively; and the CDR-L1, CDR-L2, and CDR-L3-encoding nucleic acid sequences may comprise or consist of SEQ ID NOs: 416, 417, and 418, respectively.
  • the CDR-H1, CDR-H2, and CDR-H3-encoding nucleic acid sequences may comprise or consist of SEQ ID NOs: 232, 233, and 234, respectively; and the CDR-L1, CDR-L2, and CDR-L3-encoding nucleic acid sequences may comprise or consist of SEQ ID NOs: 236, 237, and 238, respectively.
  • the CDR-H1, CDR-H2, and CDR-H3-encoding nucleic acid sequences may comprise or consist of SEQ ID NOs: 222, 223, and 224, respectively; and the CDR-L1, CDR-L2, and CDR-L3-encoding nucleic acid sequences may comprise or consist of SEQ ID NOs: 226, 227, and 228, respectively.
  • the CDR-H1, CDR-H2, and CDR-H3-encoding nucleic acid sequences may comprise or consist of SEQ ID NOs: 212, 213, and 214, respectively; and the CDR-L1, CDR-L2, and CDR-L3-encoding nucleic acid sequences may comprise or consist of SEQ ID NOs: 216, 217, and 218, respectively.
  • the % Identity of the VH-encodlng nucleic acid sequence to a VH-encoding human germline sequence may be about 80% or higher, about 85% or higher, about 90% or higher, about 93% or higher, about 95% or higher, about 96% or higher, about 97% or higher, about 98% or higher, about 98% or higher, about 99% or higher, or about 100%.
  • the % identity of the VL-encoding nucleic acid sequence to a VL-encoding human germline sequence may be about 80% or higher, about 85% or higher, about 86% or higher, about 90% or higher, about 93% or higher, about 95% or higher, about 96% or higher, about 97% or higher, about 98% or higher, about 98% or higher, or about 99% or higher, or about 100%.
  • the VH-encodlng nucleic acid sequence may comprise a human or human-like VH framework-encoding nucleic add sequence which may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human VH framework-encoding nucleic add sequence, optionally to any one of the human VH framework-encoding nucleic add sequences comprised in any one of the VH-encoding nudeic add sequences of SEQ ID NOS: 431, 421, and 411.
  • the VL- encoding nucleic add sequence may comprise a human or human-like VL frameworkencoding nucleic add sequence which may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human VL framework-encoding nucleic acid sequence, optionally to any one of the human VL framework-encoding nucleic add sequences comprised in any one of the VL-encoding nucleic add sequences of SEQ ID NOS: 435, 425, and 415.
  • the VH-encoding nucleic add sequence may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% Identical to the VH-encodlng nucleic add sequence of any of the anti-ALPPL2 antibodies described herein, such as but not limited to 8735, B506, 8416, 8380, 8001, 185, h3H2, hlE8, hlB2, 3H2, 1E8, or 182.
  • the VL-encoding nudeic add sequence may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL-encoding nucleic add sequence of any of the anti-ALPPL2 antibodies described herein, such as butnot limited to 8735, 8506, 8416, 8380, 8001, 185, h3H2, hlE8, hlB2, 3H2, 1E8, or 182.
  • the VL-encoding nucleic add sequence may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 805.
  • the VH-encoding nucleic add sequence may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 431; and the VL- encoding nucleic add sequence may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 435.
  • the VH-encoding nucleic add sequence may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 421; and the VL- encoding nucleic add sequence may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 425.
  • the VH-encoding nucleic add sequence may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 411; and the VL- encoding nucleic add sequence may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 415.
  • the VH-encoding nucleic add sequence may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 231; and the VL- encoding nucleic add sequence may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% Identical to SEQ ID NO: 235.
  • the VH-encoding nucleic add sequence may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 221; and the VL- encodlng nucleic add sequence may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 225.
  • the VH-encoding nucleic add sequence may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 211; and the VL- encoding nucleic add sequence may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% Identical to SEQ ID NO: 215.
  • the isolated polynucleotide or combination of isolated polynucleotides may encode an Ab or antigen-binding Ab fragment that is for example, but not limited to, a monoclonal Ab, a monospedfic Ab, a bispedfic Ab, a multispedfic Ab, a humanized Ab, a tetrameric Ab, a tetravalent Ab, a single chain Ab, a domain- spedfic Ab, a domain-deleted Ab, an scFc fusion protein, a chimeric Ab, a synthetic Ab, a recombinant Ab, a hybrid Ab, a mutated Ab, CDR-grafted Ab, a fragment antigen-binding (Fab), an F(ab')2, an Fab' fragment; a variable fragment (Fv), a single-chain Fv (scFv) fragment; an Fd fragment; a diabody, or a minibody.
  • Fab fragment antigen-binding
  • Fv variable fragment
  • the isolated polynucleotide or combination of isolated polynucleotides may encode encodes any of the scFvs described above or herein.
  • the linker-encoding nucleic add sequence of such a polynucleotide may comprise or consist of SEQ ID NO: 268, 68, or 259.
  • the scFv-encoding polynucleotide may comprise a nudeic add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 419, 19, 420, 20, 429, 29, 430, 30, 439, 39, 440, 40, 441, or 442.
  • the isolated polynucleotide may encode an Ab or antigenbinding Ab fragment that comprises two or more binding spedfidties.
  • the first spedfidty may be to an epitope in ALPPL2.
  • the second spedfidty may be to another epitope in ALPPL2.
  • the second spedfidty may be to an epitope In a second antigen other than ALPPL2.
  • the second antigen may be for example, but not limited to, NKG2D, 4-1BB, or Fc receptor (FcR).
  • the isolated polynucleotide or combination of isolated polynucleotides may comprise a human or human-like CHI, CH2, and/or CH3 domainencoding nucleic add sequence(s).
  • the human-like CHI, CH2, and/or CH3 domain-encoding nucleic add sequence(s) may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to (a) human CHI, CH2, and/or CH3 domainencoding nucleic add sequence(s), respectively.
  • the human or human-like CHI, CH2, and/or CH3 domainencoding nucleic add sequence(s) may be individually derived from the CHI, CH2, and/or CH3 domain-encoding nucleic add sequence(s), respectively, of a human IgM, a human IgD, a human IgG, a human IgE, or a human IgA, optionally of a human IgGl, a human IgG2, a human IgG3, or a human IgG4.
  • the isolated polynucleotide or combination of isolated polynucleotides may comprise a human or human-like fragment crystallizable (Fc) region-encoding nucleic add sequence.
  • the human or humanlike Fc region-encoding nucleic add sequence may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human Fc region-encoding nucleic add sequence.
  • the human or human-like Fc region-encoding nucleic add sequence may be or may be derived from the Fc region-encoding nucleic add sequence of a human IgM, a human IgD, a human IgG, a human IgE, or a human IgA, optionally of a human IgGl, a human IgG2, a human IgG3, or a human lgG4.
  • the encoded human or human-like Fc region may bind to an Fc receptor (FcR).
  • FcR Fc receptor
  • the FcR may be for example, but not limited to, Fc gamma receptor (FcgR), FcgRI, FcgRIIA, FcgRHBl, FcgRIIB2, FcgRIIIA, FcgRIIIB, Fc epsilon receptor (FceR), FceRI, FceRII, Fc alpha receptor (FcaR), FcaRI, Fc alpha/mu receptor (Fca/mR), or neonatal Fc receptor (FcRn).
  • FcgR Fc gamma receptor
  • FcgRI FcgRIIA
  • FcgRHBl FcgRIIB2
  • FcgRIIIA FcgRIIIB
  • Fc epsilon receptor FceR
  • FceRI FceRII
  • Fc alpha receptor FcaR
  • the isolated polynucleotide may encode any CAR described above.
  • the CAR may comprise: (a) an antigen-binding domain that binds to ALPPL2; (b) a transmembrane (TM) domain; (c) an intracellular signaling (ICS) domain;
  • the isolated polynucleotide may comprise an antigenbinding domain-encoding nucleic acid sequence may comprise or consist of any of the nucleic acid sequences encoding the anti-ALPPL2 antibody or antibody fragment described above or herein.
  • the isolated polynucleotide may comprise an antigenbinding domain-encoding nucleic acid sequence comprising or consisting of the nucleic acid sequence of any of the scFv-encoding polynucleotides described above or herein.
  • the antigen-binding domain-encoding nucleic acid sequence may comprise or consist of a nucleic acid sequence of SEQ ID NOs: 419, 19, 420, 20, 429, 29, 430, 30, 439, 39, 440, 40, 441, or 442 and in some preferred embodiments, SEQ ID NO: 439 or 440.
  • the encoded antigen-binding domain may compete for binding to ALPPL2 with a scFv comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to: (1) SEQ ID NOs: 760, 759, 750, 749, 740, 739, 730, 729, 720, 719, 319, 320, 329, 330, 339, 340, 341, or 342 or (11) the amino add sequence encoded by SEQ ID NOs: 419, 19, 420, 20, 429, 29, 430, 30, 439, 39, 440, 40, 441, or 442.
  • the isolated polynucleotide may comprise a TM domainencoding nucleic add sequence derived from the nucleic add sequence encoding the TM region, or a membrane-spanning portion thereof, of, for example, but not limited to, CD28, CD3e, CD4, CDS, CD8, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD 154, TCRa, TCRb, and CD3z.
  • the TM domainencoding nucleic add sequence may be derived from the nucleic add sequence encoding the TM region of CD28, or a membrane-spanning portion thereof.
  • the TM domainencoding nucleic add sequence may optionally comprise a nucleic add sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% Identical to SEQ ID NO: 261.
  • the isolated polynucleotide may comprise a ICS domainencoding nucleic add sequence derived from the nucleic add sequence encoding a cytoplasmic signaling sequence, or a functional fragment thereof, of, for example, but not limited to, CD3z, a lymphocyte receptor chain, a TCR/CD3 complex protein, an Fc receptor (FcR) subunit, an IL-2 receptor subunit, FcRg, FcRb, CD3g, CD3d, CD3e, CDS, CD22, CD66d, CD79a, CD79b, CD278 (ICOS), FceRI, DAP10, and DAP12.
  • the ICS domain-encoding nucleic add sequence may be derived from the nudeic add sequence encoding a cytoplasmic signaling sequence of CD3z, or a functional fragment thereof.
  • the ICS domain-encoding nucleic add sequence may optionally comprise a nucleic add sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to SEQ ID NO: 262.
  • the isolated polynucleotide may comprise a hingeencoding nucleic add sequence derived from the nucleic add sequence encoding CD28.
  • the hinge-encoding nucleic add sequence may optionally comprise a nudeic add sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to SEQ ID NO: 263.
  • the isolated polynucleotide may comprise a nucleic add sequence encoding at least one of the one or more CS domains which may be derived from the nucleic add sequence encoding a cytoplasmic signaling sequence, or functional fragment thereof, of, for example, butnot limited to, CD28, DAP10, 4-1BB (CD137), CD2, CD4, CDS, CD7, CD8a, CD8b, CDlla, CDllb, CDllc, CDlld, CD18, CD19, CD27, CD29, CD30, CD40, CD49d, CD49f, CD69, CD84, CD96 (Tactile), CD100 (SEMA4D), CD1O3, 0X40 (CD134), SLAM (SLAMF1, CD150, IPO-3), CD160 (BY55), SELPLG (CD162), DNAM1 (CD226), Ly9 (CD229), SLAMF4 (CD244, 2B4), ICOS (CD278), B
  • the CS domain-encoding nucleic acid sequence may be derived from the nucleic acid sequence encoding a cytoplasmic signaling sequence of CD28, 4- IBB, or DAP10, or functional fragment thereof.
  • the CS domain-encoding nucleic add sequence may optionally comprise a nucleic add sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to SEQ ID NO: 264, 265, or 266.
  • the CAR-encoding polynucleotide may comprise a nucleic add sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to any one of SEQ ID NOs: 451-456, 461- 466, 471-476, 51-56, 61-66, and 71-76.
  • the isolated polynucleotide of any of the above may further comprise a leader sequence (LS).
  • the LS may comprise a nucleic add sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% Identical to SEQ ID NO: 260.
  • the LS may encode a LS polypeptide comprising an amino add sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to SEQ ID NO: 160.
  • the isolated polynucleotide of any one of the above may further comprise T2A sequence and/or a sequence encoding truncated CD 19 (trCD19).
  • the T2A sequence maybe optionally at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to SEQ ID NO: 269.
  • the T2A sequence may encode a T2A polypeptide comprising an amino add sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to SEQ ID NO: 169.
  • the encoded trCD19 may optionally comprise the amino add sequence at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to SEQ ID NO: 170.
  • the trCD19-encoding nucleic add sequence may be at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to SEQ ID NO: 270.
  • the isolated CAR-encoding polynucleotide may encode an amino add at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to SEQ ID NO: 571 or 574 and/or may comprise a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to any one of SEQ ID NOs: 671, 271, 674, and 271.
  • the present invention relates to vectors and combinations of vectors comprising any of the polynucleotides or combination of polynudeotides encoding an anti-ALPPL2 agent
  • a combination of vectors encodes an anti-ALPPL2 agent such as an anti- ALPPL2 antibody
  • one vector may comprise a nucleic acid sequence encoding a VH and another vector may comprise a nucleic acid sequence encoding a VL
  • the vector or combination of vectors may comprise any polynucleotide described above.
  • the vector may be for example, but not limited to, a DNA, an RNA, a plasmid, a cosmid, a viral vector, a lentiviral vector, an adenoviral vector, or a retroviral vector.
  • the present disclosure further provides to recombinant or Isolated cells.
  • the recombinant or isolated cell may comprise: (i) any Ab or antigen-binding Ab fragment described above, (ii) any ADC described above, (iii) any CAR described above, (iv) any polynucleotide described above, and/or (v) any vector described above.
  • the recombinant or isolated cell may be or may comprise, for example, but not limited to, a non-mammalian cell, optionally a plant cell, a bacterial cell, a fungal cell, a yeast cell, a protozoa cell, or an insect cell.
  • the recombinant or isolated cell may be, for example, but not limited to, a mammalian cell, optionally a human cell, a rat cell, or a mouse cell.
  • the recombinant or isolated cell may be, for example, but not limited to, a stem cell.
  • the recombinant or isolated cell may be, for example, but not limited to, a primary cell, optionally a human primary cell or derived therefrom.
  • the recombinant or isolated cell may be, for example, but not limited to, a cell line, optionally a hybridoma cell line.
  • the recombinant or isolated cell may be, for example, but not limited to, an immune cell.
  • the recombinant or isolated cell may be MHC+ or MHC-.
  • the recombinant or isolated cell may be, for example, but not limited to, a cell line, a T cell, a T cell progenitor cell, a CD4+ T cell, a helper T cell, a regulatory T cell, a CD8+ T cell, a naive T cell, an effector T cell, a memory T cell, a stem cell memory T (TSCM) cell, a central memory T (TCM) cell, an effector memory T (TEM) cell, a terminally differentiated effector memory T cell, a tumor-infiltrating lymphocyte (TIL), an immature T cell, a mature T cell, a cytotoxic T cell, a mucosa-associated invariant T (MAIT) cell, a TH1 cell, a TH2 cell, a TH3 cell, a TH17 cell, a TH9 cell, a TH22 cell, a follicular helper T cells, and a/b T
  • TIL tumor-in
  • the recombinant or isolated cell may be a T cell or T cell progenitor cell.
  • the recombinant or isolated cell may be a T cell which has been modified such that its endogenous T cell receptor (TCR) is (i) not expressed, (li) not functionally expressed, or (111) expressed at reduced levels compared to a wild- type T cell.
  • TCR T cell receptor
  • the recombinant or isolated cell may be activated or stimulated to proliferate when the CAR binds to its target molecule.
  • the recombinant or Isolated cell may exhibit cytotoxicity against cells expressing the target molecule when the CAR binds to the target molecule.
  • administration of the recombinant or isolated cell to a subject may ameliorate a disease
  • the disease is cancer
  • the recombinant or isolated cell may increase expression of cytokines and/or chemokines when the CAR binds to its target molecule.
  • the cytokine may be IFN-g.
  • the recombinant or isolated cell may decrease expression of cytokines and/or chemokines when the CAR binds to its target
  • the cytokine may be TGF-b or IL-10.
  • the present Invention relates to populations of recombinant or Isolated cells.
  • the population may comprise at least any one recombinant or isolated cell described above.
  • the present invention relates to pharmaceutical compositions.
  • the pharmaceutical composition may comprise: (a) (a-1) any Ab or antigen-binding Ab fragment described above, (a-ii) any ADC described above, (iii) any CAR described above, (iv) any polynucleotide or combination of polynucleotides described above, (v) any vector or combination of vectors described above, (vi) any cell described above, or (vii) any population of cells; and optionally (b) a pharmaceutically acceptable excipient or carrier.
  • the present invention relates to methods of treating a subject
  • the method may be a method of treating a subject; and the method may comprise administering to the subject in need thereof a therapeutically effective amount of (i) any Ab or antigen-binding Ab fragment described above, (ii) any ADC described above, (iii) any CAR described above, (iv) any polynucleotide or combination of polynucleotides described above, (v) any vector or combination of vectors described above, (vi) any cell described above, (vii) any population of cells described above, and/or (viii) any pharmaceutical composition described above.
  • the method may be used in the treatment of, cancer.
  • the method may be used in the treatment of pancreatic cancer, testicular cancer, cervical cancer, endometrial cancer, ovarian cancer, stomach cancer, colorectal cancer, lung cancer, mesothelioma, or tongue cancer.
  • the method may be a method of treating a subject with anti-ALPPL2 agent
  • the method may comprise the steps of: (a) obtaining or having obtained a biological sample from the subject; (b) measuring the expression level of ALPPL2 in the biological sample; (c) determining whether the subject is an ALPPL2 over-expresser; and (d) If the subject Is an ALPPL2 over-expresser, administering to the subject a therapeutically effective amount of (d-i) any Ab or antigen-binding Ab fragment described above, (d-ii) any ADC described above, (d-iii) any CAR described above, (d-iv) any polynucleotide described above, (d-v) any vector described above, (d- vi) any cell described above, (d-vil) any population of cells described above, and/or (d- viii) any pharmaceutical composition described above.
  • an ALPPL2 over-expresser is a subject whose ALPPL2 expression is at least 1.5 times higher than the ALPPL2 expression of normal or healthy subjects. In certain embodiments, an ALPPL2 over-expresser Is a subject whose ALPPL2 expression Is at least 1.75 times higher than the ALPPL2 expression of normal or healthy subjects. In certain embodiments, an ALPPL2 over-expresser is a subject whose ALPPL2 expression is at least twice higher than the ALPPL2 expression of normal or healthy subjects.
  • the subject may be suffering from cancer.
  • the cancer may be for example, but not limited to, pancreatic cancer, testicular cancer, cervical cancer, endometrial cancer, ovarian cancer, stomach cancer, colorectal cancer, lung cancer, mesothelioma, or tongue cancer.
  • the present Invention relates to methods of Immune stimulation.
  • the method may be a method for stimulating an immune response in a subject, comprising administering to the subject a therapeutically effective amount of: (i) any Ab or antigen-binding Ab fragment described above, (ii) any ADC described above, (111) any CAR described above, (iv) any polynucleotide or combination of polynucleotides described above, (v) any vector or combination of vectors described above, (vi) any cell described above, (vii) any population of cells described above, and/or (viii) any pharmaceutical composition described above.
  • the present invention relates to methods of treating a disease.
  • the method may be a method of treating a disease in a subject; comprising administering to the subject in need thereof a therapeutically effective amount of (i) any Ab or antigen-binding Ab fragment described above, (ii) any ADC described above, (iii) any CAR described above, (iv) any polynucleotide or combination of polynucleotides described above, (v) any vector or combination of vectors described above, (vi) any cell described above, (vii) any population of cells described above, and/or (viii) any pharmaceutical composition described above.
  • the disease may be, for example, cancer.
  • the disease may be cancer, optionally pancreatic cancer, testicular cancer, cervical cancer, endometrial cancer, ovarian cancer, stomach cancer, colorectal cancer, lung cancer, mesothelioma, or tongue cancer.
  • the present invention further relates to methods of expanding a population of cells.
  • the method may be a method of expanding a population of cells in a subject
  • the method may comprise administering to the subject (i) any polynucleotide or combination of polynucleotides described above; (ii) any vector or combination of vectors described above; (ill) any cell described above; (iv) any population of cells described above; and/or (v) any pharmaceutical composition described above.
  • the administration may lead to a population of cells including at least one desired cell, for example, a cell that may comprise any Ab or Ab fragment described above, any ADC described above, and/or any CAR described above.
  • the cell may comprise a nucleic acid encoding such an Ab or Ab fragment; ADC, or CAR.
  • the resulting population of cells may persist in the subject for at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, at least twelve months, at least eighteen months, at least two years, or at least three years after administration.
  • the subject may be sufiferingfrom cancer.
  • the cancer may be pancreatic cancer, testicular cancer, cervical cancer, endometrial cancer, ovarian cancer, stomach cancer, colorectal cancer, lung cancer, mesothelioma, or tongue cancer.
  • any of the methods described above may further comprise administering a second agent
  • the second agent may be, but is not limited to, an anticancer drug, an anti-proliferative drug, a cytotoxic drug, an anti-angiogenic drug, an apoptotic drug, an immunostimulatory drug, an anti-microbial drug, an antibiotic drug, an antiviral drug, an anti-inflammatory drug, an ALPPL2 inhibitor, an ALPPL2 signaling inhibitor, an anti-ALPPL2 agent of the present invention, an enzyme, a hormone, a toxin, a radioisotope, a compound, a small molecule, a small molecule inhibitor, a protein, a peptide, a vector, a plasmid, a viral replicon, a viral particle, a nanoparticle, a DNA molecule, an RNA molecule, an siRNA, an shRNA, a micro RNA, an oligonucleotide, or an imaging drug.
  • an anticancer drug an anti-proliferative drug, a cytotoxic drug, an anti-
  • the present invention further relates to methods of generating a cell comprising any CAR described above.
  • the method may comprise: (i) introducing into a cell (i-a) a polynucleotide encoding at least one CAR according to any one of the foregoing or (i-b) at least one polynucleotide according to any one of the foregoing; or
  • the method may further comprise (iii) isolating the cell based on expression of said CAR and/or a selectable marker as determined via flow cytometry or immunofluorescence assays.
  • the present invention further relates to methods producing an anti-ALPPL2 agent described above or herein.
  • the method is for producing an anti-ALPPL2 Ab or Ab fragment described above or herein.
  • the method may comprise (a) culturing cells comprising any of the isolated polynucleotides or combinations of isolated polynucleotides encoding the anti-ALPPL2 Ab or Ab fragment in a condition that allows for expression of said Ab or Ab fragment; and (b) harvesting and purifying the Ab or Ab fragment from the cell culture from (a).
  • the method is for producing a recombinant or isolated cell described above or herein or population of such cells.
  • the method may comprise Introducing any of the Isolated polynucleotides or combinations of isolated polynucleotides described above or herein encoding an anti-ALPPL2 agent of interest into a cell.
  • the introducing may occur in vitro.
  • Such an in vitro introduction may be, for example, for generating recombinant cells (e. g., yeast cells, plant cells, insect cells, or mammalian cells, such as human cells; optionally CHO cells or HEK cells) encoding an anti-ALPPL2 Ab or Ab fragment for manufacturing of an ti- ALPPL2 Ab or Ab fragment
  • An in vitro introduction may alternatively be, for example, for producing cells for therapeutic use, for example cells (e.g., iPSCs, IPSC-derived cells of a cell type of Interest) expressing and/or secreting an antl-ALPPL2 agent (e.g., Ab, Ab fragment ADC, CAR).
  • the introducing may occur ex vivo.
  • Such an ex vivo introduction may be, for example, for producing cells for therapeutic use, for example cells (e.g., cells from a patient/subject, allogeneic or autologous cells, directly from a patient/subject or further processed and/or further differentiated) expressing and/or secreting an anti-ALPPL2 agent (e.g., Ab, Ab fragment ADC, CAR).
  • an anti-ALPPL2 agent e.g., Ab, Ab fragment ADC, CAR
  • the introducing may occur in vivo.
  • Such an in vivo introduction may be, for example, for producing cells in a patient/subject to be treated, that express and/or secrete an anti-ALPPL2 agent (e.g., Ab, Ab fragment ADC, CAR) for therapy.
  • an anti-ALPPL2 agent e.g., Ab, Ab fragment ADC, CAR
  • FIGS 1A-E provide exemplary schematics of chimeric antigen receptors (CARs) according to the present disclosure.
  • FIG 1A shows a general schematic of chimeric antigen receptors (CARs) of the present disclosure.
  • a CAR according to the present disclosure comprises an antigen-binding (AB) domain, transmembrane (TM) domain, and an intracellular signaling (ICS) domain.
  • FIGS IB- ID shows exemplary schematics of a CAR construct according to the present disclosure, wherein the CAR construct comprises an antigen-binding domain, a TM domain, and an ICS domain, and further comprises a hinge that joins the AB and TM domains (FIG IB) and one (FIG 1C) or two (FIG ID) costimulatory (CS) domains.
  • FIG IE shows exemplary schematics of a CAR- encoding construct that may be included in a vector and comprises a leader sequence (LS) and an exemplary CAR construct as shown in any of FIGS 1A-1D.
  • FIG IF shows exemplary schematics of a vector construct encoding a CAR according to the present disclosure, further comprising an exemplary ribosomal skip sequence (T2A) and an exemplary expression/purification marker, truncated CD19 (trCD19).
  • T2A ribosomal skip sequence
  • trCD19 truncated CD19
  • FIG 2 illustrates various exemplary antigen-binding domain constructs of CARs of some embodiments.
  • the first two examples are "hlB2scFvHL” (or “hlB2 scFv HL”) and “hlB2scFvLH” (or “hlB2 scFv LH”), which are scFvs derived from “hlB2", a humanized version of mouse anti-ALPPL2 antibody 1B2.
  • hlEBscFvHL or “hlEB scFv HL”
  • hlEBscFvLH or “hlE8 scFv LH”
  • hlEBscFvHL or “hlEB scFv HL”
  • hlEBscFvLH or “hlE8 scFv LH”
  • h3H2scFvHL or “h3H2 scFv HL”
  • h3H2scFvLH or “h3H2 scFv LH”
  • the scFvs defined by the SEQ ID NOs shown in FIG 2 use a G4S X3 linker between the VH and VL, but any suitable linker may be used.
  • a Whitlow linker may be used.
  • Such Whitlow linker-containing scFvs corresponding to e.g., "h3H2scFvHL” and “h3H2scFvLH” may be referred to herein as "h3H2scFvHL(Wh)" and "h3H2scFvLH(Wh)", respectively, and may comprise the amino acid sequences of e.g., SEQ ID NOs: 341 and 342, respectively, which may be encoded by e.g., SEQ ID NOs: 441 and 442, respectively.
  • FIGS 3A-3C contain schematics of various exemplary CAR constructs of some embodiments of the invention.
  • one of the antigen-binding domains shown in FIG 2 is used as the antigen-binding domain
  • CD28H is used as the hinge
  • CD28TM is used as the TM domain
  • CD28CS is used as the CS domain
  • CD3zICS is used as the ICS domain.
  • one of the antigen-binding domains shown in FIG 2 is used as the antigen-binding domain
  • CD28H is used as the hinge
  • CD28TM is used as the TM domain
  • 41BBCS is used as the CS domain
  • CD3zICS is used as the ICS domain.
  • CD28H is used as the hinge
  • CD28TM is used as the TM domain
  • DAP10CS is used as the CS domain
  • CD3zICS is used as the ICS domain.
  • CD28H is the hinge derived from human CD28.
  • CD28TM is the TM domain derived from human CD28.
  • CD28CS is the CS region derived from a cytoplasmic signaling sequence of human CD28.
  • CD3zICS is the ICS domain derived from a human CD 3 zeta.
  • any of the CAR constructs described in this figure or in this application may be used with LS, T2A, and/or trCD19, as shown in FIGS IE and IF.
  • FIGS IE and IF Although no connecting black lines are shown between different domains, an additional sequence may or may not be included between domains.
  • FIG 4 shows a flow chart illustrating one of many possible methods for manufacturing isolated recombinant CAR-expressing cells that may be used for in vitro or in vivo studies.
  • FIGS 5A-5D contain exemplary results from cytotoxicity assays described In Example 4.
  • Anti-ALPPL2 CART cells (“h3H2scFvHL”) ("CAR T'J or T cells mock transduced with an empty vector (“EV T”) were co-cultured with CAL27 cells (human tongue squamous cell carcinoma cell line) (FIG 5A), BxPC3 cells (human pancreatic adeno carcinoma cell line) (FIG SB), H1651 cells (colorectal cancer cell line) (FIG SC), Capan2 cells (human pancreatic ductal adenocarcinoma cell line) (FIG 5D) at various effector (CART cell) : target (tumor cell) ratios.
  • CAL27 cells human tongue squamous cell carcinoma cell line
  • BxPC3 cells human pancreatic adeno carcinoma cell line
  • H1651 cells colonrectal cancer cell line
  • Capan2 cells human pancreatic ductal adenocarcinoma cell line
  • Cytotoxicity was evaluated using a luciferase plate assay after 24 (top) or 48 (bottom) hours of co-culture. Asterisks represent significance between "CAR T” and "EV T” using a student's T test (* p ⁇ 0.05, ** p ⁇ 0.01, ***p ⁇ 0.001).
  • FIG 6 contains exemplary results from a cytokine production test described in Example 5.
  • Anti-ALPPL2 CART cells (“h3H2scFvHL”) ("CAR T") or T cells mock transduced with an empty vector (“EV T”) were co-cultured with CAL27 cells, BxPC3 cells, H1651 cells , Capan2 cells, or Capan 1 cells (another human pancreatic ductal adenocarcinoma cell line) for 24 hours, and the concentrations of IFN-g in the supernatant were compared.
  • “017” represents one experiment and "018” represents another separate experiment Error bars: standard error of the mean (SEM).
  • Statistical differences in the IFN-g levels were calculated using Student's T test (* p ⁇ 0.05, ⁇ * p ⁇ 0.01, *** p ⁇ 0.001).
  • FIGS 7A-7C contain exemplary tumor growth in an in vivo efficacy test described in Example 6.
  • NSG mice harboring Intraperitoneal Cal27 tumors were treated with human T cells expressing trCD19 butno anti-ALPPL2 ("EV T") or human T cells expressing anti-ALPPL2 CAR ("h3H2scFvHL”) ("CART").
  • FIG 7A is a series of Xenogen-IVIS® images showing the changes in the tumor burden in each treatment group.
  • FIG 7B is an exemplary graph comparing the average tumor burden in the two treatment groups using the luminescent signal intensity (in radiance (photons/second)). Error bars: standard error of the mean (SEM).
  • FIG 7C provides exemplary graphs of tumor burden in individual mice in the "EV T” group (top) and "CAR T” group (bottom) using the luminescent signal intensity (in radiance (photons/second)).
  • FIGS 8A-8B contain exemplary body weight changes in an in vivo efficacy test described in Example 6.
  • FIG 8A is an exemplary graph comparing the average body weight changes in the two treatment groups.
  • FIG 8B provides exemplary graphs of body weight changes of individual mice in the "EV T" group (top) and "CAR T” group (bottom).
  • FIGS 9A-9B contain exemplary tumor growth in an in vivo efficacy test described in Example 7.
  • NSG mice harboring intraperitoneal H1651 tumors high expressor of ALPPL2
  • EV T human T cells expressing trCD19 butno anti-ALPPL2
  • h3H2scFvHL human T cells expressing anti-ALPPL2 CAR
  • FIG 7A is a series of Xenogen-IVIS® images showing the changes in the tumor burden in each treatment group.
  • FIG 7B is an exemplary graph comparing the average tumor burden in the two treatment groups using the luminescent signal intensity (in radiance (photons/second)). Error bars: standard error of the mean (SEM).
  • FIG 7C provides exemplary graphs of tumor burden in individual mice in the "EV T” group (top) and "CAR T” group (bottom) using the luminescent signal intensity (in radiance (photons/second)).
  • FIG 10 provides exemplary binding of the 1B2-, 1E8-, or 3H2-producing hybridoma culture supernatant to ALPPL2 positive and negative cells analyzed by flow cytometry. Filled histograms show binding to ALPPL2 negative cells and open histograms show binding to ALPPL2 positive cells.
  • FIGS 11A-11B provide exemplary comparison of cell binding by 1B5 and 3H2 antibodies, measured by flow cytometry.
  • FIG 11A provides exemplary comparison of binding to human ALPPL2-expressing cells (H1651 cells and CHO cells transduced with ALPPL2) and negative control cells (CHO cells and MCF7 cells).
  • FIG 11B provides exemplary binding curves for binding to CHO-ALPPL2 cells with various antibody concentrations.
  • FIG 12 provides exemplary binding to recombinant human ALPPL2 by purified IgGs at different antibody concentrations (top) or IgG-contalnlng supernatant at different dilutions (bottom) of 1B5 and 3H2, measured by ELISA
  • FIG 13 provides exemplary binding curves obtained with 1B5 (top) and 3H2 (bottom) antibodies by BLI. The ka, kd, and KD values calculated based on the curves are also shown.
  • FIG 14 provides exemplary comparison of binding to plate-bound recombinant human ALPPL2 by different IgG antibodies before and after the affinity maturation described in Example 10, measured by ELISA
  • FIG 11A provides exemplary binding curves obtained during the initial screening for IBS, the affinity maturation parent (B001), and some of the clones obtained by affinity maturation, including those that bind better than, similarly to, or less efficiently compared to BOOL
  • FIG 11B provides exemplary binding curves for IBS and four clones (B380, B416, B506, and B735) selected from the affinity maturation products.
  • FIG 15 provides exemplary curves of monovalent binding to recombinant human ALPPL2 in solution by IgGs of IBS and four clones (B380, B416, B506, and B735), measured by ELISA
  • FIG 16 provides exemplary comparison of binding to CH0-ALPPL2 cells by IgGs of IBS and four clones (B380, B416, B506, and B735), measured by flow cytometry.
  • An aspect of the invention in general relates to the construction and use of novel ALPPL2 -binding agents.
  • the anti-ALPPL2 agent is, for example, but not limited to, anti- ALPPL2 antibodies (Abs), antigen-binding Ab fragments, multi-specific Abs, multi- specific antigen-binding Ab fragments, antibody-drug conjugates (ADCs), and chimeric antigen receptors (CARs).
  • Abs anti- ALPPL2 antibodies
  • ADCs antibody-drug conjugates
  • CARs chimeric antigen receptors
  • the antigen-binding Ab fragment comprises an antigen-binding domain.
  • the antigen-binding Ab fragment may be an antigen-binding domain.
  • the invention also provides polynucleotides encoding such an Ab, antigenbinding Ab fragment; multi-specific Ab, multi-specific antigen-binding Ab fragment; ADC, or CAR that binds to ALPPL2, vectors comprising such a polynucleotide, and cells comprising such an Ab, antigen-binding Ab fragment; multi-specific Ab, multi-specific antigen-binding Ab fragment, ADC, CAR, such a polynucleotide, or such a vector.
  • compositions comprising such an Ab, antigen-binding Ab fragment; multi-specific Ab, multi-specific antigen-binding Ab fragment, ADC, CAR, such a polynucleotide, such a vector, or such a cell.
  • the invention further provides methods of making and using an ALPPL2 -binding Ab, antigen-binding Ab fragment; multi-specific Ab, multi-specific antigen-binding Ab fragment; ADC, or CAR, or cells expressing an ALPPL2-blndlng Ab, antigen-binding Ab fragment; multi-specific Ab, multi-specific antigen-binding Ab fragment, ADC, or CAR.
  • the invention also provides methods for treating a condition associated with ALPPL2 expression in a subject; such as cancer.
  • Such anti-ALPPL2 Abs, antigen-binding Ab fragments, multi-specific Abs, multi-specific antigen-binding Ab fragments, ADCs, CARs, and cells comprising polynucleotide encoding such an ALPPL2-binding Ab, antigenbinding Ab fragment; multi-specific Ab, multi-specific antigen-binding Ab fragment; ADC, or CAR may be used to treat diseases, disorders, or conditions associated with the undesired proliferation of cells expressing ALPPL2.
  • the anti-ALPPL2 agents of the present invention bind to ALPPL2.
  • the target, or the binding target, of the anti-ALPPL2 agents of the present invention is ALPPL2.
  • the anti-ALPPL2 antibody (Ab), anti-ALPPL2 antigen-binding Ab fragment; anti-ALPPL2 multi-specific Ab, anti-ALPPL2 multi-specific antigen-binding Ab fragment, anti-ALPPL2 antibody-drug conjugate (ADC), and anti-ALPPL2 chimeric antigen receptor (CAR) of the present invention individually comprise an antigenbinding domain which binds to Alkaline phosphatase, placenta-like 2 (ALPPL2), which is also referred to as "ALPPL", “alkaline phosphatase, germ cell (APGC)", or “germ cell alkaline phosphatase (GCAP)”.
  • ALPPL2 Alkaline phosphatase
  • APGC germ cell
  • GCAP germ cell alkaline phosphatase
  • ALPPL2 is encoded by the ALPG gene on chromosome 2, with gene location 2q37.1 (NCBI).
  • Human ALPPL2 may have an amino acid sequence provided as GenBank: AIC53992.1.
  • human ALPPL2 has the amino acid sequence provided as SEQ ID NO: 101 or the equivalent residues from a non-human species, e.g., mouse, rodent; monkey, ape and the like.
  • ALPPL2 In healthy humans, the protein expression of ALPPL2 is primarily in the placenta (https;// yAvw.proteinatias.org/ ENSG00000163286-ALPPL2/tissue) and ALPPL2 regulates naive pluripotency (Bi Y. etai., Cell Rep. 2020 Mar 17;30(llj;3917-393Le5. doi: 10,1016/j.celrep.2020.02.090.).
  • ALPPL2 is upregulated in different types of cancer such as, but not limited to, pancreatic cancer, testicular cancer, cervical cancer, endometrial cancer, ovarian cancer, and stomach cancer (htq)$://www.protelnatias.Grg/ENSG00C'00163286"ALPPL2/patiiok)gy).
  • pancreatic cancer testicular cancer
  • cervical cancer endometrial cancer
  • ovarian cancer ovarian cancer
  • stomach cancer htq)$://www.protelnatias.Grg/ENSG00C'00163286"ALPPL2/patiiok)gy.
  • the anti-ALPPL2 agents of the present invention may bind to or target ALPPL2 on cancer cells of the above-mentioned cancer types.
  • anti-ALPPL2 agents may bind pancreatic cancer cells. In some embodiments, anti-ALPPL2 agents may bind testicular cancer cells. In some embodiments, anti-ALPPL2 agents may bind cervical cancer cells. In some embodiments, anti-ALPPL2 agents may bind endometrial cancer cells. In some embodiments, anti- ALPPL2 agents may bind ovarian cancer cells. In some embodiments, anti-ALPPL2 agents may bind stomach cancer cells. In some embodiments, anti-ALPPL2 agents may bind colorectal cancer cells. In some embodiments, anti-ALPPL2 agents may bind lung cancer cells. In some embodiments, anti-ALPPL2 agents may bind colorectal cancer cells. In some embodiments, anti-ALPPL2 agents may bind tongue cancer cells.
  • Anti-AI.PPL2 antibody antigen-binding fragment multi-specific antibody, multi-specific antigen-binding fragment and antibody-drug conjugate
  • the anti-ALPPL2 antibody (Ab), anti-ALPPL2 antigenbinding (AB) fragment; anti-ALPPL2 multi-specific Ab, anti-ALPPL2 multi-specific antigen-binding Ab fragment; and anti-ALPPL2 antibody-drug conjugate (ADC) of the present invention individually comprise at least one antigen-binding (AB) domain that binds to ALPPL2.
  • the ALPPL2 -binding domain may comprise the antigen-binding domain of an anti-ALPPL2 monoclonal antibody, such as a mouse antibody or a humanized version of a mouse antibody.
  • the ALPPL2 -binding domain i.e., the antigen-binding domain
  • Three novel anti-ALPPL2 monoclonal antibodies, named 1B2, 1E8, and 3H2, were generated as described in Example 1.
  • the mouse anti-ALPPL2 monoclonal antibody clone 3H2 comprises: (a) a VH sequence as set forth in SEQ ID NO: 131, which may be encoded by SEQ ID NO: 231; and (b) VL sequence as set forth in SEQ ID NO: 135, which may be encoded by SEQ ID NO: 235.
  • the CDR1, CDR 2, and CDR 3 of the VH i.e., CDR-H1, CDR-H2, and CDR-H3
  • the CDR1, CDR 2, and CDR 3 of the VL (l.e., CDR-L1, CDR-L2, and CDR-L3) comprise the amino acid sequence of SEQ ID NOs: 136, 137, and 138, respectively, which may be encoded by SEQ ID NOs: 236, 237, and 238, respectively.
  • the humanized version of 3H2 (may be referred to as h3H2) comprises: (a) a VH sequence as set forth in SEQ ID NO: 331, which may be encoded by SEQ ID NO: 431; and (b) a VL sequence as set forth in SEQ ID NO: 335, which may be encoded by SEQ ID NO: 435.
  • the CDR 1, CDR 2, and CDR 3 of the VH (l.e., CDR-H1, CDR-H2, and CDR-H3) may comprise the amino acid sequence of SEQ ID NOs: 332, 333, and 334, respectively, which may be encoded by SEQ ID NOs: 432, 433, and 434, respectively.
  • the CDR1, CDR 2, and CDR 3 of the VL may comprise the amino acid sequence of SEQ ID NOs: 336, 337, and 338, respectively, which may be encoded by SEQ ID NOs: 436, 437, and 438, respectively.
  • One of the affinity maturation products were named 1B5, which comprises: (a) a VH sequence as set forth in SEQ ID NO: 701; and (b) a VL sequence as set forth in SEQ ID NO: 705, which may be encoded by SEQ ID NO: 805.
  • the CDR 1, CDR 2, and CDR 3 of the VH i.e., CDR-H1, CDR-H2, and CDR-H3) may comprise the amino acid sequence of SEQ ID NOs: 702, 703, and 704, respectively.
  • the CDR1, CDR 2, and CDR 3 of the VL may comprise the amino acid sequence of SEQ ID NOs: 706, 707, and 708, respectively, which may be encoded by SEQ ID NOs: 806, 807, and 808, respectively.
  • Inventor performed humanization on 1B5 (specifically, humanized the VL of 1B5) as described in Example 10 and obtained B001, which comprises: (a) a VH sequence as set forth in SEQ ID NO: 711; and (b) a VL sequence as set forth in SEQ ID NO: 715.
  • the CDR 1, CDR 2, and CDR 3 of the VH i.e., CDR-H1, CDR-H2, and CDR-H3
  • the CDR1, CDR 2, and CDR 3 of the VL i.e., CDR-L1, CDR-L2, and CDR-L3
  • B380 comprises: (a) a VH sequence as set forth in SEQ ID NO: 721; and (b) a VL sequence as set forth in SEQ ID NO: 725.
  • the CDR 1, CDR 2, and CDR 3 of the VH (l.e., CDR-H1, CDR-H2, and CDR-H3) may comprise the amino acid sequence of SEQ ID NOs: 722, 723, and 724, respectively.
  • the CDR1, CDR 2, and CDR 3 of the VL i.e., CDR- Ll, CDR-L2, and CDR-L3) may comprise the amino acid sequence of SEQ ID NOs: 726, 727, and 728, respectively.
  • B416 comprises: (a) a VH sequence as set forth in SEQ ID NO: 731; and (b) a VL sequence as set forth in SEQ ID NO: 735.
  • the CDR 1, CDR 2, and CDR 3 of the VH i.e., CDR-H1, CDR-H2, and CDR-H3 may comprise the amino add sequence of SEQ ID NOs: 732, 733, and 734, respectively.
  • the CDR1, CDR 2, and CDR 3 of the VL i.e., CDR-L1, CDR-L2, and CDR-L3 may comprise the amino add sequence of SEQ ID NOs: 736, 737, and 738, respectively.
  • [230] 8506 comprises: (a) a VH sequence as set forth in SEQ ID NO: 741; and (b) a VL sequence as set forth in SEQ ID NO: 745.
  • the CDR 1, CDR 2, and CDR 3 of the VH i.e., CDR-H1, CDR-H2, and CDR-H3 may comprise the amino add sequence of SEQ ID NOs: 742, 743, and 744, respectively.
  • the CDR1, CDR 2, and CDR 3 of the VL i.e., CDR-L1, CDR-L2, and CDR-L3 may comprise the amino add sequence of SEQ ID NOs: 746, 747, and 748, respectively.
  • [231] 8735 comprises: (a) a VH sequence as setforth in SEQ ID NO: 751; and (b) a VL sequence as setforth in SEQ ID NO: 755.
  • the CDR 1, CDR 2, and CDR 3 of the VH i.e., CDR-H1, CDR-H2, and CDR-H3 may comprise the amino add sequence of SEQ ID NOs: 752, 753, and 754, respectively.
  • the CDR1, CDR 2, and CDR 3 of the VL i.e., CDR-L1, CDR-L2, and CDR-L3 may comprise the amino add sequence of SEQ ID NOs: 756, 757, and 758, respectively.
  • the ALPPL2 -binding domain (i.e., the antigenbinding domain) of the anti-ALPPL2 agent of the present invention may comprise (a) a CDR-H1, a CDR-H2, and a CDR-H3 contained in the VH of any of the anti-ALPPL2 Abs or Ab fragments described herein, and (b) a CDR-L1, a CDR-L2, and a CDR-L3 contained in the VL of any of the anti-ALPPL2 Abs or Ab fragments described herein.
  • the CDR sequences may be determined based on given VH and VL sequences or VH-encoding and VL-encoding sequences using any appropriate methods and definitions.
  • CDR sequences may be determined based on numbering schemes (e.g., most commonly EU numbering, IMGT numbering, or Rabat numbering; or alternatively Chothia numbering, Martin numbering, Gelfand numbering, Honneger's numbering) of variable regions or may be determined structurally (see e.g., IgBlast (https://www.ncbi.nhn.nih.gov/igblast/); Dondelin ger etai., Front Immunol 2018 Oct 16, ‘9: 2278; http://www.bioinf.org.Uk/abs/info.html#cdrid).
  • numbering schemes e.g., most commonly EU numbering, IMGT numbering, or Rabat numbering; or alternatively Chothia numbering, Martin numbering, Gelfand numbering, Honneger's numbering
  • IgBlast https://www.ncbi.nhn.nih.gov/igblast/
  • the ALPPL2 -binding domain (Le., the antigen-binding domain, for example the variable region of an Ab or Ab fragment or the antigen-binding domain of a CAR) may comprise (a) a VH comprising a CDR-H1, and a CDR-H2, and a CDR-H3 contained in the VH of any of the anti-ALPPL2 Abs or Ab fragments described herein; and (b) a VL comprising a CDR-L1, a aCDR-L2, and CDR-L3 contained in the VL of any of the anti-ALPPL2 Abs or Ab fragments described herein.
  • the VH comprises a human or human-like VH framework and the VL comprises a human or human-like VL framework.
  • the CDR-H1, CDR-H2, and CDR-H3 may be the CDR-H1, CDR- H2, and CDR-H3 contained In SEQ ID NO: 721 and/or may comprise the amino acid sequences set forth in SEQ ID NOs: 722, 723, and 724, respectively, and the CDR-L1, CDR-L2, and CDR-L3 may be the CDR-L1, CDR-L2, and CDR-L3 contained in SEQ ID NO: 725 and/or may comprise the amino acid sequences set forth in SEQ ID NOs: 726, 727, and 728, respectively.
  • the CDR-H1, CDR-H2, and CDR-H3 may be the CDR-H1, CDR- H2, and CDR-H3 contained in SEQ ID NO: 731 and/or may comprise the amino acid sequences set forth In SEQ ID NOs: 732, 733, and 734, respectively, and the CDR-L1, CDR-L2, and CDR-L3 may be the CDR-L1, CDR-L2, and CDR-L3 contained in SEQ ID NO: 735 and/or may comprise the amino acid sequences set forth in SEQ ID NOs: 736, 737, and 738, respectively.
  • the CDR-H1, CDR-H2, and CDR-H3 may be the CDR-H1, CDR- H2, and CDR-H3 contained in SEQ ID NO: 741 and/or may comprise the amino acid sequences set forth in SEQ ID NOs: 742, 743, and 744, respectively, and the CDR-L1, CDR-L2, and CDR-L3 may be the CDR-L1, CDR-L2, and CDR-L3 contained in SEQ ID NO: 745 and/or may comprise the amino acid sequences set forth in SEQ ID NOs: 746, 747, and 748, respectively.
  • the CDR-H1, CDR-H2, and CDR-H3 may be the CDR-H1, CDR- H2, and CDR-H3 contained in SEQ ID NO: 751 and/or may comprise the amino acid sequences set forth in SEQ ID NOs: 752, 753, and 754, respectively, and the CDR-L1, CDR-L2, and CDR-L3 may be the CDR-L1, CDR-L2, and CDR-L3 contained in SEQ ID NO: 755 and/or may comprise the amino acid sequences set forth in SEQ ID NOs: 756, 757, and 758, respectively.
  • the CDRs from the mouse antibody may be grafted into the human antibody framework. Therefore, the human-like framework may be 100% identical to a human framework.
  • the Tabhu program http://circe.med.uniromal.it/tabhu/
  • the Tabhu program http://circe.med.uniromal.it/tabhu/
  • steps which are: (i) loop grafting, (ii) estimation of the binding mode similarity between the native and human antibody, (iii) back-mutations and (iv) reevaluation of the binding mode similarity between input and humanized antibody (vicieri P. P. etal., Bioinformatics. 2015 Feb l;31(3):434-5.
  • the framework may not be 100% identical to a human framework but may still comprise significant sequence identity to a human framework.
  • the VH may comprise a human or human-like VH framework may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human VH framework
  • the VL may comprise a human or human-like VL framework may at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human VL framework
  • variable region of an anti-ALPPL2 agent disclosed herein may be altered without significantly reducing ALPPL2 binding.
  • the sequence of the variable region may be altered.
  • Antigen-Ab interactions are largely determined by six CDRs, while a person of skilled in the art will appreciate that some deviation from the exact CDR sequences may be possible. Any suitable techniques such as affinity maturation can be used to alter the CDR sequence.
  • VH CDR 3 and VL CDR 3 are generally believed as the key determinant of specificity in antigen recognition.
  • CDR-H3 diversity in the CDR 3 of VH (i.e., CDR-H3) may be particularly important for providing most antibody specificities (Xu J.L, Immunity. 2000 Jul;13(l):37-45). Therefore, one or more mutations may be incorporated in the CDR 1 and/or CDR 2 without greatly decreasing the binding affinity while achieving a more desired property of an Ab.
  • Abs or antigen-binding Ab fragments comprising CDR-H1, CDR-H2, CDR-L1, CDR-L2, and/or CDR-L3 with at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the CDR-H1, CDR-H2, CDR-L1, CDR-L2, and/or CDR-L3 disclosed herein, respectively, are also within the scope of the present invention. Furthermore, one or more mutations in CDR-H3 may be incorporated to modify, increase, or fine tune the binding or any other properties of the antigen-binding domain.
  • any one mutation may alter biochemical properties such as thermodynamic stability or immunogenicity in addition to the affinity
  • all possible mutations in any of the six CDRs, and/or any combination thereof, and/or even in the framework sequence may be tested to see if the sequence modification provides an improved or more desired overall property (Rajpal A et al., Proc Natl Acad Sci U S A 2005 Jun 14; 102(24): 8466-8471. doi: 10.1073/pnas.0503543102; Julian M. C. etal., Sci Rep. 2017; 7: 45259. Published online 2017 Mar 28. doi: 10.1038/srep45259).
  • any appropriate technique such as, but not limited to, ELISA RIA FACS, bioassay, or Western Blot assay may be used.
  • the heavy chain variable region (i.e., VH) may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 721, and the light chain variable region (i.e., VL) comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 725.
  • the VH may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 731
  • the VL comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 735.
  • the VH may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% Identical to SEQ ID NO: 741, and the VL comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 745.
  • the VH may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 751, and the VL comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 755.
  • the anti-ALPPL2 agent of the present invention may be, for example, but not limited to, a monoclonal Ab, a monospecific Ab, a bispecific Ab, a multispeclflc Ab, a humanized Ab, a tetrameric Ab, a tetravalent Ab, a single chain Ab, a domain-specific Ab, a domain-deleted Ab, an scFc fusion protein, a chimeric Ab, a synthetic Ab, a recombinant Ab, a hybrid Ab, a mutated Ab, CDR-grafted Ab, a fragment antigen-binding (Fab), an F(ab')2, an Fab' fragment; a variable fragment (Fv), a singlechain Fv (scFv) fragment; an Fd fragment; a diabody, and a minibody.
  • Fab fragment antigen-binding
  • Fv variable fragment
  • scFv singlechain Fv
  • the anti-ALPPL2 agent is or comprises a scFv comprising a VH, a linker, and a VL, where the VH and VL combinations are any of the combinations described above.
  • the scFv according to the present disclosure may comprise: a VH, a linker, and a VL, or a VL, a linker, and a VH, in the direction from the N- terminus to the C-terminus. Any appropriate linker that allows the VH and VL to form an antigen-binding site that binds to ALPPL2 may be used.
  • the linker may be a GS linker, which is one or more repeats of a sequence unit containing G (glycine) and S (serine), such as the GS linker unit of SEQ ID NO: 167.
  • G glycine
  • S serine
  • the G4SX3 linker of SEQ ID NO: 168 may be used.
  • the antigen-binding domain of the anti-ALPPL2 agent of the present invention may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to: (i) SEQ ID NOs: 760, 759, 750, 749, 740, 739, 730, 729, 720, 719, 319, 320, 329, 330, 339, 340, 341, or 342 or (ii) the amino acid sequence encoded by SEQ ID NOs: 419, 19, 420, 20, 429, 29, 430, 30, 439, 39, 440, 40, 441, or 442.
  • the anti-ALPPL2 agent of the present Invention may comprise two or more binding specificities (i.e., bispecific, trispecific, or multispecific generally).
  • the first specificity is to an epitope in ALPPL2 (first ALPPL2 epitope).
  • the anti-ALPPL2 agent of the present disclosure may have a second binding specificity to another epitope (l.e., second ALPPL2 epitope) in ALPPL2.
  • the second ALPPL2 epitope may or may not be overlapped with the first ALPPL2 epitope.
  • the second specificity may be to an epitope in a second antigen other than ALPPL2.
  • a multi-specific ALPPL2 -binding agent according to the present disclosure may bind to ALPPL2 and one or more other targets.
  • a multi-specific anti-ALPPL2 agent binds to ALPPL2 and a protein on an effector cell.
  • a multi-specific anti-ALPPL2 agent binds to ALPPL2 and a protein on a target (e.g., cancer) cell.
  • binding to a second antigen may improve functional characteristics of the anti-ALPPL2 agent; e.g., recruitment, effector functions, lysis of target cells.
  • the second antigen may be, for example without limitation, NKG2D, 4- IBB, or an Fc receptor (FcR), such as Fc gamma receptor (FcgR), FcgRI, FcgRIIA, FcgRIIBl, FcgRIIB2, FcgRIIIA, FcgRIIIB, Fc epsilon receptor (FceR), FceRI, FceRII, Fc alpha receptor (FcaR), FcaRI, Fc alpha/mu receptor (Fca/mR), or neonatal Fc receptor (FcRn).
  • FcR Fc receptor
  • FcR-medlated effects such as antibody-dependent cellular phagocytosis (ADCP) or antibody-dependent cellular cytotoxicity (ADCC) of ALPPL2 -expressing cells or cytotoxic mediator release by Fc-expressing cells.
  • ADCP antibody-dependent cellular phagocytosis
  • ADCC antibody-dependent cellular cytotoxicity
  • the FcR may be, but is not limited to, Fc gamma receptor (FcgR), FcgRI, FcgRIIA, FcgRIIBl, FcgRIIB2, FcgRIIIA, FcgRIIIB, Fc epsilon receptor (FceR), FceRI, FceRII, Fc alpha receptor (FcaR), FcaRI, Fc alpha/mu receptor (Fca/mR), or neonatal Fc receptor (FcRn).
  • FcgR Fc gamma receptor
  • FcgRI FcgRIIA
  • FcgRIIBl FcgRIIB2
  • FcgRIIIA FcgRIIIB
  • Fc epsilon receptor FceR
  • FceRI FceRII
  • Fc alpha receptor FcaR
  • Fca/mR Fc alpha/mu receptor
  • FcRn neonatal Fc receptor
  • the agent may be called bispecific.
  • Bispecific anti-ALPPL2 agents include bispecific anti-ALPPL2 Abs or antigenbinding Ab fragments.
  • the agent may be called multispecific.
  • Multispecific anti-ALPPL2 agents include multispecific anti-ALPPL2 Abs or antigen-binding Ab fragments.
  • the present invention encompasses any types of bispecific Ab-like molecules (Abs or antigen-binding Ab fragments) such as reviewed in Brinkmann U. et al., MAbs. 2017 Feb-Mar; 9(2): 182-212. Published online 2017 Jan 10. doi: 10.1080/19420862.2016.1268307; Klein C .et al., MAbs. 2016 Aug-Sep;8(6): 1010-20. doi: 10.1080/19420862.2016.1197457.
  • one of the antigen-binding domains is an anti-ALPPL2 binding domain.
  • General methods for designing and construction of bispecific or multispecific Abs or antigen-binding Ab fragments are known in the art (Brinkmann U.
  • antigen-binding Ab fragments or the antigen-binding domains having different specificities may be generated separately and then conjugated to one another, for example using sulfhydryl bonding (of, for example, the VH C-termlnus hinge regions) and/or an appropriate coupling or crosslinking agent
  • Bispecific antigen-binding Ab fragments may also be generated, for example, by using leucine zippers or by using scFv dimers (see for example, Kosteln et al.,/ Immunol. 1992 Mar 1;148(5): 1547-53).
  • Binding of the bispecific agent of the present Invention may be confirmed using any appropriate method, such as but not limited to, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), flow cytometry, bioassay, or Western blot
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • flow cytometry cytometry
  • bioassay or Western blot
  • the anti-ALPPL2 agents of the present invention may comprise a human or human-like CHI, CH2, and/or CH3 domain(s).
  • the human-like CHI, CH2, and/or CH3 domain(s) may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human CHI, CH2, and/or CH3 domaln(s), respectively.
  • the human or human-like CHI, CH2, and/or CH3 domain(s) may be individually derived from the CHI, CH2, and/or CH3 domain(s), respectively, of a human IgM, a human IgD, a human IgG, a human IgE, or a human IgA optionally of a human IgGl, a human IgG2, a human IgG3, or a human IgG4.
  • the anti-ALPPL2 agents of the present invention may comprise a human or human-like fragment crystallizable (Fc) region.
  • the human or human-like Fc region may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human Fc region.
  • the human or human-like Fc region may be derived from a human IgM, IgD, IgG, IgE, or IgA preferably IgGl, IgG2, IgG3, or IgG4.
  • the human-like Fc region may bind to an Fc receptor (FcR).
  • FcR Fc receptor
  • the FcR may be, but is not limited to, Fc gamma receptor (FcgR), FcgRI, FcgRIIA, FcgRIIBl, FcgRlIB2, FcgRIIIA FcgRIIIB, Fc epsilon receptor (FceR), FceRI, FceRII, Fc alpha receptor (FcaR), FcaRI, Fc alpha/mu receptor (Fca/mR), or neonatal Fc receptor (FcRn).
  • FcgR Fc gamma receptor
  • FcgRI FcgRIIA
  • FcgRIIBl FcgRlIB2
  • FcgRIIIA FcgRIIIB Fc epsilon receptor
  • FceR FceRI, FceRII, Fc alpha receptor (FcaR), FcaRI
  • the Ab when the anti-ALPPL2 agent is an Ab, the Ab may be of an IgM, IgD, IgG, IgE, or IgA isotype.
  • the Ab when the Ab is an IgG, and the IgG may be an IgGl, IgG2, IgG3, or IgG4.
  • Certain amino add modifications In the Fc region are known to modulate Ab effector functions and properties, such as, but not limited to, antibody-dependent cellular cytotoxidty (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement dependent cytotoxidty (CDC), and half-life (Wang X et al., Protein Cell. 2018 Jan; 9(1): 63-73; Dall'Acqua W. F. et at J Biol Chem. 2006 Aug 18;281(33):23514- 24. Epub 2006 Jun 21; Monnet C.
  • ADCC antibody-dependent cellular cytotoxidty
  • ADCP antibody-dependent cellular phagocytosis
  • CDC complement dependent cytotoxidty
  • half-life Wang X et al., Protein Cell. 2018 Jan; 9(1): 63-73; Da
  • the mutation may be symmetrical or asymmetrical
  • antibodies with Fc regions that have asymmetrical mutation(s) may provide better functions such as ADCC (Liu Z. etalj Biol Chem. 2014 Feb 7; 289(6): 3571-3590).
  • the Fc region may comprise one or more amino add substitutions.
  • the substitution maybe, for example, N297A, N297Q, D265A, L234A, L235A, C226S, C229S, P238S, E233P, L234V, G236-deleted, P238A A327Q, A327G, P329A, K322A, L234F, L235E, P331S, T394D, A330L, P331S, F243L, R292P, Y300L, V305I, P396L, S239D, I332E, S298A, E333A, K334A, L234Y, L235Q, G236W, S239M, H268D, D270E, K326D, A330M, K334E, G236A, K326W, S239D, E333S, S267E
  • the Fc region may further comprise one or more additional amino add substitutions.
  • the substitution may be, for example, but is not limited to, A330L, L234F, L235E, P331S, and/or any combination thereof (the residue numbering is according to EU numbering).
  • the Fc region may comprise one or more amino add substitutions.
  • the substitution may be, for example, but is not limited to, P238S, V234A, G237A, H268A, H268Q, H268E, V309L, N297A, N297Q, A330S, P331S, C232S, C233S, M252Y, S254T, T256E, and/or any combination thereof (the residue numbering is according to EU numbering).
  • the Fc region may further comprise one or more additional amino add substitutions.
  • the substitution may be, for example, but is not limited to, M252Y, S254T, T256E, and/or any combination thereof (the residue numbering is according to EU numbering).
  • the Fc region may comprise one or more amino add substitutions.
  • the substitution may be, for example, but is not limited to, E235Y (the residue numbering is according to EU numbering).
  • the Fc region may comprise one or more amino add substitutions.
  • the substitution may be, for example, but is not limited to, E233P, F234V, L235A, G237A, E318A S228P, L236E, S241P, L248E, T394D, M252Y, S254T, T256E, N297A, N297Q, and/or any combination thereof (the residue numbering is according to EU numbering).
  • the substitution may be, for example, S228P (the residue numbering is according to EU numbering).
  • the glycan of the human-like Fc region may be engineered to modify the effector function (for example, see Li T. et al, Proc Nad Acad Scl U S A. 2017 Mar 28;114(13):3485-3490. dol: 10.1073/pnas.l702173114. Epub 2017 Mar 13).
  • the binding property of an anti-ALPPL2 may be assessed by any appropriate methods.
  • binding to ALPPL2 may be measured by ELISA, RIA, FACS, bioassay, Western blot; or BLI.
  • the binding property may be defined for example by association rate constant (k.), equilibrium dissociation constant (kd), dissociation constant (KD), and/or half maximal effective concentration (ECso).
  • the KD of an anti-ALPPL2 agent for ALPPL2 binding may be about 10 nM or smaller.
  • the KD of an anti-ALPPL2 agent may be about 1.6 nM, about 0.8 nM, about 0.4 nM, about 0.3 nM, or about 0.2 nM.
  • the ECso of an anti-ALPPL2 agent for ALPPL2 binding may be about 1 pg/mL or smaller. In particular embodiments, the ECso of an anti- ALPPL2 agent for ALPPL2 binding may be about 0.05 pg/mL or about 0.02 pg/mL
  • binding to ALPPL2-expressing cells may be measured by flow cytometry, for example based on MFI.
  • Such binding property may be defined for example by half maximal effective concentration (ECso).
  • the ECso of an anti-ALPPL2 agent for cell binding may be about 100 nM or smaller. In particular embodiments, the ECso of an anti-ALPPL2 agent may be about 15 nM, about 10 nM, about 5 nM, about 1 nM, about 0.5 nM, or about 0.2 nM.
  • the anti-ALPPL2 agent of the present invention may be an antibody-drug conjugate (ADC).
  • ADC may comprise: (a) any Ab or antigenbinding Ab fragment described herein; and (b) a drug conjugated to the Ab or antigenbinding Ab fragment
  • the drug may be, but not limited to, an anti-cancer drug, an anti-proliferative drug, a cytotoxic drug, an anti-angiogenic drug, an apoptotic drug, an immunostimulatory drug, an anti-microbial drug, an antibiotic drug, an antiviral drug, an anti-inflammatory drug, an ALPPL2 inhibitor, an ALPPL2 signaling inhibitor, an enzyme, a hormone, a toxin, a radioisotope, a compound, a small molecule, a small molecule inhibitor, a protein, a peptide, a vector, a plasmid, a viral replicon, a viral particle, a nanoparticle, a DNA molecule, an RNA molecule, an siRNA, an shRNA, a micro RNA, an oligonucleotide, and an imaging drug.
  • an anti-cancer drug an anti-proliferative drug, a cytotoxic drug, an anti-angiogenic drug, an apoptotic drug,
  • the toxin may be a bacterial, fungal, plant, or animal toxin, or a fragment thereof.
  • examples include, but are not limited to, diphtheria A chain, diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha sardn, Aleurites fordii protein, a dianthin protein, or a Phytolacca Americana protein.
  • the anti-cancer or anti-proliferative drug may be, for example, but is not limited to, doxorubicin, daunorubicin, cucurbitacin, chaetocin, chaetoglobosin, chlamydocin, calicheamicin, nemorubicin, cryptophyscin, mensacarcin, ansamitodn, mitomydn C, geldanamydn, mechercharmydn, rebeccamydn, safradn, okilactomydn, oligomydn, actinomydn, sandramydn, hypothemydn, polyketomydn, hydroxyelliptidne, thiocolchidne, methotrexate, triptolide, taltobulin, lactacystin, dolastatin, auristatin, monomethyl auristatin E (MMAE), monomethyl
  • the radioisotope may be for example, bulls not limited to, At211, 1131, Inl31, 1125, Y90, Rel86, Rel88, Sml53, Bi212, P32, Pb212 and radioactive isotopes of Lu.
  • the drug may be, but is not limited to, MMAE or MMAF.
  • the Ab or antigen-binding Ab fragment is directly conjugated to the drug to form an ADC.
  • the Ab or antigen-binding Ab fragment is indirectly conjugated to the drug to form an ADC.
  • Any appropriate conjugation method may be used to generate an ADC (for example, Nolting B. Methods Mol Biol. 2013;1045:71-100. doi: 10.1007/978-1-62703- 541-5,5; Jain N. etal., Pharm Res. 2015 Nov;32(ll):3526-40. doi: 10.1007/sll095-015- 1657-7. Epub 2015 Mar 11; Tsuchikama K. etal., Protein Cell. 2018 Jan;9(l):33-46. doi: 10.1007/sl3238-016-0323-0. Epub 2016 Oct 14; Polakis P. et al., Pharmacol Rev. 2016 Jan;68(l):3-19. doi: 10.1124/pr.ll4.009373). Examples of methods that may be used to perform conjugation include, but are not limited to, chemical conjugation and enzymatic conjugation.
  • Chemical conjugation may utilize, for example, but is not limited to, lysine amide coupling, cysteine coupling, and/or non-natural amino add incorporation by genetic engineering.
  • Enzymatic conjugation may utilize, for example, but is not limited to, transpeptidation using sortase, transpeptidation using microbial transglutaminase, and/or N-Glycan engineering.
  • one or more of cleavable linkers may be used for conjugation.
  • the cleavable linker may enable cleavage of the drug upon responding to, for example, but not limited to, an environmental difference between the extracellular and intracellular environments (pH, redox potential, eta) or by spedfic lysosomal enzymes.
  • cleavable linker examples include, but are not limited to, hydrazone linkers, peptide linkers including cathepsin B-responsive linkers, such as valin-citrulline (vc) linker, disulfide linkers such as N-succinimidyl-4-(2 -pyridyldithio) (SPP) linker or N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB) linker, and pyrophosphate diester linkers.
  • vc cathepsin B-responsive linkers
  • disulfide linkers such as N-succinimidyl-4-(2 -pyridyldithio) (SPP) linker or N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB) linker
  • SPDB N-succinimidyl-4-(2-pyridyl
  • non-deavable linkers may be used.
  • non-cleavable linkers include thioether linkers, such as N- succinimidyl 4-(N-maleimidomethyl) cyclohexane-l-carboxylate (SMCC), and malelmidocaproyl (me) linkers.
  • thioether linkers such as N- succinimidyl 4-(N-maleimidomethyl) cyclohexane-l-carboxylate (SMCC), and malelmidocaproyl (me) linkers.
  • SMCC N- succinimidyl 4-(N-maleimidomethyl) cyclohexane-l-carboxylate
  • me malelmidocaproyl
  • Anti-ALPPI.2 Chimeric antigen receptors (CAR)
  • an anti-ALPPL2 agent according to the present disclosure may be a chimeric antigen receptor (CAR).
  • the CARs of the present invention comprise an antigen binding (AB) domain that binds to ALPPL2, a transmembrane (TM) domain, and an intracellular signaling (ICS) domain.
  • FIG 1A A schematic showing a generic CAR construct of the present invention is depicted in FIG 1A.
  • the CAR may optionally comprise a hinge that joins the antigen-binding domain and said TM domain.
  • the CAR may optionally comprise one or more costimulatory (CS) domains. Schematics showing three more generic CAR constructs of the present invention are depicted in FIGS IB- ID.
  • the CAR of the present invention comprises an antigen-binding domain which binds to ALPPL2.
  • the antigen-binding domain of the CAR may comprise any of the anti-ALPPL2 agents disclosed herein.
  • the antigen-binding domain of the CAR may comprise any of the antigen-binding domain of any of the anti-ALPPL2 agents disclosed herein.
  • the antigen-binding domain of the CAR may comprise any of the anti-ALPPL2 Abs, anti-ALPPL2 antigen-binding Ab fragments, anti-ALPPL2 multispecific Abs, anti-ALPPL2 multi-specific antigen-binding Ab fragments, and anti-ALPPL2 ADCs disclosed herein, or the antigen-binding domain thereof.
  • the antigen-binding domain of the CAR may comprise an anti-ALPPL2 scFv.
  • the scFv may comprise any of the VH and VL combinations as described above and a linker that connects the VH and VL
  • the antigen-binding domain may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to: (i) SEQ ID NOs: 760, 759, 750, 749, 740, 739, 730, 729, 720, 719, 319, 320, 329, 330, 339, 340, 341, or 342 or (11) the amino add sequence encoded by SEQ ID NOs: 419, 19, 420, 20, 429, 29, 430, 30, 439, 39, 440, 441, or 442.
  • the antigen-binding domain may compete for binding to ALPPL2 with a scFv comprising an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to: (i) SEQ ID NOs: 760, 759, 750, 749, 740, 739, 730, 729, 720, 719, 319, 320, 329, 330, 339, 340, 341, or 342 or (ii) the amino add sequence encoded by SEQ ID NOs: 419, 19, 420, 20, 429, 29, 430, 30, 439, 39, 440, 441, or 442.
  • the CAR may comprise a hinge sequence between the antigen-binding domain and the TM domain.
  • a hinge sequence is a short sequence of amino adds that facilitates flexibility (see, e.g. Woof J.M. etal., Nat Rev. Immunol., 4(2): 89-99 (2004)).
  • the hinge sequence can be any suitable sequence derived or obtained from any suitable molecule.
  • the length of the hinge sequence may be optimized based on the desired length of the extracellular portion of the CAR, which may be based on the location of the epitope within the target molecule. For example, if the epitope is in the membrane proximal region within the target molecule, longer hinges may be optimal.
  • the hinge may be derived from or include at least a portion of an immunoglobulin Fc region, for example, an IgGl Fc region, an IgG2 Fc region, an IgG3 Fc region, an IgG4 Fc region, an IgE Fc region, an IgM Fc region, or an IgA Fc region.
  • the hinge includes at least a portion of an IgGl, an IgG2, an IgG3, an IgG4, an IgE, an IgM, or an IgA immunoglobulin Fc region that falls within its CH2 and CH3 domains.
  • the hinge may also include at least a portion of a corresponding immunoglobulin hinge region.
  • the hinge is derived from or indudes at least a portion of a modified immunoglobulin Fc region, for example, a modified IgGl Fc region, a modified IgG2 Fc region, a modified IgG3 Fc region, a modified IgG4 Fc region, a modified IgE Fc region, a modified IgM Fc region, or a modified IgA Fc region.
  • the modified immunoglobulin Fc region may have one or more mutations (e.g., point mutations, insertions, deletions, duplications) resulting in one or more amino add substitutions, modifications, or deletions that cause impaired binding of the hinge to an Fc receptor (FcR).
  • the modified immunoglobulin Fc region may be designed with one or more mutations which result in one or more amino add substitutions, modifications, or deletions that cause impaired binding of the hinge to one or more FcR including, but not limited to, FcyRI, FcyR2A, FcyR2Bl, Fcy2B2, Fey 3A, Fey 3B, FCERI, FCER2, FcaRI, Fca/pR, or FcRn.
  • a portion of the immunoglobulin constant region serves as a hinge between the antigen-binding domain, for example scFv or nanobody, and the TM domain.
  • the hinge can be of a length that provides for Increased responsiveness of the CAR-expressing cell following antigen binding, as compared to in the absence of the hinge. In some examples, the hinge is at or about 12 amino acids in length or is no more than 12 amino acids in length.
  • Exemplary hinges include those having at least about 10 to 229 amino acids, about 10 to 200 amino adds, about 10 to 175 amino adds, about 10 to 150 amino acids, about 10 to 125 amino adds, about 10 to 100 amino adds, about 10 to 75 amino adds, about 10 to 50 amino adds, about 10 to 40 amino adds, about 10 to 30 amino adds, about 10 to 20 amino adds, or about 10 to 15 amino adds, and including any Integer between the endpoints of any of the listed ranges.
  • a hinge has about 12 amino adds or less, about 119 amino adds or less, or about 229 amino adds or less.
  • Exemplary hinges include a CD28 hinge, IgG4 hinge alone, IgG4 hinge linked to CH2 and CH3 domains, or IgG4 hinge linked to the CH3 domain.
  • Exemplary hinges include, but are not limited to, those described in Hudecek M. et al. (2013) Clin. Cancer Res., 19:3153, international patent application publication number W02014031687, U.S. Pat No. 8,822,647 or published App. No. US2014/0271635.
  • the hinge sequence is derived from CD8 a molecule or a CD28 molecule. In a preferred embodiment; the hinge sequence is derived from CD28. In one embodiment; the hinge comprises the amino add sequence of human CD28 hinge (SEQ ID NO: 163) or the sequence encoded by SEQ ID NO: 263. In some embodiments, the hinge has an amino add sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 163.
  • the CAR can be designed to comprise a TM domain that is fused to the antigen-binding domain of the CAR.
  • a hinge sequence may be inserted between the antigen-binding domain and the TM domain.
  • the TM domain that naturally is associated with one of the domains in the CAR is used.
  • the TM domain can be selected or modified by amino add substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
  • the TM domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Typically, the TM domain denotes a single transmembrane a helix of a transmembrane protein, also known as an integral protein. TM domains of particular use in this invention may be derived from (i.e.
  • the TM domain may be synthetic, in which case the TM domain will comprise predominantly hydrophobic residues such as leudne and valine. Preferably a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic TM domain.
  • a TM domain of the invention is thermodynamically stable in a membrane. It may be a single a helix, a transmembrane p barrel, a 0-helix of gramicidin A, or any other structure. Transmembrane helices are usually about 20 amino acids in length.
  • the TM domain in the CAR of the invention is derived from the TM region of CD28.
  • the TM domain comprises the amino acid sequence of human CD28 TM (SEQ ID NO: 161) or the sequence encoded by SEQ ID NO: 261.
  • the TM domain comprises an amino add sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 161.
  • a short oligo- or polypeptide spacer preferably between 2 and 10 amino adds in length may form the linkage between the TM domain and the ICS domaln(s) of the CAR A glydne-serine doublet may provide a suitable spacer.
  • Intracellular signaling (ICS) domain and costimulatory CCS) domain are Intracellular signaling (ICS) domain and costimulatory CCS domain
  • the ICS domain or otherwise the cytoplasmic domain of the CAR of the invention triggers or elicits activation of at least one of the normal effector functions of the cell in which the CAR has been placed.
  • effector function refers to a spedalized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines.
  • intracellular signaling domain* or "ICS domain” refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a spedalized function. While usually the entire ICS domain can be employed, in many cases it is not necessary to use the entire chain.
  • intracellular signaling domain or "ICS domain” is thus meant to include any truncated portion of the ICS domain suffident to transduce the effector function signal.
  • ICS domains for use in the CAR of the invention indude the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement; as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.
  • TCR T cell receptor
  • CS domain costimulatory domain
  • a CS domain is a domain that transduces such a secondary or costimulatory signal.
  • the CAR of the present invention may comprise two or more CS domains.
  • the CS domain(s) may be placed upstream of the ICS domain or downstream of the ICS domain.
  • FIG IB and FIG 1C Two exemplary schematics of CAR constructs of the present invention containing at least one CS domain are illustrated in FIG IB and FIG 1C.
  • T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequence: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic signaling sequences).
  • Primary cytoplasmic signaling sequences regulate primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way.
  • Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as Immunoreceptor tyrosine-based activation motifs or ITAMs. Such a cytoplasmic signaling sequence may be contained in the ICS or the CS domain of the CAR of the present invention.
  • ITAM -containing primary cytoplasmic signaling sequences that are of particular use In the Invention include those derived from an ICS domain of a lymphocyte receptor chain, a TCR/CD3 complex protein, an Fc receptor subunit; an IL-2 receptor subunit, CD3 7, FcR y, FcR f$, CD3 y, CD3 5, CD3 E, CDS, CD22, CD66d, CD79a, CD79b, CD278 (ICOS), Fc E RI, DAP10, and DAP12.
  • the ICS domain In the CAR of the Invention comprises a cytoplasmic signaling sequence derived from CD 3 zeta.
  • the ICS domain comprises the amino acid sequence of human CD3 £ ICS (SEQ ID NO: 162), or the sequence encoded by SEQ ID NO: 262.
  • the ICS domain comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 162.
  • the cytoplasmic domain of the CAR may be designed to comprise the CD3 ICS domain by itself.
  • the CD3 ( ICS domain may be combined with one or more of any other desired cytoplasmic domain(s) useful in the context of the CAR of the invention.
  • the cytoplasmic domain of the CAR can comprise a CD3 7, ICS domain and a costimulatory (CS) domain.
  • the CS region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule.
  • a costimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient response of lymphocytes to an antigen.
  • CS domains have been reported to confer differing properties.
  • the 4-1BB CS domain showed enhanced persistence in in vivo xenograph models (Milone M. C. etal. Mol Ther 2009;17:1453-1464; Song D. G. etal. Cancer Res 2011;71:4617-4627).
  • these different CS domains produce different cytokine profiles, which in turn, may produce effects on target cell-mediated cytotoxicity and the disease microenvironment
  • DAP 10 signaling in NK cells has been associated with an increase in Thl and inhibition of Th2 type cytokine production in CD8+ T cells (Barber A et al. Blood 2011;117:6571-6581).
  • co-stimulatory molecules include an MHC class I molecule, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, a Toll ligand receptor, B7-H3, BAFFR, BTLA, BLAME (SLAMF8), CD2, CD4, CDS, CD7, CDS a, CDS p, CD 11a, LFA-1 (CDlla/CD18), CDllb, CDllc, CDlld, CD18, CD19, CD19a, CD27, CD28, CD29, CD30, CD40, CD49a, CD49D, CD49f, CD69, CD84, CD96 (Tactile), CD100 (SEMA4D), CD1O3, CRTAM, 0X40 (CD134), 4-1BB (CD137), SLAM (SLAMF1, CD150, IPO- 3), CD160 (BY55), SELPLG
  • the ICS domain and the CS domain(s) of the CAR of the invention may be linked to each other in a random or specified order.
  • a short oligo- or polypeptide linker preferably between 2 and 10 amino adds in length may form the linkage.
  • a glydne-serine doublet provides a particularly suitable linker.
  • the CAR is designed to comprise a cytoplasmic signaling sequence of CD3 I, as the ICS domain and comprise a cytoplasmic signaling sequence of CD28 as the CS domain.
  • the CAR is designed to comprise a cytoplasmic signaling sequence of CD3 £ as the ICS domain and comprise a cytoplasmic signaling sequence of DAP10 as the CS domain.
  • the CAR is designed to comprise a cytoplasmic signaling sequence of CD3 I, as the ICS domain and comprise a cytoplasmic signaling sequence of 4-1BB as the CS domain.
  • Such a cytoplasmic signaling sequence of CD3 £ may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the CD3 7, ICS domain comprising the amino add sequence of human CD3z ICS (SEQ ID NO: 162).
  • Such a cytoplasmic signaling sequence of CD 3 zeta may be encoded by a nucleic add sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 262.
  • Such a cytoplasmic signaling sequence of CD28 may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of human CD28 CS domain (SEQ ID NO: 164).
  • Such a cytoplasmic signaling sequence of CD28 may be encoded by a nucleic add sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 264.
  • Such a cytoplasmic signaling sequence of DAP10 may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of human 4-1BB CS domain (SEQ ID NO: 165).
  • Such a cytoplasmic signaling sequence of 4-1BB may be encoded by a nucleic add sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 265.
  • Such a cytoplasmic signaling sequence of DAP10 may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% Identical to the sequence of human DAP10 CS domain (SEQ ID NO: 166).
  • Such a cytoplasmic signaling sequence of DAP10 may be encoded by a nucleic add sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 266.
  • the antigen-binding domain comprises the ALPPL2 -binding portion of a molecule that binds to ALPPL2 as described above
  • the TM domain of the CAR may be derived from the transmembrane portion of the molecule.
  • the CAR construct is described as "antigenbinding domain - hinge - TM domain - CS domain - ICS domain.”
  • the CARs of the present invention may comprise an amino add sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to any of the exemplary constructs below.
  • the CAR of the invention may be described as B735scFvHL- CD28H-CD28TM-CD28CS-CD3zICS and may comprise the amino add sequence as set forth in SEQ ID NO: 951.
  • the CAR of the invention may be described as B735scFvHL- CD28H-CD28TM-41BBCS-CD3zICS and may comprise the amino add sequence as set forth in SEQ ID NO: 952.
  • the CAR of the invention may be described as B735scFvHL- CD28H-CD28TM-DAP10CS-CD3zICS and may comprise the amino add sequence as set forth in SEQ ID NO: 953.
  • the CAR of the invention may be described as B735scFvLH- CD28H-CD28TM-CD28CS-CD3zICS and may comprise the amino add sequence as set forth in SEQ ID NO: 954.
  • the CAR of the invention may be described as B735scFvLH- CD28H-CD28TM-41BBCS-CD3zICS and may comprise the amino add sequence as set forth in SEQ ID NO: 955.
  • the CAR of the invention may be described as B735scFvLH- CD28H-CD28TM-DAP10CS-CD3zICS and may comprise the amino add sequence as set forth in SEQ ID NO: 956.
  • the CAR of the invention may be described as B506scFvHL- CD28H-CD28TM-CD28CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 941.
  • the CAR of the invention may be described as B506scFvHL- CD28H-CD28TM-41BBCS-CD3zlCS and may comprise the amino add sequence as set forth in SEQ ID NO: 942.
  • the CAR of the invention may be described as B506scFvHL- CD28H-CD28TM-DAP10CS-CD3zICS and may comprise the amino add sequence as set forth in SEQ ID NO: 943.
  • the CAR of the Invention may be described as B506scFvLH- CD28H-CD28TM-CD28CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 944.
  • the CAR of the invention may be described as B506scFvLH- CD28H-CD28TM-41BBCS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 945.
  • the CAR of the invention may be described as B506scFvLH- CD28H-CD28TM-DAP10CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 946.
  • the CAR of the invention may be described as B416scFvHL- CD28H-CD28TM-CD28CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 931.
  • the CAR of the Invention may be described as B416scFvHL- CD28H-CD28TM-41BBCS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 932.
  • the CAR of the invention may be described as B416scFvHL- CD28H-CD28TM-DAP10CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 933.
  • the CAR of the invention may be described as B416scFvLH- CD28H-CD28TM-CD28CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 934.
  • the CAR of the invention may be described as B416scFvLH- CD28H-CD28TM-41BBCS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 935.
  • the CAR of the invention may be described as B416scFvLH- CD28H-CD28TM-DAP10CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 936.
  • the CAR of the invention may be described as B380scFvHL- CD28H-CD28TM-CD28CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 921.
  • the CAR of the invention may be described as B380scFvHL- CD28H-CD28TM-41BBCS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 922.
  • the CAR of the invention may be described as B380scFvHL- CD28H-CD28TM-DAP10CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 923.
  • the CAR of the invention may be described as B380scFvLH- CD28H-CD28TM-CD28CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 924.
  • the CAR of the invention may be described as B380scFvLH- CD28H-CD28TM-41BBCS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 925.
  • the CAR of the Invention may be described as B380scFvLH- CD28H-CD28TM-DAP10CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 926.
  • the CAR of the invention may be described as BOOlscFvHL- CD28H-CD28TM-CD28CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 911.
  • the CAR of the invention may be described as BOOlscFvHL- CD28H-CD28TM-41BBCS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 912.
  • the CAR of the invention may be described as BOOlscFvHL- CD28H-CD28TM-DAP10CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 913.
  • the CAR of the Invention may be described as BOOlscFvLH- CD28H-CD28TM-CD28CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 914.
  • the CAR of the invention may be described as BOOlscFvLH- CD28H-CD28TM-41BBCS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 915.
  • the CAR of the invention may be described as BOOlscFvLH- CD28H-CD28TM-DAP10CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 916.
  • the CAR of the invention may be described as hlB2scFvHL- CD28H-CD28TM-CD28CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 351.
  • Such a CAR may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 451 or 51.
  • the CAR of the invention may be described as hlB2scFvHL- CD28H-CD28TM-41BBCS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 352.
  • Such a CAR may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 452 or 52.
  • the CAR of the invention may be described as hlB2scFvHL- CD28H-CD28TM-DAP10CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 353.
  • Such a CAR may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 453 or 53.
  • the CAR of the invention may be described as hlB2scFvLH- CD28H-CD28TM-CD28CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 354.
  • Such a CAR may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 454 or 54.
  • the CAR of the invention may be described as hlB2scFvLH- CD28H-CD28TM-41BBCS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 355. Such a CAR may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 455 or 55.
  • the CAR of the Invention may be described as hlB2scFvLH- CD28H-CD28TM-DAP10CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 356.
  • Such a CAR may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 456 or 56.
  • the CAR of the Invention may be described as hlEBscFvHL- CD28H-CD28TM-CD28CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 361.
  • Such a CAR may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 461 or 61.
  • the CAR of the Invention may be described as hlESscFvHL- CD28H-CD28TM-41BBCS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 362.
  • Such a CAR may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 462 or 62.
  • the CAR of the Invention may be described as hlESscFvHL- CD28H-CD28TM-DAP10CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 363.
  • Such a CAR may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 463 or 63.
  • the CAR of the Invention may be described as hlESscFvLH- CD28H-CD28TM-CD28CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 364.
  • Such a CAR may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 464 or 64.
  • the CAR of the Invention may be described as hlESscFvLH- CD28H-CD28TM-41BBCS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 365.
  • Such a CAR may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 465 or 65.
  • the CAR of the invention may be described as hlEBscFvLH- CD28H-CD28TM-DAP10CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 366.
  • Such a CAR may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 466 or 66.
  • the CAR of the invention may be described as h3H2scFvHL- CD28H-CD28TM-CD28CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 371.
  • Such a CAR may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 471 or 71.
  • the CAR of the invention may be described as h3H2scFvHL- CD28H-CD28TM-41BBCS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 372.
  • Such a CAR may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 472 or 72.
  • the CAR of the invention may be described as h3H2scFvHL- CD28H-CD28TM-DAP10CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 373.
  • Such a CAR may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 473 or 73.
  • the CAR of the invention may be described as h3H2scFvLH- CD28H-CD28TM-CD28CS-CD3zICS and may comprise the amino acid sequence as set forth In SEQ ID NO: 374.
  • Such a CAR may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 474 or 74.
  • the CAR of the invention may be described as h3H2scFvLH- CD28H-CD28TM-41BBCS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 375.
  • Such a CAR may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 475 or 75.
  • the CAR of the invention may be described as h3H2scFvLH- CD28H-CD28TM-DAP10CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 376.
  • Such a CAR may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 476 or 76.
  • FIGS 3A-3C Schematics showing examples of specific CAR constructs of some embodiments are illustrated in FIGS 3A-3C.
  • a leader sequence may be placed upstream of the polynucleotide sequences encoding the foregoing exemplary CARs.
  • the leader sequence facilitates the expression of the CAR on the cell surface.
  • the polynucleotide sequence of such a lead sequence may be as set forth in SEQ ID NO: 260, which encodes the amino acid sequence as set forth In SEQ ID NO: 160. Any other sequences that facilitate the expression of the CAR on the cell surface may be used.
  • FIG IE A general exemplary schematic of a construct for a LS-containing CAR of the present invention is shown in FIG IE.
  • the polynucleotide sequences for expressing the foregoing exemplary CARs further comprise a T2A ribosomal skip sequence (or also referred to as T2A) and/or a sequence encoding truncated CD 19 (or also referred to as trCD19).
  • T2A ribosomal skip sequence
  • trCD19 a sequence encoding truncated CD 19
  • the nucleic acid sequence for T2A may be as provided by SEQ ID NO: 269, which encodes the amino acid sequence provided by SEQ ID NO: 169.
  • trCD19 may have the sequence as provided by SEQ ID NO: 170, which may be encoded by, for example, SEQ ID NO: 270.
  • FIG IF A schematic showing such polynucleotide constructs are shown in FIG IF.
  • the present disclosure encompasses the polynucleotides encoding any of the CARs disclosed herein.
  • Exemplary CAR-encoding nucleic acid sequences include, but are not limited to, SEQ ID NOS: 451, 51, 452, 52, 453, 53, 454, 54, 455, 55, 456, 56, 461, 61, 462, 62, 463, 63, 464, 64, 465, 65, 466, 66, 471, 71, 472, 72, 473, 73, 474, 74, 475, 75, 476, 76.
  • Exemplary constructs for expressing such a CAR may be provided by, for examples, SEQ ID NOS: 671, 271, 674, and 274.
  • the CARs of the present invention may be further modified, engineered, optimized, or appended In order to provide or select for various features. These features may include, but are not limited to, efficacy, persistence, target specificity, reduced immunogenicity, multi-targeting, enhanced immune response, expansion, growth, reduced off-target effect; reduced subject toxicity, improved target cytotoxicity, improved attraction of disease alleviating immune cells, detection, selection, targeting, and the like.
  • the cells may be engineered to express another CAR, or to have a suicide mechanism, and may be modified to remove or modify expression of an endogenous receptor or molecule such as a TCR and/or MHC molecule.
  • the vector or polynucleotide encoding the CAR further encodes other genes.
  • the vector or polynucleotide may be constructed to allow for the co-expression of multiple genes using a multitude of techniques including cotransfection of two or more plasmids, the use of multiple or bidirectional promoters, or the creation of bicistronic or multicistronic vectors.
  • multicistronic vectors may include the encoding of IRES elements or 2A peptides, such as T2A, P2A, E2A, or F2A (for example, see Kim, J.H., et al., "High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice", PLoS One. 2011;6(4)J.
  • the polynucleotide or vector encoding the CAR further encodes trCD19 with the use of a T2A ribosomal skip sequence.
  • the CAR expressing cell may further comprise a disruption to one or more endogenous genes.
  • the endogenous gene encodes TCRa, TCRp, CD 52, glucocorticoid receptor (GR), deoxycytidine kinase (dCK), or an immune checkpoint protein such as, for example, programmed death- 1 (PD-1).
  • the CARs of the present invention and cells expressing these CARs may be further modified to improve efficacy against cells expressing the target molecule.
  • the cells may be cells expressing ALPPL2.
  • the cells expressing ALPPL2 may be cancer cells, vascular cells, or any other target disease-associated cells.
  • the improved efficacy may be measured by increased cytotoxicity against cells expressing the target molecule, for example cytotoxicity against cancer cells.
  • the improved efficacy may also be measured by increased production of cytotoxic mediators such as, but not limited to, IFN y, perforin, and grantyme B.
  • the improved efficacy may be shown by reduction in the signature cytokines of the diseases, or alleviated symptoms of the disease when the CAR expressing cells are administered to a subject
  • Other cytokines that may be reduced include TGF-beta, IL-6, IL-4, IL-10, and/or IL-13
  • the improved efficacy may be shown by ALPPL2 -specific immune cell responses, such as T cell cytotoxicity.
  • improved efficacy may be shown by better tumor cytotoxicity, better infiltration into the tumor, reduction of immunosuppressive mediators, reduction in weight decrease, reduction in ascites, reduction in tumor burden, and/or increased lifespan.
  • gene expression profiles may be also Investigated to evaluate the efficacy of the CAR
  • the CAR expressing cells are further modified to evade or neutralize the activity of immunosuppressive mediators, including, but not limited to prostaglandin E2 (PGE2) and adenosine.
  • this evasion or neutralization is direct
  • this evasion or neutralization is mediated via the inhibition of protein kinase A (PKA) with one or more binding partners, for example ezrin.
  • PKA protein kinase A
  • the CAR-expressing cells further express the peptide "regulatory subunit I anchoring disruptor" (RIAD).
  • RIAD is thought to inhibit the association of protein kinase A (PKA) with ezrin, which thus prevents PKA's inhibition of TCR activation (Newick K et al. Cancer Immunol Res. 2016 Jun;4(6):541-51. doi: 10.1158/2326-6066.CIR-15-0263. Epub 2016 Apr 4).
  • PKA protein kinase A
  • the CAR expressing cells of the Invention may Induce a broad immune response, consistent with epitope spreading.
  • the CAR expressing cells of the invention further comprise a homing mechanism.
  • the cell may transgenically express one or more stimulatory chemokines or cytokines or receptors thereof.
  • the cells are genetically modified to express one or more stimulatory cytokines.
  • one or more homing mechanisms are used to assist the inventive cells to accumulate more effectively to the disease site.
  • the CAR expressing cells are further modified to release inducible cytokines upon CAR activation, e.g., to attractor activate innate immune cells to a targeted cell (so-called fourth generation CARs or TRUCKS).
  • CARs may co-express homing molecules, e.g., CCR4 or CCR2b, to increase trafficking to the disease site.
  • apoptosis using, e.g., a caspase fused to a dimerization domain (see, e.g., Di et al., N Engl. J. Med. 2011 Nov. 3; 365(18):1673- 1683), can be used as a safety switch in the CAR therapy of the instant invention.
  • CAR-expressing cells can also express an inducible Caspase-9 (iCaspase-9) molecule that; upon administration of a dimerizer drug (e.g., rimiducid (also called AP1903 (Bellicum Pharmaceuticals) or AP20187 (Ariad)) leads to activation of the Caspase-9 and apoptosis of the cells.
  • a dimerizer drug e.g., rimiducid (also called AP1903 (Bellicum Pharmaceuticals) or AP20187 (Ariad)
  • iCaspase-9 molecule contains a chemical inducer of dimerization (CID) binding domain that mediates dimerization in the presence of a CID. This results in inducible and selective depletion of CAR-expressing cells.
  • CID chemical inducer of dimerization
  • the iCaspase-9 molecule is encoded by a nucleic acid molecule separate from the CAR-encoding vector(s). In some cases, the iCaspase-9 molecule is encoded by the same nucleic acid molecule as the CAR-encoding vector.
  • the iCaspase-9 can provide a safety switch to avoid any toxicity of CAR-expressing cells. See, e.g., Song etal. Cancer Gene Ther. 2008; 15(10):667-75; Clinical Trial Id. No. NCT02107963; and Di etal. N. Engl. J. Med. 2011; 365:1673-83.
  • Alternative strategies for regulating the CAR therapy of the instant invention include utilizing small molecules or antibodies that deactivate or turn off CAR activity, e.g., by deleting CAR-expressing cells, e.g., by inducing antibody dependent cell-mediated cytotoxicity (ADCC).
  • CAR-expressing cells described herein may also express an antigen that is recognized by molecules capable of inducing cell death, e.g., ADCC or compliment-induced cell death.
  • CAR expressing cells described herein may also express a receptor capable of being targeted by an antibody or antibody fragment
  • receptors include EpCAM, VEGFR, integrins (e.g., integrins avp3, a4, aI3/403, a4p7, a5pi, avp3, av), members of the TNF receptor superfamily (e.g., TRAIL-R1, TRAIL-R2), PDGF Receptor, interferon receptor, folate receptor, GPNMB, ICAM-1, HLA-DR, CEA, CA-125, MUC1, TAG-72, IL-6 receptor, 5T4, GD2, GD3, CD2, CD3, CD4, CDS, CD 11, CDlla/LFA-1, GDIS, CD18/1TGB2, CD19, CD20, CD22, CD23/lgE Receptor, CD25, CD28, CD30, CD33, CD38, CD40, CD41, CD44, CD51, CD52, CD62L,
  • CAR-expressing cells described herein may also express a truncated epidermal growth factor receptor (EGFR) which lacks signaling capacity but retains the epitope that is recognized by molecules capable of inducing ADCC, e.g., cetuximab (ERBITUX®), such that administration of cetuximab induces ADCC and subsequent depletion of the CAR-expressing cells (see, e.g., W02011/056894, and Jonnalagadda et al., Gene Ther. 2013; 20(8)853-860).
  • EGFR epidermal growth factor receptor
  • the CAR cell comprises a polynucleotide encoding a suicide polypeptide, such as for example RQR8. See, e.g., W02013153391A, which is hereby incorporated by reference in its entirety.
  • the suicide polypeptide may be expressed at the surface of a CAR cell.
  • the suicide polypeptide may also comprise a signal peptide at the amino terminus.
  • Another strategy includes expressing a highly compact marker/suicide gene that combines target epitopes from both CD32 and CD20 antigens in the CAR-expressing cells described herein, which binds rituximab, resulting in selective depletion of the CAR-expressing cells, e.g., by ADCC (see, e.g., Philip etal., Blood. 2014; 124(8)1277- 1287).
  • Other methods for depleting CAR-expressing cells described herein include administration of CAM PATH®, a monoclonal anti-CD52 antibody that selectively binds and targets mature lymphocytes, e.g., CAR-expressing cells, for destruction, e.g., by inducing ADCC.
  • the CAR-expressing cell can be selectively targeted using a CAR ligand, e.g., an anti-idiotypic antibody.
  • the anti-idiotypic antibody can cause effector cell activity, e.g., ADCC or ADC activities, thereby reducing the number of CAR-expressing cells.
  • the CAR ligand, e.g., the anti-idiotypic antibody can be coupled to an agent that induces cell killing, e.g., a toxin, thereby reducing the number of CAR-expressing cells.
  • the CAR molecules themselves can be configured such that the activity can be regulated, e.g., turned on and off, as described below.
  • a regulatable CAR where the CAR activity can be controlled is desirable to optimize the safety and efficacy of a CAR therapy.
  • a RCAR comprises a set of polypeptides, typically two in the simplest embodiments, in which the components of a standard CAR described herein, e.g., an antigen-binding domain and an ICS domain, are partitioned on separate polypeptides or members.
  • the set of polypeptides include a dimerization switch that; upon the presence of a dimerization molecule, can couple the polypeptides to one another, e.g., can couple an antigen-binding domain to an ICS domain. Additional description and exemplary configurations of such regulatable CARs are provided herein and in International Publication No. WO 2015/090229, hereby incorporated by reference in its entirety.
  • an RCAR comprises two polypeptides or members: 1) an intracellular signaling member comprising an ICS domain, e.g., a primary ICS domain described herein, and a first switch domain; 2) an antigen binding member comprising an antigen-binding domain, e.g., that specifically binds a target molecule described herein, as described herein and a second switch domain.
  • the RCAR comprises a TM domain described herein.
  • a TM domain can be disposed on the intracellular signaling member, on the antigen binding member, or on both. Unless otherwise indicated, when members or elements of an RCAR are described herein, the order can be as provided, but other orders are included as well.
  • the order is as set out in the text; but in other embodiments, the order can be different E.g., the order of elements on one side of a transmembrane region can be different from the example, e.g., the placement of a switch domain relative to an ICS domain can be different e.g., reversed.
  • the CAR expressing immune cell may only transiently express a CAR.
  • the cells of the invention may be transduced with mRNA comprising a nucleic acid sequence encoding an inventive CAR
  • the present invention also includes an RNA construct that can be directly transfected into a cell.
  • a method for generating mRNA for use in transfection involves in vitro transcription (IVT) of a template with specially designed primers, followed by polyA addition, to produce a construct containing 3' and 5' untranslated sequences ("UTRs"), a 5' cap and/or Internal Ribosome Entry Site (IRES), the nucleic acid to be expressed, and a polyA tail, typically 50-2000 bases in length.
  • RNA so produced can efficiently transfect different kinds of cells.
  • the template includes sequences for the CAR
  • an RNA CAR vector is transduced into a cell by electroporation.
  • the CAR expressing cells of the present invention may further comprise one or more CARs, in addition to the first CAR. These additional CARs may or may not be specific for the target molecule of the first CAR. In some embodiments, the one or more additional CARs may act as inhibitory or activating CARs. In some embodiments, the CAR of some embodiments Is the stimulatory or activating CAR; In other embodiments, It Is the costimulatory CAR. In some embodiments, the cells further include inhibitory CARs (iCARs, see Fedorov et al., Set Transl.
  • the antigen-binding domain of the CAR is or is part of an immunoconjugate, in which the antigen-binding domain is conjugated to one or more heterologous molecule(s), such as, but not limited to, a cytotoxic agent; an Imaging agent; a detectable moiety, a multimerization domain, or other heterologous molecule.
  • heterologous molecule(s) such as, but not limited to, a cytotoxic agent; an Imaging agent; a detectable moiety, a multimerization domain, or other heterologous molecule.
  • Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At211, 1131, 1125, Y90, Rel86, Rel88, Sml53, Bi212, P32, Pb212 and radioactive isotopes of Lu); chemotherapeutic agents; growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins.
  • radioactive isotopes e.g., At211, 1131, 1125, Y90, Rel86, Rel88, Sml53, Bi212, P32, Pb212 and radioactive isotopes of Lu
  • chemotherapeutic agents e.g., At211, 1131, 1125, Y90, Rel86, Rel88, Sml53, Bi212, P32, Pb212 and radioactive isotopes of Lu
  • growth inhibitory agents e.g., enzymes and
  • the antigen-binding domain is conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant; or animal origin, or fragments thereof), or radioactive isotopes.
  • cytotoxic agents such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant; or animal origin, or fragments thereof), or radioactive isotopes.
  • the cells of the invention may be further modified to overexpress pro-survival signals, reverse anti-survival signals, overexpress Bcl-xL, overexpress hTERT, lack Fas, or express a TGF-0 dominant negative receptor. Persistence may also be facilitated by the administration of cytokines, e.g., IL-2, IL-7, and IL-15.
  • cytokines e.g., IL-2, IL-7, and IL-15.
  • the present invention also provides vectors in which a polynucleotide encoding an anti-ALPPL2 agent of the present invention is inserted.
  • the vector may be, for example, a DNA vector or a RNA vector.
  • the vector may be, for example, but not limited to, a plasmid, a cosmid, a viral replicon, or a viral vector.
  • the viral vector may be a vector of a DNA virus, which may be an adenovirus, or an RNA virus, which may be a retrovirus.
  • Types of vectors suite for Abs, antigen-binding Ab fragments, and/or CARs are well known in the art (for example, see Rita Costa A. etal., EurJPharm Biopharm. 2010 Feb;74(2):127-38. doi: 10.1016/j.ejpb.2009.10.002. Epub 2009 Oct22; Frenzel A. etal. Front Immunol. 2013; 4: 217. Published online 2013 Jul 29. doi: 10.3389/fimmu.2013.00217).
  • insect-specific viruses When the host cells are insect cells, such as for producing Abs or antigen-binding Ab fragments, insect-specific viruses may be used.
  • insect-specific viruses include, but are not limited to, the family of Baculoviridae, particularly the Autographa califomica nuclear polyhedrosis virus(AcNPV).
  • AcNPV Autographa califomica nuclear polyhedrosis virus
  • plant-specific viruses and bacteria such as Agrobacterium tumefaciens, may be used.
  • retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells.
  • Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity. This would be particularly beneficial for expressing CAR constructs.
  • nucleic acids encoding anti-ALPPL2 agents is typically achieved by operably linking a nucleic acid encoding the anti-ALPPL2 agent polypeptide or portions thereof to a promoter, and incorporating the construct into an expression vector.
  • the vectors can be suitable for replication and integration eukaryotes, "typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired polynucleotide.
  • the expression constructs of the present invention may also be used for nucleic acid immunization and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art See, e.g., U.S. Pat Nos. 5,399,346, 5,580,859, 5,589,466, incorporated by reference herein in their entireties.
  • the invention provides a gene therapy vector.
  • the nucleic acid can be cloned into a number of types of vectors.
  • the nucleic add can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid.
  • Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequendng vectors.
  • the expression vector may be provided to a cell in the form of a viral vector.
  • Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals.
  • Viruses, which are useful as vectors include, but are not limited to, retroviruses, y-retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat No. 6,326,193).
  • retroviruses provide a convenient platform for gene delivery systems.
  • a selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo.
  • retroviral systems are known in the art In some embodiments, adenovirus vectors are used.
  • adenovirus vectors are known in the art In one embodiment lentivirus vectors are used.
  • Additional promoter elements e.g., enhancers, regulate the frequency of transcriptional initiation.
  • promoters typically contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another.
  • tk thymidine kinase
  • the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.
  • individual elements can function either cooperatively or Independently to activate transcription.
  • Various promoter sequences may be used, including, but not limited to the immediate early cytomegalovirus (CMV) promoter, the CMV-actin-globin hybrid (GAG) promotor, Elongation Growth Factor-la (EF-la), simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter.
  • CMV immediate early cytomegalovirus
  • GAG CMV-actin-globin hybrid
  • EF-la Elongation Growth Factor-la
  • SV40 simian virus 40
  • MMTV
  • inducible promoters are also contemplated as part of the invention.
  • the use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired.
  • inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.
  • the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors.
  • the selectable marker may be carried on a separate piece of DNA and used in a co- transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells.
  • Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.
  • the selectable marker gene comprises a polynucleotide encoding truncated CD19 (trCD19).
  • trCD19 truncated CD19
  • the expression of the marker may be determined via any available technique including, but not limited to, flow cytometry or immunofluorescence assays. Expression of such a marker typically indicates successful introduction and expression of the transgene(s) introduced together with the marker gene. Therefore, cells expressing the anti-ALPPL2 agent of the invention may be, for example, selected based on the expression of the marker.
  • Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences.
  • a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.
  • Suitable reporter genes may include genes encoding luciferase, ⁇ -galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82).
  • Suitable expression systems are well known and may be prepared using known techniques or obtained commercially.
  • the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter.
  • Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.
  • the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast or insect cell by any method in the art
  • the expression vector can be transferred into a host cell by physical, chemical, or biological means.
  • Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art See, for example, Sambrook etal. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.
  • Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors.
  • Viral vectors, and especially retroviral vectors have become the most widely used method for inserting genes into mammalian, e.g., human cells.
  • Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat Nos. 5,350,674 and 5,585,362.
  • Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • An exemplary colloidal system for use as a delivery vehicle tn vitro and tn vivo is a liposome (e.g., an artificial membrane vesicle).
  • an exemplary delivery vehicle is a liposome.
  • the nucleic acid may be associated with a lipid.
  • the nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid.
  • Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a "collapsed" structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape.
  • Lipids are fatty substances which may be naturally occurring or synthetic lipids.
  • lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
  • Lipids suitable for use can be obtained from commercial sources.
  • DMPC dimyristyl phosphatidylcholine
  • DCP dicetyl phosphate
  • Choi cholesterol
  • DMPG dimyristyl phosphatidylglycerol
  • Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20 degrees Celsius.
  • Liposome is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution.
  • compositions that have different structures in solution than the normal vesicular structure are also encompassed.
  • the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules.
  • lipofectamine- nudeic add complexes are also contemplated.
  • assays include, for example, "molecular biological” assays well known to those of skill in the art; such as Southern and Northern blotting, RT-PCR and PCR; "biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.
  • molecular biological assays well known to those of skill in the art
  • biochemical assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.
  • cells, cell populations, and compositions containing the cells e.g., cells comprising a polynucleotide encoding an anti-ALPPL2 agent of the present invention.
  • Cells expressing anti-ALPPL2 Abs or antigen-binding Ab fragments may be used to harvest the Abs or antigen-binding Ab fragments or may be administered to a subject/patient
  • Cells expressing anti-ALPPL2 CARs may be administered to a subject or may be incorporated in a composition to be administered to a subject
  • compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy.
  • cells may be: [i] prokaryotic cells, such as gram-negative bacteria and gram-positive bacteria; or (ii) eukaryotic cells, such as yeast, filamentous fungi, protozoa, insect cells, plant cells, and mammalian cells (reviewed in Frenzel A etal. Front Immunol. 2013; 4: 217. Published online 2013 Jul 29. doi: 10.3389/fimmu.2013.00217).
  • prokaryotic cells such as gram-negative bacteria and gram-positive bacteria
  • eukaryotic cells such as yeast, filamentous fungi, protozoa, insect cells, plant cells, and mammalian cells (reviewed in Frenzel A etal. Front Immunol. 2013; 4: 217. Published online 2013 Jul 29. doi: 10.3389/fimmu.2013.00217).
  • CHO cells or HEK cells may be used.
  • Specific examples of gram-negative bacteria that are suited for production of Ab or antigen-binding Ab fragments include, but are not limited to, E coli, Proteus mirabilis, and Pseudomonas putidas.
  • Specific examples of gram-positive bacteria include, but are not limited to, Bacillus brevis, Bacillus subtilts, Bacillus megatertum, Lactobactlluszeae casei, and Lactobacillus paracasei.
  • yeast bacteria that are suited for production of Ab or antigen-binding Ab fragments include, but are not limited to, Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Schizosaccharomyces pombe, Schwanntomyces occidentals, Kluyveromyces lactis, and Yarrowia Itpolytica.
  • filamentous fungi that are suited for production of Ab or antigen-binding Ab fragments include, but are not limited to, the genera Trichoderma and Aspergillus, A niger (subgenus A awamori), Aspergillus oryzae, and Chrysosporium lucknowense.
  • protozoa that are suited for production of Ab or antigen-binding Ab fragments include, but are not limited to, Leishmania tarentolae.
  • insect cells that are suited for production of Ab or antigen-binding Ab fragments include, but are not limited to, insect cell lines like Sf-9 and Sf-21 of Spodoptera Jrugiperda, DS2 cells of Drosophila melanogaster, High Five cells (BTI-TN-5B1-4) of Trtchopulsta nt, or Schneider2 (S2) cells of D. melanogaster. They can be efficiently transfected with insectspecific viruses from the family of Baculoviridae, particularly the Autographa califomica nuclear polyhedrosis virus (AcNPV).
  • AcNPV Autographa califomica nuclear polyhedrosis virus
  • mammalian cells that are suited for production of Ab or antigen-binding Ab fragments include, but are not limited to, Chinese hamster ovary (CHO) cells, the human embryonic retinal cell line Per.C6 [Crucell, Leiden, Netherlands], CHO-derived cell lines such as K1-, DukXBll-, Lecl3, and DG44- cell lines, mouse myeloma cells such as SP 2/0, YB 2/0, and NSO cells, GS-NSO, hybridoma cells, baby hamster kidney (BHK) cells, and the human embryonic kidney cell line HEK293, HEK293T, HEK293E, and human neuronal precursor cell line AGE1.HN (Probiogen, Berlin, Germany).
  • CHO Chinese hamster ovary
  • Per.C6 the human embryonic retinal cell line Per.C6 [Crucell, Leiden, Netherlands]
  • CHO-derived cell lines such as K1-, DukXBll
  • transgenic plants and transgenic animals may be used.
  • Exemplary plants that may be used include, but are not limited to, tabacco, maize, duckweed, Chlamydomonas reinhardtii, Nicotiana tabacum, Nicotianaben thamiana, and Nicotiana benthamiana.
  • Exemplary animals that may be used include, but are not limited to mouse, rat, and chicken.
  • the cells For expressing an anti-ALPPL2 CAR, the cells generally are eukaryotic cells, such as mammalian cells, and typically are human cells, more typically primary human cells, e.g., allogeneic or autologous donor cells.
  • the cells for introduction of the CAR may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject
  • the subject from which the cell is Isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered.
  • the subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered.
  • the cells are derived from the blood, bone marrow, lymph, or lymphoid organs, are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid cells, including monocytes, macrophages, dendritic cells, neutrophils, eosinophils, basophils, or mast cells, or lymphoid cells, including lymphocytes, typically T cells and/or NK cells.
  • Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs).
  • the cells typically are primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen.
  • the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen-specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation.
  • T cells or other cell types such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen-specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation.
  • an immortalized cell or a cell line may be used for expressing a CAR of the present disclosure.
  • Such examples include, but are not limited to, a T cell line, a CD4+ T cell line, a CD8+ T cell line, a regulatory T cell line, an NK-T cell line, an NK cell line (e.g., NK-92), a monocyte line, a macrophage line, a dendritic cell line, and a mast cell line.
  • a desired cell type for CAR expression for example T cells or NK cells may be generated from a stem cell, such as an embryonic stem cell, iPSCs, or hematopoietic stem cell.
  • the cells may be allogeneic and/or autologous.
  • the methods include off-the- shelf methods.
  • the cells are pluripotent and/or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs).
  • the methods include isolating cells from the subject; preparing, processing, culturing, and/or engineering them, as described herein, and reintroducing them into the same patient; before or after cryopreservation.
  • the cells are T cells.
  • T cells naive T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, a/p T cells, and 5/y T cells.
  • TN naive T
  • TSCM stem cell memory T
  • TCM central memory T
  • TEM effector memory T
  • TIL tumor-infiltrating lymphocytes
  • immature T cells immature T cells
  • the cells are natural killer (NK) cells, Natural Killer T (NKT) cells, cytokine-induced killer (CIK) cells, tumor-infiltrating lymphocytes (TIL), lymphokme-activated killer (LAK) cells, or the like.
  • the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils.
  • CAR-expressing phagocytic cells expressing may be able to bind to and phagocytose or nibble target cells (Morrissey M.A et al., Elife. 2018 Jun 4;7. pii: e36688. doi: 10.7554/eLife.36688).
  • the cells are derived from cell lines, e.g., T cell lines.
  • the cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat; non-human primate, and pig.
  • a source of cells can be obtained from a subject through a variety of nonlimiting methods.
  • Cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and disease sites such as tumors.
  • any number of T cell lines available and known to those skilled in the art may be used.
  • cells can be derived from a healthy donor, from a patient diagnosed with cancer or from a patient diagnosed with an infection.
  • cells can be part of a mixed population of cells which present different phenotypic characteristics.
  • the cells are primary cells, e.g., primary human cells.
  • the samples include tissue, fluid, and other samples taken directly from the subject; as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g. transduction with viral vector), washing, and/or incubation.
  • the biological sample can be a sample obtained directly from a biological source or a sample that is processed.
  • Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat; tissue and organ samples, including processed samples derived therefrom.
  • the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from a leukapheresis product
  • Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, tongue, or other organ, and/or cells derived therefrom.
  • Samples include, In the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.
  • cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis.
  • the samples contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets, and in some embodiments contains cells other than red blood cells and platelets.
  • an isolated cell according to the invention comprises a polynucleotide encoding a CAR.
  • isolation of the cells includes one or more preparation and/or non-affinity based cell separation steps.
  • cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents.
  • cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and/or resistance to particular components.
  • the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps.
  • the cells are washed with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the wash solution lacks calcium and/or magnesium and/or many or all divalent cations.
  • a washing step is accomplished a semi-automated "flow-through* centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer's Instructions.
  • a washing step Is accomplished by tangential flow filtration (TFF) according tx> the manufacturer's instructions.
  • the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca++/Mg++ free PBS.
  • components of a blood cell sample are removed and the cells directly resuspended in culture media.
  • the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. This would be particularly useful for Isolating CAR-expresslng cells.
  • the surface maker is trCD19.
  • any known method for separation based on such markers may be used.
  • the separation is affinity- or immunoaffinity-based separation.
  • the isolation in some embodiments includes separation of cells and cell populations based on the cells' expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.
  • markers typically cell surface markers
  • Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some embodiments, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.
  • multiple rounds of separation steps are carried out; where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection.
  • a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection.
  • multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.
  • T cells such as cells positive or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45R0+ T cells, are isolated by positive or negative selection techniques.
  • CD 3+ T cells can be positively selected using CD3 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander).
  • isolation is carried out by enrichment for a particular cell population by positive selection, or depletion of a particular cell population, by negative selection.
  • positive or negative selection is accomplished by Incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker+) at a relatively higher level (marker high) on the positively or negatively selected cells, respectively.
  • T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14.
  • a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells.
  • Such CD4+ and CD8+ populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and/or effector T cell subpopulations.
  • CD8+ cells are further enriched for or depleted of naive, central memory, effector memory, and/or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation.
  • enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve long-term survival, expansion, and/or engraftment following administration, which in some embodiments is particularly robust in such sub-populations. See Terakura et al. (2012) Blood. 1:72-82; Wang etal. (2012) J Immunother. 35(9):689-701.
  • combining TCM-enriched CD8+ T cells and CD4+ T cells further enhances efficacy.
  • memory T cells are presentin both CD62L+ and CD62L-subsets of CD8+ peripheral blood lymphocytes.
  • PBMC can be enriched for or depleted of CD62L-CD8+ and/or CD62L+CD8 fractions, such as using anti-CD8 and anti-CD62L antibodies.
  • the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and/or CD127; in some embodiments, it is based on negative selection for cells expressing or highly expressing CD45RA and/or granzyme B.
  • isolation of a CD8+ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment for cells expressing CD62L
  • enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD 14 and CD45RA, and a positive selection based on CD62L
  • TCM central memory T
  • the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation also is used to generate the CD4+ cell population or sub-population, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps.
  • the sample or composition of cells to be separated is incubated with small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as Dynalbeads or MACS beads).
  • the magnetically responsive material e.g., particle
  • a binding partner e.g., an antibody
  • binds to a molecule e.g., surface marker
  • the magnetic particle or bead comprises a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner.
  • a specific binding member such as an antibody or other binding partner.
  • Suitable magnetic particles include those described in Molday, U.S. Pat No. 4,452,773, and in European Patent Specification EP 452342 B, which are hereby incorporated by reference.
  • Colloidal sized particles such as those described in Owen U.S. Pat No. 4,795,698, and Liberti et al., U.S. Pat No. 5,200,084 are other examples.
  • the Incubation generally is carried out under conditions whereby the antibodies or binding partners, or molecules, such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead, specifically bind to cell surface molecules if present on cells within the sample.
  • the antibodies or binding partners, or molecules such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead, specifically bind to cell surface molecules if present on cells within the sample.
  • the sample is placed in a magnetic field, and those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from the unlabeled cells.
  • those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from the unlabeled cells.
  • positive selection cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained.
  • a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subject to further separation steps.
  • the magnetically responsive particles are coated in primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin.
  • the magnetic particles are attached to cells via a coating of primary antibodies specific for one or more markers.
  • the cells, rather than the beads are labeled with a primary antibody or binding partner, and then cell-type specific secondary antibody- or other binding partner (e.g., streptavidinj-coated magnetic particles, are added.
  • streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.
  • the magnetically responsive particles are left attached to the cells that are to be subsequently incubated, cultured and/or engineered; in some embodiments, the particles are left attached to the cells for administration to a patient
  • the magnetizable or magnetically responsive particles are removed from the cells.
  • Methods for removing magnetizable particles from cells are known and include, e.g., the use of competing non-labeled antibodies, magnetizable particles or antibodies conjugated to cleavable linkers, eta
  • the magnetizable particles are biodegradable.
  • die Isolation or separation Is carried out using a system, device, or apparatus that carries out one or more of the isolation, cell preparation, separation, processing, incubation, culture, and/or formulation steps of the methods.
  • the system is used to carry out each of these steps in a closed or sterile environment; for example, to minimize error, user handling and/or contamination.
  • the system is a system as described in International Patent Application, Publication Number W02009/072003, or US 20110003380 Al.
  • the system or apparatus carries out one or more, e.g., all, of the Isolation, processing, engineering, and formulation steps in an integrated or self- contained system, and/or in an automated or programmable fashion.
  • die system or apparatus includes a computer and/or computer program in communication with the system or apparatus, which allows a user to program, control, assess the outcome of, and/or adjust various embodiments of the processing, isolation, engineering, and formulation steps.
  • a cell population described herein is collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluidic stream.
  • a cell population described herein is collected and enriched (or depleted) via preparative scale (FACS)- sorting.
  • FACS preparative scale
  • a cell population described herein is collected and enriched (or depleted) by use of microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, e.g., WO 2010/033140, Cho etal. (2010) Lab Chip 10, 1567-1573; and Godin etal. (2008) J Biophoton. l(5):355-376. In both cases, cells can be labeled with multiple markers, allowing for the isolation of well- defined T cell subsets at high purity.
  • MEMS microelectromechanical systems
  • the antibodies or binding partners are labeled with one or more detectable marker, to facilitate separation for positive and/or negative selection.
  • separation may be based on binding to fluorescently labeled antibodies.
  • separation of cells based on binding of antibodies or other binding partners specific for one or more cell surface markers are carried in a fluidic stream, such as by fluorescence-activated cell sorting (FACS), including preparative scale (FACS) and/or microelectromechanical systems (MEMS) chips, e.g., in combination with a flow-cytometric detection system.
  • FACS fluorescence-activated cell sorting
  • MEMS microelectromechanical systems
  • the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient
  • the separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker.
  • positive selection of or enrichment for cells of a particular type refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker.
  • negative selection, removal, or depletion of cells of a particular type refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.
  • the provided methods include cultivation, Incubation, culture, and/or genetic engineering steps.
  • the cell populations are incubated in a cultureinitiating composition.
  • the incubation and/or engineering may be carried out in a culture vessel, such as a unit, chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, or other container for culture or cultivating cells.
  • the cells are incubated and/or cultured prior to or in connection with genetic engineering.
  • the incubation steps can include culture, cultivation, stimulation, activation, and/or propagation.
  • compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent
  • stimulating conditions include those designed to induce proliferation, expansion, activation, and/or survival of cells in the population, to mimic antigen exposure, and/or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor.
  • the cells of the invention can be activated and expanded, either prior to or after genetic modification of the cells, using methods as generally described, for example without limitation, in U.S. Pat Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681;
  • the conditions can include one or more of particular media, temperature, oxygen content carbon dioxide content time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.
  • agents e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.
  • T cells can be expanded in vitro or in vivo.
  • the T cells of the invention can be expanded, for example, by contact with an agent that stimulates a CD 3 TCR complex and a co-stimulatory molecule on the surface of the T cells to create an activation signal for the T cell.
  • an agent that stimulates a CD 3 TCR complex and a co-stimulatory molecule on the surface of the T cells to create an activation signal for the T cell.
  • chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitogenic lectins like phytohemagglutinin (PHA) can be used to create an activation signal for the T cell.
  • T cell populations may be stimulated in vitro by contact with, for example, an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti- CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore.
  • the T cell populations may be stimulated tn vitro by contact with Muromonab-CD3 (OKT3).
  • a ligand that binds the accessory molecule is used for co-stimulation of an accessory molecule on the surface of the T cells.
  • a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells.
  • Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640® or, X-vivo 5®, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-y, IL-4, IL-7, GM-CSF, IL-10, IL-2, IL-15, IL-21, TGF-p, and TNF, or any other additives for the growth of cells known to the skilled artisan.
  • serum e.g., fetal bovine or human serum
  • interleukin-2 IL-2
  • insulin IFN-y
  • IL-4 interleukin-7
  • GM-CSF GM-CSF
  • IL-10 interleukin-2
  • IL-15
  • T cells are stimulated in vitro by exposure to 0KT3 and IL-2.
  • Other additives for the growth of cells include, but are not limited to, surfactant, Plasmanate, and reducing agents such as N-acetyl-cysteine and 2 -mercaptoethanol.
  • Media can include RPMI 1640®, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vlvo 1®, and X-Vlvo 20®, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of T cells.
  • Antibiotics e.g., penicillin and streptomycin
  • the target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° Celsius) and atmosphere (e.g., air plus 5% C02). T cells that have been exposed to varied stimulation times may exhibit different characteristics.
  • the isolated cells of the invention can be expanded by coculturing with tissue or cells.
  • the cells can also be expanded in vivo, for example in the subjects blood after administrating the cell into the subject
  • At least one cell of the invention when cells are expanded in vivo, at least one cell of the invention may be administered to a subject and the administration may lead to an expansion of the cell in the subject resulting in a population of cells.
  • a polynucleotide or vector of the invention may be administered to a subject Once the polynucleotide or vector is taken up by a cell within the subject and the cell proliferate or expand in the subject this may result in a population of cells of the invention within the subject
  • the resulting population of cells persists in the subject for at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, at least twelve months, at least eighteen months, at least two years, or at least three years after administration.
  • the T cells are expanded by adding to the culture- initiating composition feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMC), (e.g., such that the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture (e.g. for a time sufficient to expand the numbers of T cells).
  • the non-dividing feeder cells can comprise y- irradiated PBMC feeder cells.
  • the PBMC are irradiated with y rays in the range of about 3000 to 3600 rads to prevent cell division.
  • the feeder cells are added to culture medium prior to the addition of the populations of T cells.
  • the preparation methods include steps for freezing, e.g., cryopreserving, the cells, either before or after isolation, incubation, and/or engineering.
  • the freeze and subsequent thaw step removes granulocytes and, to some extent; monocytes in the cell population.
  • the cells are suspended in a freezing solution, e.g., following a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters in some embodiments may be used.
  • a freezing solution e.g., following a washing step to remove plasma and platelets.
  • Any of a variety of known freezing solutions and parameters in some embodiments may be used.
  • PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. This is then diluted 1:1 with media so that the final concentration of DMSO and HSA are 10% and 4%, respectively.
  • the cells are then frozen to -80° Celsius at a rate of 1 degree per minute and stored in the vapor phase of a liquid nitrogen storage tank
  • Cells such as hybridomas or other recombinant cells, that are producing Abs or antigen-binding Ab fragments of the present invention may be grown using standard methods, in suitable culture medium for this purpose (such as D-MEM or RPMI-1640), or in vivo as ascites. Abs or antigen-binding Ab fragments expressed and/or secreted by the cells can be separated from the cells, culture medium, ascites fluid, or serum using conventional immunoglobulin purification procedures, such as, but not limited to, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography (Ma H. et al., Methods. 2017 Mar l;116:23-33.
  • suitable culture medium for this purpose such as D-MEM or RPMI-1640
  • Abs or antigen-binding Ab fragments expressed and/or secreted by the cells can be separated from the cells, culture medium, ascites fluid, or serum using conventional immunoglobulin purification procedures,
  • Anti-ALPPL2 agents of the present Invention (Abs, antigen-binding Ab fragments, multi-specific Abs, multi-specific antigen-binding Ab fragments, ADCs, or CARs that binds to ALPPL2), nucleic acids encoding such an agent, vectors encoding such an agent, isolated cells obtained by the methods described above, or cell lines derived from such Isolated cells, and/or pharmaceutical compositions comprising thereof can be used as a medicament in the treatment of a disease, disorder, or condition in a subject In some embodiments, such a medicament can be used for treating an ALPPL2-associated disease or condition.
  • the ALPPL2 -associated condition may be, for example, but not limited to, cancer and cancer-associated diseases and conditions.
  • the anti-ALPPL2 agents of the present invention may be used to treat a cancer.
  • ALPPL2 is upregulated in a variety of cancers, such as, but not limited to pancreatic cancer, testicular cancer, cervical cancer, endometrial cancer, ovarian cancer, stomach cancer, colorectal cancer, lung cancer, mesothelioma, and tongue cancer (https://www.protematias.org/ENSG00000163286-ALPPL2/pathology: Skinner, J.M., Whitebead, R. Virchows Archiv [Pathol Anat] 394, 109-118 (1981).; Wick M. R.
  • preferred target diseases include these cancers.
  • pancreatic cancer is a preferred target disease.
  • mesothelioma is a preferred target disease.
  • the anti-ALPPL2 agents according to the present invention may also be used to treat any other cancers in which ALPPL2 is upregulated or has a pathological role.
  • the subject referred to herein may be any living subject In a preferred embodiment; the subject is a mammal.
  • the mammal referred to herein can be any mammal.
  • the term "mammal” refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Lagomorpha, such as rabbits.
  • the mammals may be from the order Carnivora, including Felines (cats) and Canines (dogs).
  • the mammals may be from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses).
  • the mammals may be of the order Primates, Cebolds, or Simoids (monkeys) or of the order Anthropoids (humans and apes).
  • the subject; to whom the Abs, antigen-binding Ab fragments, ADCs, CAR expressing cells, cells, cell populations, or compositions are administered is a primate, such as a human.
  • the primate is a monkey or an ape.
  • the subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects.
  • the patient or subject is a validated animal model for disease, adoptive cell therapy, and/or for assessing toxic outcomes such as cytokine release syndrome (CRS).
  • CRS cytokine release syndrome
  • the subject has persistent or relapsed disease, e.g., following treatment with another immunotherapy and/or other therapy.
  • the administration effectively treats the subject despite the subject having become resistant to another therapy.
  • the subject has not relapsed but is determined to be at risk for relapse, such as at a high risk of relapse, and thus the compound or composition is administered prophylactically, e.g., to reduce the likelihood of or prevent relapse.
  • the methods include administration of an Ab, Ab fragments, ADC, or CAR expressing cell or a composition containing such an anti-ALPPL2 agent to a subject, tissue, or cell, such as one having, at risk for, or suspected of having a disease, condition or disorder associated with ALPPL2, such as cancer.
  • the anti-ALPPL2 agents and/or compositions are administered to a subject having the particular disease or condition to be treated, e.g., via adoptive cell therapy, such as adoptive T cell therapy.
  • the anti-ALPPL2 agents or compositions are administered to the subject, such as a subject having or at risk for the disease or condition.
  • the methods thereby treat; e.g., ameliorate one or more symptom of the disease or condition, for example, by reducing, inhibiting, or inactivating ALPPL2 and/or ALPPL2 -expressing cells.
  • the cells can be cells that are xenogeneic, allogeneic or autologous to the subject
  • the cell therapy e.g., adoptive cell therapy, e.g., adoptive T cell therapy
  • the cells are isolated and/or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject
  • the cells are derived from a subject; e.g., patient; in need of a treatment and the cells, following isolation and processing are administered to the same subject
  • the cell therapy e.g., adoptive cell therapy, e.g., adoptive T cell therapy
  • the cells are isolated and/or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject
  • the cells then are administered to a different subject e.g., a second subject of the same species.
  • the first and second subjects are genetically identical.
  • the first and second subjects are genetically similar.
  • the second subject expresses the same HLA class or supertype as the first subject
  • TCRs endogenous T cell receptors
  • the TCR expression may be suppressed via any appropriate technique, for example, by silencing any compartment of the endogenous TCRs using tools such as, but not limited to, an siRNA, shRNA, micro RNA, or artificial microRNA.
  • TCR gene may be disrupted or deleted via any appropriate technique, for example using the CRISPR/Cas system, transcription activator-like effector nucleases (e.g., TALENO), or Zinc finger nucleases (ZFNs).
  • TALENO transcription activator-like effector nucleases
  • ZFNs Zinc finger nucleases
  • the suppression or disruption of TCR may allow for reduction or prevention of undesired effects in which the TCRs recognize antigens in the subject as foreign and cause immune responses against the subject; an immune attack often called as graft-versus-host disease (GVHD).
  • GVHD graft-versus-host disease
  • the expression of endogenous MHC or HLA gene(s) may be suppressed or disrupted, which may be achieved via any appropriate technique, such as but not limited to, an siRNA, shRNA, micro RNA, artificial microRNA, or gene editing using the CRISPR/Cas system, transcription activator-like effector nucleases (e.g., TALEN®), or Zinc finger nucleases (ZFNs).
  • siRNA siRNA
  • shRNA RNA
  • micro RNA RNA
  • artificial microRNA or gene editing using the CRISPR/Cas system
  • transcription activator-like effector nucleases e.g., TALEN®
  • ZFNs Zinc finger nucleases
  • the suppression or disruption of MHC or HLA gene(s) may allow for reduction or prevention of undesired effects in which the subject's endogenous T cells recognize the donor cell's antigens presented on donor cell's MHC molecules as foreign and cause immune responses against the donor cells and increase the persistence of the administered cells within the subject Cells expressing anti-ALPPL2 Abs or antigen-binding Ab fragments or a composition comprising such may also be administered to a subject.
  • B cells or plasma cells expressing anti- ALPPL2 Abs or antigen-binding Ab fragments may be adoptively transferred.
  • the present invention includes antibody therapy, wherein (i) an isolated anti-ALPPL2 Ab or Ab fragment may be administered to a subject; (ii) a polynucleotide or combination of polynucleotides encoding an ALPPL2 Ab or Ab fragment or a vector or combination of vectors encoding an ALPPL2 Ab or Ab fragment may be administered to a subject to allow for expression of the encoded Ab or Ab fragment within the subject, (iii) a cell engineered to express/secrete an ALPPL2 Ab or Ab fragment may be administered to a subject
  • such an Ab or Ab fragment may bind to ALPPL2- expressing cells.
  • binding to ALPPL2 -expressing cells may cause a downstream effect; such as complement activation, ADCC, or ADCP.
  • binding to ALPPL2 -expressing cells may inhibit ALPPL2's binding to another molecule or inhibit ALPPL2 function.
  • binding to ALPPL2-expressing cells may stimulate or augment signaling and/or functions of ALPPL2.
  • binding to ALPPL2 -expressing cells may assist binding of the ALPPL2-expressing cells to another molecule or cell.
  • the present invention includes a type of cellular therapy where isolated cells are genetically modified to express a CAR against ALPPL2, and the CAR cell is infused into a subject in need thereof.
  • Such administration can promote activation of the cells (e.g., T cell activation) in a target molecule specific manner, such that the cells of the disease or disorder are targeted for destruction.
  • the cell is a T cell
  • cells, such as CAR T cells are able to replicate in vivo resulting in longterm persistence that may lead to sustained control of diseases, disorders, or conditions associated with ALPPL2, such as cancer.
  • the Isolated cells of the Invention can undergo tn vivo expansion and can persist for an extended amount of time.
  • the isolated T cells of the invention evolve into specific memory T cells that can be reactivated to inhibit growth of any additional target molecule expressing cells.
  • T cells may differentiate tn vtvo into a central memory-like state upon encounter and subsequent elimination of target cells expressing the surrogate antigen.
  • the isolated B cells may evolve into memory B cells that can be reactivated to inhibit the growth of any additional target molecule expressing cells.
  • the immune response elicited by the isolated anti-ALPPL2 agent-modified immune cells may be an active or a passive immune response.
  • the anti-ALPPL2 agent-mediated immune response may be part of an adoptive immunotherapy approach in which anti-ALPPL2 agent-modified immune cells induce an immune response specific to the antigenbinding domain of the anti-ALPPL2 agent
  • anti-ALPPL2 agent-expressing cells are modified in any number of ways, such that their therapeutic or prophylactic efficacy is Increased.
  • the anti-ALPPL2 agent may be conjugated either directly or indirectly through a linker to a targeting moiety.
  • the practice of conjugating compounds, e.g., the CAR, to targeting moieties is known in the art See, for instance, Wadwa et al.,/. Drug Targeting 3: 111 (1995), and U.S. Pat No. 5,087,616.
  • the biological activity of the engineered cell populations and/or antibodies in some embodiments is measured by any of a number of known methods.
  • Parameters to assess include specific binding of an engineered or natural T cell or other immune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry.
  • the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art; such as cytotoxicity assays described in, for example, Kochenderfer et al.,/. Immunotherapy, 32(7): 689-702 (2009), and Herman et al./.
  • the biological activity of the cells also can be measured by assaying expression and/or secretion of certain mediators, such as GM-CSF, IL-6, RANTES (CCL5), TNF-o, IL-4, IL-10, IL-13, IFN- y, granzyme B, perforin, CD 107a, or IL-2.
  • mediators such as GM-CSF, IL-6, RANTES (CCL5), TNF-o, IL-4, IL-10, IL-13, IFN- y, granzyme B, perforin, CD 107a, or IL-2.
  • the biological activity elicited by an anti-ALPPL2 agent such as an Ab or Ab fragment or a CAR-expressing cells is measured by assessing clinical outcome, such as the reduction in disease symptoms.
  • improved efficacy may be shown by better infiltration of disease-resolving immune cells into the tumor, reduced tumor sizes, or reduced ascites.
  • gene expression profiles may be also investigated to evaluate the activity.
  • Target cells Cells that may be targeted by any anti-ALPPL2 agents of present Invention include any ALPPL2-expressing cells.
  • the target cell may be present in any part of the body of a subject; including blood or lymphatic circulation, and disease-affected tissues.
  • the disease-affected tissues include, but are not limited to, pancreas, testis, cervix, endometrium, ovary, stomach, colon, rectum, lung, mesothelium, or tongue, or another oral tissue.
  • target cells may blood cells or hematopoietic cells.
  • the anti-ALPPL2 agent-expressing cells of the invention are used to treat cancer, wherein ALPPL2 is upregulated.
  • the cells of the Invention may be used to treat pancreatic cancer, testicular cancer, cervical cancer, endometrial cancer, ovarian cancer, stomach cancer, colorectal cancer, lung cancer, mesothelioma, or tongue cancer.
  • cells that are positive for ALPPL2 may be identified via known methods, for example, immunofluorescence or flow cytometry using specific antibodies, or alternatively, through cytotoxicity against target cells.
  • Methods of testing an anti- ALPPL2 agent for the ability to recognize target cells and for antigen specificity are known in the art For Instance, Clay et al.,/. Immunol., 163: 507-513 (1999), teaches methods of measuring the release of cytokines (e.g., interferon-y, granulocyte/monocyte colony stimulating factor (GM-CSF), tumor necrosis factor a (TNF-a) or interleukin 2 (IL- 2)).
  • cytokines e.g., interferon-y, granulocyte/monocyte colony stimulating factor (GM-CSF), tumor necrosis factor a (TNF-a) or interleukin 2 (IL- 2)
  • cytokines e.g., interferon-y, granulocyte/monocyte colony
  • a biopsy is the removal of tissue and/or cells from an individual. Such removal may be to collect tissue and/or cells from the individual in order to perform experimentation on the removed tissue and/or cells. This experimentation may include experiments to determine if the individual has and/or is suffering from a certain condition or disease-state.
  • the condition or disease may be, e.g., cancer.
  • the sample comprising cells of the host can be a sample comprising whole cells, lysates thereof, or a fraction of the whole cell lysates, e.g., a nuclear or cytoplasmic fraction, a whole protein fraction, or a nucleic acid fraction. If the sample comprises whole cells, the cells can be any cells of the host, e.g., the cells of any organ or tissue, including blood cells or endothelial cells.
  • compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired.
  • administration may be topical, parenteral, or enteral.
  • parenteral administration of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue, thus generally resulting in the direct administration into the blood stream, into muscle, or into an internal organ.
  • Parenteral administration thus Includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like.
  • parenteral administration is contemplated to include, but is not limited to, subcutaneous, Intraperitoneal, Intramuscular, intrastemal, Intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intrasynovial injection or infusions; and kidney dialytic infusion techniques.
  • parenteral administration of the compositions of the present invention comprises subcutaneous or Intraperitoneal administration.
  • oral refers to administration of a compound or composition to an individual by a route or mode along the alimentary canal.
  • oral routes of administration of a composition include, without limitation, swallowing liquid or solid forms of a composition from the mouth, administration of a composition through a nasojejunal or gastrostomy tube, intraduodenal administration of a composition, and rectal administration, e.g., using suppositories for the lower intestinal tract of the alimentary canal.
  • compositions of the present invention may be suited for topical, parenteral, or enteral administration.
  • compositions comprising Abs, antigen-binding Ab fragments, ADCs, or CARs, polynucleotides or vectors encoding such, or cells expressing thereof are suitable for administration via parenteral administration for example, subcutaneous, intramuscular, intraperitoneal or intravenous injection.
  • Formulations of a pharmaceutical composition suitable for parenteral administration typically generally comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.
  • the active ingredient is provided in dry (i.e. powder or granular) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.
  • a suitable vehicle e.g. sterile pyrogen-free water
  • Parenteral formulations also include aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9), but; for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water.
  • parenteral administration forms include solutions or suspensions in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired.
  • Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, or in a liposomal preparation.
  • Formulations for parenteral administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.
  • Such formulation may be, for example, made of a biodegradable, biocompatible polymer, such as, but not limited to, ethylene vinyl acetate, polyfalkyl cyanoacrylates), poly(anhydrides), poly(amides), poly(ester), poly(ester amides), pofy(phosphoesters), polygiycolic acid (PGA), collagen, polyorthoester, polylactic acid (PLA), poly(lactic-co- glycolidic acid) (PLAGA), or naturally occurring biodegradable polymers such as chitosan and hyaluronic acid-based polymers (Kamaly N. et al, Chem Rev. Author manuscript; available in PMC 2017 Jul 13).
  • a biodegradable, biocompatible polymer such as, but not limited to, ethylene vinyl acetate, polyfalkyl cyanoacrylates), poly(anhydrides), poly(amides), poly(ester), poly(ester amides), pofy(phosphoesters), polygiycolic
  • compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, semi-solids, monophasic compositions, multiphasic compositions (e.g., oil-in-water, water-ln-oll), foams, microsponges, liposomes, nanoemulsions, aerosol foams, polymers, fullerenes, and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • compositions and formulations for parenteral, intrathecal, or intraventricular administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carder compounds and other pharmaceutically acceptable carriers or excipients.
  • compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.
  • compositions of the present invention may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product
  • compositions of the present invention may be formulated to provide appropriate in vivo distribution of the active ingredient
  • concentrating the distribution of an anti-tumor drug in the tumor site is challenging, and it can be so even when a drug has a specificity to a molecule expressed by cancer cells.
  • Various strategies have been developed to address the issue and any appropriate strategies may be applied for the current invention (for example, reviewed in Rosenblum D. etal, Nat Commun. 2018 Apr 12;9(1): 1410. doi: 10.1038/s41467-018-03705-y).
  • BBB blood-brain barrier
  • Any appropriate strategies to enable BBB crossing may be utilized to for the delivery of any of the anti-ALPPL2 agents of the agents (see for example, Dong X. et al., Theranostics. 2018; 8(6): 1481-1493, for exemplary strategies).
  • compositions of the present Invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, aerosols, and enemas.
  • the compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media.
  • Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • the pharmaceutical compositions may be formulated and used as foams.
  • Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product
  • Agents that enhance uptake of oligonucleotides at the cellular level may also be added to the pharmaceutical and other compositions of the present invention.
  • cationic lipids such as lipofectin (U.S. Pat No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (WO 97/30731), also enhance the cellular uptake of oligonucleotides.
  • compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions.
  • the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or antiinflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • additional materials useful in physically formulating various dosage forms of the compositions of the present invention such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • such materials when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention.
  • the formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
  • Formulations comprising any of the anti-ALPPL2 agents of the present invention or populations of cells expressing any of the anti-ALPPL2 agents such as anti-ALPPL2 CARs of the present invention may include pharmaceutically acceptable excipient(s).
  • Excipients included in the formulations will have different purposes depending, for example, on the CAR construct; the subpopulation of cells used, and the mode of administration. Examples of generally used excipients include, without limitation: saline, buffered saline, dextrose, water-for- infection, glycerol, ethanol, and combinations thereof, stabilizing agents, solubilizing agents and surfactants, buffers and preservatives, tonicity agents, bulking agents, and lubricating agents.
  • the formulations comprising populations of the CAR-expresslng cells of the present invention will typically have been prepared and cultured in the absence of any non-human components, such as animal serum (e.g., bovine serum albumin).
  • the formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the binding molecules or cells, preferably those with activities complementary to the binding molecule or cell, where the respective activities do not adversely affect one another.
  • active ingredients are suitably present in combination in amounts that are effective for the purpose intended.
  • the pharmaceutical composition further includes other pharmaceutically active agents or drugs.
  • Such agents or drugs may be, but are not limited to, an anti-cancer drug, an anti-proliferative drug, a cytotoxic drug, an anti-angiogenic drug, an apoptotic drug, an immunostimulatory drug, an anti-microbial drug, an antibiotic drug, an antiviral drug, an anti-inflammatory drug, an ALPPL2 inhibitor, an ALPPL2 signaling inhibitor, an anti-ALPPL2 agent of the present invention, an enzyme, a hormone, a toxin, a compound, a small molecule, a small molecule inhibitor, a protein, a peptide, a vector, a plasmid, a viral replicon, a viral particle, a nanoparticle, a DNA molecule, an RNA molecule, an siRNA, an shRNA, a micro RNA, an oligonucleotide, or an imaging drug.
  • an anti-cancer drug an anti-proliferative drug, a cytotoxic drug, an anti-angiogenic drug,
  • chemotherapeutic agents e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, eta
  • the pharmaceutical composition in some embodiments can employ time- released, delayed release, and sustained release delivery systems such that the delivery of the composition occurs prior to, and with sufficient time to cause, sensitization of the site to be treated.
  • time- released, delayed release, and sustained release delivery systems such that the delivery of the composition occurs prior to, and with sufficient time to cause, sensitization of the site to be treated.
  • Many types of release delivery systems are available and known. Such systems can avoid repeated administrations of the composition, thereby increasing convenience to the subject and the physician.
  • kits comprising (a) one or more of anti-ALPPL2 agents (Abs, antigen-binding Ab fragments, ADCs, CARs), polynucleotides encoding such, vectors encoding such, cells expressing such; and (b) for example an instruction for use in treating or diagnosing a disease or condition associated with ALPPL2.
  • the kit may include a label indicating the intended use of the contents of the kit.
  • label as used herein includes any written materials, marketing materials, or recorded materials supplied on, with, in, or appended to the kit Method of administration
  • the administration route used in the method of the present invention may be any appropriate route, which depends upon whether local or systemic treatment is desired.
  • administration may be topical, parenteral, or enteral.
  • compositions comprising Abs, antigen-binding Ab fragments, ADCs, or CARs, polynucleotides or vectors encoding such, cells expressing such may be administered parenterally, for example, via subcutaneous, intramuscular, intraperitoneal or Intravenous Injection.
  • adoptive cell therapy methods for administration of cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions.
  • adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No. 2003/0170238 to Gruenberg etal; U.S. Pat No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g.,Themeli etal. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara etal. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338.
  • composition of the present Invention may be administered using any appropriate medical devices (for example, reviewed in Richter B. B Repeat J. BioDrugs (2016) 32: 425).
  • the dosage will vary and depend on, for example, the target disease, the severity of the disease, the route of administration, and pharmacokinetic factors. Dosing may be modified based on the response observed in the subject
  • appropriate dosage regimen may be determined using any appropriate methodology (for example, Bai S. et al., Clin Pharmacokinet 2012 Feb l;51(2):119-35. doi: 10.2165/11596370-000000000-00000).
  • the dosage may be from about 1 ng/kg to about 1 g/kg (of the body weight of a subject) per day. In some embodiments, the dose may be from about 10 ng/kg/day to about 900 mg/kg/day, from about 20 ng/kg/day to about 800 mg/kg/day, from about 30 ng/kg/day to about 800 mg/kg/day, from about 40 ng/kg/day to about 700 mg/kg/day, from about 50 ng/kg/day to about 600 mg/kg/day, from about 60 ng/kg/day to about 500 mg/kg/day, from about 70 ng/kg/day to about 400 mg/kg/day, from about 80 ng/kg/day to about 300 mg/kg/day, from about 90 ng/kg/day to about 200 mg/kg/day, or from about 100 ng/kg/day to about 100 mg/kg/day.
  • Dosing frequency may be, for example, three times per day, twice per day, once per day, every other day, once per week, every other week, once per three weeks, once per four weeks, once per five weeks, once per six weeks, once per seven weeks, once per eight weeks, once per nine weeks, once per ten weeks, once per three months, once per four months, once per six months, once per year, or even less frequent
  • the pharmaceutical composition in some embodiments contains cells expressing the CAR of the present invention in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount
  • Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs.
  • other dosage regimens may be useful and can be determined.
  • the desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.
  • a subject in the context of genetically engineered cells expressing an anti-ALPPL2 agent such as an Ab or Ab fragment or a CAR, a subject is administered the range of about one million to about 100 billion cells, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells,
  • the cells or population of cells can be administrated in one or more doses.
  • said effective amount of cells can be administrated as a single dose.
  • said effective amount of cells can be administrated as more than one dose over a period time. Timing of administration is within the judgment of managing physician and depends on the clinical condition of the patient
  • the cells or population of cells may be obtained from any source, such as a blood bank or a donor.
  • an effective amount means an amount which provides a therapeutic or prophylactic benefit
  • the dosage administrated will be dependent upon the age, health and weight of the recipient; kind of concurrent treatment; if any, frequency of treatment and the nature of the effect desired.
  • an effective amount of cells or composition comprising those cells are administrated parenterally.
  • administration can be an intravenous administration.
  • administration can be directly done by injection into the disease site.
  • the amount or dose of the inventive anti-ALPPL2 material administered should be sufficient to effect a therapeutic or prophylactic response in the subject or animal over a reasonable time frame.
  • the dose of the inventive anti-ALPPL2 material should be sufficient to bind to antigen, or detect; treat or prevent disease in a period of from about 2 hours or longer, e.g., about 12 to about 24 or more hours, from the time of administration. In certain embodiments, the time period could be even longer.
  • the dose will be determined by the efficacy of the particular inventive anti-ALPPL2 material and the condition of the animal (e.g., human), as well as the body weight of the animal (e.g., human) to be treated.
  • an assay which comprises, for example, comparing the extent to which target cells are lysed or, in the context of CARs, IFN-y is secreted by T cells expressing the inventive CAR, polypeptide, or protein upon administration of a given dose of such T cells to a mammal, among a set of mammals of which is each given a different dose of the T cells, could be used to determine a starting dose to be administered to a mammal.
  • the extent to which target cells are lysed or IFN-y is secreted upon administration of a certain dose can be assayed by methods known in the art
  • two or more of the anti-ALPPL2 agents or compositions of the present invention may be administered to a subject in combination or separately.
  • the anti-ALPPL2 agents or compositions of the present invention are administered as part of a combination treatment; such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as an antibody or engineered cell or receptor or agent; such as a cytotoxic or therapeutic agent
  • another therapeutic intervention such as an antibody or engineered cell or receptor or agent; such as a cytotoxic or therapeutic agent
  • the cells or antibodies in some embodiments are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order.
  • the anti-ALPPL2 agents or compositions are co-administered with another therapy sufficiently close in time such that the anti-ALPPL2 agents or compositions enhance the effect of one or more additional therapeutic agents, or vice versa.
  • the cells or antibodies are administered prior to the one or more additional therapeutic agents.
  • the anti-ALPPL2 agents such as anti-ALPPL2 Ab or Ab fragment or CAR T cells or antibodies are administered after the one or more additional therapeutic agents.
  • the compositions of the present invention may be given to a subject along with one or more of other therapies, which may be surgery, or a radiotherapy.
  • a lymphodepletlng chemotherapy Is administered to the subject prior to, concurrently with, or after administration (e.g., infusion) of CAR cells.
  • the lymphodepletlng chemotherapy is administered to the subject prior to administration of the cells.
  • the lymphodepletlng chemotherapy ends 1-4 days (e.g., 1, 2, 3, or 4 days) prior to CAR cell infusion.
  • multiple doses of CAR cells are administered, e.g., as described herein.
  • a lymphodepletlng chemotherapy is administered to the subject prior to, concurrently with, or after administration (e.g., infusion) of a CAR- expresslng cell described herein.
  • lymphodepletlon include, but may not be limited to, nonmyeloablative lymphodepletlng chemotherapy, myeloablative lymphodepletlng chemotherapy, total body irradiation, etc.
  • lymphodepletlng agents include, but are not limited to, anti-thymocyte globulin, anti- CD3 antibodies, anti-CD4 antibodies, anti-CDB antibodies, anti-CD52 antibodies, anti- CD2 antibodies, TCRcxP blockers, anti-CD20 antibodies, anti-CD19 antibodies, Bortezomib, rituximab, anti-CD154 antibodies, rapamycin, CD3 immunotoxin, fludarabine, cyclophosphamide, busulfan, melphalan, Mabthera, Tacrolimus, alefacept, alemtuzumab, 0KT3, 0KT4, 0KT8, 0KT11, fingollmod, anti-CD40 antibodies, anti-BR3 antibodies, Campath-IH, anti-CD25 antibodies, calcineurin inhibitors, mycophenolate, and steroids, which may be used alone or in combination.
  • anti-ALPPL2 agents of the present invention for example, anti-ALPPL2 Abs and antigen-binding Ab fragments, can be also useful as a diagnostic tool that may be used in vivo, ex vivo, or in vitro.
  • an anti-ALPPL2 Abs or antigen-binding Ab fragment conjugated to an imaging agent may be administered to a subject or a patient to test if a diseased cell or tissue in the patient expresses ALPPL2.
  • the diagnoses may be done using any imaging tools that can detect the imaging agent
  • a biological sample such as, but is not limited to, blood or biopsy sample, may be obtained, and an anti-ALPPL2 Abs or antigen-binding Ab fragment may be applied to the sample to test the expression ofALPPL2.
  • tests may determine whether the subject or the cell or tissue of the subject; expresses ALPPL2 or not.
  • the test may determine whether the subject, or the cell or tissue of the subject, expresses sufficient amount of ALPPL2 to be targeted by the anti-ALPPL2 therapeutic agent of the present invention.
  • the test may classify patients into different levels of ALPPL2 expression. In one aspect; a subject may be classified as an expressor or a non- expressor. In another aspect, a subject may be classified as an over-expressor, mid- expressor, or low-expressor.
  • an appropriate therapeutic approach may be determined depending on the ALPPL2 expression.
  • the expression may be determined using an anti- ALPPL2 agent of the present invention as described herein, or alternatively using any other appropriate method, such as, but not limited to, by measuring RNA expression levels or by quantifying ALPPL2 protein levels using an appropriate tool and/or technique.
  • the anti-ALPPL2 agent of the present invention may be given to an expressor but not to a non-expressor.
  • the anti-ALPPL2 agent of the present Invention may be given to an over-expressor but not to a mld-expressor or a low-expressor.
  • the anti-ALPPL2 agent of the present invention may be given to an over-expressor or a mid-expressor but not to a low-expressor. In yet another aspect; the anti-ALPPL2 agent of the present invention may be given to a mid-expressor but not to a hlgh-expressor or a low-expressor.
  • functional portion when used in reference to an Ab, antigen-binding Ab fragment; ADC, or CAR, refers to any part or fragment of the Ab, antigen-binding Ab fragment; ADC, or CAR of the invention, which part or fragment retains the biological activity of the Ab, antigen-binding Ab fragment; ADC, or CAR of which it is a part (the parent).
  • Functional portions encompass, for example, those parts of an Ab, antigen-binding Ab fragment, ADC, or CAR that retain the ability to recognize target cells, or detect; treat; or prevent a disease, to a similar extent; the same extent, or to a higher extent; as the parent
  • the functional portion can comprise, for instance, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more, of the parent
  • the functional portion can comprise additional amino acids at the amino or carboxy terminus of the portion, or at both termini, which additional amino acids are not found in the amino add sequence of the parent Ab, antigen-binding Ab fragment ADC, or CAR.
  • the additional amino adds do not interfere with the biological function of the functional portion, e.g., recognize target cells, detect treat or prevent a target disease and/or condition, eta More desirably, the additional amino adds enhance the biological activity, as compared to the biological activity of the parent Ab, antigenbinding Ab fragment ADC, or CAR.
  • the functional variant refers to an Ab, antigen-binding Ab fragment ADC, or CAR polypeptide, or protein having substantial or significant sequence identity or similarity to a parent which functional variant retains the biological activity of the Ab, antigen-binding Ab fragment ADC, or CAR of which it is a variant
  • Functional variants encompass, for example, those variants of the Ab, antigen-binding Ab fragment ADC, or CAR described herein (the parent) that retain the ability to recognize target cells to a similar extent the same extent or to a higher extent as the parent
  • the functional variant can, for Instance, be at least about 30%, 50%, 75%, 80%, 90%, 98% or more Identical In amino acid sequence to the parent
  • a functional variant can, for example, comprise the amino acid sequence of the parent with at least one conservative amino acid substitution.
  • the functional variants can comprise the amino acid sequence of the parent with at least one non-conservative amino acid substitution.
  • the non-conservative amino acid substitution may enhance the biological activity of the functional variant such that the biological activity of the functional variant is increased as compared to the parent
  • Amino acid substitutions of the inventive anti-ALPPL2 agents are preferably conservative amino acid substitutions.
  • Conservative amino acid substitutions are known in the art and Include amino acid substitutions in which one amino acid having certain physical and/or chemical properties is exchanged for another amino add that has the same or similar chemical or physical properties.
  • the conservative amino add substitution can be an addic/negatively charged polar amino add substituted for another addic/negatively charged polar amino add (e.g., Asp or Glu), an amino add with a nonpolar side chain substituted for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Vai, He, Leu, Met, Phe, Pro, Trp, Cys, Vai, etc.), a basic/positively charged polar amino acid substituted for another basic/positively charged polar amino add (e.g.
  • an uncharged amino add with a polar side chain substituted for another uncharged amino add with a polar side chain e.g., Asn, Gin, Ser, Thr, iyr, eta
  • an amino add with a ⁇ -branched side-chain substituted for another amino add with a P- branched side-chain e.g., He, Thr, and Vai
  • an amino add with an aromatic side-chain substituted for another amino add with an aromatic side chain e.g., His, Phe, Trp, and Tyr
  • amino adds may be added or removed from the sequence based on vector design.
  • the anti-ALPPL2 agents can consist essentially of the spedfied amino add sequence or sequences described herein, such that other components, e.g., other amino adds, do not materially change the biological activity of the functional variant
  • the Abs, antigen-binding Ab fragments, AD Cs, or CARs of embodiments of the invention can be of any length, i.e., can comprise any number of amino acids, provided that the Abs, antigen-binding Ab fragments, ADCs, or CARs (or functional portions or functional variants thereof) retain their biological activity, e.g., the ability to spedfically bind to antigen, detect diseased cells in a mammal, or treat or prevent disease in a mammal, eta
  • the Ab, antigen-binding Ab fragment ⁇ ADC, or CAR can be about 50 to about 5000 amino adds long, such as 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more amino adds in length.
  • the Abs, antigen-binding Ab fragments, AD Cs, or CARs of embodiments of the invention can comprise synthetic amino acids In place of one or more naturally-occurring amino acids.
  • synthetic amino acids include, for example, aminocyclohexane carboxylic acid, norleucine, a-amino n-decanoic acid, homoserine, S- acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4- amlnophenylalanine, 4-nltrophenylalanlne, 4-chlorophenylalanlne, 4- carboxyphenylalanine, p-phenylserine p-hydroxyphenylalanine, phenylglycine, a- naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic add
  • the Abs, antigen-binding Ab fragments, ADCs, or CARs of embodiments of the invention can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via, e.g., a disulfide bridge, or converted into an add addition salt and/or optionally dimerized or polymerized, or conjugated.
  • the Abs, antigen-binding Ab fragments, ADCs, or CARs of embodiments of the invention can be obtained by methods known in the art
  • the Abs, antigen-binding Ab fragments, ADCs, and CARs may be made by any suitable method of making polypeptides or proteins. Suitable methods of de novo synthesizing polypeptides and proteins are described in references, such as Chan etal., "Fmoc Solid Phase Peptide Synthesis", Orford University Press, Orford, United Kingdom, 2000; “Peptide and Protein Drug Analysis", ed. Reid, R., Marcel Dekker, Ina, 2000; “Epitope Mapping", ed.
  • polypeptides and proteins can be recombinantiy produced using the nucleic acids described herein using standard recombinant methods. See, for instance, Sambrook etal., “Molecular Cloning: A Laboratory Manual”, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; and Ausubel et al., “Current Protocols in Molecular Biology”, Greene Publishing Associates and John Wiley & Sons, N Y, 1994.
  • Abs, antigen-binding Ab fragments, or CARs of the invention can be isolated and/or purified from a source, such as a plant; a bacterium, an insect; a mammal, e.g., a rat, a human, eta Methods of isolation and purification are well-known in the art
  • the Abs, antigen-binding Ab fragments, ADCs, or CARs described herein can be commercially synthesized by companies.
  • inventive Abs, antigen-binding Ab fragments, ADCs, or CARs can be synthetic, recombinant; isolated, and/or purified.
  • 4-1BB refers to a member of the TNFR superfamily with an amino add sequence provided as GenBank Acc. No. AAA53133.1, or the equivalent residues from a non-human spedes, e.g., mouse, rodent; monkey, ape and the like.
  • the "4- IBB costimulatory domain,” also referred to as “4-1BB CS domain” or “41BBCS,” may be derived from the cytoplasmic domain of 4-1BB.
  • 41BBCS comprises the sequence provided as SEQ ID NO: 165 or the equivalent residues from a non-human species, e.g., mouse, rodent; monkey, ape and the like.
  • 41BBCS may be encoded by a nucleic add sequence provided as SEQ ID NO: 265.
  • the term "5* cap” (also termed an RNA cap, an RNA 7-methylguanosine cap or an RNA m7G cap) as used herein is a modified guanine nucleotide that has been added to the "front” or 5' end of a eukaryotic messenger RNA shortly after the start of transcription.
  • the 5' cap consists of a terminal group which is linked to the first transcribed nucleotide. Its presence is critical for recognition by the ribosome and protection from RNases. Cap addition is coupled to transcription, and occurs co- transcriptionally, such that each influences the other.
  • the 5' end of the mRNA being synthesized is bound by a cap-synthesizing complex associated with RNA polymerase.
  • This enzymatic complex catalyzes the chemical reactions that are required for mRNA capping. Synthesis proceeds as a multi- step biochemical reaction.
  • the capping moiety can be modified to modulate functionality of mRNA such as Its stability or efficiency of translation.
  • ALPPL2 is an alkaline phosphatase, also known as “alkaline phosphatase, placental like 2", “ALPG”, “ALPPL”, “alkaline phosphatase, germ cell”, or “GCAP”.
  • ALPPL2 is typically in a membrane-bound, glycosylated form.
  • ALPPL2 is encoded by the ALPG gene on chromosome 2, with gene location 2q37.1 (NCBI).
  • Human ALPPL2 may have an amino acid sequence provided as GenBank: AIC53992.1.
  • human ALPPL2 has the amino acid sequence provided as SEQ ID NO: 101 or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like.
  • allogeneic refers to any material derived from a different animal of the same species as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not Identical. In some embodiments, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically.
  • antibody or “Ab” is used herein in the broadest sense and encompasses various antibody structures, including but not limited to full-length or full- size immunoglobulins, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and/or antibody fragments (preferably those fragments that exhibit the desired antigen-binding activity, which is also referred to as "antigen-binding antibody fragments"), "typically, a full-size Ab comprises two pairs of chains, each pair comprising a heavy chain (HC) and a light chain (LC) interconnected by disulfide bonds.
  • a HC typically comprises a variable region and a constant region.
  • a LC also typically comprises a variable region and constant region.
  • variable region of a heavy chain typically comprises three complementarity-determining regions (CDRs), which are referred to herein as CDR 1, CDR 2, and CDR 3 (or referred to as CDR- Hl, CDR-H2, CDR-H3, respectively).
  • CDRs complementarity-determining regions
  • the constant region of a HC typically comprises a CHI domain, a CH2 domain, and a CH3 domain.
  • CH2 and CH3 domains form a fragment crystallizable region (Fc region), which dictates the Isotype of the Ab (IgA (further divided into IgAl and IgA2 subclasses), IgD, IgG (further divided into IgGl, IgG2, IgG3, and IgG4 subclasses), IgE, and IgM), the type of Fc receptor the Ab binds to, and therefore the effector function of the Ab.
  • Fc receptor types include, but are not limited to, FcaR (such as FcaRI), Fca/mR, FceR (such as FceRI, FceRII),and FcgR (such as FcgRI, FcgRIIA, FcgRIIBl, FcgRIIB2, FcgRIIIA, FcgRIIIB) and their associated downstream effects are well known in the art
  • the variable region of a light chain (VL) also typically comprises CDRs, which are CDR 1, CDR 2, and CDR 3 (or referred to as CDR-L1, CDR-L2, CDR-L3, respectively).
  • the constant region of a LC typically comprises a CL domain (kappa or lambda type).
  • the antigen is ALPPL2.
  • Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources.
  • a molecule comprising an antibody-derived structure that enables specific binding to an antigen is referred to "antigen-binding fragment,” “antigen-binding domain” or “antigenbinding region” of the Ab.
  • an ADC refers to a conjugate of an Ab or antigen-binding Ab fragment and a drug.
  • the drug may be attached to any part of the Ab or the antigen-binding Ab fragment via a direct or indirect attachment; such as via a linker.
  • an ADC may comprise an antibody (or antibody fragment such as a single-chain variable fragment (scFv)) linked to a payload drug (often cytotoxic).
  • the antibody causes the ADC to bind to the target cancer cells.
  • the ADC is then internalized by the cell and the drug is released into the cell.
  • Hydrophilic linkers may prevent the drug being pumped out of resistant cancer cells through MDR (multiple drug resistance) transporters.
  • the present disclosure is also related to immunoconjugates comprising an anti-ALPPL2 binding agent conjugated to a therapeutic agent, such as a cytotoxin, a drug (e.g., an Immunosuppressant) or a radiotoxin.
  • a therapeutic agent such as a cytotoxin, a drug (e.g., an Immunosuppressant) or a radiotoxin.
  • Such conjugates may be referred to as "immunoconjugates”.
  • Immunoconjugates that include one or more cytotoxins may also be referred to as "immunotoxins.”
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g., kills) cells.
  • Cytotoxins can be conjugated to antibodies according to at least some embodiments of the invention using linker technology available in the art Examples of linker types that have been used to conjugate a cytotoxin to an antibody include, but are not limited to, hydrazones, thioethers, esters, disulfides and peptide-containing linkers.
  • a linker can be chosen that is, for example, susceptible to cleavage by low pH within the lysosomal compartment or susceptible to cleavage by proteases, such as proteases preferentially expressed in tumor tissue such as cathepsins (e.g., cathepsins B, C, D).
  • proteases such as proteases preferentially expressed in tumor tissue such as cathepsins (e.g., cathepsins B, C, D).
  • Antibodies of the present invention also can be conjugated to a radioactive isotope to generate cytotoxic radiopharmaceuticals, also referred to as radioimmunoconjugates.
  • antibody fragment or "Ab fragment' as used herein refers to any portion or fragment of an Ab, including intact or full-length Abs that may be of any class or sub-class, Including IgG and sub-classes thereof, IgM, IgE, IgA and sub-classes thereof, and IgD.
  • the term encompasses molecules constructed using one or more potions or fragments of one or more Abs.
  • An Ab fragment can be immunoreactive portions of intact immunoglobulins.
  • the term is used in the broadest sense and includes polyclonal and monoclonal antibodies, including Intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, including single chain variable fragments (scFv), diabodies, and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments.
  • Fab fragment antigen binding
  • F(ab')2 fragments fragment antigen binding
  • Fab' fragments fragment antigen binding
  • Fv fragments fragment antigen binding
  • rlgG recombinant IgG fragments
  • single chain antibody fragments including single chain variable fragments (scFv), diabodies, and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments.
  • the term also encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodles, and tetrabodies, tandem dl-scFv, tandem tri- scFv.
  • the antibody fragment is a scFv.
  • Ab fragment 1 should be understood to encompass functional antibody fragments thereof.
  • a portion of an Ab fragment that comprises a structure that enables specific binding to an antigen is referred to as “antigen-binding Ab fragment;” "antigen-binding domain,” “antigen-binding region,” or “antigen-binding region” of the Ab fragment
  • a "heavy chain” or “HC” of an Ab refers to the larger of the two types of polypeptide chains present in all Ab molecules in their naturally occurring conformations.
  • a "light chain” or "LC” of an Ab refers to the smaller of the two types of polypeptide chains present in all Ab molecules in their naturally occurring conformations. Kappa and lambda light chains refer to the two major antibody light chain isotypes.
  • Anti-ALPPL2 agent' or "anti-ALPPL2 material” as used herein refers to any agents that are able to bind to and/or target ALPPL2 directly or indirectly.
  • Anti-ALPPL2 agents of the present invention include, but are not limited to, anti-ALPPL2 Abs, anti- ALPPL2 antigen-binding Ab fragments, anti-ALPPL2 multi-specific Abs, anti-ALPPL2 multi-specific antigen-binding Ab fragments, anti-ALPPL2 ADCs, and anti-ALPPL2 CARs, and polynucleotides and vectors encoding the same, and cells expressing the same.
  • anti-ALPPL2 agents may also encompass pharmaceutical compositions comprising any of the above-mentioned anti-ALPPL2 agents.
  • antigen refers to a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both.
  • antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an "antigen" as that term is used herein.
  • an antigen need not be encoded solely by a full- length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one genes and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired Immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be generated, synthesized, or can be derived from a biological sample, or might be macromolecule besides a polypeptide.
  • Such a biological sample can include, but is not limited to a tissue sample, a cancer tissue sample, a tumor tissue sample, a leukemic cell sample, an inflamed tissue sample, and a cell or a fluid with other biological components.
  • the antigen is ALPPL2.
  • antigen-binding domain refers to a portion of the anti-ALPPL2 agents, such as antl-ALPPL2 chimeric antigen receptors, of the present Invention and the portion comprises a structure that allows for specific binding of the anti-ALPPL2 agents to ALPPL2.
  • the antigen-binding domain may comprise the variable region of the Ab or a portion of the variable region, such as the CDRs.
  • the antigen-binding domain may comprise the variable region or a portion of the variable region, such as the CDRs, of the Ab that the anti-ALPPL2 agentis derived from.
  • the antigen-binding domain may be one or more extracellular domains of the CAR which have specificity for ALPPL2.
  • the antigen-binding domain When the antigen-binding domain is derived from an Ab or antigen-binding Ab fragment; the antigen-binding domain may comprise the antigenbinding domain, such as the variable region or a portion of the variable region, such as the CDRs, of the Ab or antigen-binding Ab fragment that it is derived from.
  • the antigen-binding domain of an anti-ALPPL2 agent of the present invention is scFv.
  • apheresis refers to the art-recognized extracorporeal process by which the blood of a donor or patient is removed from the donor or patient and passed through an apparatus that separates out selected particular constituents) and returns the remainder to the circulation of the donor or patient; e.g., by retransfusion.
  • an apheresis sample refers to a sample obtained using apheresis.
  • autologous or "donor-derived” as used herein refers to any material derived from the same individual to whom it is later to be re-introduced.
  • bind refers to an attractive interaction between two molecules that results in a stable association in which the molecules are in close proximity to each other.
  • the result of molecular binding is sometimes the formation of a molecular complex in which the attractive forces holding the components together are generally non-covalent; and thus are normally energetically weaker than covalent bonds.
  • cancer refers to a disease characterized by the uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers relevant to the present invention include, but are not limited, pancreatic cancer, testicular cancer, cervical cancer, endometrial cancer, ovarian cancer, stomach cancer, colorectal cancer, lung cancer, mesothelioma, and tongue cancer.
  • bispecific refers to having two binding specificities.
  • An anti-ALPPL2 bispecific Ab or a bispecific antigen-binding Ab fragment; for example, of the present invention has at least one specificity for ALPPL2.
  • the first specificity is for an epitope for ALPPL2
  • the second specificity may be for another non-overlapping or non-competing epitope for ALPPL2 or may be for a molecule other than ALPPL2.
  • the term "bispecific” is also used in the same manner for any other anti-ALPPL2 agents of the present invention, such as anti-ALPPL2 CARs.
  • CD28 refers to the protein Cluster of Differentiation 28, one of the proteins expressed on T cells that provide co-stimulatory signals required for T cell activation and survival.
  • Human CD28 protein may have at least 85, 90, 95, 96, 97, 98, 99 or 100% identity to NCBI Reference No: NP_006130 or a fragment thereof that has stimulatory activity.
  • CD28 transmembrane domain also referred to as “CD28 TM domain” or “CD28TM” refers to the amino acid residues derived from the transmembrane domain of CD28.
  • CD28TM comprises the sequence provided as SEQ ID NO: 161 or the equivalent residues from a non-human species, e.g., mouse, rodent; monkey, ape and the like.
  • CD28 TM domain may be encoded by the nucleic acid sequence provided as SEQ ID NO: 261.
  • CD28 hinge refers to amino acid residues that may be used to join two domains or two portions within a domain in CARs of some of the embodiments.
  • CD28 hinge comprises the sequence provided as SEQ ID NO: 163 or the equivalent residues from a non-human species, e.g., mouse, rodent; monkey, ape and the like.
  • CD28 hinge may be encoded by the nucleic acid sequence provided as SEQ ID NO: 263.
  • CD28 costimulatory domain also referred to as “CD28 CS domain” or “CD28CS” refers to the amino acid residues derived from the cytoplasmic domain of CD28.
  • CD28CS comprises the sequence provided as SEQ ID NO: 164 or the equivalent residues from a non-human species, e.g., mouse, rodent; monkey, ape and the like.
  • CD28 CS domain may be encoded by the nucleic acid sequence provided as SEQ ID NO: 264.
  • CD3 zeta or alternatively, “zeta,” “zeta chain,” “CD3-zeta,” “CD3z,” “TCR-zeta,” or “CD247,” is a protein encoded by the CD247 gene on chromosome 1, with gene location lq24.2, in humans.
  • CD 3 zeta together with T cell receptor (TCR) and CD 3 (a protein complex composed of a CD3 gamma, a CD3 delta, and two CD3 epsilon), forms the TCR complex.
  • Human CD3 zeta may have an amino add sequence provided as NP_000725 or NP.932170, or the equivalent residues from a non-human spedes, e.g., mouse, rodent; monkey, ape and the like.
  • the term "CD3 zeta intracellular signaling domain,” or alternatively “CD3 zeta ICS domain” or a “CD3zICS,” is defined as the amino add residues from the cytoplasmic domain of the CD3 zeta chain, or functional derivatives thereof, that are suflident to functionally transmit an initial signal necessary for T cell activation.
  • CDS zeta ICS domain Is the sequence provided as SEQ ID NO: 162.
  • “CDS zeta ICS domain” is encoded by the nucleic acid sequence provided as SEQ ID NO: 262.
  • CAR Chimeric Antigen Receptor
  • a CAR refers to a set of polypeptides, typically two In the simplest embodiments, which when In an Immune effector cell, provides the cell with specificity for a target cell, and with intracellular signal generation.
  • a CAR comprises at least an extracellular antigen-binding domain, a transmembrane domain (TM domain) and a cytoplasmic signaling domain (also referred to herein as "an Intracellular signaling domain (ICS domain)”) comprising a functional signaling domain derived from a stimulatory molecule and/or costimulatory molecule as defined below.
  • the set of polypeptides are contiguous with each other.
  • the set of polypeptides include a dimerization switch that; upon the presence of a dimerization molecule, can couple the polypeptides to one another, e.g., can couple an antigenbinding domain to an ICS domain.
  • the stimulatory molecule is the zeta chain associated with the T cell receptor complex
  • the cytoplasmic portion of a CAR further comprises a costimulatory domain (CS domain) comprising one or more functional signaling domains derived from at least one costimulatory molecule as defined below.
  • the costimulatory molecule is chosen from the costimulatory molecules described herein, e.g., 4-1BB (i.e., CD137), DAP10 and/or CD28.
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a TM domain and an ICS domain comprising a functional signaling domain derived from a stimulatory molecule. In one aspect; the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a TM domain, an ICS domain comprising a functional signaling domain derived from a stimulatory molecule, and a CS domain comprising a functional signaling domain derived from a costimulatory molecule.
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a TM domain, an ICS domain comprising a functional signaling domain derived from a stimulatory molecule, and two CS domains each of the two comprising a functional signaling domain derived from a costimulatory molecule(s) that is/are same with or different from each other.
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a TM domain, an ICS domain comprising a functional signaling domain derived from a stimulatory molecule, and at least two CS domains each comprising a functional signaling domain derived from a costimulatory moleculefs) that is/are same with or different from each other.
  • the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein.
  • the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen binding domain, wherein the leader sequence is optionally cleaved from the antigen binding domain (e.g., a scFv) during cellular processing and localization of the CAR to the cellular membrane.
  • the leader sequence comprises the amino acid sequence provided as SEQ ID NO: 160.
  • die LS may be encoded by a nucleic acid sequence provided as SEQ ID NO: 260.
  • the term "compete”, as used herein with regard to an Ab, antigen-binding Ab fragment; of antigen-binding domain of any of the anti-ALPPL2 agents of the present invention, means that a first Ab, antigen-binding Ab fragment; or antigen-binding domain, binds to an epitope in a manner sufficiently similar to the binding of a second Ab, antigen-binding Ab fragment, or antigen-binding domain, such that the result of binding of the first Ab, antigen-binding Ab fragment; or antigen-binding domain with its cognate epitope is delectably decreased in the presence of the second Ab, antigenbinding Ab fragment; or antigen-binding domain compared to the binding of the first Ab, antigen-binding Ab fragment, or antigen-binding domain in the absence of the second Ab, antigen-binding Ab fragment; or antigen-binding domain.
  • a first Ab, antigen-binding Ab fragment, or antigen-binding domain can inhibit the binding of a second Ab, antigen-binding Ab fragment, or antigenbinding domain to its epitope without that second Ab, antigen-binding Ab fragment, or antigen-binding domain inhibiting the binding of the first Ab, antigen-binding Ab fragment, or antigen-binding domain to its respective epitope.
  • each Ab, antigen-binding Ab fragment, or antigen-binding domain delectably inhibits the binding of the other Ab, antigen-binding Ab fragment; or antigen-binding domain with its cognate epitope or ligand, whether to the same, greater, or lesser extent; the two (Ab, antigen-binding Ab fragment; or antigen-binding domain) are said to "cross-compete" with each other for binding of their respective epitope(s).
  • Both competing and crosscompeting Abs, antigen-binding Ab fragments, or antigen-binding domains are encompassed by the invention.
  • CDR complementarity determining region
  • HVR hypervariable region
  • conservative amino acid substitutions are as commonly used in the art and include amino acid substitutions in which one amino acid having certain physical and/or chemical properties is exchanged for another amino add that has the same or similar chemical or physical properties.
  • the conservative amino add substitution can be an addic/negatively charged polar amino add substituted for another acidlc/negatively charged polar amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain substituted for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Vai, He, Leu, Met; Phe, Pro, Trp, Cys, Vai, eta), a basic/positively charged polar amino acid substituted for another basic/positively charged polar amino acid (e.g.
  • Non-conservative amino acid substitutions are amino acid substitutions that are not conservative amino acid substitutions.
  • costimulatory molecule refers to a cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an efficient immune response.
  • Costimulatory molecules include, but are not limited to a protein selected from the group consisting of an MHC class I molecule, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, a Toll ligand receptor, B7-H3, BAFFR, BTLA, BLAME (SLAMF8), CD2, CD4, CDS, CD7, CDBalpha, CDSbeta, CDlla, LFA-1 (CDlla/CD18),
  • MHC class I molecule TNF receptor proteins
  • Immunoglobulin-like proteins include cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, a Toll ligand receptor, B7-H3, BAFFR, BTLA, BLAME (SLAMF8), CD2, CD4, CDS, CD7, CDBalpha, CDSbet
  • CD lib CD 11c, CDlld, CD18, CD19, CD19a, CD27, CD28, CD29, CD30, CD40, CD49a, CD49D, CD49f, CD69, CD84, CD96 (Tactile), CD100 (SEMA4D), CD103, 0X40 (CD134), 4- 1BB (CD137), SLAM (SLAMF1, CD150, IPO-3), CD160 (BY55), SELPLG (CD162), DNAM1 (CD226), Ly9 (CD229), SLAMF4 (CD244, 2B4), ICOS (CD278), CEACAM1, CDS, CRTAM, DAP10, GADS, GITR, HVEM (LIGHTR), IA4, 1 CAM-1, IL2Rbeta, IL2R gamma, IL7R alpha, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS
  • each CS domain comprises a functional signaling domain derived from a costimulatory molecule.
  • the encoded CS domain comprises 4-1BB, CD28, or DAP10.
  • the CS domain comprises the amino acid sequence of CD28CS, 41BBCS, or DAP10CS (SEQ ID NO: 164, 165, or 166), or is encoded by the nucleotide sequence encoding provided as SEQ ID NOs: 264, 265, or 266.
  • cytokines refers to a broad category of small proteins that are involved in cell signaling. Generally, their release has some effect on the behavior of cells around them. Cytokines may be involved in autocrine signaling, paracrine signaling and/or endocrine signaling as immunomodulating agents. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. Cytokines are produced by a broad range of cells, including immune cells like macrophages, B lymphocytes, T lymphocytes and mast cells, as well as endothelial cells, fibroblasts, epithelial cells, and various stromal cells. “Chemokines” are a family of cytokines generally involved in mediating chemotaxis.
  • cytotoxicity generally refers to any cytocidal activity resulting from the exposure of the anti-ALPPL2 agents of the Invention or cells comprising the same to cells expressing ALPPL2. This activity may be measured by known cytotoxicity assays, including IFN-y production assays.
  • the target cell is a cancer or tumor cell
  • the term "anti-cancer cytotoxicity” or “anti-tumor cytotoxicity” may be used.
  • DAP10 refers to a protein, which In humans Is encoded by the HSCT gene. It may also be referred to as HSCT, KAP10, PIK3AP, or hematopoietic cell signal transducer. In some embodiments, DAP10 may have the sequence provided in Genbank Accession No.: Q9UBK5.1.
  • DAP10 costimulatory domain also referred to as “DAP10 CS domain” or “DAP10CS” refers to the amino add residues derived from the cytoplasmic domain of DAP10.
  • DAP10CS comprises the sequence provided as SEQ ID NO: 166 or the equivalent residues from a non-human spedes, e.g., mouse, rodent; monkey, ape and the like.
  • DAP10 CS domain may be encoded by the nucleic acid sequence provided as SEQ ID NO: 266.
  • ALPPL2 disease associated with expression of ALPPL2
  • ALPPL2- assodated disease includes, but is not limited to, a disease assodated with expression of ALPPL2 or condition assodated with cells which express ALPPL2 including, e.g., proliferative diseases such as a cancer or malignancy or a precancerous condition; or a noncancer-related indication assodated with cells which express ALPPL2.
  • proliferative diseases such as a cancer or malignancy or a precancerous condition
  • noncancer-related indication assodated with cells which express ALPPL2.
  • Examples of various cancers that express ALPPL2 include but are not limited to, pancreatic cancer, testicular cancer, cervical cancer, endometrial cancer, ovarian cancer, stomach cancer, colorectal cancer, lung cancer, mesothelioma, and tongue cancer, and the like.
  • an “effective amount” or “an amount effective to treat' refers to a dose that is adequate to prevent or treat a disease, condition, or disorder in an individual. Amounts effective for a therapeutic or prophylactic use will depend on, for example, the stage and severity of the disease or disorder being treated, the age, weight; and general state of health of the patient; another pre-existing condition, and the judgment of the prescribing physidan.
  • the size of the dose will also be determined by the active ingredient selected, method of administration, timing and frequency of administration, the existence, nature, and extent of any adverse side effects that might accompany the administration of a particular active ingredient; and the desired physiological effect It will be appredated by one of skill in the art that various diseases or disorders could require prolonged treatment involving multiple administrations, perhaps using the inventive anti-ALPPL2 agents, nucleic adds, vectors, cells, or compositions in each or various rounds of administration.
  • enteral refers to administration of a compound or composition to an individual by a route or mode along the alimentary canal.
  • oral routes of administration of a composition include, without limitation, swallowing liquid or solid forms of a composition from the mouth, administration of a composition through a nasojejunal or gastrostomy tube, intraduodenal administration of a composition, and rectal administration, e.g., using suppositories for the lower intestinal tract of the alimentary canal.
  • frame refers to the non-CDR portions of the variable region of an Ab, or in some embodiments, Antigen-binding Ab fragment or an antigen-binding domain of a CAR.
  • “Heavy chain (HC) framework” and “VH framework” are used interchangeably herein and refer to the non-CDR portion of a HC variable region, and in general, there are four framework regions (FRs) in each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4).
  • Light chain (LC) framework” and “VL framework” are used interchangeably herein and refer to the non-CDR portion of a LC variable region, and in general, there are four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4).
  • "human HC framework”, “human VH framework”, “human-like HC framework”, or “human-like VH framework” is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human HC framework.
  • human LC framework is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human LC framework.
  • genes are used broadly to refer to any segment of polynucleotide associated with a biological function.
  • genes include introns and exons as in genomic sequence, or just the coding sequences as in cDNAs and/or the regulatory sequences required for their expression.
  • gene also refers to a nucleic acid fragment that expresses mRNA or functional RNA, or encodes a specific protein, and which includes regulatory sequences.
  • spacer refers to an amino add sequence of variable length typically encoded between two or more domains or portions of a polypeptide construct to confer flexibility, improved spatial organization, proximity, eta
  • human antibody means an antibody having an amino add sequence corresponding to that of an antibody produced by a human and/or which has been made using any of the techniques for making human antibodies known to those skilled in the art or disdosed herein.
  • Human antibodies can be produced using various techniques known in the art In one embodiment; the human antibody is selected from a phage library, where that phage library expresses human antibodies (Vaughan etal., Nature Biotechnology, 14:309-314, 1996; Sheets et al., Proc. Natl Acad. Sci. (USA) 95:6157-6162, 1998; Hoogenboom and Winter,/. Mo/. Biol., 227:381, 1991; Marks et al., /.
  • Human antibodies can also be made by immunization of animals into which human immunoglobulin lod have been transgenically introduced in place of the endogenous lod, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described in U.S. Pat Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.
  • the human antibody may be prepared by Immortalizing human B lymphocytes that produce an antibody directed against a target antigen (such B lymphocytes may be recovered from an individual or from single cell cloning of the cDNA, or may have been immunized in vitro ⁇ . See, e.g., Cole et al. "Monoclonal Antibodies and Cancer Therapy", Alan R. Liss, p. 77, 1985; Boemer et al.,/. Immunol., 147 (l):86-95, 1991; and U.S. Pat No. 5,750,373.
  • humanization of an Ab refers to modification of an Ab of a nonhuman origin to increase the sequence similarity to an Ab naturally produced in humans.
  • humanized antibody refers to Abs generated via humanization of an Ab.
  • a humanized or engineered antibody has one or more amino acid residues from a source which is non-human, e.g., but not limited to mouse, rat, rabbit; non-human primate or another mammal. These human amino acid residues are often referred to as “import” residues, which are typically taken from an "Import” variable, constant or other domain of a known human sequence.
  • Known human Ig sequences are disclosed, e.g., www.ncbi.nlm.nih.gov/entrez/query.fcgi; www.atcc.org/phage/hdb.html, each entirely incorporated herein by reference.
  • Imported sequences can be used to reduce immunogenicity or reduce, enhance or modify binding, affinity, avidity, specificity, half-life, or any other suitable characteristic, as known in the art
  • part or all of the non-human or human CDR sequences are maintained while part or all of the non-human sequences of the framework and/or constant regions are replaced with human or other amino acids.
  • Antibodies can also optionally be humanized with retention of high affinity for the antigen and other favorable biological properties using three-dimensional immunoglobulin models that are known to those skilled in the art Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, framework (FR) residues can be selected and combined from the consensus and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.
  • Humanization or engineering of antibodies of the present invention can be performed using any known method, such as but not limited to those described in, for example, Winter (Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen etal., Science 239:1534 (1988)), Sims etal.,/. Immunol. 151: 2296 (1993); Chothia and Lesk,/. Mol. Biol. 196:901 (1987), Carter etal., Proc. Natl. Acad. Set USA. 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993), U.S. Pat Nos.
  • the CAR expressing cells of the invention are further transduced to express an iCAR. In one aspect; this iCAR is added to restrict the CAR expressing cell's functional activity to tumor cells.
  • immune cell refers to a cell of hematopoietic origin functionally involved in the initiation and/or execution of innate and/or adaptive immune response.
  • intracellular signaling domain refers to an intracellular portion of a molecule.
  • the intracellular signaling domain generates a signal that promotes an immune effector function of the cell transduced with a polynucleotide comprising a CAR, e.g., a CAR T cell.
  • immune effector function e.g., in a CAR T cell, include cytolytic activity and helper activity, including the secretion of cytokines.
  • ICS domains Include an ICS domain of a lymphocyte receptor chain, a TCR/CD3 complex protein, an Fc receptor subunit; an IL-2 receptor subunit; CD3 zeta, FcR gamma, FcRbeta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD 79a, CD79b, CD66d, CD278(ICOS), Fc epsilon RI, DAP10, or DAP12.
  • An "Isolated” biological component refers to a component that has been substantially separated or purified away from its environment or other biological components in the cell of the organism in which the component naturally occurs, for instance, other chromosomal and extra- chromosomal DNA and RNA, proteins, and organelles.
  • Nucleic acids and proteins that have been "isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant technology as well as chemical synthesis.
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • leader sequence or "LS” as used herein, also referred to as “signal peptide,” “signal sequence,” “targeting signal,” “localization signal,” “localization sequence,” “transit peptide,” or “leader peptide” in the art; is a short peptide present at the N- terminus of the majority of newly synthesized proteins that are destined towards the secretary pathway.
  • the core of the signal peptide may contain a long stretch of hydrophobic amino acids.
  • the signal peptide may or may not be cleaved from the mature polypeptide.
  • linker refers to a peptide linker that consists of amino acids such as glycine and/or serine residues used alone or in combination, to link variable heavy and variable light chain regions together.
  • the flexible polypeptide linker is a Gly/Ser linker and comprises one or more repeats of the amino acid sequence unit Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 167).
  • the flexible polypeptide linker includes, but is not limited to, (Gly4Ser)3, which is also referred to as G4S X3 (SEQ ID NO: 168).
  • Such a linker may be encoded for example, by the nucleic acid sequence as set forth in SEQ ID NO: 268 or 68.
  • the flexible linker may be a Whitlow linker (see e.g., Whitlow M et al., Protein Eng. 1993 Nov;6(8):989-95.).
  • a Whitlow linker may comprise the amino acid sequence of SEQ ID NO: 159 and may be encoded by e.g., the nucleic acid sequence of SEQ ID NO: 259.
  • a tag may be further included in the extracellular portion of the CAR, for example between the scFv and the hinge. In one embodiment; such a tag may be a myc tag.
  • the term "mammal” refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice, rats, and hamsters, and mammals of the order Logomorpha, such as rabbits.
  • the mammals may be from the order Carnivora, including Felines (cats) and Canines (dogs).
  • the mammals may be from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses).
  • the mammals may be of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes).
  • the term "masked CAR” refers to a CAR expressing cell that further comprises a masking peptide. This masking peptide may prevent off-target cell killing.
  • the masking peptide is often N-terminal to the CAR construct and may block the cell's ability to bind to unintended targets.
  • the masking peptide may be cleaved from the CAR expressing cell when it encounters a tumor thereby allowing the CAR expressing cell to attack its target without killing off-target cells.
  • An anti-ALPPL2 CARs of the present invention may be constructed to be a masked CAR.
  • multispecific refers to having two or more binding specificities.
  • An anti-ALPPL2 multispecific Ab or a multispecific antigen-binding Ab fragment, for example, of the present invention has at least one specificity for ALPPL2.
  • the first specificity is for an epitope for ALPPL2
  • the second (or third, fourth, and so forth) specificity may be for another epitope for ALPPL2 or may be for a molecule other than ALPPL2.
  • multispecific is also used in the same manner for any other anti-ALPPL2 agents of the present invention, such as anti-ALPPL2 CARs.
  • nucleic acid and “polynucleotide” refer to RNA or D NA that is linear or branched, single or double stranded, or a hybrid thereof. The term also encompasses RNA/DNA hybrids.
  • polynucleotides a gene or gene fragment; exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, Isolated RNA of any sequence, nucleic acid probes and primers.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracil, other sugars and linking groups such as fluororibose and thiolate, and nucleotide branches.
  • the sequence of nucleotides may be further modified after polymerization, such as by conjugation, with a labeling component Other types of modifications included in this definition are caps, substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides or solid support
  • the polynucleotides can be obtained by chemical synthesis or derived from a microorganism. [602]
  • the term "OKT3" or “Muromonab-CD3" or “Orthoclone OKT3” refers to a monoclonal anti-CD3 Ab.
  • parenteral or “parenterally” as used herein includes any route of administration of a compound or composition, characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue, thus generally resulting in the direct administration into the blood stream, into muscle, or into an internal organ.
  • Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like.
  • parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrastemal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intrasynovial injection or infusions; and kidney dialytic infusion techniques.
  • parenteral administration of the compositions of the present invention comprises subcutaneous or intraperitoneal administration.
  • pharmaceutically acceptable excipient refers to compounds or materials conventionally used in pharmaceutical compositions during formulation and/or to permit storage.
  • promoter is defined as a DNA sequence recognized by the synthetic machineiy of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence
  • the "ribosome skip sequence” refers to an amino acid sequence that; when translated, causes cleavage of a nascent polyprotein on the ribosome, allowing for coexpression of multiple genes.
  • the ribosome skip sequence may be the T2A sequence such as SEQ ID NO: 169, which may be encoded by SEQ ID NO: 269.
  • any other 2A sequences may be used. Examples of other sequences may be found elsewhere in the literature of the relevant art (for example, see Kim, J.H., et al., High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS One. 2011;6(4J).
  • the term "recombinant” means a polynucleotide, a protein, a cell, and so forth with semi-synthetic or synthetic origin which either does not occur in nature or is linked to another polynucleotide, a protein, a cell, and so forth in an arrangement not found in nature.
  • scFv single-chain Fv
  • single-chain variable fragment* refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked, e.g., via a synthetic linker, e.g., a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived.
  • a synthetic linker e.g., a short flexible polypeptide linker
  • an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-termlnal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL
  • the linker may comprise portions of the framework sequences.
  • the heavy chain variable region HC V, HCV, or VH
  • the light chain variable region LC V, LCV, or VL
  • the two domains may optionally be linked via a linker (for example, the G4S X3 linker).
  • the construct when the scFv is for example derived from h6E6, the construct may be referred to as h6E6scFvHL, h6E6HL, h6E6scFvVHVL, or h6E6VHVL
  • the heavy chain variable region may be placed downstream of the light chain variable region, and the two domains may optionally be linked via a linker (for example, the G4S X3 linker).
  • the construct when the scFv is for example derived from h6E6, the construct may be referred to as h6E6scFvLH, h6E6LH, h6E6scFvVLVH, or h6E6VLVH.
  • h6E6scFvLH when the scFv is for example derived from h6E6, the construct may be referred to as h6E6scFvLH, h6E6LH, h6E6scFvVLVH, or h6E6VLVH.
  • the same naming rules apply to other similar constructs herein.
  • signaling domain refers to the functional portion of a protein which acts by transmitting information within the cell to regulate cellular activity via defined signaling pathways by generating second messengers or functioning as effectors by responding to such messengers.
  • the term "stimulatory molecule,” refers to a molecule expressed by an immune cell (e.g., T cell, NK cell, B cell) that provides the cytoplasmic signaling sequence(s) that regulate activation of the immune cell in a stimulatory way for at least some aspect of the Immune cell signaling pathway.
  • the signal is a primary signal that is initiated by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, and which leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like.
  • a primary cytoplasmic signaling sequence (also referred to as a "primary signaling domain") that acts in a stimulatory manner may contain a signaling motif which is known as an immunoreceptor tyrosine-based activation motif or ITAM.
  • ITAM immunoreceptor tyrosine-based activation motif
  • Examples of an ITAM containing cytoplasmic signaling sequence that are of particular use in the invention include, but are not limited to, those derived from CD3 zeta, common FcR gamma (FCER1G), Fc gamma Rlla, FcR beta (Fc epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD 79b, DAP10, and DAP12.
  • the intracellular signaling domain in any one or more CARS of the invention comprises an intracellular signaling sequence, e.g., a primary signaling sequence of CD3 zeta.
  • the primary signaling sequence of human CD 3 zeta referred to as "CD3zICS" herein, is the amino add sequence provided as SEQ ID NO: 162, and may be encoded by the nudeotide sequence SEQ ID NO: 262.
  • equivalent residues from a non-human or mouse spedes e.g., rodent; monkey, ape and the like, may be utilized.
  • the term "subject” as used herein may be any living organisms, preferably a mammal.
  • the subject is a primate such as a human.
  • the primate is a monkey or an ape.
  • the subject can be male or female and can be any suitable age, including infant; juvenile, adolescent; adult; and geriatric subjects.
  • the patient or subject Is a validated animal model for disease and/or for assessing toxic outcomes.
  • the subject may also be referred to as "patient' in the art
  • the subject may have a disease or may be healthy.
  • suicide mechanism refers to a mechanism by which anti-ALPPL2 agent-expressing cells of present invention may be eradicated from a subject administered with such cells.
  • the suicide mechanism may be driven by, for example, inducible caspase 9 (Budde L E. et al., PLoS One. 2013 Dec 17;8(12):e82742. doi: 10.1371/joumal.pone.0082742. eCollection 2013), codon-optimized CD20 (Martin V. et al., Hum Gene Ther Methods.
  • the suicide mechanism may be included and utilized in CAR-expressing cells of present invention to optimize the length for the CAR- expresslng cells to stay in the system of a subject or the amount of the CAR-expressing cells, to reduce or minimize the toxicity and/or to maximize the benefit of CAR- expressing cells.
  • synthetic Ab or “synthetic antigen-binding Ab fragment” as used herein, refers to an Ab or antigen-binding Ab fragment which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein.
  • the term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino add sequence spedfying the antibody, wherein the DNA or amino add sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art
  • target refers to the molecule that an anti-ALPPL2 agents of the present invention spedfically binds to.
  • the term also encompasses cells and tissues expressing the target molecule and also diseases that are assodated with expression of the target
  • target cell refers to a cell expressing the target molecule (such as ALPPL2) of the anti-ALPPL2 agents of the present invention on the cell surface.
  • the target cell is a cancer cell or tumor cell.
  • the target cell is a vascular cell.
  • the target cell is an epithelial cell.
  • the target cell is a cell type that has a particular role in the pathology of cancer or inflammation.
  • the target cell is a cell type that has a particular role in the pathology of a disease such as but not limited to cancer.
  • target molecule refers to a molecule that is targeted by the anti-ALPPL2 agents of the present invention.
  • the antigen-binding domain of the anti-ALPPL2 agents of the present invention has a binding affinity for the target molecule.
  • the target molecule is ALPPL2.
  • trCD19 refers to a truncated version of the CD19 protein, B- lymphocyte antigen CD 19, also known as CD 19 (Cluster of Differentiation 19), which is a protein that Is encoded by the CD19 gene In humans and Is found on the surface of B- cells.
  • the trCD 19 construct is any truncated version of said protein, such that a nucleic acid sequence encoding this construct may be transduced into a host cell and expressed on the surface of this cell for the purposes of detection, selection, and/or targeting.
  • human trCD19 may comprise the amino acid sequence of SEQ ID NO: 170 or nucleotide sequence encoding such, such as SEQ ID NO: 270.
  • transfected refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell
  • a "transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid.
  • the cell includes the primary subject cell and its progeny.
  • transmembrane domain or "TM domain”
  • TM domain any three-dimensional protein structure which Is thermodynamically stable In a membrane. This may be a single alpha helix, a transmembrane beta barrel, a beta-helix of gramicidin A, or any other structure. Transmembrane helices are usually about 20 amino acids in length. Typically, the transmembrane domain denotes a single transmembrane alpha helix of a transmembrane protein, also known as an Integral protein.
  • the term “treat;* "treatment;” or “treating” generally refers to the clinical procedure for reducing or ameliorating the progression, severity, and/or duration of a disease or of a condition, or for ameliorating one or more conditions or symptoms (preferably, one or more discernible ones) of a disease.
  • the type of disease or condition to be treated may be, for example, but are not limited to, cancer and cancer- associated diseases and conditions.
  • cancer include, but are not limited to, pancreatic cancer, testicular cancer, cervical cancer, endometrial cancer, ovarian cancer, stomach cancer, colorectal cancer, lung cancer, mesothelioma, and tongue cancer.
  • the effect of the "treatment* may be evaluated by the amelioration of at least one measurable physical parameter of a disease, resulting from the administration of one or more therapies (e.g., an anti-ALPPL2 Ab or antigen-binding Ab fragment; anti-ALPPL2 ADC, or anti-ALPPL2 CAR expressing cell).
  • therapies e.g., an anti-ALPPL2 Ab or antigen-binding Ab fragment; anti-ALPPL2 ADC, or anti-ALPPL2 CAR expressing cell.
  • the parameter may be, for example, gene expression profiles, the mass of disease-affected tissues, inflammation-associated markers, cancer-associated markers, the number or frequency of disease-associated cells, tumor burden, the presence or absence of certain cytokines or chemokines or other disease-associated molecules, and may not necessarily discernible by the patient
  • "treat*, "treatment;” or “treating” may result in the inhibition of the progression of a disease, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g., stabilization of a physical parameter, or both.
  • the terms “treat”, “treatment” and “treating” refer to the reduction or stabilization of cancerous tissue or cells.
  • xenogeneic or "xeno-" refers to a graft derived from an animal of a different species.
  • mice Three mice were intraperitoneally immunized with 50 ug of human ALPPL2 (produced in CHO cells) admixed with complete Freund's adjuvant (CFA) as an adjuvant
  • CFA complete Freund's adjuvant
  • IFA incomplete Freund's adjuvant
  • Hybridomas were then screened. 50 pl of hybridoma cell culture was co-cultured with 1x10 s ALPPL2 positive cells or IxlO 5 ALPPL2 negative cells for 1 hour at 4 °C on a multi-well plate. Each well was evaluated for ALPPL2 binding using an anti-mouse Igx antibody by flow cytometry. Antibodies produced by the selected hybridomas were sequenced using a conventional method.
  • Human T cells from Donor 1 were transduced with a vector encoding anti- ALPPL2 CAR (h3H2scFvHL-CD28H-CD28TM-CD28CS-CD3zICS) or an empty vector (EV, i.e., trCD19 only) and enriched for trCD 19-positive cells as depicted in FIG 4.
  • the CAR constructs used to express CARs in this Example are SEQ ID NOs: 571 and 671 (amino acid sequence and nucleic acid sequence, respectively, for anti-ALPPL2 CAR "LS- h3H2scFvHL-CD28H-CD28TM-CD28CS-CD3zICS-T2A-trCD19").
  • Example 4 In vitro cytotoxicity by anti-ALPPL2 CAR-expressing T cells ⁇ Materials>
  • ALPPL2 positive cancer cells (Cal27, BxPC3, H1651, and Capan2) were transduced with luciferase expression vector, and luciferase positive cells were selected using puromycin and used as target cells.
  • Anti-ALPPL2 CAR expressing human T cells from Example 3 were expanded and used as effector cells.
  • T cells were plated with 5,000 luciferase-expressing ALPPL2 positive cancer cells at effector : target (E:T) ratios of 10:1, 3:1, 1:1, and 0.3:1 in 96-well light-blocking luminometer plates. After 24hrs of coculture, remaining live tumor cells were detected by luciferase activity as measured by luminescence (FIG SA, SB, SC, and 5D top). The plates were incubated an additional 24hrs to allow for a 48hr measurement (FIG SA, SB, SC, and 5D bottom).
  • FIG 5 After 24hrs and 48hrs of co-culture, a significant reduction in all ALPPL2 positive cells was observed (FIG 5). As shown in FIG 5, the reduction in cancer cells was observed with both the very low-expressor of ALPPL2 (Cal27 cells) and the high expressor pf ALPPL2 (H1692 cells).
  • Example 5 In vitro cytokine production by anti-ALPPL2 CAR-expressing T cells ⁇ Methods>
  • Anti-ALPPL2 CAR expressing human T cells and EV-transduced human T cells from Example 3 were expanded. 10 A 5 ALPPL2 positive cancer cells (CAL27, BxPC3, H1651, Capan2, and Capanl) were cultured with 10 A 5 CART cells, 10 A 5 EVT cells, or no T cells per well in a 96-well plate for 24 hours. Control wells only containing 10 A 5 CAR T cells, or EV T cells were also included. Supernatants were collected and IFN-g levels were measured by ELISA Two independent experiments referred to as "017” and "018", respectively, were performed.
  • IFN-g concentrations detected in the supernatant are shown in FIG 6. Significantly higher levels of IFN-g were observed when CAR T cells were cocultured with ALPPL2 positive cancer cells compared to when EV T cells were cocultured with ALPPL2 positive cancer cells. No IFN-g was detected from wells without a T cell.
  • Example 6 In vivo efficacy by anti-ALPPL2 CAR-expressingT cells
  • mice were then administered with 5xl0 6 human T cells expressing trCD19 but no anti- ALPPL2 (EV T) or 5xl0 6 human T cells expressing anti-ALPPL2 CAR (h3H2scFvHL- CD28CS CAR) intraperitoneally on Day 7.
  • the tumor burden of individual mice was monitored by bioluminescent imaging using the Xenogen-IVIS® Imaging System every week starting on Day 6. The weight of individual mice was recorded periodically starting on Day 0.
  • FIGS 7A-7C Exemplary changes in the tumor burden observed in the two treatment groups (EV T and CAR T) are shown in FIGS 7A-7C
  • the tumor burden was detectable (both approximately 1.2x10 9 photons/sec) on Day 6.
  • the tumor burden decreased in CAR T group by Day 13.
  • the tumor burden difference between the CAR T group and the EVT group became significant by Day 34.
  • mice in both groups continued to gain weight during the experiment No statistically significant difference was found in the average body weight between the treatment groups.
  • Example 7 In vivo efficacy by anti-ALPPL2 CAR-expressingT cells ⁇ Materials>
  • mice 2.5xl0 6 H1651-Luc cells were injected intraperitoneally into NSG mice on Day 0. Mice were then administered with 5xl0 6 human T cells expressing trCD19 butno anti- ALPPL2 (EV T) or 5xl0 6 human T cells expressing anti-ALPPL2 CAR (h3H2scFvHL- CD28CS CAR) intraperitoneally on Day 7. The tumor burden of individual mice was monitored by bioluminescent imaging using the Xenogen-IVIS® Imaging System every week starting on Day 6. The weight of individual mice was recorded periodically starting on Day 0.
  • FIGS 9A-9B Exemplary changes in the tumor burden observed in the two treatment groups (EV T and CAR T) are shown in FIGS 9A-9B.
  • the tumor burden was detectable (both approximately 1.2x10® photons/sec) on Day 6.
  • the tumor diminished almost completely in CAR T group by Day 13, and the tumor burden remained extremely low at least up to Day 34.
  • the tumor burden in CAR T group was significantly lower than that in EVT group throughout the experiment
  • Random mutations were introduced to the CDR-encoding sequences of the VH and VL of h3H2 and mouse 3H2 to generate 13 different affinity maturation libraries, each having the diversity of >10 6 .
  • the VH and VL of mouse 3H2 were included as parent sequences at least partly because mouse 3H2 seemed to provide better binding to human ALPPL2 than h3H2, when expressed as an IgG.
  • the libraries were transduced into cells and screened based on binding to soluble human ALPPL2 antigen using a mammalian display system. Selected clones were then expressed as antibodies and further tested for binding to human ALPPL2 antigen and to ALPPL2-expressing cells and the level of non-specific binding (binding to ALPPL2-negative cell lines). Antibodies that provide improved binding relative to mouse 3H2 were sequenced using a conventional method.
  • 1B5 as an IgG was obtained either as supernatant of 1B5 IgG-producing cell culture or as a protein A-purified IgG.
  • human ALPPL2-expressing cells were incubated with different concentrations of IBS or 3H2 IgG, and unbound antibodies were removed. Binding was measured by flow cytometry, and the mean fluorescence intensity (MFI) values were compared.
  • H1651 cells cell line derived from human non-small cell lung cancer, adenocarcinoma
  • CH0-ALPPL2 cells CHO cells transduced with human ALPPL2
  • MCF7 cells cell line derived from human breast adenocarcinoma
  • CHO cells were used as negative control cells.
  • Binding to recombinant human ALPPL2 by IBS IgG (supernatant or purified) in comparison to mouse 3H2 IgG was analyzed using a commercially available ELISA kit Monovalent binding to recombinant human ALPPL2 by purified IBS IgG in comparison to mouse 3H2 IgG was analyzed by Biolayer Interferometry (BLI).
  • FIG 11A Exemplary results from cell binding analyses on various cell types are provided in FIG 11A.
  • FIG 11B Exemplary results using CH0-ALPPL2 cells with more antibody concentrations are provided in FIG 11B.
  • IBS IgG had higher MFI than 3H2 IgG with H1651 cells and CHO-ALPPL2 cells, indicating improved binding to ALPPL2-expressing cells.
  • Exemplary binding results analyzed by ELISA are shown in FIG 12 (purified IgG (top) or supernatant (bottom).
  • Exemplary monovalent binding results analyzed by BLI are provided in FIG 13, with the association rate constant (ka), equilibrium dissociation constant (ka), and dissociation constant (KD) calculated for IBS and 3H2.
  • the calculated KD values Indicated that the variable region of IBS has an about 22-fold Increase in the affinity relative to the variable region of mouse 3H2.
  • IBS was humanized by humanizing the VL sequence.
  • the resulting antibody named B001, comprising the VH of SEQ ID NO: 711 (CDR1, CDR2, and CDR3 of SEQ ID NOS: 712-714) and the VL of SEQ ID NO: 715 (CDR1, CDR2, and CDR3 of SEQ ID NOS: 716-718), was found to have reduced affinity (comparison available in FIG 14 top). Therefore, further affinity maturation was performed.
  • Random mutations were introduced to either: (i) the CDR3-encoding sequence of B001 VH, while introducing no changes to the VL of B001; or (ii) the CDR3-encoding sequence of the B001 VL, while introducing no changes to the VH of B001, to generate four affinity maturation libraries, each having the diversity of >10 6 .
  • the libraries were transduced into mammalian cells and screened based on binding to soluble human ALPPL2 antigen using a mammalian display system. Selected clones were then expressed as antibodies and further tested for biding to human ALPPL2 antigen and binding to ALPPL2 -expressing cells and the level of non-specific binding (binding to ALPPL2 -negative cell lines).
  • Antibodies that provided binding at least similar to 1B5 were selected and sequenced using a conventional method.
  • FIG 14 top Exemplary binding curves from three replicate ELISA experiments to confirm efficient ALPPL2 binding to plate-bound ALPPL2 by the selected antibodies are provided in FIG 14 bottom. All four antibodies showed binding (avidity) equivalent to or greater than that of IBS.
  • All four antibodies contained one or two substitutions in the CDR3 of VH (particularly within the "AMDY" motif) relative to the VH of B001 (i.e., relative to the VH of IBS). All four antibodies comprised the VL sequence of B001 (i.e., the VH of h3H2). SEQ ID NOs corresponding to the VH, VL, and CDR amino acid sequences are summarized in Table 5. Table 6 shows % identity of the VH and VL sequences to corresponding human germline sequences for each antibody.
  • Table 5 SEQ ID NOs assigned to amino acid sequences for B380, B416, B506, and B735
  • Example 12 Cell binding analyses on B380, B416, B506, and B735 ⁇ Methods>
  • CH0-ALPPL2 cells were incubated with different concentrations of IBS, B380, B416, B506, or B735 IgG, and unbound antibodies were removed. Binding was measured by flow cytometry, and the MFI values were compared.

Abstract

The disclosure provides anti-ALPPL2 agents such as anti-ALPPL2 antibodies (Abs), antigen-binding Ab fragments, multi-specific Abs and antigen-binding Ab fragments, antibody-drug conjugates (ADCs), and chimeric antigen receptors (CARs). The disclosure also provides polynucleotides and vectors encoding, cells and pharmaceutical compositions comprising such anti-ALPPL2 agents and/or polynucleotides. The present disclosure further relates to methods of treating a subject using such anti-ALPPL2 agents and compositions, and to methods of treating, preventing, or diagnosing a disease such as cancer and methods of stimulating an immune response. Also provided are methods of producing such anti-ALPPL2 agents and cells.

Description

ANTI-ALPPL2 ANTIBODIES AND CHIMERIC ANTIGEN RECEPTORS, AND COMPOSITIONS AND METHODS OF USE
RELATED APPLICATIONS
[001] This application claims priority to U.S. Provisional Application No.: 63/337,242 filed on May 2, 2022, entitled "ANTI-ALPPL2 ANTIBODIES AND CHIMERIC ANTIGEN RECEPTORS, AND COMPOSITIONS AND METHODS OF USE", the contents of which are Incorporated by reference in their entirety herein.
REFERENCE TO AN ELECTRONIC SEQUENCE LISTING
[002] The contents of the electronic sequence listing (2953247.001013.xml; Size: 558,131 bytes; and Date of Creation: May 1, 2023) is herein incorporated by reference in its entirety.
FIELD OF INVENTION
[003] The present disclosure relates to anti-ALPPL2 agents such as anti-ALPPL2 antibodies (Abs), antigen-binding Ab fragments, multi-specific Abs and antigen-binding Ab fragments, antibody-drug conjugates (ADCs), and chimeric antigen receptors (CARs). The disclosure also relates to polynucleotides and vectors encoding, cells and pharmaceutical compositions comprising such anti-ALPPL2 agents and methods for expanding such cells. The present disclosure further relates to methods of treating a subject using such anti-ALPPL2 agents and compositions, and to methods of treating, preventing, or diagnosing a disease such as cancer and methods of stimulating an immune response. The invention also relates to methods of producing such an anti- ALPPL2 agent or composition.
BACKGROUND OF THE INVENTION
[004] Immunotherapy is a growing field, allowing for treatment of a range of diseases that did not have an effective treatment option before. Many examples of immunotherapy have been employed in oncology, including antibody therapies targeting CD20 (Non-Hodgkin's lymphoma), HER2 (HER2 -positive breast cancer), and immune checkpoints such as PD-1, PD-L1, and CTLA-4 (various cancers).
Immunotherapies are also being developed, tested, and/or marketed for non-cancer disease indications, such as autoimmune diseases (Wraith D.C. etal, Front Immunol. 2017 Nov 28;8:1668. doi: 10.3389/fimmu.2017.01668. eCollection 2017).
[005] Chimeric antigen receptor (CAR)-based cell therapy is a type of immunotherapy whereby immune cells, such as T cells, NK cells, or macrophages, are genetically modified to express a receptor that allows recognition of a specific antigen. Upon antigen recognition, the cells ere activated via signaling domains, converting them Into potent cdl killers and/or immune stimulators. In 2017, an anti-CD19 CAR T cdl product received FDA approval for B cell lymphoma, illustrating the potential of this therapeutic approach in cancer, e-g, hematological cancer [Leyftnan Y, et aL, Cancer OB Int 2018 Nov 14;18:182. dot 10.1186/S12935-018-06B5-X. eCollection 2018). While also showing great promise fir the treatment of solid tumors (Louis C(J. et aL, Blood. 2011 Dec 1; 118(23): 6050-6056. Prepublished online 2011 Oct 7. doi: 10.1182/blood-2011- 05-354449), CAR-based cell therapy has encountered additional challenges in these indications (Yong CSJd. etaL, Immunol OB Biol. 2017 Apn95(4):356-363. dot 10.1038/icb.2016.123. Epub 2016 Dec 22): the presence of an immunosuppressive tumor microenvironment; the issue of access to tumors; and a lack of the tumor- selective targeting required to minimize "off-tumoi'* toxidty.
[006] Another potential challenge of CAR-based therapy is die presence of soluble firms of a taiget protein such as those in the serum (Garda-Guerrero E et at. Front Immunol.2020; 11: 1128.). This may occur, fir example, if die taiget protein on the cell surface becomes cleaved or shed, or if the taiget cell releases exosomes containing the taiget protein. These taiget proteins outside the cell can act as a decoy fir CAR and potentially limit the effectiveness of CAR-based cell therapy. In feet; many cancer antigens are known to be shed, and fir example, a soluble version of die BCMA antigen of myeloma was found to reduce cytokine production and cytotoxicity by anti-B CMA CAR T cells (Pont M. J. etaL, Blood. 2019 Nov 7;134(19): 1585-1597. doi: 10.1182 /blood2019000050.).
[007] Alkaline phosphatase, placenta-like 2 (ALPPL2), also referred to as "ALPPL", "alkaline phosphatase, germ cell (APGC)", or "germ cell alkaline phosphatase (GCAF)", is a member of die alkaline phosphatase (AP) family and is a glycosyl phosphatidyl Inositol (GPI)-anchored membrane-bound enzyme. In healthy humans, the protein expression of ALPPL2 is primarily in the placenta (bttps://www.proteinatiaMrg/ENSG00000163286-ALPPL2/tiMue) and ALPPL2 regulates naive pluripotency (Bi Y. etaL, OU Rep. 2020 Mar 17;30(ll):3917-3931.e5. doi: 10.1016/j.cdrep2020.02.090.). APPL2 expression in the body may be altered by exposure to and/or intake of an exogenous substance (Nisio A. D. etaL, Chemosphere. 2020 Mar;242: 125208. doi: 10.1016/j.chemoephere2019.125208. Epub 20190ct24, (Kondratyev N. etaL, Cffn Eptgenetics. 2018 Oct 23;10(l): 130. dob 10.1186/sl3148- 018-0565-1, Chuang Y-H. etaL, EurJHum Genet 2017 May;25(5):608-61& doi: 10.1038/ejhg.2016.175. Epub 2017 Feb 15.) and also is upregulated in certain types of cancer. Based on the study that showed ALPPL2 is present in pancreatic cancer-derived extracellular vesicles (&g, exosomes); ALPPL2 is proposed as a potential biomarker that may be used in liquid biopsy-based diagnosis for pancreatic cancer (Sb In HS. et aL, Mol Ther Methods CHn Dev. 2019 Sep 12;15:204-210. doL* 10.1016/j.omtro.2019.08.016. eCollection 2019 Dec 13.). Another study showed that an anti-ALPPL2 antibody drug conjugate (ADC) reduced tumor yowth in a mesothelioma animal model (W02017095823). However, no group has reported generation and/or efficacy of anti- ALPPL2 CAR cells.
SUMMARY OF THE INVENTION
[008] The present disclosure provides anti-ALPPL2 agents.
[009] In some aspects, the anti-ALPPL2 anentls an antibody (Ab) or antigen-binding Ab fragment The Ab or antigen-binding Ab fragment may bind to ALPPL2 and comprise: (a) a heavy chain (HC) variable region (VH); and (b) a light chain (LC) variable region (VL). The VH may comprise: a HC complementarity determining region (CDR) 1 (also referred to as CDR-H1); a HC CDR 2 (also referred to as CDR-H2); and a HC CDR 3 (also referred to as CDR-H3). The VL may comprise: a VL CDR 1 (CDR-L1); and a VL CDR 2 (CDR-L2); a VL CDR 3 (CDR-L3).
[010] In some embodiments, the CDR-H1, CDR-H2, and CDR-H3 may be the CDR-H1, CDR-H2, and CDR-H3, respectively, comprised in the VH of any anti-ALPPL2 antibodies described herein such as but not limited to 8735, 8506, 8416, 8380, 8001, 185, h3H2, hlE8, hlB2, 3H2, 1E8, or 182; and the CDR-L1, CDR-L2, and CDR-L3 maybe the CDR-L1, CDR-L2, and CDR-L3, respectively, comprised in the VL of any anti-ALPPL2 antibodies described herein such as butnot limited to 8735, 8506, 8416, 8380, 8001, 185, h3H2, hlE8, hlB2, 3H2, 1E8, or 182.
[011] In certain embodiments, the VH may comprise the CDR-H1, CDR-H2, and CDR- H3 sequences contained in SEQ ID NO: 751, and/or the VL may comprise the CDR-L1, CDR-L2, and CDR-L3 sequences contained in SEQ ID NO: 755.
[012] In certain embodiments, the VH may comprise the CDR-H1, CDR-H2, and CDR- H3 sequences contained in SEQ ID NO: 741, and/or the VL may comprise the CDR-L1, CDR-L2, and CDR-L3 sequences contained in SEQ ID NO: 745.
[013] In certain embodiments, the VH may comprise the CDR-H1, CDR-H2, and CDR- H3 sequences contained in SEQ ID NO: 731, and/or the VL may comprise the CDR-L1, CDR-L2, and CDR-L3 sequences contained in SEQ ID NO: 735.
[014] In certain embodiments, the VH may comprise the CDR-H1, CDR-H2, and CDR- H3 sequences contained in SEQ ID NO: 721, and/or the VL may comprise the CDR-L1, CDR-L2, and CDR-L3 sequences contained in SEQ ID NO: 725.
[015] In certain embodiments, the VH may comprise the CDR-H1, CDR-H2, and CDR- H3 sequences contained in SEQ ID NO: 711, and/or the VL may comprise the CDR-L1, CDR-L2, and CDR-L3 sequences contained in SEQ ID NO: 715.
[016] In certain embodiments, the VH may comprise the CDR-H1, CDR-H2, and CDR- H3 sequences contained in SEQ ID NO: 701, and/or the VL may comprise the CDR-L1, CDR-L2, and CDR-L3 sequences contained in SEQ ID NO: 705 or encoded in SEQ ID NO: 805.
[017] In certain embodiments, the VH may comprise the CDR-H1, CDR-H2, and CDR- H3 sequences contained in SEQ ID NO: 331 or encoded in SEQ ID NO: 431, and/or the VL may comprise the CDR-L1, CDR-L2, and CDR-L3 sequences contained In SEQ ID NO: 335 or encoded in SEQ ID NO: 435.
[018] In certain embodiments, die VH may comprise the CDR-H1, CDR-H2, and CORNS sequences contained in SEQ ID NO: 321 or encoded in SEQ ID NO: 421, and/or the VL may comprise the CDR-L1, CDR-L2, and CDR-L3 sequences contained In SEQ ID NO: 325 or encoded in SEQ ID NO: 425.
[019] In certain embodiments, die VH may comprise the CDR-H1, CDR-H2, and CDR- H3 sequences contained in SEQ ID NO: 311 or encoded in SEQ ID NO: 411, and/or the VL may comprise the CDR-L1, CDR-L2, and CDR-L3 sequences contained In SEQ ID NO: 315 or encoded in SEQ ID NO: 415.
[020] In certain embodiments, die VH may comprise the CDR-H1, CDR-H2, and CORNS sequences contained in SEQ ID NO: 131 or encoded in SEQ ID NO: 231, and/or the VL may comprise the CDR-L1, CDR-L2, and CDR-L3 sequences contained In SEQ ID NO: 135 or encoded in SEQ ID NO: 235.
[021] In certain embodiments, die VH may comprise the CDR-H1, CDR-H2, and CORNS sequences contained in SEQ ID NO: 121 or encoded in SEQ ID NO: 221, and/or the VL may comprise the CDR-L1, CDR-L2, and CDR-L3 sequences contained In SEQ ID NO: 125 or encoded in SEQ ID NO: 225.
[022] In certain embodiments, die VH may comprise the CDR-H1, CDR-H2, and CORNS sequences contained in SEQ ID NO: 111 or encoded in SEQ ID NO: 211, and/or the VL may comprise the CDR-L1, CDR-L2, and CDR-L3 sequences contained In SEQ ID NO: 115 or encoded in SEQ ID NO: 215.
[023] In some embodiments, the CDR-H1, CDR-H2, and CDR-H3 may be the CDR-H1, CDR-H2, and CDR-H3, respectively, of any anti-ALPPL2 antibodies described herein such as but not limited to B735, B506, B416, B380, B001, 1B5, h3H2, hlE8, hlB2, 3H2, 1E8, or 1B2; and/or the CDR-L1, CDR-L2, and CDR-L3 may be the CDR-L1, CDR-L2, and CORLS, respectively, of any anti-ALPPL2 antibodies described herein such as but not limited to B735, B506, B416, B380, B001, IBS, h3H2, hlE8, hlB2, 3H2, 1E8, or 1B2.
[024] In certain embodiments, the CDR-H1, CDR-H2, and CDR-H3 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 752, 753, and 754, respectively; and/or the CDR-L1, CDR-L2, and CDR-L3 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 756, 757, and 758, respectively.
[025] In certain embodiments, the CDR-H1, CDR-H2, and CDR-H3 may comprise or consist of the amino acid sequence set forth In SEQ ID NOs: 742, 743, and 744, respectively; and/or the CDR-L1, CDR-L2, and CDR-L3 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 746, 747, and 748, respectively.
[026] In certain embodiments, the CDR-H1, CDR-H2, and CDR-H3 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 732, 733, and 734, respectively; and/or the CDR-L1, CDR-L2, and CDR-L3 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 736, 737, and 738, respectively.
[027] In certain embodiments, the CDR-H1, CDR-H2, and CDR-H3 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 722, 723, and 724, respectively; and/or the CDR-L1, CDR-L2, and CDR-L3 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 726, 727, and 728, respectively.
[028] In certain embodiments, the CDR-H1, CDR-H2, and CDR-H3 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 712, 713, and 714, respectively; and/or the CDR-L1, CDR-L2, and CDR-L3 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 716, 717, and 718, respectively.
[029] In certain embodiments, the CDR-H1, CDR-H2, and CDR-H3 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 702, 703, and 704, respectively; and/or the CDR-L1, CDR-L2, and CDR-L3 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 706, 707, and 708, respectively or may be encoded by SEQ ID NOs: 806, 807, and 808, respectively.
[030] In certain embodiments, the CDR-H1, CDR-H2, and CDR-H3 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 332, 333, and 334, respectively or may be encoded by SEQ ID NOs: 432, 433, and 434, respectively; and/or the CDR-L1, CDR-L2, and CDR-L3 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 336, 337, and 338, respectively or may be encoded by SEQ ID NOs: 436, 437, and 438, respectively.
[031] In certain embodiments, the CDR-H1, CDR-H2, and CDR-H3 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 322, 323, and 324, respectively or may be encoded by SEQ ID NOs: 422, 423, and 424, respectively; and/or the CDR-L1, CDR-L2, and CDR-L3 may comprise or consist of the amino add sequence set forth in SEQ ID NOs: 326, 327, and 328, respectively or may be encoded by SEQ ID NOs: 426, 427, and 428, respectively.
[032] In certain embodiments, the CDR-H1, CDR-H2, and CDR-H3 may comprise or consist of the amino add sequence set forth in SEQ ID NOs: 312, 313, and 314, respectively or may be encoded by SEQ ID NOs: 412, 413, and 414, respectively; and/or the CDR-L1, CDR-L2, and CDR-L3 may comprise or consist of the amino add sequence set forth in SEQ ID NOs: 316, 317, and 318, respectively or may be encoded by SEQ ID NOs: 416, 417, and 418, respectively.
[033] In certain embodiments, the CDR-H1, CDR-H2, and CDR-H3 may comprise or consist of the amino add sequence set forth in SEQ ID NOs: 132, 133, and 134, respectively or may be encoded by SEQ ID NOs: 232, 233, and 234, respectively; and/or the CDR-L1, CDR-L2, and CDR-L3 may comprise or consist of the amino add sequence set forth in SEQ ID NOs: 136, 137, and 138, respectively or may be encoded by SEQ ID NOs: 236, 237, and 238, respectively.
[034] In certain embodiments, the CDR-H1, CDR-H2, and CDR-H3 may comprise or consist of the amino add sequence set forth in SEQ ID NOs: 122, 123, and 124, respectively or may be encoded by SEQ ID NOs: 222, 223, and 224, respectively; and/or the CDR-L1, CDR-L2, and CDR-L3 may comprise or consist of the amino acid sequence set forth In SEQ ID NOs: 126, 127, and 128, respectively or may be encoded by SEQ ID NOs: 226, 227, and 228, respectively.
[035] In certain embodiments, the CDR-H1, CDR-H2, and CDR-H3 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 112, 113, and 114, respectively or may be encoded by SEQ ID NOs: 212, 213, and 214, respectively; and/or the CDR-L1, CDR-L2, and CDR-L3 may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 116, 117, and 118, respectively or may be encoded by SEQ ID NOs: 216, 217, and 218, respectively.
[036] In some embodiments, the % sequence identity of the VH amino acid sequence to a germline-encoded human VH sequence, optionally to the VH encoded by the human VH2-5 gene segment allele 8, may be about 80% or higher, about 85% or higher, about 90% or higher, about 93% or higher, about 95% or higher, about 96% or higher, about 97% or higher, about 98% or higher, about 98% or higher, about 99% or higher, or about 100%.
[037] In some embodiments, the % sequence identity of the VL amino acid sequence to a germline-encoded human VL, optionally to the VL encoded by the human IgKl-16 gene segment allele 1 may be about 80% or higher, about 85% or higher, about 86% or higher, about 90% or higher, about 93% or higher, about 95% or higher, about 96% or higher, about 97% or higher, about 98% or higher, about 98% or higher, or about 99% or higher, or about 100%,
[038] In some embodiments, the VH may comprise a human-like VH framework which may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human VH framework, optionally to any one of the human VH frameworks comprised in any one of the VH polypeptides of SEQ ID NO: 751, 741, 731, 721, 711, 701, 311, 321 and 331. In some embodiments, the VL may comprise a human-like VL framework which may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human VL framework, optionally to any one of the human VL frameworks comprised in any one of the VL polypeptides of SEQ ID NO: 755, 745, 735, 725, 715, 315, 325 or 335.
[039] In some embodiments, the VH and/or VL may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH and/or VH of any anti-ALPPL2 antibodies described herein such as but not limited to 8735, 8506, 8416, 8380, 8001, 185, h3H2, hlE8, hlB2, 3H2, 1E8, or 1B2 or may be encoded by a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH-encodlng sequence and/or VL-encodlng sequence of any anti-ALPPL2 antibodies described herein such as but not limited to 8735, 8506, 8416, 8380, 8001, 185, h3H2, hlE8, hlB2, 3H2, 1E8, or 182.
[040] In certain embodiments, the VH may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% Identical to SEQ ID NO: 751; and/or the VL may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 755.
[041] In certain embodiments, the VH may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 741; and/or the VL may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 745.
[042] In certain embodiments, the VH may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% Identical to SEQ ID NO: 731; and/or the VL may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 735.
[043] In certain embodiments, the VH may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 721; and/or the VL may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 725.
[044] In certain embodiments, the VH may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 711; and/or the VL may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 715.
[045] In certain embodiments, the VH may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 701; and/or the VL may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 705 or may be encoded by a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 805.
[046] In certain embodiments, the VH may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% Identical to SEQ ID NO: 111, or may be encoded by a nucleic add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 211; and/or the VL may comprise an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115, or may be encoded by a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 215.
[047] In certain embodiments, the VH may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 121, or may be encoded by a nucleic add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% Identical to SEQ ID NO: 221; and/or the VL may comprise an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 125, or may be encoded by a nucleic add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 225.
[048] In certain embodiments, the VH may comprise an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 131, or may be encoded by a nucleic add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 231; and/or the VL may comprise an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 135, or may be encoded by a nucleic add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 235.
[049] In certain embodiments, the VH may comprise an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 311, or may be encoded by a nucleic add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 411; and/or the VL may comprise an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 315, or may be encoded by a nucleic add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 415. [050] In certain embodiments, die VH may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 321, or may be encoded by a nucleic add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 421; and/or the VL may comprise an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 325, or may be encoded by a nucleic add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% Identical to SEQ ID NO: 425.
[051] In certain embodiments, die VH may comprise an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 331, or may be encoded by a nucleic add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 431; and/or the VL may comprise an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 335, or may be encoded by a nucleic add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 435.
[052] In some embodiments, the Ab or antigen-binding Ab fragment may be or may comprise or consist of any one or more of, for example, but not limited to, a monoclonal Ab, a monospedfic Ab, a bispedfic Ab, a multispedfic Ab, a humanized Ab, a tetrameric Ab, a tetravalent Ab, a single chain Ab, a domain-spedfic Ab, a domain-deleted Ab, an scFc fusion protein, a chimeric Ab, a synthetic Ab, a recombinant Ab, a hybrid Ab, a mutated Ab, CDR-grafted Ab, a fragment antigen-binding (Fab), an F(ab')2, an Fab' fragment; a variable fragment (Fv), a single-chain Fv (scFv), an Fd fragment, a diabody, or a minibody.
[053] In certain embodiments, the antibody or antigen-binding Ab fragment may comprise or consists of a scFv comprising or consisting of a VH. a VL, and a linker which joins the VH and VL, and the VH and VL may be any of the VH and VL polypeptides described above or herein.
[054] In yet further embodiments, the antibody or antigen-binding Ab fragment may comprise such a scFv, wherein the linker comprises the amino add sequence of SEQ ID NO: 168, the amino add sequence encoded by SEQ ID NO: 268 or 68, the amino add sequence of SEQ ID NO: 159, or the amino acid sequence encoded by SEQ ID NO: 259. [055] In particular embodiments, the antibody or antigen-binding Ab fragment may comprise a scFv, which comprises an amino add sequence (i) which is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 760, 759, 750, 749, 740, 739, 730, 729, 720, 719, 319, 320, 329, 330, 339, 340, 341, or 342; or (ii) encoded by a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 419, 19, 420, 20, 429, 29, 430, 30, 439, 39, 440, 40, 441, or 442. [056] In some embodiments, the Ab or antigen-binding Ab fragment may comprise two or more binding specificities. The first specificity may be to an epitope in ALPPL2. In certain embodiments, the second specificity may to another epitope in ALPPL2. In certain embodiments, the second specificity may be to an epitope in a second antigen other than ALPPL2. In particular embodiments, the second antigen may be for example, butnot limited to, NKG2D, 4-1BB, or Fc receptor (FcR).
[057] In some embodiments, the Ab or antigen-binding Ab fragment may comprise a human or human-like CHI, CH2, and/or CH3 domain(s). In certain embodiments, the human-like CHI, CH2, and/or CH3 domain(s) may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human CHI, CH2, and/or CH3 domain(s), respectively. In certain embodiments, the human or human-like CHI, CH2, and/or CH3 domain(s) is/are individually derived from the CHI, CH2, and/or CH3 domain(s), respectively, of a human IgM, a human IgD, a human IgG, a human IgE, or a human IgA optionally of a human IgGl, a human IgG2, a human IgG3, or a human IgG4.
[058] In some embodiments, the Ab or antigen-binding Ab fragment may comprise a human or human-like fragment crystallizable (Fc) region. In certain embodiments, the human or human-like Fc region may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human Fc region. In certain embodiments, the Fc region may be or may be derived from the Fc region of a human IgM, a human IgD, a human IgG, a human IgE, or a human IgA optionally of a human IgGl, a human IgG2, a human IgG3, or a human IgG4The human-like Fc region may bind to an Fc receptor (FcR). In some instances, the FcR may be for example, but not limited to, Fc gamma receptor (FcgR), FcgRl, FcgRllA, FcgRlIBl, FcgRllB2, FcgRIllA, FcgRlllB, Fc epsilon receptor (FceR), FceRI, FceRII, Fc alpha receptor (FcaR), FcaRI, Fc alpha/mu receptor (Fca/mR), and neonatal Fc receptor (FcRn).
[059] In some embodiments, the Ab or antigen-binding Ab fragment may bind to ALPPL2 with a dissociation constant (KD) of about 10 nM or smaller, about 5 nM or smaller, about 2 nM or smaller, about 1 nM or smaller, about 0.8 nM or smaller, about 0.6 nM or smaller, about 0.4 nM or smaller, about 0.3 nM or smaller, about 0.2 nM or smaller, about 0.1 nM or smaller, about 0.05 nM or smaller, about 0.02 nM or smaller, or about 0.01 nM or smaller.
[060] In some embodiments, the Ab or antigen-binding Ab fragment may bind to ALPPL2 with a dissociation constant (KD) of between about 10 nM and about 0.1 nM, between about 5 nM and about 0.2 nM, between about 10 nM and about 0.01 nM, between about 5 nM and about 0.02 nM, between about 2 nM and about 0.05 nM, between about 2 nM and about 0.1 nM, or between about 1 nM and about 0.3 nM. [061] In some embodiments, the Ab or antigen-binding Ab fragment may bind to ALPPL2 with a dissociation constant (KD) of about 1.6 nM, about 0.8 nM, about 0.4 nM, about 0.3 nM, or about 0.2 nM.
[062] In certain embodiments, die KD may be measured via enzyme-linked immunosorbent assay (ELISA) or Biolayer Interferometry (BLI).
[063] In some embodiments, the Ab or antigen-binding Ab fragment may bind to ALPPL2 with a half maximal effective concentration (ECso) of about 1 pg/mL or smaller, about 0.5 pg/mL or smaller, about 0.2 pg/mL or smaller, about 0.1 pg/mL or smaller, about 0.05 pg/mL or smaller, about 0.02 pg/mL or smaller, about 0.01 pg/mL or smaller, about 0.005 pg/mL or smaller, about 0.002 pg/mL or smaller, about 0.001 pg/mL or smaller.
[064] In some embodiments, the Ab or antigen-binding Ab fragment may bind to ALPPL2 with an ECso between about 1 pg/mL and about 0.001 pg/mL, between about 0.5 pg/mL and about 0.002 pg/mL, between about 0.2 pg/mL and about 0.005 pg/mL, between about 0.1 pg/mL and about 0.01 pg/mL, between about 0.05 pg/mL and about 0.02 pg/mL
[065] In some embodiments, the Ab or antigen-binding Ab fragment may bind to ALPPL2 with an ECso of about 0.05 pg/mL or about 0.02 pg/mL.
[066] In certain embodiments, the ECso may be measured via ELISA
[067] In some embodiments, the Ab or antigen-binding Ab fragment may bind to ALPPL2 -expressing cells, optionally ALPPL2-expressing human cancer cells, with an ECso of about 100 nM or smaller, about 50 nM or smaller, about 20 nM or smaller, about 10 nM or smaller, about 5 nM or smaller, about 2 nM or smaller, about 1 nM or smaller, about 0.5 nM or smaller, about 0.2 nM or smaller, about 0.1 nM or smaller, or about 0.05 nM or smaller.
[068] In some embodiments, the Ab or antigen-binding Ab fragment may bind to ALPPL2 -expressing cells, optionally ALPPL2-expressing human cancer cells, with an ECso of between about 100 nM and about 0.05 nM, between about 50 nM and about 0.1 nM, or between about 20 nM and about 0.2 nM.
[069] In some embodiments, the Ab or antigen-binding Ab fragment may bind to ALPPL2 -expressing cells, optionally ALPPL2-expressing human cancer cells, with an ECso of about 15 nM, about 10 nM, about 5 nM, about 1 nM, about 0.5 nM, or about 0.2 nM.
[070] In certain embodiments, the ECso may be measured using flowcytometry, further optionally based on mean fluorescence intensity (MFI).
[071] In some aspects, the anti-ALPPL2 agent of the present disclosure may be an antibody-drug conjugate (ADC). The ADC may comprise: (a) any Ab or antigen-binding Ab fragment described above; and (b) a drug conjugated to the Ab or antigen-binding Ab fragment [072] In some embodiments, the drug may be for example, but not limited to, an anticancer drug, an anti-proliferative drug, a cytotoxic drug, an anti-angiogenic drug, an apoptotic drug, an immunostimulatory drug, an anti-microbial drug, an antibiotic drug, an antiviral drug, an anti-inflammatory drug, an ALPPL2 inhibitor, an ALPPL2 signaling inhibitor, an enzyme, a hormone, a toxin, a radioisotope, a compound, a small molecule, a small molecule inhibitor, a protein, a peptide, a vector, a plasmid, a viral replicon, a viral particle, a nanoparticle, a DNA molecule, an RNA molecule, an siRNA, an shRNA, a micro RNA, an oligonucleotide, or an imaging drug.
[073] In some embodiments, the ADC may comprise a drug selected from the group consisting of doxorubicin, daunorubicin, cucurbitadn, chaetocin, chaetoglobosin, chlamydocin, calicheamicin, nemorubidn, cryptophyscin, mensacardn, ansamitodn, mitomycin C, geldanamydn, mechercharmycin, rebeccamycin, safracin, okilactomydn, ollgomycin, actinomycin, sandramydn, hypothemycln, polyketomydn, hydroxyellipticine, thiocolchicine, methotrexate, triptoHde, taltobulin, lactacystin, dolastatin, auristatin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), telomestatin, tubastatin A, combretastatin, maytansinoid, MMAD, MMAF, DM1, DM4, DTT, 16-GMB-APA-GA, 17-DMAP-GA, JW 55, pyrrolobenzodlazeplne, SN-38, Ro 5- 3335, puwainaphydn, duocarmydn, bafilomydn, taxoid, tubulysin, ferulenol, lusiol A, fumagillin, hygrolidin, glucopieriddin, amanitin, ansatrienin, dnerubin, phallacidin, phalloidin, phytosphongosine, pieriddin, poronetin, phodophyllotoxin, gramicidin A, sangulnarine, sinefungin, herboxidlene, mlcrocolln B, microcystin, muscotoxin A, tolytoxin, tripolin A, myoseverin, mytoxin B, nocuolin A, psuedolaric add B, pseurotin A, cyclopamine, curvulin, colchidne, aphidicolin, englerin, cordycepin, apoptolidin, epothilone A, limaquinone, isatropolone, isofistularin, quinaldopeptin, ixabepilone, aeroplysinin, arruginosin, agrochelin, epothilone, and a derivative of any one of the foregoing.
[074] In some aspects, the anti-ALPPL2 agent of the present disclosure is a chimeric antigen receptor (CAR). The CAR may comprise: (a) an antigen-binding domain that binds to ALPPL2, (b) a transmembrane (TM) domain, and (c) an intracellular signaling (ICS) domain. Optionally, such a CAR may further comprise (d) a hinge that joins said antigen-binding domain and said TM domain, and (e) one or more costimulatory (CS) domains.
[075] In some embodiments, the antigen-binding domain may be, comprise, or consist of any of the Abs or antigen-binding Ab fragments described above or herein.
[076] In some particular embodiments, the antigen-binding domain may be, comprise, or consist of any of the scFvs described above.
[077] In certain embodiments, the antigen-binding domain may comprise or consist of an amino acid sequence which is (i) at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 760, 759, 750, 749, 740, 739, 730, 729, 720, 719, 319, 320, 329, 330, 339, 340, 341, or 342 or (ii) encoded by a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% Identical to SEQ ID NOs: 419, 19, 420, 20, 429, 29, 430, 30, 439, 39, 440, 40, 441, or 442.
[078] In some particular embodiments, the antigen-binding domain may comprise (i) the amino acid sequence of SEQ ID NOs: 339, 340, 341, or 341 or (ii) the amino acid sequence encoded by SEQ ID NOs: 439, 39, 440, 40, 441, or 442.
[079] In yet another embodiment; the antigen-binding domain may compete for binding to ALPPL2 with a scFv comprising an amino acid sequence which is (i) at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 760, 759, 750, 749, 740, 739, 730, 729, 720, 719, 319, 320, 329, 330, 339, or 340, or (ii) encoded by a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 419, 19, 420, 20, 429, 29, 430, 30, 439, 39, 440, 40, 441, or 442.
[080] In some embodiments, the TM domain may be derived from the TM region, or a membrane-spanning portion thereof, of for example, but not limited to, CD28, CD3e, CD4, CDS, CD8, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD 154, TCRa, TCRb, or CD3z.
[081] In certain embodiments, the TM domain may be derived from the TM region of CD28, or a membrane-spanning portion thereof. The TM domain may optionally comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to the amino acid sequence: (i) set forth in SEQ ID NO: 161, or (ii) encoded by SEQ ID NO: 261.
[082] In some embodiments, the ICS domain may be derived from a cytoplasmic signaling sequence, or a functional fragment thereof, of for example, but not limited to, CD3z, a lymphocyte receptor chain, a TCR/CD3 complex protein, an Fc receptor (FcR) subunit, an IL-2 receptor subunit, FcRg, FcRb, CD3g, CD3d, CD3e, CDS, CD22, CD66d, CD79a, CD79b, CD278 (ICOS), FceRI, DAP10, or DAP12.
[083] In certain embodiments, the ICS domain may be derived from a cytoplasmic signaling sequence of CD3z, or a functional fragment thereof. The ICS domain may optionally comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% Identical to the amino add sequence: (I) set forth in SEQ ID NO: 162, or (ii) encoded by SEQ ID NO: 262.
[084] In some embodiments, the hinge may be derived from CD28. The hinge may optionally comprise an amino sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% Identical to the amino add sequence: (1) set forth in SEQ ID NO: 163, or (ii) encoded by SEQ ID NO: 263.
[085] In some embodiments, at least one of the one or more CS domains may be derived from a cytoplasmic signaling sequence, or functional fragment thereof, of for example, but not limited to, CD28, DAP10, 4- IBB (CD137), CD2, CD4, CDS, CD7, CD8a, CD8b, CD 11a, CDllb, CDllc, CDlld, CD18, CD19, CD27, CD29, CD30, CD40, CD49d, CD49f, CD69, CD84, CD96 (Tactile), CD100 (SEMA4D), CD103, 0X40 (CD134), SLAM (SLAMF1, CD150, IPO-3), CD160 (BY55), SELPLG (CD162), DNAM1 (CD226), Ly9 (CD229), SLAMF4 (CD244, 2B4), ICOS (CD278), B7-H3, BAFFR, BTLA, BLAME (SLAMF8), CEACAM1, CDS, CRTAM, GADS, GITR, HVEM (LIGHTER), IA4, ICAM-1, IL2Rb, IL2Rg, IL7Ra, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAT, LFA-1, LIGHT, LTBR, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), PAG/Cbp, PD-1, PSGL1, SLAMF6 (NTB-A, Lyl08), SLAMF7, SLP-76, TNFR2, TRANCE/RANKL, VLA1, VLA-6, or CD83 ligand.
[086] In certain embodiments, the CS domain may be derived from a cytoplasmic signaling sequence of CD28, 4-1BB, or DAP10, or functional fragment thereof. The CS domain may optionally comprise an amino sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to the amino add sequence: (1) set forth in SEQ ID NO: 164, (ii) encoded by SEQ ID NO: 264, (iii) set forth in SEQ ID NO: 165, (iv) encoded by SEQ ID NO: 265, (v) set for the in SEQ ID NO: 166, or (vi) encoded by SEQ ID NO: 266.1n certain embodiments, the CAR may comprise an amino add sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to the amino acid sequence of (i-1) B735scFvHL-CD28H- CD28TM-CD28CS-CD3zICS (SEQ ID NO: 951), (1-2) B735scFvHL-CD28H-CD28TM- 41BBCS-CD3zICS (SEQ ID NO: 952), (1-3) B735scFvHL-CD28H-CD28TM-DAP10CS- CD3zICS (SEQ ID NO: 953), (1-4) B735scFvLH-CD28H-CD28TM-CD28CS-CD3zICS (SEQ ID NO: 954), (1-5) B735scFvLH-CD28H-CD28TM-41BBCS-CD3zICS (SEQ ID NO: 955), (1- 6) B735scFvLH-CD28H-CD28TM-DAP10CS-CD3zICS (SEQ ID NO: 956), (ii-1) B506scFvHL-CD28H-CD28TM-CD28CS-CD3zICS (SEQ ID NO: 941), (11-2) B506scFvHL- CD28H-CD28TM-41BBCS-CD3zICS (SEQ ID NO: 942), (ii-3) B506scFvHL-CD28H- CD28TM-DAP10CS-CD3zICS (SEQ ID NO: 943), (ii-4) B506scFvLH-CD28H-CD28TM- CD28CS-CD3zICS (SEQ ID NO: 944), (ii-5) B506scFvLH-CD28H-CD28TM-41BBCS- CD3zICS (SEQ ID NO: 945), (ii-6) B506scFvLH-CD28H-CD28TM-DAP10CS-CD3zICS (SEQ ID NO: 946), (iii-1) B416scFvHL-CD28H-CD28TM-CD28CS-CD3zICS (SEQ ID NO: 931), (iii-2) B416scFvHL-CD28H-CD28TM-41BBCS-CD3zICS (SEQ ID NO: 932), (iii-3) B416scFvHL-CD28H-CD28TM-DAP10CS-CD3zICS (SEQ ID NO: 933), (iii-4) B416scFvLH- CD28H-CD28TM-CD28CS-CD3zICS (SEQ ID NO: 934), (iii-5) B416scFvLH-CD28H- CD28TM-41BBCS-CD3zICS (SEQ ID NO: 935), (iii-6) B416scFvLH-CD28H-CD28TM- DAP10CS-CD3zICS (SEQ ID NO: 936), (iv-1) B380scFvHL-CD28H-CD28TM-CD28CS- CD3zICS (SEQ ID NO: 921), (iv-2) B380scFvHL-CD28H-CD28TM-41BBCS-CD3zICS (SEQ ID NO: 922), (iv-3) B380scFvHL-CD28H-CD28TM-DAP10CS-CD3zICS (SEQ ID NO: 923), (iv-4) B380scFvLH-CD28H-CD28TM-CD28CS-CD3zICS (SEQ ID NO: 924), (iv-5) B380scFvLH-CD28H-CD28TM-41BBCS-CD3zICS (SEQ ID NO: 925), (iv-6) B380scFvLH- CD28H-CD28TM-DAP10CS-CD3Z1CS (SEQ ID NO: 926), (v-1) B001scFvHL-CD28H- CD28TM-CD28CS-CD3zICS (SEQ ID NO: 911), (v-2) B001scFvHL-CD28H-CD28TM- 41BBCS-CD3zICS (SEQ ID NO: 912), (v-3) B001scFvHL-CD28H-CD28TM-DAP10CS- CD3zICS (SEQ ID NO: 913), (v-4) B001scFvLH-CD28H-CD28TM-CD28CS-CD3zICS (SEQ ID NO: 914), (v-5) B001scFvLH-CD28H-CD28TM-41BBCS-CD3zICS (SEQ ID NO: 915), or (v-6) B001scFvLH-CD28H-CD28TM-DAP10CS-CD3zICS (SEQ ID NO: 916). [087] In certain embodiments, die CAR may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to the amino acid sequence of (i) hlB2scFvHL-CD28H-CD28TM-CD28CS- CDSzICS (SEQ ID NO: 351), (ii) hlB2scFvHL-CD28H-CD28TM-41BBCS-CD3zICS (SEQ ID NO: 352), (ill) hlB2scFvHL-CD28H-CD28TM-DAP10CS-CD3zICS (SEQ ID NO: 353), (iv) hlB2scFvLH-CD28H-CD28TM-CD28CS-CD3zICS (SEQ ID NO: 354), (v) hlB2scFvLH- CD28H-CD28TM-41BBCS-CD3zICS (SEQ ID NO: 355), (vi) hlB2scFvLH-CD28H-CD28TM- DAP10CS-CD3zICS (SEQ ID NO: 356), (vii) hlE8scFvHL-CD28H-CD28TM-CD28CS- CD3zICS (SEQ ID NO: 361), (vill) hlE8scFVHL-CD28H-CD28TM-41BBCS-CD3zICS (SEQ ID NO: 362), (ix) hlE8scFvHL-CD28H-CD28TM-DAP10CS-CD3zICS (SEQ ID NO: 363), (x) hlE8scFvLH-CD28H-CD28TM-CD28CS-CD3zICS (SEQ ID NO: 364), (xi) hlEBscFvLH- CD28H-CD28TM-41BBCS-CD3zICS (SEQ ID NO: 365), (xii) hlE8scFvLH-CD28H- CD28TM-DAP10CS-CD3zICS (SEQ ID NO: 366), (xlll) h3H2scFvHL-CD28H-CD28TM- CD28CS-CD3zICS (SEQ ID NO: 371), (xiv) h3H2scFvHL-CD28H-CD28TM-41BBCS- CD3zICS (SEQ ID NO: 372), (xv) h3H2scFvHL-CD28H-CD28TM-DAP10CS-CD3zICS (SEQ ID NO: 373), (xvi) h3H2scFvLH-CD28H-CD28TM-CD28CS-CD3zICS (SEQ ID NO: 374), (xvil) h3H2scFvLH-CD28H-CD28TM-41BBCS-CD3zICS (SEQ ID NO: 375), or (xvlll) h3H2scFvLH-CD28H-CD28TM-DAP10CS-CD3zICS (SEQ ID NO: 376).
[088] In certain embodiments, the CAR may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% Identical to the amino acid sequence encoded by any one of SEQ ID NOs: 451-456, 461- 466, 471-476, 51-56, 61-66, and 71-76.
[089] In some embodiments, the CAR may further comprise a cytotoxic drug conjugated to the antigen-binding domain.
[090] The present invention also relates to isolated polynucleotides encoding any of the anti-ALPPL2 agents described above and to combinations of polynucleotides which as a combination encode any of the anti-ALPPL2 agents described above.
[091] In some embodiments, the isolated polynucleotide or the combination of isolated polynucleotides may encode an antibody (Ab) or antigen-binding Ab fragment; which may comprise a VH and a VL as described above. When a combination of isolated polynucleotides encodes an antibody (Ab) or antigen-binding Ab fragment; for example, one polynucleotide may encode a VH and another polynucleotide may encode a VL, or one polynucleotide may encode a heavy chain and another polynucleotide may encode a light chain.
[092] In some embodiments, the CDR-H1, CDR-H2, and CDR-H3-encoding nucleic acid sequences may comprise or consist of the CDR-H1, CDR-H2, and CDR-H3-encoding nucleic acid sequences for any of the anti-ALPPL2 antibodies described herein, such as butnot limited to B735, B506, B416, B380, B001, 1B5, h3H2, hlE8, hlB2, 3H2, 1E8, or 1B2. In some embodiments, the CDR-L1, CDR-L2, and CDR-L3-encoding nucleic acid sequences may comprise or consist of the CDR-L1, CDR-L2, and CDR-L3-encoding nucleic acid sequences for any of the anti-ALPPL2 antibodies described herein, such as butnot limited to B735, B506, B416, B380, B001, IBS, h3H2, hlEB, hlB2, 3H2, 1E8, or 1B2.
[093] In certain embodiments, the CDR-L1, CDR-L2, and CDR-L3-encoding nucleic acid sequences may comprise or consist of SEQ ID NOs: 806, 807, and 808, respectively.
[094] In certain embodiments, the CDR-H1, CDR-H2, and CDR-H3-encoding nucleic acid sequences may comprise or consist of SEQ ID NOs: 432, 433, and 434, respectively; and the CDR-L1, CDR-L2, and CDR-L3-encoding nucleic acid sequences may comprise or consist of SEQ ID NOs: 436, 437, and 438, respectively.
[095] In certain embodiments, the CDR-H1, CDR-H2, and CDR-H3-encoding nucleic acid sequences may comprise or consist of SEQ ID NOs: 422, 423, and 424, respectively; and the CDR-L1, CDR-L2, and CDR-L3-encodlng nucleic acid sequences may comprise or consist of SEQ ID NOs: 426, 427, and 428, respectively.
[096] In certain embodiments, the CDR-H1, CDR-H2, and CDR-H3-encoding nucleic acid sequences may comprise or consist of SEQ ID NOs: 412, 413, and 414, respectively; and the CDR-L1, CDR-L2, and CDR-L3-encoding nucleic acid sequences may comprise or consist of SEQ ID NOs: 416, 417, and 418, respectively.
[097] In certain embodiments, the CDR-H1, CDR-H2, and CDR-H3-encoding nucleic acid sequences may comprise or consist of SEQ ID NOs: 232, 233, and 234, respectively; and the CDR-L1, CDR-L2, and CDR-L3-encoding nucleic acid sequences may comprise or consist of SEQ ID NOs: 236, 237, and 238, respectively.
[098] In certain embodiments, the CDR-H1, CDR-H2, and CDR-H3-encoding nucleic acid sequences may comprise or consist of SEQ ID NOs: 222, 223, and 224, respectively; and the CDR-L1, CDR-L2, and CDR-L3-encoding nucleic acid sequences may comprise or consist of SEQ ID NOs: 226, 227, and 228, respectively.
[099] In certain embodiments, the CDR-H1, CDR-H2, and CDR-H3-encoding nucleic acid sequences may comprise or consist of SEQ ID NOs: 212, 213, and 214, respectively; and the CDR-L1, CDR-L2, and CDR-L3-encoding nucleic acid sequences may comprise or consist of SEQ ID NOs: 216, 217, and 218, respectively.
[100] In some embodiments, the % Identity of the VH-encodlng nucleic acid sequence to a VH-encoding human germline sequence (e.g., the human VH2-5 gene segment allele 8) may be about 80% or higher, about 85% or higher, about 90% or higher, about 93% or higher, about 95% or higher, about 96% or higher, about 97% or higher, about 98% or higher, about 98% or higher, about 99% or higher, or about 100%.
[101] In some embodiments, the % identity of the VL-encoding nucleic acid sequence to a VL-encoding human germline sequence (e.g., the human IgKl-16 gene segment allele 1) may be about 80% or higher, about 85% or higher, about 86% or higher, about 90% or higher, about 93% or higher, about 95% or higher, about 96% or higher, about 97% or higher, about 98% or higher, about 98% or higher, or about 99% or higher, or about 100%. [102] In some embodiments, the VH-encodlng nucleic acid sequence may comprise a human or human-like VH framework-encoding nucleic add sequence which may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human VH framework-encoding nucleic add sequence, optionally to any one of the human VH framework-encoding nucleic add sequences comprised in any one of the VH-encoding nudeic add sequences of SEQ ID NOS: 431, 421, and 411. In some embodiments, the VL- encoding nucleic add sequence may comprise a human or human-like VL frameworkencoding nucleic add sequence which may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human VL framework-encoding nucleic acid sequence, optionally to any one of the human VL framework-encoding nucleic add sequences comprised in any one of the VL-encoding nucleic add sequences of SEQ ID NOS: 435, 425, and 415.
[103] In some embodiments, the VH-encoding nucleic add sequence may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% Identical to the VH-encodlng nucleic add sequence of any of the anti-ALPPL2 antibodies described herein, such as but not limited to 8735, B506, 8416, 8380, 8001, 185, h3H2, hlE8, hlB2, 3H2, 1E8, or 182. In some embodiments, the VL-encoding nudeic add sequence may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL-encoding nucleic add sequence of any of the anti-ALPPL2 antibodies described herein, such as butnot limited to 8735, 8506, 8416, 8380, 8001, 185, h3H2, hlE8, hlB2, 3H2, 1E8, or 182.
[104] In certain embodiments, the VL-encoding nucleic add sequence may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 805.
[105] In certain embodiments, the VH-encoding nucleic add sequence may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 431; and the VL- encoding nucleic add sequence may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 435.
[106] In certain embodiments, the VH-encoding nucleic add sequence may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 421; and the VL- encoding nucleic add sequence may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 425.
[107] In certain embodiments, the VH-encoding nucleic add sequence may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 411; and the VL- encoding nucleic add sequence may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 415.
[108] In certain embodiments, the VH-encoding nucleic add sequence may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 231; and the VL- encoding nucleic add sequence may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% Identical to SEQ ID NO: 235.
[109] In certain embodiments, the VH-encoding nucleic add sequence may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 221; and the VL- encodlng nucleic add sequence may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 225.
[110] In certain embodiments, the VH-encoding nucleic add sequence may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 211; and the VL- encoding nucleic add sequence may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% Identical to SEQ ID NO: 215.
[111] In some embodiments, the isolated polynucleotide or combination of isolated polynucleotides may encode an Ab or antigen-binding Ab fragment that is for example, but not limited to, a monoclonal Ab, a monospedfic Ab, a bispedfic Ab, a multispedfic Ab, a humanized Ab, a tetrameric Ab, a tetravalent Ab, a single chain Ab, a domain- spedfic Ab, a domain-deleted Ab, an scFc fusion protein, a chimeric Ab, a synthetic Ab, a recombinant Ab, a hybrid Ab, a mutated Ab, CDR-grafted Ab, a fragment antigen-binding (Fab), an F(ab')2, an Fab' fragment; a variable fragment (Fv), a single-chain Fv (scFv) fragment; an Fd fragment; a diabody, or a minibody.
[112] In particular embodiments, the isolated polynucleotide or combination of isolated polynucleotides may encode encodes any of the scFvs described above or herein.
[113] In certain embodiments, the linker-encoding nucleic add sequence of such a polynucleotide may comprise or consist of SEQ ID NO: 268, 68, or 259.
[114] In particular embodiments, the scFv-encoding polynucleotide may comprise a nudeic add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 419, 19, 420, 20, 429, 29, 430, 30, 439, 39, 440, 40, 441, or 442.
[115] In some embodiments, the isolated polynucleotide may encode an Ab or antigenbinding Ab fragment that comprises two or more binding spedfidties. The first spedfidty may be to an epitope in ALPPL2. In one aspect; the second spedfidty may be to another epitope in ALPPL2. In another aspect; the second spedfidty may be to an epitope In a second antigen other than ALPPL2. In certain embodiments, the second antigen may be for example, but not limited to, NKG2D, 4-1BB, or Fc receptor (FcR).
[116] In some embodiments, the isolated polynucleotide or combination of isolated polynucleotides may comprise a human or human-like CHI, CH2, and/or CH3 domainencoding nucleic add sequence(s). In certain embodiments, the human-like CHI, CH2, and/or CH3 domain-encoding nucleic add sequence(s) may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to (a) human CHI, CH2, and/or CH3 domainencoding nucleic add sequence(s), respectively.
[117] In some instances, the human or human-like CHI, CH2, and/or CH3 domainencoding nucleic add sequence(s) may be individually derived from the CHI, CH2, and/or CH3 domain-encoding nucleic add sequence(s), respectively, of a human IgM, a human IgD, a human IgG, a human IgE, or a human IgA, optionally of a human IgGl, a human IgG2, a human IgG3, or a human IgG4.
[118] In some embodiments, the isolated polynucleotide or combination of isolated polynucleotides may comprise a human or human-like fragment crystallizable (Fc) region-encoding nucleic add sequence. In certain embodiments, the human or humanlike Fc region-encoding nucleic add sequence may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human Fc region-encoding nucleic add sequence.
[119] In some instances, the human or human-like Fc region-encoding nucleic add sequence may be or may be derived from the Fc region-encoding nucleic add sequence of a human IgM, a human IgD, a human IgG, a human IgE, or a human IgA, optionally of a human IgGl, a human IgG2, a human IgG3, or a human lgG4.
[120] In some instances, the encoded human or human-like Fc region may bind to an Fc receptor (FcR). The FcR may be for example, but not limited to, Fc gamma receptor (FcgR), FcgRI, FcgRIIA, FcgRHBl, FcgRIIB2, FcgRIIIA, FcgRIIIB, Fc epsilon receptor (FceR), FceRI, FceRII, Fc alpha receptor (FcaR), FcaRI, Fc alpha/mu receptor (Fca/mR), or neonatal Fc receptor (FcRn).
[121] In some embodiments, the isolated polynucleotide may encode any CAR described above. The CAR may comprise: (a) an antigen-binding domain that binds to ALPPL2; (b) a transmembrane (TM) domain; (c) an intracellular signaling (ICS) domain;
(d) optionally a hinge that joins said antigen-binding domain and said TM domain; and
(e) optionally one or more costimulatory (CS) domains.
[122] In some embodiments, the isolated polynucleotide may comprise an antigenbinding domain-encoding nucleic acid sequence may comprise or consist of any of the nucleic acid sequences encoding the anti-ALPPL2 antibody or antibody fragment described above or herein.
[123] In some embodiments, the isolated polynucleotide may comprise an antigenbinding domain-encoding nucleic acid sequence comprising or consisting of the nucleic acid sequence of any of the scFv-encoding polynucleotides described above or herein. [124] In particular embodiments, the antigen-binding domain-encoding nucleic acid sequence may comprise or consist of a nucleic acid sequence of SEQ ID NOs: 419, 19, 420, 20, 429, 29, 430, 30, 439, 39, 440, 40, 441, or 442 and in some preferred embodiments, SEQ ID NO: 439 or 440.
[125] In certain embodiments, the encoded antigen-binding domain may compete for binding to ALPPL2 with a scFv comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to: (1) SEQ ID NOs: 760, 759, 750, 749, 740, 739, 730, 729, 720, 719, 319, 320, 329, 330, 339, 340, 341, or 342 or (11) the amino add sequence encoded by SEQ ID NOs: 419, 19, 420, 20, 429, 29, 430, 30, 439, 39, 440, 40, 441, or 442.
[126] In some embodiments, the isolated polynucleotide may comprise a TM domainencoding nucleic add sequence derived from the nucleic add sequence encoding the TM region, or a membrane-spanning portion thereof, of, for example, but not limited to, CD28, CD3e, CD4, CDS, CD8, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD 154, TCRa, TCRb, and CD3z. In certain embodiments, the TM domainencoding nucleic add sequence may be derived from the nucleic add sequence encoding the TM region of CD28, or a membrane-spanning portion thereof. The TM domainencoding nucleic add sequence may optionally comprise a nucleic add sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% Identical to SEQ ID NO: 261.
[127] In some embodiments, the isolated polynucleotide may comprise a ICS domainencoding nucleic add sequence derived from the nucleic add sequence encoding a cytoplasmic signaling sequence, or a functional fragment thereof, of, for example, but not limited to, CD3z, a lymphocyte receptor chain, a TCR/CD3 complex protein, an Fc receptor (FcR) subunit, an IL-2 receptor subunit, FcRg, FcRb, CD3g, CD3d, CD3e, CDS, CD22, CD66d, CD79a, CD79b, CD278 (ICOS), FceRI, DAP10, and DAP12. In certain embodiments, the ICS domain-encoding nucleic add sequence may be derived from the nudeic add sequence encoding a cytoplasmic signaling sequence of CD3z, or a functional fragment thereof. The ICS domain-encoding nucleic add sequence may optionally comprise a nucleic add sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to SEQ ID NO: 262.
[128] In some embodiments, the isolated polynucleotide may comprise a hingeencoding nucleic add sequence derived from the nucleic add sequence encoding CD28. The hinge-encoding nucleic add sequence may optionally comprise a nudeic add sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to SEQ ID NO: 263.
[129] In some embodiments, the isolated polynucleotide may comprise a nucleic add sequence encoding at least one of the one or more CS domains which may be derived from the nucleic add sequence encoding a cytoplasmic signaling sequence, or functional fragment thereof, of, for example, butnot limited to, CD28, DAP10, 4-1BB (CD137), CD2, CD4, CDS, CD7, CD8a, CD8b, CDlla, CDllb, CDllc, CDlld, CD18, CD19, CD27, CD29, CD30, CD40, CD49d, CD49f, CD69, CD84, CD96 (Tactile), CD100 (SEMA4D), CD1O3, 0X40 (CD134), SLAM (SLAMF1, CD150, IPO-3), CD160 (BY55), SELPLG (CD162), DNAM1 (CD226), Ly9 (CD229), SLAMF4 (CD244, 2B4), ICOS (CD278), B7-H3, BAFFR, BTLA, BLAME (SLAMF8), CEACAM1, CDS, CRTAM, GADS, GITR, HVEM (LIGHTER), IA4, ICAM-1, IL2Rb, IL2Rg, IL7Ra, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAT, LFA-1, LIGHT, LTBR, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), PAG/Cbp, PD-1, PSGL1, SLAMF6 (NTB-A, Lyl08), SLAMF7, SLP-76, TNFR2, TRANCE/RANKL, VLA1, VLA-6, or CD83 ligand. In certain embodiments, the CS domain-encoding nucleic acid sequence may be derived from the nucleic acid sequence encoding a cytoplasmic signaling sequence of CD28, 4- IBB, or DAP10, or functional fragment thereof. The CS domain-encoding nucleic add sequence may optionally comprise a nucleic add sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to SEQ ID NO: 264, 265, or 266.
[130] In some particular embodiments, the CAR-encoding polynucleotide may comprise a nucleic add sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to any one of SEQ ID NOs: 451-456, 461- 466, 471-476, 51-56, 61-66, and 71-76.
[131] In some embodiments, the isolated polynucleotide of any of the above may further comprise a leader sequence (LS). Optionally, the LS may comprise a nucleic add sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% Identical to SEQ ID NO: 260. Further optionally, the LS may encode a LS polypeptide comprising an amino add sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to SEQ ID NO: 160.
[132] In some embodiments, the isolated polynucleotide of any one of the above may further comprise T2A sequence and/or a sequence encoding truncated CD 19 (trCD19).
[133] In some instances, the T2A sequence maybe optionally at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to SEQ ID NO: 269. In certain instances, the T2A sequence may encode a T2A polypeptide comprising an amino add sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to SEQ ID NO: 169.
[134] In some instances, the encoded trCD19 may optionally comprise the amino add sequence at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to SEQ ID NO: 170. In certain instances, the trCD19-encoding nucleic add sequence may be at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to SEQ ID NO: 270.
[135] In particular instances, the isolated CAR-encoding polynucleotide may encode an amino add at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to SEQ ID NO: 571 or 574 and/or may comprise a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to any one of SEQ ID NOs: 671, 271, 674, and 271.
[136] The present invention relates to vectors and combinations of vectors comprising any of the polynucleotides or combination of polynudeotides encoding an anti-ALPPL2 agent When a combination of vectors encodes an anti-ALPPL2 agent such as an anti- ALPPL2 antibody, one vector may comprise a nucleic acid sequence encoding a VH and another vector may comprise a nucleic acid sequence encoding a VL
[137] In some embodiments, the vector or combination of vectors may comprise any polynucleotide described above.
[138] In some embodiments, the vector may be for example, but not limited to, a DNA, an RNA, a plasmid, a cosmid, a viral vector, a lentiviral vector, an adenoviral vector, or a retroviral vector.
[139] The present disclosure further provides to recombinant or Isolated cells.
[140] In some embodiments, the recombinant or isolated cell may comprise: (i) any Ab or antigen-binding Ab fragment described above, (ii) any ADC described above, (iii) any CAR described above, (iv) any polynucleotide described above, and/or (v) any vector described above.
[141] In some embodiments, the recombinant or isolated cell may be or may comprise, for example, but not limited to, a non-mammalian cell, optionally a plant cell, a bacterial cell, a fungal cell, a yeast cell, a protozoa cell, or an insect cell.
[142] In some embodiments, the recombinant or isolated cell may be, for example, but not limited to, a mammalian cell, optionally a human cell, a rat cell, or a mouse cell.
[143] In some embodiments, the recombinant or isolated cell may be, for example, but not limited to, a stem cell.
[144] In some embodiments, the recombinant or isolated cell may be, for example, but not limited to, a primary cell, optionally a human primary cell or derived therefrom.
[145] In some embodiments, the recombinant or isolated cell may be, for example, but not limited to, a cell line, optionally a hybridoma cell line.
[146] In some embodiments, the recombinant or isolated cell may be, for example, but not limited to, an immune cell.
[147] In some embodiments, the recombinant or isolated cell may be MHC+ or MHC-.
[148] In some embodiments, the recombinant or isolated cell may be, for example, but not limited to, a cell line, a T cell, a T cell progenitor cell, a CD4+ T cell, a helper T cell, a regulatory T cell, a CD8+ T cell, a naive T cell, an effector T cell, a memory T cell, a stem cell memory T (TSCM) cell, a central memory T (TCM) cell, an effector memory T (TEM) cell, a terminally differentiated effector memory T cell, a tumor-infiltrating lymphocyte (TIL), an immature T cell, a mature T cell, a cytotoxic T cell, a mucosa-associated invariant T (MAIT) cell, a TH1 cell, a TH2 cell, a TH3 cell, a TH17 cell, a TH9 cell, a TH22 cell, a follicular helper T cells, and a/b T cell, a g/d T cell, a Natural Killer T (NKT) cell, a cytokine-induced killer (CIK) cell, a lymphokine-activated killer (LAK) cell, a perforindeficient cell, a granzyme-deficient cell, a B cell, a myeloid cell, a monocyte, a macrophage, or a dendritic cell.
[149] In certain embodiments, the recombinant or isolated cell may be a T cell or T cell progenitor cell.
[150] In certain embodiments, the recombinant or isolated cell may be a T cell which has been modified such that its endogenous T cell receptor (TCR) is (i) not expressed, (li) not functionally expressed, or (111) expressed at reduced levels compared to a wild- type T cell.
[151] In certain embodiments, the recombinant or isolated cell may be activated or stimulated to proliferate when the CAR binds to its target molecule.
[152] In certain embodiments, the recombinant or Isolated cell may exhibit cytotoxicity against cells expressing the target molecule when the CAR binds to the target molecule.
[153] In certain embodiments, administration of the recombinant or isolated cell to a subject may ameliorate a disease In the subject In particular embodiments, the disease is cancer.
[154] In certain embodiments, the recombinant or isolated cell may increase expression of cytokines and/or chemokines when the CAR binds to its target molecule. The cytokine may be IFN-g.
[155] In certain embodiments, the recombinant or isolated cell may decrease expression of cytokines and/or chemokines when the CAR binds to its target The cytokine may be TGF-b or IL-10.
[156] The present Invention relates to populations of recombinant or Isolated cells.
[157] In some embodiments, the population may comprise at least any one recombinant or isolated cell described above.
[158] The present invention relates to pharmaceutical compositions.
[159] In some embodiments, the pharmaceutical composition may comprise: (a) (a-1) any Ab or antigen-binding Ab fragment described above, (a-ii) any ADC described above, (iii) any CAR described above, (iv) any polynucleotide or combination of polynucleotides described above, (v) any vector or combination of vectors described above, (vi) any cell described above, or (vii) any population of cells; and optionally (b) a pharmaceutically acceptable excipient or carrier.
[160] The present invention relates to methods of treating a subject
[161] In some embodiments, the method may be a method of treating a subject; and the method may comprise administering to the subject in need thereof a therapeutically effective amount of (i) any Ab or antigen-binding Ab fragment described above, (ii) any ADC described above, (iii) any CAR described above, (iv) any polynucleotide or combination of polynucleotides described above, (v) any vector or combination of vectors described above, (vi) any cell described above, (vii) any population of cells described above, and/or (viii) any pharmaceutical composition described above.
[162] In some embodiments, the method may be used in the treatment of, cancer. In certain embodiments, the method may be used in the treatment of pancreatic cancer, testicular cancer, cervical cancer, endometrial cancer, ovarian cancer, stomach cancer, colorectal cancer, lung cancer, mesothelioma, or tongue cancer.
[163] In some embodiments, the method may be a method of treating a subject with anti-ALPPL2 agent The method may comprise the steps of: (a) obtaining or having obtained a biological sample from the subject; (b) measuring the expression level of ALPPL2 in the biological sample; (c) determining whether the subject is an ALPPL2 over-expresser; and (d) If the subject Is an ALPPL2 over-expresser, administering to the subject a therapeutically effective amount of (d-i) any Ab or antigen-binding Ab fragment described above, (d-ii) any ADC described above, (d-iii) any CAR described above, (d-iv) any polynucleotide described above, (d-v) any vector described above, (d- vi) any cell described above, (d-vil) any population of cells described above, and/or (d- viii) any pharmaceutical composition described above. In certain embodiments, an ALPPL2 over-expresser is a subject whose ALPPL2 expression is at least 1.5 times higher than the ALPPL2 expression of normal or healthy subjects. In certain embodiments, an ALPPL2 over-expresser Is a subject whose ALPPL2 expression Is at least 1.75 times higher than the ALPPL2 expression of normal or healthy subjects. In certain embodiments, an ALPPL2 over-expresser is a subject whose ALPPL2 expression is at least twice higher than the ALPPL2 expression of normal or healthy subjects.
[164] In some embodiments, the subject may be suffering from cancer. The cancer may be for example, but not limited to, pancreatic cancer, testicular cancer, cervical cancer, endometrial cancer, ovarian cancer, stomach cancer, colorectal cancer, lung cancer, mesothelioma, or tongue cancer.
[165] The present Invention relates to methods of Immune stimulation.
[166] In some embodiments, the method may be a method for stimulating an immune response in a subject, comprising administering to the subject a therapeutically effective amount of: (i) any Ab or antigen-binding Ab fragment described above, (ii) any ADC described above, (111) any CAR described above, (iv) any polynucleotide or combination of polynucleotides described above, (v) any vector or combination of vectors described above, (vi) any cell described above, (vii) any population of cells described above, and/or (viii) any pharmaceutical composition described above.
[167] The present invention relates to methods of treating a disease.
[168] In some embodiments, the method may be a method of treating a disease in a subject; comprising administering to the subject in need thereof a therapeutically effective amount of (i) any Ab or antigen-binding Ab fragment described above, (ii) any ADC described above, (iii) any CAR described above, (iv) any polynucleotide or combination of polynucleotides described above, (v) any vector or combination of vectors described above, (vi) any cell described above, (vii) any population of cells described above, and/or (viii) any pharmaceutical composition described above.
[169] In some embodiments, the disease may be, for example, cancer.
[170] In certain embodiments, the disease may be cancer, optionally pancreatic cancer, testicular cancer, cervical cancer, endometrial cancer, ovarian cancer, stomach cancer, colorectal cancer, lung cancer, mesothelioma, or tongue cancer.
[171] The present invention further relates to methods of expanding a population of cells.
[172] In some embodiments, the method may be a method of expanding a population of cells in a subject
[173] In some embodiments, the method may comprise administering to the subject (i) any polynucleotide or combination of polynucleotides described above; (ii) any vector or combination of vectors described above; (ill) any cell described above; (iv) any population of cells described above; and/or (v) any pharmaceutical composition described above.
[174] In some embodiments, the administration may lead to a population of cells including at least one desired cell, for example, a cell that may comprise any Ab or Ab fragment described above, any ADC described above, and/or any CAR described above. In certain embodiments, the cell may comprise a nucleic acid encoding such an Ab or Ab fragment; ADC, or CAR.
[175] In some embodiments, the resulting population of cells may persist in the subject for at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, at least twelve months, at least eighteen months, at least two years, or at least three years after administration.
[176] In some embodiments, the subjectmay be sufiferingfrom cancer.
[177] In certain embodiments, the cancer may be pancreatic cancer, testicular cancer, cervical cancer, endometrial cancer, ovarian cancer, stomach cancer, colorectal cancer, lung cancer, mesothelioma, or tongue cancer.
[178] In some embodiments, any of the methods described above may further comprise administering a second agent
[179] In certain embodiments, the second agent may be, but is not limited to, an anticancer drug, an anti-proliferative drug, a cytotoxic drug, an anti-angiogenic drug, an apoptotic drug, an immunostimulatory drug, an anti-microbial drug, an antibiotic drug, an antiviral drug, an anti-inflammatory drug, an ALPPL2 inhibitor, an ALPPL2 signaling inhibitor, an anti-ALPPL2 agent of the present invention, an enzyme, a hormone, a toxin, a radioisotope, a compound, a small molecule, a small molecule inhibitor, a protein, a peptide, a vector, a plasmid, a viral replicon, a viral particle, a nanoparticle, a DNA molecule, an RNA molecule, an siRNA, an shRNA, a micro RNA, an oligonucleotide, or an imaging drug.
[180] The present invention further relates to methods of generating a cell comprising any CAR described above.
[181] In some embodiments, the method may comprise: (i) introducing into a cell (i-a) a polynucleotide encoding at least one CAR according to any one of the foregoing or (i-b) at least one polynucleotide according to any one of the foregoing; or
(ii) transducing a cell with the vector according to any one of the foregoing. Optionally, the method may further comprise (iii) isolating the cell based on expression of said CAR and/or a selectable marker as determined via flow cytometry or immunofluorescence assays.
[182] The present invention further relates to methods producing an anti-ALPPL2 agent described above or herein.
[183] In one aspect; the method is for producing an anti-ALPPL2 Ab or Ab fragment described above or herein. [184] In some embodiments, the method may comprise (a) culturing cells comprising any of the isolated polynucleotides or combinations of isolated polynucleotides encoding the anti-ALPPL2 Ab or Ab fragment in a condition that allows for expression of said Ab or Ab fragment; and (b) harvesting and purifying the Ab or Ab fragment from the cell culture from (a).
[185] In one aspect; the method is for producing a recombinant or isolated cell described above or herein or population of such cells.
[186] In some embodiments, the method may comprise Introducing any of the Isolated polynucleotides or combinations of isolated polynucleotides described above or herein encoding an anti-ALPPL2 agent of interest into a cell.
[187] In certain embodiments, the introducing may occur in vitro. Such an in vitro introduction may be, for example, for generating recombinant cells (e. g., yeast cells, plant cells, insect cells, or mammalian cells, such as human cells; optionally CHO cells or HEK cells) encoding an anti-ALPPL2 Ab or Ab fragment for manufacturing of an ti- ALPPL2 Ab or Ab fragment An in vitro introduction may alternatively be, for example, for producing cells for therapeutic use, for example cells (e.g., iPSCs, IPSC-derived cells of a cell type of Interest) expressing and/or secreting an antl-ALPPL2 agent (e.g., Ab, Ab fragment ADC, CAR).
[188] In certain embodiments, the introducing may occur ex vivo. Such an ex vivo introduction may be, for example, for producing cells for therapeutic use, for example cells (e.g., cells from a patient/subject, allogeneic or autologous cells, directly from a patient/subject or further processed and/or further differentiated) expressing and/or secreting an anti-ALPPL2 agent (e.g., Ab, Ab fragment ADC, CAR).
[189] In certain embodiments, the introducing may occur in vivo. Such an in vivo introduction may be, for example, for producing cells in a patient/subject to be treated, that express and/or secrete an anti-ALPPL2 agent (e.g., Ab, Ab fragment ADC, CAR) for therapy.
DETAILED DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[190] FIGS 1A-E provide exemplary schematics of chimeric antigen receptors (CARs) according to the present disclosure. FIG 1A shows a general schematic of chimeric antigen receptors (CARs) of the present disclosure. A CAR according to the present disclosure comprises an antigen-binding (AB) domain, transmembrane (TM) domain, and an intracellular signaling (ICS) domain. FIGS IB- ID shows exemplary schematics of a CAR construct according to the present disclosure, wherein the CAR construct comprises an antigen-binding domain, a TM domain, and an ICS domain, and further comprises a hinge that joins the AB and TM domains (FIG IB) and one (FIG 1C) or two (FIG ID) costimulatory (CS) domains. FIG IE shows exemplary schematics of a CAR- encoding construct that may be included in a vector and comprises a leader sequence (LS) and an exemplary CAR construct as shown in any of FIGS 1A-1D. FIG IF shows exemplary schematics of a vector construct encoding a CAR according to the present disclosure, further comprising an exemplary ribosomal skip sequence (T2A) and an exemplary expression/purification marker, truncated CD19 (trCD19). In FIG 1, different domains (e.g., antigen-binding domain, TM domain, etc) are connected via a black line, each representing a potential additional sequence that may or may not be Included between domains.
[191] FIG 2 illustrates various exemplary antigen-binding domain constructs of CARs of some embodiments. The first two examples are "hlB2scFvHL" (or "hlB2 scFv HL") and "hlB2scFvLH" (or "hlB2 scFv LH"), which are scFvs derived from "hlB2", a humanized version of mouse anti-ALPPL2 antibody 1B2. The next two examples are "hlEBscFvHL" (or "hlEB scFv HL”) and "hlEBscFvLH" (or "hlE8 scFv LH”), which are scFvs derived from "hlEB", a humanized version of mouse anti-ALPPL2 antibody 1E8. The last two examples are "h3H2scFvHL" (or "h3H2 scFv HL") and "h3H2scFvLH” (or "h3H2 scFv LH"), which are scFvs derived from "h3H2", a humanized version of mouse anti-ALPPL2 antibody 3H2. The scFvs defined by the SEQ ID NOs shown in FIG 2 use a G4S X3 linker between the VH and VL, but any suitable linker may be used. For example, a Whitlow linker may be used. Such Whitlow linker-containing scFvs corresponding to e.g., "h3H2scFvHL" and "h3H2scFvLH" may be referred to herein as "h3H2scFvHL(Wh)" and "h3H2scFvLH(Wh)", respectively, and may comprise the amino acid sequences of e.g., SEQ ID NOs: 341 and 342, respectively, which may be encoded by e.g., SEQ ID NOs: 441 and 442, respectively.
[192] FIGS 3A-3C contain schematics of various exemplary CAR constructs of some embodiments of the invention. In the exemplary constructs in FIG 3 A, one of the antigen-binding domains shown in FIG 2 is used as the antigen-binding domain, CD28H is used as the hinge, CD28TM is used as the TM domain, CD28CS is used as the CS domain, and CD3zICS is used as the ICS domain. In FIG 3B, one of the antigen-binding domains shown in FIG 2 is used as the antigen-binding domain, CD28H is used as the hinge, CD28TM is used as the TM domain, 41BBCS is used as the CS domain, and CD3zICS is used as the ICS domain. In FIG 3C, one of the antigen-binding domains shown in FIG 2 is used as the antigen-binding domain, CD28H is used as the hinge, CD28TM is used as the TM domain, DAP10CS is used as the CS domain, and CD3zICS is used as the ICS domain. CD28H is the hinge derived from human CD28. CD28TM is the TM domain derived from human CD28. CD28CS is the CS region derived from a cytoplasmic signaling sequence of human CD28. CD3zICS is the ICS domain derived from a human CD 3 zeta. Any of the CAR constructs described in this figure or in this application may be used with LS, T2A, and/or trCD19, as shown in FIGS IE and IF. In FIG 3, although no connecting black lines are shown between different domains, an additional sequence may or may not be included between domains.
[193] FIG 4 shows a flow chart illustrating one of many possible methods for manufacturing isolated recombinant CAR-expressing cells that may be used for in vitro or in vivo studies. [194] FIGS 5A-5D contain exemplary results from cytotoxicity assays described In Example 4. Anti-ALPPL2 CART cells ("h3H2scFvHL") ("CAR T'J or T cells mock transduced with an empty vector ("EV T") were co-cultured with CAL27 cells (human tongue squamous cell carcinoma cell line) (FIG 5A), BxPC3 cells (human pancreatic adeno carcinoma cell line) (FIG SB), H1651 cells (colorectal cancer cell line) (FIG SC), Capan2 cells (human pancreatic ductal adenocarcinoma cell line) (FIG 5D) at various effector (CART cell) : target (tumor cell) ratios. Cytotoxicity was evaluated using a luciferase plate assay after 24 (top) or 48 (bottom) hours of co-culture. Asterisks represent significance between "CAR T” and "EV T" using a student's T test (* p<0.05, ** p<0.01, ***p<0.001).
[195] FIG 6 contains exemplary results from a cytokine production test described in Example 5. Anti-ALPPL2 CART cells ("h3H2scFvHL") ("CAR T") or T cells mock transduced with an empty vector ("EV T") were co-cultured with CAL27 cells, BxPC3 cells, H1651 cells , Capan2 cells, or Capan 1 cells (another human pancreatic ductal adenocarcinoma cell line) for 24 hours, and the concentrations of IFN-g in the supernatant were compared. "017" represents one experiment and "018" represents another separate experiment Error bars: standard error of the mean (SEM). Statistical differences in the IFN-g levels were calculated using Student's T test (* p<0.05, ♦* p<0.01, *** p<0.001).
[196] FIGS 7A-7C contain exemplary tumor growth in an in vivo efficacy test described in Example 6. NSG mice harboring Intraperitoneal Cal27 tumors (very low expressor of ALPPL2) were treated with human T cells expressing trCD19 butno anti-ALPPL2 ("EV T") or human T cells expressing anti-ALPPL2 CAR ("h3H2scFvHL") ("CART"). FIG 7Ais a series of Xenogen-IVIS® images showing the changes in the tumor burden in each treatment group. FIG 7B is an exemplary graph comparing the average tumor burden in the two treatment groups using the luminescent signal intensity (in radiance (photons/second)). Error bars: standard error of the mean (SEM). Statistical differences between the groups were analyzed using student T test with Mann-Whitney ranking (* p<0.05). FIG 7C provides exemplary graphs of tumor burden in individual mice in the "EV T" group (top) and "CAR T" group (bottom) using the luminescent signal intensity (in radiance (photons/second)).
[197] FIGS 8A-8B contain exemplary body weight changes in an in vivo efficacy test described in Example 6. FIG 8A is an exemplary graph comparing the average body weight changes in the two treatment groups. FIG 8B provides exemplary graphs of body weight changes of individual mice in the "EV T" group (top) and "CAR T" group (bottom).
[198] FIGS 9A-9B contain exemplary tumor growth in an in vivo efficacy test described in Example 7. NSG mice harboring intraperitoneal H1651 tumors (high expressor of ALPPL2) were treated with human T cells expressing trCD19 butno anti-ALPPL2 ("EV T") or human T cells expressing anti-ALPPL2 CAR ("h3H2scFvHL") ("CART"). FIG 7Ais a series of Xenogen-IVIS® images showing the changes in the tumor burden in each treatment group. FIG 7B is an exemplary graph comparing the average tumor burden in the two treatment groups using the luminescent signal intensity (in radiance (photons/second)). Error bars: standard error of the mean (SEM). Statistical differences between the groups were analyzed using student T test with Mann-Whitney ranking (* p<0.05). FIG 7C provides exemplary graphs of tumor burden in individual mice in the "EV T" group (top) and "CAR T" group (bottom) using the luminescent signal intensity (in radiance (photons/second)).
[199] FIG 10 provides exemplary binding of the 1B2-, 1E8-, or 3H2-producing hybridoma culture supernatant to ALPPL2 positive and negative cells analyzed by flow cytometry. Filled histograms show binding to ALPPL2 negative cells and open histograms show binding to ALPPL2 positive cells.
[200] FIGS 11A-11B provide exemplary comparison of cell binding by 1B5 and 3H2 antibodies, measured by flow cytometry. FIG 11A provides exemplary comparison of binding to human ALPPL2-expressing cells (H1651 cells and CHO cells transduced with ALPPL2) and negative control cells (CHO cells and MCF7 cells). FIG 11B provides exemplary binding curves for binding to CHO-ALPPL2 cells with various antibody concentrations.
[201] FIG 12 provides exemplary binding to recombinant human ALPPL2 by purified IgGs at different antibody concentrations (top) or IgG-contalnlng supernatant at different dilutions (bottom) of 1B5 and 3H2, measured by ELISA
[202] FIG 13 provides exemplary binding curves obtained with 1B5 (top) and 3H2 (bottom) antibodies by BLI. The ka, kd, and KD values calculated based on the curves are also shown.
[203] FIG 14 provides exemplary comparison of binding to plate-bound recombinant human ALPPL2 by different IgG antibodies before and after the affinity maturation described in Example 10, measured by ELISA FIG 11A provides exemplary binding curves obtained during the initial screening for IBS, the affinity maturation parent (B001), and some of the clones obtained by affinity maturation, including those that bind better than, similarly to, or less efficiently compared to BOOL FIG 11B provides exemplary binding curves for IBS and four clones (B380, B416, B506, and B735) selected from the affinity maturation products.
[204] FIG 15 provides exemplary curves of monovalent binding to recombinant human ALPPL2 in solution by IgGs of IBS and four clones (B380, B416, B506, and B735), measured by ELISA
[205] FIG 16 provides exemplary comparison of binding to CH0-ALPPL2 cells by IgGs of IBS and four clones (B380, B416, B506, and B735), measured by flow cytometry.
DETAILED DESCRIPTION
[206] An aspect of the invention in general relates to the construction and use of novel ALPPL2 -binding agents.
[207] In one aspect; the anti-ALPPL2 agent is, for example, but not limited to, anti- ALPPL2 antibodies (Abs), antigen-binding Ab fragments, multi-specific Abs, multi- specific antigen-binding Ab fragments, antibody-drug conjugates (ADCs), and chimeric antigen receptors (CARs).
[208] In one aspect; the antigen-binding Ab fragment comprises an antigen-binding domain.
[209] In one aspect; the antigen-binding Ab fragment may be an antigen-binding domain.
[210] The invention also provides polynucleotides encoding such an Ab, antigenbinding Ab fragment; multi-specific Ab, multi-specific antigen-binding Ab fragment; ADC, or CAR that binds to ALPPL2, vectors comprising such a polynucleotide, and cells comprising such an Ab, antigen-binding Ab fragment; multi-specific Ab, multi-specific antigen-binding Ab fragment, ADC, CAR, such a polynucleotide, or such a vector. The invention also provides compositions comprising such an Ab, antigen-binding Ab fragment; multi-specific Ab, multi-specific antigen-binding Ab fragment, ADC, CAR, such a polynucleotide, such a vector, or such a cell.
[211] The invention further provides methods of making and using an ALPPL2 -binding Ab, antigen-binding Ab fragment; multi-specific Ab, multi-specific antigen-binding Ab fragment; ADC, or CAR, or cells expressing an ALPPL2-blndlng Ab, antigen-binding Ab fragment; multi-specific Ab, multi-specific antigen-binding Ab fragment, ADC, or CAR. The invention also provides methods for treating a condition associated with ALPPL2 expression in a subject; such as cancer. Such anti-ALPPL2 Abs, antigen-binding Ab fragments, multi-specific Abs, multi-specific antigen-binding Ab fragments, ADCs, CARs, and cells comprising polynucleotide encoding such an ALPPL2-binding Ab, antigenbinding Ab fragment; multi-specific Ab, multi-specific antigen-binding Ab fragment; ADC, or CAR may be used to treat diseases, disorders, or conditions associated with the undesired proliferation of cells expressing ALPPL2.
Binding target
[212] In one aspect; the anti-ALPPL2 agents of the present invention bind to ALPPL2.
[213] In one aspect, the target, or the binding target, of the anti-ALPPL2 agents of the present invention is ALPPL2.
[214] In one aspect; the anti-ALPPL2 antibody (Ab), anti-ALPPL2 antigen-binding Ab fragment; anti-ALPPL2 multi-specific Ab, anti-ALPPL2 multi-specific antigen-binding Ab fragment, anti-ALPPL2 antibody-drug conjugate (ADC), and anti-ALPPL2 chimeric antigen receptor (CAR) of the present invention individually comprise an antigenbinding domain which binds to Alkaline phosphatase, placenta-like 2 (ALPPL2), which is also referred to as "ALPPL", "alkaline phosphatase, germ cell (APGC)", or “germ cell alkaline phosphatase (GCAP)”.
[215] In humans, ALPPL2 is encoded by the ALPG gene on chromosome 2, with gene location 2q37.1 (NCBI). Human ALPPL2 may have an amino acid sequence provided as GenBank: AIC53992.1. In one aspect; human ALPPL2 has the amino acid sequence provided as SEQ ID NO: 101 or the equivalent residues from a non-human species, e.g., mouse, rodent; monkey, ape and the like.
[216] In healthy humans, the protein expression of ALPPL2 is primarily in the placenta (https;// yAvw.proteinatias.org/ ENSG00000163286-ALPPL2/tissue) and ALPPL2 regulates naive pluripotency (Bi Y. etai., Cell Rep. 2020 Mar 17;30(llj;3917-393Le5. doi: 10,1016/j.celrep.2020.02.090.). However, ALPPL2 is upregulated in different types of cancer such as, but not limited to, pancreatic cancer, testicular cancer, cervical cancer, endometrial cancer, ovarian cancer, and stomach cancer (htq)$://www.protelnatias.Grg/ENSG00C'00163286"ALPPL2/patiiok)gy). Studies further report that ALPPL2 is also upregulated in colorectal cancer, lung cancer, and mesothelioma (Skinner, J.M., Whitehead, R. Virchows Archiv [Pathol Anal] 394, 109-118 (1981).; Wick M. R. et al, Hum Pathol. 1987 Sep;18(9):946-54.; and Su Y et al., bioRxiv 2020.01.07.898122; doi: https://doi.org/10.1101/2020.01.Q7.898122. which is now Su Y et al., Cancer Res. 2020 Oct 15;80(20);4552-4564.). Examples described below additionally show that tongue cancer also express ALPPL2 (see, e.g., FIGS 5A, 6, 7A-7B).
[217] Therefore, in some embodiments, the anti-ALPPL2 agents of the present invention may bind to or target ALPPL2 on cancer cells of the above-mentioned cancer types.
[218] In some embodiments, anti-ALPPL2 agents may bind pancreatic cancer cells. In some embodiments, anti-ALPPL2 agents may bind testicular cancer cells. In some embodiments, anti-ALPPL2 agents may bind cervical cancer cells. In some embodiments, anti-ALPPL2 agents may bind endometrial cancer cells. In some embodiments, anti- ALPPL2 agents may bind ovarian cancer cells. In some embodiments, anti-ALPPL2 agents may bind stomach cancer cells. In some embodiments, anti-ALPPL2 agents may bind colorectal cancer cells. In some embodiments, anti-ALPPL2 agents may bind lung cancer cells. In some embodiments, anti-ALPPL2 agents may bind colorectal cancer cells. In some embodiments, anti-ALPPL2 agents may bind tongue cancer cells.
Anti-AI.PPL2 antibody, antigen-binding fragment multi-specific antibody, multi-specific antigen-binding fragment and antibody-drug conjugate
[219] In some embodiments, the anti-ALPPL2 antibody (Ab), anti-ALPPL2 antigenbinding (AB) fragment; anti-ALPPL2 multi-specific Ab, anti-ALPPL2 multi-specific antigen-binding Ab fragment; and anti-ALPPL2 antibody-drug conjugate (ADC) of the present invention, individually comprise at least one antigen-binding (AB) domain that binds to ALPPL2.
[220] The ALPPL2 -binding domain (i.e., the antigen-binding domain) may comprise the antigen-binding domain of an anti-ALPPL2 monoclonal antibody, such as a mouse antibody or a humanized version of a mouse antibody. In some embodiments, the ALPPL2 -binding domain (i.e., the antigen-binding domain) may comprise the antigenbinding domain of a mouse anti-ALPPL2 antibody clone 1B2, 1E8, or 3H2, or the antigen-binding domain of a humanized version of a mouse anti-ALPPL2 monoclonal antibody clone 1B2, 1E8, 3H2, or their variants. [221] Three novel anti-ALPPL2 monoclonal antibodies, named 1B2, 1E8, and 3H2, were generated as described in Example 1.
[222] The mouse anti-ALPPL2 monoclonal antibody clone 3H2 comprises: (a) a VH sequence as set forth in SEQ ID NO: 131, which may be encoded by SEQ ID NO: 231; and (b) VL sequence as set forth in SEQ ID NO: 135, which may be encoded by SEQ ID NO: 235. The CDR1, CDR 2, and CDR 3 of the VH (i.e., CDR-H1, CDR-H2, and CDR-H3) comprise the amino acid sequence of SEQ ID NOs: 132, 133, and 134, respectively, which may be encoded by SEQ ID NOs: 232, 233, and 234, respectively. The CDR1, CDR 2, and CDR 3 of the VL (l.e., CDR-L1, CDR-L2, and CDR-L3) comprise the amino acid sequence of SEQ ID NOs: 136, 137, and 138, respectively, which may be encoded by SEQ ID NOs: 236, 237, and 238, respectively.
[223] Inventor performed humanization on 1B2, 1E8, and 3H2 antibodies as described in Example 2 herein. .
[224] The humanized version of 3H2 (may be referred to as h3H2) comprises: (a) a VH sequence as set forth in SEQ ID NO: 331, which may be encoded by SEQ ID NO: 431; and (b) a VL sequence as set forth in SEQ ID NO: 335, which may be encoded by SEQ ID NO: 435. The CDR 1, CDR 2, and CDR 3 of the VH (l.e., CDR-H1, CDR-H2, and CDR-H3) may comprise the amino acid sequence of SEQ ID NOs: 332, 333, and 334, respectively, which may be encoded by SEQ ID NOs: 432, 433, and 434, respectively. The CDR1, CDR 2, and CDR 3 of the VL (i.e., CDR-L1, CDR-L2, and CDR-L3) may comprise the amino acid sequence of SEQ ID NOs: 336, 337, and 338, respectively, which may be encoded by SEQ ID NOs: 436, 437, and 438, respectively.
[225] Inventor performed affinity maturation on the VH and VL of 3H2 and h3H2 antibodies as described in Example 8.
[226] One of the affinity maturation products were named 1B5, which comprises: (a) a VH sequence as set forth in SEQ ID NO: 701; and (b) a VL sequence as set forth in SEQ ID NO: 705, which may be encoded by SEQ ID NO: 805. The CDR 1, CDR 2, and CDR 3 of the VH (i.e., CDR-H1, CDR-H2, and CDR-H3) may comprise the amino acid sequence of SEQ ID NOs: 702, 703, and 704, respectively. The CDR1, CDR 2, and CDR 3 of the VL (i.e., CDR- Ll, CDR-L2, and CDR-L3) may comprise the amino acid sequence of SEQ ID NOs: 706, 707, and 708, respectively, which may be encoded by SEQ ID NOs: 806, 807, and 808, respectively.
[227] Inventor performed humanization on 1B5 (specifically, humanized the VL of 1B5) as described in Example 10 and obtained B001, which comprises: (a) a VH sequence as set forth in SEQ ID NO: 711; and (b) a VL sequence as set forth in SEQ ID NO: 715. The CDR 1, CDR 2, and CDR 3 of the VH (i.e., CDR-H1, CDR-H2, and CDR-H3) may comprise the amino add sequence of SEQ ID NOs: 712, 713, and 714, respectively. The CDR1, CDR 2, and CDR 3 of the VL (i.e., CDR-L1, CDR-L2, and CDR-L3) may comprise the amino add sequence of SEQ ID NOs: 716, 717, and 718, respectively.
[228] Inventor then performed affinity maturation on B001 and obtained B380, B416, B506, and B735. B380 comprises: (a) a VH sequence as set forth in SEQ ID NO: 721; and (b) a VL sequence as set forth in SEQ ID NO: 725. The CDR 1, CDR 2, and CDR 3 of the VH (l.e., CDR-H1, CDR-H2, and CDR-H3) may comprise the amino acid sequence of SEQ ID NOs: 722, 723, and 724, respectively. The CDR1, CDR 2, and CDR 3 of the VL (i.e., CDR- Ll, CDR-L2, and CDR-L3) may comprise the amino acid sequence of SEQ ID NOs: 726, 727, and 728, respectively.
[229] B416 comprises: (a) a VH sequence as set forth in SEQ ID NO: 731; and (b) a VL sequence as set forth in SEQ ID NO: 735. The CDR 1, CDR 2, and CDR 3 of the VH (i.e., CDR-H1, CDR-H2, and CDR-H3) may comprise the amino add sequence of SEQ ID NOs: 732, 733, and 734, respectively. The CDR1, CDR 2, and CDR 3 of the VL (i.e., CDR-L1, CDR-L2, and CDR-L3) may comprise the amino add sequence of SEQ ID NOs: 736, 737, and 738, respectively.
[230] 8506 comprises: (a) a VH sequence as set forth in SEQ ID NO: 741; and (b) a VL sequence as set forth in SEQ ID NO: 745. The CDR 1, CDR 2, and CDR 3 of the VH (i.e., CDR-H1, CDR-H2, and CDR-H3) may comprise the amino add sequence of SEQ ID NOs: 742, 743, and 744, respectively. The CDR1, CDR 2, and CDR 3 of the VL (i.e., CDR-L1, CDR-L2, and CDR-L3) may comprise the amino add sequence of SEQ ID NOs: 746, 747, and 748, respectively.
[231] 8735 comprises: (a) a VH sequence as setforth in SEQ ID NO: 751; and (b) a VL sequence as setforth in SEQ ID NO: 755. The CDR 1, CDR 2, and CDR 3 of the VH (i.e., CDR-H1, CDR-H2, and CDR-H3) may comprise the amino add sequence of SEQ ID NOs: 752, 753, and 754, respectively. The CDR1, CDR 2, and CDR 3 of the VL (i.e., CDR-L1, CDR-L2, and CDR-L3) may comprise the amino add sequence of SEQ ID NOs: 756, 757, and 758, respectively.
[232] Therefore, in some embodiments, the ALPPL2 -binding domain (i.e., the antigenbinding domain) of the anti-ALPPL2 agent of the present invention may comprise (a) a CDR-H1, a CDR-H2, and a CDR-H3 contained in the VH of any of the anti-ALPPL2 Abs or Ab fragments described herein, and (b) a CDR-L1, a CDR-L2, and a CDR-L3 contained in the VL of any of the anti-ALPPL2 Abs or Ab fragments described herein. The CDR sequences may be determined based on given VH and VL sequences or VH-encoding and VL-encoding sequences using any appropriate methods and definitions. For example, CDR sequences may be determined based on numbering schemes (e.g., most commonly EU numbering, IMGT numbering, or Rabat numbering; or alternatively Chothia numbering, Martin numbering, Gelfand numbering, Honneger's numbering) of variable regions or may be determined structurally (see e.g., IgBlast (https://www.ncbi.nhn.nih.gov/igblast/); Dondelin ger etai., Front Immunol 2018 Oct 16, ‘9: 2278; http://www.bioinf.org.Uk/abs/info.html#cdrid).
[233] In some embodiments, the ALPPL2 -binding domain (Le., the antigen-binding domain, for example the variable region of an Ab or Ab fragment or the antigen-binding domain of a CAR) may comprise (a) a VH comprising a CDR-H1, and a CDR-H2, and a CDR-H3 contained in the VH of any of the anti-ALPPL2 Abs or Ab fragments described herein; and (b) a VL comprising a CDR-L1, a aCDR-L2, and CDR-L3 contained in the VL of any of the anti-ALPPL2 Abs or Ab fragments described herein. [234] In some embodiments, the VH comprises a human or human-like VH framework and the VL comprises a human or human-like VL framework.
[235] In some embodiments, in the antigen-binding domain of the anti-ALPPL2 agent of the present invention, the CDR-H1, CDR-H2, and CDR-H3 may be the CDR-H1, CDR- H2, and CDR-H3 contained In SEQ ID NO: 721 and/or may comprise the amino acid sequences set forth in SEQ ID NOs: 722, 723, and 724, respectively, and the CDR-L1, CDR-L2, and CDR-L3 may be the CDR-L1, CDR-L2, and CDR-L3 contained in SEQ ID NO: 725 and/or may comprise the amino acid sequences set forth in SEQ ID NOs: 726, 727, and 728, respectively.
[236] In some embodiments, in the antigen-binding domain of the anti-ALPPL2 agent of the present invention, the CDR-H1, CDR-H2, and CDR-H3 may be the CDR-H1, CDR- H2, and CDR-H3 contained in SEQ ID NO: 731 and/or may comprise the amino acid sequences set forth In SEQ ID NOs: 732, 733, and 734, respectively, and the CDR-L1, CDR-L2, and CDR-L3 may be the CDR-L1, CDR-L2, and CDR-L3 contained in SEQ ID NO: 735 and/or may comprise the amino acid sequences set forth in SEQ ID NOs: 736, 737, and 738, respectively.
[237] In some embodiments, in the antigen-binding domain of the anti-ALPPL2 agent of the present invention, the CDR-H1, CDR-H2, and CDR-H3 may be the CDR-H1, CDR- H2, and CDR-H3 contained in SEQ ID NO: 741 and/or may comprise the amino acid sequences set forth in SEQ ID NOs: 742, 743, and 744, respectively, and the CDR-L1, CDR-L2, and CDR-L3 may be the CDR-L1, CDR-L2, and CDR-L3 contained in SEQ ID NO: 745 and/or may comprise the amino acid sequences set forth in SEQ ID NOs: 746, 747, and 748, respectively.
[238] In some embodiments, in the antigen-binding domain of the anti-ALPPL2 agent of the present invention, the CDR-H1, CDR-H2, and CDR-H3 may be the CDR-H1, CDR- H2, and CDR-H3 contained in SEQ ID NO: 751 and/or may comprise the amino acid sequences set forth in SEQ ID NOs: 752, 753, and 754, respectively, and the CDR-L1, CDR-L2, and CDR-L3 may be the CDR-L1, CDR-L2, and CDR-L3 contained in SEQ ID NO: 755 and/or may comprise the amino acid sequences set forth in SEQ ID NOs: 756, 757, and 758, respectively.
[239] Generally, to humanize a mouse antibody, the CDRs from the mouse antibody may be grafted into the human antibody framework. Therefore, the human-like framework may be 100% identical to a human framework. Inventor used the Tabhu program (http://circe.med.uniromal.it/tabhu/) to perform humanization, which involves four steps, which are: (i) loop grafting, (ii) estimation of the binding mode similarity between the native and human antibody, (iii) back-mutations and (iv) reevaluation of the binding mode similarity between input and humanized antibody (Olimpieri P. P. etal., Bioinformatics. 2015 Feb l;31(3):434-5. doi: 10.1093/bioinformatics/btu667. Epub 2014 Oct 9). Therefore, in some embodiments, the framework may not be 100% identical to a human framework but may still comprise significant sequence identity to a human framework. [240] In some embodiments, (a) the VH may comprise a human or human-like VH framework may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human VH framework, and (b) the VL may comprise a human or human-like VL framework may at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human VL framework
[241] In some embodiments, the variable region of an anti-ALPPL2 agent disclosed herein may be altered without significantly reducing ALPPL2 binding. As long as the antigen-binding domain sufficiently binds to ALPPL2, the sequence of the variable region may be altered. Antigen-Ab interactions are largely determined by six CDRs, while a person of skilled in the art will appreciate that some deviation from the exact CDR sequences may be possible. Any suitable techniques such as affinity maturation can be used to alter the CDR sequence. Among the six CDRs, VH CDR 3 and VL CDR 3 are generally believed as the key determinant of specificity in antigen recognition. Particularly, diversity in the CDR 3 of VH (i.e., CDR-H3) may be particularly important for providing most antibody specificities (Xu J.L, Immunity. 2000 Jul;13(l):37-45). Therefore, one or more mutations may be incorporated in the CDR 1 and/or CDR 2 without greatly decreasing the binding affinity while achieving a more desired property of an Ab. Abs or antigen-binding Ab fragments comprising CDR-H1, CDR-H2, CDR-L1, CDR-L2, and/or CDR-L3 with at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the CDR-H1, CDR-H2, CDR-L1, CDR-L2, and/or CDR-L3 disclosed herein, respectively, are also within the scope of the present invention. Furthermore, one or more mutations in CDR-H3 may be incorporated to modify, increase, or fine tune the binding or any other properties of the antigen-binding domain. Alternatively, since any one mutation may alter biochemical properties such as thermodynamic stability or immunogenicity in addition to the affinity, all possible mutations in any of the six CDRs, and/or any combination thereof, and/or even in the framework sequence, may be tested to see if the sequence modification provides an improved or more desired overall property (Rajpal A et al., Proc Natl Acad Sci U S A 2005 Jun 14; 102(24): 8466-8471. doi: 10.1073/pnas.0503543102; Julian M. C. etal., Sci Rep. 2017; 7: 45259. Published online 2017 Mar 28. doi: 10.1038/srep45259). To test the binding of a modified Ab to the target, any appropriate technique such as, but not limited to, ELISA RIA FACS, bioassay, or Western Blot assay may be used.
[242] In some embodiments, the heavy chain variable region (i.e., VH) may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 721, and the light chain variable region (i.e., VL) comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 725. [243] In some embodiments, the VH may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 731, and the VL comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 735.
[244] In some embodiments, the VH may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% Identical to SEQ ID NO: 741, and the VL comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 745.
[245] In some embodiments, the VH may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 751, and the VL comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 755.
[246] In some embodiments, the anti-ALPPL2 agent of the present invention, may be, for example, but not limited to, a monoclonal Ab, a monospecific Ab, a bispecific Ab, a multispeclflc Ab, a humanized Ab, a tetrameric Ab, a tetravalent Ab, a single chain Ab, a domain-specific Ab, a domain-deleted Ab, an scFc fusion protein, a chimeric Ab, a synthetic Ab, a recombinant Ab, a hybrid Ab, a mutated Ab, CDR-grafted Ab, a fragment antigen-binding (Fab), an F(ab')2, an Fab' fragment; a variable fragment (Fv), a singlechain Fv (scFv) fragment; an Fd fragment; a diabody, and a minibody.
[247] In particular embodiments the anti-ALPPL2 agent is or comprises a scFv comprising a VH, a linker, and a VL, where the VH and VL combinations are any of the combinations described above. The scFv according to the present disclosure may comprise: a VH, a linker, and a VL, or a VL, a linker, and a VH, in the direction from the N- terminus to the C-terminus. Any appropriate linker that allows the VH and VL to form an antigen-binding site that binds to ALPPL2 may be used.
[248] In some embodiments, the linker may be a GS linker, which is one or more repeats of a sequence unit containing G (glycine) and S (serine), such as the GS linker unit of SEQ ID NO: 167. For example, the G4SX3 linker of SEQ ID NO: 168 may be used.
[249] In certain embodiments, the antigen-binding domain of the anti-ALPPL2 agent of the present invention may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to: (i) SEQ ID NOs: 760, 759, 750, 749, 740, 739, 730, 729, 720, 719, 319, 320, 329, 330, 339, 340, 341, or 342 or (ii) the amino acid sequence encoded by SEQ ID NOs: 419, 19, 420, 20, 429, 29, 430, 30, 439, 39, 440, 40, 441, or 442. [250] In some embodiments, the anti-ALPPL2 agent of the present Invention may comprise two or more binding specificities (i.e., bispecific, trispecific, or multispecific generally). The first specificity is to an epitope in ALPPL2 (first ALPPL2 epitope).
[251] In one aspect; the anti-ALPPL2 agent of the present disclosure may have a second binding specificity to another epitope (l.e., second ALPPL2 epitope) in ALPPL2. The second ALPPL2 epitope may or may not be overlapped with the first ALPPL2 epitope.
[252] In another aspect; the second specificity may be to an epitope in a second antigen other than ALPPL2. A multi-specific ALPPL2 -binding agent according to the present disclosure may bind to ALPPL2 and one or more other targets. In some embodiments, a multi-specific anti-ALPPL2 agent binds to ALPPL2 and a protein on an effector cell. In some embodiments, a multi-specific anti-ALPPL2 agent binds to ALPPL2 and a protein on a target (e.g., cancer) cell. In some embodiments, binding to a second antigen may improve functional characteristics of the anti-ALPPL2 agent; e.g., recruitment, effector functions, lysis of target cells.
[253] In certain embodiments, the second antigen may be, for example without limitation, NKG2D, 4- IBB, or an Fc receptor (FcR), such as Fc gamma receptor (FcgR), FcgRI, FcgRIIA, FcgRIIBl, FcgRIIB2, FcgRIIIA, FcgRIIIB, Fc epsilon receptor (FceR), FceRI, FceRII, Fc alpha receptor (FcaR), FcaRI, Fc alpha/mu receptor (Fca/mR), or neonatal Fc receptor (FcRn). For anti-ALPPL2 Abs and antigen-binding Ab fragments, having a specificity to an FcR allows FcR-medlated effects such as antibody-dependent cellular phagocytosis (ADCP) or antibody-dependent cellular cytotoxicity (ADCC) of ALPPL2 -expressing cells or cytotoxic mediator release by Fc-expressing cells.
[254] When the second epitope is in FcR, the FcR may be, but is not limited to, Fc gamma receptor (FcgR), FcgRI, FcgRIIA, FcgRIIBl, FcgRIIB2, FcgRIIIA, FcgRIIIB, Fc epsilon receptor (FceR), FceRI, FceRII, Fc alpha receptor (FcaR), FcaRI, Fc alpha/mu receptor (Fca/mR), or neonatal Fc receptor (FcRn).
[255] When the anti-ALPPL2 agent has two specificities, the agent may be called bispecific. Bispecific anti-ALPPL2 agents include bispecific anti-ALPPL2 Abs or antigenbinding Ab fragments. When the anti-ALPPL2 agent has two or more specificities, the agent may be called multispecific. Multispecific anti-ALPPL2 agents include multispecific anti-ALPPL2 Abs or antigen-binding Ab fragments.
[256] The present invention encompasses any types of bispecific Ab-like molecules (Abs or antigen-binding Ab fragments) such as reviewed in Brinkmann U. et al., MAbs. 2017 Feb-Mar; 9(2): 182-212. Published online 2017 Jan 10. doi: 10.1080/19420862.2016.1268307; Klein C .et al., MAbs. 2016 Aug-Sep;8(6): 1010-20. doi: 10.1080/19420862.2016.1197457. In a bispecific embodiment according to the present disclosure, one of the antigen-binding domains is an anti-ALPPL2 binding domain. General methods for designing and construction of bispecific or multispecific Abs or antigen-binding Ab fragments are known in the art (Brinkmann U. et al., MAbs. 2017 Feb-Mar; 9(2): 182-212. Published online 2017 Jan 10. doi: 10.1080/19420862.2016.1268307; Dimasi N. etal. Methods. 2018 Aug 11. pii: S1046- 2023(18)30149-X dol: 10.1016/j.ymeth.2018.08.004; Sedykh S. E. etal., Drug Des Devel Then 2018; 12: 195-208). Such methods include chemical conjugation, covalent attachment of fragments, and genetic engineering. For example, full-length bispecific Abs or antigen-binding Ab fragments may be generated by co-expressing two pairs of heavy and light chains, each pair having different specificities. The two pairs may be encoded in one vector, or encoded in separate vectors but expressed in the same host cell. Alternatively, antigen-binding Ab fragments or the antigen-binding domains having different specificities may be generated separately and then conjugated to one another, for example using sulfhydryl bonding (of, for example, the VH C-termlnus hinge regions) and/or an appropriate coupling or crosslinking agent Bispecific antigen-binding Ab fragments may also be generated, for example, by using leucine zippers or by using scFv dimers (see for example, Kosteln et al.,/ Immunol. 1992 Mar 1;148(5): 1547-53). Binding of the bispecific agent of the present Invention may be confirmed using any appropriate method, such as but not limited to, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), flow cytometry, bioassay, or Western blot
[257] In some embodiments, the anti-ALPPL2 agents of the present invention may comprise a human or human-like CHI, CH2, and/or CH3 domain(s).
[258] In certain embodiments, the human-like CHI, CH2, and/or CH3 domain(s) may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human CHI, CH2, and/or CH3 domaln(s), respectively.
[259] In certain embodiments, the human or human-like CHI, CH2, and/or CH3 domain(s) may be individually derived from the CHI, CH2, and/or CH3 domain(s), respectively, of a human IgM, a human IgD, a human IgG, a human IgE, or a human IgA optionally of a human IgGl, a human IgG2, a human IgG3, or a human IgG4.
[260] In some embodiments, the anti-ALPPL2 agents of the present invention may comprise a human or human-like fragment crystallizable (Fc) region.
[261] In some embodiments, the human or human-like Fc region may be at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human Fc region.
[262] In some embodiments, the human or human-like Fc region may be derived from a human IgM, IgD, IgG, IgE, or IgA preferably IgGl, IgG2, IgG3, or IgG4.
[263] In certain embodiments, the human-like Fc region may bind to an Fc receptor (FcR). The FcR may be, but is not limited to, Fc gamma receptor (FcgR), FcgRI, FcgRIIA, FcgRIIBl, FcgRlIB2, FcgRIIIA FcgRIIIB, Fc epsilon receptor (FceR), FceRI, FceRII, Fc alpha receptor (FcaR), FcaRI, Fc alpha/mu receptor (Fca/mR), or neonatal Fc receptor (FcRn).
[264] In some embodiments, when the anti-ALPPL2 agent is an Ab, the Ab may be of an IgM, IgD, IgG, IgE, or IgA isotype.
[265] In some embodiments, when the Ab is an IgG, and the IgG may be an IgGl, IgG2, IgG3, or IgG4. [266] Certain amino add modifications In the Fc region are known to modulate Ab effector functions and properties, such as, but not limited to, antibody-dependent cellular cytotoxidty (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement dependent cytotoxidty (CDC), and half-life (Wang X et al., Protein Cell. 2018 Jan; 9(1): 63-73; Dall'Acqua W. F. et at J Biol Chem. 2006 Aug 18;281(33):23514- 24. Epub 2006 Jun 21; Monnet C. etal, Front Immunol. 2015 Feb 4;6:39. doi: 10.3389/fimmu.2015.00039. eCollection 2015). The mutation may be symmetrical or asymmetrical In certain cases, antibodies with Fc regions that have asymmetrical mutation(s) (l.e., two Fc regions are not identical) may provide better functions such as ADCC (Liu Z. etalj Biol Chem. 2014 Feb 7; 289(6): 3571-3590).
[267] When the Ab is an IgGl, the Fc region may comprise one or more amino add substitutions. The substitution maybe, for example, N297A, N297Q, D265A, L234A, L235A, C226S, C229S, P238S, E233P, L234V, G236-deleted, P238A A327Q, A327G, P329A, K322A, L234F, L235E, P331S, T394D, A330L, P331S, F243L, R292P, Y300L, V305I, P396L, S239D, I332E, S298A, E333A, K334A, L234Y, L235Q, G236W, S239M, H268D, D270E, K326D, A330M, K334E, G236A, K326W, S239D, E333S, S267E, H268F, S324T, E345R, E430G, S440Y M428L, N434S, L328F, M252Y, S254T, T256E, and/or any combination thereof (the residue numbering is according to EU numbering) (Dall'Acqua W. F. et al., J Biol Chem. 2006 Aug 18;281(33):23514-24. Epub 2006 Jun 21; Wang X et al, Protein Cell. 2018 Jan; 9(1): 63-73). The Fc region may further comprise one or more additional amino add substitutions. The substitution may be, for example, but is not limited to, A330L, L234F, L235E, P331S, and/or any combination thereof (the residue numbering is according to EU numbering).
[268] When the Ab is an IgG2, the Fc region may comprise one or more amino add substitutions. The substitution may be, for example, but is not limited to, P238S, V234A, G237A, H268A, H268Q, H268E, V309L, N297A, N297Q, A330S, P331S, C232S, C233S, M252Y, S254T, T256E, and/or any combination thereof (the residue numbering is according to EU numbering). The Fc region may further comprise one or more additional amino add substitutions. The substitution may be, for example, but is not limited to, M252Y, S254T, T256E, and/or any combination thereof (the residue numbering is according to EU numbering).
[269] When the Ab is an IgG3, the Fc region may comprise one or more amino add substitutions. The substitution may be, for example, but is not limited to, E235Y (the residue numbering is according to EU numbering).
[270] When the Ab is an IgG4, the Fc region may comprise one or more amino add substitutions. The substitution may be, for example, but is not limited to, E233P, F234V, L235A, G237A, E318A S228P, L236E, S241P, L248E, T394D, M252Y, S254T, T256E, N297A, N297Q, and/or any combination thereof (the residue numbering is according to EU numbering). The substitution may be, for example, S228P (the residue numbering is according to EU numbering).
[271] In some embodiments, the glycan of the human-like Fc region may be engineered to modify the effector function (for example, see Li T. et al, Proc Nad Acad Scl U S A. 2017 Mar 28;114(13):3485-3490. dol: 10.1073/pnas.l702173114. Epub 2017 Mar 13).
[272] The binding property of an anti-ALPPL2 may be assessed by any appropriate methods.
[273] In some embodiments, binding to ALPPL2 (e.g., recombinant ALPPL2) may be measured by ELISA, RIA, FACS, bioassay, Western blot; or BLI. The binding property may be defined for example by association rate constant (k.), equilibrium dissociation constant (kd), dissociation constant (KD), and/or half maximal effective concentration (ECso).
[274] In certain embodiments, the KD of an anti-ALPPL2 agent for ALPPL2 binding may be about 10 nM or smaller. In particular embodiments, the KD of an anti-ALPPL2 agent may be about 1.6 nM, about 0.8 nM, about 0.4 nM, about 0.3 nM, or about 0.2 nM.
[275] In certain embodiments, the ECso of an anti-ALPPL2 agent for ALPPL2 binding may be about 1 pg/mL or smaller. In particular embodiments, the ECso of an anti- ALPPL2 agent for ALPPL2 binding may be about 0.05 pg/mL or about 0.02 pg/mL
[276] In some embodiments, binding to ALPPL2-expressing cells (e.g., cancer cells or recombinant cells transduced with ALPPL2) may be measured by flow cytometry, for example based on MFI. Such binding property may be defined for example by half maximal effective concentration (ECso).
[277] In certain embodiments, the ECso of an anti-ALPPL2 agent for cell binding may be about 100 nM or smaller. In particular embodiments, the ECso of an anti-ALPPL2 agent may be about 15 nM, about 10 nM, about 5 nM, about 1 nM, about 0.5 nM, or about 0.2 nM.
[278] In some embodiments, the anti-ALPPL2 agent of the present invention may be an antibody-drug conjugate (ADC). The ADC may comprise: (a) any Ab or antigenbinding Ab fragment described herein; and (b) a drug conjugated to the Ab or antigenbinding Ab fragment
[279] In some embodiments, the drug may be, but not limited to, an anti-cancer drug, an anti-proliferative drug, a cytotoxic drug, an anti-angiogenic drug, an apoptotic drug, an immunostimulatory drug, an anti-microbial drug, an antibiotic drug, an antiviral drug, an anti-inflammatory drug, an ALPPL2 inhibitor, an ALPPL2 signaling inhibitor, an enzyme, a hormone, a toxin, a radioisotope, a compound, a small molecule, a small molecule inhibitor, a protein, a peptide, a vector, a plasmid, a viral replicon, a viral particle, a nanoparticle, a DNA molecule, an RNA molecule, an siRNA, an shRNA, a micro RNA, an oligonucleotide, and an imaging drug.
[280] The toxin may be a bacterial, fungal, plant, or animal toxin, or a fragment thereof. Examples include, but are not limited to, diphtheria A chain, diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha sardn, Aleurites fordii protein, a dianthin protein, or a Phytolacca Americana protein.
[281] The anti-cancer or anti-proliferative drug may be, for example, but is not limited to, doxorubicin, daunorubicin, cucurbitacin, chaetocin, chaetoglobosin, chlamydocin, calicheamicin, nemorubicin, cryptophyscin, mensacarcin, ansamitodn, mitomydn C, geldanamydn, mechercharmydn, rebeccamydn, safradn, okilactomydn, oligomydn, actinomydn, sandramydn, hypothemydn, polyketomydn, hydroxyelliptidne, thiocolchidne, methotrexate, triptolide, taltobulin, lactacystin, dolastatin, auristatin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), telomestatin, tubastatin A, combretastatin, maytanslnoid, MMAD, MMAF, DM1, DM4, DTT, 16-GMB- APA-GA, 17-DMAP-GA, JW 55, pyrrolobenzodiazepine, SN-38, Ro 5-3335, puwainaphydn, duocarmydn, bafilomydn, taxoid, tubulysin, ferulenol, lusiol A, fumagillin, hygrolidin, glucopieriddin, amanitin, ansatrienin, dnerubin, phalladdin, phalloldln, phytosphongoslne, pieriddin, poronetin, phodophyllotoxin, gramlddln A, sanguinarine, sinefungin, herboxidiene, microcolin B, microcystin, muscotoxin A, tolytoxin, tripolin A, myoseverin, mytoxin B, nocuolin A, psuedolaric add B, pseurotin A, cyclopamine, curvulin, colchidne, aphidicolin, englerin, cordycepin, apoptolidin, epothllone A, llmaqulnone, isatropolone, isoflstularin, qulnaldopeptin, ixabepllone, aeroplysinin, arruginosin, agrochelin, epothilone, or a derivative thereof (for example, see Polakis P. et al., Pharmacol Rev. 2016 Jan;68(l):3-19. doi: 10.1124/pr.ll4.009373) (the drugs may be obtained from many vendors, including Creative Biolabs ®).
[282] The radioisotope may be for example, bulls not limited to, At211, 1131, Inl31, 1125, Y90, Rel86, Rel88, Sml53, Bi212, P32, Pb212 and radioactive isotopes of Lu.
[283] In certain embodiments, the drug may be, but is not limited to, MMAE or MMAF.
[284] In some embodiments, the Ab or antigen-binding Ab fragment is directly conjugated to the drug to form an ADC.
[285] In some embodiments, the Ab or antigen-binding Ab fragment is indirectly conjugated to the drug to form an ADC.
[286] Any appropriate conjugation method may be used to generate an ADC (for example, Nolting B. Methods Mol Biol. 2013;1045:71-100. doi: 10.1007/978-1-62703- 541-5,5; Jain N. etal., Pharm Res. 2015 Nov;32(ll):3526-40. doi: 10.1007/sll095-015- 1657-7. Epub 2015 Mar 11; Tsuchikama K. etal., Protein Cell. 2018 Jan;9(l):33-46. doi: 10.1007/sl3238-016-0323-0. Epub 2016 Oct 14; Polakis P. et al., Pharmacol Rev. 2016 Jan;68(l):3-19. doi: 10.1124/pr.ll4.009373). Examples of methods that may be used to perform conjugation include, but are not limited to, chemical conjugation and enzymatic conjugation.
[287] Chemical conjugation may utilize, for example, but is not limited to, lysine amide coupling, cysteine coupling, and/or non-natural amino add incorporation by genetic engineering. Enzymatic conjugation may utilize, for example, but is not limited to, transpeptidation using sortase, transpeptidation using microbial transglutaminase, and/or N-Glycan engineering.
[288] In certain embodiments, one or more of cleavable linkers may be used for conjugation. The cleavable linker may enable cleavage of the drug upon responding to, for example, but not limited to, an environmental difference between the extracellular and intracellular environments (pH, redox potential, eta) or by spedfic lysosomal enzymes. [289] Examples of the cleavable linker include, but are not limited to, hydrazone linkers, peptide linkers including cathepsin B-responsive linkers, such as valin-citrulline (vc) linker, disulfide linkers such as N-succinimidyl-4-(2 -pyridyldithio) (SPP) linker or N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB) linker, and pyrophosphate diester linkers.
[290] Alternatively or simultaneously, one or more of non-deavable linkers may be used. Examples of non-cleavable linkers include thioether linkers, such as N- succinimidyl 4-(N-maleimidomethyl) cyclohexane-l-carboxylate (SMCC), and malelmidocaproyl (me) linkers. Generally, non-cleavable linkers are more resistant to proteolytic degradation and more stable compared to cleavable linkers.
Anti-ALPPI.2 Chimeric antigen receptors (CAR)
[291] In some embodiments, an anti-ALPPL2 agent according to the present disclosure may be a chimeric antigen receptor (CAR). In particular, the CARs of the present invention comprise an antigen binding (AB) domain that binds to ALPPL2, a transmembrane (TM) domain, and an intracellular signaling (ICS) domain.
[292] A schematic showing a generic CAR construct of the present invention is depicted in FIG 1A.
[293] The CAR may optionally comprise a hinge that joins the antigen-binding domain and said TM domain. The CAR may optionally comprise one or more costimulatory (CS) domains. Schematics showing three more generic CAR constructs of the present invention are depicted in FIGS IB- ID.
Antigen-binding domain
[294] The CAR of the present invention comprises an antigen-binding domain which binds to ALPPL2.
[295] In some embodiments, the antigen-binding domain of the CAR may comprise any of the anti-ALPPL2 agents disclosed herein.
[296] In some embodiments, the antigen-binding domain of the CAR may comprise any of the antigen-binding domain of any of the anti-ALPPL2 agents disclosed herein.
[297] In some embodiments, the antigen-binding domain of the CAR may comprise any of the anti-ALPPL2 Abs, anti-ALPPL2 antigen-binding Ab fragments, anti-ALPPL2 multispecific Abs, anti-ALPPL2 multi-specific antigen-binding Ab fragments, and anti-ALPPL2 ADCs disclosed herein, or the antigen-binding domain thereof.
[298] In some particular embodiments, the antigen-binding domain of the CAR may comprise an anti-ALPPL2 scFv. The scFv may comprise any of the VH and VL combinations as described above and a linker that connects the VH and VL
[299] In some embodiments, the antigen-binding domain may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to: (i) SEQ ID NOs: 760, 759, 750, 749, 740, 739, 730, 729, 720, 719, 319, 320, 329, 330, 339, 340, 341, or 342 or (11) the amino add sequence encoded by SEQ ID NOs: 419, 19, 420, 20, 429, 29, 430, 30, 439, 39, 440, 441, or 442.
[300] In some embodiments, the antigen-binding domain may compete for binding to ALPPL2 with a scFv comprising an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to: (i) SEQ ID NOs: 760, 759, 750, 749, 740, 739, 730, 729, 720, 719, 319, 320, 329, 330, 339, 340, 341, or 342 or (ii) the amino add sequence encoded by SEQ ID NOs: 419, 19, 420, 20, 429, 29, 430, 30, 439, 39, 440, 441, or 442.
[301] Examples of the antigen-binding domain of the CAR, or of any other anti-ALPPL2 agents, of the present invention are depicted in FIG 2.
Hinge
[302] In some embodiments, the CAR may comprise a hinge sequence between the antigen-binding domain and the TM domain. One of the ordinary skill in the art will appredate that a hinge sequence is a short sequence of amino adds that facilitates flexibility (see, e.g. Woof J.M. etal., Nat Rev. Immunol., 4(2): 89-99 (2004)). The hinge sequence can be any suitable sequence derived or obtained from any suitable molecule.
[303] In some embodiments, the length of the hinge sequence may be optimized based on the desired length of the extracellular portion of the CAR, which may be based on the location of the epitope within the target molecule. For example, if the epitope is in the membrane proximal region within the target molecule, longer hinges may be optimal.
[304] In some embodiments, the hinge may be derived from or include at least a portion of an immunoglobulin Fc region, for example, an IgGl Fc region, an IgG2 Fc region, an IgG3 Fc region, an IgG4 Fc region, an IgE Fc region, an IgM Fc region, or an IgA Fc region. In certain embodiments, the hinge includes at least a portion of an IgGl, an IgG2, an IgG3, an IgG4, an IgE, an IgM, or an IgA immunoglobulin Fc region that falls within its CH2 and CH3 domains. In some embodiments, the hinge may also include at least a portion of a corresponding immunoglobulin hinge region. In some embodiments, the hinge is derived from or indudes at least a portion of a modified immunoglobulin Fc region, for example, a modified IgGl Fc region, a modified IgG2 Fc region, a modified IgG3 Fc region, a modified IgG4 Fc region, a modified IgE Fc region, a modified IgM Fc region, or a modified IgA Fc region. The modified immunoglobulin Fc region may have one or more mutations (e.g., point mutations, insertions, deletions, duplications) resulting in one or more amino add substitutions, modifications, or deletions that cause impaired binding of the hinge to an Fc receptor (FcR). In some embodiments, the modified immunoglobulin Fc region may be designed with one or more mutations which result in one or more amino add substitutions, modifications, or deletions that cause impaired binding of the hinge to one or more FcR including, but not limited to, FcyRI, FcyR2A, FcyR2Bl, Fcy2B2, Fey 3A, Fey 3B, FCERI, FCER2, FcaRI, Fca/pR, or FcRn.
[305] In some embodiments, a portion of the immunoglobulin constant region serves as a hinge between the antigen-binding domain, for example scFv or nanobody, and the TM domain. The hinge can be of a length that provides for Increased responsiveness of the CAR-expressing cell following antigen binding, as compared to in the absence of the hinge. In some examples, the hinge is at or about 12 amino acids in length or is no more than 12 amino acids in length. Exemplary hinges include those having at least about 10 to 229 amino acids, about 10 to 200 amino adds, about 10 to 175 amino adds, about 10 to 150 amino acids, about 10 to 125 amino adds, about 10 to 100 amino adds, about 10 to 75 amino adds, about 10 to 50 amino adds, about 10 to 40 amino adds, about 10 to 30 amino adds, about 10 to 20 amino adds, or about 10 to 15 amino adds, and including any Integer between the endpoints of any of the listed ranges. In some embodiments, a hinge has about 12 amino adds or less, about 119 amino adds or less, or about 229 amino adds or less. Exemplary hinges include a CD28 hinge, IgG4 hinge alone, IgG4 hinge linked to CH2 and CH3 domains, or IgG4 hinge linked to the CH3 domain. Exemplary hinges Include, but are not limited to, those described in Hudecek M. et al. (2013) Clin. Cancer Res., 19:3153, international patent application publication number W02014031687, U.S. Pat No. 8,822,647 or published App. No. US2014/0271635.
[306] In some embodiments, the hinge sequence is derived from CD8 a molecule or a CD28 molecule. In a preferred embodiment; the hinge sequence is derived from CD28. In one embodiment; the hinge comprises the amino add sequence of human CD28 hinge (SEQ ID NO: 163) or the sequence encoded by SEQ ID NO: 263. In some embodiments, the hinge has an amino add sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 163.
Transmembrane (TM) domain
[307] With respect to the TM domain, the CAR can be designed to comprise a TM domain that is fused to the antigen-binding domain of the CAR. A hinge sequence may be inserted between the antigen-binding domain and the TM domain. In one embodiment, the TM domain that naturally is associated with one of the domains in the CAR is used. In some instances, the TM domain can be selected or modified by amino add substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
[308] The TM domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Typically, the TM domain denotes a single transmembrane a helix of a transmembrane protein, also known as an integral protein. TM domains of particular use in this invention may be derived from (i.e. comprise at least the transmembrane regionfs) of) CD28, CD3 E, CD4, CDS, CD8, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, TCR a, TCR p, or CD3 zeta and/or TM domains containing functional variants thereof such as those retaining a substantial portion of the structural, e.g., transmembrane, properties thereof.
[309] Alternatively the TM domain may be synthetic, in which case the TM domain will comprise predominantly hydrophobic residues such as leudne and valine. Preferably a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic TM domain. A TM domain of the invention is thermodynamically stable in a membrane. It may be a single a helix, a transmembrane p barrel, a 0-helix of gramicidin A, or any other structure. Transmembrane helices are usually about 20 amino acids in length.
[310] Preferably, the TM domain in the CAR of the invention is derived from the TM region of CD28. In one embodiment, the TM domain comprises the amino acid sequence of human CD28 TM (SEQ ID NO: 161) or the sequence encoded by SEQ ID NO: 261. In some embodiments, the TM domain comprises an amino add sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 161.
[311] Optionally, a short oligo- or polypeptide spacer, preferably between 2 and 10 amino adds in length may form the linkage between the TM domain and the ICS domaln(s) of the CAR A glydne-serine doublet may provide a suitable spacer.
Intracellular signaling (ICS) domain and costimulatory CCS) domain
[312] The ICS domain or otherwise the cytoplasmic domain of the CAR of the invention triggers or elicits activation of at least one of the normal effector functions of the cell in which the CAR has been placed. The term "effector function" refers to a spedalized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Thus, the term "intracellular signaling domain* or "ICS domain" refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a spedalized function. While usually the entire ICS domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term "intracellular signaling domain" or "ICS domain" is thus meant to include any truncated portion of the ICS domain suffident to transduce the effector function signal.
[313] Preferred examples of ICS domains for use in the CAR of the invention indude the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement; as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.
[314] Signals generated through one ICS domain alone may be insufficient for full activation of a cell, and a secondary or costimulatory signal may also be required. In such cases, a costimulatory domain (CS domain) may be included in the cytoplasmic portion of a CAR A CS domain is a domain that transduces such a secondary or costimulatory signal. Optionally, the CAR of the present invention may comprise two or more CS domains. The CS domain(s) may be placed upstream of the ICS domain or downstream of the ICS domain. Two exemplary schematics of CAR constructs of the present invention containing at least one CS domain are illustrated in FIG IB and FIG 1C. [315] In some embodiments, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequence: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic signaling sequences). Primary cytoplasmic signaling sequences regulate primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as Immunoreceptor tyrosine-based activation motifs or ITAMs. Such a cytoplasmic signaling sequence may be contained in the ICS or the CS domain of the CAR of the present invention.
[316] Examples of ITAM -containing primary cytoplasmic signaling sequences that are of particular use In the Invention Include those derived from an ICS domain of a lymphocyte receptor chain, a TCR/CD3 complex protein, an Fc receptor subunit; an IL-2 receptor subunit, CD3 7, FcR y, FcR f$, CD3 y, CD3 5, CD3 E, CDS, CD22, CD66d, CD79a, CD79b, CD278 (ICOS), Fc E RI, DAP10, and DAP12.
[317] It Is particularly preferred that the ICS domain In the CAR of the Invention comprises a cytoplasmic signaling sequence derived from CD 3 zeta. In one embodiment; the ICS domain comprises the amino acid sequence of human CD3 £ ICS (SEQ ID NO: 162), or the sequence encoded by SEQ ID NO: 262. In some embodiments, the ICS domain comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 162.
[318] In a preferred embodiment; the cytoplasmic domain of the CAR may be designed to comprise the CD3 ICS domain by itself. In another preferred embodiment, the CD3 ( ICS domain may be combined with one or more of any other desired cytoplasmic domain(s) useful in the context of the CAR of the invention. For example, the cytoplasmic domain of the CAR can comprise a CD3 7, ICS domain and a costimulatory (CS) domain. The CS region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient response of lymphocytes to an antigen.
[319] Various CS domains have been reported to confer differing properties. For example, the 4-1BB CS domain showed enhanced persistence in in vivo xenograph models (Milone M. C. etal. Mol Ther 2009;17:1453-1464; Song D. G. etal. Cancer Res 2011;71:4617-4627). Additionally, these different CS domains produce different cytokine profiles, which in turn, may produce effects on target cell-mediated cytotoxicity and the disease microenvironment Indeed, DAP 10 signaling in NK cells has been associated with an increase in Thl and inhibition of Th2 type cytokine production in CD8+ T cells (Barber A et al. Blood 2011;117:6571-6581).
[320] Examples of co-stimulatory molecules include an MHC class I molecule, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, a Toll ligand receptor, B7-H3, BAFFR, BTLA, BLAME (SLAMF8), CD2, CD4, CDS, CD7, CDS a, CDS p, CD 11a, LFA-1 (CDlla/CD18), CDllb, CDllc, CDlld, CD18, CD19, CD19a, CD27, CD28, CD29, CD30, CD40, CD49a, CD49D, CD49f, CD69, CD84, CD96 (Tactile), CD100 (SEMA4D), CD1O3, CRTAM, 0X40 (CD134), 4-1BB (CD137), SLAM (SLAMF1, CD150, IPO- 3), CD160 (BY55), SELPLG (CD162), DNAM1 (CD226), Ly9 (CD229), SLAMF4 (CD244, 2B4), ICOS (CD278), CEACAM1, CDS, CRTAM, DAP10, GADS, GITR, HVEM (LIGHTR), IA4, I CAM-1, IL2R p, IL2R Y, IL7R a, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAT, LFA-1, LIGHT, LTBR, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), PAG/Cbp, PD-1, PSGL1, SLAMF6 (NTB-A, Lyl08), SLAMF7, SLP-76, TNFR2, TRANCE/RANKL, VLA1, VLA-6, a ligand that specifically binds with CD83, and the like. Thus, while the invention is exemplified primarily with regions of CD28, DAP10, and/or 4-1BB as the CS domain, other costimulatory elements are within the scope of the Invention.
[321] The ICS domain and the CS domain(s) of the CAR of the invention may be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino adds in length may form the linkage. A glydne-serine doublet provides a particularly suitable linker.
[322] In one embodiment; the CAR is designed to comprise a cytoplasmic signaling sequence of CD3 I, as the ICS domain and comprise a cytoplasmic signaling sequence of CD28 as the CS domain. In another embodiment, the CAR is designed to comprise a cytoplasmic signaling sequence of CD3 £ as the ICS domain and comprise a cytoplasmic signaling sequence of DAP10 as the CS domain. In yet another embodiment; the CAR is designed to comprise a cytoplasmic signaling sequence of CD3 I, as the ICS domain and comprise a cytoplasmic signaling sequence of 4-1BB as the CS domain. Such a cytoplasmic signaling sequence of CD3 £ may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the CD3 7, ICS domain comprising the amino add sequence of human CD3z ICS (SEQ ID NO: 162). Such a cytoplasmic signaling sequence of CD 3 zeta may be encoded by a nucleic add sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 262.
[323] Such a cytoplasmic signaling sequence of CD28 may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of human CD28 CS domain (SEQ ID NO: 164). Such a cytoplasmic signaling sequence of CD28 may be encoded by a nucleic add sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 264. Such a cytoplasmic signaling sequence of DAP10 may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of human 4-1BB CS domain (SEQ ID NO: 165). Such a cytoplasmic signaling sequence of 4-1BB may be encoded by a nucleic add sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 265. Such a cytoplasmic signaling sequence of DAP10 may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% Identical to the sequence of human DAP10 CS domain (SEQ ID NO: 166). Such a cytoplasmic signaling sequence of DAP10 may be encoded by a nucleic add sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 266.
[324] Alternatively, when the antigen-binding domain comprises the ALPPL2 -binding portion of a molecule that binds to ALPPL2 as described above, the TM domain of the CAR may be derived from the transmembrane portion of the molecule.
Exemplary CAR constructs
[325] In the following CAR examples, the CAR construct is described as "antigenbinding domain - hinge - TM domain - CS domain - ICS domain."
[326] The CARs of the present invention may comprise an amino add sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to any of the exemplary constructs below.
[327] In one embodimentthe CAR of the invention may be described as B735scFvHL- CD28H-CD28TM-CD28CS-CD3zICS and may comprise the amino add sequence as set forth in SEQ ID NO: 951.
[328] In one embodimentthe CAR of the invention may be described as B735scFvHL- CD28H-CD28TM-41BBCS-CD3zICS and may comprise the amino add sequence as set forth in SEQ ID NO: 952.
[329] In one embodimentthe CAR of the invention may be described as B735scFvHL- CD28H-CD28TM-DAP10CS-CD3zICS and may comprise the amino add sequence as set forth in SEQ ID NO: 953.
[330] In one embodimentthe CAR of the invention may be described as B735scFvLH- CD28H-CD28TM-CD28CS-CD3zICS and may comprise the amino add sequence as set forth in SEQ ID NO: 954.
[331] In one embodimentthe CAR of the invention may be described as B735scFvLH- CD28H-CD28TM-41BBCS-CD3zICS and may comprise the amino add sequence as set forth in SEQ ID NO: 955.
[332] In one embodiment the CAR of the invention may be described as B735scFvLH- CD28H-CD28TM-DAP10CS-CD3zICS and may comprise the amino add sequence as set forth in SEQ ID NO: 956.
[333] In one embodimentthe CAR of the invention may be described as B506scFvHL- CD28H-CD28TM-CD28CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 941.
[334] In one embodimentthe CAR of the invention may be described as B506scFvHL- CD28H-CD28TM-41BBCS-CD3zlCS and may comprise the amino add sequence as set forth in SEQ ID NO: 942.
[335] In one embodimentthe CAR of the invention may be described as B506scFvHL- CD28H-CD28TM-DAP10CS-CD3zICS and may comprise the amino add sequence as set forth in SEQ ID NO: 943. [336] In one embodimentthe CAR of the Invention may be described as B506scFvLH- CD28H-CD28TM-CD28CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 944.
[337] In one embodimentthe CAR of the invention may be described as B506scFvLH- CD28H-CD28TM-41BBCS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 945.
[338] In one embodiment the CAR of the invention may be described as B506scFvLH- CD28H-CD28TM-DAP10CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 946.
[339] In one embodimentthe CAR of the invention may be described as B416scFvHL- CD28H-CD28TM-CD28CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 931.
[340] In one embodiment the CAR of the Invention may be described as B416scFvHL- CD28H-CD28TM-41BBCS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 932.
[341] In one embodiment the CAR of the invention may be described as B416scFvHL- CD28H-CD28TM-DAP10CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 933.
[342] In one embodiment the CAR of the invention may be described as B416scFvLH- CD28H-CD28TM-CD28CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 934.
[343] In one embodiment the CAR of the invention may be described as B416scFvLH- CD28H-CD28TM-41BBCS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 935.
[344] In one embodiment the CAR of the invention may be described as B416scFvLH- CD28H-CD28TM-DAP10CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 936.
[345] In one embodiment the CAR of the invention may be described as B380scFvHL- CD28H-CD28TM-CD28CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 921.
[346] In one embodiment the CAR of the invention may be described as B380scFvHL- CD28H-CD28TM-41BBCS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 922.
[347] In one embodiment the CAR of the invention may be described as B380scFvHL- CD28H-CD28TM-DAP10CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 923.
[348] In one embodiment the CAR of the invention may be described as B380scFvLH- CD28H-CD28TM-CD28CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 924.
[349] In one embodiment the CAR of the invention may be described as B380scFvLH- CD28H-CD28TM-41BBCS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 925. [350] In one embodimentthe CAR of the Invention may be described as B380scFvLH- CD28H-CD28TM-DAP10CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 926.
[351] In one embodimentthe CAR of the invention may be described as BOOlscFvHL- CD28H-CD28TM-CD28CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 911.
[352] In one embodiment the CAR of the invention may be described as BOOlscFvHL- CD28H-CD28TM-41BBCS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 912.
[353] In one embodimentthe CAR of the invention may be described as BOOlscFvHL- CD28H-CD28TM-DAP10CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 913.
[354] In one embodimentthe CAR of the Invention may be described as BOOlscFvLH- CD28H-CD28TM-CD28CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 914.
[355] In one embodimentthe CAR of the invention may be described as BOOlscFvLH- CD28H-CD28TM-41BBCS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 915.
[356] In one embodimentthe CAR of the invention may be described as BOOlscFvLH- CD28H-CD28TM-DAP10CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 916.
[357] In one embodimentthe CAR of the invention may be described as hlB2scFvHL- CD28H-CD28TM-CD28CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 351. Such a CAR may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 451 or 51.
[358] In one embodimentthe CAR of the invention may be described as hlB2scFvHL- CD28H-CD28TM-41BBCS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 352. Such a CAR may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 452 or 52.
[359] In one embodimentthe CAR of the invention may be described as hlB2scFvHL- CD28H-CD28TM-DAP10CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 353. Such a CAR may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 453 or 53.
[360] In one embodimentthe CAR of the invention may be described as hlB2scFvLH- CD28H-CD28TM-CD28CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 354. Such a CAR may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 454 or 54.
[361] In one embodimentthe CAR of the invention may be described as hlB2scFvLH- CD28H-CD28TM-41BBCS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 355. Such a CAR may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 455 or 55. [362] In one embodimentthe CAR of the Invention may be described as hlB2scFvLH- CD28H-CD28TM-DAP10CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 356. Such a CAR may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 456 or 56.
[363] In one embodimentthe CAR of the Invention may be described as hlEBscFvHL- CD28H-CD28TM-CD28CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 361. Such a CAR may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 461 or 61.
[364] In one embodimentthe CAR of the Invention may be described as hlESscFvHL- CD28H-CD28TM-41BBCS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 362. Such a CAR may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 462 or 62.
[365] In one embodimentthe CAR of the Invention may be described as hlESscFvHL- CD28H-CD28TM-DAP10CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 363. Such a CAR may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 463 or 63.
[366] In one embodimentthe CAR of the Invention may be described as hlESscFvLH- CD28H-CD28TM-CD28CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 364. Such a CAR may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 464 or 64.
[367] In one embodimentthe CAR of the Invention may be described as hlESscFvLH- CD28H-CD28TM-41BBCS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 365. Such a CAR may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 465 or 65.
[368] In one embodimentthe CAR of the invention may be described as hlEBscFvLH- CD28H-CD28TM-DAP10CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 366. Such a CAR may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 466 or 66.
[369] In one embodimentthe CAR of the invention may be described as h3H2scFvHL- CD28H-CD28TM-CD28CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 371. Such a CAR may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 471 or 71.
[370] In one embodimentthe CAR of the invention may be described as h3H2scFvHL- CD28H-CD28TM-41BBCS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 372. Such a CAR may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 472 or 72.
[371] In one embodimentthe CAR of the invention may be described as h3H2scFvHL- CD28H-CD28TM-DAP10CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 373. Such a CAR may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 473 or 73.
[372] In one embodimentthe CAR of the invention may be described as h3H2scFvLH- CD28H-CD28TM-CD28CS-CD3zICS and may comprise the amino acid sequence as set forth In SEQ ID NO: 374. Such a CAR may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 474 or 74.
[373] In one embodimentthe CAR of the invention may be described as h3H2scFvLH- CD28H-CD28TM-41BBCS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 375. Such a CAR may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 475 or 75.
[374] In one embodiment the CAR of the invention may be described as h3H2scFvLH- CD28H-CD28TM-DAP10CS-CD3zICS and may comprise the amino acid sequence as set forth in SEQ ID NO: 376. Such a CAR may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 476 or 76.
[375] Schematics showing examples of specific CAR constructs of some embodiments are illustrated in FIGS 3A-3C.
[376] In some embodiments, a leader sequence (LS) may be placed upstream of the polynucleotide sequences encoding the foregoing exemplary CARs. The leader sequence facilitates the expression of the CAR on the cell surface. The polynucleotide sequence of such a lead sequence may be as set forth in SEQ ID NO: 260, which encodes the amino acid sequence as set forth In SEQ ID NO: 160. Any other sequences that facilitate the expression of the CAR on the cell surface may be used.
[377] A general exemplary schematic of a construct for a LS-containing CAR of the present invention is shown in FIG IE.
[378] In some embodiments, the polynucleotide sequences for expressing the foregoing exemplary CARs further comprise a T2A ribosomal skip sequence (or also referred to as T2A) and/or a sequence encoding truncated CD 19 (or also referred to as trCD19). The nucleic acid sequence for T2Amay be as provided by SEQ ID NO: 269, which encodes the amino acid sequence provided by SEQ ID NO: 169. trCD19 may have the sequence as provided by SEQ ID NO: 170, which may be encoded by, for example, SEQ ID NO: 270.
[379] A schematic showing such polynucleotide constructs are shown in FIG IF.
[380] When the T2A and trCD19 sequences are placed downstream of the CAR sequence, the translation will be interrupted by the T2A sequence, resulting in two separate translation products, CAR protein and trCD19 protein.
[381] The present disclosure encompasses the polynucleotides encoding any of the CARs disclosed herein.
[382] Exemplary CAR-encoding nucleic acid sequences include, but are not limited to, SEQ ID NOS: 451, 51, 452, 52, 453, 53, 454, 54, 455, 55, 456, 56, 461, 61, 462, 62, 463, 63, 464, 64, 465, 65, 466, 66, 471, 71, 472, 72, 473, 73, 474, 74, 475, 75, 476, 76.
Exemplary constructs for expressing such a CAR may be provided by, for examples, SEQ ID NOS: 671, 271, 674, and 274.
Further modification
[383] The CARs of the present invention, nucleotide sequences encoding the same, vectors encoding the same, and cells comprising nucleotide sequences encoding said CARs may be further modified, engineered, optimized, or appended In order to provide or select for various features. These features may include, but are not limited to, efficacy, persistence, target specificity, reduced immunogenicity, multi-targeting, enhanced immune response, expansion, growth, reduced off-target effect; reduced subject toxicity, improved target cytotoxicity, improved attraction of disease alleviating immune cells, detection, selection, targeting, and the like. For example, the cells may be engineered to express another CAR, or to have a suicide mechanism, and may be modified to remove or modify expression of an endogenous receptor or molecule such as a TCR and/or MHC molecule.
[384] In some embodiments, the vector or polynucleotide encoding the CAR further encodes other genes. The vector or polynucleotide may be constructed to allow for the co-expression of multiple genes using a multitude of techniques including cotransfection of two or more plasmids, the use of multiple or bidirectional promoters, or the creation of bicistronic or multicistronic vectors. The construction of multicistronic vectors may include the encoding of IRES elements or 2A peptides, such as T2A, P2A, E2A, or F2A (for example, see Kim, J.H., et al., "High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice", PLoS One. 2011;6(4)J. In a particular embodiment, the polynucleotide or vector encoding the CAR further encodes trCD19 with the use of a T2A ribosomal skip sequence.
[385] The CAR expressing cell may further comprise a disruption to one or more endogenous genes. In some embodiments, the endogenous gene encodes TCRa, TCRp, CD 52, glucocorticoid receptor (GR), deoxycytidine kinase (dCK), or an immune checkpoint protein such as, for example, programmed death- 1 (PD-1).
[386] The CARs of the present invention and cells expressing these CARs may be further modified to improve efficacy against cells expressing the target molecule. The cells may be cells expressing ALPPL2. The cells expressing ALPPL2 may be cancer cells, vascular cells, or any other target disease-associated cells. In some embodiments, the improved efficacy may be measured by increased cytotoxicity against cells expressing the target molecule, for example cytotoxicity against cancer cells. In some embodiments, the improved efficacy may also be measured by increased production of cytotoxic mediators such as, but not limited to, IFN y, perforin, and grantyme B. In some embodiments, the improved efficacy may be shown by reduction in the signature cytokines of the diseases, or alleviated symptoms of the disease when the CAR expressing cells are administered to a subject Other cytokines that may be reduced include TGF-beta, IL-6, IL-4, IL-10, and/or IL-13, the improved efficacy may be shown by ALPPL2 -specific immune cell responses, such as T cell cytotoxicity. In case of cancer, improved efficacy may be shown by better tumor cytotoxicity, better infiltration into the tumor, reduction of immunosuppressive mediators, reduction in weight decrease, reduction in ascites, reduction in tumor burden, and/or increased lifespan. In some embodiments, gene expression profiles may be also Investigated to evaluate the efficacy of the CAR
[387] In one aspect; the CAR expressing cells are further modified to evade or neutralize the activity of immunosuppressive mediators, including, but not limited to prostaglandin E2 (PGE2) and adenosine. In some embodiments, this evasion or neutralization is direct In other embodiments, this evasion or neutralization is mediated via the inhibition of protein kinase A (PKA) with one or more binding partners, for example ezrin. In a specific embodiment; the CAR-expressing cells further express the peptide "regulatory subunit I anchoring disruptor" (RIAD). RIAD is thought to inhibit the association of protein kinase A (PKA) with ezrin, which thus prevents PKA's inhibition of TCR activation (Newick K et al. Cancer Immunol Res. 2016 Jun;4(6):541-51. doi: 10.1158/2326-6066.CIR-15-0263. Epub 2016 Apr 4).
[388] In some embodiments, the CAR expressing cells of the Invention may Induce a broad immune response, consistent with epitope spreading.
[389] In some embodiments, the CAR expressing cells of the invention further comprise a homing mechanism. For example, the cell may transgenically express one or more stimulatory chemokines or cytokines or receptors thereof. In particular embodiments, the cells are genetically modified to express one or more stimulatory cytokines. In certain embodiments, one or more homing mechanisms are used to assist the inventive cells to accumulate more effectively to the disease site. In some embodiments, the CAR expressing cells are further modified to release inducible cytokines upon CAR activation, e.g., to attractor activate innate immune cells to a targeted cell (so-called fourth generation CARs or TRUCKS). In some embodiments, CARs may co-express homing molecules, e.g., CCR4 or CCR2b, to increase trafficking to the disease site.
Controlling CAR expression
[390] In some instances, it may be advantageous to regulate the activity of the CAR or CAR expressing cells CAR For example, inducing apoptosis using, e.g., a caspase fused to a dimerization domain (see, e.g., Di et al., N Engl. J. Med. 2011 Nov. 3; 365(18):1673- 1683), can be used as a safety switch in the CAR therapy of the instant invention. In another example, CAR-expressing cells can also express an inducible Caspase-9 (iCaspase-9) molecule that; upon administration of a dimerizer drug (e.g., rimiducid (also called AP1903 (Bellicum Pharmaceuticals) or AP20187 (Ariad)) leads to activation of the Caspase-9 and apoptosis of the cells. The iCaspase-9 molecule contains a chemical inducer of dimerization (CID) binding domain that mediates dimerization in the presence of a CID. This results in inducible and selective depletion of CAR-expressing cells. In some cases, the iCaspase-9 molecule is encoded by a nucleic acid molecule separate from the CAR-encoding vector(s). In some cases, the iCaspase-9 molecule is encoded by the same nucleic acid molecule as the CAR-encoding vector. The iCaspase-9 can provide a safety switch to avoid any toxicity of CAR-expressing cells. See, e.g., Song etal. Cancer Gene Ther. 2008; 15(10):667-75; Clinical Trial Id. No. NCT02107963; and Di etal. N. Engl. J. Med. 2011; 365:1673-83.
[391] Alternative strategies for regulating the CAR therapy of the instant invention include utilizing small molecules or antibodies that deactivate or turn off CAR activity, e.g., by deleting CAR-expressing cells, e.g., by inducing antibody dependent cell-mediated cytotoxicity (ADCC). For example, CAR-expressing cells described herein may also express an antigen that is recognized by molecules capable of inducing cell death, e.g., ADCC or compliment-induced cell death. For example, CAR expressing cells described herein may also express a receptor capable of being targeted by an antibody or antibody fragment Examples of such receptors include EpCAM, VEGFR, integrins (e.g., integrins avp3, a4, aI3/403, a4p7, a5pi, avp3, av), members of the TNF receptor superfamily (e.g., TRAIL-R1, TRAIL-R2), PDGF Receptor, interferon receptor, folate receptor, GPNMB, ICAM-1, HLA-DR, CEA, CA-125, MUC1, TAG-72, IL-6 receptor, 5T4, GD2, GD3, CD2, CD3, CD4, CDS, CD 11, CDlla/LFA-1, GDIS, CD18/1TGB2, CD19, CD20, CD22, CD23/lgE Receptor, CD25, CD28, CD30, CD33, CD38, CD40, CD41, CD44, CD51, CD52, CD62L, CD74, CD80, CD 125, CD147/basigin, CD152/CTLA-4, CD154/CD40L, CD195/CCR5, CD319/SLAMF7, and EGFR, and truncated versions thereof (e.g., versions preserving one or more extracellular epitopes but lacking one or more regions within the cytoplasmic domain). For example, CAR-expressing cells described herein may also express a truncated epidermal growth factor receptor (EGFR) which lacks signaling capacity but retains the epitope that is recognized by molecules capable of inducing ADCC, e.g., cetuximab (ERBITUX®), such that administration of cetuximab induces ADCC and subsequent depletion of the CAR-expressing cells (see, e.g., W02011/056894, and Jonnalagadda et al., Gene Ther. 2013; 20(8)853-860).
[392] In some embodiments, the CAR cell comprises a polynucleotide encoding a suicide polypeptide, such as for example RQR8. See, e.g., W02013153391A, which is hereby incorporated by reference in its entirety. In CAR cells comprising the polynucleotide, the suicide polypeptide may be expressed at the surface of a CAR cell. The suicide polypeptide may also comprise a signal peptide at the amino terminus. Another strategy includes expressing a highly compact marker/suicide gene that combines target epitopes from both CD32 and CD20 antigens in the CAR-expressing cells described herein, which binds rituximab, resulting in selective depletion of the CAR-expressing cells, e.g., by ADCC (see, e.g., Philip etal., Blood. 2014; 124(8)1277- 1287). Other methods for depleting CAR-expressing cells described herein include administration of CAM PATH®, a monoclonal anti-CD52 antibody that selectively binds and targets mature lymphocytes, e.g., CAR-expressing cells, for destruction, e.g., by inducing ADCC. In other embodiments, the CAR-expressing cell can be selectively targeted using a CAR ligand, e.g., an anti-idiotypic antibody. In some embodiments, the anti-idiotypic antibody can cause effector cell activity, e.g., ADCC or ADC activities, thereby reducing the number of CAR-expressing cells. In other embodiments, the CAR ligand, e.g., the anti-idiotypic antibody, can be coupled to an agent that induces cell killing, e.g., a toxin, thereby reducing the number of CAR-expressing cells. Alternatively, the CAR molecules themselves can be configured such that the activity can be regulated, e.g., turned on and off, as described below.
[393] In some embodiments, a regulatable CAR (RCAR) where the CAR activity can be controlled is desirable to optimize the safety and efficacy of a CAR therapy. In some embodiments, a RCAR comprises a set of polypeptides, typically two in the simplest embodiments, in which the components of a standard CAR described herein, e.g., an antigen-binding domain and an ICS domain, are partitioned on separate polypeptides or members. In some embodiments, the set of polypeptides include a dimerization switch that; upon the presence of a dimerization molecule, can couple the polypeptides to one another, e.g., can couple an antigen-binding domain to an ICS domain. Additional description and exemplary configurations of such regulatable CARs are provided herein and in International Publication No. WO 2015/090229, hereby incorporated by reference in its entirety.
[394] In an aspect, an RCAR comprises two polypeptides or members: 1) an intracellular signaling member comprising an ICS domain, e.g., a primary ICS domain described herein, and a first switch domain; 2) an antigen binding member comprising an antigen-binding domain, e.g., that specifically binds a target molecule described herein, as described herein and a second switch domain. Optionally, the RCAR comprises a TM domain described herein. In an embodiment, a TM domain can be disposed on the intracellular signaling member, on the antigen binding member, or on both. Unless otherwise indicated, when members or elements of an RCAR are described herein, the order can be as provided, but other orders are included as well. In other words, in an embodiment; the order is as set out in the text; but in other embodiments, the order can be different E.g., the order of elements on one side of a transmembrane region can be different from the example, e.g., the placement of a switch domain relative to an ICS domain can be different e.g., reversed.
[395] In some embodiments, the CAR expressing immune cell may only transiently express a CAR. For example, the cells of the invention may be transduced with mRNA comprising a nucleic acid sequence encoding an inventive CAR In this vein, the present invention also includes an RNA construct that can be directly transfected into a cell. A method for generating mRNA for use in transfection involves in vitro transcription (IVT) of a template with specially designed primers, followed by polyA addition, to produce a construct containing 3' and 5' untranslated sequences ("UTRs"), a 5' cap and/or Internal Ribosome Entry Site (IRES), the nucleic acid to be expressed, and a polyA tail, typically 50-2000 bases in length. RNA so produced can efficiently transfect different kinds of cells. In one embodiment; the template includes sequences for the CAR In an embodiment; an RNA CAR vector is transduced into a cell by electroporation.
Target specificity
[396] The CAR expressing cells of the present invention may further comprise one or more CARs, in addition to the first CAR. These additional CARs may or may not be specific for the target molecule of the first CAR. In some embodiments, the one or more additional CARs may act as inhibitory or activating CARs. In some embodiments, the CAR of some embodiments Is the stimulatory or activating CAR; In other embodiments, It Is the costimulatory CAR. In some embodiments, the cells further include inhibitory CARs (iCARs, see Fedorov et al., Set Transl. Medicine, 2013 Dec;5(215): 215ral72), such as a CAR recognizing an antigen other than the target molecule of the first CAR, whereby an activating signal delivered through the first CAR Is diminished or Inhibited by binding of the inhibitory CAR to its ligand, e.g., to reduce off-target effects.
[397] In some embodiments, the antigen-binding domain of the CAR is or is part of an immunoconjugate, in which the antigen-binding domain is conjugated to one or more heterologous molecule(s), such as, but not limited to, a cytotoxic agent; an Imaging agent; a detectable moiety, a multimerization domain, or other heterologous molecule. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At211, 1131, 1125, Y90, Rel86, Rel88, Sml53, Bi212, P32, Pb212 and radioactive isotopes of Lu); chemotherapeutic agents; growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins. In some embodiments, the antigen-binding domain is conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant; or animal origin, or fragments thereof), or radioactive isotopes.
[398] In some embodiments, to enhance persistence, the cells of the invention may be further modified to overexpress pro-survival signals, reverse anti-survival signals, overexpress Bcl-xL, overexpress hTERT, lack Fas, or express a TGF-0 dominant negative receptor. Persistence may also be facilitated by the administration of cytokines, e.g., IL-2, IL-7, and IL-15.
Vectors
[399] The present invention also provides vectors in which a polynucleotide encoding an anti-ALPPL2 agent of the present invention is inserted.
[400] The vector may be, for example, a DNA vector or a RNA vector. The vector may be, for example, but not limited to, a plasmid, a cosmid, a viral replicon, or a viral vector. The viral vector may be a vector of a DNA virus, which may be an adenovirus, or an RNA virus, which may be a retrovirus. Types of vectors suite for Abs, antigen-binding Ab fragments, and/or CARs are well known in the art (for example, see Rita Costa A. etal., EurJPharm Biopharm. 2010 Feb;74(2):127-38. doi: 10.1016/j.ejpb.2009.10.002. Epub 2009 Oct22; Frenzel A. etal. Front Immunol. 2013; 4: 217. Published online 2013 Jul 29. doi: 10.3389/fimmu.2013.00217).
[401] When the host cells are insect cells, such as for producing Abs or antigen-binding Ab fragments, insect-specific viruses may be used. Examples of the insect-specific viruses include, but are not limited to, the family of Baculoviridae, particularly the Autographa califomica nuclear polyhedrosis virus(AcNPV). When the host cells are plant cells, plant-specific viruses and bacteria, such as Agrobacterium tumefaciens, may be used. [402] For expressing vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity. This would be particularly beneficial for expressing CAR constructs.
[403] In brief summary, the expression of nucleic acids encoding anti-ALPPL2 agents is typically achieved by operably linking a nucleic acid encoding the anti-ALPPL2 agent polypeptide or portions thereof to a promoter, and incorporating the construct into an expression vector. The vectors can be suitable for replication and integration eukaryotes, "typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired polynucleotide.
[404] The expression constructs of the present invention may also be used for nucleic acid immunization and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art See, e.g., U.S. Pat Nos. 5,399,346, 5,580,859, 5,589,466, incorporated by reference herein in their entireties. In another embodiment, the invention provides a gene therapy vector.
[405] The nucleic acid can be cloned into a number of types of vectors. For example, the nucleic add can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequendng vectors.
[406] Further, the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, y-retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat No. 6,326,193).
[407] A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems are known in the art In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art In one embodiment lentivirus vectors are used. [408] Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or Independently to activate transcription.
[409] Various promoter sequences may be used, including, but not limited to the immediate early cytomegalovirus (CMV) promoter, the CMV-actin-globin hybrid (GAG) promotor, Elongation Growth Factor-la (EF-la), simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.
[410] In order to assess the expression of a CAR polypeptide or portions thereof, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co- transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.
[411] In some embodiments, the selectable marker gene comprises a polynucleotide encoding truncated CD19 (trCD19). When a marker such as trCD19, which can be expressed on the cell surface is used, the expression of the marker may be determined via any available technique including, but not limited to, flow cytometry or immunofluorescence assays. Expression of such a marker typically indicates successful introduction and expression of the transgene(s) introduced together with the marker gene. Therefore, cells expressing the anti-ALPPL2 agent of the invention may be, for example, selected based on the expression of the marker. [412] Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, ^-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.
Transfection/Transduction
[413] Methods of introducing and expressing genes into a cell are known in the art In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast or insect cell by any method in the art For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.
[414] For transduction of CAR constructs to obtain CAR-expressing cells, a flow chart illustrating a potential method for manufacturing isolated CAR-expressing cells is provided in FIG 4.
[415] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art See, for example, Sambrook etal. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.
[416] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat Nos. 5,350,674 and 5,585,362.
[417] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle tn vitro and tn vivo is a liposome (e.g., an artificial membrane vesicle). [418] In the case where a non-vtral delivery system Is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another aspect; the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a "collapsed" structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
[419] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St Louis, Mo.; dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, N.Y.); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol ("DMPG") and other lipids may be obtained from Avanti Polar Lipids, Ina (Birmingham, Ala.). Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20 degrees Celsius. Chloroform is used as the only solvent since it is more readily evaporated than methanol. "Liposome" is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh etal., " 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine- nudeic add complexes.
[420] Regardless of the method used to introduce exogenous nucleic adds into a host cell or otherwise expose a cell to the inhibitor of the present invention, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, "molecular biological" assays well known to those of skill in the art; such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.
Cells
[421] Also provided are cells, cell populations, and compositions containing the cells, e.g., cells comprising a polynucleotide encoding an anti-ALPPL2 agent of the present invention. Cells expressing anti-ALPPL2 Abs or antigen-binding Ab fragments, for example, may be used to harvest the Abs or antigen-binding Ab fragments or may be administered to a subject/patient Cells expressing anti-ALPPL2 CARs, for example, may be administered to a subject or may be incorporated in a composition to be administered to a subject Among the compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy.
[422] Also provided are therapeutic methods for administering the Abs or Ab fragments or the cells and compositions to subjects, e.g., patients.
Cell types
[423] Thus, also provided are cells expressing the anti-ALPPL2 agents of the present invention.
[424] For expressing an anti-ALPPL2 Ab or antigen-binding Ab fragment; any appropriate cells may be used. For example, cells may be: [i] prokaryotic cells, such as gram-negative bacteria and gram-positive bacteria; or (ii) eukaryotic cells, such as yeast, filamentous fungi, protozoa, insect cells, plant cells, and mammalian cells (reviewed in Frenzel A etal. Front Immunol. 2013; 4: 217. Published online 2013 Jul 29. doi: 10.3389/fimmu.2013.00217). In certain embodiments, CHO cells or HEK cells may be used.
[425] Specific examples of gram-negative bacteria that are suited for production of Ab or antigen-binding Ab fragments include, but are not limited to, E coli, Proteus mirabilis, and Pseudomonas putidas. Specific examples of gram-positive bacteria include, but are not limited to, Bacillus brevis, Bacillus subtilts, Bacillus megatertum, Lactobactlluszeae casei, and Lactobacillus paracasei. Specific examples of yeast bacteria that are suited for production of Ab or antigen-binding Ab fragments include, but are not limited to, Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Schizosaccharomyces pombe, Schwanntomyces occidentals, Kluyveromyces lactis, and Yarrowia Itpolytica. Specific examples of filamentous fungi that are suited for production of Ab or antigen-binding Ab fragments include, but are not limited to, the genera Trichoderma and Aspergillus, A niger (subgenus A awamori), Aspergillus oryzae, and Chrysosporium lucknowense.
Specific examples of protozoa that are suited for production of Ab or antigen-binding Ab fragments include, but are not limited to, Leishmania tarentolae. Specific examples of insect cells that are suited for production of Ab or antigen-binding Ab fragments include, but are not limited to, insect cell lines like Sf-9 and Sf-21 of Spodoptera Jrugiperda, DS2 cells of Drosophila melanogaster, High Five cells (BTI-TN-5B1-4) of Trtchopulsta nt, or Schneider2 (S2) cells of D. melanogaster. They can be efficiently transfected with insectspecific viruses from the family of Baculoviridae, particularly the Autographa califomica nuclear polyhedrosis virus (AcNPV). Specific examples of mammalian cells that are suited for production of Ab or antigen-binding Ab fragments include, but are not limited to, Chinese hamster ovary (CHO) cells, the human embryonic retinal cell line Per.C6 [Crucell, Leiden, Netherlands], CHO-derived cell lines such as K1-, DukXBll-, Lecl3, and DG44- cell lines, mouse myeloma cells such as SP 2/0, YB 2/0, and NSO cells, GS-NSO, hybridoma cells, baby hamster kidney (BHK) cells, and the human embryonic kidney cell line HEK293, HEK293T, HEK293E, and human neuronal precursor cell line AGE1.HN (Probiogen, Berlin, Germany).
[426] Alternatively, genetically modified organisms such as transgenic plants and transgenic animals may be used. Exemplary plants that may be used Include, but are not limited to, tabacco, maize, duckweed, Chlamydomonas reinhardtii, Nicotiana tabacum, Nicotianaben thamiana, and Nicotiana benthamiana. Exemplary animals that may be used include, but are not limited to mouse, rat, and chicken.
[427] For expressing an anti-ALPPL2 CAR, the cells generally are eukaryotic cells, such as mammalian cells, and typically are human cells, more typically primary human cells, e.g., allogeneic or autologous donor cells. The cells for introduction of the CAR may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject In some embodiments, the subject from which the cell is Isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered. In some embodiments, the cells are derived from the blood, bone marrow, lymph, or lymphoid organs, are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid cells, including monocytes, macrophages, dendritic cells, neutrophils, eosinophils, basophils, or mast cells, or lymphoid cells, including lymphocytes, typically T cells and/or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells typically are primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen-specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation.
[428] Alternatively, an immortalized cell or a cell line may be used for expressing a CAR of the present disclosure. Such examples include, but are not limited to, a T cell line, a CD4+ T cell line, a CD8+ T cell line, a regulatory T cell line, an NK-T cell line, an NK cell line (e.g., NK-92), a monocyte line, a macrophage line, a dendritic cell line, and a mast cell line. Furthermore, a desired cell type for CAR expression, for example T cells or NK cells may be generated from a stem cell, such as an embryonic stem cell, iPSCs, or hematopoietic stem cell.
[429] With reference to the subject to be treated with cells expressing an anti-ALPPL2 CAR, the cells may be allogeneic and/or autologous. Among the methods Include off-the- shelf methods. In some embodiments, such as for off-the-shelf technologies, the cells are pluripotent and/or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the methods include isolating cells from the subject; preparing, processing, culturing, and/or engineering them, as described herein, and reintroducing them into the same patient; before or after cryopreservation.
[430] In some embodiments, the cells are T cells. Among the sub-types and subpopulations of T cells and/or of CD4+ and/or of CD8+ T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, a/p T cells, and 5/y T cells.
[431] In some embodiments, the cells are natural killer (NK) cells, Natural Killer T (NKT) cells, cytokine-induced killer (CIK) cells, tumor-infiltrating lymphocytes (TIL), lymphokme-activated killer (LAK) cells, or the like. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils. CAR-expressing phagocytic cells expressing may be able to bind to and phagocytose or nibble target cells (Morrissey M.A et al., Elife. 2018 Jun 4;7. pii: e36688. doi: 10.7554/eLife.36688).
[432] In some embodiments, the cells are derived from cell lines, e.g., T cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat; non-human primate, and pig.
Cell acquisition for anti-ALPPL2 CAR expression
[433] For cells for expressing anti-ALPPL2 CARs, prior to expansion and genetic modification, a source of cells can be obtained from a subject through a variety of nonlimiting methods. Cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and disease sites such as tumors. In some embodiments, any number of T cell lines available and known to those skilled in the art, may be used. In some embodiments, cells can be derived from a healthy donor, from a patient diagnosed with cancer or from a patient diagnosed with an infection. In some embodiments, cells can be part of a mixed population of cells which present different phenotypic characteristics. [434] Accordingly, the cells In some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject; as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g. transduction with viral vector), washing, and/or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat; tissue and organ samples, including processed samples derived therefrom.
[435] In some embodiments, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from a leukapheresis product Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, tongue, or other organ, and/or cells derived therefrom. Samples Include, In the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.
[436] In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in some embodiments, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets, and in some embodiments contains cells other than red blood cells and platelets.
[437] Also provided herein are cell lines obtained from a transformed cell according to any of the above-described methods. Also provided herein are modified cells resistant to an immunosuppressive treatment In some embodiments, an isolated cell according to the invention comprises a polynucleotide encoding a CAR.
Cell purification
[438] In some embodiments, isolation of the cells includes one or more preparation and/or non-affinity based cell separation steps. In some examples, cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and/or resistance to particular components.
[439] In some embodiments, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and/or magnesium and/or many or all divalent cations. In some embodiments, a washing step is accomplished a semi-automated "flow-through* centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer's Instructions. In some embodiments, a washing step Is accomplished by tangential flow filtration (TFF) according tx> the manufacturer's instructions. In some embodiments, the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca++/Mg++ free PBS. In certain embodiments, components of a blood cell sample are removed and the cells directly resuspended in culture media.
[440] In some embodiments, the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. This would be particularly useful for Isolating CAR-expresslng cells. In a specific embodiment; the surface maker is trCD19. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffinity-based separation. For example, the isolation in some embodiments Includes separation of cells and cell populations based on the cells' expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.
[441] Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some embodiments, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.
[442] In some embodiments, multiple rounds of separation steps are carried out; where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.
[443] For example, in some embodiments, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45R0+ T cells, are isolated by positive or negative selection techniques. For example, CD 3+ T cells can be positively selected using CD3 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander).
[444] In some embodiments, isolation is carried out by enrichment for a particular cell population by positive selection, or depletion of a particular cell population, by negative selection. In some embodiments, positive or negative selection is accomplished by Incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker+) at a relatively higher level (marker high) on the positively or negatively selected cells, respectively.
[445] In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14. In some embodiments, a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and/or effector T cell subpopulations.
[446] In some embodiments, CD8+ cells are further enriched for or depleted of naive, central memory, effector memory, and/or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve long-term survival, expansion, and/or engraftment following administration, which in some embodiments is particularly robust in such sub-populations. See Terakura et al. (2012) Blood. 1:72-82; Wang etal. (2012) J Immunother. 35(9):689-701. In some embodiments, combining TCM-enriched CD8+ T cells and CD4+ T cells further enhances efficacy. In embodiments, memory T cells are presentin both CD62L+ and CD62L-subsets of CD8+ peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L-CD8+ and/or CD62L+CD8 fractions, such as using anti-CD8 and anti-CD62L antibodies.
[447] In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and/or CD127; in some embodiments, it is based on negative selection for cells expressing or highly expressing CD45RA and/or granzyme B. In some embodiments, isolation of a CD8+ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment for cells expressing CD62L In one aspect; enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD 14 and CD45RA, and a positive selection based on CD62L Such selections in some embodiments are carried out simultaneously and in other embodiments are carried out sequentially, in either order. In some embodiments, the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation, also is used to generate the CD4+ cell population or sub-population, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps.
[448] In some embodiments, the sample or composition of cells to be separated is incubated with small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as Dynalbeads or MACS beads). The magnetically responsive material, e.g., particle, generally Is directly or indirectly attached to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., surface marker, present on the cell, cells, or population of cells that it is desired to separate, e.g., that it is desired to negatively or positively select
[449] In some embodiments, the magnetic particle or bead comprises a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner. There are many well-known magnetically responsive materials used in magnetic separation methods. Suitable magnetic particles include those described in Molday, U.S. Pat No. 4,452,773, and in European Patent Specification EP 452342 B, which are hereby incorporated by reference. Colloidal sized particles, such as those described in Owen U.S. Pat No. 4,795,698, and Liberti et al., U.S. Pat No. 5,200,084 are other examples.
[450] The Incubation generally is carried out under conditions whereby the antibodies or binding partners, or molecules, such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead, specifically bind to cell surface molecules if present on cells within the sample.
[451] In some embodiments, the sample is placed in a magnetic field, and those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from the unlabeled cells. For positive selection, cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some embodiments, a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subject to further separation steps.
[452] In certain embodiments, the magnetically responsive particles are coated in primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin. In certain embodiments, the magnetic particles are attached to cells via a coating of primary antibodies specific for one or more markers. In certain embodiments, the cells, rather than the beads, are labeled with a primary antibody or binding partner, and then cell-type specific secondary antibody- or other binding partner (e.g., streptavidinj-coated magnetic particles, are added. In certain embodiments, streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.
[453] In some embodiments, the magnetically responsive particles are left attached to the cells that are to be subsequently incubated, cultured and/or engineered; in some embodiments, the particles are left attached to the cells for administration to a patient In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, e.g., the use of competing non-labeled antibodies, magnetizable particles or antibodies conjugated to cleavable linkers, eta In some embodiments, the magnetizable particles are biodegradable. [454] In certain embodiments, die Isolation or separation Is carried out using a system, device, or apparatus that carries out one or more of the isolation, cell preparation, separation, processing, incubation, culture, and/or formulation steps of the methods. In some embodiments, the system is used to carry out each of these steps in a closed or sterile environment; for example, to minimize error, user handling and/or contamination. In one example, the system is a system as described in International Patent Application, Publication Number W02009/072003, or US 20110003380 Al.
[455] In some embodiments, the system or apparatus carries out one or more, e.g., all, of the Isolation, processing, engineering, and formulation steps in an integrated or self- contained system, and/or in an automated or programmable fashion. In some embodiments, die system or apparatus includes a computer and/or computer program in communication with the system or apparatus, which allows a user to program, control, assess the outcome of, and/or adjust various embodiments of the processing, isolation, engineering, and formulation steps.
[456] In some embodiments, a cell population described herein is collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluidic stream. In some embodiments, a cell population described herein is collected and enriched (or depleted) via preparative scale (FACS)- sorting. In certain embodiments, a cell population described herein is collected and enriched (or depleted) by use of microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, e.g., WO 2010/033140, Cho etal. (2010) Lab Chip 10, 1567-1573; and Godin etal. (2008) J Biophoton. l(5):355-376. In both cases, cells can be labeled with multiple markers, allowing for the isolation of well- defined T cell subsets at high purity.
[457] In some embodiments, the antibodies or binding partners are labeled with one or more detectable marker, to facilitate separation for positive and/or negative selection. For example, separation may be based on binding to fluorescently labeled antibodies. In some examples, separation of cells based on binding of antibodies or other binding partners specific for one or more cell surface markers are carried in a fluidic stream, such as by fluorescence-activated cell sorting (FACS), including preparative scale (FACS) and/or microelectromechanical systems (MEMS) chips, e.g., in combination with a flow-cytometric detection system. Such methods allow for positive and negative selection based on multiple markers simultaneously.
[458] In some embodiments, the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient
[459] In any of the aforementioned separation steps, the separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.
Cell preparation and expansion
[460] In some embodiments, the provided methods Include cultivation, Incubation, culture, and/or genetic engineering steps. For example, in some embodiments, provided are methods for incubating and/or engineering the depleted cell populations and culture-initiating compositions.
[461] Thus, in some embodiments, the cell populations are incubated in a cultureinitiating composition. The incubation and/or engineering may be carried out in a culture vessel, such as a unit, chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, or other container for culture or cultivating cells.
[462] In some embodiments, the cells are incubated and/or cultured prior to or in connection with genetic engineering. The incubation steps can include culture, cultivation, stimulation, activation, and/or propagation.
[463] In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent Such conditions include those designed to induce proliferation, expansion, activation, and/or survival of cells in the population, to mimic antigen exposure, and/or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor. The cells of the invention can be activated and expanded, either prior to or after genetic modification of the cells, using methods as generally described, for example without limitation, in U.S. Pat Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681;
7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005. The conditions can include one or more of particular media, temperature, oxygen content carbon dioxide content time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.
[464] Particularly in relation to CAR-expressing cells, T cells can be expanded in vitro or in vivo. Generally, the T cells of the invention can be expanded, for example, by contact with an agent that stimulates a CD 3 TCR complex and a co-stimulatory molecule on the surface of the T cells to create an activation signal for the T cell. For example, chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitogenic lectins like phytohemagglutinin (PHA) can be used to create an activation signal for the T cell.
[465] In some embodiments, T cell populations may be stimulated in vitro by contact with, for example, an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti- CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. In some embodiments, the T cell populations may be stimulated tn vitro by contact with Muromonab-CD3 (OKT3). For co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. Conditions appropriate for T cell culture Include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640® or, X-vivo 5®, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-y, IL-4, IL-7, GM-CSF, IL-10, IL-2, IL-15, IL-21, TGF-p, and TNF, or any other additives for the growth of cells known to the skilled artisan. In a preferred embodiment, T cells are stimulated in vitro by exposure to 0KT3 and IL-2. Other additives for the growth of cells include, but are not limited to, surfactant, Plasmanate, and reducing agents such as N-acetyl-cysteine and 2 -mercaptoethanol. Media can include RPMI 1640®, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vlvo 1®, and X-Vlvo 20®, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of T cells. Antibiotics, e.g., penicillin and streptomycin, are included only In experimental cultures, not in cultures of cells that are to be infused into a subject The target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° Celsius) and atmosphere (e.g., air plus 5% C02). T cells that have been exposed to varied stimulation times may exhibit different characteristics.
[466] In some embodiments, the isolated cells of the invention can be expanded by coculturing with tissue or cells. The cells can also be expanded in vivo, for example in the subjects blood after administrating the cell into the subject
[467] In some embodiments, when cells are expanded in vivo, at least one cell of the invention may be administered to a subject and the administration may lead to an expansion of the cell in the subject resulting in a population of cells. Alternatively, a polynucleotide or vector of the invention may be administered to a subject Once the polynucleotide or vector is taken up by a cell within the subject and the cell proliferate or expand in the subject this may result in a population of cells of the invention within the subject
[468] In certain embodiments, the resulting population of cells persists in the subject for at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, at least twelve months, at least eighteen months, at least two years, or at least three years after administration.
[469] In some embodiments, the T cells are expanded by adding to the culture- initiating composition feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMC), (e.g., such that the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture (e.g. for a time sufficient to expand the numbers of T cells). In some embodiments, the non-dividing feeder cells can comprise y- irradiated PBMC feeder cells. In some embodiments, the PBMC are irradiated with y rays in the range of about 3000 to 3600 rads to prevent cell division. In some embodiments, the feeder cells are added to culture medium prior to the addition of the populations of T cells.
[470] In some embodiments, the preparation methods Include steps for freezing, e.g., cryopreserving, the cells, either before or after isolation, incubation, and/or engineering. In some embodiments, the freeze and subsequent thaw step removes granulocytes and, to some extent; monocytes in the cell population. In some embodiments, the cells are suspended in a freezing solution, e.g., following a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters in some embodiments may be used. One example involves using PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. This is then diluted 1:1 with media so that the final concentration of DMSO and HSA are 10% and 4%, respectively. The cells are then frozen to -80° Celsius at a rate of 1 degree per minute and stored in the vapor phase of a liquid nitrogen storage tank.
Isolation of Ab or antigen-binding Ab fragment from cell culture
[471] Cells, such as hybridomas or other recombinant cells, that are producing Abs or antigen-binding Ab fragments of the present invention may be grown using standard methods, in suitable culture medium for this purpose (such as D-MEM or RPMI-1640), or in vivo as ascites. Abs or antigen-binding Ab fragments expressed and/or secreted by the cells can be separated from the cells, culture medium, ascites fluid, or serum using conventional immunoglobulin purification procedures, such as, but not limited to, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography (Ma H. et al., Methods. 2017 Mar l;116:23-33. doi: 10.1016/j.ymeth.2016.11.008. Epub 2016 Nov 18; Shukla A A et al. Trends Biotechnol. 2010 May;28(5):253-61. doi: 10.1016/j.tibtech.2010.02.001. Epub 2010 Mar 19; Arora S. etal., Methods. 2017 Mar l;116:84-94. doi: 10.1016/j.ymeth.2016.12.010. Epub 2016 Dec 22).
[472] Methods for expressing, isolating, and evaluating multispecific and bispecific Abs and antigen-binding Ab fragments are also known in the art (for example, see Brinkmann U. etal., MAbs. 2017 Feb-Mar; 9(2): 182-212. Published online 2017 Jan 10. doi: 10.1080/19420862.2016.1268307; Dimasi N. etal. Methods. 2018 Aug ll. pii: S1046-2023(18)30149-X doi: 10.1016/j.ymeth.2018.08.004).
Therapeutic applications
[473] Anti-ALPPL2 agents of the present Invention (Abs, antigen-binding Ab fragments, multi-specific Abs, multi-specific antigen-binding Ab fragments, ADCs, or CARs that binds to ALPPL2), nucleic acids encoding such an agent, vectors encoding such an agent, isolated cells obtained by the methods described above, or cell lines derived from such Isolated cells, and/or pharmaceutical compositions comprising thereof can be used as a medicament in the treatment of a disease, disorder, or condition in a subject In some embodiments, such a medicament can be used for treating an ALPPL2-associated disease or condition.
Target diseases and conditions
[474] The ALPPL2 -associated condition may be, for example, but not limited to, cancer and cancer-associated diseases and conditions.
[475] In particular embodiments, the anti-ALPPL2 agents of the present invention may be used to treat a cancer. ALPPL2 is upregulated in a variety of cancers, such as, but not limited to pancreatic cancer, testicular cancer, cervical cancer, endometrial cancer, ovarian cancer, stomach cancer, colorectal cancer, lung cancer, mesothelioma, and tongue cancer (https://www.protematias.org/ENSG00000163286-ALPPL2/pathology: Skinner, J.M., Whitebead, R. Virchows Archiv [Pathol Anat] 394, 109-118 (1981).; Wick M. R. et al, Hum Pathol 1987 Sep; 18(9): 946- 54.; Su Y et al., bioRxiv 2020.01.07.898122; doi: https://doi.org/10.1101/2020.01.Q7.898122. which is now Su Y et at, Cancer Res.
2020 Oct 15;80(20):4552-4564.) (a/so see, e.g., FIGS 5A, 6, 7A-7B).
[476] Therefore, preferred target diseases include these cancers. In some embodiments, pancreatic cancer is a preferred target disease. In some embodiments, mesothelioma is a preferred target disease. The anti-ALPPL2 agents according to the present invention may also be used to treat any other cancers in which ALPPL2 is upregulated or has a pathological role.
Subject
[477] The subject referred to herein may be any living subject In a preferred embodiment; the subject is a mammal. The mammal referred to herein can be any mammal. As used herein, the term "mammal" refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Lagomorpha, such as rabbits. The mammals may be from the order Carnivora, including Felines (cats) and Canines (dogs). The mammals may be from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). The mammals may be of the order Primates, Cebolds, or Simoids (monkeys) or of the order Anthropoids (humans and apes).
[478] In some embodiments, the subject; to whom the Abs, antigen-binding Ab fragments, ADCs, CAR expressing cells, cells, cell populations, or compositions are administered is a primate, such as a human. In some embodiments, the primate is a monkey or an ape. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some examples, the patient or subject is a validated animal model for disease, adoptive cell therapy, and/or for assessing toxic outcomes such as cytokine release syndrome (CRS).
[479] In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with another immunotherapy and/or other therapy. In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another therapy. In some embodiments, the subject has not relapsed but is determined to be at risk for relapse, such as at a high risk of relapse, and thus the compound or composition is administered prophylactically, e.g., to reduce the likelihood of or prevent relapse.
[480] In some embodiments, the methods Include administration of an Ab, Ab fragments, ADC, or CAR expressing cell or a composition containing such an anti-ALPPL2 agent to a subject, tissue, or cell, such as one having, at risk for, or suspected of having a disease, condition or disorder associated with ALPPL2, such as cancer. In some embodiments, the anti-ALPPL2 agents and/or compositions are administered to a subject having the particular disease or condition to be treated, e.g., via adoptive cell therapy, such as adoptive T cell therapy. In some embodiments, the anti-ALPPL2 agents or compositions are administered to the subject, such as a subject having or at risk for the disease or condition. In some embodiments, the methods thereby treat; e.g., ameliorate one or more symptom of the disease or condition, for example, by reducing, inhibiting, or inactivating ALPPL2 and/or ALPPL2 -expressing cells.
Cell origin
[481] For purposes of the methods of anti-ALPPL2 CAR cell therapy, wherein host cells or populations of cells are administered, the cells can be cells that are xenogeneic, allogeneic or autologous to the subject
[482] In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by autologous transfer, in which the cells are isolated and/or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject Thus, in some embodiments, the cells are derived from a subject; e.g., patient; in need of a treatment and the cells, following isolation and processing are administered to the same subject
[483] In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by allogeneic transfer, in which the cells are isolated and/or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject In such embodiments, the cells then are administered to a different subject e.g., a second subject of the same species. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject
[484] In certain embodiments, where cells are T cells and are not autologous to the subject the expression of the cell's endogenous T cell receptors (TCRs) may be suppressed or disrupted. The TCR expression may be suppressed via any appropriate technique, for example, by silencing any compartment of the endogenous TCRs using tools such as, but not limited to, an siRNA, shRNA, micro RNA, or artificial microRNA. Alternatively, TCR gene may be disrupted or deleted via any appropriate technique, for example using the CRISPR/Cas system, transcription activator-like effector nucleases (e.g., TALENO), or Zinc finger nucleases (ZFNs). The suppression or disruption of TCR may allow for reduction or prevention of undesired effects in which the TCRs recognize antigens in the subject as foreign and cause immune responses against the subject; an immune attack often called as graft-versus-host disease (GVHD).
[485] In certain embodiments, where cells (donor cells) are not autologous to the subject; the expression of endogenous MHC or HLA gene(s) may be suppressed or disrupted, which may be achieved via any appropriate technique, such as but not limited to, an siRNA, shRNA, micro RNA, artificial microRNA, or gene editing using the CRISPR/Cas system, transcription activator-like effector nucleases (e.g., TALEN®), or Zinc finger nucleases (ZFNs). The suppression or disruption of MHC or HLA gene(s) may allow for reduction or prevention of undesired effects in which the subject's endogenous T cells recognize the donor cell's antigens presented on donor cell's MHC molecules as foreign and cause immune responses against the donor cells and increase the persistence of the administered cells within the subject Cells expressing anti-ALPPL2 Abs or antigen-binding Ab fragments or a composition comprising such may also be administered to a subject In some embodiments, B cells or plasma cells expressing anti- ALPPL2 Abs or antigen-binding Ab fragments may be adoptively transferred.
Functional activity
[486] In some embodiments, the present invention includes antibody therapy, wherein (i) an isolated anti-ALPPL2 Ab or Ab fragment may be administered to a subject; (ii) a polynucleotide or combination of polynucleotides encoding an ALPPL2 Ab or Ab fragment or a vector or combination of vectors encoding an ALPPL2 Ab or Ab fragment may be administered to a subject to allow for expression of the encoded Ab or Ab fragment within the subject, (iii) a cell engineered to express/secrete an ALPPL2 Ab or Ab fragment may be administered to a subject
[487] In certain embodiments, such an Ab or Ab fragment may bind to ALPPL2- expressing cells. In some instances, binding to ALPPL2 -expressing cells may cause a downstream effect; such as complement activation, ADCC, or ADCP. In some instances, binding to ALPPL2 -expressing cells may inhibit ALPPL2's binding to another molecule or inhibit ALPPL2 function. In some instances, binding to ALPPL2-expressing cells may stimulate or augment signaling and/or functions of ALPPL2. In some instances, binding to ALPPL2 -expressing cells may assist binding of the ALPPL2-expressing cells to another molecule or cell.
[488] In one embodiment; the present invention includes a type of cellular therapy where isolated cells are genetically modified to express a CAR against ALPPL2, and the CAR cell is infused into a subject in need thereof. Such administration can promote activation of the cells (e.g., T cell activation) in a target molecule specific manner, such that the cells of the disease or disorder are targeted for destruction. In the case where the cell is a T cell, cells, such as CAR T cells, are able to replicate in vivo resulting in longterm persistence that may lead to sustained control of diseases, disorders, or conditions associated with ALPPL2, such as cancer. [489] In one embodiment; the Isolated cells of the Invention can undergo tn vivo expansion and can persist for an extended amount of time. In another embodiment; where the isolated cell is a T cell, the isolated T cells of the invention evolve into specific memory T cells that can be reactivated to inhibit growth of any additional target molecule expressing cells. T cells may differentiate tn vtvo into a central memory-like state upon encounter and subsequent elimination of target cells expressing the surrogate antigen. Similarly, in certain embodiments, where the isolated cells are B cells, the isolated B cells may evolve into memory B cells that can be reactivated to inhibit the growth of any additional target molecule expressing cells.
[490] Without wishing to be bound by any particular theory, the immune response elicited by the isolated anti-ALPPL2 agent-modified immune cells may be an active or a passive immune response. In addition, the anti-ALPPL2 agent-mediated immune response may be part of an adoptive immunotherapy approach in which anti-ALPPL2 agent-modified immune cells induce an immune response specific to the antigenbinding domain of the anti-ALPPL2 agent
[491] In certain embodiments, anti-ALPPL2 agent-expressing cells are modified in any number of ways, such that their therapeutic or prophylactic efficacy is Increased. For example, the anti-ALPPL2 agent may be conjugated either directly or indirectly through a linker to a targeting moiety. The practice of conjugating compounds, e.g., the CAR, to targeting moieties is known in the art See, for instance, Wadwa et al.,/. Drug Targeting 3: 111 (1995), and U.S. Pat No. 5,087,616.
[492] Once the cells are administered to a subject (e.g., a human), the biological activity of the engineered cell populations and/or antibodies in some embodiments is measured by any of a number of known methods. Parameters to assess include specific binding of an engineered or natural T cell or other immune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art; such as cytotoxicity assays described in, for example, Kochenderfer et al.,/. Immunotherapy, 32(7): 689-702 (2009), and Herman et al./. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity of the cells also can be measured by assaying expression and/or secretion of certain mediators, such as GM-CSF, IL-6, RANTES (CCL5), TNF-o, IL-4, IL-10, IL-13, IFN- y, granzyme B, perforin, CD 107a, or IL-2.
[493] In some embodiments the biological activity elicited by an anti-ALPPL2 agent such as an Ab or Ab fragment or a CAR-expressing cells is measured by assessing clinical outcome, such as the reduction in disease symptoms. In case of cancer, improved efficacy may be shown by better infiltration of disease-resolving immune cells into the tumor, reduced tumor sizes, or reduced ascites. In some embodiments, gene expression profiles may be also investigated to evaluate the activity.
Target cells [494] Cells that may be targeted by any anti-ALPPL2 agents of present Invention include any ALPPL2-expressing cells. The target cell may be present in any part of the body of a subject; including blood or lymphatic circulation, and disease-affected tissues. For example, when the target disease is solid cancer, the disease-affected tissues Include, but are not limited to, pancreas, testis, cervix, endometrium, ovary, stomach, colon, rectum, lung, mesothelium, or tongue, or another oral tissue. Alternatively, target cells may blood cells or hematopoietic cells.
[495] Preferably, the anti-ALPPL2 agent-expressing cells of the invention are used to treat cancer, wherein ALPPL2 is upregulated. In particular, the cells of the Invention may be used to treat pancreatic cancer, testicular cancer, cervical cancer, endometrial cancer, ovarian cancer, stomach cancer, colorectal cancer, lung cancer, mesothelioma, or tongue cancer.
[496] In general, cells that are positive for ALPPL2 may be identified via known methods, for example, immunofluorescence or flow cytometry using specific antibodies, or alternatively, through cytotoxicity against target cells. Methods of testing an anti- ALPPL2 agent for the ability to recognize target cells and for antigen specificity are known in the art For Instance, Clay et al.,/. Immunol., 163: 507-513 (1999), teaches methods of measuring the release of cytokines (e.g., interferon-y, granulocyte/monocyte colony stimulating factor (GM-CSF), tumor necrosis factor a (TNF-a) or interleukin 2 (IL- 2)). In addition, CAR function can be evaluated by measurement of cellular cytotoxicity, as described in Zhao etal.,/. Immunol., 174: 4415-4423 (2005).
[497] A biopsy is the removal of tissue and/or cells from an individual. Such removal may be to collect tissue and/or cells from the individual in order to perform experimentation on the removed tissue and/or cells. This experimentation may include experiments to determine if the individual has and/or is suffering from a certain condition or disease-state. The condition or disease may be, e.g., cancer. With respect to detecting the presence of cells expressing anti-ALPPL2 agent in a host; the sample comprising cells of the host can be a sample comprising whole cells, lysates thereof, or a fraction of the whole cell lysates, e.g., a nuclear or cytoplasmic fraction, a whole protein fraction, or a nucleic acid fraction. If the sample comprises whole cells, the cells can be any cells of the host, e.g., the cells of any organ or tissue, including blood cells or endothelial cells.
Pharmaceutical compositions
[498] The compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired.
[499] In general, administration may be topical, parenteral, or enteral.
[500] As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue, thus generally resulting in the direct administration into the blood stream, into muscle, or into an internal organ. Parenteral administration thus Includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, Intraperitoneal, Intramuscular, intrastemal, Intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intrasynovial injection or infusions; and kidney dialytic infusion techniques. In a preferred embodiment; parenteral administration of the compositions of the present invention comprises subcutaneous or Intraperitoneal administration.
[501] The terms "oral", "enteral", "enterally", "orally", "non-parenteral", "non- parenterally", and the like, refer to administration of a compound or composition to an individual by a route or mode along the alimentary canal. Examples of "oral" routes of administration of a composition Include, without limitation, swallowing liquid or solid forms of a composition from the mouth, administration of a composition through a nasojejunal or gastrostomy tube, intraduodenal administration of a composition, and rectal administration, e.g., using suppositories for the lower intestinal tract of the alimentary canal.
[502] Compositions of the present invention may be suited for topical, parenteral, or enteral administration.
[503] Preferably, formulated compositions comprising Abs, antigen-binding Ab fragments, ADCs, or CARs, polynucleotides or vectors encoding such, or cells expressing thereof are suitable for administration via parenteral administration for example, subcutaneous, intramuscular, intraperitoneal or intravenous injection.
[504] Formulations of a pharmaceutical composition suitable for parenteral administration typically generally comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e. powder or granular) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. Parenteral formulations also include aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9), but; for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water. Exemplary parenteral administration forms include solutions or suspensions in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, or in a liposomal preparation. Formulations for parenteral administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release. Such formulation may be, for example, made of a biodegradable, biocompatible polymer, such as, but not limited to, ethylene vinyl acetate, polyfalkyl cyanoacrylates), poly(anhydrides), poly(amides), poly(ester), poly(ester amides), pofy(phosphoesters), polygiycolic acid (PGA), collagen, polyorthoester, polylactic acid (PLA), poly(lactic-co- glycolidic acid) (PLAGA), or naturally occurring biodegradable polymers such as chitosan and hyaluronic acid-based polymers (Kamaly N. et al, Chem Rev. Author manuscript; available in PMC 2017 Jul 13).
[505] Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, semi-solids, monophasic compositions, multiphasic compositions (e.g., oil-in-water, water-ln-oll), foams, microsponges, liposomes, nanoemulsions, aerosol foams, polymers, fullerenes, and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
[506] Compositions and formulations for parenteral, intrathecal, or intraventricular administration may Include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carder compounds and other pharmaceutically acceptable carriers or excipients.
[507] Compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
[508] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.
[509] The pharmaceutical compositions of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product
[510] The compositions of the present invention may be formulated to provide appropriate in vivo distribution of the active ingredient In many cases, concentrating the distribution of an anti-tumor drug in the tumor site is challenging, and it can be so even when a drug has a specificity to a molecule expressed by cancer cells. Various strategies have been developed to address the issue and any appropriate strategies may be applied for the current invention (for example, reviewed in Rosenblum D. etal, Nat Commun. 2018 Apr 12;9(1): 1410. doi: 10.1038/s41467-018-03705-y). For delivering a drug to the brain, the drug needs to cross the blood-brain barrier (BBB). Any appropriate strategies to enable BBB crossing may be utilized to for the delivery of any of the anti-ALPPL2 agents of the agents (see for example, Dong X. et al., Theranostics. 2018; 8(6): 1481-1493, for exemplary strategies).
[511] The compositions of the present Invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, aerosols, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
[512] In one embodiment of the present invention the pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams Include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product Agents that enhance uptake of oligonucleotides at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (U.S. Pat No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (WO 97/30731), also enhance the cellular uptake of oligonucleotides.
[513] The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or antiinflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
[514] Formulations comprising any of the anti-ALPPL2 agents of the present invention or populations of cells expressing any of the anti-ALPPL2 agents such as anti-ALPPL2 CARs of the present invention may include pharmaceutically acceptable excipient(s). Excipients included in the formulations will have different purposes depending, for example, on the CAR construct; the subpopulation of cells used, and the mode of administration. Examples of generally used excipients Include, without limitation: saline, buffered saline, dextrose, water-for- infection, glycerol, ethanol, and combinations thereof, stabilizing agents, solubilizing agents and surfactants, buffers and preservatives, tonicity agents, bulking agents, and lubricating agents. The formulations comprising populations of the CAR-expresslng cells of the present invention will typically have been prepared and cultured in the absence of any non-human components, such as animal serum (e.g., bovine serum albumin).
[515] The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the binding molecules or cells, preferably those with activities complementary to the binding molecule or cell, where the respective activities do not adversely affect one another. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs. Such agents or drugs may be, but are not limited to, an anti-cancer drug, an anti-proliferative drug, a cytotoxic drug, an anti-angiogenic drug, an apoptotic drug, an immunostimulatory drug, an anti-microbial drug, an antibiotic drug, an antiviral drug, an anti-inflammatory drug, an ALPPL2 inhibitor, an ALPPL2 signaling inhibitor, an anti-ALPPL2 agent of the present invention, an enzyme, a hormone, a toxin, a compound, a small molecule, a small molecule inhibitor, a protein, a peptide, a vector, a plasmid, a viral replicon, a viral particle, a nanoparticle, a DNA molecule, an RNA molecule, an siRNA, an shRNA, a micro RNA, an oligonucleotide, or an imaging drug. Specific examples are, for instance, but are not limited to, chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, eta
[516] The pharmaceutical composition in some embodiments can employ time- released, delayed release, and sustained release delivery systems such that the delivery of the composition occurs prior to, and with sufficient time to cause, sensitization of the site to be treated. Many types of release delivery systems are available and known. Such systems can avoid repeated administrations of the composition, thereby increasing convenience to the subject and the physician.
Kits
[517] Also provided herein are kits comprising (a) one or more of anti-ALPPL2 agents (Abs, antigen-binding Ab fragments, ADCs, CARs), polynucleotides encoding such, vectors encoding such, cells expressing such; and (b) for example an instruction for use in treating or diagnosing a disease or condition associated with ALPPL2. The kit may include a label indicating the intended use of the contents of the kit The term "label" as used herein includes any written materials, marketing materials, or recorded materials supplied on, with, in, or appended to the kit Method of administration
[518] The administration route used in the method of the present invention may be any appropriate route, which depends upon whether local or systemic treatment is desired.
[519] In general, administration may be topical, parenteral, or enteral.
[520] Preferably, formulated compositions comprising Abs, antigen-binding Ab fragments, ADCs, or CARs, polynucleotides or vectors encoding such, cells expressing such may be administered parenterally, for example, via subcutaneous, intramuscular, intraperitoneal or Intravenous Injection.
[521] In the case of adoptive cell therapy, methods for administration of cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No. 2003/0170238 to Gruenberg etal; U.S. Pat No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g.,Themeli etal. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara etal. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338.
[522] In some embodiments, the composition of the present Invention may be administered using any appropriate medical devices (for example, reviewed in Richter B. B„ J. BioDrugs (2018) 32: 425).
Dosing
[523] For administration of any of the anti-ALPPL2 agents and compositions of the present invention, the dosage will vary and depend on, for example, the target disease, the severity of the disease, the route of administration, and pharmacokinetic factors. Dosing may be modified based on the response observed in the subject
[524] For administration of any of the anti-ALPPL2 Abs, antigen-binding Ab fragments, or ADCs, or compositions comprising such, appropriate dosage regimen may be determined using any appropriate methodology (for example, Bai S. et al., Clin Pharmacokinet 2012 Feb l;51(2):119-35. doi: 10.2165/11596370-000000000-00000).
[525] In some embodiments, the dosage may be from about 1 ng/kg to about 1 g/kg (of the body weight of a subject) per day. In some embodiments, the dose may be from about 10 ng/kg/day to about 900 mg/kg/day, from about 20 ng/kg/day to about 800 mg/kg/day, from about 30 ng/kg/day to about 800 mg/kg/day, from about 40 ng/kg/day to about 700 mg/kg/day, from about 50 ng/kg/day to about 600 mg/kg/day, from about 60 ng/kg/day to about 500 mg/kg/day, from about 70 ng/kg/day to about 400 mg/kg/day, from about 80 ng/kg/day to about 300 mg/kg/day, from about 90 ng/kg/day to about 200 mg/kg/day, or from about 100 ng/kg/day to about 100 mg/kg/day. The treatment may be repeated or periodically given to a subject for days, months, or years, or until the desired effect is achieved. An exemplary dosing regimen include administering an initial dose of an anti-ALPPL2 Abs, antigen-binding Ab fragments, or ADCs of about 2 mg/kg, followed by a weekly maintenance dose of about 1 mg/kg. [526] Dosing frequency may be, for example, three times per day, twice per day, once per day, every other day, once per week, every other week, once per three weeks, once per four weeks, once per five weeks, once per six weeks, once per seven weeks, once per eight weeks, once per nine weeks, once per ten weeks, once per three months, once per four months, once per six months, once per year, or even less frequent
[527] The pharmaceutical composition in some embodiments contains cells expressing the CAR of the present invention in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and can be determined. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.
[528] In certain embodiments, in the context of genetically engineered cells expressing an anti-ALPPL2 agent such as an Ab or Ab fragment or a CAR, a subject is administered the range of about one million to about 100 billion cells, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges, and/or such a number of cells per kilogram of body weight of the subject For example, in some embodiments the administration of the cells or population of cells can comprise administration of about 103 to about 109 cells per kg body weight including all integer values of cell numbers within those ranges.
[529] The cells or population of cells can be administrated in one or more doses. In some embodiments, said effective amount of cells can be administrated as a single dose. In some embodiments, said effective amount of cells can be administrated as more than one dose over a period time. Timing of administration is within the judgment of managing physician and depends on the clinical condition of the patient The cells or population of cells may be obtained from any source, such as a blood bank or a donor. While individual needs vary, determination of optimal ranges of effective amounts of a given cell type for a particular disease or conditions within the skill of the art An effective amount means an amount which provides a therapeutic or prophylactic benefit The dosage administrated will be dependent upon the age, health and weight of the recipient; kind of concurrent treatment; if any, frequency of treatment and the nature of the effect desired. In some embodiments, an effective amount of cells or composition comprising those cells are administrated parenterally. In some embodiments, administration can be an intravenous administration. In some embodiments, administration can be directly done by injection into the disease site.
[530] For purposes of the invention, the amount or dose of the inventive anti-ALPPL2 material administered should be sufficient to effect a therapeutic or prophylactic response in the subject or animal over a reasonable time frame. For example, the dose of the inventive anti-ALPPL2 material should be sufficient to bind to antigen, or detect; treat or prevent disease in a period of from about 2 hours or longer, e.g., about 12 to about 24 or more hours, from the time of administration. In certain embodiments, the time period could be even longer. The dose will be determined by the efficacy of the particular inventive anti-ALPPL2 material and the condition of the animal (e.g., human), as well as the body weight of the animal (e.g., human) to be treated.
[531] For purposes of the invention, an assay, which comprises, for example, comparing the extent to which target cells are lysed or, in the context of CARs, IFN-y is secreted by T cells expressing the inventive CAR, polypeptide, or protein upon administration of a given dose of such T cells to a mammal, among a set of mammals of which is each given a different dose of the T cells, could be used to determine a starting dose to be administered to a mammal. The extent to which target cells are lysed or IFN-y is secreted upon administration of a certain dose can be assayed by methods known in the art
[532] In some embodiments, two or more of the anti-ALPPL2 agents or compositions of the present invention may be administered to a subject in combination or separately.
[533] In some embodiments, the anti-ALPPL2 agents or compositions of the present invention are administered as part of a combination treatment; such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as an antibody or engineered cell or receptor or agent; such as a cytotoxic or therapeutic agent The cells or antibodies in some embodiments are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order. In some contexts, the anti-ALPPL2 agents or compositions are co-administered with another therapy sufficiently close in time such that the anti-ALPPL2 agents or compositions enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cells or antibodies are administered prior to the one or more additional therapeutic agents. In some embodiments, the anti-ALPPL2 agents, such as anti-ALPPL2 Ab or Ab fragment or CAR T cells or antibodies are administered after the one or more additional therapeutic agents. Furthermore, the compositions of the present invention may be given to a subject along with one or more of other therapies, which may be surgery, or a radiotherapy. [534] In some embodiments, In CAR T therapy, a lymphodepletlng chemotherapy Is administered to the subject prior to, concurrently with, or after administration (e.g., infusion) of CAR cells. In an example, the lymphodepletlng chemotherapy is administered to the subject prior to administration of the cells. For example, the lymphodepletlng chemotherapy ends 1-4 days (e.g., 1, 2, 3, or 4 days) prior to CAR cell infusion. In embodiments, multiple doses of CAR cells are administered, e.g., as described herein. In embodiments, a lymphodepletlng chemotherapy is administered to the subject prior to, concurrently with, or after administration (e.g., infusion) of a CAR- expresslng cell described herein. Examples of lymphodepletlon Include, but may not be limited to, nonmyeloablative lymphodepletlng chemotherapy, myeloablative lymphodepletlng chemotherapy, total body irradiation, etc. Examples of lymphodepletlng agents include, but are not limited to, anti-thymocyte globulin, anti- CD3 antibodies, anti-CD4 antibodies, anti-CDB antibodies, anti-CD52 antibodies, anti- CD2 antibodies, TCRcxP blockers, anti-CD20 antibodies, anti-CD19 antibodies, Bortezomib, rituximab, anti-CD154 antibodies, rapamycin, CD3 immunotoxin, fludarabine, cyclophosphamide, busulfan, melphalan, Mabthera, Tacrolimus, alefacept, alemtuzumab, 0KT3, 0KT4, 0KT8, 0KT11, fingollmod, anti-CD40 antibodies, anti-BR3 antibodies, Campath-IH, anti-CD25 antibodies, calcineurin inhibitors, mycophenolate, and steroids, which may be used alone or in combination.
Use as a diagnostic tool
[535] The anti-ALPPL2 agents of the present invention, for example, anti-ALPPL2 Abs and antigen-binding Ab fragments, can be also useful as a diagnostic tool that may be used in vivo, ex vivo, or in vitro.
[536] For example, an anti-ALPPL2 Abs or antigen-binding Ab fragment conjugated to an imaging agent may be administered to a subject or a patient to test if a diseased cell or tissue in the patient expresses ALPPL2. The diagnoses may be done using any imaging tools that can detect the imaging agent Alternatively, a biological sample such as, but is not limited to, blood or biopsy sample, may be obtained, and an anti-ALPPL2 Abs or antigen-binding Ab fragment may be applied to the sample to test the expression ofALPPL2.
[537] These tests may determine whether the subject or the cell or tissue of the subject; expresses ALPPL2 or not In some embodiments, the test may determine whether the subject, or the cell or tissue of the subject, expresses sufficient amount of ALPPL2 to be targeted by the anti-ALPPL2 therapeutic agent of the present invention. In some embodiments, the test may classify patients into different levels of ALPPL2 expression. In one aspect; a subject may be classified as an expressor or a non- expressor. In another aspect, a subject may be classified as an over-expressor, mid- expressor, or low-expressor.
[538] In some embodiments, an appropriate therapeutic approach may be determined depending on the ALPPL2 expression. The expression may be determined using an anti- ALPPL2 agent of the present invention as described herein, or alternatively using any other appropriate method, such as, but not limited to, by measuring RNA expression levels or by quantifying ALPPL2 protein levels using an appropriate tool and/or technique. In one aspect; the anti-ALPPL2 agent of the present invention may be given to an expressor but not to a non-expressor. In another aspect the anti-ALPPL2 agent of the present Invention may be given to an over-expressor but not to a mld-expressor or a low-expressor. In another aspect; the anti-ALPPL2 agent of the present invention may be given to an over-expressor or a mid-expressor but not to a low-expressor. In yet another aspect; the anti-ALPPL2 agent of the present invention may be given to a mid-expressor but not to a hlgh-expressor or a low-expressor.
Variations
[539] Included in the scope of the invention are functional portions of the inventive anti-ALPPL2 agents described herein. The term "functional portion," when used in reference to an Ab, antigen-binding Ab fragment; ADC, or CAR, refers to any part or fragment of the Ab, antigen-binding Ab fragment; ADC, or CAR of the invention, which part or fragment retains the biological activity of the Ab, antigen-binding Ab fragment; ADC, or CAR of which it is a part (the parent). Functional portions encompass, for example, those parts of an Ab, antigen-binding Ab fragment, ADC, or CAR that retain the ability to recognize target cells, or detect; treat; or prevent a disease, to a similar extent; the same extent, or to a higher extent; as the parent In reference to the parent Ab, antigen-binding Ab fragment ADC, or CAR, the functional portion can comprise, for instance, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more, of the parent
[540] The functional portion can comprise additional amino acids at the amino or carboxy terminus of the portion, or at both termini, which additional amino acids are not found in the amino add sequence of the parent Ab, antigen-binding Ab fragment ADC, or CAR. Desirably, the additional amino adds do not interfere with the biological function of the functional portion, e.g., recognize target cells, detect treat or prevent a target disease and/or condition, eta More desirably, the additional amino adds enhance the biological activity, as compared to the biological activity of the parent Ab, antigenbinding Ab fragment ADC, or CAR.
[541] Included in the scope of the invention are functional variants of the inventive Abs, antigen-binding Ab fragments, ADCs, or CARs described herein. The term "functional variant" as used herein refers to an Ab, antigen-binding Ab fragment ADC, or CAR polypeptide, or protein having substantial or significant sequence identity or similarity to a parent which functional variant retains the biological activity of the Ab, antigen-binding Ab fragment ADC, or CAR of which it is a variant Functional variants encompass, for example, those variants of the Ab, antigen-binding Ab fragment ADC, or CAR described herein (the parent) that retain the ability to recognize target cells to a similar extent the same extent or to a higher extent as the parent In reference to the parent Ab, antigen-binding Ab fragment ADC, or CAR, the functional variant can, for Instance, be at least about 30%, 50%, 75%, 80%, 90%, 98% or more Identical In amino acid sequence to the parent
[542] A functional variant can, for example, comprise the amino acid sequence of the parent with at least one conservative amino acid substitution. Alternatively, or additionally, the functional variants can comprise the amino acid sequence of the parent with at least one non-conservative amino acid substitution. In this case, it is preferable for the non-conservative amino acid substitution to not interfere with or inhibit the biological activity of the functional variant The non-conservative amino acid substitution may enhance the biological activity of the functional variant such that the biological activity of the functional variant is increased as compared to the parent
[543] Amino acid substitutions of the inventive anti-ALPPL2 agents are preferably conservative amino acid substitutions. Conservative amino acid substitutions are known in the art and Include amino acid substitutions in which one amino acid having certain physical and/or chemical properties is exchanged for another amino add that has the same or similar chemical or physical properties. For instance, the conservative amino add substitution can be an addic/negatively charged polar amino add substituted for another addic/negatively charged polar amino add (e.g., Asp or Glu), an amino add with a nonpolar side chain substituted for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Vai, He, Leu, Met, Phe, Pro, Trp, Cys, Vai, etc.), a basic/positively charged polar amino acid substituted for another basic/positively charged polar amino add (e.g. Lys, His, Arg, etc.), an uncharged amino add with a polar side chain substituted for another uncharged amino add with a polar side chain (e.g., Asn, Gin, Ser, Thr, iyr, eta), an amino add with a ^-branched side-chain substituted for another amino add with a P- branched side-chain (e.g., He, Thr, and Vai), an amino add with an aromatic side-chain substituted for another amino add with an aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc.
[544] Also, amino adds may be added or removed from the sequence based on vector design.
[545] The anti-ALPPL2 agents can consist essentially of the spedfied amino add sequence or sequences described herein, such that other components, e.g., other amino adds, do not materially change the biological activity of the functional variant
[546] The Abs, antigen-binding Ab fragments, AD Cs, or CARs of embodiments of the invention (including functional portions and functional variants) can be of any length, i.e., can comprise any number of amino acids, provided that the Abs, antigen-binding Ab fragments, ADCs, or CARs (or functional portions or functional variants thereof) retain their biological activity, e.g., the ability to spedfically bind to antigen, detect diseased cells in a mammal, or treat or prevent disease in a mammal, eta For example, the Ab, antigen-binding Ab fragment^ ADC, or CAR can be about 50 to about 5000 amino adds long, such as 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more amino adds in length.
[547] The Abs, antigen-binding Ab fragments, AD Cs, or CARs of embodiments of the invention (including functional portions and functional variants of the invention) can comprise synthetic amino acids In place of one or more naturally-occurring amino acids. Such synthetic amino acids are known in the art, and include, for example, aminocyclohexane carboxylic acid, norleucine, a-amino n-decanoic acid, homoserine, S- acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4- amlnophenylalanine, 4-nltrophenylalanlne, 4-chlorophenylalanlne, 4- carboxyphenylalanine, p-phenylserine p-hydroxyphenylalanine, phenylglycine, a- naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic add, l,2,3,4-tetrahydroisoquinoline-3-carboxylic add, aminomalonic add, aminomalonic add monoamlde, N'-benzyl-N'-methyl-lysine, N'.N'-dlbenzyl-lysine, 6-hydroxylyslne, ornithine, a-aminocyclopentane carboxylic add, a-aminocyclohexane carboxylic add, a- aminocycloheptane carboxylic add, a-(2-amino-2-norbomane)-carboxylic add, a,y- diaminobutyric add, o,P-diaminopropionic add, homophenylalanine, and a-tert- butylglydne.
[548] The Abs, antigen-binding Ab fragments, ADCs, or CARs of embodiments of the invention (including functional portions and functional variants) can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via, e.g., a disulfide bridge, or converted into an add addition salt and/or optionally dimerized or polymerized, or conjugated.
[549] The Abs, antigen-binding Ab fragments, ADCs, or CARs of embodiments of the invention (including functional portions and functional variants thereof) can be obtained by methods known in the art The Abs, antigen-binding Ab fragments, ADCs, and CARs may be made by any suitable method of making polypeptides or proteins. Suitable methods of de novo synthesizing polypeptides and proteins are described in references, such as Chan etal., "Fmoc Solid Phase Peptide Synthesis", Orford University Press, Orford, United Kingdom, 2000; "Peptide and Protein Drug Analysis", ed. Reid, R., Marcel Dekker, Ina, 2000; "Epitope Mapping", ed. Westwood etal., " Orford University Press, Orford, United Kingdom, 2001; and U.S. Pat No. 5,449,752. Also, polypeptides and proteins can be recombinantiy produced using the nucleic acids described herein using standard recombinant methods. See, for instance, Sambrook etal., "Molecular Cloning: A Laboratory Manual", 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; and Ausubel et al., "Current Protocols in Molecular Biology", Greene Publishing Associates and John Wiley & Sons, N Y, 1994. Further, some of the Abs, antigen-binding Ab fragments, or CARs of the invention (including functional portions and functional variants thereof) can be isolated and/or purified from a source, such as a plant; a bacterium, an insect; a mammal, e.g., a rat, a human, eta Methods of isolation and purification are well-known in the art Alternatively, the Abs, antigen-binding Ab fragments, ADCs, or CARs described herein (including functional portions and functional variants thereof) can be commercially synthesized by companies. In this respect; the inventive Abs, antigen-binding Ab fragments, ADCs, or CARs can be synthetic, recombinant; isolated, and/or purified.
DEFINITIONS [550] Although various embodiments and examples of the present invention have been described referring to certain molecules, compositions, methods, or protocols, it is to be understood that the present invention is not limited to the particular molecules, compositions, methods, or protocols described herein, as theses may vary. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
[551] All references cited herein, including patent documents and non-patent documents, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention.
[552] In the specification above and in the appended claims, all transitional phrases such as "comprising," "including," "having," "containing," "involving," "composed of," and the like are to be understood to be open-ended, namely, to mean including but not limited to. Only the transitional phrases "consisting of' and "consisting essentially of' shall be closed or semi-closed transitional phrases, respectively.
[553] It must also be noted that; unless the context clearly dictates otherwise, the singular forms "a," "an," and "the" as used herein and in the appended claims include plural refence. Thus, the reference to "a cell" refers to one or more cells and equivalents thereof known to those skilled in the art; and so forth. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by a person of skilled in the art
[554] It should be understood that; unless dearly indicated otherwise, in any methods disclosed or daimed herein that comprise more than one step, the order of the steps to be performed is not restricted by the order of the steps dted.
[555] The term "4-1BB," "41BB," or "BB" as used herein refers to a member of the TNFR superfamily with an amino add sequence provided as GenBank Acc. No. AAA53133.1, or the equivalent residues from a non-human spedes, e.g., mouse, rodent; monkey, ape and the like. In one aspect; the "4- IBB costimulatory domain," also referred to as "4-1BB CS domain" or "41BBCS,” may be derived from the cytoplasmic domain of 4-1BB. In some embodiments, "41BBCS" comprises the sequence provided as SEQ ID NO: 165 or the equivalent residues from a non-human species, e.g., mouse, rodent; monkey, ape and the like. In some embodiments, "41BBCS" may be encoded by a nucleic add sequence provided as SEQ ID NO: 265.
[556] The term "5* cap" (also termed an RNA cap, an RNA 7-methylguanosine cap or an RNA m7G cap) as used herein is a modified guanine nucleotide that has been added to the "front” or 5' end of a eukaryotic messenger RNA shortly after the start of transcription. The 5' cap consists of a terminal group which is linked to the first transcribed nucleotide. Its presence is critical for recognition by the ribosome and protection from RNases. Cap addition is coupled to transcription, and occurs co- transcriptionally, such that each influences the other. Shortly after the start of transcription, the 5' end of the mRNA being synthesized Is bound by a cap-synthesizing complex associated with RNA polymerase. This enzymatic complex catalyzes the chemical reactions that are required for mRNA capping. Synthesis proceeds as a multi- step biochemical reaction. The capping moiety can be modified to modulate functionality of mRNA such as Its stability or efficiency of translation.
[557] The term "about” or "approximately" as used herein when referring to a numerical value, such as of weight, mass, volume, concentration, or time, should not be limited to the recited numerical value but rather encompasses variations of +/- 10% of a given value.
[558] "ALPPL2” as used herein, is an alkaline phosphatase, also known as "alkaline phosphatase, placental like 2", "ALPG", "ALPPL", "alkaline phosphatase, germ cell”, or "GCAP". ALPPL2 is typically in a membrane-bound, glycosylated form. In humans, ALPPL2 is encoded by the ALPG gene on chromosome 2, with gene location 2q37.1 (NCBI). Human ALPPL2 may have an amino acid sequence provided as GenBank: AIC53992.1. In one aspect, human ALPPL2 has the amino acid sequence provided as SEQ ID NO: 101 or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like.
[559] The term "allogeneic” as used herein refers to any material derived from a different animal of the same species as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not Identical. In some embodiments, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically.
[560] The term "antibody" or "Ab” is used herein in the broadest sense and encompasses various antibody structures, including but not limited to full-length or full- size immunoglobulins, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and/or antibody fragments (preferably those fragments that exhibit the desired antigen-binding activity, which is also referred to as "antigen-binding antibody fragments"), "typically, a full-size Ab comprises two pairs of chains, each pair comprising a heavy chain (HC) and a light chain (LC) interconnected by disulfide bonds. A HC typically comprises a variable region and a constant region. A LC also typically comprises a variable region and constant region. The variable region of a heavy chain (VH) typically comprises three complementarity-determining regions (CDRs), which are referred to herein as CDR 1, CDR 2, and CDR 3 (or referred to as CDR- Hl, CDR-H2, CDR-H3, respectively). The constant region of a HC typically comprises a CHI domain, a CH2 domain, and a CH3 domain. CH2 and CH3 domains form a fragment crystallizable region (Fc region), which dictates the Isotype of the Ab (IgA (further divided into IgAl and IgA2 subclasses), IgD, IgG (further divided into IgGl, IgG2, IgG3, and IgG4 subclasses), IgE, and IgM), the type of Fc receptor the Ab binds to, and therefore the effector function of the Ab. Fc receptor types include, but are not limited to, FcaR (such as FcaRI), Fca/mR, FceR (such as FceRI, FceRII),and FcgR (such as FcgRI, FcgRIIA, FcgRIIBl, FcgRIIB2, FcgRIIIA, FcgRIIIB) and their associated downstream effects are well known in the art The variable region of a light chain (VL) also typically comprises CDRs, which are CDR 1, CDR 2, and CDR 3 (or referred to as CDR-L1, CDR-L2, CDR-L3, respectively). The constant region of a LC typically comprises a CL domain (kappa or lambda type). In some embodiments, the antigen is ALPPL2. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources. A molecule comprising an antibody-derived structure that enables specific binding to an antigen is referred to "antigen-binding fragment," "antigen-binding domain" or "antigenbinding region" of the Ab.
[561] The term "antibody-drug conjugate" "Ab-drug conjugate", or "ADC" as used herein refers to a conjugate of an Ab or antigen-binding Ab fragment and a drug. The drug may be attached to any part of the Ab or the antigen-binding Ab fragment via a direct or indirect attachment; such as via a linker. In some embodiments, an ADC may comprise an antibody (or antibody fragment such as a single-chain variable fragment (scFv)) linked to a payload drug (often cytotoxic). The antibody causes the ADC to bind to the target cancer cells. In some embodiments, the ADC is then internalized by the cell and the drug is released into the cell. Because of the targeting, the side effects may be lower and may provide a wider therapeutic window. Hydrophilic linkers (e.g., PEG4Mal) may prevent the drug being pumped out of resistant cancer cells through MDR (multiple drug resistance) transporters. The present disclosure is also related to immunoconjugates comprising an anti-ALPPL2 binding agent conjugated to a therapeutic agent, such as a cytotoxin, a drug (e.g., an Immunosuppressant) or a radiotoxin. Such conjugates may be referred to as "immunoconjugates". Immunoconjugates that include one or more cytotoxins may also be referred to as "immunotoxins." A cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g., kills) cells. Cytotoxins can be conjugated to antibodies according to at least some embodiments of the invention using linker technology available in the art Examples of linker types that have been used to conjugate a cytotoxin to an antibody include, but are not limited to, hydrazones, thioethers, esters, disulfides and peptide-containing linkers. A linker can be chosen that is, for example, susceptible to cleavage by low pH within the lysosomal compartment or susceptible to cleavage by proteases, such as proteases preferentially expressed in tumor tissue such as cathepsins (e.g., cathepsins B, C, D). For further discussion of types of cytotoxins, linkers and methods for conjugating therapeutic agents to antibodies, see also Saito, G. et al. (2003) Adv. Drug Deliv. Rev. 55: 199-215; Trail, P. A etal. (2003) Cancer Immunol. Immunother. 52:328-337; Payne, G. (2003) Cancer Cell 3:207-212; Allen, T. M. (2002) Nat Rev. Cancer 2:750-763; Pastan, I. and Kreitman, R. J. (2002) Curr. Opin. Investig. Drugs 3: 1089-1091; Senter, P. D. and Springer, C. J. (2001) Adv. Drug Deliv. Rev. 53:247-264. Antibodies of the present invention also can be conjugated to a radioactive isotope to generate cytotoxic radiopharmaceuticals, also referred to as radioimmunoconjugates.
[562] The term "antibody fragment” or "Ab fragment' as used herein refers to any portion or fragment of an Ab, including intact or full-length Abs that may be of any class or sub-class, Including IgG and sub-classes thereof, IgM, IgE, IgA and sub-classes thereof, and IgD. The term encompasses molecules constructed using one or more potions or fragments of one or more Abs. An Ab fragment can be immunoreactive portions of intact immunoglobulins. The term is used in the broadest sense and includes polyclonal and monoclonal antibodies, including Intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, including single chain variable fragments (scFv), diabodies, and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term also encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodles, and tetrabodies, tandem dl-scFv, tandem tri- scFv. In a specific embodiment; the antibody fragment is a scFv.
[563] Unless otherwise stated, the term "Ab fragment1 should be understood to encompass functional antibody fragments thereof. A portion of an Ab fragment that comprises a structure that enables specific binding to an antigen is referred to as "antigen-binding Ab fragment;" "antigen-binding domain," "antigen-binding region," or "antigen-binding region" of the Ab fragment
[564] A "heavy chain" or "HC" of an Ab, as used herein, refers to the larger of the two types of polypeptide chains present in all Ab molecules in their naturally occurring conformations.
[565] A "light chain" or "LC" of an Ab, as used herein, refers to the smaller of the two types of polypeptide chains present in all Ab molecules in their naturally occurring conformations. Kappa and lambda light chains refer to the two major antibody light chain isotypes.
[566] An "anti-ALPPL2 agent' or "anti-ALPPL2 material" as used herein refers to any agents that are able to bind to and/or target ALPPL2 directly or indirectly. Anti-ALPPL2 agents of the present invention include, but are not limited to, anti-ALPPL2 Abs, anti- ALPPL2 antigen-binding Ab fragments, anti-ALPPL2 multi-specific Abs, anti-ALPPL2 multi-specific antigen-binding Ab fragments, anti-ALPPL2 ADCs, and anti-ALPPL2 CARs, and polynucleotides and vectors encoding the same, and cells expressing the same. In a broad sense, anti-ALPPL2 agents may also encompass pharmaceutical compositions comprising any of the above-mentioned anti-ALPPL2 agents.
[567] The term "antigen" or "Ag" refers to a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an "antigen" as that term is used herein. Furthermore, one skilled In the art will understand that an antigen need not be encoded solely by a full- length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one genes and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired Immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be generated, synthesized, or can be derived from a biological sample, or might be macromolecule besides a polypeptide. Such a biological sample can Include, but is not limited to a tissue sample, a cancer tissue sample, a tumor tissue sample, a leukemic cell sample, an inflamed tissue sample, and a cell or a fluid with other biological components. In some embodiments, the antigen is ALPPL2.
[568] The term "antigen-binding domain" refers to a portion of the anti-ALPPL2 agents, such as antl-ALPPL2 chimeric antigen receptors, of the present Invention and the portion comprises a structure that allows for specific binding of the anti-ALPPL2 agents to ALPPL2. When the anti-ALPPL2 agent is an Ab, the antigen-binding domain may comprise the variable region of the Ab or a portion of the variable region, such as the CDRs. When the anti-ALPPL2 agentls an antigen-binding Ab fragment or an antibodydrug conjugate, the antigen-binding domain may comprise the variable region or a portion of the variable region, such as the CDRs, of the Ab that the anti-ALPPL2 agentis derived from. When the anti-ALPPL2 agentis a chimeric antigen receptor (CAR), the antigen-binding domain may be one or more extracellular domains of the CAR which have specificity for ALPPL2. When the antigen-binding domain is derived from an Ab or antigen-binding Ab fragment; the antigen-binding domain may comprise the antigenbinding domain, such as the variable region or a portion of the variable region, such as the CDRs, of the Ab or antigen-binding Ab fragment that it is derived from. In some embodiments, the antigen-binding domain of an anti-ALPPL2 agent of the present invention is scFv.
[569] The term "apheresis" as used herein refers to the art-recognized extracorporeal process by which the blood of a donor or patient is removed from the donor or patient and passed through an apparatus that separates out selected particular constituents) and returns the remainder to the circulation of the donor or patient; e.g., by retransfusion. Thus, in the context of "an apheresis sample" refers to a sample obtained using apheresis.
[570] The term "autologous" or "donor-derived" as used herein refers to any material derived from the same individual to whom it is later to be re-introduced.
[571] The term "bind" refers to an attractive interaction between two molecules that results in a stable association in which the molecules are in close proximity to each other. The result of molecular binding is sometimes the formation of a molecular complex in which the attractive forces holding the components together are generally non-covalent; and thus are normally energetically weaker than covalent bonds.
[572] The term "cancer* refers to a disease characterized by the uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers relevant to the present invention include, but are not limited, pancreatic cancer, testicular cancer, cervical cancer, endometrial cancer, ovarian cancer, stomach cancer, colorectal cancer, lung cancer, mesothelioma, and tongue cancer.
[573] The term "bispecific" as used herein refers to having two binding specificities. An anti-ALPPL2 bispecific Ab or a bispecific antigen-binding Ab fragment; for example, of the present invention has at least one specificity for ALPPL2. When the first specificity is for an epitope for ALPPL2, the second specificity may be for another non-overlapping or non-competing epitope for ALPPL2 or may be for a molecule other than ALPPL2. The term "bispecific" is also used in the same manner for any other anti-ALPPL2 agents of the present invention, such as anti-ALPPL2 CARs.
[574] The term "CD28" refers to the protein Cluster of Differentiation 28, one of the proteins expressed on T cells that provide co-stimulatory signals required for T cell activation and survival. Human CD28 protein may have at least 85, 90, 95, 96, 97, 98, 99 or 100% identity to NCBI Reference No: NP_006130 or a fragment thereof that has stimulatory activity. The term "CD28 transmembrane domain," also referred to as "CD28 TM domain" or "CD28TM" refers to the amino acid residues derived from the transmembrane domain of CD28. In some embodiments, "CD28TM” comprises the sequence provided as SEQ ID NO: 161 or the equivalent residues from a non-human species, e.g., mouse, rodent; monkey, ape and the like. In some embodiments, "CD28 TM domain" may be encoded by the nucleic acid sequence provided as SEQ ID NO: 261. The term "CD28 hinge" as used herein refers to amino acid residues that may be used to join two domains or two portions within a domain in CARs of some of the embodiments. In some embodiments, "CD28 hinge" comprises the sequence provided as SEQ ID NO: 163 or the equivalent residues from a non-human species, e.g., mouse, rodent; monkey, ape and the like. In some embodiments, "CD28 hinge" may be encoded by the nucleic acid sequence provided as SEQ ID NO: 263. The term "CD28 costimulatory domain," also referred to as "CD28 CS domain" or "CD28CS," refers to the amino acid residues derived from the cytoplasmic domain of CD28. In some embodiments, "CD28CS" comprises the sequence provided as SEQ ID NO: 164 or the equivalent residues from a non-human species, e.g., mouse, rodent; monkey, ape and the like. In some embodiments, "CD28 CS domain" may be encoded by the nucleic acid sequence provided as SEQ ID NO: 264.
[575] The term "CD3 zeta," or alternatively, "zeta," "zeta chain," "CD3-zeta," "CD3z," "TCR-zeta," or "CD247," is a protein encoded by the CD247 gene on chromosome 1, with gene location lq24.2, in humans. CD 3 zeta, together with T cell receptor (TCR) and CD 3 (a protein complex composed of a CD3 gamma, a CD3 delta, and two CD3 epsilon), forms the TCR complex. Human CD3 zeta may have an amino add sequence provided as NP_000725 or NP.932170, or the equivalent residues from a non-human spedes, e.g., mouse, rodent; monkey, ape and the like. The term "CD3 zeta intracellular signaling domain," or alternatively "CD3 zeta ICS domain" or a "CD3zICS," is defined as the amino add residues from the cytoplasmic domain of the CD3 zeta chain, or functional derivatives thereof, that are suflident to functionally transmit an initial signal necessary for T cell activation. In one aspect; "CDS zeta ICS domain" Is the sequence provided as SEQ ID NO: 162. In one aspect, "CDS zeta ICS domain" is encoded by the nucleic acid sequence provided as SEQ ID NO: 262.
[576] The term "Chimeric Antigen Receptor" or alternatively a "CAR" refers to a set of polypeptides, typically two In the simplest embodiments, which when In an Immune effector cell, provides the cell with specificity for a target cell, and with intracellular signal generation. In some embodiments, a CAR comprises at least an extracellular antigen-binding domain, a transmembrane domain (TM domain) and a cytoplasmic signaling domain (also referred to herein as "an Intracellular signaling domain (ICS domain)") comprising a functional signaling domain derived from a stimulatory molecule and/or costimulatory molecule as defined below. In some embodiments, the set of polypeptides are contiguous with each other. In some embodiments, the set of polypeptides Include a dimerization switch that; upon the presence of a dimerization molecule, can couple the polypeptides to one another, e.g., can couple an antigenbinding domain to an ICS domain. In one aspect, the stimulatory molecule is the zeta chain associated with the T cell receptor complex In one aspect, the cytoplasmic portion of a CAR further comprises a costimulatory domain (CS domain) comprising one or more functional signaling domains derived from at least one costimulatory molecule as defined below. In one aspect, the costimulatory molecule is chosen from the costimulatory molecules described herein, e.g., 4-1BB (i.e., CD137), DAP10 and/or CD28. In one aspect; the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a TM domain and an ICS domain comprising a functional signaling domain derived from a stimulatory molecule. In one aspect; the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a TM domain, an ICS domain comprising a functional signaling domain derived from a stimulatory molecule, and a CS domain comprising a functional signaling domain derived from a costimulatory molecule. In one aspect; the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a TM domain, an ICS domain comprising a functional signaling domain derived from a stimulatory molecule, and two CS domains each of the two comprising a functional signaling domain derived from a costimulatory molecule(s) that is/are same with or different from each other. In one aspect; the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a TM domain, an ICS domain comprising a functional signaling domain derived from a stimulatory molecule, and at least two CS domains each comprising a functional signaling domain derived from a costimulatory moleculefs) that is/are same with or different from each other. In one aspect the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein. In one aspect; the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen binding domain, wherein the leader sequence is optionally cleaved from the antigen binding domain (e.g., a scFv) during cellular processing and localization of the CAR to the cellular membrane. In some embodiments, the leader sequence (LS) comprises the amino acid sequence provided as SEQ ID NO: 160. In some embodiments, die LS may be encoded by a nucleic acid sequence provided as SEQ ID NO: 260.
[577] The term "compete", as used herein with regard to an Ab, antigen-binding Ab fragment; of antigen-binding domain of any of the anti-ALPPL2 agents of the present invention, means that a first Ab, antigen-binding Ab fragment; or antigen-binding domain, binds to an epitope in a manner sufficiently similar to the binding of a second Ab, antigen-binding Ab fragment, or antigen-binding domain, such that the result of binding of the first Ab, antigen-binding Ab fragment; or antigen-binding domain with its cognate epitope is delectably decreased in the presence of the second Ab, antigenbinding Ab fragment; or antigen-binding domain compared to the binding of the first Ab, antigen-binding Ab fragment, or antigen-binding domain in the absence of the second Ab, antigen-binding Ab fragment; or antigen-binding domain. The alternative, where the binding of the second Ab, antigen-binding Ab fragment; or antigen-binding domain to its epitope is also delectably decreased in the presence of the first antibody, can, but need not be the case. That is, a first Ab, antigen-binding Ab fragment, or antigen-binding domain can inhibit the binding of a second Ab, antigen-binding Ab fragment, or antigenbinding domain to its epitope without that second Ab, antigen-binding Ab fragment, or antigen-binding domain inhibiting the binding of the first Ab, antigen-binding Ab fragment, or antigen-binding domain to its respective epitope. However, where each Ab, antigen-binding Ab fragment, or antigen-binding domain delectably inhibits the binding of the other Ab, antigen-binding Ab fragment; or antigen-binding domain with its cognate epitope or ligand, whether to the same, greater, or lesser extent; the two (Ab, antigen-binding Ab fragment; or antigen-binding domain) are said to "cross-compete" with each other for binding of their respective epitope(s). Both competing and crosscompeting Abs, antigen-binding Ab fragments, or antigen-binding domains are encompassed by the invention. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope, or portion thereof), the skilled artisan would appreciate, based upon the teachings provided herein, that such competing and/or cross-competing Abs, antigen-binding Ab fragments, or antigen-binding domains are encompassed and can be useful for the methods disclosed herein.
[578] The terms "complementarity determining region," and "CDR," synonymous with "hypervariable region" or "HVR," are known in the art to refer to non-contiguous sequences of amino acids within antibody variable regions, which confer antigen specificity and/or binding affinity. In general, there are three CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3) and three CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR-L3).
[579] The term "conservative amino acid substitutions" herein are as commonly used in the art and include amino acid substitutions in which one amino acid having certain physical and/or chemical properties is exchanged for another amino add that has the same or similar chemical or physical properties. For instance, the conservative amino add substitution can be an addic/negatively charged polar amino add substituted for another acidlc/negatively charged polar amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain substituted for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Vai, He, Leu, Met; Phe, Pro, Trp, Cys, Vai, eta), a basic/positively charged polar amino acid substituted for another basic/positively charged polar amino acid (e.g. Lys, His, Arg, eta), an uncharged amino acid with a polar side chain substituted for another uncharged amino acid with a polar side chain (e.g., Asn, Gin, Ser, Thr, Tyr, eta), an amino acid with a ^-branched side-chain substituted for another amino acid with a P- branched side-chain (e.g., He, Thr, and Vai), an amino acid with an aromatic side-chain substituted for another amino acid with an aromatic side chain (e.g., His, Phe, Trp, and Tyr), eta Non-conservative amino acid substitutions are amino acid substitutions that are not conservative amino acid substitutions.
[580] The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an efficient immune response. Costimulatory molecules Include, but are not limited to a protein selected from the group consisting of an MHC class I molecule, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, a Toll ligand receptor, B7-H3, BAFFR, BTLA, BLAME (SLAMF8), CD2, CD4, CDS, CD7, CDBalpha, CDSbeta, CDlla, LFA-1 (CDlla/CD18),
CD lib, CD 11c, CDlld, CD18, CD19, CD19a, CD27, CD28, CD29, CD30, CD40, CD49a, CD49D, CD49f, CD69, CD84, CD96 (Tactile), CD100 (SEMA4D), CD103, 0X40 (CD134), 4- 1BB (CD137), SLAM (SLAMF1, CD150, IPO-3), CD160 (BY55), SELPLG (CD162), DNAM1 (CD226), Ly9 (CD229), SLAMF4 (CD244, 2B4), ICOS (CD278), CEACAM1, CDS, CRTAM, DAP10, GADS, GITR, HVEM (LIGHTR), IA4, 1 CAM-1, IL2Rbeta, IL2R gamma, IL7R alpha, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAT, LFA-1, LIGHT, LTBR, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), PAG/Cbp, PD-1, PSGL1, SLAMF6 (NTB-A, Lyl08), SLAMF7, SLP-76, TNFR2, TRANCE/RANKL, VLA1, VLA-6, and a ligand that specifically binds with CD83. In embodiments wherein a CAR comprises one or more CS domain, each CS domain comprises a functional signaling domain derived from a costimulatory molecule. In some embodiments, the encoded CS domain comprises 4-1BB, CD28, or DAP10. In one embodiment; the CS domain comprises the amino acid sequence of CD28CS, 41BBCS, or DAP10CS (SEQ ID NO: 164, 165, or 166), or is encoded by the nucleotide sequence encoding provided as SEQ ID NOs: 264, 265, or 266.
[581] The term "cytokines" as used herein refers to a broad category of small proteins that are involved in cell signaling. Generally, their release has some effect on the behavior of cells around them. Cytokines may be involved in autocrine signaling, paracrine signaling and/or endocrine signaling as immunomodulating agents. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. Cytokines are produced by a broad range of cells, including immune cells like macrophages, B lymphocytes, T lymphocytes and mast cells, as well as endothelial cells, fibroblasts, epithelial cells, and various stromal cells. "Chemokines" are a family of cytokines generally involved in mediating chemotaxis.
[582] The term "cytotoxicity" generally refers to any cytocidal activity resulting from the exposure of the anti-ALPPL2 agents of the Invention or cells comprising the same to cells expressing ALPPL2. This activity may be measured by known cytotoxicity assays, including IFN-y production assays. When the target cell is a cancer or tumor cell, the term "anti-cancer cytotoxicity" or "anti-tumor cytotoxicity" may be used.
[583] The term "DAP10" refers to a protein, which In humans Is encoded by the HSCT gene. It may also be referred to as HSCT, KAP10, PIK3AP, or hematopoietic cell signal transducer. In some embodiments, DAP10 may have the sequence provided in Genbank Accession No.: Q9UBK5.1. The term "DAP10 costimulatory domain," also referred to as "DAP10 CS domain" or "DAP10CS," refers to the amino add residues derived from the cytoplasmic domain of DAP10. In some embodiments, "DAP10CS” comprises the sequence provided as SEQ ID NO: 166 or the equivalent residues from a non-human spedes, e.g., mouse, rodent; monkey, ape and the like. In some embodiments, "DAP10 CS domain" may be encoded by the nucleic acid sequence provided as SEQ ID NO: 266.
[584] The phrase "disease associated with expression of ALPPL2" or "ALPPL2- assodated disease" includes, but is not limited to, a disease assodated with expression of ALPPL2 or condition assodated with cells which express ALPPL2 including, e.g., proliferative diseases such as a cancer or malignancy or a precancerous condition; or a noncancer-related indication assodated with cells which express ALPPL2. Examples of various cancers that express ALPPL2 include but are not limited to, pancreatic cancer, testicular cancer, cervical cancer, endometrial cancer, ovarian cancer, stomach cancer, colorectal cancer, lung cancer, mesothelioma, and tongue cancer, and the like.
[585] An "effective amount” or "an amount effective to treat' refers to a dose that is adequate to prevent or treat a disease, condition, or disorder in an individual. Amounts effective for a therapeutic or prophylactic use will depend on, for example, the stage and severity of the disease or disorder being treated, the age, weight; and general state of health of the patient; another pre-existing condition, and the judgment of the prescribing physidan. The size of the dose will also be determined by the active ingredient selected, method of administration, timing and frequency of administration, the existence, nature, and extent of any adverse side effects that might accompany the administration of a particular active ingredient; and the desired physiological effect It will be appredated by one of skill in the art that various diseases or disorders could require prolonged treatment involving multiple administrations, perhaps using the inventive anti-ALPPL2 agents, nucleic adds, vectors, cells, or compositions in each or various rounds of administration.
[586] The terms "enteral," "enterally," "oral," "orally," "non-parenteral," "non- parenterally," and the like, refer to administration of a compound or composition to an individual by a route or mode along the alimentary canal. Examples of "oral" routes of administration of a composition include, without limitation, swallowing liquid or solid forms of a composition from the mouth, administration of a composition through a nasojejunal or gastrostomy tube, intraduodenal administration of a composition, and rectal administration, e.g., using suppositories for the lower intestinal tract of the alimentary canal.
[587] The term "framework" as used herein refers to the non-CDR portions of the variable region of an Ab, or in some embodiments, Antigen-binding Ab fragment or an antigen-binding domain of a CAR. "Heavy chain (HC) framework" and "VH framework" are used interchangeably herein and refer to the non-CDR portion of a HC variable region, and in general, there are four framework regions (FRs) in each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4). "Light chain (LC) framework" and "VL framework" are used interchangeably herein and refer to the non-CDR portion of a LC variable region, and in general, there are four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4). In some embodiments, "human HC framework”, "human VH framework", "human-like HC framework", or "human-like VH framework” is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human HC framework. In some embodiments, "human LC framework", "human VL framework”, "human-like LC framework", or "human-like VL framework" is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human LC framework.
[588] The term "gene* is used broadly to refer to any segment of polynucleotide associated with a biological function. Thus, genes include introns and exons as in genomic sequence, or just the coding sequences as in cDNAs and/or the regulatory sequences required for their expression. For example, gene also refers to a nucleic acid fragment that expresses mRNA or functional RNA, or encodes a specific protein, and which includes regulatory sequences.
[589] The term "hinge", "spacer", or "linker" refers to an amino add sequence of variable length typically encoded between two or more domains or portions of a polypeptide construct to confer flexibility, improved spatial organization, proximity, eta
[590] As used herein, "human antibody" means an antibody having an amino add sequence corresponding to that of an antibody produced by a human and/or which has been made using any of the techniques for making human antibodies known to those skilled in the art or disdosed herein. Human antibodies can be produced using various techniques known in the art In one embodiment; the human antibody is selected from a phage library, where that phage library expresses human antibodies (Vaughan etal., Nature Biotechnology, 14:309-314, 1996; Sheets et al., Proc. Natl Acad. Sci. (USA) 95:6157-6162, 1998; Hoogenboom and Winter,/. Mo/. Biol., 227:381, 1991; Marks et al., /. Mol. Biol., 222:581, 1991). Human antibodies can also be made by immunization of animals into which human immunoglobulin lod have been transgenically introduced in place of the endogenous lod, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described in U.S. Pat Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016. Alternatively, the human antibody may be prepared by Immortalizing human B lymphocytes that produce an antibody directed against a target antigen (such B lymphocytes may be recovered from an individual or from single cell cloning of the cDNA, or may have been immunized in vitro}. See, e.g., Cole et al. "Monoclonal Antibodies and Cancer Therapy", Alan R. Liss, p. 77, 1985; Boemer et al.,/. Immunol., 147 (l):86-95, 1991; and U.S. Pat No. 5,750,373.
[591] The term "humanization" of an Ab refers to modification of an Ab of a nonhuman origin to increase the sequence similarity to an Ab naturally produced in humans. The term "humanized antibody" as used herein refers to Abs generated via humanization of an Ab. Generally, a humanized or engineered antibody has one or more amino acid residues from a source which is non-human, e.g., but not limited to mouse, rat, rabbit; non-human primate or another mammal. These human amino acid residues are often referred to as "import” residues, which are typically taken from an "Import" variable, constant or other domain of a known human sequence. Known human Ig sequences are disclosed, e.g., www.ncbi.nlm.nih.gov/entrez/query.fcgi; www.atcc.org/phage/hdb.html, each entirely incorporated herein by reference. Such Imported sequences can be used to reduce immunogenicity or reduce, enhance or modify binding, affinity, avidity, specificity, half-life, or any other suitable characteristic, as known in the art Generally, part or all of the non-human or human CDR sequences are maintained while part or all of the non-human sequences of the framework and/or constant regions are replaced with human or other amino acids. Antibodies can also optionally be humanized with retention of high affinity for the antigen and other favorable biological properties using three-dimensional immunoglobulin models that are known to those skilled in the art Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, framework (FR) residues can be selected and combined from the consensus and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding. Humanization or engineering of antibodies of the present invention can be performed using any known method, such as but not limited to those described in, for example, Winter (Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen etal., Science 239:1534 (1988)), Sims etal.,/. Immunol. 151: 2296 (1993); Chothia and Lesk,/. Mol. Biol. 196:901 (1987), Carter etal., Proc. Natl. Acad. Set USA. 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993), U.S. Pat Nos. 5,723,323, 5,976,862, 5,824514, 5,817483, 5,814476, 5,763,192, 5,723,323, 5,766,886, 5,714,352, 6,204,023, 6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539; 4,816,567, each entirely incorporated herein by reference, included references cited therein. [592] The term "ICAR" Is a chimeric antigen receptor which contains Inhibitory receptor signaling domains. These domains may be based, for example, on protectin DI (PD1) or CTLA-4 (CD152). In some embodiments, the CAR expressing cells of the invention are further transduced to express an iCAR. In one aspect; this iCAR is added to restrict the CAR expressing cell's functional activity to tumor cells.
[593] The term "immune cell" refers to a cell of hematopoietic origin functionally involved in the initiation and/or execution of innate and/or adaptive immune response.
[594] The term "intracellular signaling domain" or "ICS domain" as used herein, refers to an intracellular portion of a molecule. The intracellular signaling domain generates a signal that promotes an immune effector function of the cell transduced with a polynucleotide comprising a CAR, e.g., a CAR T cell. Examples of immune effector function, e.g., in a CAR T cell, include cytolytic activity and helper activity, including the secretion of cytokines. ICS domains Include an ICS domain of a lymphocyte receptor chain, a TCR/CD3 complex protein, an Fc receptor subunit; an IL-2 receptor subunit; CD3 zeta, FcR gamma, FcRbeta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD 79a, CD79b, CD66d, CD278(ICOS), Fc epsilon RI, DAP10, or DAP12.
[595] An "Isolated" biological component (such as an Isolated protein, nucleic acid, vector, or cell) refers to a component that has been substantially separated or purified away from its environment or other biological components in the cell of the organism in which the component naturally occurs, for instance, other chromosomal and extra- chromosomal DNA and RNA, proteins, and organelles. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant technology as well as chemical synthesis. An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
[596] A "leader sequence" or "LS" as used herein, also referred to as "signal peptide," "signal sequence,” "targeting signal," "localization signal," "localization sequence,” "transit peptide," or "leader peptide" in the art; is a short peptide present at the N- terminus of the majority of newly synthesized proteins that are destined towards the secretary pathway. The core of the signal peptide may contain a long stretch of hydrophobic amino acids. The signal peptide may or may not be cleaved from the mature polypeptide.
[597] The term "linker" as used in the context of a scFv refers to a peptide linker that consists of amino acids such as glycine and/or serine residues used alone or in combination, to link variable heavy and variable light chain regions together. In one embodiment; the flexible polypeptide linker is a Gly/Ser linker and comprises one or more repeats of the amino acid sequence unit Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 167). In one embodiment; the flexible polypeptide linker includes, but is not limited to, (Gly4Ser)3, which is also referred to as G4S X3 (SEQ ID NO: 168). Such a linker may be encoded for example, by the nucleic acid sequence as set forth in SEQ ID NO: 268 or 68. In one embodiment, the flexible linker may be a Whitlow linker (see e.g., Whitlow M et al., Protein Eng. 1993 Nov;6(8):989-95.). A Whitlow linker may comprise the amino acid sequence of SEQ ID NO: 159 and may be encoded by e.g., the nucleic acid sequence of SEQ ID NO: 259. In some embodiments, when a Whitlow linker is used, a tag may be further included in the extracellular portion of the CAR, for example between the scFv and the hinge. In one embodiment; such a tag may be a myc tag.
[598] The term "mammal" refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice, rats, and hamsters, and mammals of the order Logomorpha, such as rabbits. The mammals may be from the order Carnivora, including Felines (cats) and Canines (dogs). The mammals may be from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). The mammals may be of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes).
[599] The term "masked CAR" refers to a CAR expressing cell that further comprises a masking peptide. This masking peptide may prevent off-target cell killing. The masking peptide is often N-terminal to the CAR construct and may block the cell's ability to bind to unintended targets. The masking peptide may be cleaved from the CAR expressing cell when it encounters a tumor thereby allowing the CAR expressing cell to attack its target without killing off-target cells. An anti-ALPPL2 CARs of the present invention may be constructed to be a masked CAR.
[600] The term "multispecific" as used herein refers to having two or more binding specificities. An anti-ALPPL2 multispecific Ab or a multispecific antigen-binding Ab fragment, for example, of the present invention has at least one specificity for ALPPL2. When the first specificity is for an epitope for ALPPL2, the second (or third, fourth, and so forth) specificity may be for another epitope for ALPPL2 or may be for a molecule other than ALPPL2. The term "multispecific" is also used in the same manner for any other anti-ALPPL2 agents of the present invention, such as anti-ALPPL2 CARs.
[601] The term "nucleic acid" and "polynucleotide" refer to RNA or D NA that is linear or branched, single or double stranded, or a hybrid thereof. The term also encompasses RNA/DNA hybrids. The following are non-limiting examples of polynucleotides: a gene or gene fragment; exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, Isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracil, other sugars and linking groups such as fluororibose and thiolate, and nucleotide branches. The sequence of nucleotides may be further modified after polymerization, such as by conjugation, with a labeling component Other types of modifications included in this definition are caps, substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides or solid support The polynucleotides can be obtained by chemical synthesis or derived from a microorganism. [602] The term "OKT3" or "Muromonab-CD3" or "Orthoclone OKT3" refers to a monoclonal anti-CD3 Ab.
[603] The term "parenteral" or "parenterally" as used herein includes any route of administration of a compound or composition, characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue, thus generally resulting in the direct administration into the blood stream, into muscle, or into an internal organ. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrastemal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intrasynovial injection or infusions; and kidney dialytic infusion techniques. In a preferred embodiment, parenteral administration of the compositions of the present invention comprises subcutaneous or intraperitoneal administration.
[604] The term "pharmaceutically acceptable excipient;" "pharmaceutical excipient;" "excipient;" "pharmaceutically acceptable carrier," "pharmaceutical carrier," or "carrier" as used herein refers to compounds or materials conventionally used in pharmaceutical compositions during formulation and/or to permit storage.
[605] The term "promoter", as used herein, is defined as a DNA sequence recognized by the synthetic machineiy of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence
[606] The "ribosome skip sequence" refers to an amino acid sequence that; when translated, causes cleavage of a nascent polyprotein on the ribosome, allowing for coexpression of multiple genes. In one aspect; the ribosome skip sequence may be the T2A sequence such as SEQ ID NO: 169, which may be encoded by SEQ ID NO: 269. Alternatively, any other 2A sequences may be used. Examples of other sequences may be found elsewhere in the literature of the relevant art (for example, see Kim, J.H., et al., High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS One. 2011;6(4J).
[607] The term "recombinant" means a polynucleotide, a protein, a cell, and so forth with semi-synthetic or synthetic origin which either does not occur in nature or is linked to another polynucleotide, a protein, a cell, and so forth in an arrangement not found in nature.
[608] The term "scFv," "single-chain Fv," or "single-chain variable fragment* refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked, e.g., via a synthetic linker, e.g., a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-termlnal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL The linker may comprise portions of the framework sequences. In scFvs, the heavy chain variable region (HC V, HCV, or VH) may be placed upstream of the light chain variable region (LC V, LCV, or VL), and the two domains may optionally be linked via a linker (for example, the G4S X3 linker). In this case, when the scFv is for example derived from h6E6, the construct may be referred to as h6E6scFvHL, h6E6HL, h6E6scFvVHVL, or h6E6VHVL Alternatively, the heavy chain variable region may be placed downstream of the light chain variable region, and the two domains may optionally be linked via a linker (for example, the G4S X3 linker). In this case, when the scFv is for example derived from h6E6, the construct may be referred to as h6E6scFvLH, h6E6LH, h6E6scFvVLVH, or h6E6VLVH. The same naming rules apply to other similar constructs herein.
[609] The term "signaling domain" refers to the functional portion of a protein which acts by transmitting information within the cell to regulate cellular activity via defined signaling pathways by generating second messengers or functioning as effectors by responding to such messengers.
[610] The term "stimulatory molecule," refers to a molecule expressed by an immune cell (e.g., T cell, NK cell, B cell) that provides the cytoplasmic signaling sequence(s) that regulate activation of the immune cell in a stimulatory way for at least some aspect of the Immune cell signaling pathway. In one aspect; the signal is a primary signal that is initiated by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, and which leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. A primary cytoplasmic signaling sequence (also referred to as a "primary signaling domain") that acts in a stimulatory manner may contain a signaling motif which is known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of an ITAM containing cytoplasmic signaling sequence that are of particular use in the invention include, but are not limited to, those derived from CD3 zeta, common FcR gamma (FCER1G), Fc gamma Rlla, FcR beta (Fc epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD 79b, DAP10, and DAP12. In a specific CAR of the invention, the intracellular signaling domain in any one or more CARS of the invention comprises an intracellular signaling sequence, e.g., a primary signaling sequence of CD3 zeta. In a specific CAR of the invention, the primary signaling sequence of human CD 3 zeta, referred to as "CD3zICS" herein, is the amino add sequence provided as SEQ ID NO: 162, and may be encoded by the nudeotide sequence SEQ ID NO: 262. Alternatively, equivalent residues from a non-human or mouse spedes, e.g., rodent; monkey, ape and the like, may be utilized.
[611] The term "subject” as used herein may be any living organisms, preferably a mammal. In some embodiments, the subject is a primate such as a human. In some embodiments, the primate is a monkey or an ape. The subject can be male or female and can be any suitable age, including infant; juvenile, adolescent; adult; and geriatric subjects. In some examples, the patient or subject Is a validated animal model for disease and/or for assessing toxic outcomes. The subject may also be referred to as "patient' in the art The subject may have a disease or may be healthy.
[612] The term "suicide mechanism" as used herein refers to a mechanism by which anti-ALPPL2 agent-expressing cells of present invention may be eradicated from a subject administered with such cells. The suicide mechanism may be driven by, for example, inducible caspase 9 (Budde L E. et al., PLoS One. 2013 Dec 17;8(12):e82742. doi: 10.1371/joumal.pone.0082742. eCollection 2013), codon-optimized CD20 (Martin V. et al., Hum Gene Ther Methods. 2012 Dec; 23(6): 376-386), CD34, or polypeptide RQR8 (Philip et al, and W02013153391A, which is hereby incorporated herein by reference). In some embodiments, the suicide mechanism may be included and utilized in CAR-expressing cells of present invention to optimize the length for the CAR- expresslng cells to stay in the system of a subject or the amount of the CAR-expressing cells, to reduce or minimize the toxicity and/or to maximize the benefit of CAR- expressing cells.
[613] The term "synthetic Ab" or "synthetic antigen-binding Ab fragment” as used herein, refers to an Ab or antigen-binding Ab fragment which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino add sequence spedfying the antibody, wherein the DNA or amino add sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art
[614] The term "target" as used herein refers to the molecule that an anti-ALPPL2 agents of the present invention spedfically binds to. The term also encompasses cells and tissues expressing the target molecule and also diseases that are assodated with expression of the target
[615] The term "target cell" as used herein refers to a cell expressing the target molecule (such as ALPPL2) of the anti-ALPPL2 agents of the present invention on the cell surface. In some embodiments, the target cell is a cancer cell or tumor cell. In some embodiments, the target cell is a vascular cell In some embodiments, the target cell is an epithelial cell. In some embodiments, the target cell is a cell type that has a particular role in the pathology of cancer or inflammation. In some embodiments, the target cell is a cell type that has a particular role in the pathology of a disease such as but not limited to cancer.
[616] The term "target molecule" as used herein refers to a molecule that is targeted by the anti-ALPPL2 agents of the present invention. The antigen-binding domain of the anti-ALPPL2 agents of the present invention has a binding affinity for the target molecule. In some embodiments, the target molecule is ALPPL2.
[617] The term "trCD19" refers to a truncated version of the CD19 protein, B- lymphocyte antigen CD 19, also known as CD 19 (Cluster of Differentiation 19), which is a protein that Is encoded by the CD19 gene In humans and Is found on the surface of B- cells. The trCD 19 construct is any truncated version of said protein, such that a nucleic acid sequence encoding this construct may be transduced into a host cell and expressed on the surface of this cell for the purposes of detection, selection, and/or targeting. In one aspect; human trCD19 may comprise the amino acid sequence of SEQ ID NO: 170 or nucleotide sequence encoding such, such as SEQ ID NO: 270.
[618] The term "transfected," "transformed," or "transduced" refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell A "transfected" or "transformed" or "transduced" cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.
[619] By the term "transmembrane domain" or "TM domain", what is implied is any three-dimensional protein structure which Is thermodynamically stable In a membrane. This may be a single alpha helix, a transmembrane beta barrel, a beta-helix of gramicidin A, or any other structure. Transmembrane helices are usually about 20 amino acids in length. Typically, the transmembrane domain denotes a single transmembrane alpha helix of a transmembrane protein, also known as an Integral protein.
[620] As used herein, the term "treat;* "treatment;" or "treating" generally refers to the clinical procedure for reducing or ameliorating the progression, severity, and/or duration of a disease or of a condition, or for ameliorating one or more conditions or symptoms (preferably, one or more discernible ones) of a disease. The type of disease or condition to be treated may be, for example, but are not limited to, cancer and cancer- associated diseases and conditions. Examples of cancer include, but are not limited to, pancreatic cancer, testicular cancer, cervical cancer, endometrial cancer, ovarian cancer, stomach cancer, colorectal cancer, lung cancer, mesothelioma, and tongue cancer. In specific embodiments, the effect of the "treatment* may be evaluated by the amelioration of at least one measurable physical parameter of a disease, resulting from the administration of one or more therapies (e.g., an anti-ALPPL2 Ab or antigen-binding Ab fragment; anti-ALPPL2 ADC, or anti-ALPPL2 CAR expressing cell). The parameter may be, for example, gene expression profiles, the mass of disease-affected tissues, inflammation-associated markers, cancer-associated markers, the number or frequency of disease-associated cells, tumor burden, the presence or absence of certain cytokines or chemokines or other disease-associated molecules, and may not necessarily discernible by the patient In other embodiments "treat*, "treatment;" or "treating" may result in the inhibition of the progression of a disease, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g., stabilization of a physical parameter, or both. In other embodiments the terms "treat", "treatment" and "treating" refer to the reduction or stabilization of cancerous tissue or cells. Additionally, the terms "treat;" and "prevent” as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete cure or prevention. Rather, there are vaiying degrees of treatment effects or prevention effects of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect In this respect; the Inventive methods can provide any amount of any level of treatment or prevention effects of a disease in a mammal. Furthermore, the treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions or symptoms of the disease being treated or prevented. Also, for purposes herein, "prevention" can encompass delaying the onset of the disease, or a symptom or condition thereof.
[621] The term "xenogeneic" or "xeno-" refers to a graft derived from an animal of a different species.
[622] The experimental details of experiments are described in the following examples. These examples are offered to illustrate, but not to limits the claimed invention.
EXAMPLES
Example 1: Generation of mouse anti-ALPPL2 antibodies <Methods>
[623] Three mice were intraperitoneally immunized with 50 ug of human ALPPL2 (produced in CHO cells) admixed with complete Freund's adjuvant (CFA) as an adjuvant Two weeks later, mice were intraperitoneally boosted with 50 ug of human ALPPL2 admixed with incomplete Freund's adjuvant (IFA) as an adjuvant This boost was repeated two more times at two-week Intervals. Eight weeks post initial immunization, spleen and lymph nodes were harvested, and cells were isolated and subjected to conventional hybridoma generation methods.
[624] Hybridomas were then screened. 50 pl of hybridoma cell culture was co-cultured with 1x10s ALPPL2 positive cells or IxlO5 ALPPL2 negative cells for 1 hour at 4 °C on a multi-well plate. Each well was evaluated for ALPPL2 binding using an anti-mouse Igx antibody by flow cytometry. Antibodies produced by the selected hybridomas were sequenced using a conventional method.
<Results>
[625] Three mouse anti-ALPPL2 antibodies, 1B2, 1E8, and 3H2, were obtained. SEQ ID NOs corresponding to the VH, VL, and CDR nucleic acid sequences and amino acid sequences encoding thereof are summarized in Tables 1 and 2. The binding of the 1B2,- 1E8-, or 3H2- producing hybridoma culture to ALPPL2 positive and negative cells are provided in FIG 10.
Figure imgf000108_0001
Figure imgf000109_0001
Example 2: Humanization of mouse anti-ALPPL2 antibodies <Methods>
[626] The CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences of 1B2, 1E8, and 3H2 were grafted onto human framework sequences.
<Results>
[627] Humanized 1B2, 1E8, and 3H2 (referred to herein as hlB2, hlEB, and h3H2, respectively) were obtained. SEQ ID NOs corresponding to the VH, VL, and CDR nucleic acid sequences and amino acid sequences encoding thereof are summarized in Tables 3 and 4.
Figure imgf000109_0002
Figure imgf000109_0003
Example 3: Generation and expression of CARs <Materials>
[628] Human T cells from Donor 1. [629] Empty vector (EV) encoding trCD19.
[630] Vector encoding anti-ALPPL2 CAR (h3H2) and trCD19.
<Methods>
[631] Human T cells from Donor 1 were transduced with a vector encoding anti- ALPPL2 CAR (h3H2scFvHL-CD28H-CD28TM-CD28CS-CD3zICS) or an empty vector (EV, i.e., trCD19 only) and enriched for trCD 19-positive cells as depicted in FIG 4. The CAR constructs used to express CARs in this Example are SEQ ID NOs: 571 and 671 (amino acid sequence and nucleic acid sequence, respectively, for anti-ALPPL2 CAR "LS- h3H2scFvHL-CD28H-CD28TM-CD28CS-CD3zICS-T2A-trCD19").
<Results>
[632] Expression of the CAR was confirmed.
Example 4: In vitro cytotoxicity by anti-ALPPL2 CAR-expressing T cells <Materials>
[633] Luciferase expression vector JC73
[634] Human T cells expressing trCD19 but no anti-ALPPL2 (EV, generated as in Example 3)
[635] Human T cells expressing anti-ALPPL2 CAR1 (generated as in Example 3)
[636] Human T cells expressing anti-ALPPL2 CAR2 (generated as in Example 3)
<Methods>
[637] ALPPL2 positive cancer cells (Cal27, BxPC3, H1651, and Capan2) were transduced with luciferase expression vector, and luciferase positive cells were selected using puromycin and used as target cells. Anti-ALPPL2 CAR expressing human T cells from Example 3 were expanded and used as effector cells. T cells were plated with 5,000 luciferase-expressing ALPPL2 positive cancer cells at effector : target (E:T) ratios of 10:1, 3:1, 1:1, and 0.3:1 in 96-well light-blocking luminometer plates. After 24hrs of coculture, remaining live tumor cells were detected by luciferase activity as measured by luminescence (FIG SA, SB, SC, and 5D top). The plates were incubated an additional 24hrs to allow for a 48hr measurement (FIG SA, SB, SC, and 5D bottom).
<Results>
[638] After 24hrs and 48hrs of co-culture, a significant reduction in all ALPPL2 positive cells was observed (FIG 5). As shown in FIG 5, the reduction in cancer cells was observed with both the very low-expressor of ALPPL2 (Cal27 cells) and the high expressor pf ALPPL2 (H1692 cells).
Example 5: In vitro cytokine production by anti-ALPPL2 CAR-expressing T cells <Methods>
[639] Anti-ALPPL2 CAR expressing human T cells and EV-transduced human T cells from Example 3 were expanded. 10A5 ALPPL2 positive cancer cells (CAL27, BxPC3, H1651, Capan2, and Capanl) were cultured with 10 A5 CART cells, 10 A5 EVT cells, or no T cells per well in a 96-well plate for 24 hours. Control wells only containing 10A5 CAR T cells, or EV T cells were also included. Supernatants were collected and IFN-g levels were measured by ELISA Two independent experiments referred to as "017” and "018", respectively, were performed.
<Results>
[640] Exemplary IFN-g concentrations detected in the supernatant are shown in FIG 6. Significantly higher levels of IFN-g were observed when CAR T cells were cocultured with ALPPL2 positive cancer cells compared to when EV T cells were cocultured with ALPPL2 positive cancer cells. No IFN-g was detected from wells without a T cell.
[641]
Example 6: In vivo efficacy by anti-ALPPL2 CAR-expressingT cells
<Materials>
[642] NOD scid gamma (NSG) mice
[643] CAL27 cells transduced with luciferase (CAL27-Luc)
[644] Human T cells expressing trCD19 butno anti-ALPPL2 (EVT, generated as in Example 3)
[645] Human T cells expressing anti-ALPPL2 CAR (h3H2scFvHL-CD28H-CD28TM- CD28CS-CD3zICS CAR), generated as in Example 3
<Methods>
[646] 2.5xl06 CAL27-Luc cells were injected intraperitoneally into NSG mice on Day 0. Mice were then administered with 5xl06 human T cells expressing trCD19 but no anti- ALPPL2 (EV T) or 5xl06 human T cells expressing anti-ALPPL2 CAR (h3H2scFvHL- CD28CS CAR) intraperitoneally on Day 7. The tumor burden of individual mice was monitored by bioluminescent imaging using the Xenogen-IVIS® Imaging System every week starting on Day 6. The weight of individual mice was recorded periodically starting on Day 0.
<Results>
[647] Exemplary changes in the tumor burden observed in the two treatment groups (EV T and CAR T) are shown in FIGS 7A-7C In both groups, the tumor burden was detectable (both approximately 1.2x109 photons/sec) on Day 6. The tumor burden decreased in CAR T group by Day 13. The tumor burden difference between the CAR T group and the EVT group became significant by Day 34. As shown in FIGS 8A-8B, mice in both groups continued to gain weight during the experiment No statistically significant difference was found in the average body weight between the treatment groups.
Example 7: In vivo efficacy by anti-ALPPL2 CAR-expressingT cells <Materials>
[648] NOD scld gamma (NSG) mice [649] H1651 cells transduced with luciferase (H1651-ALPPL2-Luc)
[650] Human T cells expressing trCD19 butno anti-ALPPL2 (EVT, generated as in Example 3)
[651] Human T cells expressing anti-ALPPL2 CAR (h3H2scFvHL-CD28H-CD28TM- CD28CS-CD3zICS CAR), generated as in Example 3
<Methods>
[652] 2.5xl06 H1651-Luc cells were injected intraperitoneally into NSG mice on Day 0. Mice were then administered with 5xl06 human T cells expressing trCD19 butno anti- ALPPL2 (EV T) or 5xl06 human T cells expressing anti-ALPPL2 CAR (h3H2scFvHL- CD28CS CAR) intraperitoneally on Day 7. The tumor burden of individual mice was monitored by bioluminescent imaging using the Xenogen-IVIS® Imaging System every week starting on Day 6. The weight of individual mice was recorded periodically starting on Day 0.
<Results>
[653] Exemplary changes in the tumor burden observed in the two treatment groups (EV T and CAR T) are shown in FIGS 9A-9B. In both groups, the tumor burden was detectable (both approximately 1.2x10® photons/sec) on Day 6. The tumor diminished almost completely in CAR T group by Day 13, and the tumor burden remained extremely low at least up to Day 34. The tumor burden in CAR T group was significantly lower than that in EVT group throughout the experiment
Example 8: Affinity maturation based on Clones 3H2 and h3H2 <Methods>
[654] Random mutations were introduced to the CDR-encoding sequences of the VH and VL of h3H2 and mouse 3H2 to generate 13 different affinity maturation libraries, each having the diversity of >106. The VH and VL of mouse 3H2 were included as parent sequences at least partly because mouse 3H2 seemed to provide better binding to human ALPPL2 than h3H2, when expressed as an IgG. The libraries were transduced into cells and screened based on binding to soluble human ALPPL2 antigen using a mammalian display system. Selected clones were then expressed as antibodies and further tested for binding to human ALPPL2 antigen and to ALPPL2-expressing cells and the level of non-specific binding (binding to ALPPL2-negative cell lines). Antibodies that provide improved binding relative to mouse 3H2 were sequenced using a conventional method.
<Results>
[655] Based on the initial binding test with the soluble ALPPL2, thousands of clones were selected and sequenced. IgG antibodies from >100 clones were successfully expressed, which proceeded to screening based on binding to soluble ALPPL2 and to ALPPL2-expressing cells. Nine antibodies were particularly selected for having affinity higher than mouse 3H2, all of which comprised a VL sequence derived from mouse 3H2, rather than a VL sequence derived from h3H2. One of the antibodies with the highest binding to ALPPL2 was named IBS. The VH of IBS was found to have a V (valine) to S (serine) substitution in the CDR3, and the VL of IBS was the same as mouse 3H2. SEQ ID NOs corresponding to the VH, VL, and CDR nucleic acid sequences and amino acid sequences encoding thereof are summarized in Tables 3 and 4.
Figure imgf000113_0001
Figure imgf000113_0002
Example 9: Binding analyses of IBS IgG <Methods>
[656] 1B5 as an IgG was obtained either as supernatant of 1B5 IgG-producing cell culture or as a protein A-purified IgG.
[657] To test improvement in binding of IBS IgG relative to mouse 3H2 IgG to human ALPPL2 -expressing cells, human ALPPL2-expressing cells were incubated with different concentrations of IBS or 3H2 IgG, and unbound antibodies were removed. Binding was measured by flow cytometry, and the mean fluorescence intensity (MFI) values were compared. H1651 cells (cell line derived from human non-small cell lung cancer, adenocarcinoma) and CH0-ALPPL2 cells (CHO cells transduced with human ALPPL2) were used as ALPPL2-expressing cells, and MCF7 cells (cell line derived from human breast adenocarcinoma) and CHO cells were used as negative control cells.
[658] Binding to recombinant human ALPPL2 by IBS IgG (supernatant or purified) in comparison to mouse 3H2 IgG was analyzed using a commercially available ELISA kit Monovalent binding to recombinant human ALPPL2 by purified IBS IgG in comparison to mouse 3H2 IgG was analyzed by Biolayer Interferometry (BLI).
<Results>
[659] Exemplary results from cell binding analyses on various cell types are provided in FIG 11A. Cell binding results using CH0-ALPPL2 cells with more antibody concentrations are provided in FIG 11B. IBS IgG had higher MFI than 3H2 IgG with H1651 cells and CHO-ALPPL2 cells, indicating improved binding to ALPPL2-expressing cells.
[660] Exemplary binding results analyzed by ELISA are shown in FIG 12 (purified IgG (top) or supernatant (bottom). Exemplary monovalent binding results analyzed by BLI are provided in FIG 13, with the association rate constant (ka), equilibrium dissociation constant (ka), and dissociation constant (KD) calculated for IBS and 3H2. The calculated KD values Indicated that the variable region of IBS has an about 22-fold Increase in the affinity relative to the variable region of mouse 3H2.
Example 10: Humanization of IBS and further affinity maturation <Methods>
[661] IBS was humanized by humanizing the VL sequence. The resulting antibody, named B001, comprising the VH of SEQ ID NO: 711 (CDR1, CDR2, and CDR3 of SEQ ID NOS: 712-714) and the VL of SEQ ID NO: 715 (CDR1, CDR2, and CDR3 of SEQ ID NOS: 716-718), was found to have reduced affinity (comparison available in FIG 14 top). Therefore, further affinity maturation was performed.
[662] Random mutations were introduced to either: (i) the CDR3-encoding sequence of B001 VH, while introducing no changes to the VL of B001; or (ii) the CDR3-encoding sequence of the B001 VL, while introducing no changes to the VH of B001, to generate four affinity maturation libraries, each having the diversity of >106. The libraries were transduced into mammalian cells and screened based on binding to soluble human ALPPL2 antigen using a mammalian display system. Selected clones were then expressed as antibodies and further tested for biding to human ALPPL2 antigen and binding to ALPPL2 -expressing cells and the level of non-specific binding (binding to ALPPL2 -negative cell lines). Antibodies that provided binding at least similar to 1B5 were selected and sequenced using a conventional method.
<Results>
[663] Based on the initial binding test with soluble ALPPL2, thousands of clones were selected and sequenced. IgG antibodies from >50 clones were successfully expressed, which proceeded to screening based on binding to human ALPPL2 antigen by ELISA and to ALPPL2 -expressing cells by flowcytometry. Four antibodies, named B380, B416, B506, and B735, were particularly selected for having the binding ability similar to IBS.
[664] Exemplary ELISA results from the initial screening based on binding to platebound ALPPL2 are shown in FIG 14 top. Exemplary binding curves from three replicate ELISA experiments to confirm efficient ALPPL2 binding to plate-bound ALPPL2 by the selected antibodies are provided in FIG 14 bottom. All four antibodies showed binding (avidity) equivalent to or greater than that of IBS.
[665] All four antibodies contained one or two substitutions in the CDR3 of VH (particularly within the "AMDY" motif) relative to the VH of B001 (i.e., relative to the VH of IBS). All four antibodies comprised the VL sequence of B001 (i.e., the VH of h3H2). SEQ ID NOs corresponding to the VH, VL, and CDR amino acid sequences are summarized in Table 5. Table 6 shows % identity of the VH and VL sequences to corresponding human germline sequences for each antibody.
[666] Without wishing to be bound by theory, the CDR-H3 loop of the VHs obtained by the affinity maturation in this Example appear to pair well with humanized VL to provide efficient binding.
Table 5: SEQ ID NOs assigned to amino acid sequences for B380, B416, B506, and B735
Figure imgf000115_0001
Figure imgf000115_0002
Example 11: Binding affinity analyses on B380, B416, B506, and B735
<Methods>
[667] 1B5, B380, B416, B506, and B735 were produced as purified IgGs. Monovalent binding affinity to recombinant human ALPPL2 in solution was measured using a commercially available ELISA kit
<Results>
[668] Exemplary monovalent binding curves are shown in FIG 15. Estimated affinity
(Kd) for each antibody is provided in Table 7.
Figure imgf000115_0003
Example 12: Cell binding analyses on B380, B416, B506, and B735 <Methods>
[669] CH0-ALPPL2 cells were incubated with different concentrations of IBS, B380, B416, B506, or B735 IgG, and unbound antibodies were removed. Binding was measured by flow cytometry, and the MFI values were compared.
<Results>
[670] Exemplary results from three replicate experiments of cell binding are provided in FIG 16. All four antibodies demonstrated efficient binding to CHO-ALPPL2 cells. B416, B506, and B735 appeared to bind to CHO-ALPPL2 cells particularly efficiently.
[671] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference.
APPENDIX-1: AMINO ACID AND NUCLEIC ACID SEQUENCES
ALPPL2:
Human ALPPL2 (GenBank: AIC53992.1)
(SEQID NO: 101) Protein Sequence:
MQGPWVLLLLGLRLQLSLGIIPVEEENPDFWNRQAAEALGAAKKLQPAQTAAKNLIIF LGDGMGVSTVTAARILKGQKKDKLGPETFLAMDRFPYVALSKTYSVDKHVPDSGATA TAYLCGVKGNFQTIGLSAAARFNQCNTTRGNEVISVMNRAKKAGKSVGWTTTRVQH ASPAGAYAHTVNRNWYSDADVPASARQEGCQDIATQLISNMDIDVILGGGRKYMFPM GTPDPEYPDDYSQGGTRLDGKNLVQEWLAKRQGARYVWNRTELMQASLDPSVTHL MGLFEPGDMKYEIHRDSTLDPSLMEMTEAALRLLSRNPRGFFLFVEGGRIDHGHHES RAYRALTETIMFDDA1ERAGQLTSEEDTLSLVTADHSHVFSFGGYPLRGSSIFGLAPGK ARDRKAYTVLLYGNGPGYVLKDGARPDVTESESGSPEYRQQSAVPLDGETHAGEDVA VFARGPQAHLVHGVQEQTFIAHVMAFAACLEPYTACDLAPRAGTTDAAHPGPSWPA LLPLLAGTLLLLGTATAP
Mouse anti-AI.PPI.2 Clone 1B2:
Note: Boxed sequences correspond to CDRs (CDR 1, CDR 2, and CDR 3, in the order of appearance)
Mouse antl-ALPPL2 Clone 1B2 Heavy chain variable (also referred to as 1B2 VH)
(SEQ ID NO: 111) Protein Sequence:
EVQLQQSGPELVKPGASVKISCK|ASGYSFTGYY|MHWVKQSHVKSLEWI|GRINPYNGATO|YNR NFKDKASLTVDKSSSTAYMEFHSLTSEDSAVYYlCARVYGNYPFDYWGQGhTLTVSS
(SEQ ID NO: 211) DNA Sequence:
GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCC TGCAAGlGCfrCTGGTTACTCATTCACTGGCTACTACjATGCACTGGGTGAAGCAAAGCCATGTA AAGAGCCTTGAGTGGATllGGACGTATTAATCCTTACAATGGTGCTACTAACtrACAACCGAAA TTTCAAGGACAAGGCCAGCTTGACTGTAGATAAGTCCTCCAGCACAGCCTACATGGAGTTCCA CAGCCTGACATCTGAGGACTCTGCAGTCTATTA(jTGTGCAAGAGTATATGGTAACTACCCTTT| [TGACTACTGGGGCCAAGGdACCACTCTCACAGTCTCCTCA
Mouse anti-ALPPL2 1B2 VH CDR 1 (1B2 CDR-H1)
(SEQID NO: 112) Protein Sequence:
IASGYSFTGYYI
(SEQ ID NO: 212) DNA Sequence:
IGCTTCTGGTTACTCATTCACTGGCTACTAC
Mouse anti-ALPPL2 1B2 VH CDR 2 (1B2 CDR-H2)
(SEQID NO: 113) Protein Sequence:
IGRINPYNGATN]
(SEQID NO: 213) DNA Sequence: _
IGGACGTATTAATCCTTACAATGGTGCTACTAAC
Mouse anti-ALPPL2 1B2 VH CDR 3 (1B2 CDR-H3)
(SEQID NO: 114) Protein Sequence:
ICARVYGNYPFDYWGQGI
(SEQID NO: 214) DNA Sequence: _
ITGTGCAAGAGTATATGGTAACTACCCTTTTGACTACTGGGGCCAAGGC
Mouse anti-ALPPL2 1B2 Light chain variable (1B2 VL)
(SEQID NO: 115) Protein Sequence:
DIVMSQSPSSLAVSVGEKVTMSCKS|SQSLLYSSNQKNYjLAWYQQKPGQSPKLlJlYWAS| [TR]ESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYY
Figure imgf000118_0001
]iTFGGGTKLEIK (SEQID NO: 215) DNA Sequence:
GACATTGTGATGTCACAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTT
ACTATGAGCTGCAAGTCdAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTAC TTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTCjATTTACTGGGCAl |TCCACTAGG|GAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGAT TTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTlCA^ |CAATATTATAGCTATCCG|ACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA Mouse anti-ALPPL2 1B2 VL CDR 1 (1B2 CDR-L1) (SEQID NO: 116) Protein Sequence: |SQSLLYSSNQKNY|
(SEQ ID NO: 216) DNA Sequence: _ lAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTAC Mouse anti-ALPPL2 1B2 VL CDR 2 (1B2 CDR-L2) (SEQID NO: 117) Protein Sequence: lYWASTRl
(SEQID NO: 217) DNA Sequence: [ATTTACTGGGCATCCACTAGCj
Mouse anti-ALPPL2 1B2 VL CDR 3 (1B2 CDR-L3) (SEQID NO: 118) Protein Sequence:
|QQYYSYP|
(SEQID NO: 218) DNA Sequence: ICAGCAATATTATAGCTATCCCj
Mouse anti-ALPPL2 Clone 1F8:
Mouse anti-ALPPL2 Clone 1E8 Heavy chain variable (1E8 VH)
(SEQID NO: 121) Protein Sequence:
QVQLQQPGAELVKPGASVKMSCKA|SGYTFTSYNM|NWVKQTPGQGLEWI|GAIYPGNG| |PT^YNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYY|CARAPYGNFFDYWGQGtrT LTVSS
(SEQID NO: 221) DNA Sequence:
CAGGTGCAACTGCAGCAGCCTGGGGCTGAGCTGGTGAAGCCTGGGGCCTCAGTGAAG ATGTCCTGCAAGGCltTCTGGCTACACATTTACCAGTTACAATATGlAACTGGGTTAAG CAGACACCTGGACAGGGCCTGGAATGGATliGGAGCTATTTATCCAGGAAATGGTGATl lACTTCCtTACAATCAGAAGTTCAAAGGCAAGGCCACATTGACTGCAGACAAATCCTCC AGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACQ |GTGCAAGAGCCCCGTATGGTAATTTCTTTGACTACTGGGGCCAAGGC|ACCACTCTCAC AGTCTCCTCA
Mouse anti-ALPPL2 Clone 1E8 VH CDR 1 (1E8 CDR-H1)
(SEQID NO: 122) Protein Sequence:
ISGYTFTSYNM]
(SEQID NO: 222) DNA Sequence: _
ITCTGGCTACACATTTACCAGTTACAATAT^
Mouse anti-ALPPL2 Clone 1E8 VH CDR 2 (1E8 CDR-H2)
(SEQID NO: 123) Protein Sequence:
|GAIYPGNGDTS]
(SEQID NO: 223) DNA Sequence: _
IGGAGCTATTTATCCAGGAAATGGTGATACTTCC
Mouse anti-ALPPL2 Clone 1E8 VH CDR 3 (1E8 CDR-H3)
(SEQID NO: 124) Protein Sequence:
ICARAPYGNFFDYWGQGl
(SEQID NO: 224) DNA Sequence: _ iTGTGCAAGAGCCCCGTATGGTAATTTCTTTGACTACTGGGGCCAAGGd Mouse anti-ALPPL2 Clone 1E8 Light chain variable (1E8 VL) (SEQID NO: 125) Protein Sequence: DIVMTQSHKFMSTSVGDRVSITCKAlSQDVSfAVjAWYQQKPGQSPKLljYWASTR|HTG VPDRFTGSGSGTDYTLIISSVQAEDLALYYClQQHYS'llHTFGGGTKLEIK (SEQID NO: 225) DNA Sequence: GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTC
AGCATCACCTGCAAGGCdAGTCAGGATGTGAGTACTGCTGf^GCCTGGTATCAACAA AAACCAGGGCAATCTCCTAAACTACTGjATTTACTGGGCATCCACCCGdCACACTGGA GTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTATACTCTCATTATCAGCA GTGTGCAGGCTGAAGACCTGGCACTTTATTACTG'llCAGCAACATTATAGCAC'llCACA CGTTCGGAGGGGGGACCAAGCTGGAAATAAAA Mouse anti-ALPPL2 Clone 1E8 VL CDR 1 (1E8 CDR-L1) (SEQID NO: 126) Protein Sequence:
[SQDVSTAVl
(SEQ ID NO: 226) DNA Sequence:
[AGTCAGGATGTGAGTACTGCTGTA
Mouse anti-ALPPL2 Clone 1E8 VL CDR 2 (1E8 CDR-L2)
(SEQID NO: 127) Protein Sequence: lYWASTRl
(SEQID NO: 227) DNA Sequence: lATTTACTGGGCATCCACCCG^
Mouse anti-ALPPL2 Clone 1E8 VL CDR 3 (1E8 CDR-L3) (SEQID NO: 128) Protein Sequence:
(SEQID NO: 228) DNA Sequence: ICAGCAACATTATAGCACll
Mouse anti-ALPPL2 Clone 3H2:
Mouse anti-ALPPL2 Clone 3H2 Heavy chain variable (3H2 VH)
(SEQID NO: 131) Protein Sequence:
QVTLKESGPGILQPSQTLSLTCSF|SGFSLSTSGMG|VSWIRQPSGKGLEWLA|HIYWDDD YNPSLKSRLTISKDTSSNQVFLKITSVDTADTATYYlCARRPITTWAPYFYAMDYW GTSVTVSS
(SEQID NO: 231) DNA Sequence:
CAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATTGCAGCCCTCCCAGACCCTCAGTC TGACTTGTTCTTTCtrCTGGGTTTTCACTGAGCACTTCTGGTATGGGTlGTGAGCTGGA TTCGTCAGCCTTCAGGAAAGGGTCTGGAGTGGCTGGCA|CACATTTACTGGGATGATG| lACAAGCGdTATAACCCATCCCTGAAGAGCCGGCTCACAATCTCCAAGGATACCTCCAG CAACCAGGTATTCCTCAAGATCACCAGTGTGGACACTGCAGATACTGCCACATACTAC TGTGCTCGAAGACCTATTACTACGGTAGTAGCTCCATATTTCTATGCTATGGACTAC TGGGGTCAA|GGAACCTCAGTCACCGTCTCCTCA
Mouse anti-ALPPL2 Clone 3H2 VH CDR 1 (3H2 CDR-H1)
(SEQID NO: 132) Protein Sequence:
|SGFSLSTSGMCj
(SEQID NO: 232) DNA Sequence: _
|TCTGGGTTTTCACTGAGCACTTCTGGTATGGGT|
Mouse anti-ALPPL2 Clone 3H2 VH CDR 2 (3H2 CDR-H2)
(SEQID NO: 133) Protein Sequence:
HIYWDDDKR]
(SEQ ID NO: 233) DNA Sequence:
ICACATTTACTGGGATGATGACAAGCGC
Mouse anti-ALPPL2 Clone 3H2 VH CDR 3 (3H2 CDR-H3)
(SEQID NO: 134) Protein Sequence:
ICARRPITTWAPYFYAMDYWGQl
(SEQID NO: 234) DNA Sequence: _
ITGTGCTCGAAGACCTATTACTACGGTAGTAGCTCCATATTTCTATGCTATGGACTACI ITGGGGTCAAI
Mouse anti-ALPPL2 Clone 3H2 Light drain variable (3H2 VL)
(SEQID NO: 135) Protein Sequence:
DIVMTQSQKFMSTSVGDRVSVTCKA|SQNVATljVAWYQQKPGQSPKALl|YSASY|RYSG VPDRFTGSGSGTDFTLTISNVQSEDLAEYFdQQYNSYP|FTFGSGTKLEIK
(SEQID NO: 235) DNA Sequence:
GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTC AGCGTCACCTGCAAGGCdAGTCAGAATGTGGCTACTACgGTGGCCTGGTATCAACAG AAACCAGGGCAATCTCCTAAAGCACTGATltTACTCGGCATCCTAClCGGTACAGTGGA GTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCA ATGTGCAGTCTGAAGACTTGGCAGAGTATTTCTGTlCAGCAATATAACAGCTATCCAh’ TCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA Mouse anti-ALPPL2 Clone 3H2 VL CDR 1 (3H2 CDR-L1) (SEQID NO: 136) Protein Sequence:
|SQNVATT|
(SEQ ID NO: 236) DNA Sequence: lAGTCAGAATGTGGCTACTACT
Mouse anti-ALPPL2 Clone 3H2 VL CDR 2 (3H2 CDR-L2) (SEQID NO: 137) Protein Sequence: lYSASYj
(SEQID NO: 237) DNA Sequence: iTACTCGGCATCCTAd
Mouse anti-ALPPL2 Clone 3H2 VL CDR 3 (3H2 CDR-L3) (SEQID NO: 138) Protein Sequence:
(SEQID NO: 238) DNA Sequence: ICAGCAATATAACAGCTATCCA
Humanized anti-ALPPL2 Clone 1R2 fh1H2):
Note: Boxed sequences correspond to CDRs (CDR 1, CDR 2, and CDR 3, in the order of appearance)
Humanized anti-ALPPL2 Clone 1B2 Heavy chain variable (hlB2 VH)
(SEQID NO: 311) Protein Sequence:
QVQLVQSGAEVKKPGASVKVSCK|ASGYSFTGYY|MHWVKQAPGQGLEWM|GRINPYNG| IAT^YNQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYICARVYGNYPFDYWGQGIT TLTVSS
(SEQID NO: 411) DNA Sequence:
CAGGTCCAGCTGGTACAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCTTCAGTGAAG GTATCCTGCAAGfGCTTCTGGTTACTCATTCACTGGCTACTA^ATGCACTGGGTGAAG CAAGCCCCTGGACAGGGCCTTGAGTGGATGlGGACGTATTAATCCTTACAATGGTGC'll lACTAACtTACAACCAAAAATTCCAGGGCAGGGTCACCATGACTAGAGATACGTCCATC AGCACAGCCTACATGGAGCTCAGCAGGCTGAGATCTGACGACACTGCAGTCTATTAC TGTGCAAGAGTATATGGTAACTACCCTTTTGACTACTGGGGCCAAGGdACCACTCTC ACAGTCTCCTCA
Humanized anti-ALPPL2 Clone 1B2 VH CDR 1 (hlB2 CDR-H1)
(SEQID NO: 312) Protein Sequence:
[ASGYSFTGYYl
(SEQID NO: 412) DNA Sequence: _
IGCTTCTGGTTACTCATTCACTGGCTACTAC Humanized anti-ALPPL2 Clone 1B2 VH CDR 2 (hlB2 CDR-H2) (SEQID NO: 313) Protein Sequence:
|GRINPYNGATN|
(SEQID NO: 413) DNA Sequence: _ iGGACGTATTAATCCTTACAATGGTGCTACTAACl Humanized anti-ALPPL2 Clone 1B2 VH CDR 3 (hlB2 CDR-H3) (SEQID NO: 314) Protein Sequence:
ICARVYGNYPFDYWGQGI
(SEQID NO: 414) DNA Sequence: _
ITGTGCAAGAGTATATGGTAACTACCCTTTTGACTACTGGGGCCAAGGC Humanized anti-ALPPL2 Clone 1B2 Light chain variable (hlB2 VL) (SEQID NO: 315) Protein Sequence:
Figure imgf000124_0001
(SEQID NO: 415) DNA Sequence: GACATTGTGATGACACAGTCTCCAGACTCCCTAGCTGTGTCACTTGGAGAGAGGGCT
ACTATCAACTGCAAGTCdAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTAC TTGGCCTGGTTCCAGCAGAAACCAGGGCAGCCTCCTAAACTGCTG|ATTTACTGGGCA| TCCACTAGGIGAATCTGGGGTCCCTGATCGCTTCAGTGGCAGTGGATCTGGGACAGAT TTCACTCTCACCATCAGCAGTCTGCAGGCTGAAGACGTGGCAGTTTATTACTGTlCAGd |AATATTATAGCACTCCG|ACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA Humanized anti-ALPPL2 Clone 1B2 VL CDR 1 (H1B2 CDR-L1) (SEQ ID NO: 316) Protein Sequence:
ISQSLLYSSNQKNY]
(SEQ ID NO: 416) DNA Sequence: _ lAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTAd Humanized anti-ALPPL2 Clone 1B2 VL CDR 2 (H1B2 CDR-L2) (SEQ ID NO: 317) Protein Sequence: lYWASTRl
(SEQ ID NO: 417) DNA Sequence: lATTTACTGGGCATCCACTAGd Humanized anti-ALPPL2 Clone 1B2 VL CDR 3 (H1B2 CDR-L3) (SEQ ID NO: 318) Protein Sequence: |QQYYSTP|
(SEQ ID NO: 418) DNA Sequence: ICAGCAATATTATAGCACTCCd Humanized anti-ALPPL2 1B2 scFv VH-G4S3-VL (H1B2 scFv HL, or HlB2scFvHL) (SEQ ID NO: 319) Protein Sequence: QVQLVQSGAEVKKPGASVKVSCK1ASGYSFTGYY|MHWVKQAPGQGLEWM|GRINPYNG| [ATN|YNQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYY|CARVYGNYPFDYWGQG|T TLTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSlSQSLLYSSNQKNYl LAWFQQKPGQPPKLL|IYWASTR]ESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC|Q^ lYYSTPtTFGGGTKLEIK
(SEQ ID NO: 419) DNA Sequence:
CAGGTCCAGCTGGTACAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCTTCAGTGAAG GTATCCTGCAAG|GCTTCTGGTTACTCATTCACTGGCTACTAC|ATGCACTGGGTGAAG CAAGCCCCTGGACAGGGCCTTGAGTGGATGlGGACGTATTAATCCTTACAATGGTGC'll ^CTAA^TACAACCAAAAATTCCAGGGCAGGGTCACCATGACTAGAGATACGTCCATC AGCACAGCCTACATGGAGCTCAGCAGGCTGAGATCTGACGACACTGCAGTCTATTAC TGTGCAAGAGTATATGGTAACTACCCTTTTGACTACTGGGGCCAAGGdACCACTCTC ACAGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCCG ACATTGTGATGACACAGTCTCCAGACTCCCTAGCTGTGTCACTTGGAGAGAGGGCTA CTATCAACTGCAAGTCdAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTAC|T TGGCCTGGTTCCAGCAGAAACCAGGGCAGCCTCCTAAACTGCTGlATTTACTGGGCA'll |CCACTAGG|GAATCTGGGGTCCCTGATCGCTTCAGTGGCAGTGGATCTGGGACAGATT TCACTCTCACCATCAGCAGTCTGCAGGCTGAAGACGTGGCAGTTTATTACTGl|CAGC| |AATATTATAGCACTCCG|ACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID NO: 19) Alternative DNA Sequence:
CAGGTCCAGCTGGTACAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCTTCAGTGAAG GTATCCTGCAAGlGCTTCTGGTTACTCATTCACTGGCTACTAdATGCACTGGGTGAAG CAAGCCCCTGGACAGGGCCTTGAGTGGATGlGGACGTATTAATCCTTACAATGGTGCT lACTAACtTACAACCAAAAATTCCAGGGCAGGGTCACCATGACTAGAGATACGTCCATC AGCACAGCCTACATGGAGCTCAGCAGGCTGAGATCTGACGACACTGCAGTCTATTAC TGTGCAAGAGTATATGGTAACTACCCTTTTGACTACTGGGGCCAAGGdACCACTCTC ACAGTCTCCTCAGGTGGAGGAGGTTCTGGAGGCGGTGGGTCCGGGGGAGGTGGCTCC GACATTGTGATGACACAGTCTCCAGACTCCCTAGCTGTGTCACTTGGAGAGAGGGCT ACTATCAACTGCAAGTCC|AGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTAC TTGGCCTGGTTCCAGCAGAAACCAGGGCAGCCTCCTAAACTGCTG|ATTTACTGGGCA| |TCCACTAGG|GAATCTGGGGTCCCTGATCGCTTCAGTGGCAGTGGATCTGGGACAGAT TTCACTCTCACCATCAGCAGTCTGCAGGCTGAAGACGTGGCAGTTTATTACTG’llCAGd |AATATTATAGCACTCCG|ACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA Humanized anti-ALPPL2 1B2 scFv VL-G4S3-VH (H1B2 scFv LH, or HlB2scFvLH) (SEQID NO: 320) Protein Sequence: DIVMTQSPDSLAVSLGERATINCKS|SQSLLYSSNQKNY|LAWFQQKPGQPPKLL|IYWAS| &SGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC|QQYYSTP|TFGGGTKLEIKGGGGS GGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCK1ASGYSFTGYY1MHWVKQAPGQGLE WM|GRINPYNGATN|YNQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYY|CARVYGN| lYPFDYWGQCjTTLTVSS
(SEQID NO: 420) DNA Sequence: GACATTGTGATGACACAGTCTCCAGACTCCCTAGCTGTGTCACTTGGAGAGAGGGCT ACTATCAACTGCAAGTCdAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTAC TTGGCCTGGTTCCAGCAGAAACCAGGGCAGCCTCCTAAACTGCTGIATTTACTGGGCAI |TCCACTAGG|GAATCTGGGGTCCCTGATCGCTTCAGTGGCAGTGGATCTGGGACAGAT TTCACTCTCACCATCAGCAGTCTGCAGGCTGAAGACGTGGCAGTTTATTACTGT|CAGCj lAATATTATAGCACTCCGlACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAGGTGGTG GTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCCCAGGTCCAGCTGGTACAGTC TGGAGCTGAGGTGAAGAAGCCTGGGGCTTCAGTGAAGGTATCCTGCAAG|GCTTCTGG| TTACTCATTCACTGGCTACfAdATGCACTGGGTGAAGCAAGCCCCTGGACAGGGCCTT GAGTGGATGfcGACGTATTAATCCTTACAATGGTGCTACTAA^rACAACCAAAAATTC CAGGGCAGGGTCACCATGACTAGAGATACGTCCATCAGCACAGCCTACATGGAGCTC AGCAGGCTGAGATCTGACGACACTGCAGTCTATTACfrGTGCAAGAGTATATGGTAAC TACCCTTTTGACTACTGGGGCCAAGGClACCACTCTCACAGTCTCCTCA (SEQID NO: 20) Alternative DNA Sequence: GACATTGTGATGACACAGTCTCCAGACTCCCTAGCTGTGTCACTTGGAGAGAGGGCT ACTATCAACTGCAAGTCC|AGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTAC TTGGCCTGGTTCCAGCAGAAACCAGGGCAGCCTCCTAAACTGCTGIATTTACTGGGCAI |TCCACTAGG|GAATCTGGGGTCCCTGATCGCTTCAGTGGCAGTGGATCTGGGACAGAT TTCACTCTCACCATCAGCAGTCTGCAGGCTGAAGACGTGGCAGTTTATTACTG’llCAGCl |AATATTATAGCACTCCG|ACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAGGTGGAG GAGGTTCTGGAGGCGGTGGGTCCGGGGGAGGTGGCTCCCAGGTCCAGCTGGTACAGT CTGGAGCTGAGGTGAAGAAGCCTGGGGCTTCAGTGAAGGTATCCTGCAAGlGCTTCTGl GTTACTCATTCACTGGCTACTAdATGCACTGGGTGAAGCAAGCCCCTGGACAGGGCC TTGAGTGGATGlGGACGTATTAATCCTTACAATGGTGCTACTAACtrACAACCAAAAAT TCCAGGGCAGGGTCACCATGACTAGAGATACGTCCATCAGCACAGCCTACATGGAGC
TCAGCAGGCTGAGATCTGACGACACTGCAGTCTATTAdTGTGCAAGAGTATATGGTA ACTACCCTTTTGACTACTGGGGCCAAGGdACCACTCTCACAGTCTCCTCA
Humanized anti-ALPPL2 Clone 1E8 Qi IE 81:
Note: Boxed sequences correspond to CDRs (CDR 1, CDR 2, and CDR 3, in the order of appearance)
Humanized anti-ALPPL2 Clone 1E8 Heavy chain variable (hlE8 VH)
(SEQID NO: 321) Protein Sequence:
QVQLVQSGAEVKKPGASVKVSCKA|SGYTFTSYNM|NWVKQAPGQGLEWM|GAIYPGNG| [PT^NQKFQGRVTMTADTSTSTVYMELSSLRSEDTAVYY|CARAPYGNFFDYWGQG|T TLTVSS
(SEQID NO: 421) DNA Sequence:
CAGGTGCAACTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAG GTGTCCTGCAAGGCT|TCTGGCTACACATTTACCAGTTACAATATCjAACTGGGTTAAG CAGGCACCTGGACAGGGCCTGGAATGGATGlGGAGCTATTTATCCAGGAAATGGTGA'll lACTTCCtTACAATCAGAAGTTCCAAGGCAGGGTCACAATGACTGCAGACACATCCACC AGCACAGTCTACATGGAGCTCAGCAGCCTGCGATCTGAGGACACTGCGGTCTATTAC TGTGCAAGAGCCCCGTATGGTAATTTCTTTGACTACTGGGGCCAAGGdACCACTCTC ACAGTCTCCTCA
Humanized anti-ALPPL2 Clone 1E8 VH CDR 1 (hlE8 CDR-H1)
(SEQID NO: 322) Protein Sequence:
ISGYTFTSYNM]
(SEQID NO: 422) DNA Sequence: _
IlCTGGCTACACATTTACCAGTTACAATAf^ Humanized anti-ALPPL2 Clone 1E8 VH CDR 2 (hlE8 CDR-H2) (SEQID NO: 323) Protein Sequence: iGAIYPGNGDTSj
(SEQID NO: 423) DNA Sequence: _
IGGAGCTATTTATCCAGGAAATGGTGATACTTCC Humanized anti-ALPPL2 Clone 1E8 VH CDR 3 (hlE8 CDR-H3) (SEQID NO: 324) Protein Sequence:
ICARAPYGNFFDYWGQGI
(SEQID NO: 424) DNA Sequence: _
ITGTGCAAGAGCCCCGTATGGTAATTTCTTTGACTACTGGGGCCAAGGC Humanized anti-ALPPL2 Clone 1E8 Light chain variable (hlE8 VL) (SEQID NO: 325) Protein Sequence: DIVMTQSPDSLAVSLGERATINCKA|SQDVSTAV|AWYQQKPGQPPKLL|iYWASTR|ESGV PDRFSGSGSGTDFTLTISSLQAEDVAVYYClQQYYS'llHTFGGGTKLEIK (SEQID NO: 425) DNA Sequence: GACATTGTGATGACCCAGTCTCCCGACAGCCTGGCCGTATCACTAGGAGAAAGGGCC ACCATCAACTGCAAGGCC|AGTCAGGATGTGAGTACTGCTGTA|GCCTGGTATCAACAA AAACCAGGGCAACCTCCTAAACTACTG|ATTTACTGGGCATCCACCCGdGAAAGTGGA
GTCCCTGATCGCTTCAGCGGCAGTGGATCTGGGACAGATTTTACTCTCACTATCAGCA GTTTGCAGGCTGAAGACGTGGCAGTTTATTACTGljCAGCAATATTATAGCAClICACA CGTTCGGAGGGGGGACCAAGCTGGAAATAAAA Humanized anti-ALPPL2 Clone 1E8 VL CDR 1 (H1E8 CDR-L1) (SEQID NO: 326) Protein Sequence: SQDVSTAV
(SEQ ID NO: 426) DNA Sequence:
[AGTCAGGATGTGAGTACTGCTGTA
Humanized anti-ALPPL2 Clone 1E8 VL CDR 2 (H1E8 CDR-L2)
(SEQID NO: 327) Protein Sequence: lYWASTR
(SEQID NO: 427) DNA Sequence: lATTTACTGGGCATCCACCCG^ Humanized anti-ALPPL2 Clone 1E8 VL CDR 3 (H1E8 CDR-L3) (SEQID NO: 328) Protein Sequence: QQYYST
(SEQID NO: 428) DNA Sequence: CAGCAATATTATAGCACT
Humanized anti-ALPPL2 1E8 scFv VH-G4S3-VL (hlE8 scFv HL, or hlEBscFvHL) (SEQID NO: 329) Protein Sequence: QVQLVQSGAEVKKPGASVKVSCKA|SGYTFTSYNM|NWVKQAPGQGLEWM|GAIYPGNG| |DT^yNQKFQGRVTMTADTSTSTVYMELSSLRSEDTAVYY|CARAPYGNFFDYWGQG|T TLTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKA|SQDVSTAV|AWYQ QKPGQPPKLL|lYWASTR|ESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC|QQYYS’llHT FGGGTKLEIK
(SEQID NO: 429) DNA Sequence: CAGGTGCAACTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAG GTGTCCTGCAAGGCTfTCTGGCTACACATTTACCAGTTACAATATGjAACTGGGTTAAG CAGGCACCTGGACAGGGCCTGGAATGGATGlGGAGCTATTTATCCAGGAAATGGTGA'll lACTTCCtTACAATCAGAAGTTCCAAGGCAGGGTCACAATGACTGCAGACACATCCACC AGCACAGTCTACATGGAGCTCAGCAGCCTGCGATCTGAGGACACTGCGGTCTATTAC TGTGCAAGAGCCCCGTATGGTAATTTCTTTGACTACTGGGGCCAAGGCIACCACTCTC ACAGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCCG ACATTGTGATGACCCAGTCTCCCGACAGCCTGGCCGTATCACTAGGAGAAAGGGCCAC CATCAACTGCAAGGC0AGTCAGGATGTGAGTACTGCTGT^GCCTGGTATCAACAAAA ACCAGGGCAACCTCCTAAACTACTG|ATTTACTGGGCATCCACCCGG|GAAAGTGGAGT CCCTGATCGCTTCAGCGGCAGTGGATCTGGGACAGATTTTACTCTCACTATCAGCAGT TTGCAGGCTGAAGACGTGGCAGTTTATTACTGT|CAGCAATATTATAGCACljCACACG TTCGGAGGGGGGACCAAGCTGGAAATAAAA
(SEQID NO: 29) Alternative DNA Sequence: CAGGTGCAACTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAG GTGTCCTGCAAGGCT|TCTGGCTACACATTTACCAGTTACAATATCjAACTGGGTTAAG CAGGCACCTGGACAGGGCCTGGAATGGATGlGGAGCTATTTATCCAGGAAATGGTGATl lACTTCdTACAATCAGAAGTTCCAAGGCAGGGTCACAATGACTGCAGACACATCCACC AGCACAGTCTACATGGAGCTCAGCAGCCTGCGATCTGAGGACACTGCGGTCTATTAC TGTGCAAGAGCCCCGTATGGTAATTTCTTTGACTACTGGGGCCAAGGCIACCACTCTC ACAGTCTCCTCAGGTGGAGGAGGTTCTGGAGGCGGTGGGTCCGGGGGAGGTGGCTCC GACATTGTGATGACCCAGTCTCCCGACAGCCTGGCCGTATCACTAGGAGAAAGGGCC ACCATCAACTGCAAGGCC|AGTCAGGATGTGAGTACTGCTGTA|GCCTGGTATCAACAA AAACCAGGGCAACCTCCTAAACTACTGjATTTACTGGGCATCCACCCGCjGAAAGTGGA GTCCCTGATCGCTTCAGCGGCAGTGGATCTGGGACAGATTTTACTCTCACTATCAGCA GTTTGCAGGCTGAAGACGTGGCAGTTTATTACTGljCAGCAATATTATAGCACTlCACA CGTTCGGAGGGGGGACCAAGCTGGAAATAAAA
Humanized anti-ALPPL2 1E8 scFv VL-G4S3-VH (H1E8 scFv LH, or HlESscFvLH) (SEQID NO: 330) Protein Sequence: DIVMTQSPDSLAVSLGERATINCKA|SQDVSTAVjAWYOQKPGOPPKLL|iYWASTR|ESGV PDRFSGSGSGTDFTLTISSLQAEDVAVYYC|QQYYSTjHTFGGGTKLEIKGGGGSGGGGSG GGGSQVQLVQSGAEVKKPGASVKVSCKA^GYTFTSYNM|NWVKQAPGQGLEWM|GAIY| |PGNGDTStYNQKFQGRVTMTADTSTSTVYMELSSLRSEDTAVYY|CARAPYGNFFDYWj (GQ^TTLTVSS
(SEQID NO: 430) DNA Sequence:
GACATTGTGATGACCCAGTCTCCCGACAGCCTGGCCGTATCACTAGGAGAAAGGGCC ACCATCAACTGCAAGGCC|AGTCAGGATGTGAGTACTGCTGTA|GCCTGGTATCAACAA AAACCAGGGCAACCTCCTAAACTACTGjATTTACTGGGCATCCACCCGCjGAAAGTGGA GTCCCTGATCGCTTCAGCGGCAGTGGATCTGGGACAGATTTTACTCTCACTATCAGCA GTTTGCAGGCTGAAGACGTGGCAGTTTATTACTGljCAGCAATATTATAGCACT|CACA CGTTCGGAGGGGGGACCAAGCTGGAAATAAAAGGTGGTGGTGGTTCTGGCGGCGGCG GCTCCGGTGGTGGTGGTTCCCAGGTGCAACTGGTGCAGTCTGGGGCTGAGGTGAAGA AGCCTGGGGCCTCAGTGAAGGTGTCCTGCAAGGCltTCTGGCTACACATTTACCAGTlI |ACAATATG|AACTGGGTTAAGCAGGCACCTGGACAGGGCCTGGAATGGATG|GGAGCTA| |TTTATCCAGGAAATGGTGATACTTCC|TACAATCAGAAGTTCCAAGGCAGGGTCACAA TGACTGCAGACACATCCACCAGCACAGTCTACATGGAGCTCAGCAGCCTGCGATCTGA GGACACTGCGGTCTATTACfrGTGCAAGAGCCCCGTATGGTAATTTCTTTGACTACTGl IGGGCCAAGG^ACCACTCTCACAGTCTCCTCA (SEQID NO: 30) Alternative DNA Sequence:
GACATTGTGATGACCCAGTCTCCCGACAGCCTGGCCGTATCACTAGGAGAAAGGGCC ACCATCAACTGCAAGGCdAGTCAGGATGTGAGTACTGCTGT^GCCTGGTATCAACAA AAACCAGGGCAACCTCCTAAACTACTGjATTTACTGGGCATCCACCCGCjGAAAGTGGA GTCCCTGATCGCTTCAGCGGCAGTGGATCTGGGACAGATTTTACTCTCACTATCAGCA GTTTGCAGGCTGAAGACGTGGCAGTTTATTACTGljCAGCAATATTATAGCACTlCACA CGTTCGGAGGGGGGACCAAGCTGGAAATAAAAGGTGGAGGAGGTTCTGGAGGCGGTG GGTCCGGGGGAGGTGGCTCCCAGGTGCAACTGGTGCAGTCTGGGGCTGAGGTGAAGA AGCCTGGGGCCTCAGTGAAGGTGTCCTGCAAGGClfrCTGGCTACACATTTACCAGTlj |ACAATATG|AACTGGGTTAAGCAGGCACCTGGACAGGGCCTGGAATGGATG|GGAGCTA| iTTTATCCAGGAAATGGTGATACTTCdTACAATCAGAAGTTCCAAGGCAGGGTCACAA TGACTGCAGACACATCCACCAGCACAGTCTACATGGAGCTCAGCAGCCTGCGATCTGA GGACACTGCGGTCTATTAqrGTGCAAGAGCCCCGTATGGTAATTTCTTTGACTACTGl
GGGCCAAGG^ACCACTCTCACAGTCTCCTCA
Humanized anti-ALPPL2 Clone 3H2 (h3H2):
Note: Boxed sequences correspond to CDRs (CDR 1, CDR 2, and CDR 3, in the order of appearance)
Humanized anti-ALPPL2 Clone 3H2 Heavy chain variable (h3H2 VH)
(SEQID NO: 331) Protein Sequence:
QVTLKESGPALVKPTQTLTLTCTF|SGFSLSTSGMG|VSWIRQPPGKALEWLA|HIYWDD| |DKgYNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTWAPYFYAMD| IYWGQIGTSVTVSS
(SEQID NO: 431) DNA Sequence:
CAGGTTACTCTGAAAGAGTCTGGCCCTGCGCTAGTGAAGCCCACCCAGACCCTCACTC TGACTTGTACTTTCh’CTGGGTTTTCACTGAGCACTTCTGGTATGGG'ljGTGAGCTGGA TTCGTCAGCCTCCAGGAAAGGCTCTGGAGTGGCTGGCA|CACATTTACTGGGATGATG| |ACAAGCGC|TATAACCCATCCCTGAAGAGCCGGCTCACAATCACCAAGGATACCTCCAA GAACCAGGTAGTCCTCACGATGACCAATATGGACCCTGTAGATACTGCCACATACTA CfrGTGCTCGAAGACCTATTACTACGGTAGTAGCTCCATATTTCTATGCTATGGACTA ICTGGGGTCAAIGGAACCTCAGTCACCGTCTCCTCA
Humanized anti-ALPPL2 Clone 3H2 VH CDR 1 (h3H2 CDR-H1)
(SEQID NO: 332) Protein Sequence:
[SGFSLSTSGM
(SEQID NO: 432) DNA Sequence: _
IlCTGGGTTTTCACTGAGCACTTCTGGTATGGGT
Humanized anti-ALPPL2 Clone 3H2 VH CDR 2 (h3H2 CDR-H2)
(SEQID NO: 333) Protein Sequence:
HIYWDDDKRI
(SEQ ID NO: 433) DNA Sequence:
ICACATTTACTGGGATGATGACAAGCGC
Humanized anti-ALPPL2 Clone 3H2 VH CDR 3 (h3H2 CDR-H3)
(SEQID NO: 334) Protein Sequence:
ICARRPITTWAPYFYAMDYWGQI
(SEQID NO: 434) DNA Sequence: _
ITGTGCTCGAAGACCTATTACTACGGTAGTAGCTCCATATTTCTATGCTATGGACTAC iTGGGGTCAAl
Humanized anti-ALPPL2 Clone 3H2 Light chain variable (h3H2 VL)
(SEQID NO: 335) Protein Sequence:
DIQMTQSPSSLSASVGDRVTITCKA|SQNVATTjVAWFQQKPGKAPKSLl|YSASY|LQSGVP SRFSGSGSGTDFTLTISSLQPEDFATYYdQQYNSYPlFTFGSGTKLEIK
(SEQID NO: 435) DNA Sequence:
GACATTCAGATGACCCAGTCTCCAAGCTCCCTGTCCGCATCAGTAGGAGACAGGGTCA CCATCACCTGCAAGGCCjAGTCAGAATGTGGCTACTACTjGTGGCCTGGTTTCAACAGA AACCAGGGAAAGCTCCTAAAAGCCTGATT|TACAGCGCATCCTAC|TTGCAGAGTGGAG TCCCTAGTCGCTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAG TCTGCAGCCTGAAGACTTTGCAACGTATTACTGTlCAGCAATATAACAGCTATCCAh’T
CACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA
Humanized anti-ALPPL2 Clone 3H2 VL CDR 1 (H3H2 CDR-L1)
(SEQ ID NO: 336) Protein Sequence:
ISQNVATTI
(SEQID NO: 436) DNA Sequence:
IAGTCAGAATGTGGCTACTACTI
Humanized anti-ALPPL2 Clone 3H2 VL CDR 2 (H3H2 CDR-L2)
(SEQID NO: 337) Protein Sequence:
IYSASYI
(SEQID NO: 437) DNA Sequence: iTACAGCGCATCCTAd
Humanized anti-ALPPL2 Clone 3H2 VL CDR 3 (H3H2 CDR-L3)
(SEQID NO: 338) Protein Sequence:
(SEQID NO: 438) DNA Sequence:
|CAGCAATATAACAGCTATCCA
Humanized anti-ALPPL2 3H2 scFv VH-G4S3-VL (h3H2 scFvHL, or H3H2scFvHL) (SEQID NO: 339) Protein Sequence:
QVTLKESGPALVKPTQTLTLTCTF|SGFSLSTSGMG|VSWIRQPPGKALEWLA|HIYWDD| |DKgYNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTWAPYFYAMD| |YWGQ|GTSVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKA|SQNVA'l1 @VAWFQQKPGKAPKSLI|YSASY|LQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC|Q^ |YNSYP|FTFGSGTKLEIK
(SEQID NO: 439) DNA Sequence:
CAGGTTACTCTGAAAGAGTCTGGCCCTGCGCTAGTGAAGCCCACCCAGACCCTCACTC TGACTTGTACTTTCfrCTGGGTTTTCACTGAGCACTTCTGGTATGGGllGTGAGCTGGA TTCGTCAGCCTCCAGGAAAGGCTCTGGAGTGGCTGGCA|CACATTTACTGGGATGATG| |ACAAGCGC|TATAACCCATCCCTGAAGAGCCGGCTCACAATCACCAAGGATACCTCCAA GAACCAGGTAGTCCTCACGATGACCAATATGGACCCTGTAGATACTGCCACATACTA CtTGTGCTCGAAGACCTATTACTACGGTAGTAGCTCCATATTTCTATGCTATGGACTA ICTGGGGTCAAIGGAACCTCAGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGC GGCTCCGGTGGTGGTGGTTCCGACATTCAGATGACCCAGTCTCCAAGCTCCCTGTCCG
CATCAGTAGGAGACAGGGTCACCATCACCTGCAAGGC(jAGTCAGAATGTGGCTACTA| ^GTGGCCTGGTTTCAACAGAAACCAGGGAAAGCTCCTAAAAGCCTGATTfrACAGCGl ICATCCTAdTTGCAGAGTGGAGTCCCTAGTCGCTTCAGCGGCAGTGGATCTGGGACAG ATTTCACTCTCACCATCAGCAGTCTGCAGCCTGAAGACTTTGCAACGTATTACTG'IQ iGCAATATAACAGCTATCCAh’TCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA (SEQID NO: 39) Alternative DNA Sequence:
CAGGTTACTCTGAAAGAGTCTGGCCCTGCGCTAGTGAAGCCCACCCAGACCCTCACTC TGACTTGTACTTTCh'CTGGGTTTTCACTGAGCACTTCTGGTATGGG’ljGTGAGCTGGA TTCGTCAGCCTCCAGGAAAGGCTCTGGAGTGGCTGGCA|CACATTTACTGGGATGATG| [ACAAGCGyTATAACCCATCCCTGAAGAGCCGGCTCACAATCACCAAGGATACCTCCAA GAACCAGGTAGTCCTCACGATGACCAATATGGACCCTGTAGATACTGCCACATACTA CtTGTGCTCGAAGACCTATTACTACGGTAGTAGCTCCATATTTCTATGCTATGGACTA ICTGGGGTCAAIGGAACCTCAGTCACCGTCTCCTCAGGTGGAGGAGGTTCTGGAGGCGG TGGGTCCGGGGGAGGTGGCTCCGACATTCAGATGACCCAGTCTCCAAGCTCCCTGTCC GCATCAGTAGGAGACAGGGTCACCATCACCTGCAAGGCC|AGTCAGAATGTGGCTACT| lACT^GTGGCCTGGTTTCAACAGAAACCAGGGAAAGCTCCTAAAAGCCTGATTtTACAGCl |GCATCCTAdTTGCAGAGTGGAGTCCCTAGTCGCTTCAGCGGCAGTGGATCTGGGACA GATTTCACTCTCACCATCAGCAGTCTGCAGCCTGAAGACTTTGCAACGTATTACTGTg lAGCAATATAACAGCTATCCAhTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA Humanized anti-ALPPL2 3H2 scFv VL-G4S3-VH (h3H2 scFvLH, or H3H2scFvLH) (SEQID NO: 340) Protein Sequence:
DIQMTQSPSSLSASVGDRVTITCKAISQNVATT^AWFQQKPGKAPKSLIIYSASYILQSGVP SRFSGSGSGTDFTLTISSLQPEDFATYYC|QQYNSYP|FTFGSGTKLEIKGGGGSGGGGSGG GGSQVTLKESGPALVKPTQTLTLTCTF|SGFSLSTSGMG|VSWIRQPPGKALEWLA|HIYW| |DDDKR1YNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTWAPYFYA|
GTSVTVSS
(SEQID NO: 440) DNA Sequence: GACATTCAGATGACCCAGTCTCCAAGCTCCCTGTCCGCATCAGTAGGAGACAGGGTCA CCATCACCTGCAAGGCCjAGTCAGAATGTGGCTACTAC'ljGTGGCCTGGTTTCAACAGA AACCAGGGAAAGCTCCTAAAAGCCTGATltTACAGCGCATCCTAC|TTGCAGAGTGGAG TCCCTAGTCGCTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAG TCTGCAGCCTGAAGACTTTGCAACGTATTACTGT|CAGCAATATAACAGCTATCCA|TT CACGTTCGGCTCGGGGACAAAGTTGGAAATAAAAGGTGGTGGTGGTTCTGGCGGCGG CGGCTCCGGTGGTGGTGGTTCCCAGGTTACTCTGAAAGAGTCTGGCCCTGCGCTAGTG AAGCCCACCCAGACCCTCACTCTGACTTGTACTTTCfrCTGGGTTTTCACTGAGCACTT CTGGTATGGG^GTGAGCTGGATTCGTCAGCCTCCAGGAAAGGCTCTGGAGTGGCTGG CAjCACATTTACTGGGATGATGACAAGCGCtrATAACCCATCCCTGAAGAGCCGGCTCA CAATCACCAAGGATACCTCCAAGAACCAGGTAGTCCTCACGATGACCAATATGGACCC TGTAGATACTGCCACATACTACtrGTGCTCGAAGACCTATTACTACGGTAGTAGCTCC ATATTTCTATGCTATGGACTACTGGGGTCAA|GGAACCTCAGTCACCGTCTCCTCA (SEQID NO: 40) Alternative DNA Sequence:
GACATTCAGATGACCCAGTCTCCAAGCTCCCTGTCCGCATCAGTAGGAGACAGGGTCA CCATCACCTGCAAGGCClAGTCAGAATGTGGCTACTAC'ljGTGGCCTGGTTTCAACAGA AACCAGGGAAAGCTCCTAAAAGCCTGATlfrACAGCGCATCCTAC|TTGCAGAGTGGAG TCCCTAGTCGCTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAG TCTGCAGCCTGAAGACTTTGCAACGTATTACTGTICAGCAATATAACAGCTATCCAITT CACGTTCGGCTCGGGGACAAAGTTGGAAATAAAAGGTGGAGGAGGTTCTGGAGGCGG TGGGTCCGGGGGAGGTGGCTCCCAGGTTACTCTGAAAGAGTCTGGCCCTGCGCTAGT GAAGCCCACCCAGACCCTCACTCTGACTTGTACTTTCffCTGGGTTTTCACTGAGCACT TCTGGTATGGGl^GTGAGCTGGATTCGTCAGCCTCCAGGAAAGGCTCTGGAGTGGCTG GCAjCACATTTACTGGGATGATGACAAGCG^TATAACCCATCCCTGAAGAGCCGGCTC ACAATCACCAAGGATACCTCCAAGAACCAGGTAGTCCTCACGATGACCAATATGGAC CCTGTAGATACTGCCACATACTAGtTGTGCTCGAAGACCTATTACTACGGTAGTAGCT CCATATTTCTATGCTATGGACTACTGGGGTCAA|GGAACCTCAGTCACCGTCTCCTCA Humanized anti-ALPPL2 3H2 scFv VH-Whitiow-VL (H3H2 scFv HL (Wh), or H3H2scFvHL(Wh))
(SEQID NO: 341) Protein Sequence:
QVTLKESGPALVKPTQTLTLTCTF|SGFSLSTSGMG|VSWIRQPPGKALEWLA|HIYWDD| |DK§YNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTWAPYFYAMD| |YWGQ|GTSVTVSSGSTSGSGKPGSGEGSTKGDIQMTQSPSSLSASVGDRVTITCKA|SQNV| IAT^VAWFOQKPGKAPKSLIIYSASYILOSGVPSRFSGSGSGTDFTLTISSLOPEDFATYYC
7TFGSGTKLEIK
(SEQID NO: 441) DNA Sequence: CAGGTTACTCTGAAAGAGTCTGGCCCTGCGCTAGTGAAGCCCACCCAGACCCTCACTC TGACTTGTACTTTCfrCTGGGTTTTCACTGAGCACTTCTGGTATGGGljGTGAGCTGGA TTCGTCAGCCTCCAGGAAAGGCTCTGGAGTGGCTGGCAjCACATTTACTGGGATGATGl lACAAGCGdTATAACCCATCCCTGAAGAGCCGGCTCACAATCACCAAGGATACCTCCAA GAACCAGGTAGTCCTCACGATGACCAATATGGACCCTGTAGATACTGCCACATACTA CfrGTGCTCGAAGACCTATTACTACGGTAGTAGCTCCATATTTCTATGCTATGGACTA CTGGGGTCAAIGGAACCTCAGTCACCGTCTCCTCAGGCTCAACTTCCGGTTCAGGTAAA CCTGGAAGCGGGGAGGGTAGCACAAAGGGGGACATTCAGATGACCCAGTCTCCAAGC TCCCTGTCCGCATCAGTAGGAGACAGGGTCACCATCACCTGCAAGGCCIAGTCAGAAT GTGGCTACTAC^GTGGCCTGGTTTCAACAGAAACCAGGGAAAGCTCCTAAAAGCCTG ATT|TACAGCGCATCCTACtrTGCAGAGTGGAGTCCCTAGTCGCTTCAGCGGCAGTGGA TCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAGCCTGAAGACTTTGCAACGT ATTACTG’dCAGCAATATAACAGCTATCCAh'TCACGTTCGGCTCGGGGACAAAGTTGG AAATAAAA
Humanized anti-ALPPL2 3H2 scFv VL-Whitiow-VH (H3H2 scFv LH (Wh), or h3H2scFvLH(Wh))
(SEQID NO: 342) Protein Sequence:
DIQMTQSPSSLSASVGDRVTITCKA|SQNyAT7jVAWFQQKPGKAPKSLl|YSASY|LQSGVP SRFSGSGSGTDFTLTISSLQPEDFATYYdQQYNSYP|FTFGSGTKLEIKGSTSGSGKPGSGE GSTKGQVTLKESGPALVKPTQTLTLTCTFtSGFSLSTSGM^VSWIRQPPGKALEWLA^ |YWDDDKR|YNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYY|CARRPITTWAPYF1 IYAMDYWGQIGTSVTVSS
(SEQID NO: 442) DNA Sequence: GACATTCAGATGACCCAGTCTCCAAGCTCCCTGTCCGCATCAGTAGGAGACAGGGTCA CCATCACCTGCAAGGCCjAGTCAGAATGTGGCTACTACTjGTGGCCTGGTTTCAACAGA AACCAGGGAAAGCTCCTAAAAGCCTGATltTACAGCGCATCCTAClTTGCAGAGTGGAG TCCCTAGTCGCTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAG TCTGCAGCCTGAAGACTTTGCAACGTATTACTGTlCAGCAATATAACAGCTATCCAhT CACGTTCGGCTCGGGGACAAAGTTGGAAATAAAAGGCTCAACTTCCGGTTCAGGTAA ACCTGGAAGCGGGGAGGGTAGCACAAAGGGGCAGGTTACTCTGAAAGAGTCTGGCCC TGCGCTAGTGAAGCCCACCCAGACCCTCACTCTGACTTGTACTTTCfrCTGGGTTTTCA CTGAGCACTTCTGGTATGGGT^GTGAGCTGGATTCGTCAGCCTCCAGGAAAGGCTCTG GAGTGGCTGGCA|CACATTTACTGGGATGATGACAAGCG3TATAACCCATCCCTGAAG AGCCGGCTCACAATCACCAAGGATACCTCCAAGAACCAGGTAGTCCTCACGATGACCA ATATGGACCCTGTAGATACTGCCACATACTACtrGTGCTCGAAGACCTATTACTACGG TAGTAGCTCCATATTTCTATGCTATGGACTACTGGGGTCAA|GGAACCTCAGTCACCG TCTCCTCA
Anti-ALPPL2 CARsj
CAR subparts:
Lead sequence (LS)
(SEQ ID NO: 160) Protein Sequence:
METPAQLLFLLLLWLPDTTG
(SEQ ID NO: 260) DNA Sequence:
ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCG GA
Human CD28 transmembrane domain (CD28 TM domain, or CD28TM) (SEQ ID NO: 161) Protein Sequence:
FWVLVWGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 261) DNA Sequence: TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACA GTGGCCTTTATTATTTTCTGGGTG
Human CDS zeta intracellular signaling domain (CDSz ICS domain, CDS £ ICS, or CDSzICS)
(SEQ ID NO: 162) Protein Sequence:
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP R
(SEQ ID NO: 262) DNA Sequence: AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAG CTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGA CGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAA GGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGG ATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGT ACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC Human CD28 hinge (CD28 hinge, or CD28H)
(SEQ ID NO: 163) Protein Sequence:
LEVKGKHLCPSPLFPGPSKP
(SEQ ID NO: 263) DNA Sequence:
CTCGAGGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGC CC
Human CD28 costimulatory domain (CD28 CS domain, or CD28CS) (SEQ ID NO: 164) Protein Sequence: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKL (SEQ ID NO: 264) DNA Sequence: AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGC CCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCT ATCGCTCCAAGCTT
Human 4-1BB costimulatory domain (4-1BB CS domain, 41BB CS domain, or 41BBCS) (SEQID NO: 165) Protein Sequence:
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQID NO: 265) DNA Sequence:
AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTA CAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGA GGATGTGAACTG
Human DAP10 costimulatory domain (DAP10 CS domain, or DAP10CS) (SEQID NO: 166) Protein Sequence:
LCARPRRSPAQEDGKVYINMPGRG
(SEQID NO: 266) DNA Sequence:
CTGTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGATGGCAAAGTCTACATCAACA TGCCAGGCAGGGGC
Glycine-Serine linker unit (Gly-Gly-Gly-Gly-Ser linker unit; or GS linker unit) (SEQID NO: 167) Protein Sequence:
GGGGS
(SEQID NO: 267) DNA Sequence:
GGTGGTGGTGGTTCT
(Gly-Gly-Gly-Gly-Ser)X3 linker ((Gly4Ser)3 linker, (G4S)3 linker, G4SX3 linker, or G4SX3 linker)
(SEQID NO: 168) Protein Sequence:
GGGGSGGGGSGGGGS
(SEQID NO: 268) DNA Sequence:
GGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCC (SEQID NO: 68) Alternative DNA Sequence:
GGTGGAGGAGGTTCTGGAGGCGGTGGGTCCGGGGGAGGTGGCTCC
T2A ribosomal skip sequence (T2A) (SEQID NO: 169) Protein Sequence: AGAKRSGSGEGRGSLLTCGDVEENPGPR (SEQID NO: 269) DNA Sequence: GCCGGCGCCAAAAGGTCTGGCTCCGGTGAGGGCAGAGGAAGTCTTCTAACATGCGGT GACGTGGAGGAGAATCCCGGCCCTAGA truncated CD 19 (trCD19)
(SEQID NO: 170) Protein Sequence:
MPPPRLLFFLLFLTPMEVRPEEPLWKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPL KPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNV EGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPC LPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELK DDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLR TGGWKVSAVTLAYLIFCLCSLVGILHLQRALVLRRKRKRMT (SEQID NO: 270) DNA Sequence:
ATGCCACCTCCTCGCCTCCTCTTCTTCCTCCTCTTCCTCACCCCCATGGAAGTCAGGCC CGAGGAACCTCTAGTGGTGAAGGTGGAAGAGGGAGATAACGCTGTGCTGCAGTGCCT CAAGGGGACCTCAGATGGCCCCACTCAGCAGCTGACCTGGTCTCGGGAGTCCCCGCTT AAACCCTTCTTAAAACTCAGCCTGGGGCTGCCAGGCCTGGGAATCCACATGAGGCCCC TGGCCATCTGGCTTTTCATCTTCAACGTCTCTCAACAGATGGGGGGCTTCTACCTGTG CCAGCCGGGGCCCCCCTCTGAGAAGGCCTGGCAGCCTGGCTGGACAGTCAATGTGGAG GGCAGCGGGGAGCTGTTCCGGTGGAATGTTTCGGACCTAGGTGGCCTGGGCTGTGGC CTGAAGAACAGGTCCTCAGAGGGCCCCAGCTCCCCTTCCGGGAAGCTCATGAGCCCCA AGCTGTATGTGTGGGCCAAAGACCGCCCTGAGATCTGGGAGGGAGAGCCTCCGTGTC TCCCACCGAGGGACAGCCTGAACCAGAGCCTCAGCCAGGACCTCACCATGGCCCCTGG CTCCACACTCTGGCTGTCCTGTGGGGTACCCCCTGACTCTGTGTCCAGGGGCCCCCTC TCCTGGACCCATGTGCACCCCAAGGGGCCTAAGTCATTGCTGAGCCTAGAGCTGAAGG ACGATCGCCCGGCCAGAGATATGTGGGTAATGGAGACGGGTCTGTTGTTGCCCCGGG CCACAGCTCAAGACGCTGGAAAGTATTATTGTCACCGTGGCAACCTGACCATGTCATT CCACCTGGAGATCACTGCTCGGCCAGTACTATGGCACTGGCTGCTGAGGACTGGTGGC TGGAAGGTCTCAGCTGTGACTTTGGCTTATCTGATCTTCTGCCTGTGTTCCCTTGTGG GCATTCTTCATCTTCAAAGAGCCCTGGTCCTGAGGAGGAAAAGAAAGCGAATGACTT AA
Whitlow linker
(SEQID NO: 159) Protein Sequence:
GSTSGSGKPGSGEGSTKG
(SEQID NO: 259) DNA Sequence:
GGCTCAACTTCCGGTTCAGGTAAACCTGGAAGCGGGGAGGGTAGCACAAAGGGG
Anti-ALPPL2 CARs
CARs comprising hlRZscFvHL:
HlB2scFvHL-CD28H-CD28TM-CD28CS-CD3zICS
(SEQ ID NO: 351) Protein Sequence:
QVQLVQSGAEVKKPGASVKVSCK|ASGYSFTGYY|MHWVKQAPGQGLEWM|GRINPYNG [ATN|YNQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYY|CARVYGNYPFDYWGQG|T TLTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKS|SQSLLYSSNQKNY| LAWFQQKPGQPPKHJIYWASTRIESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCIQQ] [YYST^FGGGTKLEIKLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFII FWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKLRVKFSRSADAP AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
(SEQ ID NO: 451) DNA Sequence: CAGGTCCAGCTGGTACAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCTTCAGTGAAG GTATCCTGCAAG|GCTTCTGGTTACTCATTCACTGGCTACTAC|ATGCACTGGGTGAAG CAAGCCCCTGGACAGGGCCTTGAGTGGATGlGGACGTATTAATCCTTACAATGGTGC'll lACTAACtrACAACCAAAAATTCCAGGGCAGGGTCACCATGACTAGAGATACGTCCATC AGCACAGCCTACATGGAGCTCAGCAGGCTGAGATCTGACGACACTGCAGTCTATTAC TGTGCAAGAGTATATGGTAACTACCCTTTTGACTACTGGGGCCAAGGdACCACTCTC ACAGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCCG ACATTGTGATGACACAGTCTCCAGACTCCCTAGCTGTGTCACTTGGAGAGAGGGCTA CTATCAACTGCAAGTCdAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTAClT TGGCCTGGTTCCAGCAGAAACCAGGGCAGCCTCCTAAACTGCTGlATTTACTGGGCAT |CCACTAGG|GAATCTGGGGTCCCTGATCGCTTCAGTGGCAGTGGATCTGGGACAGATT TCACTCTCACCATCAGCAGTCTGCAGGCTGAAGACGTGGCAGTTTATTACTGT|CAGC| |AATATTATAGCACTCCG|ACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACTCGAGG TGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTG GGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGC CTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACAT GAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCA CCACGCGACTTCGCAGCCTATCGCTCCAAGCTTAGAGTGAAGTTCAGCAGGAGCGCAG ACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACG AAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGG AAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGA TAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAA GGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCC CTTCACATGCAGGCCCTGCCCCCTCGC
(SEQ ID NO: 51) Alternative DNA Sequence: CAGGTCCAGCTGGTACAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCTTCAGTGAAG GTATCCTGCAAG|GCTTCTGGTTACTCATTCACTGGCTACTA^ATGCACTGGGTGAAG CAAGCCCCTGGACAGGGCCTTGAGTGGATGIGGACGTATTAATCCTTACAATGGTGC’II lACTAACtrACAACCAAAAATTCCAGGGCAGGGTCACCATGACTAGAGATACGTCCATC AGCACAGCCTACATGGAGCTCAGCAGGCTGAGATCTGACGACACTGCAGTCTATTAC TGTGCAAGAGTATATGGTAACTACCCTTTTGACTACTGGGGCCAAGGdACCACTCTC ACAGTCTCCTCAGGTGGAGGAGGTTCTGGAGGCGGTGGGTCCGGGGGAGGTGGCTCC GACATTGTGATGACACAGTCTCCAGACTCCCTAGCTGTGTCACTTGGAGAGAGGGCT ACTATCAACTGCAAGTCC|AGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTAC TTGGCCTGGTTCCAGCAGAAACCAGGGCAGCCTCCTAAACTGCTG|ATTTACTGGGCA| |TCCACTAGG|GAATCTGGGGTCCCTGATCGCTTCAGTGGCAGTGGATCTGGGACAGAT TTCACTCTCACCATCAGCAGTCTGCAGGCTGAAGACGTGGCAGTTTATTACTGTlCAGCl |AATATTATAGCACTCCG|ACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACTCGAGG TGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTG GGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGC CTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACAT GAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCA CCACGCGACTTCGCAGCCTATCGCTCCAAGCTTAGAGTGAAGTTCAGCAGGAGCGCAG ACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACG AAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGG AAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGA TAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAA GGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCC CTTCACATGCAGGCCCTGCCCCCTCGC hlB2scFvHL-CD28H-CD28TM-41BBCS-CD3zICS (SEQID NO: 352) Protein Sequence:
QVQLVQSGAEVKKPGASVKVSCK|ASGYSFTGYY|MHWVKQAPGQGLEWM|GRINPYNG| |AT5YNQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYY|CARVYGNYPFDYWGQG|T TLTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKS|SQSLLYSSNQKNY| LAWFQQKPGQPPKLL|IYWASTR]ESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC|Q^ ^ST^TFGGGTKLEIKLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFII FWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAY QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQID NO: 452) DNA Sequence:
CAGGTCCAGCTGGTACAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCTTCAGTGAAG GTATCCTGCAAG|GCTTCTGGTTACTCATTCACTGGCTACTA(jATGCACTGGGTGAAG CAAGCCCCTGGACAGGGCCTTGAGTGGATGlGGACGTATTAATCCTTACAATGGTGC'll lACTAACtTACAACCAAAAATTCCAGGGCAGGGTCACCATGACTAGAGATACGTCCATC AGCACAGCCTACATGGAGCTCAGCAGGCTGAGATCTGACGACACTGCAGTCTATTAC TGTGCAAGAGTATATGGTAACTACCCTTTTGACTACTGGGGCCAAGGdACCACTCTC ACAGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCCG ACATTGTGATGACACAGTCTCCAGACTCCCTAGCTGTGTCACTTGGAGAGAGGGCTA CTATCAACTGCAAGTCdAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTAC|T TGGCCTGGTTCCAGCAGAAACCAGGGCAGCCTCCTAAACTGCTGlATTTACTGGGCAT CCACTAGGIGAATCTGGGGTCCCTGATCGCTTCAGTGGCAGTGGATCTGGGACAGATT TCACTCTCACCATCAGCAGTCTGCAGGCTGAAGACGTGGCAGTTTATTACTGljCAGC| |AATATTATAGCACTCCG|ACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACTCGAGG TGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTG GGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGC CTTTATTATTTTCTGGGTGAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACA ACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATT TCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAG ACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACG AAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGG AAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGA TAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAA GGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCC CTTCACATGCAGGCCCTGCCCCCTCGC (SEQID NO: 52) Alternative DNA Sequence: CAGGTCCAGCTGGTACAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCTTCAGTGAAG GTATCCTGCAAGlGCTTCTGGTTACTCATTCACTGGCTACTA^ATGCACTGGGTGAAG CAAGCCCCTGGACAGGGCCTTGAGTGGATGlGGACGTATTAATCCTTACAATGGTGC’ll lACTAACtrACAACCAAAAATTCCAGGGCAGGGTCACCATGACTAGAGATACGTCCATC AGCACAGCCTACATGGAGCTCAGCAGGCTGAGATCTGACGACACTGCAGTCTATTAC TGTGCAAGAGTATATGGTAACTACCCTTTTGACTACTGGGGCCAAGGdACCACTCTC ACAGTCTCCTCAGGTGGAGGAGGTTCTGGAGGCGGTGGGTCCGGGGGAGGTGGCTCC GACATTGTGATGACACAGTCTCCAGACTCCCTAGCTGTGTCACTTGGAGAGAGGGCT ACTATCAACTGCAAGTCC|AGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTAC TTGGCCTGGTTCCAGCAGAAACCAGGGCAGCCTCCTAAACTGCTG|ATTTACTGGGCA| |TCCACTAGG|GAATCTGGGGTCCCTGATCGCTTCAGTGGCAGTGGATCTGGGACAGAT TTCACTCTCACCATCAGCAGTCTGCAGGCTGAAGACGTGGCAGTTTATTACTGTlCAGd |AATATTATAGCACTCCG|ACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACTCGAGG TGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTG GGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGC CTTTATTATTTTCTGGGTGAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACA ACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATT TCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAG ACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACG AAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGG AAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGA TAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAA GGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCC CTTCACATGCAGGCCCTGCCCCCTCGC HlB2scFvHL-CD28H-CD28TM-DAP10CS-CD3zICS (SEQID NO: 353) Protein Sequence: OVOLVQSGAEVKKPGASVKVSCK|ASGYSFTGYY|MHWVKQAPGQGLEWM|GRINPYNG| [ATN|YNQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYY|CARVYGNYPFDYWGQG|T TLTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKS|SQSLLYSSNQKNY| LAWFQQKPGQPPKLIJIYWASTRIESGVPDRFSGSGSGTDFTLTISSLOAEDVAVYYCIQQ |YYSTPtrFGGGTKLEIKLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFII FWVLCARPRRSPAQEDGKVYINMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRRE EYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG HDGLYQGLSTATKDTYDALHMQALPPR
(SEQID NO: 453) DNA Sequence:
CAGGTCCAGCTGGTACAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCTTCAGTGAAG GTATCCTGCAAG|GCTTCTGGTTACTCATTCACTGGCTACTAC|ATGCACTGGGTGAAG CAAGCCCCTGGACAGGGCCTTGAGTGGATGlGGACGTATTAATCCTTACAATGGTGC'll lACTAACtrACAACCAAAAATTCCAGGGCAGGGTCACCATGACTAGAGATACGTCCATC AGCACAGCCTACATGGAGCTCAGCAGGCTGAGATCTGACGACACTGCAGTCTATTAC TGTGCAAGAGTATATGGTAACTACCCTTTTGACTACTGGGGCCAAGGdACCACTCTC ACAGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCCG ACATTGTGATGACACAGTCTCCAGACTCCCTAGCTGTGTCACTTGGAGAGAGGGCTA CTATCAACTGCAAGTCC|AGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTAC|T TGGCCTGGTTCCAGCAGAAACCAGGGCAGCCTCCTAAACTGCTGlATTTACTGGGCAT |CCACTAGG|GAATCTGGGGTCCCTGATCGCTTCAGTGGCAGTGGATCTGGGACAGATT
TCACTCTCACCATCAGCAGTCTGCAGGCTGAAGACGTGGCAGTTTATTACTGT|CAG(j |AATATTATAGCACTCCG|ACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACTCGAGG TGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTG GGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGC CTTTATTATTTTCTGGGTGCTGTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGAT GGCAAAGTCTACATCAACATGCCAGGCAGGGGCAGAGTGAAGTTCAGCAGGAGCGCA GACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGAC GAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGG GAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAG ATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCA AGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGC CCTTCACATGCAGGCCCTGCCCCCTCGC
(SEQID NO: 53) Alternative DNA Sequence:
CAGGTCCAGCTGGTACAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCTTCAGTGAAG GTATCCTGCAAG|GCTTCTGGTTACTCATTCACTGGCTACTA(jATGCACTGGGTGAAG CAAGCCCCTGGACAGGGCCTTGAGTGGATGlGGACGTATTAATCCTTACAATGGTGC'll lACTAACtrACAACCAAAAATTCCAGGGCAGGGTCACCATGACTAGAGATACGTCCATC AGCACAGCCTACATGGAGCTCAGCAGGCTGAGATCTGACGACACTGCAGTCTATTAC TGTGCAAGAGTATATGGTAACTACCCTTTTGACTACTGGGGCCAAGGdACCACTCTC ACAGTCTCCTCAGGTGGAGGAGGTTCTGGAGGCGGTGGGTCCGGGGGAGGTGGCTCC GACATTGTGATGACACAGTCTCCAGACTCCCTAGCTGTGTCACTTGGAGAGAGGGCT ACTATCAACTGCAAGTCdAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTAC TTGGCCTGGTTCCAGCAGAAACCAGGGCAGCCTCCTAAACTGCTGIATTTACTGGGCAI TCCACTAGGjGAATCTGGGGTCCCTGATCGCTTCAGTGGCAGTGGATCTGGGACAGAT TTCACTCTCACCATCAGCAGTCTGCAGGCTGAAGACGTGGCAGTTTATTACTGTlCAGd |AATATTATAGCACTCCG|ACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACTCGAGG TGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTG GGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGC CTTTATTATTTTCTGGGTGCTGTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGAT GGCAAAGTCTACATCAACATGCCAGGCAGGGGCAGAGTGAAGTTCAGCAGGAGCGCA GACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGAC GAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGG GAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAG ATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCA AGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGC CCTTCACATGCAGGCCCTGCCCCCTCGC
CARs comprising hlBZscFvLH:
HlB2scFvLH-CD28H-CD28TM-CD28CS-CD3zICS
(SEQID NO: 354) Protein Sequence:
DIVMTQSPDSLAVSLGERATINCKS|SQSLLYSSNQKNYjLAWFOQKPGOPPKLL|lYWAS| &SGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC|QQYYSTP|TFGGGTKLEIKGGGGS GGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKlASGYSFTGYYlMHWVKQAPGQGLE WM|GRINPYNGATN|YNQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYY|CARVYGN| |YPFDYWGQG|TTLTVSSLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFI IFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKLRVKFSRSADA PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQID NO: 454) DNA Sequence: GACATTGTGATGACACAGTCTCCAGACTCCCTAGCTGTGTCACTTGGAGAGAGGGCT ACTATCAACTGCAAGTCC|AGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTAC TTGGCCTGGTTCCAGCAGAAACCAGGGCAGCCTCCTAAACTGCTG|ATTTACTGGGCA| |TCCACTAGG|GAATCTGGGGTCCCTGATCGCTTCAGTGGCAGTGGATCTGGGACAGAT TTCACTCTCACCATCAGCAGTCTGCAGGCTGAAGACGTGGCAGTTTATTACTGlICAGCi |AATATTATAGCACTCCG|ACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAGGTGGTG GTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCCCAGGTCCAGCTGGTACAGTC TGGAGCTGAGGTGAAGAAGCCTGGGGCTTCAGTGAAGGTATCCTGCAAG|GCTTCTGG| |TTACTCATTCACTGGCTACfA^ATGCACTGGGTGAAGCAAGCCCCTGGACAGGGCCTT GAGTGGATGlGGACGTATTAATCCTTACAATGGTGCTACTAACfrACAACCAAAAATTC CAGGGCAGGGTCACCATGACTAGAGATACGTCCATCAGCACAGCCTACATGGAGCTC AGCAGGCTGAGATCTGACGACACTGCAGTCTATTAGfrGTGCAAGAGTATATGGTAACl TACCCTTTTGACTACTGGGGCCAAGGClACCACTCTCACAGTCTCCTCACTCGAGGTGA AAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGT GCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTT TATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAA
CATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCAC GCGACTTCGCAGCCTATCGCTCCAAGCTTAGAGTGAAGTTCAGCAGGAGCGCAGACGC CCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGA GAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAG CCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAG ATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGG CACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTC ACATGCAGGCCCTGCCCCCTCGC
(SEQID NO: 54) Alternative DNA Sequence: GACATTGTGATGACACAGTCTCCAGACTCCCTAGCTGTGTCACTTGGAGAGAGGGCT ACTATCAACTGCAAGTCC|AGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTAC TTGGCCTGGTTCCAGCAGAAACCAGGGCAGCCTCCTAAACTGCTGIATTTACTGGGCAI ITCCACTAGGIGAATCTGGGGTCCCTGATCGCTTCAGTGGCAGTGGATCTGGGACAGAT TTCACTCTCACCATCAGCAGTCTGCAGGCTGAAGACGTGGCAGTTTATTACTGlICAGd AATATTATAGCACTCCGlACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAGGTGGAG GAGGTTCTGGAGGCGGTGGGTCCGGGGGAGGTGGCTCCCAGGTCCAGCTGGTACAGT CTGGAGCTGAGGTGAAGAAGCCTGGGGCTTCAGTGAAGGTATCCTGCAAG|GCTTCTG| GTTACTCATTCACTGGCTACTACjATGCACTGGGTGAAGCAAGCCCCTGGACAGGGCC TTGAGTGGATG|GGACGTATTAATCCTTACAATGGTGCTACTAACtrACAACCAAAAAT TCCAGGGCAGGGTCACCATGACTAGAGATACGTCCATCAGCACAGCCTACATGGAGC TCAGCAGGCTGAGATCTGACGACACTGCAGTCTATTACh'GTGCAAGAGTATATGGTA ACTACCCTTTTGACTACTGGGGCCAAGGdACCACTCTCACAGTCTCCTCACTCGAGGT GAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGG GTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCC TTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATG AACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACC ACGCGACTTCGCAGCCTATCGCTCCAAGCTTAGAGTGAAGTTCAGCAGGAGCGCAGA CGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGA AGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGA AAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGAT AAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAG GGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCC TTCACATGCAGGCCCTGCCCCCTCGC hlB2scFvLH-CD28H-CD28TM-41BBCS-CD3zICS (SEQID NO: 355) Protein Sequence: DIVMTQSPDSLAVSLGERATINCKS|SQSLLYSSNQKNYjLAWFQQKPGQPPKLL|lYWAS|TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC|QQYYSTP|TFGGGTKLEIKGGGGS GGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCK1ASGYSFTGYY|MHWVKQAPGQGLE WMlGRINPYNGATNlYNQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYlCARVYGNl lYPFDYWGQCjTTLTVSSLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFI IFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPA YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQID NO: 455) DNA Sequence: GACATTGTGATGACACAGTCTCCAGACTCCCTAGCTGTGTCACTTGGAGAGAGGGCT ACTATCAACTGCAAGTCdAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTAC TTGGCCTGGTTCCAGCAGAAACCAGGGCAGCCTCCTAAACTGCTG|ATTTACTGGGCA| |TCCACTAGG|GAATCTGGGGTCCCTGATCGCTTCAGTGGCAGTGGATCTGGGACAGAT TTCACTCTCACCATCAGCAGTCTGCAGGCTGAAGACGTGGCAGTTTATTACTGTlCAGd |AATATTATAGCACTCCG|ACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAGGTGGTG GTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCCCAGGTCCAGCTGGTACAGTC TGGAGCTGAGGTGAAGAAGCCTGGGGCTTCAGTGAAGGTATCCTGCAAG|GCTTCTGG| |TTACTCATTCACTGGCTACTA(jATGCACTGGGTGAAGCAAGCCCCTGGACAGGGCCTT GAGTGGATGlGGACGTATTAATCCTTACAATGGTGCTACTAACtrACAACCAAAAATTC CAGGGCAGGGTCACCATGACTAGAGATACGTCCATCAGCACAGCCTACATGGAGCTC TACCCTTTTGACTACTGGGGCCAAGGdACCACTCTCACAGTCTCCTCACTCGAGGTGA AAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGT GCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTT TATTATTTTCTGGGTGAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACC ATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCC AGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACG CCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAG AGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAA GCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAA GATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGG GCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTT CACATGCAGGCCCTGCCCCCTCGC
(SEQID NO: 55) Alternative DNA Sequence: GACATTGTGATGACACAGTCTCCAGACTCCCTAGCTGTGTCACTTGGAGAGAGGGCT ACTATCAACTGCAAGTCC|AGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTAC TTGGCCTGGTTCCAGCAGAAACCAGGGCAGCCTCCTAAACTGCTG|ATTTACTGGGCA| |TCCACTAGG|GAATCTGGGGTCCCTGATCGCTTCAGTGGCAGTGGATCTGGGACAGAT TTCACTCTCACCATCAGCAGTCTGCAGGCTGAAGACGTGGCAGTTTATTACTGT|CAGd |AATATTATAGCACTCCG|ACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAGGTGGAG GAGGTTCTGGAGGCGGTGGGTCCGGGGGAGGTGGCTCCCAGGTCCAGCTGGTACAGT CTGGAGCTGAGGTGAAGAAGCCTGGGGCTTCAGTGAAGGTATCCTGCAAG|GCTTCTG| GTTACTCATTCACTGGCTACTAdATGCACTGGGTGAAGCAAGCCCCTGGACAGGGCC TTGAGTGGATG|GGACGTATTAATCCTTACAATGGTGCTACTAAC|TACAACCAAAAAT TCCAGGGCAGGGTCACCATGACTAGAGATACGTCCATCAGCACAGCCTACATGGAGC TCAGCAGGCTGAGATCTGACGACACTGCAGTCTATTACh'GTGCAAGAGTATATGGTA ACTACCCTTTTGACTACTGGGGCCAAGGdACCACTCTCACAGTCTCCTCACTCGAGGT GAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGG GTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCC TTTATTATTTTCTGGGTGAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAA CCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTT CCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGAC GCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAA GAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAA AGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATA AGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGG GGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCT TCACATGCAGGCCCTGCCCCCTCGC HlB2scFvLH-CD28H-CD28TM-DAP10CS-CD3zICS
(SEQID NO: 356) Protein Sequence:
DIVMTQSPDSLAVSLGERATINCKS|SQSLLYSSNQKNYjLAWFQQKPGQPPKLL|lYWAS| |TR1ESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC|QQYYSTP|TFGGGTKLEIKGGGGS GGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCK|ASGYSFTGYY|MHWVKQAPGQGLE WMlGRINPYNGATNlYNQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYlCARVYGNl YPFDYWGQdTTLTVSSLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFI IFWVLCARPRRSPAQEDGKVYINMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRR EEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALPPR
(SEQID NO: 456) DNA Sequence:
GACATTGTGATGACACAGTCTCCAGACTCCCTAGCTGTGTCACTTGGAGAGAGGGCT ACTATCAACTGCAAGTCdAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTAC TTGGCCTGGTTCCAGCAGAAACCAGGGCAGCCTCCTAAACTGCTG|ATTTACTGGGCA| |TCCACTAGG|GAATCTGGGGTCCCTGATCGCTTCAGTGGCAGTGGATCTGGGACAGAT TTCACTCTCACCATCAGCAGTCTGCAGGCTGAAGACGTGGCAGTTTATTACTG’llCAGCl lAATATTATAGCACTCCGlACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAGGTGGTG GTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCCCAGGTCCAGCTGGTACAGTC TGGAGCTGAGGTGAAGAAGCCTGGGGCTTCAGTGAAGGTATCCTGCAAG|GCTTCTGG| TTACTCATTCACTGGCTACTA^ATGCACTGGGTGAAGCAAGCCCCTGGACAGGGCCTT GAGTGGATGlGGACGTATTAATCCTTACAATGGTGCTACTAA^rACAACCAAAAATTC CAGGGCAGGGTCACCATGACTAGAGATACGTCCATCAGCACAGCCTACATGGAGCTC AGCAGGCTGAGATCTGACGACACTGCAGTCTATTACfrGTGCAAGAGTATATGGTAAC TACCCTTTTGACTACTGGGGCCAAGGCJACCACTCTCACAGTCTCCTCACTCGAGGTGA AAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGT GCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTT TATTATTTTCTGGGTGCTGTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGATGGC AAAGTCTACATCAACATGCCAGGCAGGGGCAGAGTGAAGTTCAGCAGGAGCGCAGAC GCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAA GAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAA AGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATA AGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGG GGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCT
TCACATGCAGGCCCTGCCCCCTCGC
(SEQID NO: 56) Alternative DNA Sequence: GACATTGTGATGACACAGTCTCCAGACTCCCTAGCTGTGTCACTTGGAGAGAGGGCT ACTATCAACTGCAAGTCdAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTAC TTGGCCTGGTTCCAGCAGAAACCAGGGCAGCCTCCTAAACTGCTGIATTTACTGGGCAI |TCCACTAGG|GAATCTGGGGTCCCTGATCGCTTCAGTGGCAGTGGATCTGGGACAGAT TTCACTCTCACCATCAGCAGTCTGCAGGCTGAAGACGTGGCAGTTTATTACTG’llCAGd lAATATTATAGCACTCCGlACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAGGTGGAG GAGGTTCTGGAGGCGGTGGGTCCGGGGGAGGTGGCTCCCAGGTCCAGCTGGTACAGT CTGGAGCTGAGGTGAAGAAGCCTGGGGCTTCAGTGAAGGTATCCTGCAAG|GCTTCTG| GTTACTCATTCACTGGCTACTAdATGCACTGGGTGAAGCAAGCCCCTGGACAGGGCC TTGAGTGGATGlGGACGTATTAATCCTTACAATGGTGCTACTAACh’ACAACCAAAAAT TCCAGGGCAGGGTCACCATGACTAGAGATACGTCCATCAGCACAGCCTACATGGAGC TCAGCAGGCTGAGATCTGACGACACTGCAGTCTATTACfrGTGCAAGAGTATATGGTA ACTACCCTTTTGACTACTGGGGCCAAGGqACCACTCTCACAGTCTCCTCACTCGAGGT GAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGG GTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCC TTTATTATTTTCTGGGTGCTGTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGATG GCAAAGTCTACATCAACATGCCAGGCAGGGGCAGAGTGAAGTTCAGCAGGAGCGCAG ACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACG AAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGG AAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGA TAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAA GGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCC CTTCACATGCAGGCCCTGCCCCCTCGC
CARs comprising hlEBscFvHL: hlE8scFvHL-CD28H-CD28TM-CD28CS-CD3zICS
(SEQID NO: 361) Protein Sequence:
QVQLVQSGAEVKKPGASVKVSCKA|SGYTFTSYNM|NWVKQAPGQGLEWM|GAIYPGNG| |DT^YNQKFQGRVTMTADTSTSTVYMELSSLRSEDTAVYY|CARAPYGNFFDYWGQG|T TLTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKA^QDVSTA^AWYQ QKPGQPPKLLjlYWASTR|ESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC|QQYYST|HT FGGGTKLEIKLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFIIFWVRS KRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKLRVKFSRSADAPAYQQG QNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAY SEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
(SEQID NO: 461) DNA Sequence: CAGGTGCAACTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAG GTGTCCTGCAAGGCTfTCTGGCTACACATTTACCAGTTACAATATgAACTGGGTTAAG CAGGCACCTGGACAGGGCCTGGAATGGATG|GGAGCTATTTATCCAGGAAATGGTGAT| lACTTCCtrACAATCAGAAGTTCCAAGGCAGGGTCACAATGACTGCAGACACATCCACC AGCACAGTCTACATGGAGCTCAGCAGCCTGCGATCTGAGGACACTGCGGTCTATTAC TGTGCAAGAGCCCCGTATGGTAATTTCTTTGACTACTGGGGCCAAGGdACCACTCTC ACAGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCCG ACATTGTGATGACCCAGTCTCCCGACAGCCTGGCCGTATCACTAGGAGAAAGGGCCAC CATCAACTGCAAGGCdAGTCAGGATGTGAGTACTGCTGT^GCCTGGTATCAACAAAA ACCAGGGCAACCTCCTAAACTACTG|ATTTACTGGGCATCCACCCGG|GAAAGTGGAGT CCCTGATCGCTTCAGCGGCAGTGGATCTGGGACAGATTTTACTCTCACTATCAGCAGT TTGCAGGCTGAAGACGTGGCAGTTTATTACTGTICAGCAATATTATAGCACTICACACG TTCGGAGGGGGGACCAAGCTGGAAATAAAACTCGAGGTGAAAGGGAAACACCTTTGT CCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTG GAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGA GGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCC CGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTAT CGCTCCAAGCTTAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGG GCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTT TGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGA ACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACA GTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACC AGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCC CCCTCGC
(SEQID NO: 61) Alternative DNA Sequence: CAGGTGCAACTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAG GTGTCCTGCAAGGCT|TCTGGCTACACATTTACCAGTTACAATATCjAACTGGGTTAAG CAGGCACCTGGACAGGGCCTGGAATGGATGlGGAGCTATTTATCCAGGAAATGGTGATl lACTTCdrACAATCAGAAGTTCCAAGGCAGGGTCACAATGACTGCAGACACATCCACC AGCACAGTCTACATGGAGCTCAGCAGCCTGCGATCTGAGGACACTGCGGTCTATTAC TGTGCAAGAGCCCCGTATGGTAATTTCTTTGACTACTGGGGCCAAGGCIACCACTCTC ACAGTCTCCTCAGGTGGAGGAGGTTCTGGAGGCGGTGGGTCCGGGGGAGGTGGCTCC GACATTGTGATGACCCAGTCTCCCGACAGCCTGGCCGTATCACTAGGAGAAAGGGCC ACCATCAACTGCAAGGCC|AGTCAGGATGTGAGTACTGCTGTA|GCCTGGTATCAACAA AAACCAGGGCAACCTCCTAAACTACTGjATTTACTGGGCATCCACCCGCjGAAAGTGGA GTCCCTGATCGCTTCAGCGGCAGTGGATCTGGGACAGATTTTACTCTCACTATCAGCA GTTTGCAGGCTGAAGACGTGGCAGTTTATTACTGljCAGCAATATTATAGCACTlCACA CGTTCGGAGGGGGGACCAAGCTGGAAATAAAACTCGAGGTGAAAGGGAAACACCTTT GTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGG TGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGT GAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCG CCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCT ATCGCTCCAAGCTTAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCA GGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGT TTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAA GAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTA CAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTA CCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTG CCCCCTCGC hlE8scFvHL-CD28H-CD28TM-41BBCS-CD3zICS (SEQID NO: 362) Protein Sequence: QVQLVQSGAEVKKPGASVKVSCKA|SGYTFTSYNM|NWVKQAPGQGLEWM|GAIYPGNG| [DT^NQKFQGRVTMTADTSTSTVYMELSSLRSEDTAVYY|CARAPYGNFFDYWGQG|T TLTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKAlSQDVSTA^AWYQ QKPGQPPKLL|lYWASTR|ESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC|QQYYS’llHT FGGGTKLEIKLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFIIFWVKR GRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQN QLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSE IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQID NO: 462) DNA Sequence: CAGGTGCAACTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAG GTGTCCTGCAAGGCT|TCTGGCTACACATTTACCAGTTACAATATG}AACTGGGTTAAG CAGGCACCTGGACAGGGCCTGGAATGGATGiGGAGCTATTTATCCAGGAAATGGTGATl lACTTCCtTACAATCAGAAGTTCCAAGGCAGGGTCACAATGACTGCAGACACATCCACC AGCACAGTCTACATGGAGCTCAGCAGCCTGCGATCTGAGGACACTGCGGTCTATTAC TGTGCAAGAGCCCCGTATGGTAATTTCTTTGACTACTGGGGCCAAGGdACCACTCTC ACAGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCCG ACATTGTGATGACCCAGTCTCCCGACAGCCTGGCCGTATCACTAGGAGAAAGGGCCAC CATCAACTGCAAGGCdAGTCAGGATGTGAGTACTGCTGfgGCCTGGTATCAACAAAA ACCAGGGCAACCTCCTAAACTACTGlATTTACTGGGCATCCACCCGGlGAAAGTGGAGT CCCTGATCGCTTCAGCGGCAGTGGATCTGGGACAGATTTTACTCTCACTATCAGCAGT TTGCAGGCTGAAGACGTGGCAGTTTATTACTGT|CAGCAATATTATAGCAC^CACACG TTCGGAGGGGGGACCAAGCTGGAAATAAAACTCGAGGTGAAAGGGAAACACCTTTGT CCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTG GAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGA AACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTAC AAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAG GATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGG GCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTT TGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGA ACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACA GTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACC AGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCC CCCTCGC
(SEQID NO: 62) Alternative DNA Sequence: CAGGTGCAACTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAG GTGTCCTGCAAGGCT|TCTGGCTACACATTTACCAGTTACAATATG|AACTGGGTTAAG CAGGCACCTGGACAGGGCCTGGAATGGATGlGGAGCTATTTATCCAGGAAATGGTGA'll |ACTTCCtrACAATCAGAAGTTCCAAGGCAGGGTCACAATGACTGCAGACACATCCACC AGCACAGTCTACATGGAGCTCAGCAGCCTGCGATCTGAGGACACTGCGGTCTATTAC TGTGCAAGAGCCCCGTATGGTAATTTCTTTGACTACTGGGGCCAAGGdACCACTCTC ACAGTCTCCTCAGGTGGAGGAGGTTCTGGAGGCGGTGGGTCCGGGGGAGGTGGCTCC GACATTGTGATGACCCAGTCTCCCGACAGCCTGGCCGTATCACTAGGAGAAAGGGCC ACCATCAACTGCAAGGCC|AGTCAGGATGTGAGTACTGCTGTA|GCCTGGTATCAACAA AAACCAGGGCAACCTCCTAAACTACTG|ATTTACTGGGCATCCACCCGC1GAAAGTGGA GTCCCTGATCGCTTCAGCGGCAGTGGATCTGGGACAGATTTTACTCTCACTATCAGCA GTTTGCAGGCTGAAGACGTGGCAGTTTATTACTGljCAGCAATATTATAGCACT|CACA CGTTCGGAGGGGGGACCAAGCTGGAAATAAAACTCGAGGTGAAAGGGAAACACCTTT GTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGG TGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGT GAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGT ACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGG AGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCA GGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGT TTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAA GAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTA CAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTA CCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTG CCCCCTCGC
HlE8scFvHL-CD28H-CD28TM-DAP10CS-CD3zICS
(SEQID NO: 363) Protein Sequence:
QVQLVQSGAEVKKPGASVKVSCKA|SGYTFTSYNM|NWVKQAPGQGLEWM|GAIYPGNG| jDT^yNQKFQGRVTMTADTSTSTVYMELSSLRSEDTAVYYlCARAPYGNFFDYWGQGlT TLTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKA^QDVSTAVlAWYQ QKPGQPPKLlJlYWASTRlESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYClQQYYSTjHT FGGGTKLEIKLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFIIFWVLC ARPRRSPAQEDGKVYINMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPR (SEQID NO: 463) DNA Sequence: CAGGTGCAACTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAG GTGTCCTGCAAGGCT|TCTGGCTACACATTTACCAGTTACAATATG|AACTGGGTTAAG CAGGCACCTGGACAGGGCCTGGAATGGATGlGGAGCTATTTATCCAGGAAATGGTGA'll lACTTCCtrACAATCAGAAGTTCCAAGGCAGGGTCACAATGACTGCAGACACATCCACC AGCACAGTCTACATGGAGCTCAGCAGCCTGCGATCTGAGGACACTGCGGTCTATTAC TGTGCAAGAGCCCCGTATGGTAATTTCTTTGACTACTGGGGCCAAGGC|ACCACTCTC ACAGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCCG ACATTGTGATGACCCAGTCTCCCGACAGCCTGGCCGTATCACTAGGAGAAAGGGCCAC CATCAACTGCAAGGCCIAGTCAGGATGTGAGTACTGCTGT^GCCTGGTATCAACAAAA ACCAGGGCAACCTCCTAAACTACTG|ATTTACTGGGCATCCACCCGG|GAAAGTGGAGT CCCTGATCGCTTCAGCGGCAGTGGATCTGGGACAGATTTTACTCTCACTATCAGCAGT TTGCAGGCTGAAGACGTGGCAGTTTATTACTGT|CAGCAATATTATAGCACT]CACACG TTCGGAGGGGGGACCAAGCTGGAAATAAAACTCGAGGTGAAAGGGAAACACCTTTGT CCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTG GAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGCT GTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGATGGCAAAGTCTACATCAACATG CCAGGCAGGGGCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAG GGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTT TTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAG AACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTAC AGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTAC CAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGC CCCCTCGC
(SEQID NO: 63) Alternative DNA Sequence: CAGGTGCAACTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAG GTGTCCTGCAAGGCT|TCTGGCTACACATTTACCAGTTACAATATG}AACTGGGTTAAG CAGGCACCTGGACAGGGCCTGGAATGGATGIGGAGCTATTTATCCAGGAAATGGTGATI |ACTTCCtrACAATCAGAAGTTCCAAGGCAGGGTCACAATGACTGCAGACACATCCACC AGCACAGTCTACATGGAGCTCAGCAGCCTGCGATCTGAGGACACTGCGGTCTATTAC TGTGCAAGAGCCCCGTATGGTAATTTCTTTGACTACTGGGGCCAAGGdACCACTCTC ACAGTCTCCTCAGGTGGAGGAGGTTCTGGAGGCGGTGGGTCCGGGGGAGGTGGCTCC GACATTGTGATGACCCAGTCTCCCGACAGCCTGGCCGTATCACTAGGAGAAAGGGCC ACCATCAACTGCAAGGCC|AGTCAGGATGTGAGTACTGCTGTA|GCCTGGTATCAACAA AAACCAGGGCAACCTCCTAAACTACTGlATTTACTGGGCATCCACCCGdGAAAGTGGA GTCCCTGATCGCTTCAGCGGCAGTGGATCTGGGACAGATTTTACTCTCACTATCAGCA GTTTGCAGGCTGAAGACGTGGCAGTTTATTACTGTlCAGCAATATTATAGCAC'llCACA CGTTCGGAGGGGGGACCAAGCTGGAAATAAAACTCGAGGTGAAAGGGAAACACCTTT GTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGG TGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGT GCTGTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGATGGCAAAGTCTACATCAAC ATGCCAGGCAGGGGCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAG CAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGAT GTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGG AAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCC TACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTT TACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCC TGCCCCCTCGC
CARs comprising hlESscFvLH: hlE8scFvLH-CD28H-CD28TM-CD28CS-CD3zICS
(SEQID NO: 364) Protein Sequence:
DIVMTQSPDSLAVSLGERATINCKA|SQDVSTAV|AWYOQKPGOPPKLL5YWASTR1ESGV PDRFSGSGSGTDFTLTISSLQAEDVAVYYC|QQYYSTjHTFGGGTKLEIKGGGGSGGGGSG GGGSQVQLVQSGAEVKKPGASVKVSCKA^GYTFTSYNM|NWVKQAPGQGLEWM|GAIY| |PGNGDTS|YNQKFQGRVTMTADTSTSTVYMELSSLRSEDTAVYY|CARAPYGNFFDYW| ^Q^TTLTVSSLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFIIFWVRS KRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKLRVKFSRSADAPAYQQG QNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAY SEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQID NO: 464) DNA Sequence: GACATTGTGATGACCCAGTCTCCCGACAGCCTGGCCGTATCACTAGGAGAAAGGGCC ACCATCAACTGCAAGGCC|AGTCAGGATGTGAGTACTGCTGTA|GCCTGGTATCAACAA AAACCAGGGCAACCTCCTAAACTACTG|ATTTACTGGGCATCCACCCGC1GAAAGTGGA GTCCCTGATCGCTTCAGCGGCAGTGGATCTGGGACAGATTTTACTCTCACTATCAGCA GTTTGCAGGCTGAAGACGTGGCAGTTTATTACTGljCAGCAATATTATAGCACT|CACA CGTTCGGAGGGGGGACCAAGCTGGAAATAAAAGGTGGTGGTGGTTCTGGCGGCGGCG GCTCCGGTGGTGGTGGTTCCCAGGTGCAACTGGTGCAGTCTGGGGCTGAGGTGAAGA AGCCTGGGGCCTCAGTGAAGGTGTCCTGCAAGGCltrCTGGCTACACATTTACCAGTTl |ACAATATG|AACTGGGTTAAGCAGGCACCTGGACAGGGCCTGGAATGGATG|GGAGCTA| |TTTATCCAGGAAATGGTGATACTTCC|TACAATCAGAAGTTCCAAGGCAGGGTCACAA TGACTGCAGACACATCCACCAGCACAGTCTACATGGAGCTCAGCAGCCTGCGATCTGA GGACACTGCGGTCTATTACfrGTGCAAGAGCCCCGTATGGTAATTTCTTTGACTACTGl IGGGCCAAGG^ACCACTCTCACAGTCTCCTCACTCGAGGTGAAAGGGAAACACCTTTG TCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGT GGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGC CCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCT ATCGCTCCAAGCTTAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCA GGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGT TTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAA GAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTA CAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTA CCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTG CCCCCTCGC
(SEQID NO: 64) Alternative DNA Sequence: GACATTGTGATGACCCAGTCTCCCGACAGCCTGGCCGTATCACTAGGAGAAAGGGCC ACCATCAACTGCAAGGCC|AGTCAGGATGTGAGTACTGCTGTA|GCCTGGTATCAACAA AAACCAGGGCAACCTCCTAAACTACTGlATTTACTGGGCATCCACCCGdGAAAGTGGA GTCCCTGATCGCTTCAGCGGCAGTGGATCTGGGACAGATTTTACTCTCACTATCAGCA GTTTGCAGGCTGAAGACGTGGCAGTTTATTACTGTlCAGCAATATTATAGCAC'llCACA CGTTCGGAGGGGGGACCAAGCTGGAAATAAAAGGTGGAGGAGGTTCTGGAGGCGGTG GGTCCGGGGGAGGTGGCTCCCAGGTGCAACTGGTGCAGTCTGGGGCTGAGGTGAAGA AGCCTGGGGCCTCAGTGAAGGTGTCCTGCAAGGClfrCTGGCTACACATTTACCAGTll |ACAATAT^AACTGGGTTAAGCAGGCACCTGGACAGGGCCTGGAATGGATG|GGAGCTA| |TTTATCCAGGAAATGGTGATACTTCC|TACAATCAGAAGTTCCAAGGCAGGGTCACAA TGACTGCAGACACATCCACCAGCACAGTCTACATGGAGCTCAGCAGCCTGCGATCTGA GGACACTGCGGTCTATTACtrGTGCAAGAGCCCCGTATGGTAATTTCTTTGACTACTGl iGGGCCAAGGdACCACTCTCACAGTCTCCTCACTCGAGGTGAAAGGGAAACACCTTTG TCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGT GGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGC CCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCT ATCGCTCCAAGCTTAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCA GGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGT TTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAA GAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTA CAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTA CCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTG CCCCCTCGC hlE8scFvLH-CD28H-CD28TM-41BBCS-CD3zICS (SEQID NO: 365) Protein Sequence: DIVMTQSPDSLAVSLGERATINCKA|SQDVSTAV|AWYQQKPGQPPKLL|iYWASTR|ESGV PDRFSGSGSGTDFTLTISSLQAEDVAVYYC}QQYYST)HTFGGGTKLEIKGGGGSGGGGSG GGGSQVQLVQSGAEVKKPGASVKVSCKA^GYTFTSYNM|NWVKQAPGQGLEWM^AIY| |PGNGDTS|YNQKFQGRVTMTADTSTSTVYMELSSLRSEDTAVYY|CARAPYGNFFDYW| ^Q^TTLTVSSLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFIIFWVKR GRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQN QLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSE IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQID NO: 465) DNA Sequence: GACATTGTGATGACCCAGTCTCCCGACAGCCTGGCCGTATCACTAGGAGAAAGGGCC ACCATCAACTGCAAGGCdAGTCAGGATGTGAGTACTGCTGT^GCCTGGTATCAACAA AAACCAGGGCAACCTCCTAAACTACTGjATTTACTGGGCATCCACCCGCjGAAAGTGGA GTCCCTGATCGCTTCAGCGGCAGTGGATCTGGGACAGATTTTACTCTCACTATCAGCA GTTTGCAGGCTGAAGACGTGGCAGTTTATTACTGljCAGCAATATTATAGCACTlCACA CGTTCGGAGGGGGGACCAAGCTGGAAATAAAAGGTGGTGGTGGTTCTGGCGGCGGCG GCTCCGGTGGTGGTGGTTCCCAGGTGCAACTGGTGCAGTCTGGGGCTGAGGTGAAGA AGCCTGGGGCCTCAGTGAAGGTGTCCTGCAAGGClfrCTGGCTACACATTTACCAGTlj |ACAATATG|AACTGGGTTAAGCAGGCACCTGGACAGGGCCTGGAATGGATG|GGAGCTA| iTTTATCCAGGAAATGGTGATACTTCdTACAATCAGAAGTTCCAAGGCAGGGTCACAA TGACTGCAGACACATCCACCAGCACAGTCTACATGGAGCTCAGCAGCCTGCGATCTGA GGACACTGCGGTCTATTACtrGTGCAAGAGCCCCGTATGGTAATTTCTTTGACTACTGl GGGCCAAGGQACCACTCTCACAGTCTCCTCACTCGAGGTGAAAGGGAAACACCTTTG TCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGT GGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTA CAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGA GGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAG GGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTT TTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAG AACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTAC AGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTAC CAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGC CCCCTCGC
(SEQID NO: 65) Alternative DNA Sequence:
GACATTGTGATGACCCAGTCTCCCGACAGCCTGGCCGTATCACTAGGAGAAAGGGCC ACCATCAACTGCAAGGCC|AGTCAGGATGTGAGTACTGCTGTA|GCCTGGTATCAACAA AAACCAGGGCAACCTCCTAAACTACTGjATTTACTGGGCATCCACCCGCjGAAAGTGGA GTCCCTGATCGCTTCAGCGGCAGTGGATCTGGGACAGATTTTACTCTCACTATCAGCA GTTTGCAGGCTGAAGACGTGGCAGTTTATTACTGljCAGCAATATTATAGCACTjCACA CGTTCGGAGGGGGGACCAAGCTGGAAATAAAAGGTGGAGGAGGTTCTGGAGGCGGTG GGTCCGGGGGAGGTGGCTCCCAGGTGCAACTGGTGCAGTCTGGGGCTGAGGTGAAGA AGCCTGGGGCCTCAGTGAAGGTGTCCTGCAAGGCTfrCTGGCTACACATTTACCAGTll |ACAATATGjAACTGGGTTAAGCAGGCACCTGGACAGGGCCTGGAATGGATG|GGAGCTAj |TTTATCCAGGAAATGGTGATACTTCC|TACAATCAGAAGTTCCAAGGCAGGGTCACAA TGACTGCAGACACATCCACCAGCACAGTCTACATGGAGCTCAGCAGCCTGCGATCTGA GGACACTGCGGTCTATTACfrGTGCAAGAGCCCCGTATGGTAATTTCTTTGACTACTGl IGGGCCAAGG^ACCACTCTCACAGTCTCCTCACTCGAGGTGAAAGGGAAACACCTTTG TCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGT GGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTA CAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGA GGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAG GGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTT TTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAG AACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTAC AGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTAC CAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGC CCCCTCGC
HlE8scFvLH-CD28H-CD28TM-DAP10CS-CD3zICS
(SEQID NO: 366) Protein Sequence:
DIVMTQSPDSLAVSLGERATINCKAlSQDVSfAVjAWYQQKPGQPPKLljYWASTRlESGV PDRFSGSGSGTDFTLTISSLQAEDVAVYYClQQYYS’ljHTFGGGTKLEIKGGGGSGGGGSG GGGSQVQLVQSGAEVKKPGASVKVSCKA^GYTFTSYNM|NWVKQAPGQGLEWM|GAIY| PGNGDTS|YNQKFQGRVTMTADTSTSTVYMELSSLRSEDTAVYY|CARAPYGNFFDYW GQGITTLTVSSLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFIIFWVLC ARPRRSPAQEDGKVYINMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPR
(SEQID NO: 466) DNA Sequence:
GACATTGTGATGACCCAGTCTCCCGACAGCCTGGCCGTATCACTAGGAGAAAGGGCC ACCATCAACTGCAAGGCdAGTCAGGATGTGAGTACTGCTGT^GCCTGGTATCAACAA AAACCAGGGCAACCTCCTAAACTACTGjATTTACTGGGCATCCACCCGCjGAAAGTGGA GTCCCTGATCGCTTCAGCGGCAGTGGATCTGGGACAGATTTTACTCTCACTATCAGCA GTTTGCAGGCTGAAGACGTGGCAGTTTATTACTG'llCAGCAATATTATAGCAC'llCACA CGTTCGGAGGGGGGACCAAGCTGGAAATAAAAGGTGGTGGTGGTTCTGGCGGCGGCG GCTCCGGTGGTGGTGGTTCCCAGGTGCAACTGGTGCAGTCTGGGGCTGAGGTGAAGA AGCCTGGGGCCTCAGTGAAGGTGTCCTGCAAGGClfrCTGGCTACACATTTACCAGTlI |ACAATATG(AACTGGGTTAAGCAGGCACCTGGACAGGGCCTGGAATGGATG|GGAGCTAj |TTTATCCAGGAAATGGTGATACTTCC|TACAATCAGAAGTTCCAAGGCAGGGTCACAA TGACTGCAGACACATCCACCAGCACAGTCTACATGGAGCTCAGCAGCCTGCGATCTGA GGACACTGCGGTCTATTACtrGTGCAAGAGCCCCGTATGGTAATTTCTTTGACTACTGl IGGGCCAAGG^ACCACTCTCACAGTCTCCTCACTCGAGGTGAAAGGGAAACACCTTTG TCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGT GGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG CTGTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGATGGCAAAGTCTACATCAACA TGCCAGGCAGGGGCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGC AGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATG TTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGA AGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCT ACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTT ACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCT GCCCCCTCGC
(SEQID NO: 66) Alternative DNA Sequence: GACATTGTGATGACCCAGTCTCCCGACAGCCTGGCCGTATCACTAGGAGAAAGGGCC ACCATCAACTGCAAGGCdAGTCAGGATGTGAGTACTGCTGT^GCCTGGTATCAACAA AAACCAGGGCAACCTCCTAAACTACTGjATTTACTGGGCATCCACCCGCjGAAAGTGGA GTCCCTGATCGCTTCAGCGGCAGTGGATCTGGGACAGATTTTACTCTCACTATCAGCA GTTTGCAGGCTGAAGACGTGGCAGTTTATTACTGljCAGCAATATTATAGCACTlCACA CGTTCGGAGGGGGGACCAAGCTGGAAATAAAAGGTGGAGGAGGTTCTGGAGGCGGTG GGTCCGGGGGAGGTGGCTCCCAGGTGCAACTGGTGCAGTCTGGGGCTGAGGTGAAGA AGCCTGGGGCCTCAGTGAAGGTGTCCTGCAAGGClfrCTGGCTACACATTTACCAGTlj |ACAATATG|AACTGGGTTAAGCAGGCACCTGGACAGGGCCTGGAATGGATG|GGAGCTA| iTTTATCCAGGAAATGGTGATACTTCdTACAATCAGAAGTTCCAAGGCAGGGTCACAA TGACTGCAGACACATCCACCAGCACAGTCTACATGGAGCTCAGCAGCCTGCGATCTGA GGACACTGCGGTCTATTACtrGTGCAAGAGCCCCGTATGGTAATTTCTTTGACTACTGl GGGCCAAGGQACCACTCTCACAGTCTCCTCACTCGAGGTGAAAGGGAAACACCTTTG TCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGT GGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG CTGTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGATGGCAAAGTCTACATCAACA TGCCAGGCAGGGGCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGC AGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATG TTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGA AGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCT ACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTT ACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCT GCCCCCTCGC
CARs comprising h3H2scFvHL: h3H2scFvHL-CD28H-CD28TM-CD28CS-CD3zICS
(SEQID NO: 371) Protein Sequence:
QVTLKESGPALVKPTQTLTLTCTF|SGFSLSTSGM(jVSWIRQPPGKALEWLA|HIYWDD| iKl PSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTWAPYFYAMD|
_ }TSVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKA|SQNVAT| @VAWFQQKPGKAPKSLI|YSASY|LQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC|Q^ ^NSYgFTFGSGTKLEIKLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFI IFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKLRVKFSRSADA PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQID NO: 471) DNA Sequence: CAGGTTACTCTGAAAGAGTCTGGCCCTGCGCTAGTGAAGCCCACCCAGACCCTCACTC TGACTTGTACTTTCfrCTGGGTTTTCACTGAGCACTTCTGGTATGGGljGTGAGCTGGA TTCGTCAGCCTCCAGGAAAGGCTCTGGAGTGGCTGGCAjCACATTTACTGGGATGATGl lACAAGCGdTATAACCCATCCCTGAAGAGCCGGCTCACAATCACCAAGGATACCTCCAA GAACCAGGTAGTCCTCACGATGACCAATATGGACCCTGTAGATACTGCCACATACTA CtrGTGCTCGAAGACCTATTACTACGGTAGTAGCTCCATATTTCTATGCTATGGACTA |CTGGGGTCAA|GGAACCTCAGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGC GGCTCCGGTGGTGGTGGTTCCGACATTCAGATGACCCAGTCTCCAAGCTCCCTGTCCG CATCAGTAGGAGACAGGGTCACCATCACCTGCAAGGC(jAGTCAGAATGTGGCTACTA| 0GTGGCCTGGTTTCAACAGAAACCAGGGAAAGCTCGTAAAAGCCTGATTfrACAGCG| |CATCCTAC|TTGCAGAGTGGAGTCCCTAGTCGCTTCAGCGGCAGTGGATCTGGGACAG ATTTCACTCTCACCATCAGCAGTCTGCAGCCTGAAGACTTTGCAACGTATTACTG'O iGCAATATAACAGCTATCCAh’TCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACT CGAGGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCC TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACA GTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGAC TACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATG CCCCACCACGCGACTTCGCAGCCTATCGCTCCAAGCTTAGAGTGAAGTTCAGCAGGAG CGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTA GGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATG GGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAG AAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGG GGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACG ACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQID NO: 71) Alternative DNA Sequence:
CAGGTTACTCTGAAAGAGTCTGGCCCTGCGCTAGTGAAGCCCACCCAGACCCTCACTC TGACTTGTACTTTCtrCTGGGTTTTCACTGAGCACTTCTGGTATGGGT^GTGAGCTGGA TTCGTCAGCCTCCAGGAAAGGCTCTGGAGTGGCTGGCA|CACATTTACTGGGATGATG| ACAAGCGdTATAACCCATCCCTGAAGAGCCGGCTCACAATCACCAAGGATACCTCCAA GAACCAGGTAGTCCTCACGATGACCAATATGGACCCTGTAGATACTGCCACATACTA CTGGGGTCAAjGGAACCTCAGTCACCGTCTCCTCAGGTGGAGGAGGTTCTGGAGGCGG TGGGTCCGGGGGAGGTGGCTCCGACATTCAGATGACCCAGTCTCCAAGCTCCCTGTCC GCATCAGTAGGAGACAGGGTCACCATCACCTGCAAGGCC|AGTCAGAATGTGGCTACT| |AC^GTGGCCTGGTTTCAACAGAAACCAGGGAAAGCTCCTAAAAGCCTGATlfrACAGC| |GCATCCTAC|TTGCAGAGTGGAGTCCCTAGTCGCTTCAGCGGCAGTGGATCTGGGACA GATTTCACTCTCACCATCAGCAGTCTGCAGCCTGAAGACTTTGCAACGTATTACTGTg lAGCAATATAACAGCTATCCAtTTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAAC TCGAGGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCC
CTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAAC AGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGA CTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTAT GCCCCACCACGCGACTTCGCAGCCTATCGCTCCAAGCTTAGAGTGAAGTTCAGCAGGA GCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCT AGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGAT GGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCA GAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAG GGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTAC GACGCCCTTCACATGCAGGCCCTGCCCCCTCGC H3H2scFvHL-CD28H-CD28TM-41BBCS-CD3zICS
(SEQID NO: 372) Protein Sequence:
QVTLKESGPALVKPTQTLTLTCTF|SGFSLSTSGMG|VSWIRQPPGKALEWLA|HIYWDD| |DKR|YNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTWAPYFYAMD| _ s® GTSVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKA^QNVA^ @VAWFQQKPGKAPKSLI|YSASY|LQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC|Q^ |YNSYP|FTFGSGTKLEIKLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFI IFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPA YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQID NO: 472) DNA Sequence: CAGGTTACTCTGAAAGAGTCTGGCCCTGCGCTAGTGAAGCCCACCCAGACCCTCACTC TGACTTGTACTTTCtrCTGGGTTTTCACTGAGCACTTCTGGTATGGGTjGTGAGCTGGA TTCGTCAGCCTCCAGGAAAGGCTCTGGAGTGGCTGGCA|CACATTTACTGGGATGATG| |ACAAGCGC|TATAACCCATCCCTGAAGAGCCGGCTCACAATCACCAAGGATACCTCCAA GAACCAGGTAGTCCTCACGATGACCAATATGGACCCTGTAGATACTGCCACATACTA CtTGTGCTCGAAGACCTATTACTACGGTAGTAGCTCCATATTTCTATGCTATGGACTA ICTGGGGTCAAIGGAACCTCAGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGC
GGCTCCGGTGGTGGTGGTTCCGACATTCAGATGACCCAGTCTCCAAGCTCCCTGTCCG CATCAGTAGGAGACAGGGTCACCATCACCTGCAAGGCdAGTCAGAATGTGGCTACTAl ^gGTGGCCTGGTTTCAACAGAAACCAGGGAAAGCTCCTAAAAGCCTGATTfTACAGCGl ICATCCTAdTTGCAGAGTGGAGTCCCTAGTCGCTTCAGCGGCAGTGGATCTGGGACAG ATTTCACTCTCACCATCAGCAGTCTGCAGCCTGAAGACTTTGCAACGTATTACTG1@ GCAATATAACAGCTATCCATrTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACT CGAGGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCC TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACA GTGGCCTTTATTATTTTCTGGGTGAAACGGGGCAGAAAGAAACTCCTGTATATATTC AAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGC CGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGC GCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAG GACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGG GGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGA AAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGG GCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACG ACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQID NO: 72) Alternative DNA Sequence: CAGGTTACTCTGAAAGAGTCTGGCCCTGCGCTAGTGAAGCCCACCCAGACCCTCACTC TGACTTGTACTTTCtrCTGGGTTTTCACTGAGCACTTCTGGTATGGGTjGTGAGCTGGA TTCGTCAGCCTCCAGGAAAGGCTCTGGAGTGGCTGGCAjCACATTTACTGGGATGATGl |ACAAGCGC|TATAACCCATCCCTGAAGAGCCGGCTCACAATCACCAAGGATACCTCCAA GAACCAGGTAGTCCTCACGATGACCAATATGGACCCTGTAGATACTGCCACATACTA CfrGTGCTCGAAGACCTATTACTACGGTAGTAGCTCCATATTTCTATGCTATGGACTA ICTGGGGTCAAIGGAACCTCAGTCACCGTCTCCTCAGGTGGAGGAGGTTCTGGAGGCGG TGGGTCCGGGGGAGGTGGCTCCGACATTCAGATGACCCAGTCTCCAAGCTCCCTGTCC GCATCAGTAGGAGACAGGGTCACCATCACCTGCAAGGCClAGTCAGAATGTGGCTAC'll [ACgGTGGCCTGGTTTCAACAGAAACCAGGGAAAGCTCCTAAAAGCCTGATlfrACAGCl iGCATCCTAdTTGCAGAGTGGAGTCCCTAGTCGCTTCAGCGGCAGTGGATCTGGGACA GATTTCACTCTCACCATCAGCAGTCTGCAGCCTGAAGACTTTGCAACGTATTACTGT§ lAGCAATATAACAGCTATCCAhTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAAC TCGAGGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCC CTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAAC AGTGGCCTTTATTATTTTCTGGGTGAAACGGGGCAGAAAGAAACTCCTGTATATATT CAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTG CCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAG CGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTA GGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATG GGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAG AAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGG GGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACG ACGCCCTTCACATGCAGGCCCTGCCCCCTCGC h3H2scFvHL-CD28H-CD28TM-DAP10CS-CD3zICS (SEQID NO: 373) Protein Sequence: QVTLKESGPALVKPTQTLTLTCTF|SGFSLSTSGMG|VSWIRQPPGKALEWLA|HIYWDD| |PKgYNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTWAPYFYAMD| |YWGQ|GTSVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKA|SQNVA'l1 3VAWFQQKPGKAPKSLI|YSASY1LOSGVPSRFSGSGSGTDFTLTISSLOPEDFATYYC|Q5 VNSYP|FTFGSGTKLEIKLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFI IFWVLCARPRRSPAQEDGKVYINMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRR EEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALPPR
(SEQID NO: 473) DNA Sequence:
CAGGTTACTCTGAAAGAGTCTGGCCCTGCGCTAGTGAAGCCCACCCAGACCCTCACTC TGACTTGTACTTTCtrCTGGGTTTTCACTGAGCACTTCTGGTATGGGTjGTGAGCTGGA TTCGTCAGCCTCCAGGAAAGGCTCTGGAGTGGCTGGCA|CACATTTACTGGGATGATG| lACAAGCGdTATAACCCATCCCTGAAGAGCCGGCTCACAATCACCAAGGATACCTCCAA GAACCAGGTAGTCCTCACGATGACCAATATGGACCCTGTAGATACTGCCACATACTA CtTGTGCTCGAAGACCTATTACTACGGTAGTAGCTCCATATTTCTATGCTATGGACTA |CTGGGGTCAA|GGAACCTCAGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGC GGCTCCGGTGGTGGTGGTTCCGACATTCAGATGACCCAGTCTCCAAGCTCCCTGTCCG CATCAGTAGGAGACAGGGTCACCATCACCTGCAAGGC(jAGTCAGAATGTGGCTACTA| ^gGTGGCCTGGTTTCAACAGAAACCAGGGAAAGCTCCTAAAAGCCTGATTfTACAGCGl |CATCCTAC|TTGCAGAGTGGAGTCCCTAGTCGCTTCAGCGGCAGTGGATCTGGGACAG
ATTTCACTCTCACCATCAGCAGTCTGCAGCCTGAAGACTTTGCAACGTATTACTG'lQ iGCAATATAACAGCTATCCAh’TCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACT CGAGGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCC
TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACA GTGGCCTTTATTATTTTCTGGGTGCTGTGCGCACGCCCACGCCGCAGCCCCGCCCAAG
AAGATGGCAAAGTCTACATCAACATGCCAGGCAGGGGCAGAGTGAAGTTCAGCAGGA GCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCT AGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGAT GGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCA GAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAG GGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTAC GACGCCCTTCACATGCAGGCCCTGCCCCCTCGC
(SEQID NO: 73) Alternative DNA Sequence:
CAGGTTACTCTGAAAGAGTCTGGCCCTGCGCTAGTGAAGCCCACCCAGACCCTCACTC TGACTTGTACTTTCtrCTGGGTTTTCACTGAGCACTTCTGGTATGGGTjGTGAGCTGGA TTCGTCAGCCTCCAGGAAAGGCTCTGGAGTGGCTGGCA|CACATTTACTGGGATGATG| |ACAAGCGC|TATAACCCATCCCTGAAGAGCCGGCTCACAATCACCAAGGATACCTCCAA GAACCAGGTAGTCCTCACGATGACCAATATGGACCCTGTAGATACTGCCACATACTA GtTGTGCTCGAAGACCTATTACTACGGTAGTAGCTCCATATTTCTATGCTATGGACTA ICTGGGGTCAAIGGAACCTCAGTCACCGTCTCCTCAGGTGGAGGAGGTTCTGGAGGCGG TGGGTCCGGGGGAGGTGGCTCCGACATTCAGATGACCCAGTCTCCAAGCTCCCTGTCC GCATCAGTAGGAGACAGGGTCACCATCACCTGCAAGGCClAGTCAGAATGTGGCTAC'll lA^TGGCCTGGTTTCAACAGAAACCAGGGAAAGCTCCTAAAAGCCTGATTh’ACAGCl iGCATCCTAdTTGCAGAGTGGAGTCCCTAGTCGCTTCAGCGGCAGTGGATCTGGGACA
GATTTCACTCTCACCATCAGCAGTCTGCAGCCTGAAGACTTTGCAACGTATTACTGT@ AGCAATATAACAGCTATCCAfTTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAAC TCGAGGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCC CTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAAC AGTGGCCTTTATTATTTTCTGGGTGCTGTGCGCACGCCCACGCCGCAGCCCCGCCCAA GAAGATGGCAAAGTCTACATCAACATGCCAGGCAGGGGCAGAGTGAAGTTCAGCAGG AGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATC TAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGA TGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGC AGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGA GGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCT ACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC
CARs comprising h3H2scFvLH:
H3H2scFvLH-CD28H-CD28TM-CD28CS-CD3zICS
(SEQID NO: 374) Protein Sequence:
DIQMTQSPSSLSASVGDRVTITCKA|SQNVAf^VAWFQQKPGKAPKSLl|YSASY|LQSGVP SRFSGSGSGTDFTLTISSLQPEDFATYYC •|SQEQEYN®SYP|FTFGSGTKLEIKGGGGSGGGGSGG GGSQVTLKESGPALVKPTQTLTLTCTF^GFSLSTSGMGlVSWIRQPPGKALEWLAlHIYWj |DDDKR|YNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTWAPYFYA|
GTSVTVSSLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFIIF WVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKLRVKFSRSADAP AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQID NO: 474) DNA Sequence: GACATTCAGATGACCCAGTCTCCAAGCTCCCTGTCCGCATCAGTAGGAGACAGGGTCA CCATCACCTGCAAGGCClAGTCAGAATGTGGCTACTAC'ljGTGGCCTGGTTTCAACAGA AACCAGGGAAAGCTCCTAAAAGCCTGATTfTACAGCGCATCCTAC|TTGCAGAGTGGAG TCCCTAGTCGCTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAG TCTGCAGCCTGAAGACTTTGCAACGTATTACTGT|CAGCAATATAACAGCTATCCA|TT CACGTTCGGCTCGGGGACAAAGTTGGAAATAAAAGGTGGTGGTGGTTCTGGCGGCGG CGGCTCCGGTGGTGGTGGTTCCCAGGTTACTCTGAAAGAGTCTGGCCCTGCGCTAGTG AAGCCCACCCAGACCCTCACTCTGACTTGTACTTTdTCTGGGTTTTCACTGAGCACTT CTGGTATGGGTjGTGAGCTGGATTCGTCAGCCTCCAGGAAAGGCTCTGGAGTGGCTGG CAjCACATTTACTGGGATGATGACAAGCG^TATAACCCATCCCTGAAGAGCCGGCTCA CAATCACCAAGGATACCTCCAAGAACCAGGTAGTCCTCACGATGACCAATATGGACCC TGTAGATACTGCCACATACTACtTGTGCTCGAAGACCTATTACTACGGTAGTAGCTCC ATATTTCTATGCTATGGACTACTGGGGTCAAIGGAACCTCAGTCACCGTCTCCTCACTC GAGGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCT TTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAG TGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACT ACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGC CCCACCACGCGACTTCGCAGCCTATCGCTCCAAGCTTAGAGTGAAGTTCAGCAGGAGC GCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAG GACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGG GGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGA AAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGG GCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACG ACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQID NO: 74) Alternative DNA Sequence:
GACATTCAGATGACCCAGTCTCCAAGCTCCCTGTCCGCATCAGTAGGAGACAGGGTCA CCATCACCTGCAAGGCdAGTCAGAATGTGGCTACTAC'ljGTGGCCTGGTTTCAACAGA AACCAGGGAAAGCTCCTAAAAGCCTGATlfTACAGCGCATCCTAC|TTGCAGAGTGGAG TCCCTAGTCGCTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAG TCTGCAGCCTGAAGACTTTGCAACGTATTACTGT|CAGCAATATAACAGCTATCCAh’T CACGTTCGGCTCGGGGACAAAGTTGGAAATAAAAGGTGGAGGAGGTTCTGGAGGCGG TGGGTCCGGGGGAGGTGGCTCCCAGGTTACTCTGAAAGAGTCTGGCCCTGCGCTAGT GAAGCCCACCCAGACCCTCACTCTGACTTGTACTTTCfrCTGGGTTTTCACTGAGCACT TCTGGTATGGG^GTGAGCTGGATTCGTCAGCCTCCAGGAAAGGCTCTGGAGTGGCTG GCAjCACATTTACTGGGATGATGACAAGCG^TATAACCCATCCCTGAAGAGCCGGCTC ACAATCACCAAGGATACCTCCAAGAACCAGGTAGTCCTCACGATGACCAATATGGAC CCTGTAGATACTGCCACATACTAdTGTGCTCGAAGACCTATTACTACGGTAGTAGCT CCATATTTCTATGCTATGGACTACTGGGGTCAAIGGAACCTCAGTCACCGTCTCCTCAC TCGAGGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCC CTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAAC AGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGA CTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTAT GCCCCACCACGCGACTTCGCAGCCTATCGCTCCAAGCTTAGAGTGAAGTTCAGCAGGA GCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCT AGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGAT GGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCA GAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAG GGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTAC GACGCCCTTCACATGCAGGCCCTGCCCCCTCGC H3H2scFvLH-CD28H-CD28TM-41BBCS-CD3zICS (SEQID NO: 375) Protein Sequence:
DIQMTQSPSSLSASVGDRVTITCKA|SQNyATTjVAWFQQKPGKAPKSLl|YSASY|LQSGVP SRFSGSGSGTDFTLTISSLQPEDFATYYC|QQYNSYP|FTFGSGTKLEIKGGGGSGGGGSGG GGSQVTLKESGPALVKPTQTLTLTCTF^GFSLSTSGMG|VSWIRQPPGKALEWLA|HIYW| |DDDKR|YNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTWAPYFYA| IMDYWGQIGTSVTVSSLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFIIF WVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQ QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMA EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQID NO: 475) DNA Sequence:
GACATTCAGATGACCCAGTCTCCAAGCTCCCTGTCCGCATCAGTAGGAGACAGGGTCA CCATCACCTGCAAGGCCIAGTCAGAATGTGGCTACTACT^GTGGCCTGGTTTCAACAGA AACCAGGGAAAGCTCCTAAAAGCCTGATltrACAGCGCATCCTAC|TTGCAGAGTGGAG TCCCTAGTCGCTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAG TCTGCAGCCTGAAGACTTTGCAACGTATTACTGT|CAGCAATATAACAGCTATCCAh’T CACGTTCGGCTCGGGGACAAAGTTGGAAATAAAAGGTGGTGGTGGTTCTGGCGGCGG CGGCTCCGGTGGTGGTGGTTCCCAGGTTACTCTGAAAGAGTCTGGCCCTGCGCTAGTG AAGCCCACCCAGACCCTCACTCTGACTTGTACTTTCfrCTGGGTTTTCACTGAGCACTT CTGGTATGGG^GTGAGCTGGATTCGTCAGCCTCCAGGAAAGGCTCTGGAGTGGCTGG CA|CACATTTACTGGGATGATGACAAGCGdrATAACCCATCCCTGAAGAGCCGGCTCA CAATCACCAAGGATACCTCCAAGAACCAGGTAGTCCTCACGATGACCAATATGGACCC TGTAGATACTGCCACATACTAC|TGTGCTCGAAGACCTATTACTACGGTAGTAGCTCC GAGGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCT TTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAG TGGCCTTTATTATTTTCTGGGTGAAACGGGGCAGAAAGAAACTCCTGTATATATTCA AACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCC GATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGC GCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAG GACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGG GGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGA AAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGG GCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACG ACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQID NO: 75) Alternative DNA Sequence: GACATTCAGATGACCCAGTCTCCAAGCTCCCTGTCCGCATCAGTAGGAGACAGGGTCA CCATCACCTGCAAGGCCjAGTCAGAATGTGGCTACTAC'ljGTGGCCTGGTTTCAACAGA AACCAGGGAAAGCTCCTAAAAGCCTGATlfrACAGCGCATCCTAdTTGCAGAGTGGAG TCCCTAGTCGCTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAG TCTGCAGCCTGAAGACTTTGCAACGTATTACTGTlCAGCAATATAACAGCTATCCAh’T CACGTTCGGCTCGGGGACAAAGTTGGAAATAAAAGGTGGAGGAGGTTCTGGAGGCGG TGGGTCCGGGGGAGGTGGCTCCCAGGTTACTCTGAAAGAGTCTGGCCCTGCGCTAGT GAAGCCCACCCAGACCCTCACTCTGACTTGTACTTTCfrCTGGGTTTTCACTGAGCACT TCTGGTATGGGI^GTGAGCTGGATTCGTCAGCCTCCAGGAAAGGCTCTGGAGTGGCTG GCAjCACATTTACTGGGATGATGACAAGCG^TATAACCCATCCCTGAAGAGCCGGCTC ACAATCACCAAGGATACCTCCAAGAACCAGGTAGTCCTCACGATGACCAATATGGAC CCTGTAGATACTGCCACATACTAC|TGTGCTCGAAGACCTATTACTACGGTAGTAGCT CCATATTTCTATGCTATGGACTACTGGGGTCAA|GGAACCTCAGTCACCGTCTCCTCAC TCGAGGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCC CTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAAC AGTGGCCTTTATTATTTTCTGGGTGAAACGGGGCAGAAAGAAACTCCTGTATATATT CAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTG CCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAG CGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTA GGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATG GGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAG AAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGG GGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACG ACGCCCTTCACATGCAGGCCCTGCCCCCTCGC H3H2scFvLH-CD28H-CD28TM-DAP10CS-CD3zICS (SEQID NO: 376) Protein Sequence:
Figure imgf000167_0001
DDDKR|YNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTWAPYFYA| MDYWGQIGTSVTVSSLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFIIF WVLCARPRRSPAQEDGKVYINMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRREE YDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR
(SEQID NO: 476) DNA Sequence: GACATTCAGATGACCCAGTCTCCAAGCTCCCTGTCCGCATCAGTAGGAGACAGGGTCA CCATCACCTGCAAGGCdAGTCAGAATGTGGCTACTAC'ljGTGGCCTGGTTTCAACAGA AACCAGGGAAAGCTCCTAAAAGCCTGATltrACAGCGCATCCTAClTTGCAGAGTGGAG TCCCTAGTCGCTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAG TCTGCAGCCTGAAGACTTTGCAACGTATTACTGT|CAGCAATATAACAGCTATCCA|TT CACGTTCGGCTCGGGGACAAAGTTGGAAATAAAAGGTGGTGGTGGTTCTGGCGGCGG CGGCTCCGGTGGTGGTGGTTCCCAGGTTACTCTGAAAGAGTCTGGCCCTGCGCTAGTG AAGCCCACCCAGACCCTCACTCTGACTTGTACTTTCfrCTGGGTTTTCACTGAGCACTT CTGGTATGGG^GTGAGCTGGATTCGTCAGCCTCCAGGAAAGGCTCTGGAGTGGCTGG CA|CACATTTACTGGGATGATGACAAGCGCtrATAACCCATCCCTGAAGAGCCGGCTCA CAATCACCAAGGATACCTCCAAGAACCAGGTAGTCCTCACGATGACCAATATGGACCC TGTAGATACTGCCACATACTACh'GTGCTCGAAGACCTATTACTACGGTAGTAGCTCC ATATTTCTATGCTATGGACTACTGGGGTCAA|GGAACCTCAGTCACCGTCTCCTCACTC GAGGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCT TTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAG TGGCCTTTATTATTTTCTGGGTGCTGTGCGCACGCCCACGCCGCAGCCCCGCCCAAGA AGATGGCAAAGTCTACATCAACATGCCAGGCAGGGGCAGAGTGAAGTTCAGCAGGAG CGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTA GGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATG GGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAG AAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGG GGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACG ACGCCCTTCACATGCAGGCCCTGCCCCCTCGC
(SEQID NO: 76) DNA Sequence: GACATTCAGATGACCCAGTCTCCAAGCTCCCTGTCCGCATCAGTAGGAGACAGGGTCA CCATCACCTGCAAGGCCIAGTCAGAATGTGGCTACTACT^GTGGCCTGGTTTCAACAGA AACCAGGGAAAGCTCCTAAAAGCCTGATltrACAGCGCATCCTAC|TTGCAGAGTGGAG TCCCTAGTCGCTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAG TCTGCAGCCTGAAGACTTTGCAACGTATTACTGT|CAGCAATATAACAGCTATCCAh’T CACGTTCGGCTCGGGGACAAAGTTGGAAATAAAAGGTGGAGGAGGTTCTGGAGGCGG TGGGTCCGGGGGAGGTGGCTCCCAGGTTACTCTGAAAGAGTCTGGCCCTGCGCTAGT GAAGCCCACCCAGACCCTCACTCTGACTTGTACTTTCfrCTGGGTTTTCACTGAGCACT TCTGGTATGGCgGTGAGCTGGATTCGTCAGCCTCCAGGAAAGGCTCTGGAGTGGCTG GCA|CACATTTACTGGGATGATGACAAGCG5TATAACCCATCCCTGAAGAGCCGGCTC ACAATCACCAAGGATACCTCCAAGAACCAGGTAGTCCTCACGATGACCAATATGGAC CCTGTAGATACTGCCACATACTACh'GTGCTCGAAGACCTATTACTACGGTAGTAGCT CCATATTTCTATGCTATGGACTACTGGGGTCAA|GGAACCTCAGTCACCGTCTCCTCAC TCGAGGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCC CTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAAC AGTGGCCTTTATTATTTTCTGGGTGCTGTGCGCACGCCCACGCCGCAGCCCCGCCCAA GAAGATGGCAAAGTCTACATCAACATGCCAGGCAGGGGCAGAGTGAAGTTCAGCAGG AGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATC TAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGA TGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGC AGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGA GGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCT ACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC
Anti-ALPPI.2 CARs with LS. T2A. and trCD19:
LS-h3H2scFvHL-CD28H-CD28TM-CD28CS-CD3zICS-T2A-trCD19 (SEQ ID NO: 571) Protein Sequence:
METPAQLLFLLLLWLPDTTGQVTLKESGPALVKPTQTLTLTCTF|SGFSLSTSGMG|VSW IRQPPGKALEWLA|HIYWDDDKR|YNPSLKSRLTITKDTSKNQWLTMTNMDPVDTAT YY|CARRPITTWAPYFYAMDYWGQ|GTSVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSL SASVGDRVTITCKA|SQNVATgVAWFQQKPGKAPKSLl|YSASY|LQSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCIQQYNSYPIFTFGSGTKLEIKLEVKGKHLCPSPLFPGPSKPFWV LVWGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPR DFAAYRSKLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK PQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA LHMQALPPRAGAKRSGSGEGRGSLLTCGDVEENPGPRMPPPRLLFFLLFLTPMEVRPE EPLWKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAI WLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGL KNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGS TLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPR ATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVSAVTLAYLIFCLCSL VGILHLQRALVLRRKRKRMT
(SEQ ID NO: 671) DNA Sequence:
ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCG GACAGGTTACTCTGAAAGAGTCTGGCCCTGCGCTAGTGAAGCCCACCCAGACCCTCAC TCTGACTTGTACTTTCfrCTGGGTTTTCACTGAGCACTTCTGGTATGGG^GTGAGCTG GATTCGTCAGCCTCCAGGAAAGGCTCTGGAGTGGCTGGCAlCACATTTACTGGGATGA |TGACAAGCG^rATAACCCATCCCTGAAGAGCCGGCTCACAATCACCAAGGATACCTCC AAGAACCAGGTAGTCCTCACGATGACCAATATGGACCCTGTAGATACTGCCACATAC TACfrGTGCTCGAAGACCTATTACTACGGTAGTAGCTCCATATTTCTATGCTATGGACl ITACTGGGGTCAAIGGAACCTCAGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGC GGCGGCTCCGGTGGTGGTGGTTCCGACATTCAGATGACCCAGTCTCCAAGCTCCCTGT CCGCATCAGTAGGAGACAGGGTCACCATCACCTGCAAGGCdAGTCAGAATGTGGCTAl |CTACT|GTGGCCTGGTTTCAACAGAAACCAGGGAAAGCTCCTAAAAGCCTGATT|TACA| IGCGCATCCTA^TTGCAGAGTGGAGTCCCTAGTCGCTTCAGCGGCAGTGGATCTGGGA CAGATTTCACTCTCACCATCAGCAGTCTGCAGCCTGAAGACTTTGCAACGTATTACTG TjCAGCAATATAACAGCTATCCAhTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAA ACTCGAGGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAG CCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTA ACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGT GACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCT ATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAAGCTTAGAGTGAAGTTCAGCAG GAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAAT CTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAG ATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTG CAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGG AGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACC TACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGCCGGCGCCAAAAGGTCTGGCT CCGGTGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCC CTAGAATGCCACCTCCTCGCCTCCTCTTCTTCCTCCTCTTCCTCACCCCCATGGAAGTC AGGCCCGAGGAACCTCTAGTGGTGAAGGTGGAAGAGGGAGATAACGCTGTGCTGCAG TGCCTCAAGGGGACCTCAGATGGCCCCACTCAGCAGCTGACCTGGTCTCGGGAGTCCC CGCTTAAACCCTTCTTAAAACTCAGCCTGGGGCTGCCAGGCCTGGGAATCCACATGAG GCCCCTGGCCATCTGGCTTTTCATCTTCAACGTCTCTCAACAGATGGGGGGCTTCTAC CTGTGCCAGCCGGGGCCCCCCTCTGAGAAGGCCTGGCAGCCTGGCTGGACAGTCAATG TGGAGGGCAGCGGGGAGCTGTTCCGGTGGAATGTTTCGGACCTAGGTGGCCTGGGCT GTGGCCTGAAGAACAGGTCCTCAGAGGGCCCCAGCTCCCCTTCCGGGAAGCTCATGAG CCCCAAGCTGTATGTGTGGGCCAAAGACCGCCCTGAGATCTGGGAGGGAGAGCCTCCG TGTCTCCCACCGAGGGACAGCCTGAACCAGAGCCTCAGCCAGGACCTCACCATGGCCC CTGGCTCCACACTCTGGCTGTCCTGTGGGGTACCCCCTGACTCTGTGTCCAGGGGCCC CCTCTCCTGGACCCATGTGCACCCCAAGGGGCCTAAGTCATTGCTGAGCCTAGAGCTG AAGGACGATCGCCCGGCCAGAGATATGTGGGTAATGGAGACGGGTCTGTTGTTGCCC CGGGCCACAGCTCAAGACGCTGGAAAGTATTATTGTCACCGTGGCAACCTGACCATG TCATTCCACCTGGAGATCACTGCTCGGCCAGTACTATGGCACTGGCTGCTGAGGACTG GTGGCTGGAAGGTCTCAGCTGTGACTTTGGCTTATCTGATCTTCTGCCTGTGTTCCCT TGTGGGCATTCTTCATCTTCAAAGAGCCCTGGTCCTGAGGAGGAAAAGAAAGCGAAT GACTTAA
(SEQID NO: 271) Alternative DNA Sequence:
ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCG GACAGGTTACTCTGAAAGAGTCTGGCCCTGCGCTAGTGAAGCCCACCCAGACCCTCAC TCTGACTTGTACTTTCfrCTGGGTTTTCACTGAGCACTTCTGGTATGGG^GTGAGCTG GATTCGTCAGCCTCCAGGAAAGGCTCTGGAGTGGCTGGCA|CACATTTACTGGGATGA |TGACAAGCG(trATAACCCATCCCTGAAGAGCCGGCTCACAATCACCAAGGATACCTCC AAGAACCAGGTAGTCCTCACGATGACCAATATGGACCCTGTAGATACTGCCACATAC TACtrGTGCTCGAAGACCTATTACTACGGTAGTAGCTCCATATTTCTATGCTATGGAC ITACTGGGGTCAAIGGAACCTCAGTCACCGTCTCCTCAGGTGGAGGAGGTTCTGGAGGC GGTGGGTCCGGGGGAGGTGGCTCCGACATTCAGATGACCCAGTCTCCAAGCTCCCTGT CCGCATCAGTAGGAGACAGGGTCACCATCACCTGCAAGGCC|AGTCAGAATGTGGCTA| ^TACTjGTGGCCTGGTTTCAACAGAAACCAGGGAAAGCTCCTAAAAGCCTGATllTACAl |GCGCATCCTA^TTGCAGAGTGGAGTCCCTAGTCGCTTCAGCGGCAGTGGATCTGGGA CAGATTTCACTCTCACCATCAGCAGTCTGCAGCCTGAAGACTTTGCAACGTATTACTG TlCAGCAATATAACAGCTATCCAhTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAA ACTCGAGGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAG CCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTA ACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGT GACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCT ATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAAGCTTAGAGTGAAGTTCAGCAG GAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAAT CTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAG ATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTG CAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGG AGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACC TACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGCCGGCGCCAAAAGGTCTGGCT CCGGTGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCC CTAGAATGCCACCTCCTCGCCTCCTCTTCTTCCTCCTCTTCCTCACCCCCATGGAAGTC AGGCCCGAGGAACCTCTAGTGGTGAAGGTGGAAGAGGGAGATAACGCTGTGCTGCAG TGCCTCAAGGGGACCTCAGATGGCCCCACTCAGCAGCTGACCTGGTCTCGGGAGTCCC CGCTTAAACCCTTCTTAAAACTCAGCCTGGGGCTGCCAGGCCTGGGAATCCACATGAG GCCCCTGGCCATCTGGCTTTTCATCTTCAACGTCTCTCAACAGATGGGGGGCTTCTAC CTGTGCCAGCCGGGGCCCCCCTCTGAGAAGGCCTGGCAGCCTGGCTGGACAGTCAATG TGGAGGGCAGCGGGGAGCTGTTCCGGTGGAATGTTTCGGACCTAGGTGGCCTGGGCT GTGGCCTGAAGAACAGGTCCTCAGAGGGCCCCAGCTCCCCTTCCGGGAAGCTCATGAG CCCCAAGCTGTATGTGTGGGCCAAAGACCGCCCTGAGATCTGGGAGGGAGAGCCTCCG TGTCTCCCACCGAGGGACAGCCTGAACCAGAGCCTCAGCCAGGACCTCACCATGGCCC CTGGCTCCACACTCTGGCTGTCCTGTGGGGTACCCCCTGACTCTGTGTCCAGGGGCCC CCTCTCCTGGACCCATGTGCACCCCAAGGGGCCTAAGTCATTGCTGAGCCTAGAGCTG AAGGACGATCGCCCGGCCAGAGATATGTGGGTAATGGAGACGGGTCTGTTGTTGCCC CGGGCCACAGCTCAAGACGCTGGAAAGTATTATTGTCACCGTGGCAACCTGACCATG TCATTCCACCTGGAGATCACTGCTCGGCCAGTACTATGGCACTGGCTGCTGAGGACTG GTGGCTGGAAGGTCTCAGCTGTGACTTTGGCTTATCTGATCTTCTGCCTGTGTTCCCT TGTGGGCATTCTTCATCTTCAAAGAGCCCTGGTCCTGAGGAGGAAAAGAAAGCGAAT GACTTAA LS-h3H2scFvLH-CD28H-CD28TM-CD28CS-CD3zICS-T2A-trCD19 (SEQID NO: 574) Protein Sequence: METPAQLLFLLLLWLPDTTGDIQMTQSPSSLSASVGDRVTITCKA|SQNVATT1VAWFQ QKPGKAPKSLl|YSASYtQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC|QQYNSYP|FT FGSGTKLEIKGGGGSGGGGSGGGGSQVTLKESGPALVKPTQTLTLTCTF^GFSLSTSGM! QVSWIRQPPGKALEWLA|HIYWDDDKR|YNPSLKSRLTITKDTSKNQVVLTMTNMDPV DTATYY|CARRPITTWAPYFYAMDYWGQ|GTSVTVSSLEVKGKHLCPSPLFPGPSKPFW VLVWGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPP RDFAAYRSKLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPRAGAKRSGSGEGRGSLLTCGDVEENPGPRMPPPRLLFFLLFLTPMEVRP EEPLWKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPL AIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCG LKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPG STLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPR ATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVSAVTLAYLIFCLCSL VGILHLQRALVLRRKRKRMT (SEQID NO: 674) DNA Sequence: ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCG GAGACATTCAGATGACCCAGTCTCCAAGCTCCCTGTCCGCATCAGTAGGAGACAGGGT CACCATCACCTGCAAGGCdAGTCAGAATGTGGCTACTAC^GTGGCCTGGTTTCAACA GAAACCAGGGAAAGCTCCTAAAAGCCTGATllTACAGCGCATCCTACtrTGCAGAGTGG AGTCCCTAGTCGCTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGC AGTCTGCAGCCTGAAGACTTTGCAACGTATTACTGT|CAGCAATATAACAGCTATCCA TTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAAGGTGGTGGTGGTTCTGGCGGC GGCGGCTCCGGTGGTGGTGGTTCCCAGGTTACTCTGAAAGAGTCTGGCCCTGCGCTAG TGAAGCCCACCCAGACCCTCACTCTGACTTGTACTTTCfrCTGGGTTTTCACTGAGCACl TTCTGGTATGGGTjGTGAGCTGGATTCGTCAGCCTCCAGGAAAGGCTCTGGAGTGGCT GGCA|CACATTTACTGGGATGATGACAAGCG5TATAACCCATCCCTGAAGAGCCGGCT CACAATCACCAAGGATACCTCCAAGAACCAGGTAGTCCTCACGATGACCAATATGGA CCCTGTAGATACTGCCACATACTACtrGTGCTCGAAGACCTATTACTACGGTAGTAGC TCCATATTTCTATGCTATGGACTACTGGGGTCAAlGGAACCTCAGTCACCGTCTCCTCA CTCGAGGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGC CCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAA CAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTG ACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTA TGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAAGCTTAGAGTGAAGTTCAGCAGG AGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATC TAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGA TGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGC AGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGA GGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCT ACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGCCGGCGCCAAAAGGTCTGGCTC CGGTGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCC TAGAATGCCACCTCCTCGCCTCCTCTTCTTCCTCCTCTTCCTCACCCCCATGGAAGTCA GGCCCGAGGAACCTCTAGTGGTGAAGGTGGAAGAGGGAGATAACGCTGTGCTGCAGT GCCTCAAGGGGACCTCAGATGGCCCCACTCAGCAGCTGACCTGGTCTCGGGAGTCCCC GCTTAAACCCTTCTTAAAACTCAGCCTGGGGCTGCCAGGCCTGGGAATCCACATGAGG CCCCTGGCCATCTGGCTTTTCATCTTCAACGTCTCTCAACAGATGGGGGGCTTCTACC TGTGCCAGCCGGGGCCCCCCTCTGAGAAGGCCTGGCAGCCTGGCTGGACAGTCAATGT GGAGGGCAGCGGGGAGCTGTTCCGGTGGAATGTTTCGGACCTAGGTGGCCTGGGCTG TGGCCTGAAGAACAGGTCCTCAGAGGGCCCCAGCTCCCCTTCCGGGAAGCTCATGAGC CCCAAGCTGTATGTGTGGGCCAAAGACCGCCCTGAGATCTGGGAGGGAGAGCCTCCG TGTCTCCCACCGAGGGACAGCCTGAACCAGAGCCTCAGCCAGGACCTCACCATGGCCC CTGGCTCCACACTCTGGCTGTCCTGTGGGGTACCCCCTGACTCTGTGTCCAGGGGCCC CCTCTCCTGGACCCATGTGCACCCCAAGGGGCCTAAGTCATTGCTGAGCCTAGAGCTG AAGGACGATCGCCCGGCCAGAGATATGTGGGTAATGGAGACGGGTCTGTTGTTGCCC CGGGCCACAGCTCAAGACGCTGGAAAGTATTATTGTCACCGTGGCAACCTGACCATG TCATTCCACCTGGAGATCACTGCTCGGCCAGTACTATGGCACTGGCTGCTGAGGACTG GTGGCTGGAAGGTCTCAGCTGTGACTTTGGCTTATCTGATCTTCTGCCTGTGTTCCCT TGTGGGCATTCTTCATCTTCAAAGAGCCCTGGTCCTGAGGAGGAAAAGAAAGCGAAT GACTTAA
(SEQ ID NO: 274) Alternative DNA Sequence: ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCG GAGACATTCAGATGACCCAGTCTCCAAGCTCCCTGTCCGCATCAGTAGGAGACAGGGT CACCATCACCTGCAAGGCdAGTCAGAATGTGGCTACTAC^GTGGCCTGGTTTCAACA GAAACCAGGGAAAGCTCCTAAAAGCCTGATTlTACAGCGCATCCTACtrTGCAGAGTGG AGTCCCTAGTCGCTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGC AGTCTGCAGCCTGAAGACTTTGCAACGTATTACTGTICAGCAATATAACAGCTATCCA TTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAAGGTGGAGGAGGTTCTGGAGGC GGTGGGTCCGGGGGAGGTGGCTCCCAGGTTACTCTGAAAGAGTCTGGCCCTGCGCTA GTGAAGCCCACCCAGACCCTCACTCTGACTTGTACTTTCfrCTGGGTTTTCACTGAGCA CTTCTGGTATGGGljGTGAGCTGGATTCGTCAGCCTCCAGGAAAGGCTCTGGAGTGGC TGGCAjCACATTTACTGGGATGATGACAAGCGCfrATAACCCATCCCTGAAGAGCCGGC TCACAATCACCAAGGATACCTCCAAGAACCAGGTAGTCCTCACGATGACCAATATGG ACCCTGTAGATACTGCCACATACTAC^GTGCTCGAAGACCTATTACTACGGTAGTAGl CTCCATATTTCTATGCTATGGACTACTGGGGTCAA|GGAACCTCAGTCACCGTCTCCTC ACTCGAGGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAG CCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTA ACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGT GACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCT ATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAAGCTTAGAGTGAAGTTCAGCAG GAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAAT CTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAG ATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTG CAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGG AGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACC TACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGCCGGCGCCAAAAGGTCTGGCT CCGGTGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCC CTAGAATGCCACCTCCTCGCCTCCTCTTCTTCCTCCTCTTCCTCACCCCCATGGAAGTC AGGCCCGAGGAACCTCTAGTGGTGAAGGTGGAAGAGGGAGATAACGCTGTGCTGCAG TGCCTCAAGGGGACCTCAGATGGCCCCACTCAGCAGCTGACCTGGTCTCGGGAGTCCC CGCTTAAACCCTTCTTAAAACTCAGCCTGGGGCTGCCAGGCCTGGGAATCCACATGAG
GCCCCTGGCCATCTGGCTTTTCATCTTCAACGTCTCTCAACAGATGGGGGGCTTCTAC CTGTGCCAGCCGGGGCCCCCCTCTGAGAAGGCCTGGCAGCCTGGCTGGACAGTCAATG TGGAGGGCAGCGGGGAGCTGTTCCGGTGGAATGTTTCGGACCTAGGTGGCCTGGGCT GTGGCCTGAAGAACAGGTCCTCAGAGGGCCCCAGCTCCCCTTCCGGGAAGCTCATGAG CCCCAAGCTGTATGTGTGGGCCAAAGACCGCCCTGAGATCTGGGAGGGAGAGCCTCCG TGTCTCCCACCGAGGGACAGCCTGAACCAGAGCCTCAGCCAGGACCTCACCATGGCCC CTGGCTCCACACTCTGGCTGTCCTGTGGGGTACCCCCTGACTCTGTGTCCAGGGGCCC CCTCTCCTGGACCCATGTGCACCCCAAGGGGCCTAAGTCATTGCTGAGCCTAGAGCTG AAGGACGATCGCCCGGCCAGAGATATGTGGGTAATGGAGACGGGTCTGTTGTTGCCC CGGGCCACAGCTCAAGACGCTGGAAAGTATTATTGTCACCGTGGCAACCTGACCATG TCATTCCACCTGGAGATCACTGCTCGGCCAGTACTATGGCACTGGCTGCTGAGGACTG GTGGCTGGAAGGTCTCAGCTGTGACTTTGGCTTATCTGATCTTCTGCCTGTGTTCCCT TGTGGGCATTCTTCATCTTCAAAGAGCCCTGGTCCTGAGGAGGAAAAGAAAGCGAAT GACTTAA
APPENDIX-2: AMINO ACID AND NUCLEIC ACID SEQUENCES
(continued)
Affinity matured anti-ALPPL2 antibodles|
Anti-ALPPL2 Clone IBS (humanized VH + mouse VL):
Anti-ALPPL2 Clone IBS Heavy chain variable region (also referred to as IBS VH)
(SEQ ID NO: 701) Protein Sequence:
QVTLKESGPALVKPTQTLTLTCTflSGFSLSTSGM(jVSWIROPPGKALEWLA|HIYWDDDKR|YNPSLK
SRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFYAMDYWGQ|GTSVTVSS
Anti-ALPPL2 Clone IBS VH CDR 1 (IBS CDR-H1) (SEQ ID NO: 702) Protein Sequence: |SGFSLSTSGMCj
Anti-ALPPL2 Clone IBS VH CDR 2 (IBS CDR-H2) (SEQ ID NO: 703) Protein Sequence: |HIYWDDDKR|
Anti-ALPPL2 Clone IBS VH CDR 3 (IBS CDR-H3) (SEQ ID NO: 704) Protein Sequence: ICARRPITTSVAPYFYAMDYWGQI
Anti-ALPPL2 Clone 1B5 Light chain variable (IBS VL)
(SEQ ID NO: 705) Protein Sequence:
DIVMTQSQKFMSTSVGDRVSVTCKAlSQNVATTjVAWYQQKPGQSPKALllYSASYlRYSG VPDRFTGSGSGTDFTLTISNVQSEDLAEYFdQQYNSYP|FTFGSGTKLEIK
(SEQ ID NO: 805) DNA Sequence:
GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTC AGCGTCACCTGCAAGGCCjAGTCAGAATGTGGCTACTAClXlTGGCCTGGTATCAACAG AAACCAGGGCAATCTCCTAAAGCACTGATTfTACTCGGCATCCTAC|CGGTACAGTGGA GTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCA ATGTGCAGTCTGAAGACTTGGCAGAGTATTTCTGljCAGCAATATAACAGCTATCCAh’ TCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA
Anti-ALPPL2 Clone IBS VL CDR 1 (IBS CDR-L1)
(SEQ ID NO: 706) Protein Sequence:
[SQNVATTl
(SEQ ID NO: 806) DNA Sequence: lAGTCAGAATGTGGCTACTACll
Anti-ALPPL2 Clone IBS VL CDR 2 (IBS CDR-L2)
(SEQ ID NO: 707) Protein Sequence:
IYSASYI
(SEQ ID NO: 807) DNA Sequence: iTACTCGGCATCCTACl Anti-ALPPL2 Clone IBS VL CDR 3 (IBS CDR-L3) (SEQ ID NO: 708) Protein Sequence:
(SEQ ID NO: 808) DNA Sequence: ICAGCAATATAACAGCTATCCA
Humanized anti-ALPPL2 Clone B001 :
Humanized anti-ALPPL2, Clone B001, Heavy chain variable region (also referred to as B001 VH)
(SEQ ID NO: 711) Protein Sequence:
QVTLKESGPALVKPTQTLTLTCTFtSGFSLSTSGMGtVSWIRQPPGKALEWLA>iIYWDDDKR)YN
PSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFYAMDYWGQ|GTSVTV ss
Humanized anti-ALPPL2, Clone B001 VH CDR 1 (B001 CDR-H1)
(SEQID NO: 712) Protein Sequence:
ISGFSLSTSGM^
Humanized anti-ALPPL2J Clone B001, VH CDR 2 (B001 CDR-H2)
(SEQID NO: 713) Protein Sequence:
HIYWDDDKR]
Humanized anti-ALPPL2, Clone B001, VH CDR 3 (B001 CDR-H3)
(SEQID NO: 714) Protein Sequence:
ICARRPITTSVAPYFYAMDYWG^
Humanized anti-ALPPL2, Clone B001, Light chain variable (B001 VL)
(SEQID NO: 715) Protein Sequence:
DIQMTQSPSSLSASVGDRVTITCKA|SQNVAf^VAWFQQKPGQAPKSLl|YSASY|RQSGV
PSRFSGSGSGTDFTLTISSLQSEDFATYFdQQYNSYPlFTFGSGTKLEIK
Humanized anti-ALPPL2, Clone B001, VL CDR 1 (B001 CDR-L1)
(SEQID NO: 716) Protein Sequence:
Humanized anti-ALPPL2, Clone B001, VL CDR 2 (B001 CDR-L2) (SEQID NO: 717) Protein Sequence: lYSASYj
Humanized anti-ALPPL2, Clone B001, VL CDR 3 (B001 CDR-L3) (SEQID NO: 718) Protein Sequence:
Humanized anti-ALPPL2, B001 scFv, VH-G4S3-VL (B001 scFvHL, or BOOlscFvHL)
(SEQID NO: 719) Protein Sequence:
QVTLKESGPALVKPTQTLTLTCTF|SGFSLSTSGMG|VSWIRQPPGKALEWLA|HIYWDD|
|[)KR}YNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYY|CARRPITTSVAPYFYAMD|
|YWGQ|GTSVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKA|SQNVA,I1
@VAWFQQKPGQAPKSLI|YSASY|RQSGVPSRFSGSGSGTDFTLTISSLQSEDFATYFC|Q^ IYNSYPIFTFGSGTKLEIK Humanized anti-ALPPL2, B001 scFv, VL-G4S3-VH (B001 scFvLH, or BOOlscFvLH)
(SEQID NO: 720) Protein Sequence:
DIQMTQSPSSLSASVGDRVTITCKAlSQNVATTjVAWFQQKPGQAPKSLllYSASYlRQSGV
PSRFSGSGSGTDFTLTISSLQSEDFATYFC •|SQMQYNBSYEP|rFTFGSGTKLEIKGGGGSGGGGSG GGGSQVTLKESGPALVKPTQTLTLTCTFPGFSLSTSGMG|VSWIRQPPGKALEWLA|nY] |WDDDKR1YNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFY| mm GTSVTVSS
Humanized anti-ALPPL2. Clone B380:
Humanized anti-ALPPL2, Clone B380, Heavy chain variable region (also referred to as B380 VH)
(SEQ ID NO: 721) Protein Sequence:
QVTLKESGPALVKPTQTLTLTCTFtSGFSLSTSGMGtVSWIRQPPGKALEWLA>iIYWDDDKR)YN PSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFYSMDYWGQ|GTSVTVS s
Humanized anti-ALPPL2, Clone B380 VH CDR 1 (B380 CDR-H1) (SEQID NO: 722) Protein Sequence:
ISGFSLSTSGMCj
Humanized anti-ALPPL2, Clone B380, VH CDR 2 (B380 CDR-H2) (SEQID NO: 723) Protein Sequence:
Humanized anti-ALPPL2, Clone B380, VH CDR 3 (B380 CDR-H3) (SEQID NO: 724) Protein Sequence:
ICARRPITTSVAPYFYSMDYWGQ]
Humanized anti-ALPPL2, Clone B380, Light chain variable (B380 VL)
(SEQID NO: 725) Protein Sequence:
DIQMTQSPSSLSASVGDRVTITCKAlSQNVAf^VAWFQQKPGQAPKSLl|YSASY|RQSGV
PSRFSGSGSGTDFTLTISSLQSEDFATYFdQQYNSY^FTFGSGTKLEIK
Humanized anti-ALPPL2, Clone B380, VL CDR 1 (B380 CDR-L1) (SEQID NO: 726) Protein Sequence: jSQNVATlI
Humanized anti-ALPPL2, Clone B380, VL CDR 2 (B380 CDR-L2) (SEQID NO: 727) Protein Sequence:
|YSASY|
Humanized anti-ALPPL2, Clone B380, VL CDR 3 (B380 CDR-L3) (SEQID NO: 728) Protein Sequence:
Humanized anti-ALPPL2, B380 scFv, VH-G4S3-VL (B380 scFv HL, or B380scFvHL)
(SEQID NO: 729) Protein Sequence:
QVTLKESGPALVKPTQTLTLTCTF|SGFSLSTSGMG|VSWIRQPPGKALEWLA|HIYWDD|
|DKR|YNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYY|CARRPITTSVAPYFYSMD| |YWGQ|GTSVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKA|SQNVAT
@VAWFQQKPGQAPKSLI|YSASY}RQSGVPSRFSGSGSGTDFTLTISSLQSEDFATYFC|QQ
|YNSYP|FTFGSGTKLEIK
Humanized anti-ALPPL2, B380 scFv, VL-G4S3-VH (B380 scFv LH, or
B380scFvLH)
(SEQID NO: 730) Protein Sequence:
DIQMTQSPSSLSASVGDRVTITCKA|SQNVAfgVAWFQQKPGQAPKSLl|YSASY|RQSGV
PSRFSGSGSGTDFTLTISSLQSEDFATYFC|QQYNSYP|FTFGSGTKLEIKGGGGSGGGGSG GGGSQVTLKESGPALVKPTQTLTLTCTF^GFSLSTSGMGlVSWIRQPPGKALEWLA^iY] |WDDDKR1YNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFY|
GTSVTVSS
Humanized anti-ALPPL2. Clone B416:
Humanized anti-ALPPL2, Clone B416, Heavy chain variable region (also referred to as 8416 VH)
(SEQ ID NO: 731) Protein Sequence:
QVTLKESGPALVKPTQTLTLTCTFtSGFSLSTSGMGtVSWIRQPPGKALEWLA>iIYWDDDKR)YN PSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFYSYDYWGQ|GTSVTVS s
Humanized anti-ALPPL2, Clone B416 VH CDR 1 (B416 CDR-H1) (SEQID NO: 732) Protein Sequence:
ISGFSLSTSGMCj
Humanized anti-ALPPL2, Clone B416, VH CDR 2 (B416 CDR-H2) (SEQID NO: 733) Protein Sequence:
Humanized anti-ALPPL2, Clone B416, VH CDR 3 (B416 CDR-H3) (SEQID NO: 734) Protein Sequence:
ICARRPITTSVAPYFYSYDYWGQ]
Humanized anti-ALPPL2, Clone B416, Light chain variable (B416 VL)
(SEQID NO: 735) Protein Sequence:
DIQMTQSPSSLSASVGDRVTITCKAlSQNVAf^VAWFQQKPGQAPKSLl|YSASY|RQSGV
PSRFSGSGSGTDFTLTISSLQSEDFATYFdQQYNSY^FTFGSGTKLEIK
Humanized anti-ALPPL2, Clone B416, VL CDR 1 (B416 CDR-L1) (SEQID NO: 736) Protein Sequence: jSQNVATlI
Humanized anti-ALPPL2, Clone B416, VL CDR 2 (B416 CDR-L2) (SEQID NO: 737) Protein Sequence:
|YSASY|
Humanized anti-ALPPL2, Clone B416, VL CDR 3 (B416 CDR-L3) (SEQID NO: 738) Protein Sequence:
Humanized anti-ALPPL2, B416 scFv, VH-G4S3-VL (B416 scFv HL, or B416scFvHL)
(SEQID NO: 739) Protein Sequence:
QVTLKESGPALVKPTQTLTLTCTF|SGFSLSTSGMG|VSWIRQPPGKALEWLA|HIYWDD|
|DKR|YNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYY|CARRPITTSVAPYFYSYDY| VAWFQQKPGQAPKSLI|YSASY|RQSGVPSRFSGSGSGTDFTLTISSLQSEDFATYF
|NSYP|FTFGSGTKLEIK
Humanized anti-ALPPL2, B416 scFv, VL-G4S3-VH (B416 scFv LH, or B416scFvLH)
(SEQID NO: 740) Protein Sequence:
DIQMTQSPSSLSASVGDRVTITCKA|SQNVAfgVAWFQQKPGQAPKSLl|YSASY|RQSGV PSRFSGSGSGTDFTLTISSLQSEDFATYFC|QQYNSYP|FTFGSGTKLEIKGGGGSGGGGSG GGGSQVTLKESGPALVKPTQTLTLTCTF^GFSLSTSGMGlVSWIRQPPGKALEWLA^iY] |WDDDKR1YNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFY| bTSVTVSS
Humanized anti-ALPPL2. Clone BS06:
Humanized anti-ALPPL2, Clone B506, Heavy chain variable region (also referred to as
B506 VH)
(SEQ ID NO: 741) Protein Sequence:
QVTLKESGPALVKPTQTLTLTCTFtSGFSLSTSGMGtVSWIRQPPGKALEWLA>iIYWDDDKR)YN PSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFYAYDYWGQ|GTSVTVS s
Humanized anti-ALPPL2, Clone B506 VH CDR 1 (B506 CDR-H1) (SEQID NO: 742) Protein Sequence:
ISGFSLSTSGMCj
Humanized anti-ALPPL2, Clone B506, VH CDR 2 (B506 CDR-H2) (SEQID NO: 743) Protein Sequence: HIYWDDDKRI
Humanized anti-ALPPL2, Clone B506, VH CDR 3 (B506 CDR-H3) (SEQID NO: 744) Protein Sequence: ICARRPITTSVAPYFYAYDYWG^
Humanized anti-ALPPL2, Clone B506, Light chain variable (B506 VL)
(SEQID NO: 745) Protein Sequence:
DIQMTQSPSSLSASVGDRVTITCKAlSQNVAfgVAWFQQKPGQAPKSLllYSASYlRQSGV
PSRFSGSGSGTDFTLTISSLQSEDFATYFC|QQYNSYP|FTFGSGTKLEIK
Humanized anti-ALPPL2, Clone B506, VL CDR 1 (B506 CDR-L1) (SEQID NO: 746) Protein Sequence:
ISQNVATTI
Humanized anti-ALPPL2, Clone B506, VL CDR 2 (B506 CDR-L2) (SEQID NO: 747) Protein Sequence:
|YSASY|
Humanized anti-ALPPL2, Clone B506, VL CDR 3 (B506 CDR-L3) (SEQID NO: 748) Protein Sequence:
IQQYNSYPI
Humanized anti-ALPPL2, B506 scFv, VH-G4S3-VL (B506 scFv HL, or
B506scFvHL)
(SEQID NO: 749) Protein Sequence:
QVTLKESGPALVKPTQTLTLTCTF|SGFSLSTSGMG|VSWIRQPPGKALEWLA|HIYWDD| > >
IK1 PSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFYAYD| }TSVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKA|SQNVAT| @VAWFQQKPGQAPKSLI|YSASY}RQSGVPSRFSGSGSGTDFTLTISSLQSEDFATYFC|Q^ |YNSYP|FTFGSGTKLEIK
Humanized anti-ALPPL2, B506 scFv, VL-G4S3-VH (B506 scFv LH, or B506scFvLH)
(SEQID NO: 750) Protein Sequence: >
DIQMTQSPSSLSASVGDRVTITCKA|SQNVATTjVAWFQQKPGQAPKSLl|YSASY|RQSGV PSRFSGSGSGTDFTLTISSLQSEDFATYFC|QQYNSYP|FTFGSGTKLEIKGGGGSGGGGSG GGGSQVTLKESGPALVKPTQTLTLTCTF^GFSLSTSGMG|VSWIRQPPGKALEWLAfnY] YNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYYICARRPITTSVAPYFYI IAYDYWGQIGTSVTVSS
Humanized anti-ALPPL2. Clone B735:
Humanized anti-ALPPL2, Clone B735, Heavy chain variable region (also referred to as 8735 VH)
(SEQ ID NO: 751) Protein Sequence:
QVTLKESGPALVKPTQTLTLTCTFtSGFSLSTSGMGtVSWIRQPPGKALEWLA>iIYWDDDKR)YN
PSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFYSFDYWGQ|GTSVTVS s
Humanized anti-ALPPL2, Clone B735 VH CDR 1 (B735 CDR-H1) (SEQID NO: 752) Protein Sequence:
ISGFSLSTSGMCj
Humanized anti-ALPPL2, Clone B735, VH CDR 2 (B735 CDR-H2) (SEQID NO: 753) Protein Sequence: HIYWDDDKR]
Humanized anti-ALPPL2, Clone B735, VH CDR 3 (B735 CDR-H3) (SEQID NO: 754) Protein Sequence: ICARRPITTSVAPYFYSFDYWGQ
Humanized anti-ALPPL2, Clone B735, Light chain variable (B735 VL)
(SEQID NO: 755) Protein Sequence:
DIQMTQSPSSLSASVGDRVTITCKAlSQNVAf^VAWFQQKPGQAPKSLl|YSASY|RQSGV
PSRFSGSGSGTDFTLTISSLQSEDFATYFdQQYNSY^FTFGSGTKLEIK
Humanized anti-ALPPL2, Clone B735, VL CDR 1 (B735 CDR-L1) (SEQID NO: 756) Protein Sequence: jSQNVATTl
Humanized anti-ALPPL2, Clone B735, VL CDR 2 (B735 CDR-L2) (SEQID NO: 757) Protein Sequence:
|YSASY|
Humanized anti-ALPPL2, Clone B735, VL CDR 3 (B735 CDR-L3) (SEQID NO: 758) Protein Sequence:
Humanized anti-ALPPL2, B735 scFv, VH-G4S3-VL (B735 scFvHL, or B735scFvHL)
(SEQID NO: 759) Protein Sequence:
QVTLKESGPALVKPTQTLTLTCTF|SGFSLSTSGMG|VSWIRQPPGKALEWLA|HIYWDD|
|DKR|YNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYY|CARRPITTSVAPYFYSFDY| VAWFQQKPGQAPKSLI|YSASY|RQSGVPSRFSGSGSGTDFTLTISSLQSEDFATYF
|NSYP|FTFGSGTKLEIK
Humanized anti-ALPPL2, B735 scFv, VL-G4S3-VH (B735 scFvLH, or
B735scFvLH)
(SEQID NO: 760) Protein Sequence:
DIQMTQSPSSLSASVGDRVTITCKA|SQNVAfgVAWFQQKPGQAPKSLl|YSASY|RQSGV
PSRFSGSGSGTDFTLTISSLQSEDFATYFC|QQYNSYP|FTFGSGTKLEIKGGGGSGGGGSG GGGSQVTLKESGPALVKPTQTLTLTCTF^GFSLSTSGMGlVSWIRQPPGKALEWLA^iY] |WDDDKR1YNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFY|
GTSVTVSS
APPENDIX-3: AMINO ACID AND NUCLEIC ACID SEQUENCES
(continued)
Anti-ALPPLZ CARs using affinity matured VH and VI
CARs comprising BOOlscFvHL:
B001scFvHL-CD28H-CD28TM-CD28CS-CD3zICS
(SEQID NO: 911) Protein Sequence:
QVTLKESGPALVKPTQTLTLTCTF|SGFSLSTSGMG|VSWIROPPGKALEWLA|HIYWDD| |DKgYNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFYAMD| IYWGQ|GTSVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKA|SQNVAT1 0VAWFQQKPGQAPKSLI|YSASY|RQSGVPSRFSGSGSGTDFTLTISSLQSEDFATYFC|QQ |YNSYP|FTFGSGTKLEIKLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFI IFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKLRVKFSRSADA PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
B001scFvHL-CD28H-CD28TM-41BBCS-CD3zICS
(SEQID NO: 912) Protein Sequence:
QVTLKESGPALVKPTQTLTLTCTF|SGFSLSTSGMG|VSWIRQPPGKALEWLA|HIYWDD|
Y |DWKRG|YQNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFYAMD| GTSVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKAlSQNVATl
TVAWFQQKPGQAPKSLI|YSASY|RQSGVPSRFSGSGSGTDFTLTISSLQSEDFATYFC|Q^ |YNSYP|FTFGSGTKLEIKLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFI IFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPA YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
B001scFvHL-CD28H-CD28TM-DAP10CS-CD3zICS
(SEQID NO: 913) Protein Sequence:
QVTLKESGPALVKPTQTLTLTCTF|SGFSLSTSGMG|VSWIRQPPGKALEWLA|HIYWDD| [DKR}YNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFYAMD| |YWGQ|GTSVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKA|SQNVA,I1 @VAWFQQKPGQAPKSLI|YSASY|RQSGVPSRFSGSGSGTDFTLTISSLQSEDFATYFC|Q^ ^NSYgFTFGSGTKLEIKLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFI IFWVLCARPRRSPAQEDGKVYINMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRR EEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALPPR CARs comprising BOOlscFvLH:
B001scFvLH-CD28H-CD28TM-CD28CS-CD3zICS
(SEQID NO: 914) Protein Sequence:
DIQMTQSPSSLSASVGDRVTITCKA|SQNVAf^VAWFQQKPGQAPKSLl|YSASY|RQSGV
PSRFSGSGSGTDFTLTISSLQSEDFATYFC •|SQMQYNBSYEP|rFTFGSGTKLEIKGGGGSGGGGSG GGGSQVTLKESGPALVKPTQTLTLTCTFPGFSLSTSGMG|VSWIRQPPGKALEWLA|nY] |WDDDKR|YNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFY|
GTSVTVSSLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFI
IFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKLRVKFSRSADA PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
B001scFvLH-CD28H-CD28TM-41BBCS-CD3zICS
(SEQID NO: 915) Protein Sequence:
DIQMTQSPSSLSASVGDRVTITCKAlSQNVATTjVAWFQQKPGQAPKSLllYSASYlRQSGV PSRFSGSGSGTDFTLTISSLQSEDFATYFC|QQYNSYP|FTFGSGTKLEIKGGGGSGGGGSG GGGSQVTLKESGPALVKPTQTLTLTCTF^GFSLSTSGMG|VSWIRQPPGKALEWLApiY] WDDDKR|YNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYY|CARRPITTSVAPYFY| AMDYWGQIGTSVTVSSLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFI IFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPA YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
B001scFvLH-CD28H-CD28TM-DAP10CS-CD3zICS
(SEQID NO: 916) Protein Sequence:
DIQMTQSPSSLSASVGDRVTITCKA1SQNVAT^VAWFQQKPGQAPKSLI|YSASY|RQSGV PSRFSGSGSGTDFTLTISSLQSEDFATYFC|QQYNSYP|FTFGSGTKLEIKGGGGSGGGGSG GGGSQVTLKESGPALVKPTQTLTLTCTF^GFSLSTSGMG|VSWIRQPPGKALEWLA|HiYl WDDDKR|YNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFY| AMDYWGQIGTSVTVSSLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFI IFWVLCARPRRSPAQEDGKVYINMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRR EEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALPPR
CARs comprising B380scFvHL:
B380scFvHL-CD28H-CD28TM-CD28CS-CD3zI0S
(SEQID NO: 921) Protein Sequence:
QVTLKESGPALVKPTQTLTLTCTF|SGFSLSTSGM(jVSWIRQPPGKALEWLA|HIYWDD| iKl PSLKSRLTITKDTSKNQWLTMTNMDPVDTATYYlCARRPITTSVAPYFYSMDl _ }TSVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKA|SQNVAT| @VAWFQQKPGQAPKSLI|YSASY}RQSGVPSRFSGSGSGTDFTLTISSLQSEDFATYFC|Q^ ^NSYgFTFGSGTKLEIKLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFI IFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKLRVKFSRSADA PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
B380scFvHL-CD28H-CD28TM-41BBCS-CD3zICS
(SEQID NO: 922) Protein Sequence:
QVTLKESGPALVKPTQTLTLTCTF|SGFSLSTSGMG|VSWIRQPPGKALEWLA|HIYWDD| |DKgYNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFYSMD| |YWGQ|GTSVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKA|SQNVA,I1 @VAWFQQKPGQAPKSLI|YSASY|RQSGVPSRFSGSGSGTDFTLTISSLQSEDFATYFC|Q^ ^NSY^FTFGSGTKLEIKLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFI IFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPA YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
B380scFvHL-CD28H-CD28TM-DAP10CS-CD3zICS
(SEQID NO: 923) Protein Sequence:
QVTLKESGPALVKPTQTLTLTCTF|SGFSLSTSGMG|VSWIRQPPGKALEWLA|HIYWDD| |DK§YNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFYSMD| |YWGQ|GTSVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKA
@VAWFQQKPGQAPKSLI|YSASY|RQSGVPSRFSGSGSGTDFTLTISSLQSEDFATYFC|Q^ |YNSYP|FTFGSGTKLEIKLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFI IFWVLCARPRRSPAQEDGKVYINMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRR EEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALPPR
CARs comprising B380scFvLH:
B380scFvLH-CD28H-CD28TM-CD28CS-CD3zICS
(SEQID NO: 924) Protein Sequence:
DIQMTQSPSSLSASVGDRVTITCKAlSQNVATTjVAWFQQKPGQAPKSLllYSASYlRQSGV
PSRFSGSGSGTDFTLTISSLQSEDFATYFC •|SQMQYNBSYEP|rFTFGSGTKLEIKGGGGSGGGGSG
GGGSQVTLKESGPALVKPTQTLTLTCTFPGFSLSTSGMG|VSWIRQPPGKALEWLA|nY]
|WDDDKR|YNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFY|
GTSVTVSSLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFII
FWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKLRVKFSRSADAP AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
B380scFvLH-CD28H-CD28TM-41BBCS-CD3zICS
(SEQID NO: 925) Protein Sequence:
DIQMTQSPSSLSASVGDRVTITCKAlSQNVATTjVAWFQQKPGQAPKSLllYSASYlRQSGV PSRFSGSGSGTDFTLTISSLQSEDFATYFC|QQYNSYP|FTFGSGTKLEIKGGGGSGGGGSG GGGSQVTLKESGPALVKPTQTLTLTCTF^GFSLSTSGMG|VSWIRQPPGKALEWLApiY] |WDDDKR|YNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY1CARRPITTSVAPYFY| ISMDYWGQIGTSVTVSSLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFII FWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAY QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
B380scFvLH-CD28H-CD28TM-DAP10CS-CD3zICS
(SEQID NO: 926) Protein Sequence:
DIQMTQSPSSLSASVGDRVTITCKA1SQNVAT^VAWFQQKPGQAPKSLI|YSASY|RQSGV PSRFSGSGSGTDFTLTISSLQSEDFATYFC|QQYNSYP|FTFGSGTKLEIKGGGGSGGGGSG GGGSQVTLKESGPALVKPTQTLTLTCTF^GFSLSTSGMG|VSWIRQPPGKALEWLA|HiYl |WDDDKR|YNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFY| ISMDYWGQIGTSVTVSSLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFII FWVLCARPRRSPAQEDGKVYINMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRRE EYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG HDGLYQGLSTATKDTYDALHMQALPPR
CARs comprising B416scFvHL:
B416scFvHL-CD28H-CD28TM-CD28CS-CD3zICS
(SEQID NO: 931) Protein Sequence:
QVTLKESGPALVKPTQTLTLTCTF|SGFSLSTSGM(jVSWIRQPPGKALEWLA|HIYWDD| DKR|YNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFYSYDY| WGQjGTSVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKAjSQNVA'nl VAWFQQKPGQAPKSLI|YSASY|RQSGVPSRFSGSGSGTDFTLTISSLQSEDFATYFC|QQYI ^SYBFTFGSGTKLEIKLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFII FWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKLRVKFSRSADAP AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
B416scFvHL-CD28H-CD28TM-41BBCS-CD3zICS
(SEQID NO: 932) Protein Sequence:
QVTLKESGPALVKPTQTLTLTCTF|SGFSLSTSGMG|VSWIRQPPGKALEWLA|HIYWDD| |DKR1YNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFYSYDY| |WGQ|GTSVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKA|SQNVATT| VAWFQQKPGQAPKSLI|YSASY|RQSGVPSRFSGSGSGTDFTLTISSLQSEDFATYFC|QQYI ^SYgFTFGSGTKLEIKLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFII FWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAY
QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
B416scFvHL-CD28H-CD28TM-DAP10CS-CD3zICS
(SEQID NO: 933) Protein Sequence:
QVTLKESGPALVKPTQTLTLTCTF|SGFSLSTSGMG|VSWIRQPPGKALEWLA|HIYWDD| DKR|YNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFYSYDY| WG^GTSVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKAlSQNVATTl VAWFQQKPGQAPKSLI^SAS^RQSGVPSRFSGSGSGTDFTLTISSLQSEDFATYFC^Q^ ^gFTFGSGTKLEIKLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFII FWVLCARPRRSPAQEDGKVYINMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRRE
EYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG HDGLYQGLSTATKDTYDALHMQALPPR
CARs comprising B416scFvLH:
B416scFvLH-CD28H-CD28TM-CD28CS-CD3zICS
(SEQID NO: 934) Protein Sequence:
DIQMTQSPSSLSASVGDRVTITCKA|SQNVAf^VAWFQQKPGQAPKSLl|YSASY|RQSGV PSRFSGSGSGTDFTLTISSLQSEDFATYFC •|SQMQYNBSYEP|rFTFGSGTKLEIKGGGGSGGGGSG GGGSQVTLKESGPALVKPTQTLTLTCTFPGFSLSTSGMG|VSWIRQPPGKALEWLA|nY] |WDDDKR|YNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFY| ISYDYWGQIGTSVTVSSLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFII FWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKLRVKFSRSADAP AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
B416scFvLH-CD28H-CD28TM-41BBCS-CD3zICS
(SEQID NO: 935) Protein Sequence:
DIQMTQSPSSLSASVGDRVTITCKAlSQNVAf^VAWFQQKPGQAPKSLllYSASYlRQSGV PSRFSGSGSGTDFTLTISSLQSEDFATYFdQQYNSYP|FTFGSGTKLEIKGGGGSGGGGSG GGGSQVTLKESGPALVKPTQTLTLTCTF^GFSLSTSGMG|VSWIRQPPGKALEWLApiY] ^VDDDKR|YNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFY| ISYDYWGQIGTSVTVSSLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFII FWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAY QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
B416scFvLH-CD28H-CD28TM-DAP10CS-CD3zICS
(SEQID NO: 936) Protein Sequence:
DIQMTQSPSSLSASVGDRVTITCKA|SQNVAT7jVAWFQQKPGQAPKSLl[YSASY|RQSGV PSRFSGSGSGTDFTLTISSLQSEDFATYFC|QQYNSYP|FTFGSGTKLEIKGGGGSGGGGSG GGGSQVTLKESGPALVKPTQTLTLTCTF^GFSLSTSGMG|VSWIRQPPGKALEWLA|HiY| |WDDDKR|YNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFY| ISYDYWGQIGTSVTVSSLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFII FWVLCARPRRSPAQEDGKVYINMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRRE EYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG
HDGLYQGLSTATKDTYDALHMQALPPR
CARs comprising B506scFvHL:
B506scFvHL-CD28H-CD28TM-CD28CS-CD3zICS
(SEQID NO: 941) Protein Sequence:
QVTLKESGPALVKPTQTLTLTCTF|SGFSLSTSGM(jVSWIRQPPGKALEWLA|HIYWDD| iKl PSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFYAYD|
_ }TSVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKA|SQNVAT| @VAWFQQKPGQAPKSLI|YSASY}RQSGVPSRFSGSGSGTDFTLTISSLQSEDFATYFC|Q^ ^NSYgFTFGSGTKLEIKLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFI IFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKLRVKFSRSADA PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
B506scFvHL-CD28H-CD28TM-41BBCS-CD3zICS
(SEQID NO: 942) Protein Sequence:
QVTLKESGPALVKPTQTLTLTCTF|SGFSLSTSGMG|VSWIRQPPGKALEWLA|HIYWDD| |DKgYNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFYAYD| |YWGQ|GTSVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKA|SQNVA’l1 @VAWFQQKPGQAPKSLI|YSASY|RQSGVPSRFSGSGSGTDFTLTISSLQSEDFATYFC|Q^ ^NSY^FTFGSGTKLEIKLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFI IFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPA YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
B506scFvHL-CD28H-CD28TM-DAP10CS-CD3zICS
(SEQID NO: 943) Protein Sequence:
QVTLKESGPALVKPTQTLTLTCTF|SGFSLSTSGMG|VSWIRQPPGKALEWLA|HIYWDD| |DK§YNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFYAYD| |YWGQ|GTSVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKA
@VAWFQQKPGQAPKSLI|YSASY|RQSGVPSRFSGSGSGTDFTLTISSLQSEDFATYFC|Q^ |YNSYP|FTFGSGTKLEIKLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFI IFWVLCARPRRSPAQEDGKVYINMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRR EEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALPPR
CARs comprising BSOfiscFvLH:
B506scFvLH-CD28H-CD28TM-CD28CS-CD3zICS
(SEQID NO: 944) Protein Sequence:
DIQMTQSPSSLSASVGDRVTITCKA|SQNVAf^VAWFQQKPGQAPKSLl|YSASY|RQSGV PSRFSGSGSGTDFTLTISSLQSEDFATYFC •|SQMQYNBSYEP|rFTFGSGTKLEIKGGGGSGGGGSG GGGSQVTLKESGPALVKPTQTLTLTCTFPGFSLSTSGMG|VSWIRQPPGKALEWLA|nY] WDDDKR|YNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFY| AYDYWGQIGTSVTVSSLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFII FWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKLRVKFSRSADAP AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
B506scFvLH-CD28H-CD28TM-41BBCS-CD3zICS
(SEQID NO: 945) Protein Sequence:
DIQMTQSPSSLSASVGDRVTITCKAlSQNVAf^VAWFQQKPGQAPKSLllYSASYlRQSGV PSRFSGSGSGTDFTLTISSLQSEDFATYFdQQYNSYP|FTFGSGTKLEIKGGGGSGGGGSG GGGSQVTLKESGPALVKPTQTLTLTCTF^GFSLSTSGMG|VSWIRQPPGKALEWLApiY] WDDDKR|YNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFY| AYDYWGQIGTSVTVSSLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFII FWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAY QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
B506scFvLH-CD28H-CD28TM-DAP10CS-CD3zICS
(SEQID NO: 946) Protein Sequence:
DIQMTQSPSSLSASVGDRVTITCKA|SQNVAT7jVAWFQQKPGQAPKSLl[YSASY|RQSGV PSRFSGSGSGTDFTLTISSLQSEDFATYFC|QQYNSYP|FTFGSGTKLEIKGGGGSGGGGSG GGGSQVTLKESGPALVKPTQTLTLTCTF^GFSLSTSGMG|VSWIRQPPGKALEWLA|HiY| WDDDKR|YNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFY| AYDYWGQIGTSVTVSSLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFII FWVLCARPRRSPAQEDGKVYINMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRRE EYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG
HDGLYQGLSTATKDTYDALHMQALPPR
CARs comprising B735scFvHL:
B735scFvHL-CD28H-CD28TM-CD28CS-CD3zICS
(SEQID NO: 951) Protein Sequence:
QVTLKESGPALVKPTQTLTLTCTF|SGFSLSTSGM(jVSWIRQPPGKALEWLA|HIYWDD| DKR|YNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFYSFDY| WGQjGTSVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKAjSQNVA'nl VAWFQQKPGQAPKSLI|YSASY|RQSGVPSRFSGSGSGTDFTLTISSLQSEDFATYFC|QQYI ^SYBFTFGSGTKLEIKLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFII FWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKLRVKFSRSADAP AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
B735scFvHL-CD28H-CD28TM-41BBCS-CD3zICS
(SEQID NO: 952) Protein Sequence:
QVTLKESGPALVKPTQTLTLTCTF|SGFSLSTSGMG|VSWIRQPPGKALEWLA|HIYWDD| |DKR1YNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFYSFDY| |WGQ|GTSVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKA|SQNVATT| VAWFQQKPGQAPKSLI|YSASY|RQSGVPSRFSGSGSGTDFTLTISSLQSEDFATYFC|QQYI ^SYgFTFGSGTKLEIKLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFII FWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAY
QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
B735scFvHL-CD28H-CD28TM-DAP10CS-CD3zICS
(SEQID NO: 953) Protein Sequence:
QVTLKESGPALVKPTQTLTLTCTF|SGFSLSTSGMG|VSWIRQPPGKALEWLA|HIYWDD| DKR|YNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFYSFDY| WG^GTSVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKAlSQNVATTl VAWFQQKPGQAPKSLI^SAS^RQSGVPSRFSGSGSGTDFTLTISSLQSEDFATYFC^Q^ ^gFTFGSGTKLEIKLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFII FWVLCARPRRSPAQEDGKVYINMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRRE EYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG HDGLYQGLSTATKDTYDALHMQALPPR
CARs comprising B735scFvLH:
B735scFvLH-CD28H-CD28TM-CD28CS-CD3zICS
(SEQID NO: 954) Protein Sequence:
DIQMTQSPSSLSASVGDRVTITCKA|SQNVAf^VAWFQQKPGQAPKSLl|YSASY|RQSGV PSRFSGSGSGTDFTLTISSLQSEDFATYFC •|SQMQYNBSYEP|rFTFGSGTKLEIKGGGGSGGGGSG GGGSQVTLKESGPALVKPTQTLTLTCTFPGFSLSTSGMG|VSWIRQPPGKALEWLA|nY] |WDDDKR|YNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFY| ISFDYWGQIGTSVTVSSLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFII FWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKLRVKFSRSADAP AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
B735scFvLH-CD28H-CD28TM-41BBCS-CD3zICS
(SEQID NO: 955) Protein Sequence:
DIQMTQSPSSLSASVGDRVTITCKAlSQNVAf^VAWFQQKPGQAPKSLllYSASYlRQSGV PSRFSGSGSGTDFTLTISSLQSEDFATYFdQQYNSYP|FTFGSGTKLEIKGGGGSGGGGSG GGGSQVTLKESGPALVKPTQTLTLTCTF^GFSLSTSGMG|VSWIRQPPGKALEWLApiY] ^VDDDKR|YNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFY| ISFDYWGQIGTSVTVSSLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFII FWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAY QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
B735scFvLH-CD28H-CD28TM-DAP10CS-CD3zICS
(SEQID NO: 956) Protein Sequence:
DIQMTQSPSSLSASVGDRVTITCKA|SQNVAT7jVAWFQQKPGQAPKSLl[YSASY|RQSGV PSRFSGSGSGTDFTLTISSLQSEDFATYFC|QQYNSYP|FTFGSGTKLEIKGGGGSGGGGSG GGGSQVTLKESGPALVKPTQTLTLTCTF^GFSLSTSGMG|VSWIRQPPGKALEWLA|HiY| |WDDDKR|YNPSLKSRLTITKDTSKNQWLTMTNMDPVDTATYY|CARRPITTSVAPYFY| ISFDYWGQIGTSVTVSSLEVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFII FWVLCARPRRSPAQEDGKVYINMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRRE EYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG
HDGLYQGLSTATKDTYDALHMQALPPR

Claims

What is claimed is:
1. An antibody (Ab) or antigen-binding Ab fragment thereof, wherein the Ab or Ab fragment binds to alkaline phosphatase, placenta-like 2 (ALPPL2), optionally having SEQ ID NO: 101, and comprises:
(a) a heavy chain variable region (VH) comprising a VH complementarity determining region 1 (CDR-H1), a VH complementarity determining region 2 (CDR-H2), and a VH complementarity determining region 3 (CDR-H3); and/or
(b) a light chain variable region (VL) comprising a VL complementarity determining region 1 (CDR-L1), a VL complementarity determining region 2 (CDR-L2), and, a VL complementarity determining region 3 (CDR-L3), wherein:
(I) (i) the VH comprises the CDR-H1 , CDR-H2, and CDR-H3 sequences contained in SEQ ID NO: 751 , and/or the VL comprises the CDR- L1, CDR-L2, and CDR-L3 sequences contained in SEQ ID NO: 755,
(ii) the VH comprises the CDR-H1, CDR-H2, and CDR-H3 sequences contained in SEQ ID NO: 741 , and/or the VL comprises the CDR- L1, CDR-L2, and CDR-L3 sequences contained in SEQ ID NO: 745,
(Hi) the VH comprises the CDR-H1, CDR-H2, and CDR-H3 sequences contained in SEQ ID NO: 731 , and/or the VL comprises the CDR- L1, CDR-L2, and CDR-L3 sequences contained in SEQ ID NO: 735,
(iv) the VH comprises the CDR-H1 , CDR-H2, and CDR-H3 sequences contained in SEQ ID NO: 721 , and/or the VL comprises the CDR- L1, CDR-L2, and CDR-L3 sequences contained in SEQ ID NO: 725,
(v) the VH comprises the CDR-H1, CDR-H2, and CDR-H3 sequences contained in SEQ ID NO: 711, and/or the VL comprises the CDR- L1, CDR-L2, and CDR-L3 sequences contained in SEQ ID NO: 715,
(vi) the VH comprises the CDR-H1 , CDR-H2, and CDR-H3 sequences contained in SEQ ID NO: 701 , and/or the VL comprises the CDR- L1, CDR-L2, and CDR-L3 sequences contained in SEQ ID NO: 705 or encoded in SEQ ID NO: 805,
(vii) the VH comprises the CDR-H1 , CDR-H2, and CDR-H3 sequences contained in SEQ ID NO: 331 or encoded in SEQ ID NO: 431, and/or the VL comprises the CDR-L1 , CDR-L2, and CDR-L3 sequences contained in SEQ ID NO: 335 or encoded in SEQ ID NO: 435,
(viii) the VH comprises the CDR-H1 , CDR-H2, and CDR-H3 sequences contained in SEQ ID NO: 321 or encoded in SEQ ID NO: 421, and/or the VL comprises the CDR-L1 , CDR-L2, and CDR-L3 sequences contained in SEQ ID NO: 325 or encoded in SEQ ID NO: 425,
(ix) the VH comprises the CDR-H1 , CDR-H2, and CDR-H3 sequences contained in SEQ ID NO: 311 or encoded in SEQ ID NO: 411, and/or the VL comprises the CDR-L1 , CDR-L2, and CDR-L3 sequences contained in SEQ ID NO: 315 or encoded in SEQ ID NO: 415,
(x) the VH comprises the CDR-H1, CDR-H2, and CDR-H3 sequences contained in SEQ ID NO: 131 or encoded in SEQ ID NO: 231, and/or the VL comprises the CDR-L1 , CDR-L2, and CDR-L3 sequences contained in SEQ ID NO: 135 or encoded in SEQ ID NO: 235,
(xi) the VH comprises the CDR-H1 , CDR-H2, and CDR-H3 sequences contained in SEQ ID NO: 121 or encoded in SEQ ID NO: 221, and/or the VL comprises the CDR-L1 , CDR-L2, and CDR-L3 sequences contained in SEQ ID NO: 125 or encoded in SEQ ID NO: 225, or
(xii) the VH comprises the CDR-H1 , CDR-H2, and CDR-H3 sequences contained in SEQ ID NO: 111 or encoded in SEQ ID NO: 211 , and/or the VL comprises the CDR-L1 , CDR-L2, and CDR-L3 sequences contained in SEQ ID NO: 115 or encoded in SEQ ID NO: 215; and/or
(II) (I) the CDR-H1 , CDR-H2, and CDR-H3 comprise or consist of the amino add sequence set forth in SEQ ID NOs: 752, 753, and 754, respectively; and/or the CDR-L1 , CDR-L2, and CDR-L3 comprise or consist of the amino add sequence set forth in SEQ ID NOs: 756, 757, and 758, respectively,
(ii) the CDR-H1 , CDR-H2, and CDR-H3 comprise or consist of the amino add sequence set forth in SEQ ID NOs: 742, 743, and 744, respectively; and/or the CDR-L1 , CDR-L2, and CDR-L3 comprise or consist of the amino add sequence set forth in SEQ ID NOs: 746, 747, and 748, respectively,
(iii) the CDR-H1 , CDR-H2, and CDR-H3 comprise or consist of the amino add sequence set forth in SEQ ID NOs: 732, 733, and 734, respectively; and/or the CDR-L1 , CDR-L2, and CDR-L3 comprise or consist of the amino add sequence set forth in SEQ ID NOs: 736, 737, and 738, respectively,
(iv) the CDR-H1 , CDR-H2, and CDR-H3 comprise or consist of the amino add sequence set forth in SEQ ID NOs: 722, 723, and 724, respectively; and/or the CDR-L1 , CDR-L2, and CDR-L3 comprise or consist of the amino add sequence set forth in SEQ ID NOs: 726, 727, and 728, respectively,
(v) the CDR-H1 , CDR-H2, and CDR-H3 comprise or consist of the amino add sequence set forth in SEQ ID NOs: 712, 713, and 714, respectively; and/or the CDR-L1 , CDR-L2, and CDR-L3 comprise or consist of the amino add sequence set forth in SEQ ID NOs: 716, 717, and 718, respectively,
(vi) the CDR-H1 , CDR-H2, and CDR-H3 comprise or consist of the amino add sequence set forth in SEQ ID NOs: 702, 703, and 704, respectively; and/or the CDR-L1 , CDR-L2, and CDR-L3 comprise or consist of the amino add sequence set forth in SEQ ID NOs: 706, 707, and 708, respectively or are encoded by SEQ ID NOs: 806, 807, and 808, respectively,
(vii) the CDR-H1 , CDR-H2, and CDR-H3 comprise or consist of the amino add sequence set forth in SEQ ID NOs: 332, 333, and 334, respectively or are encoded by SEQ ID NOs: 432, 433, and 434, respectively; and/or the CDR-L1 , CDR-L2, and CDR-L3 comprise or consist of the amino add sequence set forth in SEQ ID NOs: 336, 337, and 338, respectively or are encoded by SEQ ID NOs: 436, 437, and 438, respectively,
(viii) the CDR-H1 , CDR-H2, and CDR-H3 comprise or consist of the amino add sequence set forth in SEQ ID NOs: 322, 323, and 324, respectively or are encoded by SEQ ID NOs: 422, 423, and 424, respectively; and/or the CDR-L1 , CDR-L2, and CDR-L3 comprise or consist of the amino add sequence set forth in SEQ ID NOs: 326, 327, and 328, respectively or are encoded by SEQ ID NOs: 426, 427, and 428, respectively,
(lx) the CDR-H1 , CDR-H2, and CDR-H3 comprise or consist of the amino add sequence set forth in SEQ ID NOs: 312, 313, and 314, respectively or are encoded by SEQ ID NOs: 412, 413, and 414, respectively; and/or the CDR-L1 , CDR-L2, and CDR-L3 comprise or consist of the amino add sequence set forth in SEQ ID NOs: 316, 317, and 318, respectively or are encoded by SEQ ID NOs: 416, 417, and 418, respectively,
(x) the CDR-H1 , CDR-H2, and CDR-H3 comprise or consist of the amino add sequence set forth in SEQ ID NOs: 132, 133, and 134, respectively or are encoded by SEQ ID NOs: 232, 233, and 234, respectively; and/or the CDR-L1 , CDR-L2, and CDR-L3 comprise or consist of the amino add sequence set forth in SEQ ID NOs: 136, 137, and 138, respectively or are encoded by SEQ ID NOs: 236, 237, and 238, respectively,
(xi) the CDR-H1 , CDR-H2, and CDR-H3 comprise or consist of the amino add sequence set forth in SEQ ID NOs: 122, 123, and 124, respectively or are encoded by SEQ ID NOs: 222, 223, and 224, respectively; and/or the CDR-L1 , CDR-L2, and CDR-L3 comprise or consist of the amino add sequence set forth in SEQ ID NOs: 126, 127, and 128, respectively or are encoded by SEQ ID NOs: 226, 227, and 228, respectively, or
(xii) the CDR-H1 , CDR-H2, and CDR-H3 comprise or consist of the amino add sequence set forth in SEQ ID NOs: 112, 113, and 114, respectively or are encoded by SEQ ID NOs: 212, 213, and 214, respectively; and/or the CDR-L1 , CDR-L2, and CDR-L3 comprise or consist of the amino add sequence set forth in SEQ ID NOs: 116, 117, and 18, respectively or are encoded by SEQ ID NOs: 216, 217, and 218, respectively, or an affinity matured variant of any of the foregoing.
2. The Ab or Ab fragment of claim 1, wherein:
(I) (a) the % sequence identity of the VH to a germline-encoded human
VH, optionally to the VH encoded by the human VH2-5 gene segment allele 8, is about 80% or higher, about 85% or higher, about 90% or higher, about 93% or higher, about 95% or higher, about 96% or higher, about 97% or higher, about 98% or higher, about 98% or higher, about 99% or higher, or about 100%, and/or (b) the % sequence identity of the VL to a germline-encoded human VL, optionally to the VL encoded by the human lgK1 -16 gene segment allele 1 is about 80% or higher, about 85% or higher, about 86% or higher, about 90% or higher, about 93% or higher, about 95% or higher, about 96% or higher, about 97% or higher, about 98% or higher, about 98% or higher, or about 99% or higher, or about 100%; and/or
(II) (a) the VH comprises a human or human-like VH framework which is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human VH framework, optionally to any one of the human VH frameworks comprised in any one of the VH polypeptides of SEQ ID NO: 751 , 741 , 731 , 721 , 711 , 701 , 311 , 321 and 331 , and/or
(b) the VL comprises a human or human-like VL framework which is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human VL framework, optionally to any one of the human VL frameworks comprised in any one of the VL polypeptides of SEQ ID NO: 755, 745, 735, 725, 715, 315, 325 or 335, optionally wherein:
(i) the VH comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 751 ; and/or the VL comprises an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 755,
(ii) the VH comprises an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 741 ; and/or the VL comprises an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 745,
(iii) the VH comprises an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 731 ; and/or the VL comprises an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 735 ,
(iv) the VH comprises an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 721 ; and/or the VL comprises an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 725,
(v) the VH comprises an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 711; and/or the VL comprises an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 715, (vi) the VH comprises an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 701 ; and/or the VL comprises an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 705,
(vii) the VH comprises an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 331 or is encoded by a nudeic add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 431 ; and/or the VL comprises an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 335 or is encoded by a nudeic add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 435,
(viii) the VH comprises an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 321 or is encoded by a nudeic add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 421 ; and/or the VL comprises an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 325 or is encoded by a nudeic add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 425,
(ix) the VH comprises an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 311 or is encoded by a nudeic add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 411 ; and/or the VL comprises an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 315 or is encoded by a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 415,
(x) the VH comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 131 or is encoded by a nucleic add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 231 ; and/or the VL comprises an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 135 or is encoded by a nudeic add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 235,
(xi) the VH comprises an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 121 or is encoded by a nudeic add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 221 ; and/or the VL comprises an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 125 or is encoded by a nudeic add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 225, and/or
(xii) the VH comprises an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 111 or is encoded by a nudeic add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 211 ; and/or the VL comprises an amino add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115 or is encoded by a nudeic add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 215.
3. The Ab or Ab fragment of claim 1 or 2, which comprises or consists of any one or more of a monodonal Ab, a monospedfic Ab, a bispecific Ab, a multispedfic Ab, a humanized Ab, a tetrameric Ab, a tetravalent Ab, a single chain Ab, a domain-specific Ab, a domain-deleted Ab, an scFc fusion protein, a chimeric Ab, a synthetic Ab, a recombinant Ab, a hybrid Ab, a mutated Ab, CDR-grafted Ab, a fragment antigen-binding (Fab), an F(ab')2, an Fab' fragment, a variable fragment (Fv), a single-chain Fv (scFv), an Fd fragment, a diabody, and a minibody.
4. The Ab or Ab fragment of any one of claims 1-3, which comprises or consists of a scFv comprising the VH, the VL, and a linker which joins the VH and the VL, optionally wherein: the scFv comprises or consists of the VH, the linker, and the VL from the direction from the N-terminus to the C-terminus; or the scFv comprises the VL, the linker, and the VH from the direction from the N- terminus to the C-terminus, optionally wherein the linker comprises:
(I) the amino add sequence of SEQ ID NO: 168;
(II) the amino add sequence encoded by SEQ ID NO: 268 or 68;
(III) the amino add sequence of SEQ ID NO: 159; or
(IV) the amino add sequence encoded by SEQ ID NO: 259, further optionally wherein the scFv comprises an amino add sequence:
(i) which is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 760, 759, 750, 749, 740, 739, 730, 729, 720, 719, 319, 320, 329, 330, 339, 340, 341, or 342; and/or
(ii) encoded by a nudeic add sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 419, 19, 420, 20, 429, 29, 430, 30, 439, 39, 440, 40, 441, or 442.
S. The Ab or Ab fragment of any one of claims 1-4, comprising at least a first binding specificity and a second binding specificity, wherein the first specificity is to an epitope in ALPPL2, optionally wherein the second specificity is: (I) to another epitope in ALPPL2; or (ii) an epitope in a second antigen other than ALPPL2, optionally wherein the second antigen is selected from the group consisting of NKG2D, 4-1 BB, and Fc receptor (FcR).
6. The Ab or Ab fragment of any one of claims 1-5, comprising: (I) a human or human-like CH1 , CH2, and/or CH3 domain(s); and/or
(II) a human or human-like fragment crystallizable (Fc) region, optionally wherein: in (I), the human-like CH1 , CH2, and/or CHS domain(s) is/are at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human CH1, CH2, and/or CHS domain(s), respectively; in (II), the human-like Fc region is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human Fc region; in (I), the human or human-like CH1 , CH2, and/or CHS domain(s) is/are individually derived from the CH1, CH2, and/or CHS domain(s), respectively, of a human IgM, a human IgD, a human IgG, a human IgE, or a human IgA, optionally of a human lgG1 , a human lgG2, a human IgGS, or a human lgG4; and/or in (II), (i) the Fc region is or is derived from the Fc region of a human IgM, a human IgD, a human IgG, a human IgE, or a human IgA, optionally of a human lgG1, a human lgG2, a human IgGS, or a human lgG4; and/or (ii) the human or human-like Fc region binds to an FcR selected from the group consisting of Fc gamma receptor (FcgR), FcgRI, FcgRI I A, FcgRHBI , FcgRIIB2, FcgRI 11 A, FcgRIIIB, Fc epsilon receptor (FceR), FceRI, FceRII, Fc alpha receptor (FcaR), FcaRI, Fc alpha/mu receptor (Fca/mR), and neonatal Fc receptor (FcRn).
7. The Ab or Ab fragment of any one of claims 1-6, which comprises one or more of the following features:
(a) binds to ALPPL2 with a dissociation constant (KD) of:
(i) about 10 nM or smaller, about 5 nM or smaller, about 2 nM or smaller, about 1 nM or smaller, about 0.8 nM or smaller, about 0.6 nM or smaller, about 0.4 nM or smaller, about 0.3 nM or smaller, about 0.2 nM or smaller, about 0.1 nM or smaller, about 0.05 nM or smaller, about 0.02 nM or smaller, or about 0.01 nM or smaller,
(II) between about 10 nM and about 0.01 nM, between about 5 nM and about 0.02 nM, between about 2 nM and about 0.05 nM, between about 2 nM and about 0.1 nM, or between about 1 nM and about 0.3 nM, and/or
(Hi) about 1.6 nM, about 0.8 nM, about 0.4 nM, about 0.3 nM, or about 0.2 nM, optionally wherein the KD is measured via enzyme-linked immunosorbent assay (ELISA) or Biolayer Interferometry (BLI); and/or
(b) binds to ALPPL2 with a half maximal effective concentration (ECso) of: (i) about 1 pg/mL or smaller, about 0.5 pg/mL or smaller, about 0.2 pg/mL or smaller, about 0.1 pg/mL or smaller, about 0.05 pg/mL or smaller, about 0.02 pg/mL or smaller, about 0.01 pg/mL or smaller, about 0.005 pg/mL or smaller, about 0.002 pg/mL or smaller, about 0.001 pg/mL or smaller,
(II) between about 1 pg/mL and about 0.001 pg/mL, between about 0.5 pg/mL and about 0.002 pg/mL, between about 0.2 pg/mL and about 0.005 pg/mL, between about 0.1 pg/mL and about 0.01 pg/mL, between about 0.05 pg/mL and about 0.02 pg/mL, and/or (Hi) about 0.05 pg/mL or about 0.02 pg/mL, optionally wherein the ECso is measured via ELISA; and/or
(c) binds to ALPPL2-expressing cells, optionally ALPPL2-expressing human cancer cells, with a half maximal effective concentration (ECso) of:
(i) about 100 nM or smaller, about 50 nM or smaller, about 20 nM or smaller, about 10 nM or smaller, about 5 nM or smaller, about 2 nM or smaller, about 1 nM or smaller, about 0.5 nM or smaller, about 0.2 nM or smaller, about 0.1 nM or smaller, or about 0.05 nM or smaller,
(ii) between about 100 nM and about 0.05 nM, between about 50 nM and about 0.1 nM, or between about 20 nM and about 0.2 nM, and/or
(iii) about 15 nM, about 10 nM, about 5 nM, about 1 nM, about 0.5 nM, or about 0.2 nM, optionally wherein the ECso is measured using flowcytometry, further optionally based on mean fluorescence intensity (MFI).
8. An antibody-drug conjugate (ADC) comprising:
(a) the Ab or Ab fragment of any one of claims 1-7; and
(b) a drug conjugated to the Ab or Ab fragment, optionally wherein the drug is selected from the group consisting of an anticancer drug, an anti-proliferative drug, a cytotoxic drug, an anti-angiogenic drug, an apoptotic drug, an immunostimulatory drug, an anti-microbial drug, an antibiotic drug, an antiviral drug, an anti-inflammatory drug, an ALPPL2 inhibitor, an ALPPL2 signaling inhibitor, an enzyme, a hormone, a toxin, a radio isotope, a compound, a small molecule, a small molecule inhibitor, a protein, a peptide, a vector, a plasmid, a viral replicon, a viral particle, a nanoparticle, a DMA molecule, an RNA molecule, an siRNA, an shRNA, a micro RNA, an oligonucleotide, and an imaging drug, and/or optionally wherein the drug is selected from doxorubicin, daunorubidn, cucurbitadn, chaetodn, chaetoglobosin, chlamydodn, calicheamidn, nemorubicin, cryptophysdn, mensacarcin, ansamitodn, mitomydn C, geldanamydn, mechercharmydn, rebeccamydn, safradn, okilactomycin, oligomydn, actinomycin, sandramydn, hypothemydn, polyketomydn, hydroxyelliptidne, thiocolchicine, methotrexate, triptolide, taltobulin, lactacystin, dolastatin, auristatin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), telomestatin, tubastatin A, combretastatin, maytansinoid, MMAD, MMAF, DM1, DM4, DTT, 16-GMB-APA-GA, 17-DMAP- GA, JW55, pyrrolobenzodiazepine, SN-38, Ro 5-3335, puwainaphydn, duocarmydn, bafilomydn, taxoid, tubulysin, ferulenol, lusiol A, fumagillin, hygrolidin, glucopieriddin, amanitin, ansatrienin, dnerubin, phalladdin, phalloidin, phytosphongosine, pieriddin, poronetin, phodophyllotoxin, gramiddin A, sanguinarine, sinefungin, herboxidiene, microcolin B, microcystin, muscotoxin A, tolytoxin, tripolin A, myoseverin, mytoxin B, nocuolin A, psuedolaric add B, pseurotin A, cydopamine, curvulin, colchidne, aphidicolin, englerin, cordycepin, apoptolidin, epothilone A, limaquinone, isatropolone, isofistularin, quinaldopeptin, ixabepilone, aeroplysinin, arruginosin, agrochelin, or epothilone, or a derivative thereof.
9. A chimeric antigen receptor (CAR) comprising:
(a) an antigen-binding domain that binds to ALPPL2, optionally having SEQ ID NO: 101;
(b) a transmembrane (TM) domain;
(c) an intracellular signaling (ICS) domain;
(d) optionally a hinge that joins said antigen-binding domain and said TM domain; and
(e) optionally one or more costimulatory (CS) domains.
10. The CAR of claim 9, wherein the antigen-binding domain is the Ab or Ab fragment according to any one of claims 1-8, optionally wherein the antigenbinding domain comprises an scFv according to claim 4, further optionally wherein the antigen-binding domain comprises:
(I) the amino add sequence of SEQ ID NOs: 760, 759, 750, 749, 740, 739, 730, 729, 720, 719, 339, 340, 341, 342, 330, 329, 320, or 319; and/or
(ii) the amino add sequence encoded by SEQ ID NOs: 439, 39, 440, 40, 441, 442, 430, 30, 429, 29, 420, 20, 419, or 19; and/or an amino add sequence which is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to:
(ii-1) SEQ ID NOs: 760, 759, 750, 749, 740, 739, 730, 729, 720, 719, 319, 320, 329, 330, 339, 340, 341, or 342; and/or
(ii-2) the amino add sequence encoded by SEQ ID NOs: 419, 19, 420, 20, 429, 29, 430, 30, 439, 39, 440, 40, 441, or 442.
11. The CAR of claim 9 or 10, wherein: (I) the TM domain is derived from the TM region, or a membrane-spanning portion thereof, of a protein selected from the group consisting of CD28, CD3e, CD4, CDS, CDS, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, TCRa, TCRb, and CD3z, optionally wherein the TM domain is derived from the TM region of CD28, or a membrane-spanning portion thereof, optionally comprising an amino add sequence which is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to the amino add sequence:
(i) set forth in SEQ ID NO: 161, and/or
(ii) encoded by SEQ ID NO: 261 ;
(II) the ICS domain is derived from a cytoplasmic signaling sequence, or a functional fragment thereof, of a protein selected from the group consisting of CD3z, a lymphocyte receptor chain, a TCR/CD3 complex protein, an Fc receptor (FcR) subunit, an IL-2 receptor subunit, FcRg, FcRb, CD3g, CD3d, CD3e, CDS, CD22, CD66d, CD79a, CD79b, CD278 (ICOS), FceRI, DAP10, and DAP12, optionally wherein the ICS domain is derived from a cytoplasmic signaling sequence of CD3z, or a functional fragment thereof, optionally comprising an amino add sequence which is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to the amino add sequence:
(i) set forth in SEQ ID NO: 162, and/or
(ii) encoded by SEQ ID NO: 262;
(III) the CAR comprises a hinge optionally derived from CD28, further optionally wherein the hinge comprises an amino sequence which is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to the amino add sequence:
(i) set forth in SEQ ID NO: 163, and/or
(ii) encoded by SEQ ID NO: 263; and/or
(IV) the CAR comprises one or more CS domains, optionally wherein at least one of the one or more CS domains is derived from a cytoplasmic signaling sequence, or functional fragment thereof, of a protein selected from the group consisting of CD28, DAP10, 4-1 BB (CD137), CD2, CD4, CDS, CD7, CD8a, CD8b, CD11a, CD11b, CD11c, CD11d, CD18, CD19, CD27, CD29, CD30, CD40, CD49d, CD49f, CD69, CD84, CD96 (Tactile), CD100 (SEMA4D), CD103, 0X40 (CD134), SLAM (SLAMF1, CD150, IPO- 3), CD160 (BY55), SELPLG (CD162), DNAM1 (CD226), Ly9 (CD229), SLAMF4 (CD244, 2B4), ICOS (CD278), B7-H3, BAFFR, BTLA, BLAME (SLAMF8), CEACAM1, CDS, CRTAM, GADS, GITR, HVEM (LIGHTER), IA4, ICAM-1, IL2Rb, IL2Rg, IL7Ra, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAT, LFA-1, LIGHT, LTBR, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKpBO (KLRF1), PAG/Cbp, PD-1, PSGL1, SLAMF6 (NTB-A, Ly108), SLAMF7, SLP-76, TNFR2, TRANCE/RANKL, VLA1, VLA-6, and CD83 ligand, optionally wherein at least one of the one or more CS domain is derived from a cytoplasmic signaling sequence of CD28, 4-1 BB, or DAP10, or functional fragment thereof, optionally comprising an amino sequence which is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to the amino add sequence:
(i) set forth in SEQ ID NO: 164,
(ii) encoded by SEQ ID NO: 264,
(iii) set forth in SEQ ID NO: 165,
(iv) encoded by SEQ ID NO: 265,
(v) set forth in SEQ ID NO: 166, and/or
(vi) encoded by SEQ ID NO: 266.
12. The CAR of any one of daims 9-11 , which comprises:
(I) an amino add sequence which is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to the amino add sequence of:
(i-1) B735scFvHL-CD28H-CD28TM-CD28CS-CD3zlCS (SEQ ID NO:
951),
(i-2) B735scFvHL-CD28H-CD28TM-41BBCS-CD3zlCS (SEQ ID NO:
952),
(i-3) B735scFvHL-CD28H-CD28TM-DAP10CS-CD3zlCS (SEQ ID NO:
953),
(i-4) B735scFvLH-CD28H-CD28TM-CD28CS-CD3zlCS (SEQ ID NO:
954),
(i-5) B735scFvLH-CD28H-CD28TM-41BBCS-CD3zlCS (SEQ ID NO:
955),
(i-6) B735scFvLH-CD28H-CD28TM-DAP10CS-CD3zlCS (SEQ ID NO:
956),
(ii-1) B506scFvHL-CD28H-CD28TM-CD28CS-CD3zlCS (SEQ ID NO:
941),
(ii-2) B506scFvHL-CD28H-CD28TM-41BBCS-CD3zlCS (SEQ ID NO:
942),
(ii-3) B506scFvHL-CD28H-CD28TM-DAP10CS-CD3zlCS (SEQ ID NO:
943),
(ii-4) B506scFvLH-CD28H-CD28TM-CD28CS-CD3zlCS (SEQ ID NO:
944),
(ii-5) B506scFvLH-CD28H-CD28TM-41BBCS-CD3zlCS (SEQ ID NO:
945),
(ii-6) B506scFvLH-CD28H-CD28TM-DAP10CS-CD3zlCS (SEQ ID NO:
946), (iii-1) B416scFvHL-CD28H-CD28TM-CD28CS-CD3zlCS (SEQ ID NO:
931),
(iii-2) B416scFvHL-CD28H-CD28TM-41BBCS-CD3zlCS (SEQ ID NO:
932),
(iii-3) B416scFvHL-CD28H-CD28TM-DAP10CS-CD3zlCS (SEQ ID
NO: 933),
(iii-4) B416scFvLH-CD28H-CD28TM-CD28CS-CD3zlCS (SEQ ID NO:
934),
(iii-5) B416scFvLH-CD28H-CD28TM-41BBCS-CD3zlCS (SEQ ID NO:
935),
(iii-6) B416scFvLH-CD28H-CD28TM-DAP10CS-CD3zlCS (SEQ ID
NO: 936),
(iv-1) B380scFvHL-CD28H-CD28TM-CD28CS-CD3zlCS (SEQ ID NO:
921),
(iv-2) B380scFvHL-CD28H-CD28TM-41 BBCS-CD3zlCS (SEQ ID NO:
922),
(iv-3) B380scFvHL-CD28H-CD28TM-DAP10CS-CD3zlCS (SEQ ID
NO: 923),
(iv-4) B380scFvLH-CD28H-CD28TM-CD28CS-CD3zlCS (SEQ ID NO:
924),
(iv-5) B380scFvLH-CD28H-CD28TM-41BBCS-CD3zlCS (SEQ ID NO:
925),
(iv-6) B380scFvLH-CD28H-CD28TM-DAP10CS-CD3zlCS (SEQ ID
NO: 926),
(v-1) B001scFvHL-CD28H-CD28TM-CD28CS-CD3zlCS (SEQ ID NO:
911),
(v-2) B001scFvHL-CD28H-CD28TM-41BBCS-CD3zlCS (SEQ ID NO:
912),
(v-3) B001scFvHL-CD28H-CD28TM-DAP10CS-CD3zlCS (SEQ ID NO:
913),
(v-4) B001scFvLH-CD28H-CD28TM-CD28CS-CD3zlCS (SEQ ID NO:
914),
(v-5) B001scFvLH-CD28H-CD28TM-41BBCS-CD3zlCS (SEQ ID NO:
915),
(v-6) B001scFvLH-CD28H-CD28TM-DAP10CS-CD3zlCS (SEQ ID NO:
916),
(vi-1) h1B2scFvHL-CD28H-CD28TM-CD28CS-CD3zlCS (SEQ ID NO:
351),
(vi-2) h1B2scFvHL-CD28H-CD28TM-41BBCS-CD3zlCS (SEQ ID NO:
352),
(vi-3) h1B2scFvHL-CD28H-CD28TM-DAP10CS-CD3zlCS (SEQ ID
NO: 353), (vi-4) h1B2scFvLH-CD28H-CD28TM-CD28CS-CD3zlCS (SEQ ID NO:
354),
(vi-5) h1B2scFvLH-CD28H-CD28TM-41BBCS-CD3zlCS (SEQ ID NO:
355),
(vi-6) h1B2scFvLH-CD28H-CD28TM-DAP10CS-CD3zlCS (SEQ ID
NO: 356),
(vii-1) h1E8scFvHL-CD28H-CD28TM-CD28CS-CD3zlCS (SEQ ID NO:
361),
(vii-2) h1E8scFvHL-CD28H-CD28TM-41BBCS-CD3zlCS (SEQ ID NO:
362),
(vii-3) h1E8scFvHL-CD28H-CD28TM-DAP10CS-CD3zlCS (SEQ ID
NO: 363),
(vii-4) h1E8scFvLH-CD28H-CD28TM-CD28CS-CD3zlCS (SEQ ID NO:
364),
(vii-5) h1E8scFvLH-CD28H-CD28TM-41BBCS-CD3zlCS (SEQ ID NO:
365),
(vii-6) h1E8scFvLH-CD28H-CD28TM-DAP10CS-CD3zlCS (SEQ ID
NO: 366),
(viii-1) h3H2scFvHL-CD28H-CD28TM-CD28CS-CD3zlCS (SEQ ID NO:
371),
(viii-2) h3H2scFvHL-CD28H-CD28TM-41BBCS-CD3zlCS (SEQ ID NO:
372),
(viii-3) h3H2scFvHL-CD28H-CD28TM-DAP10CS-CD3zlCS (SEQ ID
NO: 373),
(viii-4) h3H2scFvLH-CD28H-CD28TM-CD28CS-CD3zlCS (SEQ ID NO:
374),
(viii-5) h3H2scFvLH-CD28H-CD28TM-41BBCS-CD3zlCS (SEQ ID NO:
375), or
(viii-6) h3H2scFvLH-CD28H-CD28TM-DAP10CS-CD3zlCS (SEQ ID NO: 376);
(II) the amino add sequence encoded by any one of SEQ ID NOs 451- 456, 461-466, 471-476, 51-56, 61-66, and 71-76; and/or
(III) a cytotoxic drug, optionally conjugated to the antigen-binding domain.
13. An isolated polynucleotide or a combination of isolated polynucleotides encoding the Ab or Ab fragment of any one of claims 1-7, optionally wherein:
(i) the CDR-L1 , CDR-L2, and CDR-L3-encoding nucleic acid sequences comprise or consist of SEQ ID NOs: 806, 807, and 808, respectively,
(ii) the CDR-H1, CDR-H2, and CDR-H3-encoding nucleic add sequences comprise or consist of SEQ ID NOs: 432, 433, and 434, respectively; and the CDR-L1 , CDR-L2, and CDR-L3-encoding nucleic add sequences comprise or consist of SEQ ID NOs: 436, 437, and 438, respectively,
(iii) the CDR-H1, CDR-H2, and CDR-H3-encoding nucleic acid sequences comprise or consist of SEQ ID NOs: 422, 423, and 424, respectively; and the CDR-L1 , CDR-L2, and CDR-L3-encoding nucleic add sequences comprise or consist of SEQ ID NOs: 426, 427, and 428, respectively,
(iv) the CDR-H1, CDR-H2, and CDR-H3-encoding nudeic add sequences comprise or consist of SEQ ID NOs: 412, 413, and 414, respectively; and the CDR-L1 , CDR-L2, and CDR-L3-encoding nucleic add sequences comprise or consist of SEQ ID NOs: 416, 417, and 418, respectively,
(v) the CDR-H1, CDR-H2, and CDR-H3-encoding nudeicadd sequences comprise or consist of SEQ ID NOs: 232, 233, and 234, respectively; and the CDR-L1 , CDR-L2, and CDR-L3-encoding nudeic add sequences comprise or consist of SEQ ID NOs: 236, 237, and 238, respectively,
(vi) the CDR-H1 , CDR-H2, and CDR-H3-encoding nudeic add sequences comprise or consist of SEQ ID NOs: 222, 223, and 224, respectively; and the CDR-L1 , CDR-L2, and CDR-L3-encoding nudeic add sequences comprise or consist of SEQ ID NOs: 226, 227, and 228, respectively, or
(vii) the CDR-H1 , CDR-H2, and CDR-H3-encoding nudeic add sequences comprise or consist of SEQ ID NOs: 212, 213, and 214, respectively; and the CDR-L1 , CDR-L2, and CDR-L3-encoding nudeic add sequences comprise or consist SEQ ID NOs: 216, 217, and 218, respectively, optionally wherein:
(I) (a) the % identity of the VH-encoding nudeic add sequence to a VH- encoding human germline sequence, optionally to the human VH2- 5 gene segment allele 8, is about 80% or higher, about 85% or higher, about 90% or higher, about 93% or higher, about 95% or higher, about 96% or higher, about 97% or higher, about 98% or higher, about 98% or higher, about 99% or higher, or about 100%, and/or
(b) the % identity of the VL-encoding nudeic add sequence to a VL- encoding human germline sequence, optionally to the human lgK1 - 16 gene segment allele 1 , is about 80% or higher, about 85% or higher, about 86% or higher, about 90% or higher, about 93% or higher, about 95% or higher, about 96% or higher, about 97% or higher, about 98% or higher, about 98% or higher, or about 99% or higher, or about 100%; and/or (II) (a) the VH-encoding nucleic acid sequence comprises a human or human-like VH framework-encoding nucleic add sequence which is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human VH framework-encoding nudeic add sequence, optionally to any one of the human VH frameworkencoding nudeic add sequences comprised in any one of the VH- encoding nudeic add sequences of SEQ ID NOS: 431, 421, and 411 , and/or
(b) the VL-encoding nudeic add sequence comprises a human or human-like VL framework-encoding nudeic add sequence which is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human VL framework-encoding nudeic add sequence, optionally to any one of the human VL framework-encoding nudeic add sequences comprised in any one of the VL-encoding nudeic add sequences of SEQ ID NOS: 435, 425, and 415, optionally wherein:
(i) the VH-encoding nudeic add sequence is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 431 ; and/or the VL-encoding nudeic add sequence is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 435,
(ii) the VH-encoding nudeic add sequence is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 421 ; and/or the VL-encoding nudeic add sequence is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 425,
(iii) the VH-encoding nudeic add sequence is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 411 ; and/or the VL-encoding nudeic add sequence is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 415,
(iv) the VH-encoding nudeic add sequence is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 231 ; and/or the VL-encoding nucleic acid sequence is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 235,
(v) the VH-encoding nucleic acid sequence is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 221 ; and/or the VL-encoding nucleic acid sequence is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 225, or
(vi) the VH-encoding nucleic add sequence is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 211 ; and/or the VL-encoding nudeic add sequence is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 215.
14. The isolated polynudeotide of claim 13, wherein the Ab or Ab fragment is selected from the group consisting of a monodonal Ab, a monospecific Ab, a bispecific Ab, a multispecific Ab, a humanized Ab, a tetrameric Ab, a tetravalent Ab, a single chain Ab, a domain-specific Ab, a domain-deleted Ab, an scFc fusion protein, a chimeric Ab, a synthetic Ab, a recombinant Ab, a hybrid Ab, a mutated Ab, CDR-grafted Ab, a fragment antigen-binding (Fab), an F(ab’)2, an Fab’ fragment, a variable fragment (Fv), a single-chain Fv (scFv) fragment, an Fd fragment, a diabody, and a minibody, optionally wherein the polynudeotide or the combination of isolated polynudeotides is a polynudeotide encoding the scFv of claim 4, further optionally wherein:
(i) the linker-encoding nudeic add sequence comprises or consists of any one of SEQ ID NOS: 268, 68, and 259 and/or
(ii) the polynudeotide comprises a nudeic add sequence which is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 419, 19, 420, 20, 429, 29, 430, 30, 439, 39, 440, 40, 441 , or 442.
15. The isolated polynucleotide or a combination of isolated polynucleotides of claim 13 or 14, wherein the Ab or Ab fragment comprises at least a first and second binding specificities, wherein the first specificity is to an epitope in ALPPL2, optionally wherein the second specificity is to another epitope in ALPPL2or an epitope in a second antigen other than ALPPL2, further wherein the second antigen is selected from the group consisting of NKG2D, 4-1 BB, and FcR.
16. The isolated polynudeotide or a combination of isolated polynudeotides of any one of claims 13-15, which comprises:
(I) a human or human-like CH1 , CH2, and/or CHS domain-encoding nudeic add sequence(s); and/or
(II) a human or human-like fragment crystallizable (Fc) region-encoding nudeic add sequence, optionally wherein: in (I), the human-like CH1 , CH2, and/or CH3 domain-encoding nudeic add sequence(s) is/are at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human CH1 , CH2, and/or CH3 domainencoding nudeic add sequence(s), respectively; in (II), the human or human-like Fc region-encoding nudeic add sequence is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a human Fc region-encoding nudeic add sequence; in (I), the human or human-like CH1 , CH2, and/or CHS domain-encoding nudeic add sequence(s) is/are individually derived from the CH1, CH2, and/or CH3 domain-encoding nudeic add sequence(s), respectively, of a human IgM, a human IgD, a human IgG, a human IgE, ora human IgA, optionally of a human IgG 1 , a human lgG2, a human lgG3, or a human lgG4; and/or in (II), (i) the human or human-like Fc region-encoding nudeic add sequence is or is derived from the Fc region-encoding nudeic add sequence of a human IgM, a human IgD, a human IgG, a human IgE, or a human IgA, optionally of a human lgG1, a human lgG2, a human lgG3, ora human lgG4; and/or
(ii) the encoded human or human-like Fc region binds to an FcR selected from the group consisting of Fc gamma receptor (FcgR), FcgRI, FcgRHA, FcgRHBI, FcgRIIB2, FcgRIIIA, FcgRIIIB, Fc epsilon receptor (FceR), FceRI, FceRII, Fc alpha receptor (FcaR), FcaRI, Fc alpha/mu receptor (Fca/mR), and neonatal Fc receptor (FcRn).
17. An isolated polynucleotide encoding the CAR of any one of claims 9-12, optionally wherein:
(I) the antigen-binding domain-encoding nucleic add sequence comprises or consists of the nudeic add sequence of the polynudeotide of any one of daims 13-16, optionally wherein the antigen-binding domain-encoding nucleic acid sequence comprises or consists of the nucleic add sequence of the polynudeotide of daim 14, further wherein the antigen-binding domain-encoding nudeic add sequence comprises or consists of SEQ ID NO: 419, 19, 420, 20, 429, 29, 430, 30, 439, 39, 440, 40, 441, or 442 or an amino add sequence which is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to:
(i) SEQ ID NOs: 760, 759, 750, 749, 740, 739, 730, 729, 720, 719, 319, 320, 329, 330, 339, 340, 341, or 342; or
(ii) the amino add sequence encoded by SEQ ID NOs: 419, 19, 420, 20, 429, 29, 430, 30, 439, 39, 440, 40, 441, or 442;
(II) the TM domain-encoding nudeic add sequence is derived from the nudeic add sequence encoding the TM region, or a membrane-spanning portion thereof, of a protein selected from the group consisting of CD28, CD3e, CD4, CDS, CDS, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, TCRa, TCRb, and CD3z, optionally wherein the TM domain-encoding nudeic add sequence is derived from the nudeic add sequence encoding the TM region of CD28, or a membrane-spanning portion thereof, optionally wherein the isolated polynudeotide comprises a nudeic add sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to SEQ ID NO: 261;
(III) the ICS domain-encoding nudeic add sequence is derived from the nudeic add sequence encoding a cytoplasmic signaling sequence, or a functional fragment thereof, of a protein selected from the group consisting of CD3z, a lymphocyte receptor chain, a TCR/CD3 complex protein, an Fc receptor (FcR) subunit, an IL-2 receptor subunit, FcRg, FcRb, CD3g, CD3d, CD3e, CDS, CD22, CD66d, CD79a, CD79b, CD278 (ICOS), FceRI, DAP10, and DAP12, further optionally derived from the nudeic add sequence encoding a cytoplasmic signaling sequence of CD3z, or a functional fragment thereof, optionally wherein the isolated polynudeotide comprises a nudeic add sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to SEQ ID NO: 262;
(IV) the CAR comprises a hinge, optionally wherein the hinge-encoding nudeic add sequence is derived from the nudeic add sequence encoding CD28, further optionally wherein the isolated polynudeotide comprises a nudeic add sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to SEQ ID NO: 263; and/or
(V), the CAR comprises one or more CS domain, optionally wherein the nudeic add sequence encoding at least one of the one or more CS domains is derived from the nudeic add sequence encoding a cytoplasmic signaling sequence, or functional fragment thereof, of a protein selected from the group consisting of CD28, DAP10, 4-1 BB (CD137), CD2, CD4, CDS, CD7, CD8a, CD8b, CD11a, CD11b, CD11c, CD11d, CD18, CD19, CD27, CD29, CD30, CD40, CD49d, CD49f, CD69, CD84, CD96 (Tactile), CD100 (SEMA4D), CD103, 0X40 (CD134), SLAM (SLAMF1, CD150, IPO- 3), CD160 (BY55), SELPLG (CD162), DNAM1 (CD226), Ly9 (CD229), SLAMF4 (CD244, 2B4), ICOS (CD278), B7-H3, BAFFR, BTLA, BLAME (SLAMF8), CEACAM1, CDS, CRTAM, GADS, GITR, HVEM (LIGHTER), IA4, ICAM-1, IL2Rb, IL2Rg, IL7Ra, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAT, LFA-1, LIGHT, LTBR, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKpBO (KLRF1), PAG/Cbp, PD-1, PSGL1, SLAMF6 (NTB-A, Ly108), SLAMF7, SLP-76, TNFR2, TRANCE/RANKL, VLA1, VLA-6, and CD83 ligand, optionally wherein the CS domain-encoding nudeic add sequence is derived from the nudeic add sequence encoding a cytoplasmic signaling sequence of CD28, 4-1 BB, or DAP10, or functional fragment thereof, further optionally wherein the isolated polynudeotide comprises a nudeic add sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to SEQ ID NO: 264, 265, or 266.
18. The isolated polynucleotide of claim 17, which:
(I) is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to any one of SEQ ID NOs: 451-456, 461- 466, 471-476, 51-56, 61-66, and 71-76;
(II) further comprises a leader sequence (LS), optionally wherein the LS comprises a nucleic acid sequence which is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to SEQ ID NO: 260 or encodes a LS polypeptide comprising an amino add sequence which is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to SEQ ID NO: 160;
(III) further comprises a T2A sequence and/or a sequence encoding truncated CD19 (trCD19), optionally wherein the T2A sequence comprises a nudeic add sequence which is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to SEQ ID NO: 269 and/or encodes a T2A polypeptide comprising an amino add sequence which is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to SEQ ID NO: 169, further optionally wherein the trCD19 comprises an amino add sequence which is at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to SEQ ID NO: 170 and/or wherein the trCD19-encoding nucleic acid sequence is at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to SEQ ID NO: 270; and/or
(IV) encodes an amino add which is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to SEQ ID NO: 571 or 574 and/or comprises a nudeic add sequence which is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% at least 99%, or 100% identical to any one of SEQ ID NOs: 671 , 271 , 674, and 271.
19. A vector or a combination of vectors comprising the polynucleotide or combination of polynucleotides according to any one of claims 13-18, optionally wherein the vector is selected from a DNA, an RNA, a plasmid, a cosmid, a viral replicon, a viral vector, a lenti viral vector, an adenoviral vector, or a retroviral vector.
20. A recombinant or isolated cell comprising:
(i) the Ab or Ab fragment of any one of claims 1-7,
(ii) the ADC of claim 8,
(iii) the CAR of any one of claims 9-12,
(iv) the polynudeotide or combination of polynudeotides of any one of claims 13-18, and/or
(v) the vector or combination of vectors of claim 19.
21. The recombinant or isolated cell of claim 20, which is or comprises:
(I) a non-mammalian cell, optionally selected from a plant cell, a bacterial cell, a fungal cell, a yeast cell, a protozoa cell, or an insect cell;
(II) a mammalian cell, optionally selected from a human cell, a rat cell, or a mouse cell;
(III) a stem cell;
(IV) a primary cell, optionally a human primary cell or derived therefrom;
(V) a cell line, optionally a hybridoma cell line;
(VI) an immune cell;
(VII) MHC* or MHC-;
(VIII) selected from the group consisting of a cell line, a T cell, a T cell progenitor cell, a CD4+ T cell, a helper T cell, a regulatory T cell, a CD8+ T cell, a naive T cell, an effector! cell, a memory T cell, a stem cell memory T (TSCM) cell, a central memory T (TCM) cell, an effector memory T (TEM) cell, a terminally differentiated effector memory T cell, a tumor-infiltrating lymphocyte (TIL), an immature T cell, a mature T cell, a cytotoxic ! cell, a mucosa-associated invariant ! (MAI!) cell, a !H1 cell, a TH2 cell, a THS cell, a TH17 cell, a TH9 cell, a TH22 cell, a follicular helper! cells, and a/b T cell, a g/d T cell, a Natural Killer ! (NKT) cell, a cytokine-induced killer (CIK) cell, a lymphokine-activated killer (LAK) cell, a perforin-deficient cell, a granzyme-deficient cell, a B cell, a myeloid cell, a monocyte, a macrophage, and a dendritic cell;
(IX) a T cell or T cell progenitor cell or NK cell; and/or
(X) a T cell which has been modified such that its endogenous T cell receptor (ICR) is
(i) not expressed,
(ii) not functionally expressed, or
(iii) expressed at reduced levels compared to a wild-type T cell.
22. The recombinant or isolated cell of claim 20 or 21, wherein:
(I) the cell is activated or stimulated to proliferate when
(i) the Ab or Ab fragment,
(ii) the ADC, or
(iii) the CAR, binds to its target molecule;
(II) the cell exhibits cytotoxicity against cells expressing the target molecule when
(i) the Ab or Ab fragment,
(ii) the ADC, or
(iii) the CAR, binds to the target molecule;
(III) administration of the cell to a subject ameliorates a disease, optionally cancer, in the subject when
(i) the Ab or Ab fragment,
(ii) the ADC, or
(iii) the CAR binds to its target molecule;
(IV) the cell increases expression of a cytokine and/or a chemokine when
(i) the Ab or Ab fragment,
(ii) the ADC, or
(iii) the CAR binds to its target molecule, optionally wherein the cytokine is IFN-g; and/or
(V) the cell decreases expression of a cytokine and/or a chemokine when
(i) the Ab or Ab fragment,
(ii) the ADC, or
(iii) the CAR binds to its target, optionally wherein the cytokine is TGF-b or IL-10.
23. A population of cells comprising at least one recombinant or isolated cell of any one of claims 20-22.
24. A pharmaceutical composition comprising:
(a) (i) the Ab or Ab fragment of any one of claims 1-7,
(ii) the ADC of claim 8,
(iii) the CAR of any one of claims 9-12,
(iv) the polynudeotide or combination of polynudeotides of any one of claims 13-18,
(v) the vector or combination of vectors of claim 19,
(vi) the cell of any one of claims 20-22, and/or
(vii) the population of cells of claim 23; and optionally
(b) a pharmaceutically acceptable exdpient or carrier.
25. A method of treating a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of:
(i) the Ab or Ab fragment of any one of claims 1-7,
(ii) the ADC of claim 8,
(iii) the CAR of any one of claims 9-12,
(iv) the polynucleotide or combination of polynucleotides of any one of claims 13-18,
(v) the vector or combination of vectors of claim 19,
(vi) the cell of any one of claims 20-22,
(vii) the population of cells of claim 23, and/or
(viii) the pharmaceutical composition of claim 24, optionally wherein method is used in the treatment of cancer, further optionally wherein the cancer is selected from pancreatic cancer, testicular cancer, cervical cancer, endometrial cancer, ovarian cancer, stomach cancer, colorectal cancer, lung cancer, mesothelioma, or tongue cancer, and further optionally wherein the method further comprises administering a second agent, further optionally wherein the second agent is selected from the group consisting of an anti-cancer drug, an anti-proliferative drug, a cytotoxic drug, an anti-angiogenic drug, an apoptotic drug, an immunostimulatory drug, an anti-microbial drug, an antibiotic drug, an antiviral drug, an anti-inflammatory drug, an ALPPL2 inhibitor, an ALPPL2 signaling inhibitor, an enzyme, a hormone, a toxin, a radio isotope, a compound, a small molecule, a small molecule inhibitor, a protein, a peptide, a vector, a plasmid, a viral replicon, a viral particle, a nanopartide, a DNA molecule, an RNA molecule, an siRNA, an shRNA, a micro RNA, an oligonudeotide, and an imaging drug.
26. A method of treating a subject with anti-ALPPL2 agent wherein APPL2 optionally has SEQ ID NO: 101, the method comprising the steps of (a) obtaining or having obtained a biological sample from the subject,
(b) measuring the expression level of ALPPL2 in the biological sample,
(c) determining whether the subject is an ALPPL2 over-expresser, and
(d) if the subject is an ALPPL2 over-expresser, administering to the subject a therapeutically effective amount of
(i) the Ab or Ab fragment of any one of claims 1-7,
(ii) the ADC of claim 8,
(iii) the CAR of any one of claims 9-12,
(iv) the polynucleotide or combination of polynucleotides of any one of claims 13-18,
(v) the vector or combination of vectors of claim 19,
(vi) the cell of any one of claims 20-22,
(vii) the population of cells of claim 23, and/or (viii) the pharmaceutical composition of claim 24, optionally wherein the subject is suffering from cancer, further optionally wherein the cancer is selected from pancreatic cancer, testicular cancer, cervical cancer, endometrial cancer, ovarian cancer, stomach cancer, colorectal cancer, lung cancer, mesothelioma, or tongue cancer, and further optionally wherein the method further comprises administering a second agent, further optionally wherein the second agent is selected from the group consisting of an anti-cancer drug, an anti-proliferative drug, a cytotoxic drug, an anti-angiogenic drug, an apoptotic drug, an immunostimulatory drug, an anti-microbial drug, an antibiotic drug, an antiviral drug, an anti-inflammatory drug, an ALPPL2 inhibitor, an ALPPL2 signaling inhibitor, an enzyme, a hormone, a toxin, a radio isotope, a compound, a small molecule, a small molecule inhibitor, a protein, a peptide, a vector, a plasmid, a viral replicon, a viral particle, a nanopartide, a DNA molecule, an RNA molecule, an siRNA, an shRNA, a micro RNA, an oligonudeotide, and an imaging drug.
27. A method for stimulating an immune response in a subject, comprising administering to the subject a therapeutically effective amount of
(i) the Ab or Ab fragment of any one of claims 1-7,
(ii) the ADC of claim 8,
(iii) the CAR of any one of claims 9-12,
(iv) the polynudeotide or combination of polynudeotides of any one of claims 13-18,
(v) the vector or combination of vectors of claim 19,
(vi) the cell of any one of claims 20-22,
(vii) the population of cells of claim 23, and/or (viii) the pharmaceutical composition of claim 24, optionally wherein the method further comprises administering a second agent, further optionally wherein the second agent is selected from the group consisting of an anti-cancer drug, an anti-proliferative drug, a cytotoxic drug, an anti-angiogenic drug, an apoptotic drug, an immunostimulatory drug, an anti-microbial drug, an antibiotic drug, an antiviral drug, an anti-inflammatory drug, an ALPPL2 inhibitor, an ALPPL2 signaling inhibitor, an enzyme, a hormone, a toxin, a radio isotope, a compound, a small molecule, a small molecule inhibitor, a protein, a peptide, a vector, a plasmid, a viral replicon, a viral particle, a nanoparticle, a DNA molecule, an RNA molecule, an siRNA, an shRNA, a micro RNA, an oligonucleotide, and an imaging drug.
28. A method of treating a disease in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of:
(i) the Ab or Ab fragment of any one of claims 1-7,
(ii) the ADC of claim 8,
(iii) the CAR of any one of claims 9-12,
(iv) the polynucleotide or combination of polynucleotides of any one of claims 13-18,
(v) the vector or combination of vectors of claim 19,
(vi) the cell of any one of claims 20-22,
(vii) the population of cells of claim 23, and/or (viii) the pharmaceutical composition of claim 24, optionally wherein the disease comprises cancer or an infectious condition, optionally wherein cancer is selected from pancreatic cancer, testicular cancer, cervical cancer, endometrial cancer, ovarian cancer, stomach cancer, colorectal cancer, lung cancer, mesothelioma, or tongue cancer, and further optionally wherein the method further comprises administering a second agent, further optionally wherein the second agent is selected from the group consisting of an anti-cancer drug, an anti-proliferative drug, a cytotoxic drug, an anti-angiogenic drug, an apoptotic drug, an immunostimulatory drug, an anti-microbial drug, an antibiotic drug, an antiviral drug, an anti-inflammatory drug, an ALPPL2 inhibitor, an ALPPL2 signaling inhibitor, an enzyme, a hormone, a toxin, a radio isotope, a compound, a small molecule, a small molecule inhibitor, a protein, a peptide, a vector, a plasmid, a viral replicon, a viral partide, a nanopartide, a DNA molecule, an RNA molecule, an siRNA, an shRNA, a micro RNA, an oligonudeotide, and an imaging drug.
29. A method of producing the Ab or Ab fragment of any one of claims 1-7, comprising: (a) culturing cells comprising the isolated polynucleotide or combination of isolated polynucleotides of any one of claims 13-16 in a condition that allows for expression of said Ab or Ab fragment, and
(b) harvesting and purifying the Ab or Ab fragment from the cell culture from (a).
30. A method of producing the recombinant or isolated cells of claims 20-22 or a population of such cells, comprising introducing the isolated polynucleotides or combination of isolated polynucleotides of any one of claims 13-16 or the isolated polynucleotide of claim 17 or 18 into a cell, optionally wherein the introducing occurs in vitro, ex vivo, or in vivo.
PCT/US2023/066496 2022-05-02 2023-05-02 Anti-alppl2 antibodies and chimeric antigen receptors, and compositions and methods of use WO2023215746A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202263337242P 2022-05-02 2022-05-02
US63/337,242 2022-05-02

Publications (3)

Publication Number Publication Date
WO2023215746A2 true WO2023215746A2 (en) 2023-11-09
WO2023215746A3 WO2023215746A3 (en) 2023-12-07
WO2023215746A9 WO2023215746A9 (en) 2024-02-15

Family

ID=88647141

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2023/066496 WO2023215746A2 (en) 2022-05-02 2023-05-02 Anti-alppl2 antibodies and chimeric antigen receptors, and compositions and methods of use

Country Status (1)

Country Link
WO (1) WO2023215746A2 (en)

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MX369626B (en) * 2012-05-24 2019-11-13 Mountgate Group Ltd Compositions and methods related to prevention and treatment of rabies infection.
DK3383920T3 (en) * 2015-11-30 2024-04-02 Univ California DELIVERY OF TUMOR-SPECIFIC PAYLOAD AND IMMUNE ACTIVATION USING A HUMAN ANTIBODY DIRECTED AGAINST A HIGHLY SPECIFIC TUMOR CELL SURFACE ANTIGEN
SG11201912069VA (en) * 2017-06-14 2020-01-30 Adicet Bio Inc Antibodies capable of binding HLA-A2/TyrD in an HLA restricted manner and uses thereof

Also Published As

Publication number Publication date
WO2023215746A3 (en) 2023-12-07
WO2023215746A9 (en) 2024-02-15

Similar Documents

Publication Publication Date Title
JP6793800B2 (en) CD123-specific chimeric antigen receptor-redirected T cells and how to use them
JP7303749B2 (en) Chimeric antigen receptor targeting TIM-1
US20200325223A1 (en) Methods of treatments using antigen-binding proteins targeting cd56
TW202016132A (en) Methods of treating cancers and enhancing efficacy of t cell redirecting therapeutics
US20210196755A1 (en) Compositions and methods for treating antibody resistance
EP3875484A1 (en) Cll1-targeting antibody and application thereof
JP2022530542A (en) Chimeric receptor and how to use it
EP3938405A1 (en) Anti-adam12 antibodies and chimeric antigen receptors, and compositions and methods comprising
WO2023056429A1 (en) Anti-nmdar2b antibodies, antibody-drug conjugates, and chimeric antigen receptors, and compositions and methods of use
US11505599B2 (en) T cell receptor-like antibodies specific for Foxp3-derived peptides
US10934337B2 (en) Anti-BCMA chimeric antigen receptors
KR20220057598A (en) Dimeric antigen receptor (DAR) that binds BCMA
US20240000938A1 (en) Adoptive cell therapy for treatment of cancer associated with loss of heterozygosity
RU2725807C2 (en) Soluble universal reinforcing adcc synthetic fused gene, peptide technology and use thereof
WO2023215746A2 (en) Anti-alppl2 antibodies and chimeric antigen receptors, and compositions and methods of use
WO2022170033A1 (en) Engineered natural ligand-based car: directed evolution of the stress-receptor nkp30
RU2809160C2 (en) Types of combination therapy using chimeric antigen receptors and pd-1 inhibitors

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23800172

Country of ref document: EP

Kind code of ref document: A2