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WO2019246593A2 - Compositions and methods to target cll-1 and cd123 for the treatment of acute myeloid leukemia and related disorders - Google Patents

Compositions and methods to target cll-1 and cd123 for the treatment of acute myeloid leukemia and related disorders Download PDF

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WO2019246593A2
WO2019246593A2 PCT/US2019/038596 US2019038596W WO2019246593A2 WO 2019246593 A2 WO2019246593 A2 WO 2019246593A2 US 2019038596 W US2019038596 W US 2019038596W WO 2019246593 A2 WO2019246593 A2 WO 2019246593A2
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car
cells
seq
cell
domain
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WO2019246593A3 (en
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Cameron J. Turtle
Sajid Mahmood
Kevin A. Hay
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Fred Hutchinson Cancer Research Center
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2851—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
    • 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
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • 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
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16044—Chimeric viral vector comprising heterologous viral elements for production of another viral vector

Definitions

  • the current disclosure provides compositions and methods to target C-type lectin-like molecule-1 (CLL-1) and interleukin-3 receptor (CD123) for the treatment of acute myeloid leukemia and related disorders.
  • CLL-1 C-type lectin-like molecule-1
  • CD123 interleukin-3 receptor
  • the disclosure describes multiple strategies to target CLL-1 and CD123 utilizing multiple chimeric antigen receptor (CAR) construct system designs.
  • AML Acute myeloid leukemia
  • CAR constructs encode proteins including several distinct subcomponents that allow the genetically modified T cells to recognize and kill cancer cells.
  • the subcomponents include at least an extracellular component and an intracellular component.
  • the extracellular component includes a binding domain that specifically binds a marker that is preferentially present on the surface of cancer cells. When the binding domain binds such markers, the intracellular component signals the T cell to destroy the bound cell.
  • CAR constructs can additionally include a transmembrane domain that can link the extracellular component to the intracellular component, and other subcomponents that can increase the CAR’s function.
  • a transmembrane domain that can link the extracellular component to the intracellular component
  • other subcomponents that can increase the CAR’s function.
  • linker sequences such as a spacer region
  • CAR-T CAR-expressing T cells
  • One of the keys to successful targeted immunotherapy is the choice of the target(s) on the cancer cell.
  • An ideal target marker is immunogenic, plays a critical role in proliferation and differentiation (driver mutations), and is expressed only on the surface of all malignant cells and malignant stem cells. Further, a large portion of patients should test positive for the marker on their cancer cells (Cheever, et al., 2009. Clin. Cancer Res. 15(17): 5323-8337) in order for the therapy to reach the broadest number of patients.
  • CLL-1 and CD123 C-type lectin-like molecule-1 (CLL-1) and interleukin-3 receptor (CD123) for the treatment of acute myeloid leukemia (AML) and related disorders.
  • CLL-1 and CD123 were selected to target following the use of flow cytometry to study the immunophenotype of leukemic blasts isolated from AML patients. It was found that in all studied patients, CLL-1 and/or CD 123 was expressed by over 99.9% of AML blasts.
  • the current disclosure provides multiple strategies to target CLL-1 and/or CD123 based on the genetic modification of cells to express one or more chimeric antigen receptors (CAR).
  • CAR chimeric antigen receptors
  • the cells express two different CAR constructs, one targeting CLL-1 and one targeting CD123 (the“dual CAR” approach);
  • the cells express two CAR constructs with multimerizing domains, with one targeting CLL-1 and one targeting CD123; both targeting CLL-1 ; or both targeting CD123 (the“dimerized CAR” approach); and
  • the cells express a single CAR construct containing at least two binding domains in the extracellular component wherein at least one binding domain specifically binds CLL-1 and at least one binding domain specifically binds CD123 (the“tandem” approach).
  • the strategies depicted in FIG. 1 can be utilized alone or in various combinations.
  • the“dimerized CAR” approach if a genetically modified cell expresses multimerizing binding domains that all bind the same cancer antigen (e.g., CLL-1), these cells would be used within a dual population approach, for example a second population of cells expressing multimerizing binding domains that all bind CD123.
  • CLL-1 cancer antigen
  • the strategies disclosed herein provide several important advantages. First, by targeting two cancer antigens also known as cancer markers, patients that may lack one or the other will still receive the benefit of the treatment as it is unlikely that patients would lack both markers based off the phenotypic assessment of AML blasts. Second, by linking at least two CAR molecules together through co-expression and/or multimerization, activation signals that the cell receives when the now-paired binding domains bind targeted cancer cell markers can be increased.
  • compositions and methods disclosed herein provide effective therapeutics for a broader patient population, induce stronger cell activation signals and/or provide improved therapeutic effects.
  • homo- or heterodimerization of CARs can improve function and T cell signaling, leading to improved clinical outcomes.
  • FIG. 1 Exemplary CLL-1/CD123 dual targeting approaches.
  • the Dual Population includes two separate populations of engineered cells, each with a different CAR.
  • Dual CARs include genetically modified cells expressing two CAR constructs (either expressed from the same or separate vectors), one that binds CLL-1 and one that binds CD123.
  • Dimerized CARs include at least two CAR molecules expressed by the same genetically modified cell either from the same or separate vectors that multimerize.
  • Tandem CARs (right panel) include a single CAR that contains two antigen binding domains, one that specifically binds CLL-1 and one that specifically binds CD123.
  • FIGs. 2A-2D Interleukin-3 receptor (CD123) target.
  • CD123 Interleukin-3 receptor
  • CD123 interleukin-3 receptor subunit a
  • NLG N-linked glycosylation
  • ECD extracellular domain
  • hsCD123 Homo sapiens CD123
  • SP stands for signal peptide
  • TM transmembrane domain
  • CPD cytoplasmic domain.
  • Sequences for SP (SEQ ID NO: 30), hsCD123 (SEQ ID NO: 2), ECD (SEQ ID NO: 3), TM (SEQ ID NO: 4), and CPD (SEQ ID NO: 5) from the CD123 target molecule are also labeled.
  • FIG. 3 Annotated sequence of C-type lectin domain family 12 member A (CLL-1) (SEQ ID NO: 6) wherein hsCLL-1 stands for Homo sapiens CLL-1. Sequences for CPD (SEQ ID NO: 7), TM (SEQ ID NO: 8), ECD (SEQ ID NO: 9), and C-type lectin (SEQ ID NO: 126) from the CLL- 1 target molecule are also labeled
  • FIG. 4 Annotated sequence of schematics of disclosed chimeric antigen receptor (CAR) (SEQ ID NO: 10) including a single-chain variable fragment (scFv), linker, transmembrane domain, and intracellular signaling components. Sequences described within this example CAR include the SP (SEQ ID NO: 31), scFv (SEQ ID NO: 1 1), lgG4 (SEQ ID NO: 38), TM_CD28 (SEQ ID NO: 39), 4-1 BB (SEQ ID NO: 41), O ⁇ 3z (SEQ ID NO: 43), T2A (SEQ ID NO: 36), EGFR (SEQ ID NO: 45), and TM_EGFR (SEQ ID NO: 46).
  • SP SEQ ID NO: 31
  • scFv SEQ ID NO: 1 1
  • lgG4 SEQ ID NO: 38
  • TM_CD28 SEQ ID NO: 39
  • 4-1 BB SEQ ID NO: 41
  • O ⁇ 3z SEQ ID NO: 43
  • FIGs. 5A-5D Vector map for a CLL-1 CAR, demonstrating an scFv region in a V L -V H orientation and a spacer region with an lgG4 hinge region.
  • the CAR molecule is expressed separately from the tEGFR (truncated human EGFR) transduction marker allowing for monitoring by flow cytometry by incorporation of a T2A sequence in the CAR construct.
  • T cells transduced with the CLL-1 CAR demonstrate specific lysis of CLL-1 positive (pos or +) HL60 AML cell lines but show no activity against CLL-1 negative (neg or -) K562 cells.
  • T cells transduced with the CD123 CAR demonstrate specific lysis of CD123pos KG1a AML cell lines, respectively, but show no activity against CD123neg K562 cells.
  • a carboxyfluorescein succinimidyl ester (CFSE) dilution demonstrates proliferation of CLL-1 and CD123 CAR-T cells after stimulation with AML cell lines that express (Positive) their target antigen, but not antigen-negative cells (Negative) or without stimulation (None).
  • FIGs. 6A, 6B Results of CLL-1 CAR spacer analysis.
  • CD19 serves as a positive control for the experiment.
  • the panel on the right side demonstrates CFSE proliferation of CLL1-short, CLL1-intermediate, CLL1- long in the presence of target cells.
  • Interferon (IFN)- y, Interleukin (IL)-2 and tumor necrosis factor (TNF)-a were all assessed against target cells K19 and KCLL1.
  • FIGs. 7A, 7B Results of CD123 CAR spacer analysis.
  • the panel on the right side demonstrates CFSE proliferation of CD123-short, CD123- intermediate, CD123-long in the presence of target cells.
  • FIGs. 8A, 8B (8A) Results of the analysis of the tandem CAR which contains the CLL1 and CD123 scFvs in various orientations, CD123-G4Sx3-CLL1 and CLL1-G4Sx4-CD123 against target cells expressing CD19, CD123, CLL-1 , or both CD123 and CLL-1. Lysis of target cells was assessed for various EffectorTarget ratios. The higher lysis with the lower ratio of E:T indicates high potency of the T cells expressing that CAR-T construct.
  • FIG. 9. CFSE Proliferation of CD123-short and CD123-reverse (V L V H ) constructs versus various target K562 cells transduced with CD19 (K19), CLL1 (KCLL1) and CD123 (K123).
  • FIG. 10 Chromium 51 cytotoxicity release assay of (top panel) CD123-reverse (VLVH) and (bottom panel) CLL1 -reverse (VLVH) constructs versus various target K562 cells transduced with CD19 (K19), CLL1 (KCLL1) and CD123 (K123).
  • FIG. 11 A, 11 B. (11 A) The dock and lock (DNL) dimerization domain is a hyperstable disulfide crosslinked dimerization domain used in a variety of secreted protein constructs.
  • DNL refers to any form of dock and lock dimerization domain including, for example, PRKAR1A (DNL_5HVZ), PRKAR1 E (DNL_5HVZ_E*), and PRKAR1 R (DNL_5HVZ_R*).
  • PRKAR1A NNL_5HVZ
  • PRKAR1 E NNL_5HVZ_E*
  • PRKAR1 R NNL_5HVZ_R*
  • the lgG4 hinge is a short linker that can enforce homodimerization.
  • FIG. 12A, 12B Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrating the DNL dimerization domain’s ability to form homodimers targeting CD123 and heterodimers targeting CLL-1 and CD123 (reduced (R) and non-reduced (NR)).
  • the DNL dimerization domain is well-suited to form disulfide crosslinked homodimers and heterodimers.
  • FIGs. 13A, 13B Engineered DNL Heterodimer.
  • 14A Structural model of DNL heterodimer highlighting the internal salt bridge.
  • 14B SDS-PAGE showing expression of the heterodimer facilitated by the engineered DNL dimerization domain under NR and R conditions. This shows that coexpression of the two monomers results in a 1 : 1 complex of monomer 1 and monomer 2.
  • FIGs. 14A-14C Diagrams of various locations in which a dimerization domain can be inserted within a CAR construct.
  • FIGs. 15A-15C Designs for CLL-1 and CD123 targeting CAR.
  • 15A Compared to frequently used CARs which use the lgG4 hinge region as a spacer region, this depicted design uses a longer dimerization domain. The presence of a disulfide linked dimer ensures homodimer formation. The second target can stay conserved.
  • the VH stands for the heavy chain variable region and the VL stands for the light chain variable region of an scFv.
  • the dimerization domain is located intracellularly, but the distance from the binding domain to the cell membrane is conserved using a spacer region (labeled as the Linker).
  • This design ensures dimerization using a dimerization domain that does not require disulfide bond formation and hence can be used as part of the CPD.
  • This depicted construct enables the design of a heterodimer with mutated dimerization domains enabling dimerization of the two CARs including scFvl and scFv2.
  • FIGs. 16A-16J Annotated sequences of CAR constructs.
  • the structure for this CAR construct can also be applied to target the CD 123 receptor by using a scFv or any other binding agent that targets CD123. With the DNL dimerization domain located extracellularly, the receptors can be simultaneously expressed as soluble proteins and biophysically characterized using Biacore, crystallography, or other analysis tools.
  • the structure for this CAR construct can also be applied to target the CD123 receptor by using an scFv or any other binding agent that targets CD123. With the DNL dimerization domain located intracellularly, extracellular linkers located on the wild type receptors can be maintained.
  • the diagram depicts a DNL_S31 R Linker intracellularly, but it could be placed either intracellularly or extracellularly as described in FIGs. 14A-14C.
  • the depicted CAR construct which targets CLL-1 (SEQ ID NO: 16) can be paired with a similar CAR construct that targets CD123 to enforce a heterodimeric receptor.
  • FIGs. 17A-17F lgG4-hinge containing scFv.
  • 17A Schematic of the lgG4-hinge containing scFv that is thought to form obligate homodimers on the T cell surface. Expressing the extracellular portion of the receptor as a soluble protein revealed some heterogeneity that has implications for clinical CAR-T cells using this format of receptor as shown in FIGs. 17C-17F.
  • FIGs. 18A-18C DNL containing scFv.
  • FIGs. 19A-19D DNL containing scFv with mutations to favor heterodimerization.
  • FIGs. 20A, 20B [0032] FIGs. 20A, 20B.
  • FIGs. 21A, 21 B (21A) SDS-PAGE gel showing purification of the human CD123 ectodomain under NR and reducing (Red) conditions. (21 B) SDS-PAGE gel showing purification of the human CLL-1 ectodomain under NR and R conditions. NiPD stands for Nickel Pull Down.
  • FIGs. 22A-22F Comparison of bicistronic and DNL CAR constructs directed against CLL- 1 and CD123.
  • T cells were transduced with viral vector encoding a CAR directed against both anti-CLL-1 and CD123 CAR (bicistronic) or CLL-1-DNLE and CD123 DNLR DNLCAR.
  • CAR-T cells were sorted and expanded as per rapid expansion protocol (REP).
  • REP rapid expansion protocol
  • CAR-T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and cultured with irradiated targets or media control as labeled. CFSE fluorescence was measured by flow cytometry after four days of co-culture.
  • CFSE fluorescence was measured by flow cytometry after four days of co-culture.
  • Bicsitronic and (22D) DNL CAR-T cells were exposed to various chromium 51 -labelled targets as listed within the FIG. Four hours following co-culture, chromium 51 levels were measured to quantify the degree of cytotoxicity.
  • FIGs. 23A, 23B Accumulation of inhibitory immunoreceptors (TIM3, LAG3 and PD1) following repetitive stimulation of dual-targeting CAR.
  • CLL-1-, CD123 short and bicistronic CD123 targeting CAR were co-cultured with irradiated CD123-expressing K562 cell lines (K123) for 5 days (Stim 1), then re-exposed to K123 cells for 5 days (Stim 2) and then a further 5 days (Stim 3). After each stimulation, percentage of immunoinhibitory receptor-expressing CAR-T cells were assessed by flow cytometry by comparison with fluorescence minus one (FMO) expression.
  • FMO fluorescence minus one
  • FIG. 24 Expression of Nur77 following exposure to CLL-1 and CD123 targets.
  • CD123 and CLL-1 -targeting bicstronic and DNL CAR-T cells were exposed to irradiated CLL-1 and CD123 expressing targets for 5 days three times (Stim 1 , Stim 2 and Stim 3) and Nur77 expression was then measured by intranuclear flow cytometry staining as shown.
  • FIG. 25 Exemplary clinical applications of the compositions provided herein.
  • FIG. 26 Exemplary sequences supporting the disclosure (SEQ ID NOs: 1-205, 260, and 267).
  • AML acute myeloid leukemia
  • AML is a malignancy of clonal, proliferative myeloid blast cells.
  • AML is also known as acute myelocytic leukemia, acute myelogenous leukemia, acute granulocytic leukemia, and acute nonlymphocytic leukemia.
  • CAR molecules are proteins including several distinct subcomponents that allow the genetically modified T cells to recognize and kill cancer cells.
  • the subcomponents include at least an extracellular component and an intracellular component.
  • the extracellular component includes a binding domain that specifically binds a marker that is preferentially present on the surface of cancer cells.
  • the binding domain is typically a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but it can be based on other formats which include an antibody like antigen binding site.
  • the intracellular component When the binding domain binds such markers, the intracellular component signals the T cell to destroy the bound cell.
  • the intracellular components provide such activation signals based on the inclusion of an effector domain.
  • First generation CARs utilized the cytoplasmic region of O ⁇ 3z as an effector domain.
  • Second generation CARs utilized O ⁇ 3z in combination with cluster of differentiation 28 (CD28) or 4-1 BB (CD137), while third generation CARs have utilized ⁇ 3z in combination with CD28 and 401 BB within intracellular effector domains.
  • CARs generally also include one or more linker sequences that are used for a variety of purposes within the molecule.
  • a transmembrane domain can be used to link the extracellular component of the CAR to the intracellular component.
  • a flexible linker sequence often referred to as a spacer region that is membrane-proximal to the binding domain can be used to create additional distance between a binding domain and the cellular membrane. This can be beneficial to reduce steric hindrance to binding based on proximity to the membrane.
  • a common spacer region used for this purpose is the lgG4 linker. More compact spacers or longer spacers can be used, depending on the targeted cell marker.
  • Other potential CAR subcomponents are described in more detail elsewhere herein.
  • the key to successful targeted immunotherapy is in the choice of the target cancer cell marker.
  • An ideal target marker is immunogenic, plays a critical role in proliferation and differentiation, is expressed only on the surface of all malignant cells and malignant stem cells, and a large portion of patients should test positive for the marker (Cheever, et al. , 2009. Clin. Cancer Res. 15(17): 5323-8337). While the CD19-specific CAR-T cell therapy has been successful for ALL and NHL, identification of a marker for AML has proven more difficult. Many markers expressed by AML myeloblasts, or blasts, and leukemic stem cells overlap with normal hematopoiesis.
  • B-cell lineage-restricted antigen CD19
  • CD19 B-cell lineage-restricted antigen
  • CLL-1 C-type lectin-like molecule-1
  • CD123 interleukin-3 receptor
  • AML-related disorders where CLL-1 and/or CD123 provide therapeutic targets include: blastic plasmacytoid dendritic cell neoplasm (BPDCN), myelodysplastic syndromes (MDS), natural killer cell lymphomas, hairy cell leukemia, acute lymphocytic leukemia (ALL; also known as acute lymphoblastic lymphoma), chronic myelocytic leukemia (CML), other leukemias, hematological cancers or tumors, Hodgkin’s lymphoma (HL), B-cell HL, non-Hodgkin lymphoma (NHL), mantle cell lymphoma (MCL), T cell lymphoma, multiple myeloma (refractory, relapsed, etc.), systemic mastocytosis (SM), hypereosinophilic syndrome (HES), myelofibrosis, anemia, systemic lupus erythematosus (SLE), psoriasis,
  • the current disclosure provides multiple strategies to target CLL-1 and/or CD123 based on the genetic modification of cells to express one or more CAR molecules.
  • a depiction of exemplary disclosed strategies is provided in FIG. 1 showing genetically modifying cells to create a population of genetically modified cells wherein:
  • the cells express two different CAR constructs, one targeting CLL-1 and one targeting CD123 (the“dual CAR” approach);
  • the cells express two CAR constructs with multimerizing domains, with at least one targeting CLL-1 and at least one targeting CD123; all targeting CLL-1 ; or all targeting CD213 (the “dimerized CAR” approach); and
  • the cells express CAR having at least two binding domains in the extracellular component wherein at least one binding domain specifically binds CLL-1 and at least one binding domain specifically binds CD123 (the“tandem” approach).
  • strategy (i) is practiced with strategies (ii), (iii), and/or (iv).
  • strategy (ii) is practiced with strategy (iii).
  • strategy (i) is practiced with strategy (iv).
  • strategy (iii) is practiced with strategy (i).
  • strategy (i), (ii), and (iii) are practiced together.
  • strategy (ii), (iii), and (iv) are practiced together.
  • strategy (i), (iii), and (iv) are practiced together.
  • dimerization of CAR results based on inclusion of complementary forms of the protein kinase cAMP-dependent type I regulatory subunit a or b (PRKAR1A or PRKAR1 B) dimerization domains within expressed CAR molecules.
  • such multimerization or dimerization domains can be placed at any position within an expressed CAR, so long as the placement does not significantly interfere with binding to targeted cell markers.
  • multimerization or dimerization domains can be placed at any position within an expressed CAR, so long as the placement does not significantly interfere with cell activation following binding to targeted cell markers.
  • a “complete homomultimer” includes copies of identical monomer subunits (CAR), but for, in certain circumstances, inclusion of complementary multimerization domains.
  • a partial homomultimer has identical subcomponents of a CAR, but are not completely identical.
  • a partial homomultimer may be identical but for the inclusion of different transmembrane domains, spacer regions, effector domains, signaling domains and/or co-stimulatory domains.
  • a heteromultimer includes binding domains that bind different cancer antigen epitopes.
  • the different cancer antigen epitopes are located on the same cancer antigen.
  • the different cancer antigen epitopes are located on different cancer antigens (i.e. , CLL-1 and CD123).
  • the strategies disclosed herein provide several important advantages. First, by targeting two cancer markers, patients that may lack one or the other will still receive the benefit of the treatment as it is unlikely that patients would lack both markers. Second, by linking at least two CARs together through co-expression and/or multimerization, activation signals that the cell receives when the now-paired binding domains bind targeted cancer cell markers can be increased.
  • compositions and methods disclosed herein provide therapeutic efficacy for a broader patient population, induce stronger cell activation signals, and/or provide improved therapeutic effects.
  • homo- or heterodimerization of CAR can improve function and T cell signaling, leading to improved clinical outcomes.
  • Binding domains include any substance that binds to a cellular marker to form a complex.
  • the choice of binding domain can depend upon the type and number of cellular markers that define the surface of a target cell.
  • Examples of binding domains include cellular marker ligands, receptor ligands, antibodies, peptides, peptide aptamers, receptors (e.g., T cell receptors), or combinations and engineered fragments or formats thereof.
  • Antibodies are one example of binding domains and include whole antibodies or binding fragments of an antibody, e.g., Fv, Fab, Fab', F(ab')2, and single chain (sc) forms and fragments thereof that bind specifically to a cellular marker.
  • Antibodies or antigen binding fragments can include all or a portion of polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, synthetic antibodies, non-human antibodies, recombinant antibodies, chimeric antibodies, bispecific antibodies, mini bodies, and linear antibodies.
  • Functional fragments thereof include a single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) of camelid derived nanobody, and the like.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • VHH variable domain
  • scFvs can be prepared according to methods known in the art (see, for example, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
  • ScFv molecules can be produced by linking VL and VH regions of an antibody together using flexible polypeptide linkers. If a short polypeptide linker is employed (e.g., between 5-10 amino acids) intrachain folding is prevented. Interchain folding is also required to bring the two variable regions together to form a functional epitope binding site.
  • linker orientations and sizes see, e.g., Hollinger et al. 1993 Proc Natl Acad. Sci. U.S.A. 90:6444- 6448, US 2005/0100543, US 2005/0175606, US 2007/0014794, W02006/020258, and W02007/024715.
  • An scFv can include a linker of at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more amino acid residues between its VL and VH regions.
  • the linker sequence may include any naturally occurring amino acid.
  • linker sequences that are used to connect the VL and VH of an scFv are five to 35 amino acids in length.
  • a VL-VH linker includes from five to 35, ten to 30 amino acids or from 15 to 25 amino acids. Variation in the linker length may retain or enhance activity, giving rise to superior efficacy in activity studies.
  • the linker sequence includes the amino acids glycine and serine.
  • the linker sequence includes sets of glycine and serine repeats such as from one to ten repeats of (Gly x Ser y ) n , wherein x and y are independently an integer from 0 to 10 provided that x and y are not both 0 and wherein n is an integer of 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10) and wherein linked VH-VL regions form a functional immunoglobulin-like binding domain (e.g., scFv, scTCR).
  • a functional immunoglobulin-like binding domain e.g., scFv, scTCR
  • linker is (Gly4Ser)4 (SEQ ID NO: 212) or (Gly4Ser) 3 (SEQ ID NO: 213).
  • such linkers can also be used to link T cell receptor V c/ p and C o/ p chains (e.g., V a -C a , Vp-Cp, V a -Vp).
  • Additional examples include scFv-based grababodies and soluble VH domain antibodies. These antibodies form binding regions using only heavy chain variable regions. See, for example, Jespers et al., Nat. Biotechnol. 22: 1161 , 2004; Cortez- Retamozo et al., Cancer Res. 64:2853, 2004; Baral et al., Nature Med. 12:580, 2006; and Barthelemy et al., J. Biol. Chem. 283:3639, 2008.
  • the binding domain it is beneficial for the binding domain to be derived from the same species it will ultimately be used in.
  • the antigen binding domain may include a human antibody, humanized antibody, or a fragment or engineered form thereof.
  • Antibodies from human origin or humanized antibodies have lowered or no immunogenicity in humans and have a lower number of non-immunogenic epitopes compared to non-human antibodies.
  • Antibodies and their engineered fragments will generally be selected to have a reduced level or no antigenicity in human subjects.
  • the binding domain includes a humanized antibody or an engineered fragment thereof.
  • a non-human antibody is humanized, where one or more amino acid residues of the antibody are modified to increase similarity to an antibody naturally produced in a human or fragment thereof. These nonhuman amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain.
  • humanized antibodies or antibody fragments include one or more CDRs from nonhuman immunoglobulin molecules and framework regions wherein the amino acid residues including the framework are derived completely or mostly from human germline.
  • the antigen binding domain is humanized.
  • a humanized antibody can be produced using a variety of techniques known in the art, including CDR-grafting (see, e.g., European Patent No. EP 239,400; WO 91/09967; and US 5,225,539, US 5,530,101 , and US 5,585,089), veneering or resurfacing (see, e.g., EP 592, 106 and EP 519,596; Padlan, 1991 , Molecular Immunology, 28(4/5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805- 814; and Roguska et al., 1994, PNAS, 91 :969-973), chain shuffling (see, e.g., US.
  • CDR-grafting see, e.g., European Patent No. EP 239,400; WO 91/09967; and US 5,225,539, US 5,530,101 , and US 5,585,089)
  • veneering or resurfacing see,
  • framework substitutions are identified by methods well-known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for cellular marker binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., US 5,585,089; and Riechmann et al. , 1988, Nature, 332:323).
  • Antibodies that specifically bind a particular cellular marker can be prepared using methods of obtaining monoclonal antibodies, methods of phage display, methods to generate human or humanized antibodies, or methods using a transgenic animal or plant engineered to produce antibodies as is known to those of ordinary skill in the art (see, for example, US 6, 291 , 161 and US 6,291 , 158).
  • Phage display libraries of partially or fully synthetic antibodies are available and can be screened for an antibody or fragment thereof that can bind to a cellular marker. For example, binding domains may be identified by screening a Fab phage library for Fab fragments that specifically bind to a cellular marker of interest (see Hoet et al., Nat. Biotechnol. 23:344, 2005).
  • Phage display libraries of human antibodies are also available. Additionally, traditional strategies for hybridoma development using a cellular marker of interest as an immunogen in convenient systems (e.g., mice, HuMAb mouse® (GenPharm Int’l. Inc., Mountain View, CA), TC mouse® (Kirin Pharma Co. Ltd., Tokyo, JP), KM-mouse® (Medarex, Inc., Princeton, NJ), llamas, chicken, rats, hamsters, rabbits, etc.) can be used to develop binding domains. In particular embodiments, antibodies specifically bind to a cellular marker preferentially expressed by a particular cancer cell type and do not cross react with nonspecific components or unrelated targets. Once identified, the amino acid sequence of the antibody and gene sequence encoding the antibody can be isolated and/or determined.
  • An alternative source of binding domains includes sequences that encode random peptide libraries or sequences that encode an engineered diversity of amino acids in loop regions of alternative non-antibody scaffolds, such as scTCR (see, e.g., Lake et al., Int. Immunol.11 :745, 1999; Maynard et al., J. Immunol. Methods 306:51 , 2005; US 8,361 ,794), fibrinogen domains (see, e.g., Shoesl et al., Science 230: 1388, 1985), Kunitz domains (see, e.g., US 6,423,498), designed ankyrin repeat proteins (DARPins; Binz et al., J. Mol.
  • DARPins ankyrin repeat proteins
  • V-like domains see, e.g., US 2007/0065431
  • C-type lectin domains Zaelensky and Gready, FEBS J. 272:6179, 2005; Beavil et al., Proc. Nat'l. Acad. Sci. (USA) 89:753, 1992 and Sato et al., Proc. Nat'l. Acad. Sci.
  • mAb2 or Fc-region with antigen binding domain FcabTM (F-Star Biotechnology, Cambridge UK; see, e.g., WO 2007/098934 and WO 2006/072620), armadillo repeat proteins (see, e.g., Madhurantakam et al., Protein Sci. 21 : 1015, 2012; WO 2009/040338), affilin (Ebersbach et al., J. Mol. Biol. 372: 172, 2007), affibody, avimers, knottins, fynomers, atrimers, cytotoxic T-lymphocyte associated protein-4 (Weidle et al., Cancer Gen. Proteo.
  • Peptide aptamers include a peptide loop (which is specific for a cellular marker) attached at both ends to a protein scaffold. This double structural constraint increases the binding affinity of peptide aptamers to levels comparable to antibodies.
  • the variable loop length is typically 8 to 20 amino acids and the scaffold can be any protein that is stable, soluble, small, and non-toxic.
  • Peptide aptamer selection can be made using different systems, such as the yeast two-hybrid system (e.g., Gal4 yeast-two-hybrid system), or the LexA interaction trap system.
  • a binding domain is a sc T cell receptor (scTCR) including Va/b and Ca/b chains (e.g., Va-Ca, /b-Ob, Va-C/b) or including a Va-Ca, /b-Ob, Va- /b pair specific for a cellular marker of interest (e.g., peptide-MHC complex).
  • scTCR sc T cell receptor
  • engineered CAR include a sequence that is at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to an amino acid sequence of a known or identified TCR Va, ⁇ /b, Ca, or Ob, wherein each CDR includes zero changes or at most one, two, or three changes, from a TCR or fragment or derivative thereof that specifically binds to the targeted cellular marker.
  • engineered CAR include Va, nb, Ca, or Ob regions derived from or based on a Va, nb, Ca, or Ob of a known or identified TCR (e.g., a high-affinity TCR) and includes one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions or non-conservative amino acid substitutions), or a combination of the above-noted changes, when compared with the Va, nb, Ca, or Ob of a known or identified TCR.
  • TCR e.g., a high-affinity TCR
  • amino acid substitutions e.g., conservative amino acid substitutions or non-conservative amino acid substitutions
  • An insertion, deletion or substitution may be anywhere in a Va, nb, Ca, or Ob region, including at the amino- or carboxy-terminus or both ends of these regions, provided that each CDR includes zero changes or at most one, two, or three changes and provides a target binding domain containing a modified Va, nb, Ca, or Ob region can still specifically bind its target with an affinity and action similar to wild type.
  • the scFv that binds CLL-1 can be SC02357.
  • the variable light chain of SC02357 includes a CDRL1 sequence including QSISSYLN (SEQ ID NO: 261), a CDRL2 sequence including LLIYAASSLQS (SEQ ID NO: 262), and a CDRL3 sequence including QQSYSTPP (SEQ ID NO: 263), and a variable heavy chain including a CDRH1 sequence including GSISSSNWWS (SEQ ID NO: 264), a CDRH2 sequence including WIGEIYHSGSPDY (SEQ ID NO: 265), and a CDRH3 sequence including KVSTGGFFDY (SEQ ID NO: 266).
  • the binding domain of a CAR can be an scFv that binds CLL-1 with a VH including the amino acid sequence described in SEQ ID NO: 13 and a VL including the amino acid sequence described in SEQ ID NO: 14.
  • the binding domain of a CAR can be an scFv that binds CLL-1 with a VL encoded by the nucleic acid sequence described in SEQ ID NO: 27 and a VH encoded by the nucleic acid sequence described in SEQ ID NO: 26.
  • the binding domain that binds CLL-1 is derived from at least one of M26, M31 , G4, M22, M29, M2, M5, or G12, for example, the CDRs of M26, M31 , G4, M22, M29, M2, M5, or G12.
  • the binding domain includes a variable light chain region including the sequence:
  • the binding domain includes a variable light chain including a CDRL1 sequence including DYYMH (SEQ ID NO: 216), a CDRL2 sequence including RINPYAGAAFYSQNFKD (SEQ ID NO: 217), and a CDRL3 sequence including ERGADLEGYAMDY (SEQ ID NO: 218), and a variable heavy chain including a CDRH1 sequence including RASQSVSTSSYN YM H (SEQ ID NO: 219), a CDRH2 sequence including YASNLES (SEQ ID NO: 220), and a CDRH3 sequence including QHSWEIPLT (SEQ ID NO: 221).
  • the binding domain of the M26 antibody includes a variable light chain including a CDRL1 sequence including QELSGY (SEQ ID NO: 222), a CDRL2 sequence including AAS, and a CDRL3 sequence including LQYAIYPYT (SEQ ID NO: 223), and a variable heavy chain including a CDRH1 sequence including GYTFTSYF (SEQ ID NO: 224), a CDRH2 sequence including INPYNDGS (SEQ ID NO: 225), and a CDRH3 sequence including TRDDGYYGYAMDY (SEQ ID NO: 226).
  • a variable light chain including a CDRL1 sequence including QELSGY (SEQ ID NO: 222), a CDRL2 sequence including AAS, and a CDRL3 sequence including LQYAIYPYT (SEQ ID NO: 223)
  • the binding domain of the M31 antibody includes a variable light chain including a CDRL1 sequence including ESVDSYGNSF (SEQ ID NO: 227), a CDRL2 sequence including LAS, and a CDRL3 sequence including QQNNYDPWT (SEQ ID NO: 228), and a variable heavy chain including a CDRH1 sequence including GYTFTSYV (SEQ ID NO: 229), a CDRH2 sequence including INPYNDGT (SEQ ID NO: 230), and a CDRH3 sequence including ARPIYFDNDYFDY (SEQ ID NO: 231).
  • the binding domain of the G4 antibody includes a variable light chain including a CDRL1 sequence including HDISNY (SEQ ID NO: 232), a CDRL2 sequence including YTS, and a CDRL3 sequence including QQGKTLLWT (SEQ ID NO: 233), and a variable heavy chain including a CDRH1 sequence including GYSFTGYT (SEQ ID NO: 234), a CDRH2 sequence including INPYNDGT (SEQ ID NO: 230), and a CDRH3 sequence including ARTDDYDDYTMDY (SEQ ID NO: 235).
  • the binding domain is human or humanized.
  • CLL-1 and associated binding domains see US 9751946, US 8536310, US 9163090, US 9914777, US 9908946, US 9145588, US 9248181 , US 9248182, PCT /N L2016/050507 , PCT/NL2013/050693, EP2147594B1 , JP5749161 , EP20170170370, and PCT/EP2016/051470.
  • the scFv that binds CD123 can be KLON120.
  • the binding domain of a CAR can be an scFv that binds CD123 with a VH including the amino acid sequence described in SEQ ID NO: 19 and a VL including the amino acid sequence described in SEQ ID NO: 18.
  • the binding domain of a CAR can be an scFv that binds CD123 with a VL encoded by the nucleic acid sequence described in SEQ ID NO: 22 and a VH encoded by the nucleic acid sequence described in SEQ ID NO: 23.
  • the antibody includes a variable light chain region including the sequence:
  • the binding domain that binds CD123 is derived from at least one of IMGN632 (ImmunoGen), ADAMTS2 (Abnova), 7G3, 32716, 32701 , 32703, 26292 (US 8163279), for example, the CDRs of IMGN632, ADAMTS2, 7G3, 32716, 32701 , 32703, or 26292.
  • the binding domain for the 26292 antibody includes a variable light chain including a CDRL1 sequence including RASKSISKDLA (SEQ ID NO: 236), a CDRL2 sequence including SGSTLQS (SEQ ID NO: 237), and a CDRL3 sequence including QQHNKYPYT (SEQ ID NO: 238), and a variable heavy chain including a CDRH 1 sequence including SYWMN (SEQ ID NO: 239), a CDRH2 sequence including RIDPYDSETHYNQKFKD (SEQ ID NO: 240), and a CDRH3 sequence including GNWDDY (SEQ ID NO: 241).
  • a variable light chain including a CDRL1 sequence including RASKSISKDLA (SEQ ID NO: 236), a CDRL2 sequence including SGSTLQS (SEQ ID NO: 237), and a CDRL3 sequence including QQHNKYPYT (SEQ ID NO: 238)
  • the binding domain for the 32703 antibody includes a variable light chain including a CDRL1 sequence including RSNKSLLHSNGNTYLY (SEQ ID NO: 242), a CDRL2 sequence including RMSNLAS (SEQ ID NO: 243), a CDRL3 sequence including MQHLEYPYT (SEQ ID NO: 244), and a variable heavy chain including a CDRH1 sequence including NYWMN (SEQ ID NO: 245), a CDRH2 sequence including RIDPSDSESHYNQKFKD (SEQ ID NO: 246), and a CDRH3 sequence including YDYDDTMDY (SEQ ID NO: 247).
  • a CDRL1 sequence including RSNKSLLHSNGNTYLY SEQ ID NO: 242
  • RMSNLAS SEQ ID NO: 243
  • MQHLEYPYT SEQ ID NO: 244
  • the binding domain for the 32701 antibody includes a variable light chain including a CDRL1 sequence including RASESVDNYGNTFMH (SEQ ID NO: 248), a CDRL2 sequence including RASNLES (SEQ ID NO: 249), a CDRL3 sequence including QQSKEDPPT (SEQ ID NO: 250), and a variable heavy chain including a CDRH1 sequence including NYGMN (SEQ ID NO: 251), a CDRH2 sequence including WMNTNTGEPTSLEDFKG (SEQ ID NO: 252), and a CDRH3 sequence including SGGYDPMDY (SEQ ID NO: 253).
  • the binding domain for the 32716 antibody includes a variable light chain including a CDRL1 sequence including RASESVDNYGNTFMH (SEQ ID NO: 248), a CDRL2 sequence including RASNLES (SEQ ID NO: 249), a CDRL3 sequence including QQSKEDPPT (SEQ ID NO: 250), and a variable heavy chain including a CDRH1 sequence including NYGMN (SEQ ID NO: 251), a CDRH2 sequence including WINTYTGESTYSADFKG (SEQ ID NO: 254), and a CDRH3 sequence including SGGYDPMDY (SEQ ID NO: 253).
  • the binding domain is human or humanized. For more information regarding binding domains that bind CD123, see US 8163279.
  • CD123 WO2013173820A2 , PCT/IB2008/002930, PCT/US2015/031580, PCT/US1988/00001 1 , EP19890907981 , PCT/US2014/028961 , US 12/082940, US 1 1/271381 , US 7763242, EP2063907, JP5550905, and US 8188231 B2.
  • binding domains of each of the targeting CAR include SC02357 and KLON120.
  • binding domains of each co-targeting CAR include scFvs derived from antibodies that specifically bind CLL-1 and CD123 to target acute myeloid leukemia (AML).
  • CAR can completely or partially homodimerize.
  • binding domains of each of the homodimerized CAR are both SC02357.
  • binding domains of each of the homodimerized CAR are both KLON120.
  • binding domains of each of the homodimerized CAR are both scFvs derived from antibodies that specifically bind CLL-1.
  • binding domains of each of the homodimerized CAR are both scFvs derived from antibodies that specifically bind CD123.
  • CAR can multimerize with binding domains that are different but that both specifically bind the same marker (i.e., CLL-1 or CD123).
  • multimerized CAR include three SC02357 binding domains. In particular embodiments, multimerized CAR include three KLON120 binding domains. In particular embodiments, multimerized CAR include four SC02357 binding domains. In particular embodiments, multimerized CAR include four KLON120 binding domains. In particular embodiments, multimerized CAR include five SC02357 binding domains. In particular embodiments, multimerized CAR include five KLON120 binding domains.
  • CAR can multimerize with binding domains specific to distinct antigens.
  • multimerized CAR include one SC02357 and two KLON120 binding domains.
  • multimerized CAR include two SC02357 and one KLON120 binding domains.
  • multimerized CAR include scFvs derived from antibodies that specifically bind CLL-1 and CD123 as the binding domains.
  • binding domains can adopt a variety of engineered formats including, for example, Fab fragments, scFv, scFv-based grababodies, and soluble VH domain antibodies.
  • a binding domain of a CAR includes or is a sequence that is at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to an amino acid sequence of a light chain variable region (VL) or to a heavy chain variable region (VH), or both, wherein each CDR includes zero changes or at most one, two, or three changes, from a monoclonal antibody or fragment or derivative thereof that specifically binds to a cellular marker of interest.
  • VL light chain variable region
  • VH heavy chain variable region
  • a VL region in a binding domain of the present disclosure is derived from or based on a VL of a known monoclonal antibody and contains one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions), or a combination of the above-noted changes, when compared with the VL of the known monoclonal antibody.
  • one or more e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10
  • amino acid substitutions e.g., conservative amino acid substitutions
  • An insertion, deletion or substitution may be anywhere in the VL region, including at the amino- or carboxy-terminus or both ends of this region, provided that each CDR includes zero changes or at most one, two, or three changes and provided a binding domain containing the modified VL region can still specifically bind its target with an affinity similar to the wild type binding domain.
  • a binding domain VH region of the present disclosure can be derived from or based on a VH of a known monoclonal antibody and can contain one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions or non-conservative amino acid substitutions), or a combination of the above-noted changes, when compared with the VH of a known monoclonal antibody.
  • one or more e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10
  • amino acid substitutions e.g., conservative amino acid substitutions or non-conservative amino acid substitutions
  • An insertion, deletion or substitution may be anywhere in the VH region, including at the amino- or carboxy-terminus or both ends of this region, provided that each CDR includes zero changes or at most one, two, or three changes and provided a binding domain containing the modified VH region can still specifically bind its target with an affinity similar to the wild type binding domain.
  • the boundaries of a given CDR or FR may vary depending on the scheme used for identification.
  • the Kabat scheme is based on structural alignments
  • the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, "30a,” and deletions appearing in some antibodies.
  • the two schemes place certain insertions and deletions ("indels") at different positions, resulting in differential numbering.
  • the Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.
  • the antibody CDR sequences disclosed herein are according to Kabat numbering.
  • Intracellular Signaling Components The intracellular or otherwise the cytoplasmic signaling components of a CAR are responsible for activation of the cell in which the CAR is expressed.
  • the term“intracellular signaling components” or“intracellular components” is thus meant to include any portion of the intracellular domain sufficient to transduce an activation signal.
  • Intracellular components of expressed CAR can include effector domains.
  • An effector domain is an intracellular portion of a fusion protein or receptor that can directly or indirectly promote a biological or physiological response in a cell when receiving the appropriate signal.
  • an effector domain is part of a protein or protein complex that receives a signal when bound, or it binds directly to a target molecule, which triggers a signal from the effector domain.
  • An effector domain may directly promote a cellular response when it contains one or more signaling domains or motifs, such as an immunoreceptor tyrosine-based activation motif (ITAM).
  • ITAM immunoreceptor tyrosine-based activation motif
  • an effector domain will indirectly promote a cellular response by associating with one or more other proteins that directly promote a cellular response, such as co stimulatory domains.
  • Effector domains can provide for activation of at least one function of a modified cell upon binding to the cellular marker expressed by a cancer cell. Activation of the modified cell can include one or more of differentiation, proliferation and/or activation or other effector functions.
  • an effector domain can include an intracellular signaling component including a T cell receptor and a co-stimulatory domain which can include the cytoplasmic sequence from co-receptor or co-stimulatory molecule.
  • An effector domain can include one, two, three or more receptor signaling domains, intracellular signaling components (e.g., cytoplasmic signaling sequences), co-stimulatory domains, or combinations thereof.
  • exemplary effector domains include signaling and stimulatory domains selected from: 4-1 BB (CD137), CARD11 , CD3y, CD36, CD3s, O ⁇ 3z, CD27, CD28, CD79A, CD79B, DAP10, FcRa, FcRb (FcsRI b), FcRy, Fyn, HVEM (LIGHTR), ICOS, LAG 3, LAT, Lck, LRP, NKG2D, NOTCH1 , pTa, PTCH2, 0X40, ROR2, Ryk, SLAMF1 , Slp76, TCRa, p ⁇ b, TRIM, Wnt, Zap70, or any combination thereof.
  • exemplary effector domains include signaling and co-stimulatory domains selected from: CD86, FcyRIla, DAP12, CD30, CD40, PD-1 , lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1 , GITR, BAFFR, SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8a, O ⁇ 8b, IL2Rb, I L2 R g , IL7Ra, ITGA4, VLA1 , CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1 d, ITGAE, CD103, ITGAL, CD1 1 a, ITGAM, CD1 1 b, ITGAX, CD11 c, ITGB1 , CD29, ITGB2, CD18,
  • Intracellular signaling component sequences that act in a stimulatory manner may include iTAMs.
  • iTAMs including primary cytoplasmic signaling sequences include those derived from CD3y, CD36, CD3s, O ⁇ 3z, CD5, CD22, CD66d, CD79a, CD79b, and common FcRy (FCER1 G), FcyRIla, FcRb (Fes Rib), DAP10, and DAP12.
  • variants of O ⁇ 3z retain at least one, two, three, or all ITAM regions.
  • an effector domain includes a cytoplasmic portion that associates with a cytoplasmic signaling protein, wherein the cytoplasmic signaling protein is a lymphocyte receptor or signaling domain thereof, a protein including a plurality of ITAMs, a co stimulatory domain, or any combination thereof.
  • intracellular signaling components include the cytoplasmic sequences of the O ⁇ 3z chain, and/or co- receptors that act in concert to initiate signal transduction following binding domain engagement.
  • a co-stimulatory domain is domain whose activation can be required for an efficient lymphocyte response to cellular marker binding. Some molecules are interchangeable as intracellular signaling components or co-stimulatory domains. Examples of costimulatory domains include CD27, CD28, 4-1 BB (CD 137), 0X40, CD30, CD40, PD-1 , ICOS, lymphocyte function- associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83.
  • CD27 co-stimulation has been demonstrated to enhance expansion, effector function, and survival of human CART cells in vitro and augments human T cell persistence and anti-cancer activity in vivo (Song et al. Blood. 2012; 1 19(3):696-706).
  • co-stimulatory domain molecules include CDS, ICAM-1 , GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8a, CD8 , IL2R , IL2Ry, IL7Ra, ITGA4, VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDIId, ITGAE, CD103, ITGAL, CDIIa, ITGAM, CDI lb, ITGAX, CDIIc, ITGBI, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), NKG2D, CEACAM1 , CRTAM, Ly9 (CD22), CD19
  • the amino acid sequence of the intracellular signaling component includes a variant of O ⁇ 3z (SEQ ID NOs: 43 and 44) and a portion of the 4-1 BB (SEQ ID NO: 41 and 42) intracellular signaling component.
  • the intracellular signaling component includes (i) all or a portion of the signaling domain of O ⁇ 3z, (ii) all or a portion of the signaling domain of 4-1 BB, or (iii) all or a portion of the signaling domain of O ⁇ 3z and 4-1 BB.
  • Intracellular components may also include one or more of a protein of a Wnt signaling pathway (e.g., LRP, Ryk, or ROR2), NOTCH signaling pathway (e.g., NOTCH1 , NOTCH2, NOTCH3, or NOTCH4), Hedgehog signaling pathway (e.g., PTCH or SMO), receptor tyrosine kinases (RTKs) (e.g., epidermal growth factor (EGF) receptor family, fibroblast growth factor (FGF) receptor family, hepatocyte growth factor (HGF) receptor family, insulin receptor (IR) family, platelet-derived growth factor (PDGF) receptor family, vascular endothelial growth factor (VEGF) receptor family, tropomycin receptor kinase (Trk) receptor family, ephrin (Eph) receptor family, AXL receptor family, leukocyte tyrosine kinase (LTK) receptor family, tyrosine kinase with immunoglobul
  • Linkers can be any portion of a CAR molecule that serves to connect two other subcomponents of the molecule. Some linkers serve no purpose other than to link other components while many linkers serve an additional purpose. Linkers in the context of linking VL and VH of antibody derived binding domains of scFv are described above. Linkers can also include spacer regions, and junction amino acids.
  • Spacer regions are a type of linker region that are used to create appropriate distances and/or flexibility from other linked components.
  • the length of a spacer region can be customized for individual cellular markers on unwanted cells to optimize unwanted cell recognition and destruction.
  • the spacer can be of a length that provides for increased responsiveness of the cell following antigen binding, as compared to in the absence of the spacer.
  • a spacer region length can be selected based upon the location of a cellular marker epitope, affinity of a binding domain for the epitope, and/or the ability of the modified cells expressing the molecule to proliferate in vitro and/or in vivo in response to cellular marker recognition. Spacer regions can also allow for high expression levels in modified cells.
  • Exemplary spacers include those having 10 to 250 amino acids, 10 to 200 amino acids, 10 to 150 amino acids, 10 to 100 amino acids, 10 to 50 amino acids, or 10 to 25 amino acids.
  • a spacer region is 12 amino acids, 20 amino acids, 21 amino acids, 26 amino acids, 27 amino acids, 45 amino acids, or 50 amino acids.
  • the spacer region is selected from the group including all or a portion of a hinge region sequence from lgG1 , lgG2, lgG3, lgG4 or IgD alone or in combination with all or a portion of a CH2 region; all or a portion of a CH3 region; or all or a portion of a CH2 region and all or a portion of a CH3 region.
  • Exemplary spacers include lgG4 hinge alone, lgG4 hinge linked to CH2 and CH3 domains, or lgG4 hinge linked to the CH3 domain.
  • the spacer includes an lgG4 linker of the amino acid sequence: ESKYGPPCPPC (SEQ ID NO: 257).
  • the spacer includes a hinge of an amino acid sequence listed in FIG. 26 (SEQ ID NO: 38, 62, 65, 66, and 129). Hinge regions can be modified to avoid undesirable structural interactions such as dimerization with unintended partners.
  • a spacer region includes a hinge region that a type II C-lectin interdomain (stalk) region or a cluster of differentiation (CD) molecule stalk region.
  • a“wild type immunoglobulin hinge region” refers to a naturally occurring upper and middle hinge amino acid sequences interposed between and connecting the CH1 and CH2 domains (for IgG, IgA, and IgD) or interposed between and connecting the CH1 and CH3 domains (for IgE and IgM) found in the heavy chain of an antibody.
  • A“stalk region” of a type II C-lectin or CD molecule refers to the portion of the extracellular domain of the type 11 C-lectin or CD molecule that is located between the C-type lectin-like domain (CTLD; e.g., similar to CTLD of natural killer cell receptors) and the hydrophobic portion (transmembrane domain).
  • C-type lectin-like domain C-type lectin-like domain
  • hydrophobic portion transmembrane domain
  • AAC50291.1 corresponds to amino acid residues 34-179, but the CTLD corresponds to amino acid residues 61-176, so the stalk region of the human CD94 molecule includes amino acid residues 34-60, which are located between the hydrophobic portion (transmembrane domain) and CTLD (see Boyington et al., Immunity 10: 15, 1999; for descriptions of other stalk regions, see also Beavil et al. , Proc. Nat'l. Acad. Sci. USA 89: 153, 1992; and Figdor et al., Nat. Rev. Immunol. 2:1 1 , 2002).
  • These type II C-lectin or CD molecules may also have junction amino acids (described below) between the stalk region and the transmembrane region or the CTLD.
  • the 233 amino acid human NKG2A protein (GenBank Accession No. P26715.1) has a hydrophobic portion (transmembrane domain) ranging from amino acids 71-93 and an extracellular domain ranging from amino acids 94-233.
  • the CTLD includes amino acids 119-231 and the stalk region includes amino acids 99-1 16, which may be flanked by additional junction amino acids.
  • Other type II C-lectin or CD molecules, as well as their extracellular ligand-binding domains, stalk regions, and CTLDs are known in the art (see, e.g., GenBank Accession Nos.
  • a multimerization domain can function as a spacer region.
  • a conventional spacer region can be used in conjunction with a multimerization domain.
  • the spacer region can also be the multimerization domain.
  • the spacer region includes a dock and lock (DNL) dimerization domain of the amino acid sequences (SEQ ID NOs: 48, 49, 50, 53, and 55) and nucleic acid sequences listed in FIG. 26 (SEQ ID NOs: 51 , 52, 54, and 56).
  • DNL dock and lock
  • Exemplary spacers also include those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153 or WO2014031687.
  • the spacer region can be a CD28 linker of the amino acid sequence PSPLFPGPSKP (SEQ ID NO: 57).
  • the spacer region is (GGGGS) n wherein n is an integer including, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or more.
  • the spacer region is (EAAAK) n (SEQ ID NO: 258) wherein n is an integer including 1 , 2, 3, 4, 5, 6, 7, 8, 9, or more.
  • a long spacer is greater than 1 19 amino acids (e.g., 229 amino acids) an intermediate spacer is 13-119 amino acids, and a short spacer is 12 amino acids or less.
  • a short spacer region includes the portion of the lgG4 hinge region encoded by SEQ ID NO: 64).
  • An example of an intermediate spacer region includes all or a portion of a lgG4 hinge region sequence and a CH3 region (e.g., SEQ ID NO: 65).
  • An example of a long spacer includes all or a portion of a lgG4 hinge region sequence, a CH2 region, and a CH3 region (e.g., SEQ ID NO: 66).
  • short spacer sequences are preferred.
  • an extracellular component of a fusion protein optionally includes an extracellular, non-signaling spacer or linker region, which, for example, can position the binding domain away from the host cell (e.g., T cell) surface to enable proper cell/cell contact, antigen binding and activation (Patel et al., Gene Therapy 6: 412-419 (1999)).
  • an extracellular, non-signaling spacer or linker region which, for example, can position the binding domain away from the host cell (e.g., T cell) surface to enable proper cell/cell contact, antigen binding and activation (Patel et al., Gene Therapy 6: 412-419 (1999)).
  • an extracellular spacer region of a fusion binding protein is generally located between a hydrophobic portion or transmembrane domain and the extracellular binding domain, and the spacer region length may be varied to maximize antigen recognition (e.g., tumor recognition) based on the selected target molecule, selected binding epitope, or antigen-binding domain size and affinity (see, e.g., Guest et al., J. Immunother. 28:203-11 (2005); PCT Publication No. WO 2014/031687).
  • a spacer region includes an immunoglobulin hinge region.
  • An immunoglobulin hinge region may be a wild-type immunoglobulin hinge region or an altered wild-type immunoglobulin hinge region.
  • an immunoglobulin hinge region is a human immunoglobulin hinge region.
  • An immunoglobulin hinge region may be an IgG, IgA, IgD, IgE, or IgM hinge region.
  • An IgG hinge region may be an lgG1 , lgG2, lgG3, or lgG4 hinge region.
  • An exemplary altered lgG4 hinge region is described in PCT Publication No. WO 2014/031687.
  • an altered lgG4 hinge region includes an amino acid sequence as set forth in SEQ ID NO:38.
  • Other examples of hinge regions used in the fusion binding proteins described herein include the hinge region present in the extracellular regions of type 1 membrane proteins, such as CD8a, CD4, CD28 and CD7, which may be wild-type or variants thereof.
  • an extracellular spacer region includes all or a portion of an Fc domain selected from: a CH1 domain, a CH2 domain, a CH3 domain, a CH4 domain, or any combination thereof (see, e.g., PCT Publication WO 2014/031687).
  • the Fc domain or portion thereof may be wildtype of altered (e.g., to reduce antibody effector function).
  • the extracellular component includes an immunoglobulin hinge region, a CH2 domain, a CH3 domain, or any combination thereof disposed between the binding domain and the hydrophobic portion.
  • the extracellular component includes an lgG1 hinge region, an lgG1 CH2 domain, and an lgG1 CH3 domain.
  • the lgG1 CH2 domain includes (i) a N297Q mutation, (ii) substitution of the first six amino acids (APEFLG) with APPVA, or both of (i) and (ii).
  • the immunoglobulin hinge region, Fc domain or portion thereof, or both are human.
  • transmembrane domains within a CAR molecule often serving to connect the extracellular component and intracellular component through the cell membrane.
  • the transmembrane domain can anchor the expressed molecule in the modified cell’s membrane.
  • the transmembrane domain can be derived either from a natural and/or a synthetic source. When the source is natural, the transmembrane domain can be derived from any membrane-bound or transmembrane protein.
  • Transmembrane domains can include at least the transmembrane region(s) of the a, b or z chain of a T-cell receptor, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22; CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.
  • a transmembrane domain may include at least the transmembrane region(s) of, e.g., KIRDS2, 0X40, CD2, CD27, LFA-1 (CD 11a, CD18), ICOS (CD278), 4-1 BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R , I L2 R g , IL7R a, ITGA1 , VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDI Id, ITGAE, CD103, ITGAL, CDI la, ITGAM, CDI lb, ITGAX, CDI lc, ITGB1 , CD29, ITGB2, CD18, ITGB7, TNFR2, DNAM
  • a variety of human hinges can be employed as well including the human Ig (immunoglobulin) hinge (e.g., an lgG4 hinge, an IgD hinge), a GS linker (e.g., a GS linker described herein), a KIR2DS2 hinge or a CD8a hinge.
  • human Ig immunoglobulin
  • a GS linker e.g., a GS linker described herein
  • KIR2DS2 hinge e.g., a KIR2DS2 hinge or a CD8a hinge.
  • a transmembrane domain has a three-dimensional structure that is thermodynamically stable in a cell membrane, and generally ranges in length from 15 to 30 amino acids.
  • the structure of a transmembrane domain can include an a helix, a b barrel, a b sheet, a b helix, or any combination thereof.
  • a transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid within the extracellular region of the CAR (e.g., up to 15 amino acids of the extracellular region) and/or one or more additional amino acids within the intracellular region of the CAR (e.g., up to 15 amino acids of the intracellular components).
  • the transmembrane domain is from the same protein that the signaling domain, co-stimulatory domain or the hinge domain is derived from.
  • the transmembrane domain is not derived from the same protein that any other domain of the CAR is derived from.
  • the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other unintended members of the receptor complex.
  • the transmembrane domain is capable of homodimerization with another CAR on the cell surface of a CAR-expressing cell.
  • the amino acid sequence of the transmembrane domain may be modified or substituted so as to minimize interactions with the binding domains of the native binding partner present in the same CAR-expressing cell.
  • the transmembrane domain includes the amino acid sequence of the CD28 transmembrane domain (SEQ ID NOs: 39 and 40).
  • junction amino acids can be a linker which can be used to connect the sequences of CAR domains when the distance provided by a spacer is not needed and/or wanted. Junction amino acids are short amino acid sequences that can be used to connect co-stimulatory intracellular signaling components. In particular embodiments, junction amino acids are 9 amino acids or less.
  • junction amino acids can be a short oligo- or protein linker, preferably between 2 and 9 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, or 9 amino acids) in length to form the linker.
  • a glycine-serine doublet can be used as a suitable junction amino acid linker.
  • a single amino acid e.g., an alanine, a glycine, can be used as a suitable junction amino acid.
  • Multimerization Domains Protein biological activities depend upon their tertiary and quaternary structure. The quaternary structure requires the physical and chemical interaction of different protein subunits or polypeptides.
  • A“multimerization domain” is a domain that causes two or more proteins (monomers) to interact with each other through covalent and/or non-covalent association(s). Multimerization domains present in proteins can result in protein interactions that form dimers, trimers, tetramers, pentamers, hexamers, heptamers, etc., depending on the number of units/monomers incorporated into the multimer, and/or homomultimers or heteromultimers, depending on whether the binding monomers are the same type or a different type.
  • the multimerization domain is a dimerization domain that allows binding of two monomers to form a dimer.
  • the dimerization domain includes dimerization motifs derived from PRKAR1A (SEQ ID NOs: 48 and 50; also referred to as DNL_5HVZ and DNL Linker).
  • the dimerization domain includes dimerization motifs derived from PRKAR1 B (SEQ ID NO: 67).
  • the dimerization domain includes dimerization motifs derived from PRKAR1 R (SEQ ID NOs: 49 and 55; also referred to as DNL_5HVZ_R*, DNL_S31 R Linker, and DNLR).
  • the dimerization domain includes dimerization motifs derived from PRKAR1 E (SEQ ID NO: 53; also referred to DNL_5HVZ_E* and DNLE).
  • homodimerization can be accomplished using dimerization motifs derived from PRKAR1A and PRKAR1A (SEQ ID NOs: 48 and 50) as dimerization domains.
  • homodimerization can be accomplished using dimerization motifs derived from PRKAR1 B and PRKAR1 B (SEQ ID NO: 67) as dimerization domains.
  • heterodimerization can be accomplished using dimerization motifs derived from PRKAR1 R (SEQ ID NOs: 49 and 55) and PRKAR1 E (SEQ ID NO: 53) as dimerization domains.
  • PRKAR1 R SEQ ID NOs: 49 and 55
  • PRKAR1 E SEQ ID NO: 53
  • These constructs allow for dimerization of CAR through a series of human disulfide cross-linked dimerization motifs using constructs derived from the cAMP-dependent protein kinase type I regulatory subunit a (PRKAR1A) (SEQ ID NO: 60) or b (PRKAR1 B) (SEQ ID NO: 61).
  • a dimerization and docking domain can be derived from the cAMP-dependent protein kinase (PKA) regulatory subunits and can be paired with an anchoring domain (AD).
  • the AD can be derived from a specific region found in various A-kinase anchoring proteins (AKAPs) that mediates association with the R subunits of PKA.
  • AKAPs A-kinase anchoring proteins
  • the dimerization domain on one CAR is a DDD (DDD1 and DDD2) (SEQ ID NOs: 68 and 69) and the dimerization domain on the second CAR is an anchoring domain (AD) AD (AD1 and AD2) (SEQ ID NOs: 70 and 71) to facilitate a stably tethered structure.
  • DDD1 and DDD2 DDD1 and DDD2
  • AD1 and AD2 anchoring domain
  • the DDD (DDD1 and DDD2) (SEQ ID NOs: 68 and 69) are derived from the regulatory subunits of a cAMP-dependent protein kinase (PKA), and the AD (AD1 and AD2) (SEQ ID NOs: 70 and 71)
  • PKA cAMP-dependent protein kinase
  • AD1 and AD2 SEQ ID NOs: 70 and 71
  • DDDs and ADs are known and can be used such as: the 4-helix bundle type DDD domains obtained from p53, DCoH (pterin 4 a carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor 1 a (TCF1)) and HNF-1 (hepatocyte nuclear factor 1).
  • TCF1 carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor 1 a
  • HNF-1 hepatocyte nuclear factor 1
  • the binding of the DDD to AD is further stabilized with a subsequent reaction to covalently secure the two components of the assembly, for example via disulfide bridges, which occurs very efficiently as the initial binding interactions orient the reactive thiol groups to ligate site-specifically.
  • disulfide bridges By placing cysteine residues at strategic locations in both the DDD and AD sequences as shown for DDD2 and AD2 in SEQ ID NOs: 69 and 71 , the binding interaction can be made covalent via disulfide bridges, thereby forming a stably tethered structure.
  • the stably tethered structure also retains the full functional properties of the two precursors.
  • the X-type four-helix bundle dimerization domain that is a structural characteristic of the DDD (Newlon, et al. EMBO J. 2001 ; 20: 1651-1662; Newlon, et al. Nature Struct Biol. 1999; 3: 222-227) can be helpful in forming multimeric structures.
  • this X-type four-helix bundle enables the binding of each dimer to two p53 peptides derived from the c-terminal regulatory domain (residues 367-388) with micromolar affinity (Rustandi, et al. Biochemistry. 1998; 37: 1951- 1960).
  • HNF-1a HNF-1a
  • DCoH dimerization cofactor for HNF-1a
  • HNF-p1 dimerization cofactor for HNF-1a
  • these naturally occurring systems can be used to provide stable multimeric structures with multiple functions or binding specificities.
  • complementary binding domains can dimerize.
  • the binding domain is a transmembrane polypeptide derived from a FcsRI chain.
  • a CAR can include a part of a FcsRI a chain and another CAR can include a part of an FcsRI b chain such that said FcsRI chains spontaneously dimerize together to form a dimeric CAR.
  • CAR can include a part of a FcsRI a chain and a part of a FcsRI g chain such that said FcsRI chains spontaneously trimerize together to form a trimeric CAR
  • the multi-chain CAR can include a part of FcsRI a chain, a part of FcsRI b chain and a part of FcsRI g chain such that said FcsRI chains spontaneously tetramerize together to form a tetrameric CAR.
  • a multimerization domain can be derived from binding events such as those between an enzyme and its substrate/inhibitor, for example, cutinase and phosphonates (Hodneland, et al. Proc Natl Acd Sci USA. 2002; 99: 5048-5052), may also be utilized to generate the two associating components (the“docking” step), which are subsequently stabilized covalently (the“lock” step).
  • cutinase and phosphonates Hodneland, et al. Proc Natl Acd Sci USA. 2002; 99: 5048-5052
  • a multimerization domain can be induced using a third molecule or chemical inducer.
  • This method of dimerization requires that one CAR include a chemical inducer of dimerization binding domain 1 (CBD1) and the second CAR include the second chemical inducer of dimerization binding domain (CBD2), wherein CBD1 and CBD2 are capable of simultaneously binding to a chemical inducer of dimerization (CID).
  • CBD1 may include a rapamycin binding domain of FK-binding protein 12 (FKBP12) (SEQ ID NO: 72) and CBD2 may include a FKBP12-Rapamycin Binding (FRB) domain of mTOR (SEQ ID NO: 73).
  • the CID can include rapamycin or a derivative thereof which is capable of causing CBD1 and CBD2 to heterodimerize.
  • CBD1 and CBD2 are a FK506 (Tacrolimus) binding domain of FKBP12 and a cyclosporin binding domain of cylcophilin A
  • the CID can include a FK506/cyclosporin fusion protein.
  • CBD1 and CBD2 are FKBP12 binding domains including a F36V mutation, the CID can be AP1903.
  • CBD1 and CBD2 are an oestrogen-binding domain (EBD) and a streptavidin binding domain
  • the CID can be an estrone/biotin fusion protein.
  • CBD1 and CBD2 are a glucocorticoid-binding domain (GBD) and a dihydrofolate reductase (DHFR) binding domain
  • the CID can be a dexamethasone/methotrexate fusion molecule.
  • CBD1 and CBD2 are an O 6 - alkylguanine-DNA alkyltransferase (AGT) binding domain and a DHFR binding domain
  • the CID can be an 0 6 -benzylguanine derivative/methotrexate fusion molecule.
  • CBD1 and CBD2 are a retinoic acid receptor domain and an ecodysone receptor domain
  • the CID can include RSL1.
  • CID binding domains can also be used to alter the affinity to the CID. For instance, altering amino acids at positions 2095, 2098, and 2101 of FRB can alter binding to Rapamycin (Bayle et al, Chemistry & Biology 13, 99-107, 2006).
  • multimerization domains can be derived from binding events such as those between receptor dimer pair such as the interleukin-8 receptor (IL-8R), integrin heterodimers such as LFA-I and GPIIIb/llla, dimeric ligand polypeptides such as nerve growth factor (NGF), neurotrophin-3 (NT-3), interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, PDGF members, and brain-derived neurotrophic factor (BDNF) (Arakawa et al., J Biol.
  • IL-8R interleukin-8 receptor
  • integrin heterodimers such as LFA-I and GPIIIb/llla
  • dimeric ligand polypeptides such as nerve growth factor (NGF), neurotrophin-3 (NT-3), interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, PDGF members, and brain-derived neuro
  • dimerization domains can include protein sequence motifs such as coiled coils, acid patches, zinc fingers, calcium hands, a CH1-CL pair, an "interface” with an engineered “knob” and/or “protruberance” (US 5821333), leucine zippers (US 5932448), SH2 and SH3 (Vidal et al., Biochemistry, 43:7336- 44, 2004), PTB (Zhou et al., Nature, 378:584- 592, 1995), WW (Sudol Prog Biochys MoL Bio, 65: 113-132, 1996), PDZ (Kim et al., Nature, 378: 85- 88, 1995; Komau et al., Science, 269: 1737-1740, 1995) and WD40 (Hu et al., J Biol Chem., 273:33489- 33494, 1998).
  • protein sequence motifs such as coiled coils, acid patches, zinc fingers, calcium hands,
  • the sequence corresponding to a dimerization domain includes the leucine zipper domain of Jun (SEQ ID NO: 74), the dimerization domain of Fos (SEQ ID NO: 75), a consensus sequence for a WW motif (SEQ ID NO: 76), the dimerization domain of the SH2B adapter protein from GenBank Accession no. AAF73912.1 (Nishi et al., Mol Cell Biol, 25: 2607-2621 , 2005; SEQ ID NO: 77), the SH3 domain of IB1 from GenBank Accession no.
  • C4b multimerization domains can also be used. Particular C4b multimerization domains that can be used are provided in FIG. 26 as SEQ ID NOs: 84 - 116.
  • the C4b multimerization domain will be a multimerization domain which includes (i) glycine at position 12, (ii) alanine at position 28, (iii) leucines at positions 29, 34, 36, and/or 41 ; (iv) tyrosine at position 32; (v) lysine at position 33; and/or (vi) cysteine at positions 6 and 18.
  • the C4b multimerization domain will be a multimerization domain which includes (i) glycine at position 12, (ii) alanine at position 28, (iii) leucines at positions 29, 34, 36, and 41 ; (iv) tyrosine at position 32; (v) lysine at position 33; and (vi) cysteine at positions 6 and 18.
  • C4b multimerization domains can include any of SEQ ID NOs: 84-1 16 with an N-terminal deletion of at least 1 consecutive amino acid residue (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10 consecutive amino acid residues) in length. Additional embodiments can include a C-terminal deletion of at least 1 consecutive amino acid residue (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10 consecutive amino acid residues) in length.
  • Particular C4b multimerization domain embodiments will retain or will be modified to include at least 1 of the following residues: A6; E1 1 ; A13; D21 ; C22; P25; A27; E28; L29; R30; T31 ; L32; L33; E34; I35; K37; L38; L40; E41 ; I42; Q43; K44; L45; E48; L49; or Q50.
  • dextrameric and ferritin-based multimerization can be used.
  • An exemplary ferritin fusion sequence is described in PMID 26279189.
  • additional methods of causing dimerization can be utilized. Additional modifications to generate a dimerization domain in a CAR could include: generating a second interchain disulfide bond in the C-terminus domain by introducing a second cysteine residue into both CAR; swapping interacting residues in each of the CAR constructs in the C- terminus domains (“knob-in-hole”); and fusing the variable domains of the CARs directly to ⁇ 3z ( ⁇ 3z fusion) (Schmitt ei al., Hum. Gene Ther. 2009. 20: 1240-1248).
  • CAR constructs can include one or more tag cassettes, transduction markers, and/or suicide switches.
  • the transduction marker and/or suicide switch is within the same construct but is expressed as a separate molecule on the cell surface.
  • Tag cassettes and transduction markers can be used to activate, promote proliferation of, detect, enrich for, isolate, track, deplete and/or eliminate genetically modified cells in vitro, in vivo and/or ex vivo.
  • Tag cassette refers to a unique synthetic peptide sequence affixed to, fused to, or that is part of a CAR, to which a cognate binding molecule (e.g., ligand, antibody, or other binding partner) is capable of specifically binding where the binding property can be used to activate, promote proliferation of, detect, enrich for, isolate, track, deplete and/or eliminate the tagged protein and/or cells expressing the tagged protein.
  • Transduction markers can serve the same purposes but are derived from naturally occurring molecules and are often expressed using a skipping element that separates the transduction marker from the rest of the CAR molecule.
  • Tag cassettes that bind cognate binding molecules include, for example, His tag (SEQ ID NO: 58), Flag tag (SEQ ID NO: 1 17), Xpress tag (SEQ ID NO: 1 18), Avi tag (SEQ ID NO: 1 19), Calmodulin tag (SEQ ID NO: 120), Polyglutamate tag, HA tag (SEQ ID NO: 121), Myc tag (SEQ ID NO: 122), Softag 1 (SEQ ID NO: 123), Softag 3 (SEQ ID NO: 124), and V5 tag (SEQ ID NO: 125).
  • a CAR includes a Myc tag (SEQ ID NO: 122).
  • Conjugate binding molecules that specifically bind tag cassette sequences disclosed herein are commercially available.
  • His tag antibodies are commercially available from suppliers including Life Technologies, Pierce Antibodies, and GenScript.
  • Flag tag antibodies are commercially available from suppliers including Pierce Antibodies, GenScript, and Sigma- Aldrich.
  • Xpress tag antibodies are commercially available from suppliers including Pierce Antibodies, Life Technologies and GenScript.
  • Avi tag antibodies are commercially available from suppliers including Pierce Antibodies, IsBio, and Genecopoeia.
  • Calmodulin tag antibodies are commercially available from suppliers including Santa Cruz Biotechnology, Abeam, and Pierce Antibodies.
  • HA tag antibodies are commercially available from suppliers including Pierce Antibodies, Cell Signal and Abeam.
  • Myc tag antibodies are commercially available from suppliers including Santa Cruz Biotechnology, Abeam, and Cell Signal.
  • Transduction markers may be selected from at least one of a truncated CD19 (tCD19; see Budde et al. , Blood 122: 1660, 2013); a truncated human EGFR (tEGFR; see Wang et al., Blood 1 18: 1255, 201 1); an extracellular domain of human CD34; and/or RQR8 which combines target epitopes from CD34 (see Fehse et al, Mol. Therapy 1 ( 5 Pt 1); 448-456, 2000) and CD20 antigens (see Philip et al, Blood 124: 1277-1278).
  • tCD19 see Budde et al. , Blood 122: 1660, 2013
  • tEGFR truncated human EGFR
  • RQR8 which combines target epitopes from CD34 (see Fehse et al, Mol. Therapy 1 ( 5 Pt 1); 448-456, 2000) and CD20 antigens (see Philip et al
  • a polynucleotide encoding an iCaspase9 construct may be inserted into a CAR nucleotide construct as a suicide switch.
  • Control features may be present in multiple copies in a CAR or can be expressed as distinct molecules with the use of a skipping element.
  • a CAR can have one, two, three, four or five tag cassettes and/or one, two, three, four, or five transduction markers could also be expressed.
  • embodiments can include a CAR construct having two Myc tag cassettes, or a His tag and an HA tag cassette, or a HA tag and a Softag 1 tag cassette, or a Myc tag and a SBP tag cassette.
  • CAR that will multimerize following expression include different tag cassettes.
  • a transduction marker includes tEFGR. Exemplary transduction markers and cognate pairs are described in US 13/463,247.
  • One advantage of including at least one control feature in a CAR is that CAR expressing cells administered to a subject can be depleted using the cognate binding molecule to a tag cassette.
  • the present disclosure provides a method for depleting a modified cell expressing a CAR by using an antibody specific for the tag cassette, using an cognate binding molecule specific for the control feature, or by using a second modified cell expressing a CAR and having specificity for the control feature. Elimination of modified cells may be accomplished using depletion agents specific for a control feature.
  • modified cells expressing a chimeric molecule may be detected or tracked in vivo by using antibodies that bind with specificity to a control feature (e.g., anti-Tag antibodies), or by other cognate binding molecules that specifically bind the control feature, which binding partners for the control feature are conjugated to a fluorescent dye, radio-tracer, iron- oxide nanoparticle or other imaging agent known in the art for detection by X-ray, CT-scan, MRI- scan, PET-scan, ultrasound, flow-cytometry, near infrared imaging systems, or other imaging modalities (see, e.g., Yu, et al., Theranostics 2:3, 2012).
  • a control feature e.g., anti-Tag antibodies
  • binding partners for the control feature are conjugated to a fluorescent dye, radio-tracer, iron- oxide nanoparticle or other imaging agent known in the art for detection by X-ray, CT-scan, MRI- scan, PET-scan, ultrasound, flow-cytometry, near infrared imaging
  • modified cells expressing at least one control feature with a CAR can be, e.g., more readily identified, isolated, sorted, induced to proliferate, tracked, and/or eliminated as compared to a modified cell without a tag cassette.
  • an anti-CLL-1 CAR includes the components SC02357-- short spacer -28TM-41 BB-3Z; SC02357-lgG4 hinge -28TM-41 BB-3Z; or anti-CLL_scFV-hsCD28_TM-hs4-1 BB_CPD-hsCD3 ⁇ CPD-T2A-hsEGFRt.
  • an anti-CD123 CAR includes the components KLON120- short spacer-28TM-41 BB- ⁇ 3z; KLON120-lgG4 Ii ⁇ b-28TM-41 BB-003z; or Anti-CD123_scFV- hsCD28_TM-hs41 BB_CPD-hsCD3z_CPD-T2A-hsEGFRt wherein KLON 120 is the scFv targeting CD123 and can be selected from SEQ ID NO: 138 or from a construct of VL of scFv targeting CD123 (SEQ ID NO: 18) and VH of scFv targeting CD123 (SEQ ID NO: 19) connected by a linker selected from SEQ ID NOs: 206-213 and 258-259.
  • the short spacer can be selected from SEQ ID NO: 38, 206-213, and 257-259.
  • the transmembrane domain is derived from CD28 which can be selected from SEQ ID NO: 39 and SEQ ID NO: 40.
  • 41 BB and O ⁇ 3z make up the intracellular domain.
  • 41 BB can be selected from SEQ ID NO: 41 and SEQ ID NO: 42.
  • O ⁇ 3z can be selected from SEQ ID NO: 43 and SEQ ID NO: 44.
  • T2A can be selected from SEQ ID NO: 36 and SEQ ID NO: 37 and hsEGFRt is SEQ ID NO: 47.
  • the extracellular component of an anti-CLL-1 and anti-CD123 tandem CAR includes 5’-CLL-1-linker-CD123-3’, 5’CD123-linker-CLL-1-3’, 5’-CLL1-G4Sx1- CD123-3’, 5’-CD123-G4Sx1-CLL1-3’, 5’-CD123-G4Sx3-CLL1-3’, 5’-CLL1-G4Sx4-CD123-3’, 5’- CD123-G4Sx4-CLL1 -3’ , 5’-CLL1-EAAAKx3-CD123-3’, and 5’-CD123-EAAAKx3-CLL1 wherein CLL-1 stands for the scFv targeting CLL-1 (SEQ ID NO: 139), CD123 stands for the scFv targeting CD123 (SEQ ID NO: 138), a linker can be selected from SEQ ID NOs: 206-213, and G4Sx1 (
  • a dimerized CAR construct is anti-CLL-1 and anti- CD123 including the VL of the scFv that binds CLL-1 (SEQ ID NO: 14) linked to the VH of the scFv that binds CLL-1 (SEQ ID NO: 13) by a (GGGGS) 4 linker (SEQ ID NO: 212).
  • This binding domain is attached to a multimerization domain that acts as a spacer region including the DNL_5HVZ (PRKAR1 E) multimerization domain (SEQ ID NO: 53), and a CD28 transmembrane domain (SEQ ID NOs: 39 or 40).
  • the CLL-1-targeting CAR is linked to the CD123-targeting CAR through the multimerization domain that serves as a spacer region.
  • the VL of the scFv that binds CD123 (SEQ ID NO: 18) is linked to the VH of the scFv that binds CD123 (SEQ ID NO: 19) by a (GGGGS) 4 linker (SEQ ID NO: 212)
  • This binding domain is attached to the CD28 transmembrane domain (SEQ ID NOs: 39 or 40) by a multimerization domain that acts as a spacer region that includes the DNL_5HVZ (PRKAR1 R) multimerization domain (SEQ ID NO: 55) which connects the CLL-1 targeting CAR to the CD123 targeting CAR through the complimentary binding of PRKAR1 R and PRKAR1 E.
  • the dimerized CAR construct KLON120-PRKAR1AR-28TM- 41 BB-3Z forms heterodimers with SC02357-PRKAR1AE-28TM-41 BB-3Z, wherein SC02357 is an scFv that binds CLL-1 (CD371) and PRKAR1AR and PRKAR1AE are dimerization domains derived from PRKAR1 dimerization domains with a mutation at a core serine or cysteine residue to either an arginine or glutamic acid.
  • the CAR construct KLON120- PRKAR1 AE-28TM-41 BB-3Z forms heterodimers with SC02357-PRKAR1AR-28TM-41 BB ⁇ .
  • the dimerized CAR construct KLON120-PRKAR1A-28TM- 41 BB-3Z forms homodimers, wherein KLON120 is an scFv that binds CD123, PRKAR1A is a dimerization domain, 28TM is the CD28 transmembrane domain, 41 BB is the 4-1 BB co stimulatory domain, and 3Z is the O ⁇ 3z domain.
  • the CAR construct SC02357-PRKAR1 A-28TM-41 BB-3Z forms homodimers, wherein SC02357 is an scFv that binds CLL-1.
  • the CAR construct KLON120-PRKAR1AR-28TM-41 BB-3Z forms heterodimers with SC02357-PRKAR1AE-28TM-41 BB-3Z, wherein SC02357 is an scFv that binds CLL-1 (CD371) and PRKAR1AR and PRKAR1AE are dimerization domains derived from PRKAR1 dimerization domains with a mutation at a core serine or cysteine residue to either an arginine or glutamic acid.
  • the CAR construct KLON120- PRKAR1 AE-28TM-41 BB-3Z forms heterodimers with SC02357-PRKAR1AR-28TM-41 BB-3Z.
  • multimerizing constructs include
  • hsCD123_scFV-DNL-hsCD28_TM-hs4-1 BB_CPD-hsCD3 ⁇ CPD-T2A-hsEGFRt (MDT-000828); hsCD123_scFV-DNL_5HVZ-His (MDT-000830);
  • CLL-1 (rev) or CD123(rev) refers to a reverse orientation of depicted V L and V H domains of an scFv;
  • T refers to a T2A ribosomal skip sequence;
  • intDS refers to an intermediate length spacer of 1 19 aa in length, containing the lgG4 hinge and CH3 domains;
  • t19 refers to truncated CD19;
  • 1ST and 2ST refer to the STREP-TAG ® II sequences, wherein 1ST is one sequence and 2ST is two sequences.
  • the present invention also includes cells genetically modified to express CAR molecules as described herein.
  • genetically modified cells include lymphocytes.
  • genetically modified cells include T-cells, B cells, natural killer (NK) cells, monocytes/macrophages and hematopoeitic stem cells (HSCs).
  • T-cell receptor TCR
  • TCRa and TOBb T-cell receptor a and b
  • gd T-cells represent a small subset of T-cells that possess a distinct T-cell receptor (TCR) on their surface.
  • TCR T-cell receptor
  • the TCR is made up of one g-chain and one d-chain. This group of T-cells is much less common (2% of total T-cells) than the ab T-cells.
  • CD3 is expressed on all mature T cells. Activated T-cells express 4-1 BB (CD137), CD69, and CD25. CD5 and transferrin receptor are also expressed on T-cells.
  • T-cells can further be classified into helper cells (CD4+ T-cells) and cytotoxic T-cells (CTLs, CD8+ T-cells), which include cytolytic T-cells.
  • T helper cells assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and activation of cytotoxic T-cells and macrophages, among other functions. These cells are also known as CD4+ T-cells because they express the CD4 protein on their surface.
  • Helper T-cells become activated when they are presented with peptide antigens by MHC class II molecules that are expressed on the surface of antigen presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response.
  • APCs antigen presenting cells
  • Cytotoxic T-cells destroy virally infected cells and tumor cells, and are also implicated in transplant rejection. These cells are also known as CD8+ T-cells because they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to antigen associated with MHC class I, which is present on the surface of nearly every cell of the body.
  • Central memory T-cells refers to an antigen experienced CTL that expresses CD62L or CCR7 and CD45RO on the surface thereof, and does not express or has decreased expression of CD45RA as compared to naive cells.
  • central memory cells are positive for expression of CD62L, CCR7, CD25, CD127, CD45RO, and CD95, and have decreased expression of CD45RA as compared to naive cells.
  • Effective memory T-cell refers to an antigen experienced T- cell that does not express or has decreased expression of CD62L on the surface thereof as compared to central memory cells and does not express or has decreased expression of CD45RA as compared to a naive cell.
  • effector memory cells are negative for expression of CD62L and CCR7, compared to naive cells or central memory cells, and have variable expression of CD28 and CD45RA.
  • Effector T-cells are positive for granzyme B and perforin as compared to memory or naive T-cells.
  • naive T-cells refers to a non-antigen experienced T cell that expresses CD62L and CD45RA and does not express CD45RO as compared to central or effector memory cells.
  • naive CD8+ T lymphocytes are characterized by the expression of phenotypic markers of naive T-cells including CD62L, CCR7, CD28, CD127, and CD45RA.
  • Natural killer cells also known as NK cells, K cells, and killer cells
  • NK cells are activated in response to interferons or macrophage-derived cytokines. They serve to contain viral infections while the adaptive immune response is generating antigen-specific cytotoxic T cells that can clear the infection.
  • NK cells express CD8, CD16 and CD56 but do not express CD3.
  • Macrophages (and their precursors, monocytes) reside in every tissue of the body (in certain instances as microglia, Kupffer cells and osteoclasts) where they engulf apoptotic cells, pathogens and other non-self-components.
  • Monocytes/macrophages express CD1 1 b, F4/80; CD68; CD11 c; IL-4Ra; and/or CD163.
  • Immature dendritic cells engulf antigens and other non-self- components in the periphery and subsequently, in activated form, migrate to T-cell areas of lymphoid tissues where they provide antigen presentation to T cells.
  • Dendritic cells express CD1 a, CD1 b, CD1c, CD1d, CD21 , CD35, CD39, CD40, CD86, CD101 , CD148, CD209, and DEC-205.
  • Hematopoietic Stem/Progenitor Cells or HSPC refer to hematopoietic stem cells and/or hematopoietic progenitor cells.
  • Hematopoietic stem cells refer to undifferentiated hematopoietic cells that are capable of self-renewal either in vivo, essentially unlimited propagation in vitro, and capable of differentiation to other cell types including immune effector cells (e.g., T cells, NK cells).
  • immune effector cells e.g., T cells, NK cells.
  • a hematopoietic progenitor cell is a cell derived from hematopoietic stem cells or fetal tissue that is capable of further differentiation into mature cells types.
  • hematopoietic progenitor cells are CD24'° Lin- CD117 + hematopoietic progenitor cells.
  • Hematopoietic progenitor cells include embryonic stem cells.
  • Embryonic stem cells or "ES cells” refer to undifferentiated embryonic stem cells that have the ability to integrate into and become part of the germ line of a developing embryo. Embryonic stem cells are capable of differentiating into hematopoietic progenitor cells, and any tissue or organ.
  • HSPC can self-renew or can differentiate into (i) myeloid progenitor cells which ultimately give rise to monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, or dendritic cells; or (ii) lymphoid progenitor cells which ultimately give rise to T-cells, B-cells, and NK-cells.
  • HSPC can be positive for a specific marker expressed in increased levels on HSPC relative to other types of hematopoietic cells.
  • markers include CD34, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD1 17, CD133, CD166, HLA DR, or a combination thereof.
  • the HSPC can be negative for an expressed marker relative to other types of hematopoietic cells.
  • markers include Lin, CD38, or a combination thereof.
  • the HSPC are CD34 + cells.
  • Cells to be genetically modified according to the teachings of the current disclosure can be patient-derived cells (autologous) or, when appropriate can be allogeneic.
  • the methods include isolating cells from a subject, preparing, processing, culturing, and/or engineering them, as described herein, and re-introducing them into the same patient, before or after cryopreservation.
  • Particular embodiments can use an off-the-shelf HSPC cell source.
  • the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or 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, or other organ, and/or cells derived therefrom.
  • PBMCs peripheral blood mononuclear cells
  • 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, HSPC, red blood cells, and/or platelets, and in some aspects contains cells other than red blood cells and platelets and further processing is necessary.
  • Sources of HSPC include umbilical cord blood, placental blood, and peripheral blood (see U.S. Patent Nos. 5,004,681 ; 7,399,633; and 7, 147,626; Craddock, et al., 1997, Blood 90(12):4779-4788; Jin, et at., 2008, Journal of Translational Medicine 6:39; Pelus, 2008, Curr. Opin. Hematol.
  • Sources of HSPC also include bone marrow (see Kodo, et al., 1984, J. Clin. Invest. 73: 1377-1384), embryonic cells, aortal-gonadal-mesonephros derived cells, lymph, liver, thymus, and spleen from age-appropriate donors.
  • 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.
  • the isolation can include one or more of various cell preparation and separation steps, including separation based on one or more properties, such as size, density, sensitivity or resistance to particular reagents, and/or affinity, e.g., immunoaffinity, to antibodies or other binding partners.
  • the isolation is carried out using the same apparatus or equipment sequentially in a single process stream and/or simultaneously.
  • the isolation, culture, and/or engineering of the different populations is carried out from the same starting composition or material, such as from the same sample.
  • one or more of the cell populations enriched, isolated and/or selected from a sample by the provided methods are cells that are positive for (marker+) or express high levels (markerhigh) of one or more particular markers, such as surface markers, or that are negative for (marker-) or express relatively low levels (markerlow) of one or more markers.
  • the cell populations (such as T cells or HSPCs) are enriched for cells that are positive or expressing high surface levels of cell markers described elsewhere herein.
  • the isolating, incubating, expansion, and/or engineering steps are carried out in a sterile or contained environment and/or in an automated fashion, such as controlled by a computer attached to a device in which the steps are performed.
  • the stimulating conditions for the incubation or engineering of T cells include conditions whereby T cells of the culture-initiating composition proliferate or expand.
  • the incubation is carried out in the presence of an agent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex, such as a O ⁇ 3z chain, or capable of activating signaling through such a complex or component.
  • the incubation is carried out in the presence of an anti- CD3 antibody, and anti-CD28 antibody, anti-4-1 BB antibody, for example, such antibodies coupled to or present on the surface of a solid support, such as a bead, and/or a cytokine, such as IL-2, IL-15, IL-7, and/or IL-21.
  • an anti- CD3 antibody, and anti-CD28 antibody, anti-4-1 BB antibody for example, such antibodies coupled to or present on the surface of a solid support, such as a bead, and/or a cytokine, such as IL-2, IL-15, IL-7, and/or IL-21.
  • HSPCs can be expanded in order to increase the number of HSPC. Isolation and/or expansion methods are described in, for example, US 7,399,633; US 5,004,681 ; US 2010/0183564; W02006/047569; W02007/095594; WO 2011/127470; and WO 2011/127472; Vamum-Finney, et at., 1993, Blood 101 : 1784-1789; Delaney, et ai, 2005, Blood 06:2693-2699; Ohishi, et ai, 2002, J. Clin. Invest. 110: 1165-1 174; Delaney, et at., 2010, Nature Med. 16(2): 232-236; and Chapter 2 of Regenerative Medicine, Department of Health and Human Services, August 2006, and the references cited therein. Each of the referenced methods of collection, isolation, and expansion can be used in particular embodiments of the disclosure.
  • Particular methods of expanding HSPC include expansion of HSPC with a Notch agonist, such as the extracellular binding ligands Delta and Serrate (e.g., Jagged), RBP Jxl Suppressor of Hairless, Deltex, Fringe, or fragments thereof which promote Notch pathway activation.
  • Notch agonists include any compound that binds to or otherwise interacts with Notch proteins or other proteins in the Notch pathway such that Notch pathway activity is promoted.
  • Additional culture conditions for HSPC can include expansion in the presence of one more growth factors, such as: angiopoietin-like proteins (Angptls, e.g., Angptl2, Angptl3, Angptl7, Angpt15, and Mfap4); erythropoietin; fibroblast growth factor-1 (FGF-1); Flt-3 ligand (Flt-3L); granulocyte colony stimulating factor (G-CSF); granulocyte-macrophage colony stimulating factor (GM-CSF); insulin growth factor-2 (IFG-2); interleukin-3 (IL-3); interleukin-6 (IL-6); interleukin-7 (IL-7); interleukin-11 (IL-1 1); stem cell factor (SCF; also known as the c-kit ligand or mast cell growth factor); thrombopoietin (TPO); and analogs thereof (wherein the analogs include any structural variants of the growth factors having the biological
  • Desired genes encoding CAR disclosed herein can be introduced into cells by any method known in the art, including transfection, electroporation, microinjection, lipofection, calcium phosphate mediated transfection, infection with a viral or bacteriophage vector including the gene sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, sheroplast fusion, in vivo nanoparticle-mediated delivery, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see e.g., Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen, et ai, 1993, Meth. Enzymol.
  • the technique can provide for the stable transfer of the gene to the cell, so that the gene is expressible by the cell and, in certain instances, preferably heritable and expressible by its cell progeny.
  • the term “gene” refers to a nucleic acid sequence (used interchangeably with polynucleotide or nucleotide sequence) that encodes a as described herein. This definition includes various sequence polymorphisms, mutations, and/or sequence variants wherein such alterations do not substantially affect the function of the encoded CAR.
  • the term“gene” may include not only coding sequences but also regulatory regions such as promoters, enhancers, and termination regions. The term further can include all introns and other DNA sequences spliced from an mRNA transcript, along with variants resulting from alternative splice sites. Gene sequences encoding the molecule can be DNA or RNA that directs the expression of the chimeric molecule.
  • nucleic acid sequences may be a DNA strand sequence that is transcribed into RNA or an RNA sequence that is translated into protein.
  • the nucleic acid sequences include both the full-length nucleic acid sequences as well as non-full-length sequences derived from the full- length protein.
  • the sequences can also include degenerate codons of the native sequence or sequences that may be introduced to provide codon preference in a specific cell type. Portions of complete gene sequences are referenced throughout the disclosure as is understood by one of ordinary skill in the art.
  • Gene sequences encoding CAR can be readily prepared by synthetic or recombinant methods from the relevant amino acid sequences and other description provided herein.
  • the gene sequence encoding any of these sequences can also have one or more restriction enzyme sites at the 5' and/or 3' ends of the coding sequence in order to provide for easy excision and replacement of the gene sequence encoding the sequence with another gene sequence encoding a different sequence.
  • the gene sequence encoding the sequences can be codon optimized for expression in mammalian cells.
  • Encoding refers to the property of specific sequences of nucleotides in a gene, such as a cDNA, or an mRNA, to serve as templates for synthesis of other macromolecules such as a defined sequences of amino acids.
  • a gene codes for a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • a "gene sequence encoding a protein” includes all nucleotide sequences that are degenerate versions of each other and that code for the same amino acid sequence or amino acid sequences of substantially similar form and function.
  • Polynucleotide gene sequences encoding more than one portion of an expressed CAR can be operably linked to each other and relevant regulatory sequences. For example, there can be a functional linkage between a regulatory sequence and an exogenous nucleic acid sequence resulting in expression of the latter.
  • a first nucleic acid sequence can be operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary or helpful, join coding regions, into the same reading frame
  • a polynucleotide can include a polynucleotide that encodes a self-cleaving polypeptide, wherein the polynucleotide encoding the self-cleaving polypeptide is located between the polynucleotide encoding the CAR construct and a polynucleotide encoding a transduction marker (e.g., tEGFR).
  • a transduction marker e.g., tEGFR
  • Exemplary self-cleaving polypeptides include 2A peptide from porcine teschovirus-1 (P2A), Thosea asigna virus (T2A), equine rhinitis A virus (E2A), foot-and-mouth disease virus (F2A), or variant thereof (see FIG. 18). Further exemplary nucleic acid and amino acid sequences of 2A peptides are set forth in, for example, Kim et al. ( PLOS One 6:e18556 (201 1).
  • a "vector” is a nucleic acid molecule that is capable of transporting another nucleic acid.
  • Vectors may be, e.g., plasmids, cosmids, viruses, or phage.
  • An "expression vector” is a vector that is capable of directing the expression of a protein encoded by one or more genes carried by the vector when it is present in the appropriate environment.
  • Retroviruses are viruses having an RNA genome.
  • Gammaretrovirus refers to a genus of the retroviridae family.
  • Exemplary gammaretroviruses include mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis viruses.
  • Retroviral vectors can be used.
  • the gene to be expressed is cloned into the retroviral vector for its delivery into cells.
  • a retroviral vector includes all of the cis-acting sequences necessary for the packaging and integration of the viral genome, i.e. , (a) a long terminal repeat (LTR), or portions thereof, at each end of the vector; (b) primer binding sites for negative and positive strand DNA synthesis; and (c) a packaging signal, necessary for the incorporation of genomic RNA into virions.
  • LTR long terminal repeat
  • retroviral vectors More detail about retroviral vectors can be found in Boesen, et al., 1994, Bi ot herapy 6:291-302; Clowes, et ai, 1994, J. Clin. Invest. 93:644-651 ; Kiem, et ai, 1994, Blood 83: 1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4: 129-141 ; and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:1 10-1 14.
  • Adenoviruses, adena-associated viruses (AAV) and alphaviruses can also be used.
  • Lentivirus refers to a genus of retroviruses that are capable of infecting dividing and non-dividing cells.
  • HIV human immunodeficiency virus: including HIV type 1 , and HIV type 2
  • equine infectious anemia virus feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).
  • HIV human immunodeficiency virus: including HIV type 1 , and HIV type 2
  • equine infectious anemia virus HIV
  • feline immunodeficiency virus (FIV) feline immunodeficiency virus
  • BIV bovine immune deficiency virus
  • SIV simian immunodeficiency virus
  • Suitable viral vectors include vectors based on RNA viruses, such as retrovirus-derived vectors, e.g., Moloney murine leukemia virus (MLV)-derived vectors, and include more complex retrovirus-derived vectors, e.g., lentivirus-derived vectors. HIV-1- derived vectors belong to this category.
  • retrovirus-derived vectors e.g., Moloney murine leukemia virus (MLV)-derived vectors
  • retrovirus-derived vectors e.g., Moloney murine leukemia virus (MLV)-derived vectors
  • retrovirus-derived vectors e.g., Moloney murine leukemia virus (MLV)-derived vectors
  • retrovirus-derived vectors e.g., Moloney murine leukemia virus (MLV)-derived vectors
  • retrovirus-derived vectors e.g., Moloney murine leukemia virus (MLV)-derived vectors
  • retrovirus-derived vectors e.g.
  • lentivirus vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and Maedi-Visna virus ovine lentivirus.
  • Methods of using retroviral and lentiviral viral vectors and packaging cells for transducing mammalian host cells with viral particles including CAR transgenes are described in, e.g., US 8,1 19,772; Walchli, et ai., 201 1 , PLoS One 6:327930; Zhao, et ai., 2005, J. Immunol. 174:4415; Engels, et ai., 2003, Hum. Gene Ther.
  • Retroviral and lentiviral vector constructs and expression systems are also commercially available.
  • codon-optimized sequences encoding the CLL-1 -specific VH- VL and VL-VH SCFVS or CD123-specific VH-VL and VL-VH SCFVS can be synthesized and cloned into the epHIV7 lentiviral plasmid backbone used in previous CD19 CAR-T cell clinical trials.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • Cas CRISPR-associated protein
  • CRISPR-Cas systems and components thereof are described in, for example, US8697359, US8771945, US8795965, US8865406, US8871445, US8889356, US8889418, US8895308, US8906616, US8932814, US8945839, US8993233 and US8999641 and applications related thereto; and WO2014/018423, WO2014/093595, WO2014/093622 , WO2014/093635, WO2014/093655, WO2014/093661 , WO2014/093694, WO2014/093701 , WO2014/093709, WO2014/093712, WO2014/093718, WO2014/145599, WO2014/204723, W02014/204724, WO2014/204725, WO2014/204726, WO2014/204727, WO2014/204728, WO2014/204729, WO2015/065964, WO2015/0893
  • ZFNs zinc finger nucleases
  • DSBs double stranded breaks
  • TALENs transcription activator like effector nucleases
  • TALE transcription activator-like effector
  • TALENs are used to edit genes and genomes by inducing double DSBs in the DNA, which induce repair mechanisms in cells.
  • two TALENs must bind and flank each side of the target DNA site for the DNA cleavage domain to dimerize and induce a DSB.
  • MegaTALs have a sc rare-cleaving nuclease structure in which a TALE is fused with the DNA cleavage domain of a meganuclease.
  • Meganucleases also known as homing endonucleases, are single peptide chains that have both DNA recognition and nuclease function in the same domain. In contrast to the TALEN, the megaTAL only requires the delivery of a single peptide chain for functional activity.
  • Nanoparticles that result in selective in vivo genetic modification of targeted cell types have been described and can be used within the teachings of the current disclosure.
  • the nanoparticles can be those described in WO20141531 14, W02017181 110, and WO201822672.
  • genetically- modified cells can be harvested from a culture medium, and washed and concentrated into a carrier in a therapeutically-effective amount.
  • exemplary carriers include saline, buffered saline, physiological saline, water, Hanks' solution, Ringer's solution, Nonnosol-R (Abbott Labs), PLASMA-LYTE A ® (Baxter Laboratories, Inc., Morton Grove, IL), glycerol, ethanol, and combinations thereof.
  • carriers can be supplemented with human serum albumin (HSA) or other human serum components or fetal bovine serum.
  • HAS human serum albumin
  • a carrier for infusion includes buffered saline with 5% HAS or dextrose.
  • Additional isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol.
  • Carriers can include buffering agents, such as citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and/or trimethylamine salts.
  • buffering agents such as citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and/or trimethylamine salts.
  • Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which helps to prevent cell adherence to container walls.
  • Typical stabilizers can include polyhydric sugar alcohols; amino acids, such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, and cyclitols, such as inositol; PEG; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate
  • compositions or formulations can include a local anesthetic such as lidocaine to ease pain at a site of injection.
  • Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides, hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol.
  • Therapeutically effective amounts of cells within compositions or formulations can be greater than 10 2 cells, greater than 10 3 cells, greater than 10 4 cells, greater than 10 5 cells, greater than 10 6 cells, greater than 10 7 cells, greater than 10 8 cells, greater than 10 9 cells, greater than 10 10 cells, or greater than 10 11 .
  • cells are generally in a volume of a liter or less, 500 mis or less, 250 mis or less or 100 mis or less.
  • density of administered cells is typically greater than 10 4 cells/ml, 10 7 cells/ml or 10 8 cells/ml.
  • compositions include one genetically-modified cell type (e.g., modified T cells, NK cells, or stem cells).
  • Formulations can include different types of genetically-modified cells (e.g.,T cells, NK cells, and/or stem cells in combination).
  • Different types of genetically-modified cells or cell subsets can be provided in different ratios e.g., a 1 : 1 :1 ratio, 2: 1 : 1 ratio, 1 :2: 1 ratio, 1 :1 :2 ratio, 5: 1 : 1 ratio, 1 :5: 1 ratio, 1 : 1 :5 ratio, 10: 1 :1 ratio, 1 : 10: 1 ratio, 1 : 1 : 10 ratio, 2:2: 1 ratio, 1 :2:2 ratio, 2: 1 :2 ratio, 5:5:1 ratio, 1 :5:5 ratio, 5: 1 :5 ratio, 10: 10:1 ratio, 1 : 10: 10 ratio, 10:1 : 10 ratio, etc.
  • ratios can also apply to numbers of cells expressing the same or different CAR components. If only two of the cell types are combined or only 2 combinations of expressed CAR components are included within a formulation, the ratio can include any 2 number combination that can be created from the 3 number combinations provided above.
  • the combined cell populations are tested for efficacy and/or cell proliferation in vitro, in vivo and/or ex vivo, and the ratio of cells that provides for efficacy and/or proliferation of cells is selected. Particular embodiments include a 1 : 1 ratio of CD4 T cells and CD8 T cells.
  • compositions and formulations disclosed herein can be prepared for administration by, e.g., injection, infusion, perfusion, or lavage.
  • the compositions and formulations can further be formulated for bone marrow, intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, and/or subcutaneous injection.
  • Nanoparticle Formulations Nanoparticles that result in in vivo genetic modification of cells can be formulated alone or in combination into compositions for administration to subjects.
  • Compositions include nanoparticles formulated with at least one pharmaceutically acceptable carrier.
  • compositions can be formulated as aqueous solutions, such as in buffers including Hanks' solution, Ringer's solution, or physiological saline.
  • aqueous solutions can include formulatory agents such as suspending, stabilizing, and/or dispersing agents.
  • the formulation can be in lyophilized and/or powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • solvents for example, dichloromethane, chloroform, ethyl acetate, triacetin, N-methyl pyrrolidone, tetrahydrofuran, phenol, or combinations thereof
  • Other useful solvents include water, ethanol, dimethyl sulfoxide (DMSO), N-methyl-2-pyrrolidone (NMP), acetone, methanol, isopropyl alcohol (I PA), ethyl benzoate, and benzyl benzoate.
  • Exemplary release modifiers can include surfactants, detergents, internal phase viscosity enhancers, complexing agents, surface active molecules, co-solvents, chelators, stabilizers, derivatives of cellulose, (hydroxypropyl)methyl cellulose (HPMC), HPMC acetate, cellulose acetate, pluronics (e.g., F68/F127), polysorbates, Span® (Croda Americas, Wilmington, Delaware), poly(vinyl alcohol) (PVA), Brij® (Croda Americas, Wilmington, Delaware), sucrose acetate isobutyrate (SAIB), salts, and buffers.
  • surfactants e.g., hydroxypropyl)methyl cellulose (HPMC), HPMC acetate, cellulose acetate, pluronics (e.g., F68/F127), polysorbates, Span® (Croda Americas, Wilmington, Delaware), poly(vinyl alcohol) (PVA), Brij® (Croda Americas, Wilmington, Delaware), suc
  • compositions disclosed herein can advantageously include any other pharmaceutically acceptable carriers which include those that do not produce significantly adverse, allergic, or other untoward reactions that outweigh the benefit of administration.
  • exemplary pharmaceutically acceptable carriers and formulations are disclosed in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990.
  • formulations can be prepared to meet sterility, pyrogenicity, general safety, and purity standards as required by the US FDA Office of Biological Standards and/or other relevant foreign regulatory agencies.
  • Methods disclosed herein include treating subjects (humans, veterinary animals (dogs, cats, reptiles, birds, etc.) livestock (horses, cattle, goats, pigs, chickens, etc.) and research animals (monkeys, rats, mice, fish, etc.) with compositions and formulations disclosed herein. Treating subjects includes delivering therapeutically effective amounts. Therapeutically effective amounts include those that provide effective amounts, prophylactic treatments and/or therapeutic treatments.
  • an "effective amount” is the amount of a composition necessary to result in a desired physiological change in the subject.
  • an effective amount can provide an immunogenic anti-cancer.
  • Effective amounts are often administered for research purposes.
  • Effective amounts disclosed herein can cause a stati stically-signifi cant effect in an animal model or in vitro assay relevant to the assessment of a cancer’s development or progression.
  • An immunogenic composition can be provided in an effective amount, wherein the effective amount stimulates an immune response.
  • a prophylactic treatment includes a treatment administered to a subject who does not display signs or symptoms of a cancer or displays only early signs or symptoms of a cancer such that treatment is administered for the purpose of diminishing or decreasing the risk of developing the cancer further.
  • a prophylactic treatment functions as a preventative treatment against a cancer.
  • prophylactic treatments reduce, delay, or prevent metastasis from a primary a cancer tumor site from occurring.
  • a "therapeutic treatment” includes a treatment administered to a subject who displays symptoms or signs of a cancer and is administered to the subject for the purpose of diminishing or eliminating those signs or symptoms of the cancer.
  • the therapeutic treatment can reduce, control, or eliminate the presence or activity of the cancer and/or reduce control or eliminate side effects of the cancer.
  • prophylactic treatment or therapeutic treatment are not mutually exclusive, and in particular embodiments, administered dosages may accomplish more than one treatment type.
  • therapeutically effective amounts provide anti-cancer effects.
  • Anti-cancer effects include a decrease in the number of cancer cells, decrease in the number of metastases, a decrease in tumor volume, an increase in life expectancy, induced chemo- or radiosensitivity in cancer cells, inhibited angiogenesis near cancer cells, inhibited cancer cell proliferation, inhibited tumor growth, prevented or reduced metastases, prolonged subject life, reduced cancer-associated pain, and/or reduced relapse or re-occurrence of cancer following treatment.
  • a "tumor” can be liquid or solid depending on the cell origin.
  • a solid tumor is a swelling or lesion formed by an abnormal growth of cells (called neoplastic cells or tumor cells).
  • a "tumor cell” is an abnormal cell that grows by a rapid, uncontrolled cellular proliferation and continues to grow after the stimuli that initiated the new growth cease and can be considered a solid tumor or liquid tumor in the art depending on the cell origin. Tumors show partial or complete lack of structural organization and functional coordination with the normal tissue, and usually form a distinct mass of tissue, which may be benign, pre-malignant or malignant. Liquid tumors also refer to the total mass of circulating neoplastic cells, for examples in hematopoietic malignancies such as leukemia.
  • Particular embodiments of the disclosure are used to treat AML, BPDCN, MDS, natural killer cell lymphomas, hairy cell leukemia, ALL, CML, other leukemias, hematological cancers or tumors, (HL, B-cell HL, NHL, MCL, T cell lymphoma, multiple myeloma (refractory, relapsed, etc.), SM, HES, myelofibrosis, anemia, SLE, psoriasis, and scleroderma.
  • therapeutically effective amounts can be initially estimated based on results from in vitro assays and/or animal model studies. Such information can be used to more accurately determine useful doses in subjects of interest.
  • the actual dose amount administered to a particular subject can be determined by a physician, veterinarian or researcher taking into account parameters such as physical and physiological factors including target, body weight, severity of condition, type of cancer, stage of cancer, previous or concurrent therapeutic interventions, idiopathy of the subject and route of administration.
  • Therapeutically effective amounts to administer can include greater than 10 2 cells, greater than 10 3 cells, greater than 10 4 cells, greater than 10 5 cells, greater than 10 6 cells, greater than 10 7 cells, greater than 10 8 cells, greater than 10 9 cells, greater than 10 10 cells, or greater than 10 11 .
  • Useful doses can range from 0.1 to 5 pg/kg or from 0.5 to 1 pg /kg.
  • a dose can include 1 pg /kg, 15 pg /kg, 30 pg /kg, 50 pg/kg, 55 pg/kg, 70 pg/kg, 90 pg/kg, 150 pg/kg, 350 pg/kg, 500 pg/kg, 750 pg/kg, 1000 pg/kg, 0.1 to 5 mg/kg or from 0.5 to 1 mg/kg.
  • a dose can include 1 mg/kg, 10 mg/kg, 30 mg/kg, 50 mg/kg, 70 mg/kg, 100 mg/kg, 300 mg/kg, 500 mg/kg, 700 mg/kg, 1000 mg/kg or more.
  • Therapeutically effective amounts can be achieved by administering single or multiple doses during the course of a treatment regimen (e.g., daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months or yearly).
  • a treatment regimen e.g., daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months or yearly.
  • compositions and formulations disclosed herein can be administered by, e.g., injection, infusion, perfusion, or lavage and can more particularly include administration through one or more bone marrow, intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, and/or subcutaneous infusions and/or bolus injections.
  • cells or nanoparticle-based formulations are administered to a patient in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities.
  • cells or nanoparticle-based formulations may be used in combination with chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as alemtuzumab, anti-CD3 antibodies or other antibody therapies, cytoxin, fludarabine, cyclophosphamide, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation.
  • immunosuppressive agents such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies
  • immunoablative agents such as alemtuzumab, anti-CD3 antibodies or other antibody therapies, cytoxin
  • cells or nanoparticle-based formulations may be used in conjunction with cyclophosphamide.
  • cells or nanoparticle-based formulations may be used in conjunction with fludarabine.
  • cells or nanoparticle-based formulations may be used in conjunction with cyclophosphamide and fludarabine.
  • FIG. 25 illustrates three exemplary clinical applications of treatment strategies disclosed herein.
  • An initial clinical trial could include patients having R/R AML who have a suitable allogenic donor identified.
  • chemotherapy can be administered to a patient having R/R AML for a first time period. Then, chemotherapy can be reduced and the patient can be infused with anti-CLL-1/anti-CD123 CAR-T cells for a second time period. Upon myeloid recovery, chemotherapy can then be administered to the patient for a third time period.
  • chemotherapy can be administered to a patient having R/R AML for a first time period. Then, chemotherapy can be reduced and the patient can be infused with CLL-1/CD123 CAR-T cells for a second time period. Upon myeloid recovery, a control feature (tag cassette, transduction marker and/or suicide switch) can be activated in the CLL- 1/CD123 CAR-T cells and chemotherapy can then be administered to the patient for a third time period.
  • a control feature tag cassette, transduction marker and/or suicide switch
  • chemotherapy can be administered to a patient having R/R AML for a first time period. Then, chemotherapy can be reduced and the patient can be infused with CLL-1/CD123 CAR-T cells for a second time period.
  • a control feature can be activated in the CLL-1/CD123 CAR-T cells, the patient can then undergo myeloablation followed by a bone marrow transplant (BMT).
  • BMT bone marrow transplant
  • a chimeric antigen receptor (CAR) system including a cell genetically modified to express at least two CAR constructs wherein each expressed CAR construct includes
  • the extracellular component of at least one CAR construct expressed by the cell includes a binding domain that specifically binds CLL-1 ; and wherein the extracellular component of a second CAR construct expressed by the cell includes a binding domain that specifically binds CD123;
  • intracellular components of the CAR constructs include an effector domain.
  • binding domain that specifically binds CLL- 1 is a single chain variable fragment (sFv) including the variable light chain and the variable heavy chain complementarity determining regions (CDRs) of SC02357 as set forth in SEC ID NO: 261-266, according to Kabat numbering.
  • sFv single chain variable fragment
  • CDRs variable heavy chain complementarity determining regions
  • binding domain that specifically binds CD123 is a single chain variable fragment (scFv) including the variable light chain and the variable heavy chain of KLON120, as set forth in SEC ID NO: 19 and SEC ID NO: 18.
  • scFv single chain variable fragment
  • variable heavy chain of KLON120 and the variable light chain of KLON120 are linked through a Gly-Ser linker including SEC ID NO: 212.
  • a chimeric antigen receptor (CAR) system of any of embodiments 1-5 including a cell genetically modified to express a CAR construct including an extracellular component linked to an intracellular component through a transmembrane domain;
  • the extracellular component includes a binding domain that specifically binds CLL-1 and a binding domain that specifically binds CD123
  • intracellular components of the CAR construct include an effector domain.
  • the cell is a T cell selected from a CD3 T cell, a CD4 T cell, a CD8 T cell, a central memory T cell, an effector memory T cell, and/or a naive T cell.
  • the CAR system of any of embodiments 1-10 including CD4 T cells and CD8 T cells.
  • the CAR system of embodiment 11 including a 1 :1 ratio of CD4 T cells to CD8 T cells.
  • the CAR system of any of embodiments 1-12 including at least two cell types genetically modified ex vivo to express a CAR construct of the system.
  • a chimeric antigen receptor (CAR) system including cells genetically modified to express at least one CAR, wherein binding domains of CAR within the system specifically bind CLL-1 or CD123, and wherein each CAR in the system:
  • extracellular components within the system include:
  • the intracellular component includes an effector domain.
  • the CAR system of any of embodiments 15-17 wherein at least two CAR within the system are expressed by a same genetically modified cell and wherein the first CAR includes a binding domain that specifically binds CLL-1 and the second CAR includes a binding domain that specifically binds CLL-1.
  • the CAR system of any of embodiments 15-17 wherein at least two CAR within the system are expressed by a same genetically modified cell and wherein the first CAR includes a binding domain that specifically binds CD123 and the second CAR includes a binding domain that specifically binds CD123.
  • the CAR system of any of embodiments 15-21 wherein the binding domain of at least one CAR within the system is derived from a binding domain that specifically binds CLL-1 and the binding domain of at least one CAR within the system is derived from a binding domain that specifically binds CD123.
  • binding domain that specifically binds CLL-1 is a single chain variable fragment (sFv) including the variable light chain and the variable heavy chain CDRs of SC02357 as set forth in SEQ ID NO: 261-266, according to Kabat numbering.
  • sFv single chain variable fragment
  • binding domain that specifically binds CD123 is a single chain variable fragment (scFv) including the variable light chain and the variable heavy chain of KLON120, as set forth in SEQ ID NO: 19 and SEQ ID NO: 18.
  • scFv single chain variable fragment
  • binding domains include a binding domain as set forth in SEQ ID NO: 138 and a binding domain selected from SEQ ID NO 139 and SEQ ID NO: 267.
  • a binding domain within the system that specifically binds CLL-1 is derived from the CDRs of M26, M31 , G4, M22, M29, M2, M5, or G12 according to Kabat numbering.
  • the CAR system of any of embodiments 15-32, wherein a binding domain within the system that specifically binds CD123 is derived from the CDRs of IMGN632, ADAMTS2, 7G3, 32716, 32701 , 32703, or 26292 according to Kabat numbering.
  • a CAR within the system includes SC02357-lgG4 hinge-28TM-41 BB-3Z; KLON120-lgG4 hinge-28TM-41 BB-3Z; anti- CD123_scFV-hsCD28_TM-hs4-1 BB_CPD-hsCD3 ⁇ CPD-T2A-hsEGFRt and/or anti- 0 ⁇ _30Rn-I ⁇ 30028_TM- ⁇ 34-1 BB_0R0- ⁇ 3003z_0R0-T2A- ⁇ 3E0RR ⁇ .
  • SC02357 includes a single chain variable fragment that specifically binds CLL-1 including the variable light chain and the variable heavy chain CDRs of SC02357 as set forth in SEQ ID NO: 261-266, according to Kabat numbering.
  • KLON120 includes a single chain variable fragment that specifically binds CD123 including the variable heavy chain and the variable light chain of KLON 120 as set forth in SEQ ID NO: 19 and SEQ ID NO: 18.
  • variable heavy chain of KLON120 and the variable light chain of KLON120 are linked through a Gly-Ser linker including SEQ ID NO: 212.
  • the CAR system of any of embodiments 34-38, wherein the lgG4 hinge can be selected from SEQ ID NO: 38 or SEQ ID NO: 257.
  • the multimerization domains include a PRKAR1A dimerization domain, PRKAR1 E (SEQ ID NO: 53), and/or a PRKAR1 R dimerization domain.
  • the CAR system of embodiment 41 wherein the PRKAR1 A dimerization domain is selected from SEQ ID NO: 48 and SEQ ID NO: 50.
  • the CAR system of embodiments 41 or 42, wherein the PRKAR1 R dimerization domain is selected from SEQ ID NO: 49 and SEQ ID NO: 55.
  • the CAR system of any of embodiments 40-44 wherein the multimerization domains are selected from the leucine zipper domain of Jun (SEQ ID NO: 74), the dimerization domain of Fos (SEQ ID NO: 75), a consensus sequence for a WW motif (SEQ ID NO: 76), the dimerization domain of the SH2B adapter protein (SEQ ID NO: 77), the SH3 domain of IB1 (SEQ ID NO: 78), the PTB domain of human DOK-7 (SEQ ID NO: 79), the PDZ-like domain of SATB1 (SEQ ID NO: 80), the WD40 repeats of APAF (SEQ ID NO: 81), the PAS motif of the dioxin receptor (SEQ ID NO: 82), the EF hand motif of parvalbumin (SEQ ID NO: 83), and a C4b multimerization domain selected from SEQ ID NOs: 84 - 116 and/or ferritin.
  • PRKAR1 AR-28TM-41 BB-3Z KLON120-PRKAR1 AE-28TM-41 BB-3Z ; MDT-000828 (SEQ ID NO: 17); MDT-000830 (SEQ ID NO: 20); MDT-000831 (SEQ ID NO: 24); and/or MDT- 000832 (SEQ ID NO: 28).
  • PRKAR1A is selected from SEQ ID NO: 48 and SEQ ID NO: 50.
  • PRKAR1AE is SEQ ID NO: 53.
  • PRKAR1AR is selected from SEQ ID NO: 49 and SEQ ID NO: 55.
  • the CAR system of any of embodiments 15-55 further including a spacer region between the binding domain and the transmembrane domain.
  • the CAR system of any of embodiments 15-58 further including a control feature selected from a tag cassette, a transduction marker, and/or a suicide switch.
  • the CAR system of any of embodiments 15-59, wherein the genetically-modified cells are T cells, natural killer cells, monocytes/macrophages, hematopoietic stem cells or hematopoietic progenitor cells.
  • T cells are selected from CD3 T cells, CD4 T cell, CD8 T cells, central memory T cells, effector memory T cells, and/or naive T cells.
  • the CAR system of embodiment 61 including CD4 T cells and CD8 T cells.
  • the CAR system of embodiment 62 including a 1 : 1 ratio of CD4 T cells to CD8 T cells.
  • the CAR system of embodiment 64 wherein the at least two cell types include T cells and natural killer cells, T cells and monocyte/macrophages, T cells and hematopoietic stem cells, T cells and hematopoietic progenitor cells, natural killer cells and monocyte/macrophages, natural killer cells and hematopoietic stem cells, natural killer cells and hematopoietic progenitor cells, monocyte/macrophages and hematopoietic stem cells, monocyte/macrophages and hematopoietic progenitor cells, or hematopoietic stem cells and hematopoietic progenitor cells.
  • the at least two cell types include T cells and natural killer cells, T cells and monocyte/macrophages, T cells and hematopoietic stem cells, T cells and hematopoietic progenitor cells, natural killer cells and monocyte/macrophages, natural killer cells and hematopoietic stem cells, natural killer cells
  • the cell of embodiment 70 wherein the cell is a CD4 T cell or a CD8 T cell.
  • composition of embodiment 72 wherein the cells are T cells, natural killer cells, monocyte/macrophages, hematopoietic stem cells or hematopoietic progenitor cells.
  • composition of embodiment 73 wherein the T cells are selected from CD3 T cells, CD4 T cells, CD8 T cells, central memory T cells, effector memory T cells, and/or naive T cells.
  • the composition of embodiment 74, wherein the T cells are CD4 T cells and CD8 T cells.
  • the composition of any of embodiments 72-76, wherein the cells include a formulation of at least two cell types genetically modified ex vivo to express the CAR system.
  • composition of embodiment 77 wherein the at least two cell types include T cells and natural killer cells, T cells and monocyte/macrophages, T cells and hematopoietic stem cells, T cells and hematopoietic progenitor cells, natural killer cells and monocyte/macrophages, natural killer cells and hematopoietic stem cells, natural killer cells and hematopoietic progenitor cells, monocyte/macrophages and hematopoietic stem cells, monocyte/macrophages and hematopoietic progenitor cells, or hematopoietic stem cells and hematopoietic progenitor cells.
  • a method of treating a subject in need thereof including administering a therapeutically effective amount of a composition of any of embodiments 72-78 to the subject wherein the therapeutically effective amount results in in vivo expression of the CAR system of any of embodiments 1-66 within the subject thereby treating the subject in need thereof.
  • Example 1 CAR Engineering, Lentivirus Production, T Cell Transduction, and Readout Assays. Patients were treated using a CD19 CAR including a short lgG4-hinge only spacer, CD28 transmembrane (TM) domain, 4-1 BB co-stimulatory domain, and a ⁇ 3z signaling domain that is separated by a T2A ribosomal skip sequence from a truncated human EGFR (tEGFR), which serves as a marker of transgene expression (see Turtle et al., Sci Transl Med. 2016;8(355):355ra1 16-355ra1 16; Turtle et al., J Clin Invest.
  • tEGFR truncated human EGFR
  • the epHIV7 lentiviral backbone used in the CD19 CAR was employed to design novel CAR constructs that when introduced into primary T cells redirect the specificity of the T cells to either CLL-1 or CD123.
  • CLL-1 -CAR were generated that include a human CLL-1 scFv (SC02357) derived from a phage display library.
  • CD123-CAR were generated that include a mouse scFv (Klon120) derived from a phage display library from mouse spleen RNA.
  • CLL-1- and CD123- CAR including the following in their extracellular domain were also generated: i) an lgG4 hinge“short” spacer (12 amino acids); ii) an lgG4 hinge and CH3 domain containing an“intermediate” spacer (1 19 amino acids); and iii) an lgG4 hinge, CH2, and CH3 domain containing a“long” spacer (229 amino acids).
  • FIGs. 6A, 6B depict the results of the CLL-1-CAR spacer analysis. As shown, it was found that the CLL-1 scFv proliferates and produces cytokines optimally on a short spacer. More particularly, FIG. 6A demonstrates that CLL1-short, CLL1-int, and CLL1-long CD8 CAR-T cells are effective at specifically lysing K562 cells transduced with CLL1 (K-CLL1) but not CD19 (K-19) at varying effector to target ratios.
  • FIG. 6B demonstrates the specific production of the cytokines IL-2, TNF-a, and IFN-g by CLL1 -short, CLL1 -int, and CLL1-long CD8 CAR-T cells when co-cultured with K-CLL1 , with minimal cytokine production when co-cultured with K-19 cells.
  • FIGs. 7A, 7B depict the results of the CD123-CAR spacer analysis. As shown, it was found that the CD123 scFv proliferates and produces cytokines optimally on a short spacer. More particularly, FIG. 7A demonstrates that CD123-short, CD123-int, and CD123-long CD8 CAR-T cells are effective at specifically lysing K562 cells transduced with CD123 (K-123) but not CD19 (K-19) at varying effector to target ratios. It can be seen that CFSE labelled CD123-short, CD123- int, and CD123-long CD8 CAR-T cells have specific proliferation in response to K-123 but not K- 19 cells.
  • FIG. 7B demonstrates the specific production of the cytokines IL-2, TNF-a, and IFN-g by CD123- short, CD123-int, and CD123-long CD8 CAR-T cells when co-cultured with K-123, with minimal cytokine production when co-cultured with K-19 cells.
  • CD19-short CAR activity is shown by way of comparison.
  • Tandem CLL-1/CD123 CAR including the following extracellular domains were generated: CLL1-G4Sx1-CD123, CD123- G4Sx1-CLL1 , CD123-G4Sx3-CLL1 , CLL1-G4Sx4-CD123, CD123-G4Sx4-CLL1 , CLL1-
  • each tandem CLL-1/CD123 CAR includes a different order of its CLL-1 and CD123 scFvs and/or different linker modules between its CLL-1 and CD123 scFvs.
  • FIG. 8A demonstrates that CD123-G4Sx3-CLL1 and CLL1-CLL1-G4Sx4-CD123 tandem CD8 CAR-T cells are effective at specifically lysing K562 cells transduced with CLL1 (K-CLL1), CD123 (K- 123), or both CLL1 and CD123 (K-123CLL1) but not CD19 (K-19) at varying effector to target ratios.
  • CD8+ T cells can be enriched by immunomagnetic positive selection using CD8+ microbeads (MILTENYI BIOTECTM) and cryopreserved. Thawed CD8+ T cells can be activated on day zero with anti-CD3/CD28 paramagnetic beads (LIFE SCIENCESTM) in medium with IL-15 1 ng/mL. On day one, cultures can be transduced with the CAR lentiviral supernatant. Paramagnetic beads can be removed on day five, and on day seven, tEGFR+ transduced T cells can be flow sorted and expanded for seven additional days in IL-15.
  • MILTENYI BIOTECTM CD8+ microbeads
  • IFE SCIENCESTM anti-CD3/CD28 paramagnetic beads
  • CAR-T cells can be assayed for their proliferative capacity and ability to induce target cell cytotoxicity after stimulation with antigen-positive (K562/CLL-1) or antigen negative control (K562) target cells.
  • K562/CLL-1 stimulation the percentage of proliferating CAR-T cells can be assessed by CFSE dilution and the percentage of K562/CLL-1 target cells lysed can be assessed by flow cytometry.
  • Example 4 The capacity of humanized MISTRG/IL6 mice to allow engraftment of primary AML blasts and human hematopoiesis can be harnessed, and the capacity of CLL-1- and CD123- specific Tandem CAR-, Dual CAR-, Dual Population CAR, and multimerizing CAR cells (developed above) that are manufactured from AML patients to eliminate autologous AML blasts can be compared.
  • Antibodies and antibody fragments targeting specific antigens are referred to, herein, as the antibody name, antibody fragment name, antigen target, and“anti-“ the antigen target.
  • molecules targeting CD123 are referred to as KLON120, anti-CD123, CD123, scFv targeting CD123, CD123_VH and CD123_VL, etc.
  • amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e. , substitutions of similarly charged or uncharged amino acids.
  • a conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains.
  • Naturally occurring amino acids are generally divided into conservative substitution families as follows: Group 1 : Alanine (Ala), Glycine (Gly), Serine (Ser), and Threonine (Thr); Group 2: (acidic): Aspartic acid (Asp), and Glutamic acid (Glu); Group 3: (acidic; also classified as polar, negatively charged residues and their amides): Asparagine (Asn), Glutamine (Gin), Asp, and Glu; Group 4: Gin and Asn; Group 5: (basic; also classified as polar, positively charged residues): Arginine (Arg), Lysine (Lys), and Histidine (His); Group 6 (large aliphatic, nonpolar residues): Isoleucine (lie), Leucine (Leu), Methionine (Met), Valine (Val) and Cysteine (Cys); Group 7 (uncharged polar): Tyrosine (Tyr), Gly, Asn, Gin, Cys, Ser, and Thr; Group
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1), 105-32). Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982).
  • amino acid substitutions may be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • variants of gene sequences can include codon optimized variants, sequence polymorphisms, splice variants, and/or mutations that do not affect the function of an encoded product to a statistically-significant degree.
  • Variants of the protein, nucleic acid, and gene sequences disclosed herein also include sequences with at least 70% sequence identity, 80% sequence identity, 85% sequence, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity to the protein, nucleic acid, or gene sequences disclosed herein.
  • % sequence identity refers to a relationship between two or more sequences, as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between protein, nucleic acid, or gene sequences as determined by the match between strings of such sequences.
  • Identity (often referred to as “similarity") can be readily calculated by known methods, including those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H.
  • Variants also include nucleic acid molecules that hybridizes under stringent hybridization conditions to a sequence disclosed herein and provide the same function as the reference sequence.
  • Exemplary stringent hybridization conditions include an overnight incubation at 42 °C in a solution including 50% formamide, 5XSSC (750 mM NaCI, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5XDenhardt's solution, 10% dextran sulfate, and 20 pg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1XSSC at 50 °C.
  • 5XSSC 750 mM NaCI, 75 mM trisodium citrate
  • 50 mM sodium phosphate pH 7.6
  • 5XDenhardt's solution 10% dextran sulfate
  • 20 pg/ml denatured, sheared salmon sperm DNA followed by washing the filters in 0.1XSSC at 50 °C
  • Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature.
  • washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5XSSC).
  • Variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments.
  • Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations.
  • the inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
  • binds refers to an association of a binding domain (of, for example, a CAR binding domain or a nanoparticle selected cell targeting ligand) to its cognate binding molecule with an affinity or K a (i.e., an equilibrium association constant of a particular binding interaction with units of 1/M) equal to or greater than 10 5 M -1 , while not significantly associating with any other molecules or components in a relevant environment sample.“Specifically binds” is also referred to as“binds” herein. Binding domains may be classified as "high affinity” or "low affinity”.
  • high affinity binding domains refer to those binding domains with a K a of at least 10 7 M 1 , at least 10 8 M 1 , at least 10 9 M 1 , at least 10 10 M 1 , at least 10 11 M 1 , at least 10 12 M 1 , or at least 10 13 M 1 .
  • low affinity binding domains refer to those binding domains with a K a of up to 10 7 M 1 , up to 10 6 M 1 , up to 10 5 M 1 .
  • affinity may be defined as an equilibrium dissociation constant (K d ) of a particular binding interaction with units of M (e.g., 10 5 M to 10 13 M).
  • a binding domain may have "enhanced affinity," which refers to a selected or engineered binding domains with stronger binding to a cognate binding molecule than a wild type (or parent) binding domain.
  • enhanced affinity may be due to a Ka (equilibrium association constant) for the cognate binding molecule that is higher than the reference binding domain or due to a K d (dissociation constant) for the cognate binding molecule that is less than that of the reference binding domain, or due to an off- rate (K 0ff ) for the cognate binding molecule that is less than that of the reference binding domain.
  • assays are known for detecting binding domains that specifically bind a particular cognate binding molecule as well as determining binding affinities, such as Western blot, ELISA, and BIACORE ® analysis (see also, e.g., Scatchard, et al., 1949, Ann. N. Y. Acad. Sci. 51:660; and US 5,283, 173, US 5,468,614, or the equivalent).
  • each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component.
  • the terms“include” or“including” should be interpreted to recite: “comprise, consist of, or consist essentially of.”
  • the transition term“comprise” or“comprises” means includes, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts.
  • the transitional phrase “consisting of’ excludes any element, step, ingredient or component not specified.
  • the transition phrase “consisting essentially of” limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment. A material effect would cause a statistically significant reduction in the dimerization of CAR expressed by a genetically modified cell.
  • the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ⁇ 20% of the stated value; ⁇ 19% of the stated value; ⁇ 18% of the stated value; ⁇ 17% of the stated value; ⁇ 16% of the stated value; ⁇ 15% of the stated value; ⁇ 14% of the stated value; ⁇ 13% of the stated value; ⁇ 12% of the stated value; ⁇ 1 1 % of the stated value; ⁇ 10% of the stated value; ⁇ 9% of the stated value; ⁇ 8% of the stated value; ⁇ 7% of the stated value; ⁇ 6% of the stated value; ⁇ 5% of the stated value; ⁇ 4% of the stated value; ⁇ 3% of the stated value; ⁇ 2% of the stated value; or ⁇ 1 % of the stated value.

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Abstract

Compositions and methods to target C-type lectin-like molecule-1 (CLL-1) and interleukin-3 receptor (CD123) for the treatment of acute myeloid leukemia and related disorders are described. The disclosure describes multiple strategies to target CLL-1 and CD123 utilizing multiple chimeric antigen receptor (CAR) system designs.

Description

COMPOSITIONS AND METHODS TO TARGET CLL-1 AND CD123 FOR THE TREATMENT OF ACUTE MYELOID LEUKEMIA AND RELATED DISORDERS
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional Patent Application No. 62/688,674 filed June 22, 2018 the entire contents of which are incorporated by reference herein.
FIELD OF THE DISCLOSURE
[0002] The current disclosure provides compositions and methods to target C-type lectin-like molecule-1 (CLL-1) and interleukin-3 receptor (CD123) for the treatment of acute myeloid leukemia and related disorders. The disclosure describes multiple strategies to target CLL-1 and CD123 utilizing multiple chimeric antigen receptor (CAR) construct system designs.
BACKGROUND OF THE DISCLOSURE
[0003] According to the World Health Organization, cancer is the second leading cause of death globally, and was responsible for an estimated 9.6 million deaths in 2018. Acute myeloid leukemia (AML) is a malignancy of clonal, proliferative myeloid blast cells. High complete remission rates for patients with AML can be achieved with conventional chemotherapy at rates of 60% to 80% for younger adults and 40% to 60% for adults greater than age 60 (Dohner, et al., 2015. Nat Engl. J. Med. 373(12): 1 136-1 152). Unfortunately, because of the chemorefractoriness of leukemic stem cells, relapse after conventional therapy is common (Eppert, et al., 201 1. Nat. Med. 17(9): 1086-1093) and current treatment options for relapsed/refractory (R/R) AML are dismal, resulting in less than 30% overall survival at 12 months.
[0004] In recent years, more targeted therapies have emerged to specifically target cancer cells by identifying and exploiting specific molecular changes seen primarily in those cells. For example, significant progress has been made in genetically engineering T cells of the immune system to target and kill unwanted cell types, such as cancer cells. Many of these T cells have been genetically engineered to express a chimeric antigen receptor (CAR). CAR constructs encode proteins including several distinct subcomponents that allow the genetically modified T cells to recognize and kill cancer cells. The subcomponents include at least an extracellular component and an intracellular component. The extracellular component includes a binding domain that specifically binds a marker that is preferentially present on the surface of cancer cells. When the binding domain binds such markers, the intracellular component signals the T cell to destroy the bound cell. CAR constructs can additionally include a transmembrane domain that can link the extracellular component to the intracellular component, and other subcomponents that can increase the CAR’s function. For example, the inclusion of one or more linker sequences, such as a spacer region, can allow the CAR to have additional conformational flexibility, often increasing the binding domain’s ability to bind the targeted cell marker.
[0005] Clinical trials with CAR-expressing T cells (CAR-T) have shown positive responses in patients with refractory large B-cell lymphoma when conventional treatments had failed (Neelapu, et al 2017 N Engl J Med 377:2531-2544). However, while successfully genetically engineer CAR- T cells result in cancer cell destruction, they have failed to provide prolonged anti-cancer activity in vivo for some indications. For example, in some cases, T cells have not received a strong enough signal when the binding domain binds the targeted cancer cell marker and does not activate the cytotoxic (killer) T cell response. Further, toxicities have been reported from some CAR-T studies, and additional theoretical toxicities exist. Many of the toxicities are thought to be due to "on-target off-cancer" effects when the targeted cancer cell marker is also expressed by non-cancerous tissues.
[0006] One of the keys to successful targeted immunotherapy is the choice of the target(s) on the cancer cell. An ideal target marker is immunogenic, plays a critical role in proliferation and differentiation (driver mutations), and is expressed only on the surface of all malignant cells and malignant stem cells. Further, a large portion of patients should test positive for the marker on their cancer cells (Cheever, et al., 2009. Clin. Cancer Res. 15(17): 5323-8337) in order for the therapy to reach the broadest number of patients.
[0007] Unfortunately for AML and related disorders, before the current disclosure, no single myeloid lineage-restricted marker had been identified that is expressed on all myeloblasts, or blasts, to enable effective targeting of AML. Further, AML is clonally heterogeneous, rendering single marker targeting ineffective for its treatment, due to the high likelihood that marker-negative blasts will escape single marker targeted therapy (see Buckley SA et al., ASH Education Program Book. 2015;2015(1):584-595).
SUMMARY OF THE DISCLOSURE
[0008] The current disclosure provides compositions and methods to target C-type lectin-like molecule-1 (CLL-1) and interleukin-3 receptor (CD123) for the treatment of acute myeloid leukemia (AML) and related disorders. CLL-1 and CD123 were selected to target following the use of flow cytometry to study the immunophenotype of leukemic blasts isolated from AML patients. It was found that in all studied patients, CLL-1 and/or CD 123 was expressed by over 99.9% of AML blasts.
[0009] The current disclosure provides multiple strategies to target CLL-1 and/or CD123 based on the genetic modification of cells to express one or more chimeric antigen receptors (CAR). A depiction of exemplary disclosed strategies is provided in FIG. 1 showing genetically modifying cells to create a population of genetically modified cells wherein:
(i) different cells within the population express a CLL-1 targeting CAR or a CD123-targeting CAR (the“dual population” approach);
(ii) the cells express two different CAR constructs, one targeting CLL-1 and one targeting CD123 (the“dual CAR” approach);
(iii) the cells express two CAR constructs with multimerizing domains, with one targeting CLL-1 and one targeting CD123; both targeting CLL-1 ; or both targeting CD123 (the“dimerized CAR” approach); and
(iv) the cells express a single CAR construct containing at least two binding domains in the extracellular component wherein at least one binding domain specifically binds CLL-1 and at least one binding domain specifically binds CD123 (the“tandem” approach).
[0010] The strategies depicted in FIG. 1 can be utilized alone or in various combinations. Regarding the“dimerized CAR” approach, if a genetically modified cell expresses multimerizing binding domains that all bind the same cancer antigen (e.g., CLL-1), these cells would be used within a dual population approach, for example a second population of cells expressing multimerizing binding domains that all bind CD123.
[0011] The strategies disclosed herein provide several important advantages. First, by targeting two cancer antigens also known as cancer markers, patients that may lack one or the other will still receive the benefit of the treatment as it is unlikely that patients would lack both markers based off the phenotypic assessment of AML blasts. Second, by linking at least two CAR molecules together through co-expression and/or multimerization, activation signals that the cell receives when the now-paired binding domains bind targeted cancer cell markers can be increased.
[0012] In particular embodiments, the compositions and methods disclosed herein provide effective therapeutics for a broader patient population, induce stronger cell activation signals and/or provide improved therapeutic effects. In particular embodiments, homo- or heterodimerization of CARs can improve function and T cell signaling, leading to improved clinical outcomes.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1. Exemplary CLL-1/CD123 dual targeting approaches. As labeled, the Dual Population (left panel) includes two separate populations of engineered cells, each with a different CAR. Dual CARs include genetically modified cells expressing two CAR constructs (either expressed from the same or separate vectors), one that binds CLL-1 and one that binds CD123. Dimerized CARs include at least two CAR molecules expressed by the same genetically modified cell either from the same or separate vectors that multimerize. Tandem CARs (right panel) include a single CAR that contains two antigen binding domains, one that specifically binds CLL-1 and one that specifically binds CD123. Each of these approaches can be practiced alone or in various combinations
[0014] FIGs. 2A-2D. Interleukin-3 receptor (CD123) target. (2A) Structure of CD123 bound to interleukin 3 (IL-3). (2B) Structural alignment between antibody bound CD123 and IL-3 bound CD123. There is a conformation shift due to antibody or IL-3 binding. (2C) Structural representation of CD123 bound to anti-CD123 antibody Fab. (2D) Annotated sequence of interleukin-3 receptor subunit a (CD123) (SEQ ID NO: 1) wherein, NLG stands for N-linked glycosylation, ECD stands for extracellular domain, hsCD123 stands for Homo sapiens CD123, SP stands for signal peptide, TM stands for transmembrane domain, and CPD stands for cytoplasmic domain. Sequences for SP (SEQ ID NO: 30), hsCD123 (SEQ ID NO: 2), ECD (SEQ ID NO: 3), TM (SEQ ID NO: 4), and CPD (SEQ ID NO: 5) from the CD123 target molecule are also labeled.
[0015] FIG. 3. Annotated sequence of C-type lectin domain family 12 member A (CLL-1) (SEQ ID NO: 6) wherein hsCLL-1 stands for Homo sapiens CLL-1. Sequences for CPD (SEQ ID NO: 7), TM (SEQ ID NO: 8), ECD (SEQ ID NO: 9), and C-type lectin (SEQ ID NO: 126) from the CLL- 1 target molecule are also labeled
[0016] FIG. 4. Annotated sequence of schematics of disclosed chimeric antigen receptor (CAR) (SEQ ID NO: 10) including a single-chain variable fragment (scFv), linker, transmembrane domain, and intracellular signaling components. Sequences described within this example CAR include the SP (SEQ ID NO: 31), scFv (SEQ ID NO: 1 1), lgG4 (SEQ ID NO: 38), TM_CD28 (SEQ ID NO: 39), 4-1 BB (SEQ ID NO: 41), Oϋ3z (SEQ ID NO: 43), T2A (SEQ ID NO: 36), EGFR (SEQ ID NO: 45), and TM_EGFR (SEQ ID NO: 46).
[0017] FIGs. 5A-5D. (5A) Vector map for a CLL-1 CAR, demonstrating an scFv region in a VL-VH orientation and a spacer region with an lgG4 hinge region. The CAR molecule is expressed separately from the tEGFR (truncated human EGFR) transduction marker allowing for monitoring by flow cytometry by incorporation of a T2A sequence in the CAR construct. (5B) T cells transduced with the CLL-1 CAR demonstrate specific lysis of CLL-1 positive (pos or +) HL60 AML cell lines but show no activity against CLL-1 negative (neg or -) K562 cells. (5C) T cells transduced with the CD123 CAR demonstrate specific lysis of CD123pos KG1a AML cell lines, respectively, but show no activity against CD123neg K562 cells. (5D) A carboxyfluorescein succinimidyl ester (CFSE) dilution demonstrates proliferation of CLL-1 and CD123 CAR-T cells after stimulation with AML cell lines that express (Positive) their target antigen, but not antigen-negative cells (Negative) or without stimulation (None).
[0018] FIGs. 6A, 6B. Results of CLL-1 CAR spacer analysis. (6A) Left panel: T cells engineered with chimeric antigen receptors with extracellular binding domains specific to CD19 or CLL1 with varying spacer lengths (short, intermediate, and long) were incubated with various target K562 cells transduced with CD19 (K19 - black with circle points), CLL1 (KCLL1 -grey with square points) with a series of EffectorTarget ratio. CD19 serves as a positive control for the experiment. The panel on the right side demonstrates CFSE proliferation of CLL1-short, CLL1-intermediate, CLL1- long in the presence of target cells. (6B) Cytokine profile of the CLL1 CAR constructs with varying spacer length against target cells of 6A. Interferon (IFN)- y, Interleukin (IL)-2 and tumor necrosis factor (TNF)-a were all assessed against target cells K19 and KCLL1.
[0019] FIGs. 7A, 7B. Results of CD123 CAR spacer analysis. (7A) Left panel: T cells engineered with chimeric antigen receptors with extracellular binding domains specific to CD19 or CD123 with varying spacer lengths (short, intermediate, and long) were incubated with various target K562 cells transduced with CD19 (K19 - black with circle points), CD123 (K123-grey with triangle points) at decreasing EffectorTarget ratio. CD19 serves as a positive control for the experiment. The panel on the right side demonstrates CFSE proliferation of CD123-short, CD123- intermediate, CD123-long in the presence of target cells. (7B) Cytokine profile of the CD123CAR constructs with varying spacer length against target cells of 7A. IFN-g, IL-2 and TNF-a were all assessed against target cells K19 and K123.
[0020] FIGs. 8A, 8B. (8A) Results of the analysis of the tandem CAR which contains the CLL1 and CD123 scFvs in various orientations, CD123-G4Sx3-CLL1 and CLL1-G4Sx4-CD123 against target cells expressing CD19, CD123, CLL-1 , or both CD123 and CLL-1. Lysis of target cells was assessed for various EffectorTarget ratios. The higher lysis with the lower ratio of E:T indicates high potency of the T cells expressing that CAR-T construct. (8B) CFSE Proliferation of tandem CAR construct (CD123-G4Sx3-CLL1 (upper panel) and CLL1-G4Sx4-CD123 (lower panel) against target cells K-123CLL1 , K-123, K-CLL1 , K-19 (negative control).
[0021] FIG. 9. CFSE Proliferation of CD123-short and CD123-reverse (VLVH) constructs versus various target K562 cells transduced with CD19 (K19), CLL1 (KCLL1) and CD123 (K123).
[0022] FIG. 10. Chromium51 cytotoxicity release assay of (top panel) CD123-reverse (VLVH) and (bottom panel) CLL1 -reverse (VLVH) constructs versus various target K562 cells transduced with CD19 (K19), CLL1 (KCLL1) and CD123 (K123). [0023] FIG. 11 A, 11 B. (11 A) The dock and lock (DNL) dimerization domain is a hyperstable disulfide crosslinked dimerization domain used in a variety of secreted protein constructs. As used herein, DNL refers to any form of dock and lock dimerization domain including, for example, PRKAR1A (DNL_5HVZ), PRKAR1 E (DNL_5HVZ_E*), and PRKAR1 R (DNL_5HVZ_R*). (11 B) The lgG4 hinge is a short linker that can enforce homodimerization.
[0024] FIG. 12A, 12B. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrating the DNL dimerization domain’s ability to form homodimers targeting CD123 and heterodimers targeting CLL-1 and CD123 (reduced (R) and non-reduced (NR)). The DNL dimerization domain is well-suited to form disulfide crosslinked homodimers and heterodimers. (12B) The disulfide crosslinked homodimer targeting CD123 fails to form with high fidelity with an lgG4 hinge.
[0025] FIGs. 13A, 13B. Engineered DNL Heterodimer. (14A) Structural model of DNL heterodimer highlighting the internal salt bridge. (14B) SDS-PAGE showing expression of the heterodimer facilitated by the engineered DNL dimerization domain under NR and R conditions. This shows that coexpression of the two monomers results in a 1 : 1 complex of monomer 1 and monomer 2.
[0026] FIGs. 14A-14C. Diagrams of various locations in which a dimerization domain can be inserted within a CAR construct. (14A) Dimerization domains can be located extracellularly in addition to a spacer region. (14B) Dimerization domains can be located extracellularly to replace a spacer region. (14C) Dimerization domains can be located intracellularly membrane proximal to the intracellular signaling components.
[0027] FIGs. 15A-15C. Designs for CLL-1 and CD123 targeting CAR. (15A) Compared to frequently used CARs which use the lgG4 hinge region as a spacer region, this depicted design uses a longer dimerization domain. The presence of a disulfide linked dimer ensures homodimer formation. The second target can stay conserved. The VH stands for the heavy chain variable region and the VL stands for the light chain variable region of an scFv. (15B) The dimerization domain is located intracellularly, but the distance from the binding domain to the cell membrane is conserved using a spacer region (labeled as the Linker). This design ensures dimerization using a dimerization domain that does not require disulfide bond formation and hence can be used as part of the CPD. (15C) This depicted construct enables the design of a heterodimer with mutated dimerization domains enabling dimerization of the two CARs including scFvl and scFv2.
[0028] FIGs. 16A-16J. Annotated sequences of CAR constructs. (16A) Annotated sequence for CAR construct targeting CLL-1 with an extracellular DNL dimerization domain (SEQ ID NO: 12). The structure for this CAR construct can also be applied to target the CD 123 receptor by using a scFv or any other binding agent that targets CD123. With the DNL dimerization domain located extracellularly, the receptors can be simultaneously expressed as soluble proteins and biophysically characterized using Biacore, crystallography, or other analysis tools. (16B) Annotated sequence for a CAR construct targeting CLL-1 with an intracellular DNL dimerization domain (SEQ ID NO: 15). The structure for this CAR construct can also be applied to target the CD123 receptor by using an scFv or any other binding agent that targets CD123. With the DNL dimerization domain located intracellularly, extracellular linkers located on the wild type receptors can be maintained. (16C) Annotated sequence for a CAR construct that heterodimerizes to target CD123 and CLL-1. The diagram depicts a DNL_S31 R Linker intracellularly, but it could be placed either intracellularly or extracellularly as described in FIGs. 14A-14C. The depicted CAR construct which targets CLL-1 (SEQ ID NO: 16) can be paired with a similar CAR construct that targets CD123 to enforce a heterodimeric receptor. Sequences for Signal Peptide (SP) (SEQ ID NO: 31), VH_CLL1 (SEQ ID NO: 13), Linker (SEQ ID NO: 213), VL_CLL1 (SEQ ID NO: 14), DNL Linker (SEQ ID NO: 48) and DNL_S31 R Linker (SEQ ID NO: 49), TM_CD28 (SEQ ID NO: 39), 4-1 BB (SEQ ID NO: 41), CD3z is Oϋ3z (SEQ ID NO: 43), T2A (SEQ ID NO: 36), and CD28 Linker (SEQ ID NO: 57) are noted. (16D) Annotated amino acid sequence for parental CAR T cell Vector (MDT- 000828) (SEQ ID NO:17). (16E) Amino acid sequence for MDT-000830 which is a portion of a CAR designed to target CD123 as a homodimer (SEQ ID NO: 20). (16F) Nucleotide sequence encoding MDT-000830 (SEQ ID NO: 21). (16G) Amino acid sequence for MDT-000831 which is a portion of a dual targeting CAR designed to target CLL-1 as a heterodimer (SEQ ID NO:24). (16H) Nucleotide sequence encoding MDT-000831 (SEQ ID NO:_25). (161) Amino acid sequence for MDT-000832 which is a portion of a dual targeting CAR designed to target CD123 as a heterodimer (SEQ ID NO:_28). (16J) Nucleotide sequence encoding MDT-000832 (SEQ ID NO: 29)
[0029] FIGs. 17A-17F. lgG4-hinge containing scFv. (17A) Schematic of the lgG4-hinge containing scFv that is thought to form obligate homodimers on the T cell surface. Expressing the extracellular portion of the receptor as a soluble protein revealed some heterogeneity that has implications for clinical CAR-T cells using this format of receptor as shown in FIGs. 17C-17F. (17B) Preliminary crystals of the monomeric fraction of the scFv-based receptor which is shown in FIG. 17E. (17C) Analytical size exclusion column showing the monomeric fraction (lagging peak) and the dimeric fraction (leading peak), highlighting the observation that the lgG4 hinge does not readily form homodimeric receptors in solution. (17D) SDS-PAGE gel showing that the lgG4 hinge containing scFv does not readily form a disulfide cross-linked homodimer. (17E) Analytical size exclusion column showing an overlay of monomeric and dimeric soluble receptors which were purified separately. (17F) SDS-PAGE gel showing the monomeric and dimeric soluble receptors from FIG. 17E.
[0030] FIGs. 18A-18C. DNL containing scFv. (18A) Schematic of the DNL containing scFv that forms obligate homodimers on the T cell surface. (18B) Analytical size exclusion column showing purification of the soluble homodimeric DNL CAR. (18C) SDS-PAGE gel showing that the DNL containing scFv readily forms a disulfide cross-linked homodimer which is in stark contrast to FIG. 17D.
[0031] FIGs. 19A-19D. DNL containing scFv with mutations to favor heterodimerization. (19A) Schematic of the DNL containing scFvs that harbor mutations in the DNL domain to favor heterodimerization. (19B) Preliminary crystals of the soluble heterodimeric DNL receptor targeting CLL-1 and CD123. (19C) Analytical size exclusion column showing purification of the soluble heterodimeric DNL CAR. (19D) SDS-PAGE gel showing that the soluble heterodimeric DNL CAR readily forms a disulfide cross-linked heterodimer which is in stark contrast to FIG. 17D.
[0032] FIGs. 20A, 20B. (20A) SDS-PAGE gel showing a comparison (from left to right) of the soluble lgG4 hinge CAR (MDT-000829), the homodimeric DNL CAR (MDT-000830), and the heterodimeric DNLR/DNLE CAR (MDT-000831/000832), all under non-reducing and reducing conditions. (20B) Analytical size exclusion column showing an overlay of the three proteins described in FIG. 20A.
[0033] FIGs. 21A, 21 B. (21A) SDS-PAGE gel showing purification of the human CD123 ectodomain under NR and reducing (Red) conditions. (21 B) SDS-PAGE gel showing purification of the human CLL-1 ectodomain under NR and R conditions. NiPD stands for Nickel Pull Down.
[0034] FIGs. 22A-22F. Comparison of bicistronic and DNL CAR constructs directed against CLL- 1 and CD123. T cells were transduced with viral vector encoding a CAR directed against both anti-CLL-1 and CD123 CAR (bicistronic) or CLL-1-DNLE and CD123 DNLR DNLCAR. Following transduction, CAR-T cells were sorted and expanded as per rapid expansion protocol (REP). (22A) Schematic of DNL DNL-E (top) and DNL-R (middle) CAR constructs and bicistronic CAR (bottom) is shown. (22B) On day 12 following expansion CAR-T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and cultured with irradiated targets or media control as labeled. CFSE fluorescence was measured by flow cytometry after four days of co-culture. (22C) Bicsitronic and (22D) DNL CAR-T cells were exposed to various chromium51-labelled targets as listed within the FIG. Four hours following co-culture, chromium51 levels were measured to quantify the degree of cytotoxicity. (22E) Enzyme-linked immunosorbent assay of interleukin-2 (-) and (22F) interferon-g (IFN- g) levels following 24-hours of exposure to various targets as listed on the x-axis. ns = not significant. * p<0.05. **p<0.01.
[0035] FIGs. 23A, 23B. Accumulation of inhibitory immunoreceptors (TIM3, LAG3 and PD1) following repetitive stimulation of dual-targeting CAR. (23A) CLL-1-, CD123 short and bicistronic CD123 targeting CAR were co-cultured with irradiated CD123-expressing K562 cell lines (K123) for 5 days (Stim 1), then re-exposed to K123 cells for 5 days (Stim 2) and then a further 5 days (Stim 3). After each stimulation, percentage of immunoinhibitory receptor-expressing CAR-T cells were assessed by flow cytometry by comparison with fluorescence minus one (FMO) expression. (23B) DNL and bicistronic CAR-T cells were exposed three times (Stim 1 , 2 and 3) every 5 days to irradiated K562 cells which express both CLL-1 and CD123 (KBoth). After each stimulation, percentage of immunoinhibitory receptor-expressing CAR-T cells were assessed by flow cytometry by comparison with FMO expression.
[0036] FIG. 24. Expression of Nur77 following exposure to CLL-1 and CD123 targets. CD123 and CLL-1 -targeting bicstronic and DNL CAR-T cells were exposed to irradiated CLL-1 and CD123 expressing targets for 5 days three times (Stim 1 , Stim 2 and Stim 3) and Nur77 expression was then measured by intranuclear flow cytometry staining as shown.
[0037] FIG. 25. Exemplary clinical applications of the compositions provided herein.
[0038] FIG. 26. Exemplary sequences supporting the disclosure (SEQ ID NOs: 1-205, 260, and 267).
DETAILED DESCRIPTION
[0039] According to the World Health Organization, cancer is the second leading cause of death globally, and was responsible for an estimated 9.6 million deaths in 2018. Acute myeloid leukemia (AML) is a malignancy of clonal, proliferative myeloid blast cells. AML is also known as acute myelocytic leukemia, acute myelogenous leukemia, acute granulocytic leukemia, and acute nonlymphocytic leukemia.
[0040] High complete remission rates for patients with AML can be achieved with conventional chemotherapy at rates of 60% to 80% for younger adults and 40% to 60% for adults greater than age 60 (Dohner, et al., 2015. Nat Engl. J. Med. 373(12): 1136-1 152). Unfortunately, because of the chemorefractoriness of leukemic stem cells, relapse after conventional therapy is common (Eppert, et al., 201 1. Nat. Med. 17(9): 1086-1093) and current treatment options for relapsed/refractory (R/R) AML are dismal, resulting in less than 30% overall survival at 12 months.
[0041] In recent years, more targeted therapies have emerged to specifically target cancer cells by identifying and exploiting specific molecular changes seen primarily in those cells. For example, significant progress has been made in genetically engineering T cells of the immune system to target and kill unwanted cell types, such as cancer cells. Many of these T cells have been genetically engineered to express chimeric antigen receptor (CAR) constructs. [0042] CAR molecules are proteins including several distinct subcomponents that allow the genetically modified T cells to recognize and kill cancer cells. The subcomponents include at least an extracellular component and an intracellular component. The extracellular component includes a binding domain that specifically binds a marker that is preferentially present on the surface of cancer cells. The binding domain is typically a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but it can be based on other formats which include an antibody like antigen binding site.
[0043] When the binding domain binds such markers, the intracellular component signals the T cell to destroy the bound cell. The intracellular components provide such activation signals based on the inclusion of an effector domain. First generation CARs utilized the cytoplasmic region of Oϋ3z as an effector domain. Second generation CARs utilized Oϋ3z in combination with cluster of differentiation 28 (CD28) or 4-1 BB (CD137), while third generation CARs have utilized ΰϋ3z in combination with CD28 and 401 BB within intracellular effector domains.
[0044] CARs generally also include one or more linker sequences that are used for a variety of purposes within the molecule. For example, a transmembrane domain can be used to link the extracellular component of the CAR to the intracellular component. A flexible linker sequence often referred to as a spacer region that is membrane-proximal to the binding domain can be used to create additional distance between a binding domain and the cellular membrane. This can be beneficial to reduce steric hindrance to binding based on proximity to the membrane. A common spacer region used for this purpose is the lgG4 linker. More compact spacers or longer spacers can be used, depending on the targeted cell marker. Other potential CAR subcomponents are described in more detail elsewhere herein.
[0045] Clinical trials with CAR-expressing T cells have shown positive responses in patients with refractory large B-cell lymphoma when conventional treatments had failed (Neelapu, et al 2017 N Engl J Med 377:2531-2544). However, while CAR constructs can successfully genetically engineer T cells to result in cancer cell destruction, they have failed to provide prolonged anti cancer activity in vivo for some indications. For example, in some cases, T cells have not received a strong enough signal when the binding domain binds the targeted cancer cell marker. Further, toxicities have been reported from some CAR studies, and additional theoretical toxicities exist. Many of the toxicities are thought to be due to "on-target off-cancer" or“on-target off-tumor” effects when the targeted cancer cell marker is also expressed by non-cancerous tissues.
[0046] The key to successful targeted immunotherapy is in the choice of the target cancer cell marker. An ideal target marker is immunogenic, plays a critical role in proliferation and differentiation, is expressed only on the surface of all malignant cells and malignant stem cells, and a large portion of patients should test positive for the marker (Cheever, et al. , 2009. Clin. Cancer Res. 15(17): 5323-8337). While the CD19-specific CAR-T cell therapy has been successful for ALL and NHL, identification of a marker for AML has proven more difficult. Many markers expressed by AML myeloblasts, or blasts, and leukemic stem cells overlap with normal hematopoiesis. Furthermore, while the B-cell lineage-restricted antigen, CD19, is a good target for the treatment of B-cell malignancies, no single myeloid lineage-restricted antigen has been identified that is expressed on all blasts to enable effective targeting of AML. AML is clonally heterogeneous, rendering single marker targeting ineffective for the treatment of AML, due to the high likelihood that antigen-negative blasts will escape single marker targeted therapy (see Buckley SA et al., ASH Education Program Book. 2015;2015(1):584-595).
[0047] Leading to aspects of the current disclosure, flow cytometry was used to study the immunophenotype of leukemic blasts isolated from AML patients and it was found that in all examined patients, C-type lectin-like molecule-1 (CLL-1) and/or interleukin-3 receptor (CD123) were expressed by over 99.9% of AML blasts. GenBank Accession Nos. for CLL-1 include: 94557293, 94557290, 333609225, and 663429587. GenBank Accession Nos. for CD123 include: 1034676276, 1034674220, 530423009, 530423007, 530420931 , 530420929, 391224440, and 13324710.
[0048] Other AML-related disorders where CLL-1 and/or CD123 provide therapeutic targets include: blastic plasmacytoid dendritic cell neoplasm (BPDCN), myelodysplastic syndromes (MDS), natural killer cell lymphomas, hairy cell leukemia, acute lymphocytic leukemia (ALL; also known as acute lymphoblastic lymphoma), chronic myelocytic leukemia (CML), other leukemias, hematological cancers or tumors, Hodgkin’s lymphoma (HL), B-cell HL, non-Hodgkin lymphoma (NHL), mantle cell lymphoma (MCL), T cell lymphoma, multiple myeloma (refractory, relapsed, etc.), systemic mastocytosis (SM), hypereosinophilic syndrome (HES), myelofibrosis, anemia, systemic lupus erythematosus (SLE), psoriasis, and systemic sclerosis (scleroderma).
[0049] The current disclosure provides multiple strategies to target CLL-1 and/or CD123 based on the genetic modification of cells to express one or more CAR molecules. A depiction of exemplary disclosed strategies is provided in FIG. 1 showing genetically modifying cells to create a population of genetically modified cells wherein:
(i) different cells within the population express a CLL-1 targeting CAR or a CD123-targeting CAR (the“dual population” approach);
(ii) the cells express two different CAR constructs, one targeting CLL-1 and one targeting CD123 (the“dual CAR” approach);
(iii) the cells express two CAR constructs with multimerizing domains, with at least one targeting CLL-1 and at least one targeting CD123; all targeting CLL-1 ; or all targeting CD213 (the “dimerized CAR” approach); and
(iv) the cells express CAR having at least two binding domains in the extracellular component wherein at least one binding domain specifically binds CLL-1 and at least one binding domain specifically binds CD123 (the“tandem” approach).
[0050] The strategies depicted in FIG. 1 can be utilized alone or in various combinations. Regarding the“dimerized CAR” approach, if a genetically modified cell expresses multimerizing binding domains that all bind the same cancer antigen (e.g., CLL-1), these cells would be used within a dual population approach, for example with a second population of cells expressing multimerizing binding domains that all bind CD123. In particular embodiments, strategy (i) is practiced with strategies (ii), (iii), and/or (iv). In particular embodiments, strategy (ii) is practiced with strategy (iii). In particular embodiments, strategy (i) is practiced with strategy (iv). In particular embodiments, strategy (iii) is practiced with strategy (i). In particular embodiments, strategy (i), (ii), and (iii) are practiced together. In particular embodiments, strategy (ii), (iii), and (iv) are practiced together. In particular embodiments, strategy (i), (iii), and (iv) are practiced together.
[0051] In particular embodiments, dimerization of CAR results based on inclusion of complementary forms of the protein kinase cAMP-dependent type I regulatory subunit a or b (PRKAR1A or PRKAR1 B) dimerization domains within expressed CAR molecules. In particular embodiments, such multimerization or dimerization domains can be placed at any position within an expressed CAR, so long as the placement does not significantly interfere with binding to targeted cell markers. In particular embodiments, multimerization or dimerization domains can be placed at any position within an expressed CAR, so long as the placement does not significantly interfere with cell activation following binding to targeted cell markers.
[0052] With respect to multimerization of CAR molecules, and as used herein, a “complete homomultimer” includes copies of identical monomer subunits (CAR), but for, in certain circumstances, inclusion of complementary multimerization domains. A partial homomultimer has identical subcomponents of a CAR, but are not completely identical. For example, a partial homomultimer may be identical but for the inclusion of different transmembrane domains, spacer regions, effector domains, signaling domains and/or co-stimulatory domains. A heteromultimer includes binding domains that bind different cancer antigen epitopes. In particular embodiments, the different cancer antigen epitopes are located on the same cancer antigen. In particular embodiments, the different cancer antigen epitopes are located on different cancer antigens (i.e. , CLL-1 and CD123).
[0053] The strategies disclosed herein provide several important advantages. First, by targeting two cancer markers, patients that may lack one or the other will still receive the benefit of the treatment as it is unlikely that patients would lack both markers. Second, by linking at least two CARs together through co-expression and/or multimerization, activation signals that the cell receives when the now-paired binding domains bind targeted cancer cell markers can be increased.
[0054] In particular embodiments, the compositions and methods disclosed herein provide therapeutic efficacy for a broader patient population, induce stronger cell activation signals, and/or provide improved therapeutic effects. In particular embodiments, homo- or heterodimerization of CAR can improve function and T cell signaling, leading to improved clinical outcomes.
[0055] The following aspects and options related to the current disclosure are now described in additional detail as follows: (i) CLL-1 and CD123 Binding Domains; (ii) Intracellular Signaling Components; (iii) Linkers; (iv) Multimerization Domains; (v) Control Features Including Tag Cassettes, Transduction Markers, and Suicide Switches; (vi) Exemplary Constructs; (vii) Immune Cells; (viii) Methods to Collect and Modify Cells Ex Vivo and In Vivo; (ix) Ex Vivo Manufactured Cell Formulations; (x) Nanoparticle Formulations; (xi) Methods of Use; (xii) Exemplary Embodiments; (xiii) Experimental Examples; and (xiv) Closing Paragraphs.
[0056] (i) CLL-1 and CD123 Binding Domains. Binding domains include any substance that binds to a cellular marker to form a complex. The choice of binding domain can depend upon the type and number of cellular markers that define the surface of a target cell. Examples of binding domains include cellular marker ligands, receptor ligands, antibodies, peptides, peptide aptamers, receptors (e.g., T cell receptors), or combinations and engineered fragments or formats thereof.
[0057] Antibodies are one example of binding domains and include whole antibodies or binding fragments of an antibody, e.g., Fv, Fab, Fab', F(ab')2, and single chain (sc) forms and fragments thereof that bind specifically to a cellular marker. Antibodies or antigen binding fragments can include all or a portion of polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, synthetic antibodies, non-human antibodies, recombinant antibodies, chimeric antibodies, bispecific antibodies, mini bodies, and linear antibodies. Functional fragments thereof, include a single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) of camelid derived nanobody, and the like.
[0058] In some instances, scFvs can be prepared according to methods known in the art (see, for example, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). ScFv molecules can be produced by linking VL and VH regions of an antibody together using flexible polypeptide linkers. If a short polypeptide linker is employed (e.g., between 5-10 amino acids) intrachain folding is prevented. Interchain folding is also required to bring the two variable regions together to form a functional epitope binding site. For examples of linker orientations and sizes see, e.g., Hollinger et al. 1993 Proc Natl Acad. Sci. U.S.A. 90:6444- 6448, US 2005/0100543, US 2005/0175606, US 2007/0014794, W02006/020258, and W02007/024715.
[0059] An scFv can include a linker of at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more amino acid residues between its VL and VH regions. In particular embodiments, the linker sequence may include any naturally occurring amino acid. Generally, linker sequences that are used to connect the VL and VH of an scFv are five to 35 amino acids in length. In particular embodiments, a VL-VH linker includes from five to 35, ten to 30 amino acids or from 15 to 25 amino acids. Variation in the linker length may retain or enhance activity, giving rise to superior efficacy in activity studies.
[0060] In some embodiments, the linker sequence includes the amino acids glycine and serine. In particular embodiments, the linker sequence includes sets of glycine and serine repeats such as from one to ten repeats of (GlyxSery)n, wherein x and y are independently an integer from 0 to 10 provided that x and y are not both 0 and wherein n is an integer of 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10) and wherein linked VH-VL regions form a functional immunoglobulin-like binding domain (e.g., scFv, scTCR). Particular examples include (Gly4Ser)n (SEQ ID NO: 206), (Gly3Ser)n(Gly4Ser)n (SEQ ID NO: 207), (Gly3Ser)n(Gly2Ser)n (SEQ ID NO: 208), (Gly3Ser)n(Gly4Ser)i (SEQ ID NO: 209), (Gly4Ser)i (SEQ ID NO: 210), (Gly3Ser)i (SEQ ID NO: 211), or (Gly2Ser)i. In particular embodiments, the linker is (Gly4Ser)4 (SEQ ID NO: 212) or (Gly4Ser)3 (SEQ ID NO: 213). As indicated through reference to scTCR above, such linkers can also be used to link T cell receptor Vc/p and Co/p chains (e.g., Va-Ca, Vp-Cp, Va-Vp).
[0061] Additional examples include scFv-based grababodies and soluble VH domain antibodies. These antibodies form binding regions using only heavy chain variable regions. See, for example, Jespers et al., Nat. Biotechnol. 22: 1161 , 2004; Cortez- Retamozo et al., Cancer Res. 64:2853, 2004; Baral et al., Nature Med. 12:580, 2006; and Barthelemy et al., J. Biol. Chem. 283:3639, 2008.
[0062] In some instances, it is beneficial for the binding domain to be derived from the same species it will ultimately be used in. For example, for use in humans, it may be beneficial for the antigen binding domain to include a human antibody, humanized antibody, or a fragment or engineered form thereof. Antibodies from human origin or humanized antibodies have lowered or no immunogenicity in humans and have a lower number of non-immunogenic epitopes compared to non-human antibodies. Antibodies and their engineered fragments will generally be selected to have a reduced level or no antigenicity in human subjects.
[0063] In particular embodiments, the binding domain includes a humanized antibody or an engineered fragment thereof. In particular embodiments, a non-human antibody is humanized, where one or more amino acid residues of the antibody are modified to increase similarity to an antibody naturally produced in a human or fragment thereof. These nonhuman amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. As provided herein, humanized antibodies or antibody fragments include one or more CDRs from nonhuman immunoglobulin molecules and framework regions wherein the amino acid residues including the framework are derived completely or mostly from human germline. In one aspect, the antigen binding domain is humanized. A humanized antibody can be produced using a variety of techniques known in the art, including CDR-grafting (see, e.g., European Patent No. EP 239,400; WO 91/09967; and US 5,225,539, US 5,530,101 , and US 5,585,089), veneering or resurfacing (see, e.g., EP 592, 106 and EP 519,596; Padlan, 1991 , Molecular Immunology, 28(4/5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805- 814; and Roguska et al., 1994, PNAS, 91 :969-973), chain shuffling (see, e.g., US. 5,565,332), and techniques disclosed in, e.g., US 2005/0042664, US 2005/0048617, US 6,407,213, US 5,766,886, WO 9317105, Tan et al., J. Immunol., 169:1 119-25 (2002), Caldas et al., Protein Eng., 13(5): 353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16): 10678-84 (1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto et al., Cancer Res., 55 (23 Supp):5973s-5977s (1995), Couto et al., Cancer Res., 55(8): 1717-22 (1995), Sandhu J S, Gene, 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994). Often, framework residues in the framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, for example improve, cellular marker binding. These framework substitutions are identified by methods well-known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for cellular marker binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., US 5,585,089; and Riechmann et al. , 1988, Nature, 332:323).
[0064] Antibodies that specifically bind a particular cellular marker can be prepared using methods of obtaining monoclonal antibodies, methods of phage display, methods to generate human or humanized antibodies, or methods using a transgenic animal or plant engineered to produce antibodies as is known to those of ordinary skill in the art (see, for example, US 6, 291 , 161 and US 6,291 , 158). Phage display libraries of partially or fully synthetic antibodies are available and can be screened for an antibody or fragment thereof that can bind to a cellular marker. For example, binding domains may be identified by screening a Fab phage library for Fab fragments that specifically bind to a cellular marker of interest (see Hoet et al., Nat. Biotechnol. 23:344, 2005). Phage display libraries of human antibodies are also available. Additionally, traditional strategies for hybridoma development using a cellular marker of interest as an immunogen in convenient systems (e.g., mice, HuMAb mouse® (GenPharm Int’l. Inc., Mountain View, CA), TC mouse® (Kirin Pharma Co. Ltd., Tokyo, JP), KM-mouse® (Medarex, Inc., Princeton, NJ), llamas, chicken, rats, hamsters, rabbits, etc.) can be used to develop binding domains. In particular embodiments, antibodies specifically bind to a cellular marker preferentially expressed by a particular cancer cell type and do not cross react with nonspecific components or unrelated targets. Once identified, the amino acid sequence of the antibody and gene sequence encoding the antibody can be isolated and/or determined.
[0065] An alternative source of binding domains includes sequences that encode random peptide libraries or sequences that encode an engineered diversity of amino acids in loop regions of alternative non-antibody scaffolds, such as scTCR (see, e.g., Lake et al., Int. Immunol.11 :745, 1999; Maynard et al., J. Immunol. Methods 306:51 , 2005; US 8,361 ,794), fibrinogen domains (see, e.g., Weisel et al., Science 230: 1388, 1985), Kunitz domains (see, e.g., US 6,423,498), designed ankyrin repeat proteins (DARPins; Binz et al., J. Mol. Biol. 332:489, 2003 and Binz et al., Nat. Biotechnol. 22:575, 2004), fibronectin binding domains (adnectins or monobodies; Richards et al. , J. Mol. Biol. 326: 1475, 2003; Parker et al., Protein Eng. Des. Selec. 18:435, 2005 and Hackel et al. (2008) J. Mol. Biol. 381 :1238-1252), cysteine-knot miniproteins (Vita et al., 1995, Proc. Nat'l. Acad. Sci. (USA) 92:6404-6408; Martin et al., 2002, Nat. Biotechnol. 21 :71 , 2002 and Huang et al. (2005) Structure 13:755, 2005), tetratricopeptide repeat domains (Main et al., Structure 11 :497, 2003 and Cortajarena et al. , ACS Chem. Biol. 3: 161 , 2008), leucine-rich repeat domains (Stumpp et al., J. Mol. Biol. 332:471 , 2003), lipocalin domains (see, e.g., WO 2006/095164, Beste et al. , Proc. Nat'l. Acad. Sci. (USA) 96: 1898, 1999 and Schonfeld et al. , Proc. Nat'l. Acad. Sci. (USA) 106:8198, 2009), V-like domains (see, e.g., US 2007/0065431), C-type lectin domains (Zelensky and Gready, FEBS J. 272:6179, 2005; Beavil et al., Proc. Nat'l. Acad. Sci. (USA) 89:753, 1992 and Sato et al., Proc. Nat'l. Acad. Sci. (USA) 100:7779, 2003), mAb2 or Fc-region with antigen binding domain (Fcab™ (F-Star Biotechnology, Cambridge UK; see, e.g., WO 2007/098934 and WO 2006/072620), armadillo repeat proteins (see, e.g., Madhurantakam et al., Protein Sci. 21 : 1015, 2012; WO 2009/040338), affilin (Ebersbach et al., J. Mol. Biol. 372: 172, 2007), affibody, avimers, knottins, fynomers, atrimers, cytotoxic T-lymphocyte associated protein-4 (Weidle et al., Cancer Gen. Proteo. 10:155, 2013), or the like (Nord et al., Protein Eng. 8:601 , 1995; Nord et al., Nat. Biotechnol. 15:772, 1997; Nord et al., Euro. J. Biochem. 268:4269, 2001 ; Binz et al., Nat. Biotechnol. 23:1257, 2005; Boersma and Pluckthun, Curr. Opin. Biotechnol. 22:849, 2011).
[0066] Peptide aptamers include a peptide loop (which is specific for a cellular marker) attached at both ends to a protein scaffold. This double structural constraint increases the binding affinity of peptide aptamers to levels comparable to antibodies. The variable loop length is typically 8 to 20 amino acids and the scaffold can be any protein that is stable, soluble, small, and non-toxic. Peptide aptamer selection can be made using different systems, such as the yeast two-hybrid system (e.g., Gal4 yeast-two-hybrid system), or the LexA interaction trap system.
[0067] In particular embodiments, a binding domain is a sc T cell receptor (scTCR) including Va/b and Ca/b chains (e.g., Va-Ca, /b-Ob, Va-C/b) or including a Va-Ca, /b-Ob, Va- /b pair specific for a cellular marker of interest (e.g., peptide-MHC complex).
[0068] In particular embodiments, engineered CAR include a sequence that is at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to an amino acid sequence of a known or identified TCR Va, \/b, Ca, or Ob, wherein each CDR includes zero changes or at most one, two, or three changes, from a TCR or fragment or derivative thereof that specifically binds to the targeted cellular marker.
[0069] In particular embodiments, engineered CAR include Va, nb, Ca, or Ob regions derived from or based on a Va, nb, Ca, or Ob of a known or identified TCR (e.g., a high-affinity TCR) and includes one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions or non-conservative amino acid substitutions), or a combination of the above-noted changes, when compared with the Va, nb, Ca, or Ob of a known or identified TCR. An insertion, deletion or substitution may be anywhere in a Va, nb, Ca, or Ob region, including at the amino- or carboxy-terminus or both ends of these regions, provided that each CDR includes zero changes or at most one, two, or three changes and provides a target binding domain containing a modified Va, nb, Ca, or Ob region can still specifically bind its target with an affinity and action similar to wild type.
[0070] In particular embodiments, the scFv that binds CLL-1 can be SC02357. In particular embodiments, the variable light chain of SC02357 includes a CDRL1 sequence including QSISSYLN (SEQ ID NO: 261), a CDRL2 sequence including LLIYAASSLQS (SEQ ID NO: 262), and a CDRL3 sequence including QQSYSTPP (SEQ ID NO: 263), and a variable heavy chain including a CDRH1 sequence including GSISSSNWWS (SEQ ID NO: 264), a CDRH2 sequence including WIGEIYHSGSPDY (SEQ ID NO: 265), and a CDRH3 sequence including KVSTGGFFDY (SEQ ID NO: 266). [0071] In particular embodiments, the binding domain of a CAR can be an scFv that binds CLL-1 with a VH including the amino acid sequence described in SEQ ID NO: 13 and a VL including the amino acid sequence described in SEQ ID NO: 14. In particular embodiments, the binding domain of a CAR can be an scFv that binds CLL-1 with a VL encoded by the nucleic acid sequence described in SEQ ID NO: 27 and a VH encoded by the nucleic acid sequence described in SEQ ID NO: 26.
[0072] In particular embodiments, the binding domain that binds CLL-1 is derived from at least one of M26, M31 , G4, M22, M29, M2, M5, or G12, for example, the CDRs of M26, M31 , G4, M22, M29, M2, M5, or G12.
[0073] In particular embodiments, the binding domain includes a variable light chain region including the sequence:
METDTLLLWVLLLWVPGSTGDASQIVLSQSPAILSASPGEKVTMTCRASSSINYMHWYQQKPG SSPKPWIFATSNLASGVPSRFSGSGSGTSYSLTISRVEAEDAATYYCQQWRSDRALTFGAGTK LELIRAAAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 214), and a variable heavy chain region including the sequence:
METDTLLLWVLLLWVPGSTGDASQVQLQQPGAELVKPGTSVKLSCKASGYTFTRYWMHWVK
QRPGQGLEWIGMIHPSSGSTSYNEKVKNKATLTVDRSSTTAYMQLSSLTSEDSAVYYCARDG
DYYYGTGDYWGQGTTLTVSSARTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN
AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY
TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD
KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 215).
[0074] In particular embodiments, the binding domain includes a variable light chain including a CDRL1 sequence including DYYMH (SEQ ID NO: 216), a CDRL2 sequence including RINPYAGAAFYSQNFKD (SEQ ID NO: 217), and a CDRL3 sequence including ERGADLEGYAMDY (SEQ ID NO: 218), and a variable heavy chain including a CDRH1 sequence including RASQSVSTSSYN YM H (SEQ ID NO: 219), a CDRH2 sequence including YASNLES (SEQ ID NO: 220), and a CDRH3 sequence including QHSWEIPLT (SEQ ID NO: 221).
[0075] In particular embodiments, the binding domain of the M26 antibody includes a variable light chain including a CDRL1 sequence including QELSGY (SEQ ID NO: 222), a CDRL2 sequence including AAS, and a CDRL3 sequence including LQYAIYPYT (SEQ ID NO: 223), and a variable heavy chain including a CDRH1 sequence including GYTFTSYF (SEQ ID NO: 224), a CDRH2 sequence including INPYNDGS (SEQ ID NO: 225), and a CDRH3 sequence including TRDDGYYGYAMDY (SEQ ID NO: 226).
[0076] In particular embodiments, the binding domain of the M31 antibody includes a variable light chain including a CDRL1 sequence including ESVDSYGNSF (SEQ ID NO: 227), a CDRL2 sequence including LAS, and a CDRL3 sequence including QQNNYDPWT (SEQ ID NO: 228), and a variable heavy chain including a CDRH1 sequence including GYTFTSYV (SEQ ID NO: 229), a CDRH2 sequence including INPYNDGT (SEQ ID NO: 230), and a CDRH3 sequence including ARPIYFDNDYFDY (SEQ ID NO: 231).
[0077] In particular embodiments, the binding domain of the G4 antibody includes a variable light chain including a CDRL1 sequence including HDISNY (SEQ ID NO: 232), a CDRL2 sequence including YTS, and a CDRL3 sequence including QQGKTLLWT (SEQ ID NO: 233), and a variable heavy chain including a CDRH1 sequence including GYSFTGYT (SEQ ID NO: 234), a CDRH2 sequence including INPYNDGT (SEQ ID NO: 230), and a CDRH3 sequence including ARTDDYDDYTMDY (SEQ ID NO: 235). In particular embodiments, the binding domain is human or humanized. For more information regarding CLL-1 and associated binding domains see US 9751946, US 8536310, US 9163090, US 9914777, US 9908946, US 9145588, US 9248181 , US 9248182, PCT /N L2016/050507 , PCT/NL2013/050693, EP2147594B1 , JP5749161 , EP20170170370, and PCT/EP2016/051470.
[0078] In particular embodiments, the scFv that binds CD123 can be KLON120.
[0079] In particular embodiments, the binding domain of a CAR can be an scFv that binds CD123 with a VH including the amino acid sequence described in SEQ ID NO: 19 and a VL including the amino acid sequence described in SEQ ID NO: 18. In particular embodiments, the binding domain of a CAR can be an scFv that binds CD123 with a VL encoded by the nucleic acid sequence described in SEQ ID NO: 22 and a VH encoded by the nucleic acid sequence described in SEQ ID NO: 23.
[0080] In particular embodiments, the antibody includes a variable light chain region including the sequence:
DIQMTQSPSSMSASVGERVTITCRASQDINSYLSWFQQKPGKSPKTLIYRVNRLVDGVPSRFS GSGSGQDYSLTISSLEPEDMGIYYCLQYDAFPYTFGQGTKLEIKR (SEQ ID NO: 255), and a variable heavy chain region including the sequence
QVQLVQSGAEVVKPGASVKMSCKASGYTFTSSIMHWMKQKPGQGLEWIGYIRPYNDGTRYN QKFQGKATLTSDRSSSTANMELNSLTSEDSAVYYCAREGGNDY (SEQ ID NO: 256).
[0081] In particular embodiments, the binding domain that binds CD123 is derived from at least one of IMGN632 (ImmunoGen), ADAMTS2 (Abnova), 7G3, 32716, 32701 , 32703, 26292 (US 8163279), for example, the CDRs of IMGN632, ADAMTS2, 7G3, 32716, 32701 , 32703, or 26292.
[0082] In particular embodiments, the binding domain for the 26292 antibody includes a variable light chain including a CDRL1 sequence including RASKSISKDLA (SEQ ID NO: 236), a CDRL2 sequence including SGSTLQS (SEQ ID NO: 237), and a CDRL3 sequence including QQHNKYPYT (SEQ ID NO: 238), and a variable heavy chain including a CDRH 1 sequence including SYWMN (SEQ ID NO: 239), a CDRH2 sequence including RIDPYDSETHYNQKFKD (SEQ ID NO: 240), and a CDRH3 sequence including GNWDDY (SEQ ID NO: 241).
[0083] In particular embodiments, the binding domain for the 32703 antibody includes a variable light chain including a CDRL1 sequence including RSNKSLLHSNGNTYLY (SEQ ID NO: 242), a CDRL2 sequence including RMSNLAS (SEQ ID NO: 243), a CDRL3 sequence including MQHLEYPYT (SEQ ID NO: 244), and a variable heavy chain including a CDRH1 sequence including NYWMN (SEQ ID NO: 245), a CDRH2 sequence including RIDPSDSESHYNQKFKD (SEQ ID NO: 246), and a CDRH3 sequence including YDYDDTMDY (SEQ ID NO: 247).
[0084] In particular embodiments, the binding domain for the 32701 antibody includes a variable light chain including a CDRL1 sequence including RASESVDNYGNTFMH (SEQ ID NO: 248), a CDRL2 sequence including RASNLES (SEQ ID NO: 249), a CDRL3 sequence including QQSKEDPPT (SEQ ID NO: 250), and a variable heavy chain including a CDRH1 sequence including NYGMN (SEQ ID NO: 251), a CDRH2 sequence including WMNTNTGEPTSLEDFKG (SEQ ID NO: 252), and a CDRH3 sequence including SGGYDPMDY (SEQ ID NO: 253).
[0085] In particular embodiments, the binding domain for the 32716 antibody includes a variable light chain including a CDRL1 sequence including RASESVDNYGNTFMH (SEQ ID NO: 248), a CDRL2 sequence including RASNLES (SEQ ID NO: 249), a CDRL3 sequence including QQSKEDPPT (SEQ ID NO: 250), and a variable heavy chain including a CDRH1 sequence including NYGMN (SEQ ID NO: 251), a CDRH2 sequence including WINTYTGESTYSADFKG (SEQ ID NO: 254), and a CDRH3 sequence including SGGYDPMDY (SEQ ID NO: 253). In particular embodiments, the binding domain is human or humanized. For more information regarding binding domains that bind CD123, see US 8163279.
[0086] The following publications can be referenced to derive additional binding domains for CD123: WO2013173820A2 , PCT/IB2008/002930, PCT/US2015/031580, PCT/US1988/00001 1 , EP19890907981 , PCT/US2014/028961 , US 12/082940, US 1 1/271381 , US 7763242, EP2063907, JP5550905, and US 8188231 B2.
[0087] In particular embodiments, binding domains of each of the targeting CAR include SC02357 and KLON120. In particular embodiments, binding domains of each co-targeting CAR include scFvs derived from antibodies that specifically bind CLL-1 and CD123 to target acute myeloid leukemia (AML).
[0088] As indicated, in particular embodiments, CAR can completely or partially homodimerize. In particular embodiments, binding domains of each of the homodimerized CAR are both SC02357. In particular embodiments, binding domains of each of the homodimerized CAR are both KLON120. In particular embodiments, binding domains of each of the homodimerized CAR are both scFvs derived from antibodies that specifically bind CLL-1. In particular embodiments, binding domains of each of the homodimerized CAR are both scFvs derived from antibodies that specifically bind CD123. In particular embodiments, CAR can multimerize with binding domains that are different but that both specifically bind the same marker (i.e., CLL-1 or CD123).
[0089] In particular embodiments, multimerized CAR include three SC02357 binding domains. In particular embodiments, multimerized CAR include three KLON120 binding domains. In particular embodiments, multimerized CAR include four SC02357 binding domains. In particular embodiments, multimerized CAR include four KLON120 binding domains. In particular embodiments, multimerized CAR include five SC02357 binding domains. In particular embodiments, multimerized CAR include five KLON120 binding domains.
[0090] In particular embodiments, CAR can multimerize with binding domains specific to distinct antigens. In particular embodiments, multimerized CAR include one SC02357 and two KLON120 binding domains. In particular embodiments, multimerized CAR include two SC02357 and one KLON120 binding domains. In particular embodiments, multimerized CAR include scFvs derived from antibodies that specifically bind CLL-1 and CD123 as the binding domains.
[0091] As indicated previously, binding domains can adopt a variety of engineered formats including, for example, Fab fragments, scFv, scFv-based grababodies, and soluble VH domain antibodies.
[0092] In particular embodiments, a binding domain of a CAR includes or is a sequence that is at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to an amino acid sequence of a light chain variable region (VL) or to a heavy chain variable region (VH), or both, wherein each CDR includes zero changes or at most one, two, or three changes, from a monoclonal antibody or fragment or derivative thereof that specifically binds to a cellular marker of interest.
[0093] In particular embodiments, a VL region in a binding domain of the present disclosure is derived from or based on a VL of a known monoclonal antibody and contains one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions), or a combination of the above-noted changes, when compared with the VL of the known monoclonal antibody. An insertion, deletion or substitution may be anywhere in the VL region, including at the amino- or carboxy-terminus or both ends of this region, provided that each CDR includes zero changes or at most one, two, or three changes and provided a binding domain containing the modified VL region can still specifically bind its target with an affinity similar to the wild type binding domain.
[0094] In particular embodiments, a binding domain VH region of the present disclosure can be derived from or based on a VH of a known monoclonal antibody and can contain one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions or non-conservative amino acid substitutions), or a combination of the above-noted changes, when compared with the VH of a known monoclonal antibody. An insertion, deletion or substitution may be anywhere in the VH region, including at the amino- or carboxy-terminus or both ends of this region, provided that each CDR includes zero changes or at most one, two, or three changes and provided a binding domain containing the modified VH region can still specifically bind its target with an affinity similar to the wild type binding domain.
[0095] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by: Kabat et al. (1991) "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (Kabat numbering scheme); Al-Lazikani et al. (1997) J Mol Biol 273: 927-948 (Chothia numbering scheme); Maccallum et al. (1996) J Mol Biol 262: 732-745 (Contact numbering scheme); Martin et al. (1989) Proc. Natl. Acad. Sci., 86: 9268-9272 (AbM numbering scheme); Lefranc M P et al. (2003) Dev Comp Immunol 27(1): 55-77 (IMGT numbering scheme); and Honegger and Pluckthun (2001) J Mol Biol 309(3): 657-670 ("Aho" numbering scheme). The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, "30a," and deletions appearing in some antibodies. The two schemes place certain insertions and deletions ("indels") at different positions, resulting in differential numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. In particular embodiments, the antibody CDR sequences disclosed herein are according to Kabat numbering.
[0096] (ii) Intracellular Signaling Components. The intracellular or otherwise the cytoplasmic signaling components of a CAR are responsible for activation of the cell in which the CAR is expressed. The term“intracellular signaling components” or“intracellular components” is thus meant to include any portion of the intracellular domain sufficient to transduce an activation signal. Intracellular components of expressed CAR can include effector domains. An effector domain is an intracellular portion of a fusion protein or receptor that can directly or indirectly promote a biological or physiological response in a cell when receiving the appropriate signal. In certain embodiments, an effector domain is part of a protein or protein complex that receives a signal when bound, or it binds directly to a target molecule, which triggers a signal from the effector domain. An effector domain may directly promote a cellular response when it contains one or more signaling domains or motifs, such as an immunoreceptor tyrosine-based activation motif (ITAM). In other embodiments, an effector domain will indirectly promote a cellular response by associating with one or more other proteins that directly promote a cellular response, such as co stimulatory domains.
[0097] Effector domains can provide for activation of at least one function of a modified cell upon binding to the cellular marker expressed by a cancer cell. Activation of the modified cell can include one or more of differentiation, proliferation and/or activation or other effector functions. In particular embodiments, an effector domain can include an intracellular signaling component including a T cell receptor and a co-stimulatory domain which can include the cytoplasmic sequence from co-receptor or co-stimulatory molecule.
[0098] An effector domain can include one, two, three or more receptor signaling domains, intracellular signaling components (e.g., cytoplasmic signaling sequences), co-stimulatory domains, or combinations thereof. Exemplary effector domains include signaling and stimulatory domains selected from: 4-1 BB (CD137), CARD11 , CD3y, CD36, CD3s, Oϋ3z, CD27, CD28, CD79A, CD79B, DAP10, FcRa, FcRb (FcsRI b), FcRy, Fyn, HVEM (LIGHTR), ICOS, LAG 3, LAT, Lck, LRP, NKG2D, NOTCH1 , pTa, PTCH2, 0X40, ROR2, Ryk, SLAMF1 , Slp76, TCRa, p^b, TRIM, Wnt, Zap70, or any combination thereof. In particular embodiments, exemplary effector domains include signaling and co-stimulatory domains selected from: CD86, FcyRIla, DAP12, CD30, CD40, PD-1 , lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1 , GITR, BAFFR, SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8a, Oϋ8b, IL2Rb, I L2 R g , IL7Ra, ITGA4, VLA1 , CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1 d, ITGAE, CD103, ITGAL, CD1 1 a, ITGAM, CD1 1 b, ITGAX, CD11 c, ITGB1 , CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE/RANKL, DNAM 1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM 1 , CRTAM, Ly9 (CD229), PSGL1 , CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, GADS, PAG/Cbp, NKp44, NKp30, or NKp46.
[0099] Intracellular signaling component sequences that act in a stimulatory manner may include iTAMs. Examples of iTAMs including primary cytoplasmic signaling sequences include those derived from CD3y, CD36, CD3s, Oϋ3z, CD5, CD22, CD66d, CD79a, CD79b, and common FcRy (FCER1 G), FcyRIla, FcRb (Fes Rib), DAP10, and DAP12. In particular embodiments, variants of Oϋ3z retain at least one, two, three, or all ITAM regions.
[00100] In particular embodiments, an effector domain includes a cytoplasmic portion that associates with a cytoplasmic signaling protein, wherein the cytoplasmic signaling protein is a lymphocyte receptor or signaling domain thereof, a protein including a plurality of ITAMs, a co stimulatory domain, or any combination thereof.
[00101] Additional examples of intracellular signaling components include the cytoplasmic sequences of the Oϋ3z chain, and/or co- receptors that act in concert to initiate signal transduction following binding domain engagement.
[00102] A co-stimulatory domain is domain whose activation can be required for an efficient lymphocyte response to cellular marker binding. Some molecules are interchangeable as intracellular signaling components or co-stimulatory domains. Examples of costimulatory domains include CD27, CD28, 4-1 BB (CD 137), 0X40, CD30, CD40, PD-1 , ICOS, lymphocyte function- associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83. For example, CD27 co-stimulation has been demonstrated to enhance expansion, effector function, and survival of human CART cells in vitro and augments human T cell persistence and anti-cancer activity in vivo (Song et al. Blood. 2012; 1 19(3):696-706). Further examples of such co-stimulatory domain molecules include CDS, ICAM-1 , GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8a, CD8 , IL2R , IL2Ry, IL7Ra, ITGA4, VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDIId, ITGAE, CD103, ITGAL, CDIIa, ITGAM, CDI lb, ITGAX, CDIIc, ITGBI, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), NKG2D, CEACAM1 , CRTAM, Ly9 (CD229), PSGL1 , CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1 , CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, and CD19a.
[00103] In particular embodiments, the amino acid sequence of the intracellular signaling component includes a variant of Oϋ3z (SEQ ID NOs: 43 and 44) and a portion of the 4-1 BB (SEQ ID NO: 41 and 42) intracellular signaling component.
[00104] In particular embodiments, the intracellular signaling component includes (i) all or a portion of the signaling domain of Oϋ3z, (ii) all or a portion of the signaling domain of 4-1 BB, or (iii) all or a portion of the signaling domain of Oϋ3z and 4-1 BB.
[00105] Intracellular components may also include one or more of a protein of a Wnt signaling pathway (e.g., LRP, Ryk, or ROR2), NOTCH signaling pathway (e.g., NOTCH1 , NOTCH2, NOTCH3, or NOTCH4), Hedgehog signaling pathway (e.g., PTCH or SMO), receptor tyrosine kinases (RTKs) (e.g., epidermal growth factor (EGF) receptor family, fibroblast growth factor (FGF) receptor family, hepatocyte growth factor (HGF) receptor family, insulin receptor (IR) family, platelet-derived growth factor (PDGF) receptor family, vascular endothelial growth factor (VEGF) receptor family, tropomycin receptor kinase (Trk) receptor family, ephrin (Eph) receptor family, AXL receptor family, leukocyte tyrosine kinase (LTK) receptor family, tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE) receptor family, receptor tyrosine kinase-like orphan (ROR) receptor family, discoidin domain (DDR) receptor family, rearranged during transfection (RET) receptor family, tyrosine-protein kinase-like (PTK7) receptor family, related to receptor tyrosine kinase (RYK) receptor family, or muscle specific kinase (MuSK) receptor family); G-protein-coupled receptors, GPCRs (Frizzled or Smoothened); serine/threonine kinase receptors (BMPR or TGFR); or cytokine receptors (IL1 R, IL2R, IL7R, or IL15R).
[00106] (iii) Linkers. As used herein, a linker can be any portion of a CAR molecule that serves to connect two other subcomponents of the molecule. Some linkers serve no purpose other than to link other components while many linkers serve an additional purpose. Linkers in the context of linking VL and VH of antibody derived binding domains of scFv are described above. Linkers can also include spacer regions, and junction amino acids.
[00107] Spacer regions are a type of linker region that are used to create appropriate distances and/or flexibility from other linked components. In particular embodiments, the length of a spacer region can be customized for individual cellular markers on unwanted cells to optimize unwanted cell recognition and destruction. The spacer can be of a length that provides for increased responsiveness of the cell following antigen binding, as compared to in the absence of the spacer. In particular embodiments, a spacer region length can be selected based upon the location of a cellular marker epitope, affinity of a binding domain for the epitope, and/or the ability of the modified cells expressing the molecule to proliferate in vitro and/or in vivo in response to cellular marker recognition. Spacer regions can also allow for high expression levels in modified cells.
[00108] Exemplary spacers include those having 10 to 250 amino acids, 10 to 200 amino acids, 10 to 150 amino acids, 10 to 100 amino acids, 10 to 50 amino acids, or 10 to 25 amino acids. In particular embodiments, a spacer region is 12 amino acids, 20 amino acids, 21 amino acids, 26 amino acids, 27 amino acids, 45 amino acids, or 50 amino acids. [00109] In particular embodiments, the spacer region is selected from the group including all or a portion of a hinge region sequence from lgG1 , lgG2, lgG3, lgG4 or IgD alone or in combination with all or a portion of a CH2 region; all or a portion of a CH3 region; or all or a portion of a CH2 region and all or a portion of a CH3 region.
[00110] Exemplary spacers include lgG4 hinge alone, lgG4 hinge linked to CH2 and CH3 domains, or lgG4 hinge linked to the CH3 domain. In particular embodiments, the spacer includes an lgG4 linker of the amino acid sequence: ESKYGPPCPPC (SEQ ID NO: 257). In particular embodiments, the spacer includes a hinge of an amino acid sequence listed in FIG. 26 (SEQ ID NO: 38, 62, 65, 66, and 129). Hinge regions can be modified to avoid undesirable structural interactions such as dimerization with unintended partners.
[00111] In particular embodiments, a spacer region includes a hinge region that a type II C-lectin interdomain (stalk) region or a cluster of differentiation (CD) molecule stalk region. As used herein, a“wild type immunoglobulin hinge region” refers to a naturally occurring upper and middle hinge amino acid sequences interposed between and connecting the CH1 and CH2 domains (for IgG, IgA, and IgD) or interposed between and connecting the CH1 and CH3 domains (for IgE and IgM) found in the heavy chain of an antibody.
[00112] A“stalk region” of a type II C-lectin or CD molecule refers to the portion of the extracellular domain of the type 11 C-lectin or CD molecule that is located between the C-type lectin-like domain (CTLD; e.g., similar to CTLD of natural killer cell receptors) and the hydrophobic portion (transmembrane domain). For example, the extracellular domain of human CD94 (GenBank Accession No. AAC50291.1) corresponds to amino acid residues 34-179, but the CTLD corresponds to amino acid residues 61-176, so the stalk region of the human CD94 molecule includes amino acid residues 34-60, which are located between the hydrophobic portion (transmembrane domain) and CTLD (see Boyington et al., Immunity 10: 15, 1999; for descriptions of other stalk regions, see also Beavil et al. , Proc. Nat'l. Acad. Sci. USA 89: 153, 1992; and Figdor et al., Nat. Rev. Immunol. 2:1 1 , 2002). These type II C-lectin or CD molecules may also have junction amino acids (described below) between the stalk region and the transmembrane region or the CTLD. In another example, the 233 amino acid human NKG2A protein (GenBank Accession No. P26715.1) has a hydrophobic portion (transmembrane domain) ranging from amino acids 71-93 and an extracellular domain ranging from amino acids 94-233. The CTLD includes amino acids 119-231 and the stalk region includes amino acids 99-1 16, which may be flanked by additional junction amino acids. Other type II C-lectin or CD molecules, as well as their extracellular ligand-binding domains, stalk regions, and CTLDs are known in the art (see, e.g., GenBank Accession Nos. NP 001993.2; AAH07037.1 ; NP 001773.1 ; AAL65234.1 ; CAA04925.1 ; for the sequences of human CD23, CD69, CD72, NKG2A, and NKG2D and their descriptions, respectively).
[00113] In particular embodiments disclosed herein, a multimerization domain can function as a spacer region. In particular embodiments, a conventional spacer region can be used in conjunction with a multimerization domain. In particular embodiments, the spacer region can also be the multimerization domain. In particular embodiments, the spacer region includes a dock and lock (DNL) dimerization domain of the amino acid sequences (SEQ ID NOs: 48, 49, 50, 53, and 55) and nucleic acid sequences listed in FIG. 26 (SEQ ID NOs: 51 , 52, 54, and 56).
[00114] Exemplary spacers also include those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153 or WO2014031687. In particular embodiments, the spacer region can be a CD28 linker of the amino acid sequence PSPLFPGPSKP (SEQ ID NO: 57). In particular embodiments, the spacer region is (GGGGS)n wherein n is an integer including, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or more. In particular embodiments, the spacer region is (EAAAK)n (SEQ ID NO: 258) wherein n is an integer including 1 , 2, 3, 4, 5, 6, 7, 8, 9, or more.
[00115] In particular embodiments, a long spacer is greater than 1 19 amino acids (e.g., 229 amino acids) an intermediate spacer is 13-119 amino acids, and a short spacer is 12 amino acids or less. In one example a short spacer region includes the portion of the lgG4 hinge region encoded by SEQ ID NO: 64). An example of an intermediate spacer region includes all or a portion of a lgG4 hinge region sequence and a CH3 region (e.g., SEQ ID NO: 65). An example of a long spacer includes all or a portion of a lgG4 hinge region sequence, a CH2 region, and a CH3 region (e.g., SEQ ID NO: 66). In particular embodiments of the present disclosure, short spacer sequences are preferred.
[00116] As further description regarding spacer regions, an extracellular component of a fusion protein optionally includes an extracellular, non-signaling spacer or linker region, which, for example, can position the binding domain away from the host cell (e.g., T cell) surface to enable proper cell/cell contact, antigen binding and activation (Patel et al., Gene Therapy 6: 412-419 (1999)). As indicated, an extracellular spacer region of a fusion binding protein is generally located between a hydrophobic portion or transmembrane domain and the extracellular binding domain, and the spacer region length may be varied to maximize antigen recognition (e.g., tumor recognition) based on the selected target molecule, selected binding epitope, or antigen-binding domain size and affinity (see, e.g., Guest et al., J. Immunother. 28:203-11 (2005); PCT Publication No. WO 2014/031687). In certain embodiments, a spacer region includes an immunoglobulin hinge region. An immunoglobulin hinge region may be a wild-type immunoglobulin hinge region or an altered wild-type immunoglobulin hinge region. In certain embodiments, an immunoglobulin hinge region is a human immunoglobulin hinge region. An immunoglobulin hinge region may be an IgG, IgA, IgD, IgE, or IgM hinge region. An IgG hinge region may be an lgG1 , lgG2, lgG3, or lgG4 hinge region. An exemplary altered lgG4 hinge region is described in PCT Publication No. WO 2014/031687. In certain embodiments, an altered lgG4 hinge region includes an amino acid sequence as set forth in SEQ ID NO:38. Other examples of hinge regions used in the fusion binding proteins described herein include the hinge region present in the extracellular regions of type 1 membrane proteins, such as CD8a, CD4, CD28 and CD7, which may be wild-type or variants thereof.
[00117] In certain embodiments, an extracellular spacer region includes all or a portion of an Fc domain selected from: a CH1 domain, a CH2 domain, a CH3 domain, a CH4 domain, or any combination thereof (see, e.g., PCT Publication WO 2014/031687). The Fc domain or portion thereof may be wildtype of altered (e.g., to reduce antibody effector function). In certain embodiments, the extracellular component includes an immunoglobulin hinge region, a CH2 domain, a CH3 domain, or any combination thereof disposed between the binding domain and the hydrophobic portion. In certain embodiments, the extracellular component includes an lgG1 hinge region, an lgG1 CH2 domain, and an lgG1 CH3 domain. In further embodiments, the lgG1 CH2 domain includes (i) a N297Q mutation, (ii) substitution of the first six amino acids (APEFLG) with APPVA, or both of (i) and (ii). In certain embodiments, the immunoglobulin hinge region, Fc domain or portion thereof, or both are human.
[00118] As indicated, transmembrane domains within a CAR molecule, often serving to connect the extracellular component and intracellular component through the cell membrane. The transmembrane domain can anchor the expressed molecule in the modified cell’s membrane.
[00119] The transmembrane domain can be derived either from a natural and/or a synthetic source. When the source is natural, the transmembrane domain can be derived from any membrane-bound or transmembrane protein. Transmembrane domains can include at least the transmembrane region(s) of the a, b or z chain of a T-cell receptor, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22; CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. In particular embodiments, a transmembrane domain may include at least the transmembrane region(s) of, e.g., KIRDS2, 0X40, CD2, CD27, LFA-1 (CD 11a, CD18), ICOS (CD278), 4-1 BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R , I L2 R g , IL7R a, ITGA1 , VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDI Id, ITGAE, CD103, ITGAL, CDI la, ITGAM, CDI lb, ITGAX, CDI lc, ITGB1 , CD29, ITGB2, CD18, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1 , CRT AM, Ly9(CD229), , PSGL1 , CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1 , CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKG2D, or NKG2C. In particular embodiments, a variety of human hinges can be employed as well including the human Ig (immunoglobulin) hinge (e.g., an lgG4 hinge, an IgD hinge), a GS linker (e.g., a GS linker described herein), a KIR2DS2 hinge or a CD8a hinge.
[00120] In particular embodiments, a transmembrane domain has a three-dimensional structure that is thermodynamically stable in a cell membrane, and generally ranges in length from 15 to 30 amino acids. The structure of a transmembrane domain can include an a helix, a b barrel, a b sheet, a b helix, or any combination thereof.
[00121] A transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid within the extracellular region of the CAR (e.g., up to 15 amino acids of the extracellular region) and/or one or more additional amino acids within the intracellular region of the CAR (e.g., up to 15 amino acids of the intracellular components). In one aspect, the transmembrane domain is from the same protein that the signaling domain, co-stimulatory domain or the hinge domain is derived from. In another aspect, the transmembrane domain is not derived from the same protein that any other domain of the CAR is derived from. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other unintended members of the receptor complex. In one aspect, the transmembrane domain is capable of homodimerization with another CAR on the cell surface of a CAR-expressing cell. In a different aspect, the amino acid sequence of the transmembrane domain may be modified or substituted so as to minimize interactions with the binding domains of the native binding partner present in the same CAR-expressing cell. In particular embodiments, the transmembrane domain includes the amino acid sequence of the CD28 transmembrane domain (SEQ ID NOs: 39 and 40).
[00122] Junction amino acids can be a linker which can be used to connect the sequences of CAR domains when the distance provided by a spacer is not needed and/or wanted. Junction amino acids are short amino acid sequences that can be used to connect co-stimulatory intracellular signaling components. In particular embodiments, junction amino acids are 9 amino acids or less.
[00123] Junction amino acids can be a short oligo- or protein linker, preferably between 2 and 9 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, or 9 amino acids) in length to form the linker. In particular embodiments, a glycine-serine doublet can be used as a suitable junction amino acid linker. In particular embodiments, a single amino acid, e.g., an alanine, a glycine, can be used as a suitable junction amino acid.
[00124] (iv) Multimerization Domains. Protein biological activities depend upon their tertiary and quaternary structure. The quaternary structure requires the physical and chemical interaction of different protein subunits or polypeptides. A“multimerization domain” is a domain that causes two or more proteins (monomers) to interact with each other through covalent and/or non-covalent association(s). Multimerization domains present in proteins can result in protein interactions that form dimers, trimers, tetramers, pentamers, hexamers, heptamers, etc., depending on the number of units/monomers incorporated into the multimer, and/or homomultimers or heteromultimers, depending on whether the binding monomers are the same type or a different type.
[00125] In particular embodiments, the multimerization domain is a dimerization domain that allows binding of two monomers to form a dimer. In particular embodiments, the dimerization domain includes dimerization motifs derived from PRKAR1A (SEQ ID NOs: 48 and 50; also referred to as DNL_5HVZ and DNL Linker). In particular embodiments, the dimerization domain includes dimerization motifs derived from PRKAR1 B (SEQ ID NO: 67). In particular embodiments, the dimerization domain includes dimerization motifs derived from PRKAR1 R (SEQ ID NOs: 49 and 55; also referred to as DNL_5HVZ_R*, DNL_S31 R Linker, and DNLR). In particular embodiments, the dimerization domain includes dimerization motifs derived from PRKAR1 E (SEQ ID NO: 53; also referred to DNL_5HVZ_E* and DNLE). In particular embodiments, homodimerization can be accomplished using dimerization motifs derived from PRKAR1A and PRKAR1A (SEQ ID NOs: 48 and 50) as dimerization domains. In particular embodiments, homodimerization can be accomplished using dimerization motifs derived from PRKAR1 B and PRKAR1 B (SEQ ID NO: 67) as dimerization domains. In particular embodiments, heterodimerization can be accomplished using dimerization motifs derived from PRKAR1 R (SEQ ID NOs: 49 and 55) and PRKAR1 E (SEQ ID NO: 53) as dimerization domains. These constructs allow for dimerization of CAR through a series of human disulfide cross-linked dimerization motifs using constructs derived from the cAMP-dependent protein kinase type I regulatory subunit a (PRKAR1A) (SEQ ID NO: 60) or b (PRKAR1 B) (SEQ ID NO: 61).
[00126] In particular embodiments, a dimerization and docking domain (DDD) can be derived from the cAMP-dependent protein kinase (PKA) regulatory subunits and can be paired with an anchoring domain (AD). The AD can be derived from a specific region found in various A-kinase anchoring proteins (AKAPs) that mediates association with the R subunits of PKA. In particular embodiments, the dimerization domain on one CAR is a DDD (DDD1 and DDD2) (SEQ ID NOs: 68 and 69) and the dimerization domain on the second CAR is an anchoring domain (AD) AD (AD1 and AD2) (SEQ ID NOs: 70 and 71) to facilitate a stably tethered structure. In particular embodiments, the DDD (DDD1 and DDD2) (SEQ ID NOs: 68 and 69) are derived from the regulatory subunits of a cAMP-dependent protein kinase (PKA), and the AD (AD1 and AD2) (SEQ ID NOs: 70 and 71) However, one skilled in the art will realize that other DDDs and ADs are known and can be used such as: the 4-helix bundle type DDD domains obtained from p53, DCoH (pterin 4 a carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor 1 a (TCF1)) and HNF-1 (hepatocyte nuclear factor 1). Other AD sequences of potential use may be found in US 2003/0232420A1. In particular embodiments, the binding of the DDD to AD is further stabilized with a subsequent reaction to covalently secure the two components of the assembly, for example via disulfide bridges, which occurs very efficiently as the initial binding interactions orient the reactive thiol groups to ligate site-specifically. By placing cysteine residues at strategic locations in both the DDD and AD sequences as shown for DDD2 and AD2 in SEQ ID NOs: 69 and 71 , the binding interaction can be made covalent via disulfide bridges, thereby forming a stably tethered structure. The stably tethered structure also retains the full functional properties of the two precursors.
[00127] The X-type four-helix bundle dimerization domain that is a structural characteristic of the DDD (Newlon, et al. EMBO J. 2001 ; 20: 1651-1662; Newlon, et al. Nature Struct Biol. 1999; 3: 222-227) can be helpful in forming multimeric structures. For S100B, this X-type four-helix bundle enables the binding of each dimer to two p53 peptides derived from the c-terminal regulatory domain (residues 367-388) with micromolar affinity (Rustandi, et al. Biochemistry. 1998; 37: 1951- 1960). Similarly, the N-terminal dimerization domain of HNF-1a (HNF-p1) was shown to associate with a dimer of DCoH (dimerization cofactor for HNF-1) via a dimer of HNF-p1 (Rose, et al. Nature Struct Biol. 2000; 7: 744-748). In particular embodiments, these naturally occurring systems can be used to provide stable multimeric structures with multiple functions or binding specificities.
[00128] In particular embodiments, complementary binding domains can dimerize. In particular embodiments, the binding domain is a transmembrane polypeptide derived from a FcsRI chain. In particular embodiments, a CAR can include a part of a FcsRI a chain and another CAR can include a part of an FcsRI b chain such that said FcsRI chains spontaneously dimerize together to form a dimeric CAR. In particular embodiments, CAR can include a part of a FcsRI a chain and a part of a FcsRI g chain such that said FcsRI chains spontaneously trimerize together to form a trimeric CAR, and in another embodiment the multi-chain CAR can include a part of FcsRI a chain, a part of FcsRI b chain and a part of FcsRI g chain such that said FcsRI chains spontaneously tetramerize together to form a tetrameric CAR.
[00129] In particular embodiments, a multimerization domain can be derived from binding events such as those between an enzyme and its substrate/inhibitor, for example, cutinase and phosphonates (Hodneland, et al. Proc Natl Acd Sci USA. 2002; 99: 5048-5052), may also be utilized to generate the two associating components (the“docking” step), which are subsequently stabilized covalently (the“lock” step).
[00130] In particular embodiments, a multimerization domain can be induced using a third molecule or chemical inducer. This method of dimerization requires that one CAR include a chemical inducer of dimerization binding domain 1 (CBD1) and the second CAR include the second chemical inducer of dimerization binding domain (CBD2), wherein CBD1 and CBD2 are capable of simultaneously binding to a chemical inducer of dimerization (CID). CBD1 may include a rapamycin binding domain of FK-binding protein 12 (FKBP12) (SEQ ID NO: 72) and CBD2 may include a FKBP12-Rapamycin Binding (FRB) domain of mTOR (SEQ ID NO: 73). In this case, the CID can include rapamycin or a derivative thereof which is capable of causing CBD1 and CBD2 to heterodimerize. If CBD1 and CBD2 are a FK506 (Tacrolimus) binding domain of FKBP12 and a cyclosporin binding domain of cylcophilin A, the CID can include a FK506/cyclosporin fusion protein. If CBD1 and CBD2 are FKBP12 binding domains including a F36V mutation, the CID can be AP1903. If CBD1 and CBD2 are an oestrogen-binding domain (EBD) and a streptavidin binding domain, the CID can be an estrone/biotin fusion protein. If CBD1 and CBD2 are a glucocorticoid-binding domain (GBD) and a dihydrofolate reductase (DHFR) binding domain, the CID can be a dexamethasone/methotrexate fusion molecule. If CBD1 and CBD2 are an O6- alkylguanine-DNA alkyltransferase (AGT) binding domain and a DHFR binding domain, the CID can be an 06-benzylguanine derivative/methotrexate fusion molecule. If CBD1 and CBD2 are a retinoic acid receptor domain and an ecodysone receptor domain, the CID can include RSL1. Use of the CID binding domains can also be used to alter the affinity to the CID. For instance, altering amino acids at positions 2095, 2098, and 2101 of FRB can alter binding to Rapamycin (Bayle et al, Chemistry & Biology 13, 99-107, 2006).
[00131] In particular embodiments, multimerization domains can be derived from binding events such as those between receptor dimer pair such as the interleukin-8 receptor (IL-8R), integrin heterodimers such as LFA-I and GPIIIb/llla, dimeric ligand polypeptides such as nerve growth factor (NGF), neurotrophin-3 (NT-3), interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, PDGF members, and brain-derived neurotrophic factor (BDNF) (Arakawa et al., J Biol. Chem., 269:27833-27839, 1994; Radziejewski et al., Biochem, 32: 1350, 1993) and variants of some of these domains with modified affinities, such as those described in W02012001647A2.
[00132] In particular embodiments, dimerization domains can include protein sequence motifs such as coiled coils, acid patches, zinc fingers, calcium hands, a CH1-CL pair, an "interface" with an engineered "knob" and/or "protruberance" (US 5821333), leucine zippers (US 5932448), SH2 and SH3 (Vidal et al., Biochemistry, 43:7336- 44, 2004), PTB (Zhou et al., Nature, 378:584- 592, 1995), WW (Sudol Prog Biochys MoL Bio, 65: 113-132, 1996), PDZ (Kim et al., Nature, 378: 85- 88, 1995; Komau et al., Science, 269: 1737-1740, 1995) and WD40 (Hu et al., J Biol Chem., 273:33489- 33494, 1998).
[00133] In particular embodiments, the sequence corresponding to a dimerization domain includes the leucine zipper domain of Jun (SEQ ID NO: 74), the dimerization domain of Fos (SEQ ID NO: 75), a consensus sequence for a WW motif (SEQ ID NO: 76), the dimerization domain of the SH2B adapter protein from GenBank Accession no. AAF73912.1 (Nishi et al., Mol Cell Biol, 25: 2607-2621 , 2005; SEQ ID NO: 77), the SH3 domain of IB1 from GenBank Accession no. AAD22543.1 (Kristensen el al., EMBO J., 25: 785-797, 2006; SEQ ID NO: 78), the PTB domain of human DOK-7 from GenBank Accession no. NP_005535.1 (Wagner et al., Cold Spring Harb Perspect Biol. 5: a008987, 2013; SEQ ID NO: 79), the PDZ-like domain of SATB1 from UniProt Accession No. Q01826 (Galande et al., Mol Cell Biol. Aug; 21 : 5591-5604, 2001 ; SEQ ID NO: 80), the WD40 repeats of APAF from UniProt Accession No. 014727 (Jorgensen et al., 2009. PLOS One. 4(12):e8463; SEQ ID NO: 81), the PAS motif of the dioxin receptor from UniProt Accession No. I6L9E7 (Pongratz et al., Mol Cell Biol, 18:4079^1088, 1998; SEQ ID NO: 82) and the EF hand motif of parvalbumin from UniProt Accession No. P20472 (Jamalian et al., Int J Proteomics, 2014: 153712, 2014; SEQ ID NO: 83).
[00134] C4b multimerization domains can also be used. Particular C4b multimerization domains that can be used are provided in FIG. 26 as SEQ ID NOs: 84 - 116. In particular embodiments, the C4b multimerization domain will be a multimerization domain which includes (i) glycine at position 12, (ii) alanine at position 28, (iii) leucines at positions 29, 34, 36, and/or 41 ; (iv) tyrosine at position 32; (v) lysine at position 33; and/or (vi) cysteine at positions 6 and 18. In particular embodiments, the C4b multimerization domain will be a multimerization domain which includes (i) glycine at position 12, (ii) alanine at position 28, (iii) leucines at positions 29, 34, 36, and 41 ; (iv) tyrosine at position 32; (v) lysine at position 33; and (vi) cysteine at positions 6 and 18.
[00135] C4b multimerization domains can include any of SEQ ID NOs: 84-1 16 with an N-terminal deletion of at least 1 consecutive amino acid residue (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10 consecutive amino acid residues) in length. Additional embodiments can include a C-terminal deletion of at least 1 consecutive amino acid residue (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10 consecutive amino acid residues) in length.
[00136] Particular C4b multimerization domain embodiments will retain or will be modified to include at least 1 of the following residues: A6; E1 1 ; A13; D21 ; C22; P25; A27; E28; L29; R30; T31 ; L32; L33; E34; I35; K37; L38; L40; E41 ; I42; Q43; K44; L45; E48; L49; or Q50. Further embodiments will retain or will be modified to include A6; E1 1 ; A13; D21 ; C22; P25; A27; E28; L29; R30; T31 ; L32; L33; E34; I35; K37; L38; L40; E41 ; I42; Q43; K44; L45; E48; L49; and Q50. Particular C4b multimerization domain embodiments will include the amino acid sequence "AELR".
[00137] In particular embodiments, dextrameric and ferritin-based multimerization can be used. An exemplary ferritin fusion sequence is described in PMID 26279189.
[00138] In particular embodiments, additional methods of causing dimerization can be utilized. Additional modifications to generate a dimerization domain in a CAR could include: generating a second interchain disulfide bond in the C-terminus domain by introducing a second cysteine residue into both CAR; swapping interacting residues in each of the CAR constructs in the C- terminus domains (“knob-in-hole”); and fusing the variable domains of the CARs directly to ΰϋ3z (ΰϋ3z fusion) (Schmitt ei al., Hum. Gene Ther. 2009. 20: 1240-1248).
[00139] (v) Control Features Including Tag Cassettes, Transduction Markers, and Suicide Switches. In particular embodiments, CAR constructs can include one or more tag cassettes, transduction markers, and/or suicide switches. In some embodiments, the transduction marker and/or suicide switch is within the same construct but is expressed as a separate molecule on the cell surface. Tag cassettes and transduction markers can be used to activate, promote proliferation of, detect, enrich for, isolate, track, deplete and/or eliminate genetically modified cells in vitro, in vivo and/or ex vivo. "Tag cassette" refers to a unique synthetic peptide sequence affixed to, fused to, or that is part of a CAR, to which a cognate binding molecule (e.g., ligand, antibody, or other binding partner) is capable of specifically binding where the binding property can be used to activate, promote proliferation of, detect, enrich for, isolate, track, deplete and/or eliminate the tagged protein and/or cells expressing the tagged protein. Transduction markers can serve the same purposes but are derived from naturally occurring molecules and are often expressed using a skipping element that separates the transduction marker from the rest of the CAR molecule.
[00140] Tag cassettes that bind cognate binding molecules include, for example, His tag (SEQ ID NO: 58), Flag tag (SEQ ID NO: 1 17), Xpress tag (SEQ ID NO: 1 18), Avi tag (SEQ ID NO: 1 19), Calmodulin tag (SEQ ID NO: 120), Polyglutamate tag, HA tag (SEQ ID NO: 121), Myc tag (SEQ ID NO: 122), Softag 1 (SEQ ID NO: 123), Softag 3 (SEQ ID NO: 124), and V5 tag (SEQ ID NO: 125). In particular embodiments, a CAR includes a Myc tag (SEQ ID NO: 122).
[00141] Conjugate binding molecules that specifically bind tag cassette sequences disclosed herein are commercially available. For example, His tag antibodies are commercially available from suppliers including Life Technologies, Pierce Antibodies, and GenScript. Flag tag antibodies are commercially available from suppliers including Pierce Antibodies, GenScript, and Sigma- Aldrich. Xpress tag antibodies are commercially available from suppliers including Pierce Antibodies, Life Technologies and GenScript. Avi tag antibodies are commercially available from suppliers including Pierce Antibodies, IsBio, and Genecopoeia. Calmodulin tag antibodies are commercially available from suppliers including Santa Cruz Biotechnology, Abeam, and Pierce Antibodies. HA tag antibodies are commercially available from suppliers including Pierce Antibodies, Cell Signal and Abeam. Myc tag antibodies are commercially available from suppliers including Santa Cruz Biotechnology, Abeam, and Cell Signal.
[00142] Transduction markers may be selected from at least one of a truncated CD19 (tCD19; see Budde et al. , Blood 122: 1660, 2013); a truncated human EGFR (tEGFR; see Wang et al., Blood 1 18: 1255, 201 1); an extracellular domain of human CD34; and/or RQR8 which combines target epitopes from CD34 (see Fehse et al, Mol. Therapy 1 ( 5 Pt 1); 448-456, 2000) and CD20 antigens (see Philip et al, Blood 124: 1277-1278).
[00143] In particular embodiments, a polynucleotide encoding an iCaspase9 construct (iCasp9) may be inserted into a CAR nucleotide construct as a suicide switch.
[00144] Control features may be present in multiple copies in a CAR or can be expressed as distinct molecules with the use of a skipping element. For example, a CAR can have one, two, three, four or five tag cassettes and/or one, two, three, four, or five transduction markers could also be expressed. For example, embodiments can include a CAR construct having two Myc tag cassettes, or a His tag and an HA tag cassette, or a HA tag and a Softag 1 tag cassette, or a Myc tag and a SBP tag cassette. In particular embodiments, CAR that will multimerize following expression include different tag cassettes. In particular embodiments, a transduction marker includes tEFGR. Exemplary transduction markers and cognate pairs are described in US 13/463,247.
[00145] One advantage of including at least one control feature in a CAR is that CAR expressing cells administered to a subject can be depleted using the cognate binding molecule to a tag cassette. In certain embodiments, the present disclosure provides a method for depleting a modified cell expressing a CAR by using an antibody specific for the tag cassette, using an cognate binding molecule specific for the control feature, or by using a second modified cell expressing a CAR and having specificity for the control feature. Elimination of modified cells may be accomplished using depletion agents specific for a control feature.
[00146] In certain embodiments, modified cells expressing a chimeric molecule may be detected or tracked in vivo by using antibodies that bind with specificity to a control feature (e.g., anti-Tag antibodies), or by other cognate binding molecules that specifically bind the control feature, which binding partners for the control feature are conjugated to a fluorescent dye, radio-tracer, iron- oxide nanoparticle or other imaging agent known in the art for detection by X-ray, CT-scan, MRI- scan, PET-scan, ultrasound, flow-cytometry, near infrared imaging systems, or other imaging modalities (see, e.g., Yu, et al., Theranostics 2:3, 2012).
[00147] Thus, modified cells expressing at least one control feature with a CAR can be, e.g., more readily identified, isolated, sorted, induced to proliferate, tracked, and/or eliminated as compared to a modified cell without a tag cassette.
[00148] (vi) Exemplary Constructs. In particular embodiments, an anti-CLL-1 CAR includes the components SC02357-- short spacer -28TM-41 BB-3Z; SC02357-lgG4 hinge -28TM-41 BB-3Z; or anti-CLL_scFV-hsCD28_TM-hs4-1 BB_CPD-hsCD3^CPD-T2A-hsEGFRt.
[00149] In particular embodiments, an anti-CD123 CAR includes the components KLON120- short spacer-28TM-41 BB-ΰϋ3z; KLON120-lgG4 Ii^b-28TM-41 BB-003z; or Anti-CD123_scFV- hsCD28_TM-hs41 BB_CPD-hsCD3z_CPD-T2A-hsEGFRt wherein KLON 120 is the scFv targeting CD123 and can be selected from SEQ ID NO: 138 or from a construct of VL of scFv targeting CD123 (SEQ ID NO: 18) and VH of scFv targeting CD123 (SEQ ID NO: 19) connected by a linker selected from SEQ ID NOs: 206-213 and 258-259. The short spacer can be selected from SEQ ID NO: 38, 206-213, and 257-259. The transmembrane domain is derived from CD28 which can be selected from SEQ ID NO: 39 and SEQ ID NO: 40. 41 BB and Oϋ3z make up the intracellular domain. 41 BB can be selected from SEQ ID NO: 41 and SEQ ID NO: 42. Oϋ3z can be selected from SEQ ID NO: 43 and SEQ ID NO: 44. T2A can be selected from SEQ ID NO: 36 and SEQ ID NO: 37 and hsEGFRt is SEQ ID NO: 47.
[00150] In particular embodiments, the extracellular component of an anti-CLL-1 and anti-CD123 tandem CAR includes 5’-CLL-1-linker-CD123-3’, 5’CD123-linker-CLL-1-3’, 5’-CLL1-G4Sx1- CD123-3’, 5’-CD123-G4Sx1-CLL1-3’, 5’-CD123-G4Sx3-CLL1-3’, 5’-CLL1-G4Sx4-CD123-3’, 5’- CD123-G4Sx4-CLL1 -3’ , 5’-CLL1-EAAAKx3-CD123-3’, and 5’-CD123-EAAAKx3-CLL1 wherein CLL-1 stands for the scFv targeting CLL-1 (SEQ ID NO: 139), CD123 stands for the scFv targeting CD123 (SEQ ID NO: 138), a linker can be selected from SEQ ID NOs: 206-213, and G4Sx1 (SEQ ID NO: 210), G4Sx3 (SEQ ID NO: 213), G4Sx4(SEQ ID NO: 212), and EAAAKx3 (SEQ ID NO:259) are linkers. In particular embodiments, a dimerized CAR construct is anti-CLL-1 and anti- CD123 including the VL of the scFv that binds CLL-1 (SEQ ID NO: 14) linked to the VH of the scFv that binds CLL-1 (SEQ ID NO: 13) by a (GGGGS)4 linker (SEQ ID NO: 212). This binding domain is attached to a multimerization domain that acts as a spacer region including the DNL_5HVZ (PRKAR1 E) multimerization domain (SEQ ID NO: 53), and a CD28 transmembrane domain (SEQ ID NOs: 39 or 40). The CLL-1-targeting CAR is linked to the CD123-targeting CAR through the multimerization domain that serves as a spacer region. The VL of the scFv that binds CD123 (SEQ ID NO: 18) is linked to the VH of the scFv that binds CD123 (SEQ ID NO: 19) by a (GGGGS)4 linker (SEQ ID NO: 212) This binding domain is attached to the CD28 transmembrane domain (SEQ ID NOs: 39 or 40) by a multimerization domain that acts as a spacer region that includes the DNL_5HVZ (PRKAR1 R) multimerization domain (SEQ ID NO: 55) which connects the CLL-1 targeting CAR to the CD123 targeting CAR through the complimentary binding of PRKAR1 R and PRKAR1 E.
[00151] In particular embodiments, the dimerized CAR construct KLON120-PRKAR1AR-28TM- 41 BB-3Z forms heterodimers with SC02357-PRKAR1AE-28TM-41 BB-3Z, wherein SC02357 is an scFv that binds CLL-1 (CD371) and PRKAR1AR and PRKAR1AE are dimerization domains derived from PRKAR1 dimerization domains with a mutation at a core serine or cysteine residue to either an arginine or glutamic acid. In particular embodiments, the CAR construct KLON120- PRKAR1 AE-28TM-41 BB-3Z forms heterodimers with SC02357-PRKAR1AR-28TM-41 BB^.
[00152] In particular embodiments, the dimerized CAR construct KLON120-PRKAR1A-28TM- 41 BB-3Z forms homodimers, wherein KLON120 is an scFv that binds CD123, PRKAR1A is a dimerization domain, 28TM is the CD28 transmembrane domain, 41 BB is the 4-1 BB co stimulatory domain, and 3Z is the Oϋ3z domain. In particular embodiments, the CAR construct SC02357-PRKAR1 A-28TM-41 BB-3Z forms homodimers, wherein SC02357 is an scFv that binds CLL-1. In particular embodiments, the CAR construct KLON120-PRKAR1AR-28TM-41 BB-3Z forms heterodimers with SC02357-PRKAR1AE-28TM-41 BB-3Z, wherein SC02357 is an scFv that binds CLL-1 (CD371) and PRKAR1AR and PRKAR1AE are dimerization domains derived from PRKAR1 dimerization domains with a mutation at a core serine or cysteine residue to either an arginine or glutamic acid. In particular embodiments, the CAR construct KLON120- PRKAR1 AE-28TM-41 BB-3Z forms heterodimers with SC02357-PRKAR1AR-28TM-41 BB-3Z.
In particular embodiments, multimerizing constructs include
hsCD123_scFV-DNL-hsCD28_TM-hs4-1 BB_CPD-hsCD3^CPD-T2A-hsEGFRt (MDT-000828); hsCD123_scFV-DNL_5HVZ-His (MDT-000830);
hsCLL_scFV-DNL_E*-His (MDT-000831); and hsCLL_scFV-DNL_E*-His (MDT-000832).
[00153] In particular embodiments CLL-1 (rev) or CD123(rev) refers to a reverse orientation of depicted VL and VH domains of an scFv; T refers to a T2A ribosomal skip sequence; intDS refers to an intermediate length spacer of 1 19 aa in length, containing the lgG4 hinge and CH3 domains; t19 refers to truncated CD19; and 1ST and 2ST refer to the STREP-TAG® II sequences, wherein 1ST is one sequence and 2ST is two sequences.
[00154] (vii) Immune Cells. The present invention also includes cells genetically modified to express CAR molecules as described herein. In particular embodiments, genetically modified cells include lymphocytes. In particular embodiments, genetically modified cells include T-cells, B cells, natural killer (NK) cells, monocytes/macrophages and hematopoeitic stem cells (HSCs).
[00155] Several different subsets of T-cells have been discovered, each with a distinct function. For example, a majority of T-cells have a T-cell receptor (TCR) existing as a complex of several proteins. The actual T-cell receptor is composed of two separate peptide chains, which are produced from the independent T-cell receptor a and b (TCRa and TOBb) genes and are called a- and b-TCR chains.
[00156] gd T-cells represent a small subset of T-cells that possess a distinct T-cell receptor (TCR) on their surface. In gd T-cells, the TCR is made up of one g-chain and one d-chain. This group of T-cells is much less common (2% of total T-cells) than the ab T-cells.
[00157] CD3 is expressed on all mature T cells. Activated T-cells express 4-1 BB (CD137), CD69, and CD25. CD5 and transferrin receptor are also expressed on T-cells.
[00158] T-cells can further be classified into helper cells (CD4+ T-cells) and cytotoxic T-cells (CTLs, CD8+ T-cells), which include cytolytic T-cells. T helper cells assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and activation of cytotoxic T-cells and macrophages, among other functions. These cells are also known as CD4+ T-cells because they express the CD4 protein on their surface. Helper T-cells become activated when they are presented with peptide antigens by MHC class II molecules that are expressed on the surface of antigen presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response.
[00159] Cytotoxic T-cells destroy virally infected cells and tumor cells, and are also implicated in transplant rejection. These cells are also known as CD8+ T-cells because they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to antigen associated with MHC class I, which is present on the surface of nearly every cell of the body.
[00160] "Central memory" T-cells (or "TCM") as used herein refers to an antigen experienced CTL that expresses CD62L or CCR7 and CD45RO on the surface thereof, and does not express or has decreased expression of CD45RA as compared to naive cells. In particular embodiments, central memory cells are positive for expression of CD62L, CCR7, CD25, CD127, CD45RO, and CD95, and have decreased expression of CD45RA as compared to naive cells.
[00161] "Effector memory" T-cell (or "TEM") as used herein refers to an antigen experienced T- cell that does not express or has decreased expression of CD62L on the surface thereof as compared to central memory cells and does not express or has decreased expression of CD45RA as compared to a naive cell. In particular embodiments, effector memory cells are negative for expression of CD62L and CCR7, compared to naive cells or central memory cells, and have variable expression of CD28 and CD45RA. Effector T-cells are positive for granzyme B and perforin as compared to memory or naive T-cells.
[00162] "Naive" T-cells as used herein refers to a non-antigen experienced T cell that expresses CD62L and CD45RA and does not express CD45RO as compared to central or effector memory cells. In particular embodiments, naive CD8+ T lymphocytes are characterized by the expression of phenotypic markers of naive T-cells including CD62L, CCR7, CD28, CD127, and CD45RA.
[00163] Natural killer cells (also known as NK cells, K cells, and killer cells) are activated in response to interferons or macrophage-derived cytokines. They serve to contain viral infections while the adaptive immune response is generating antigen-specific cytotoxic T cells that can clear the infection. NK cells express CD8, CD16 and CD56 but do not express CD3.
[00164] Macrophages (and their precursors, monocytes) reside in every tissue of the body (in certain instances as microglia, Kupffer cells and osteoclasts) where they engulf apoptotic cells, pathogens and other non-self-components. Monocytes/macrophages express CD1 1 b, F4/80; CD68; CD11 c; IL-4Ra; and/or CD163.
[00165] Immature dendritic cells (i.e., pre-activation) engulf antigens and other non-self- components in the periphery and subsequently, in activated form, migrate to T-cell areas of lymphoid tissues where they provide antigen presentation to T cells. Dendritic cells express CD1 a, CD1 b, CD1c, CD1d, CD21 , CD35, CD39, CD40, CD86, CD101 , CD148, CD209, and DEC-205.
[00166] Hematopoietic Stem/Progenitor Cells or HSPC refer to hematopoietic stem cells and/or hematopoietic progenitor cells.
[00167] Hematopoietic stem cells refer to undifferentiated hematopoietic cells that are capable of self-renewal either in vivo, essentially unlimited propagation in vitro, and capable of differentiation to other cell types including immune effector cells (e.g., T cells, NK cells).
[00168] A hematopoietic progenitor cell is a cell derived from hematopoietic stem cells or fetal tissue that is capable of further differentiation into mature cells types. In certain embodiments, hematopoietic progenitor cells are CD24'° Lin- CD117+ hematopoietic progenitor cells. Hematopoietic progenitor cells include embryonic stem cells.
[00169] Embryonic stem cells or "ES cells" refer to undifferentiated embryonic stem cells that have the ability to integrate into and become part of the germ line of a developing embryo. Embryonic stem cells are capable of differentiating into hematopoietic progenitor cells, and any tissue or organ.
[00170] Thus, HSPC can self-renew or can differentiate into (i) myeloid progenitor cells which ultimately give rise to monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, or dendritic cells; or (ii) lymphoid progenitor cells which ultimately give rise to T-cells, B-cells, and NK-cells.
[00171] HSPC can be positive for a specific marker expressed in increased levels on HSPC relative to other types of hematopoietic cells. For example, such markers include CD34, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD1 17, CD133, CD166, HLA DR, or a combination thereof. Also, the HSPC can be negative for an expressed marker relative to other types of hematopoietic cells. For example, such markers include Lin, CD38, or a combination thereof. Preferably, the HSPC are CD34+ cells.
[00172] Cells to be genetically modified according to the teachings of the current disclosure can be patient-derived cells (autologous) or, when appropriate can be allogeneic.
[00173] (viii) Methods to Collect and Modify Cells Ex Vivo and In Vivo. Methods of cell collection and culture conditions are known by those skilled in the art. In some embodiments, the methods include isolating cells from a subject, preparing, processing, culturing, and/or engineering them, as described herein, and re-introducing them into the same patient, before or after cryopreservation. Particular embodiments can use an off-the-shelf HSPC cell source.
[00174] In some aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or 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, or other organ, and/or cells derived therefrom. In particular embodiments, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in particular embodiments, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, HSPC, red blood cells, and/or platelets, and in some aspects contains cells other than red blood cells and platelets and further processing is necessary.
[00175] Sources of HSPC include umbilical cord blood, placental blood, and peripheral blood (see U.S. Patent Nos. 5,004,681 ; 7,399,633; and 7, 147,626; Craddock, et al., 1997, Blood 90(12):4779-4788; Jin, et at., 2008, Journal of Translational Medicine 6:39; Pelus, 2008, Curr. Opin. Hematol. 15(4):285-292; Papayannopoulou, et al., 1998, Blood 91 (7):2231-2239; Tricot, et al., 2008, Haematologica 93(11 ): 1739-1742; and Weaver et al., 2001 , Bone Marrow Transplantation 27(2):S23-S29). Methods regarding collection, anti-coagulation and processing, etc. of blood samples can be found in, for example, Alsever, et al., 1941 , N. Y. St. J. Med. 41 : 126; De Gowin, et al., 1940, J. Am. Med. Ass. 1 14:850; Smith, et al., 1959, J. Thorac. Cardiovasc. Surg. 38:573; Rous and Turner, 1916, J. Exp. Med. 23:219; and Hum, 1968, Storage of Blood, Academic Press, New York, pp. 26-160. Sources of HSPC also include bone marrow (see Kodo, et al., 1984, J. Clin. Invest. 73: 1377-1384), embryonic cells, aortal-gonadal-mesonephros derived cells, lymph, liver, thymus, and spleen from age-appropriate donors.
[00176] 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. The isolation can include one or more of various cell preparation and separation steps, including separation based on one or more properties, such as size, density, sensitivity or resistance to particular reagents, and/or affinity, e.g., immunoaffinity, to antibodies or other binding partners. In particular embodiments, the isolation is carried out using the same apparatus or equipment sequentially in a single process stream and/or simultaneously. In particular embodiments, the isolation, culture, and/or engineering of the different populations is carried out from the same starting composition or material, such as from the same sample.
[00177] In particular embodiments, one or more of the cell populations enriched, isolated and/or selected from a sample by the provided methods are cells that are positive for (marker+) or express high levels (markerhigh) of one or more particular markers, such as surface markers, or that are negative for (marker-) or express relatively low levels (markerlow) of one or more markers. In particular embodiments, the cell populations (such as T cells or HSPCs) are enriched for cells that are positive or expressing high surface levels of cell markers described elsewhere herein.
[00178] In some aspects, the isolating, incubating, expansion, and/or engineering steps are carried out in a sterile or contained environment and/or in an automated fashion, such as controlled by a computer attached to a device in which the steps are performed.
[00179] The stimulating conditions for the incubation or engineering of T cells include conditions whereby T cells of the culture-initiating composition proliferate or expand. For example, in particular embodiments, the incubation is carried out in the presence of an agent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex, such as a Oϋ3z chain, or capable of activating signaling through such a complex or component. In particular embodiments, the incubation is carried out in the presence of an anti- CD3 antibody, and anti-CD28 antibody, anti-4-1 BB antibody, for example, such antibodies coupled to or present on the surface of a solid support, such as a bead, and/or a cytokine, such as IL-2, IL-15, IL-7, and/or IL-21.
[00180] Following isolation and/or enrichment, HSPCs can be expanded in order to increase the number of HSPC. Isolation and/or expansion methods are described in, for example, US 7,399,633; US 5,004,681 ; US 2010/0183564; W02006/047569; W02007/095594; WO 2011/127470; and WO 2011/127472; Vamum-Finney, et at., 1993, Blood 101 : 1784-1789; Delaney, et ai, 2005, Blood 06:2693-2699; Ohishi, et ai, 2002, J. Clin. Invest. 110: 1165-1 174; Delaney, et at., 2010, Nature Med. 16(2): 232-236; and Chapter 2 of Regenerative Medicine, Department of Health and Human Services, August 2006, and the references cited therein. Each of the referenced methods of collection, isolation, and expansion can be used in particular embodiments of the disclosure.
[00181] Particular methods of expanding HSPC include expansion of HSPC with a Notch agonist, such as the extracellular binding ligands Delta and Serrate (e.g., Jagged), RBP Jxl Suppressor of Hairless, Deltex, Fringe, or fragments thereof which promote Notch pathway activation. Notch agonists include any compound that binds to or otherwise interacts with Notch proteins or other proteins in the Notch pathway such that Notch pathway activity is promoted. For information regarding expansion of HSPC using Notch agonists, see sec. 5.1 and 5.3 of US 7,399,633; US 5,780,300; US 5,648,464; US 5,849,869; US 5,856,441 ; WO 1992/119734; Schlondorfi and Blobel, 1999, J. Cell Sci. 112:3603-3617; Olkkonen and Stenmark, 1997, Int. Rev. Cytol. 176: 1- 85; Kopan, et ai., 2009, Cell 137:216-233; Rebay, et ai., 1991 , Cell 67:687-699, and Jarriault, et a!., 1998, Mol. Cell. Biol. 18:7423-7431.
[00182] Additional culture conditions for HSPC can include expansion in the presence of one more growth factors, such as: angiopoietin-like proteins (Angptls, e.g., Angptl2, Angptl3, Angptl7, Angpt15, and Mfap4); erythropoietin; fibroblast growth factor-1 (FGF-1); Flt-3 ligand (Flt-3L); granulocyte colony stimulating factor (G-CSF); granulocyte-macrophage colony stimulating factor (GM-CSF); insulin growth factor-2 (IFG-2); interleukin-3 (IL-3); interleukin-6 (IL-6); interleukin-7 (IL-7); interleukin-11 (IL-1 1); stem cell factor (SCF; also known as the c-kit ligand or mast cell growth factor); thrombopoietin (TPO); and analogs thereof (wherein the analogs include any structural variants of the growth factors having the biological activity of the naturally occurring growth factor; see, e.g., WO 2007/1145227 and US 2010/0183564). The amount or concentration of growth factors suitable for expanding HSPC depends on the activity of the growth factor preparation, and the species correspondence between the growth factors and HSPC, etc.
[00183] The collection and processing of other cell types described herein are understood by one of ordinary skill in the art.
[00184] Desired genes encoding CAR disclosed herein can be introduced into cells by any method known in the art, including transfection, electroporation, microinjection, lipofection, calcium phosphate mediated transfection, infection with a viral or bacteriophage vector including the gene sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, sheroplast fusion, in vivo nanoparticle-mediated delivery, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see e.g., Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen, et ai, 1993, Meth. Enzymol. 217:618-644; Cline, 1985, Pharmac. Ther. 29:69-92) and may be used, provided that the necessary developmental and physiological functions of the recipient cells are not unduly disrupted. The technique can provide for the stable transfer of the gene to the cell, so that the gene is expressible by the cell and, in certain instances, preferably heritable and expressible by its cell progeny.
[00185] The term “gene” refers to a nucleic acid sequence (used interchangeably with polynucleotide or nucleotide sequence) that encodes a as described herein. This definition includes various sequence polymorphisms, mutations, and/or sequence variants wherein such alterations do not substantially affect the function of the encoded CAR. The term“gene” may include not only coding sequences but also regulatory regions such as promoters, enhancers, and termination regions. The term further can include all introns and other DNA sequences spliced from an mRNA transcript, along with variants resulting from alternative splice sites. Gene sequences encoding the molecule can be DNA or RNA that directs the expression of the chimeric molecule. These nucleic acid sequences may be a DNA strand sequence that is transcribed into RNA or an RNA sequence that is translated into protein. The nucleic acid sequences include both the full-length nucleic acid sequences as well as non-full-length sequences derived from the full- length protein. The sequences can also include degenerate codons of the native sequence or sequences that may be introduced to provide codon preference in a specific cell type. Portions of complete gene sequences are referenced throughout the disclosure as is understood by one of ordinary skill in the art.
[00186] Gene sequences encoding CAR can be readily prepared by synthetic or recombinant methods from the relevant amino acid sequences and other description provided herein. In embodiments, the gene sequence encoding any of these sequences can also have one or more restriction enzyme sites at the 5' and/or 3' ends of the coding sequence in order to provide for easy excision and replacement of the gene sequence encoding the sequence with another gene sequence encoding a different sequence. In embodiments, the gene sequence encoding the sequences can be codon optimized for expression in mammalian cells.
[00187] "Encoding” refers to the property of specific sequences of nucleotides in a gene, such as a cDNA, or an mRNA, to serve as templates for synthesis of other macromolecules such as a defined sequences of amino acids. Thus, a gene codes for a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. A "gene sequence encoding a protein" includes all nucleotide sequences that are degenerate versions of each other and that code for the same amino acid sequence or amino acid sequences of substantially similar form and function.
[00188] Polynucleotide gene sequences encoding more than one portion of an expressed CAR can be operably linked to each other and relevant regulatory sequences. For example, there can be a functional linkage between a regulatory sequence and an exogenous nucleic acid sequence resulting in expression of the latter. For another example, a first nucleic acid sequence can be operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary or helpful, join coding regions, into the same reading frame
[00189] In any of the embodiments described herein, a polynucleotide can include a polynucleotide that encodes a self-cleaving polypeptide, wherein the polynucleotide encoding the self-cleaving polypeptide is located between the polynucleotide encoding the CAR construct and a polynucleotide encoding a transduction marker (e.g., tEGFR). Exemplary self-cleaving polypeptides include 2A peptide from porcine teschovirus-1 (P2A), Thosea asigna virus (T2A), equine rhinitis A virus (E2A), foot-and-mouth disease virus (F2A), or variant thereof (see FIG. 18). Further exemplary nucleic acid and amino acid sequences of 2A peptides are set forth in, for example, Kim et al. ( PLOS One 6:e18556 (201 1).
[00190] A "vector" is a nucleic acid molecule that is capable of transporting another nucleic acid. Vectors may be, e.g., plasmids, cosmids, viruses, or phage. An "expression vector" is a vector that is capable of directing the expression of a protein encoded by one or more genes carried by the vector when it is present in the appropriate environment.
[00191] "Retroviruses" are viruses having an RNA genome. "Gammaretrovirus" refers to a genus of the retroviridae family. Exemplary gammaretroviruses include mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis viruses.
[00192] Retroviral vectors (see Miller, et ai, 1993, Meth. Enzymol. 217:581-599) can be used. In such embodiments, the gene to be expressed is cloned into the retroviral vector for its delivery into cells. In particular embodiments, a retroviral vector includes all of the cis-acting sequences necessary for the packaging and integration of the viral genome, i.e. , (a) a long terminal repeat (LTR), or portions thereof, at each end of the vector; (b) primer binding sites for negative and positive strand DNA synthesis; and (c) a packaging signal, necessary for the incorporation of genomic RNA into virions. More detail about retroviral vectors can be found in Boesen, et al., 1994, Bi ot herapy 6:291-302; Clowes, et ai, 1994, J. Clin. Invest. 93:644-651 ; Kiem, et ai, 1994, Blood 83: 1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4: 129-141 ; and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:1 10-1 14. Adenoviruses, adena-associated viruses (AAV) and alphaviruses can also be used. See Kozarsky and Wlson, 1993, Current Opinion in Genetics and Development 3:499-503, Rosenfeld, et ai., 1991 , Science 252:431-434; Rosenfeld, et ai., 1992, Cell 68: 143-155; Mastrangeli, et ai., 1993, J. Clin. Invest. 91 :225-234; Walsh, et ai., 1993, Proc. Soc. Exp. Bioi. Med. 204:289-300; and Lundstrom, 1999, J. Recept. Signal Transduct. Res. 19: 673-686. Other methods of gene delivery include use of mammalian artificial chromosomes (Vos, 1998, Curr. Op. Genet. Dev. 8:351-359); liposomes (Tarahovsky and Ivanitsky, 1998, Biochemistry (Mosc) 63:607-618); ribozymes (Branch and Klotman, 1998, Exp. Nephrol. 6:78-83); and triplex DNA (Chan and Glazer, 1997, J. Mol. Med. 75:267-282).
[00193] "Lentivirus" refers to a genus of retroviruses that are capable of infecting dividing and non-dividing cells. Several examples of lentiviruses include HIV (human immunodeficiency virus: including HIV type 1 , and HIV type 2); equine infectious anemia virus; feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).
[00194] There are a large number of available viral vectors suitable within the current disclosure, including those identified for human gene therapy applications (see Pfeifer and Verma, 2001 , Ann. Rev. Genomics Hum. Genet. 2: 177). Suitable viral vectors include vectors based on RNA viruses, such as retrovirus-derived vectors, e.g., Moloney murine leukemia virus (MLV)-derived vectors, and include more complex retrovirus-derived vectors, e.g., lentivirus-derived vectors. HIV-1- derived vectors belong to this category. Other examples include lentivirus vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and Maedi-Visna virus (ovine lentivirus). Methods of using retroviral and lentiviral viral vectors and packaging cells for transducing mammalian host cells with viral particles including CAR transgenes are described in, e.g., US 8,1 19,772; Walchli, et ai., 201 1 , PLoS One 6:327930; Zhao, et ai., 2005, J. Immunol. 174:4415; Engels, et ai., 2003, Hum. Gene Ther. 14: 1155; Frecha, et ai., 2010, Mol. Ther. 18:1748; and Verhoeyen, et ai., 2009, Methods Mol. Biol. 506:97. Retroviral and lentiviral vector constructs and expression systems are also commercially available.
[00195] In particular embodiments, codon-optimized sequences encoding the CLL-1 -specific VH- VL and VL-VH SCFVS or CD123-specific VH-VL and VL-VH SCFVS can be synthesized and cloned into the epHIV7 lentiviral plasmid backbone used in previous CD19 CAR-T cell clinical trials.
[00196] Targeted genetic engineering approaches may also be utilized. The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated protein) nuclease system is an engineered nuclease system used for genetic engineering that is based on a bacterial system. Information regarding CRISPR-Cas systems and components thereof are described in, for example, US8697359, US8771945, US8795965, US8865406, US8871445, US8889356, US8889418, US8895308, US8906616, US8932814, US8945839, US8993233 and US8999641 and applications related thereto; and WO2014/018423, WO2014/093595, WO2014/093622 , WO2014/093635, WO2014/093655, WO2014/093661 , WO2014/093694, WO2014/093701 , WO2014/093709, WO2014/093712, WO2014/093718, WO2014/145599, WO2014/204723, W02014/204724, WO2014/204725, WO2014/204726, WO2014/204727, WO2014/204728, WO2014/204729, WO2015/065964, WO2015/089351 , WO2015/089354, WO2015/089364, WO2015/089419, WO2015/089427, WO2015/089462, WO2015/089465, WO2015/089473 and WO2015/089486, W02016205711 , WO2017/106657, WO2017/127807 and applications related thereto.
[00197] Particular embodiments utilize zinc finger nucleases (ZFNs) as gene editing agents. ZFNs are a class of site-specific nucleases engineered to bind and cleave DNA at specific positions. ZFNs are used to introduce double stranded breaks (DSBs) at a specific site in a DNA sequence which enables the ZFNs to target unique sequences within a genome in a variety of different cells.
[00198] For additional information regarding ZFNs and ZFNs useful within the teachings of the current disclosure, see, e.g., US 6,534,261 ; US 6,607,882; US 6,746,838; US 6,794, 136; US
6,824,978; 6,866,997; US 6,933,1 13; 6,979,539; US 7,013,219; US 7,030,215; US 7,220,719; US 7,241 ,573; US 7,241 ,574; US 7,585,849; US 7,595,376; US 6,903, 185; US 6,479,626; US 2003/0232410 and US 2009/0203140 as well as Gaj et al., Nat Methods, 2012, 9(8):805-7;
Ramirez et al., Nucl Acids Res, 2012, 40(12):5560-8; Kim et al., Genome Res, 2012, 22(7): 1327- 33; Urnov et al., Nature Reviews Genetics, 2010, 1 1 :636-646; Miller, et al. Nature biotechnology 25, 778-785 (2007); Bibikova, et al. Science 300, 764 (2003); Bibikova, et al. Genetics 161 , 1 169- 1175 (2002); Wolfe, et al. Annual review of biophysics and biomolecular structure 29, 183-212 (2000); Kim, et al. Proceedings of the National Academy of Sciences of the United States of America 93, 1 156-1 160 (1996); and Miller, et al. The EMBO journal 4, 1609-1614 (1985).
[00199] Particular embodiments can use transcription activator like effector nucleases (TALENs) as gene editing agents. TALENs refer to fusion proteins including a transcription activator-like effector (TALE) DNA binding protein and a DNA cleavage domain. TALENs are used to edit genes and genomes by inducing double DSBs in the DNA, which induce repair mechanisms in cells. Generally, two TALENs must bind and flank each side of the target DNA site for the DNA cleavage domain to dimerize and induce a DSB. For additional information regarding TALENs, see US 8,440,431 ; US 8,440,432; US 8,450,471 ; US 8,586,363; and US 8,697,853; as well as Joung and Sander, Nat Rev Mol Cell Biol, 2013, 14(l):49-55; Beurdeley et al., Nat Commun, 2013, 4: 1762; Scharenberg et al., Curr Gene Ther, 2013, 13(4):291-303; Gaj et al., Nat Methods, 2012, 9(8):805-7; Miller, et al. Nature biotechnology 29, 143-148 (2011); Christian, et al. Genetics 186, 757-761 (2010); Boch, et al. Science 326, 1509-1512 (2009); and Moscou, & Bogdanove, Science 326, 1501 (2009).
[00200] Particular embodiments can utilize MegaTALs as gene editing agents. MegaTALs have a sc rare-cleaving nuclease structure in which a TALE is fused with the DNA cleavage domain of a meganuclease. Meganucleases, also known as homing endonucleases, are single peptide chains that have both DNA recognition and nuclease function in the same domain. In contrast to the TALEN, the megaTAL only requires the delivery of a single peptide chain for functional activity.
[00201] Nanoparticles that result in selective in vivo genetic modification of targeted cell types have been described and can be used within the teachings of the current disclosure. In particular embodiments, the nanoparticles can be those described in WO20141531 14, W02017181 110, and WO201822672.
[00202] (ix) Ex Vivo Manufactured Cell Formulations. In particular embodiments, genetically- modified cells can be harvested from a culture medium, and washed and concentrated into a carrier in a therapeutically-effective amount. Exemplary carriers include saline, buffered saline, physiological saline, water, Hanks' solution, Ringer's solution, Nonnosol-R (Abbott Labs), PLASMA-LYTE A® (Baxter Laboratories, Inc., Morton Grove, IL), glycerol, ethanol, and combinations thereof.
[00203] In particular embodiments, carriers can be supplemented with human serum albumin (HSA) or other human serum components or fetal bovine serum. In particular embodiments, a carrier for infusion includes buffered saline with 5% HAS or dextrose. Additional isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol.
[00204] Carriers can include buffering agents, such as citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and/or trimethylamine salts.
[00205] Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which helps to prevent cell adherence to container walls. Typical stabilizers can include polyhydric sugar alcohols; amino acids, such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, and cyclitols, such as inositol; PEG; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, a-monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (i.e., <10 residues); proteins such as HSA, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose and glucose; disaccharides such as lactose, maltose and sucrose; trisaccharides such as raffinose, and polysaccharides such as dextran.
[00206] Where necessary or beneficial, compositions or formulations can include a local anesthetic such as lidocaine to ease pain at a site of injection.
[00207] Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides, hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol.
[00208] Therapeutically effective amounts of cells within compositions or formulations can be greater than 102 cells, greater than 103 cells, greater than 104 cells, greater than 105 cells, greater than 106 cells, greater than 107 cells, greater than 108 cells, greater than 109 cells, greater than 1010 cells, or greater than 1011.
[00209] In compositions and formulations disclosed herein, cells are generally in a volume of a liter or less, 500 mis or less, 250 mis or less or 100 mis or less. Hence the density of administered cells is typically greater than 104 cells/ml, 107 cells/ml or 108 cells/ml.
[00210] As indicated, compositions include one genetically-modified cell type (e.g., modified T cells, NK cells, or stem cells). Formulations can include different types of genetically-modified cells (e.g.,T cells, NK cells, and/or stem cells in combination).
[00211] Different types of genetically-modified cells or cell subsets (e.g., modified T cells, NK cells, and/or stem cells) can be provided in different ratios e.g., a 1 : 1 :1 ratio, 2: 1 : 1 ratio, 1 :2: 1 ratio, 1 :1 :2 ratio, 5: 1 : 1 ratio, 1 :5: 1 ratio, 1 : 1 :5 ratio, 10: 1 :1 ratio, 1 : 10: 1 ratio, 1 : 1 : 10 ratio, 2:2: 1 ratio, 1 :2:2 ratio, 2: 1 :2 ratio, 5:5:1 ratio, 1 :5:5 ratio, 5: 1 :5 ratio, 10: 10:1 ratio, 1 : 10: 10 ratio, 10:1 : 10 ratio, etc. These ratios can also apply to numbers of cells expressing the same or different CAR components. If only two of the cell types are combined or only 2 combinations of expressed CAR components are included within a formulation, the ratio can include any 2 number combination that can be created from the 3 number combinations provided above. In embodiments, the combined cell populations are tested for efficacy and/or cell proliferation in vitro, in vivo and/or ex vivo, and the ratio of cells that provides for efficacy and/or proliferation of cells is selected. Particular embodiments include a 1 : 1 ratio of CD4 T cells and CD8 T cells.
[00212] The cell-based compositions and formulations disclosed herein can be prepared for administration by, e.g., injection, infusion, perfusion, or lavage. The compositions and formulations can further be formulated for bone marrow, intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, and/or subcutaneous injection.
[00213] (x) Nanoparticle Formulations. Nanoparticles that result in in vivo genetic modification of cells can be formulated alone or in combination into compositions for administration to subjects. Compositions include nanoparticles formulated with at least one pharmaceutically acceptable carrier.
[00214] For injection, compositions can be formulated as aqueous solutions, such as in buffers including Hanks' solution, Ringer's solution, or physiological saline. The aqueous solutions can include formulatory agents such as suspending, stabilizing, and/or dispersing agents. Alternatively, the formulation can be in lyophilized and/or powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
[00215] The use of different solvents (for example, dichloromethane, chloroform, ethyl acetate, triacetin, N-methyl pyrrolidone, tetrahydrofuran, phenol, or combinations thereof) can alter nanoparticle size and structure in order to modulate release characteristics. Other useful solvents include water, ethanol, dimethyl sulfoxide (DMSO), N-methyl-2-pyrrolidone (NMP), acetone, methanol, isopropyl alcohol (I PA), ethyl benzoate, and benzyl benzoate.
[00216] Exemplary release modifiers can include surfactants, detergents, internal phase viscosity enhancers, complexing agents, surface active molecules, co-solvents, chelators, stabilizers, derivatives of cellulose, (hydroxypropyl)methyl cellulose (HPMC), HPMC acetate, cellulose acetate, pluronics (e.g., F68/F127), polysorbates, Span® (Croda Americas, Wilmington, Delaware), poly(vinyl alcohol) (PVA), Brij® (Croda Americas, Wilmington, Delaware), sucrose acetate isobutyrate (SAIB), salts, and buffers.
[00217] Any composition disclosed herein can advantageously include any other pharmaceutically acceptable carriers which include those that do not produce significantly adverse, allergic, or other untoward reactions that outweigh the benefit of administration. Exemplary pharmaceutically acceptable carriers and formulations are disclosed in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990. Moreover, formulations can be prepared to meet sterility, pyrogenicity, general safety, and purity standards as required by the US FDA Office of Biological Standards and/or other relevant foreign regulatory agencies.
[00218] (xi) Methods of Use. Methods disclosed herein include treating subjects (humans, veterinary animals (dogs, cats, reptiles, birds, etc.) livestock (horses, cattle, goats, pigs, chickens, etc.) and research animals (monkeys, rats, mice, fish, etc.) with compositions and formulations disclosed herein. Treating subjects includes delivering therapeutically effective amounts. Therapeutically effective amounts include those that provide effective amounts, prophylactic treatments and/or therapeutic treatments.
[00219] An "effective amount" is the amount of a composition necessary to result in a desired physiological change in the subject. For example, an effective amount can provide an immunogenic anti-cancer. Effective amounts are often administered for research purposes. Effective amounts disclosed herein can cause a stati stically-signifi cant effect in an animal model or in vitro assay relevant to the assessment of a cancer’s development or progression. An immunogenic composition can be provided in an effective amount, wherein the effective amount stimulates an immune response.
[00220] A "prophylactic treatment" includes a treatment administered to a subject who does not display signs or symptoms of a cancer or displays only early signs or symptoms of a cancer such that treatment is administered for the purpose of diminishing or decreasing the risk of developing the cancer further. Thus, a prophylactic treatment functions as a preventative treatment against a cancer. In particular embodiments, prophylactic treatments reduce, delay, or prevent metastasis from a primary a cancer tumor site from occurring.
[00221] A "therapeutic treatment" includes a treatment administered to a subject who displays symptoms or signs of a cancer and is administered to the subject for the purpose of diminishing or eliminating those signs or symptoms of the cancer. The therapeutic treatment can reduce, control, or eliminate the presence or activity of the cancer and/or reduce control or eliminate side effects of the cancer.
[00222] Function as an effective amount, prophylactic treatment or therapeutic treatment are not mutually exclusive, and in particular embodiments, administered dosages may accomplish more than one treatment type.
[00223] In particular embodiments, therapeutically effective amounts provide anti-cancer effects. Anti-cancer effects include a decrease in the number of cancer cells, decrease in the number of metastases, a decrease in tumor volume, an increase in life expectancy, induced chemo- or radiosensitivity in cancer cells, inhibited angiogenesis near cancer cells, inhibited cancer cell proliferation, inhibited tumor growth, prevented or reduced metastases, prolonged subject life, reduced cancer-associated pain, and/or reduced relapse or re-occurrence of cancer following treatment.
[00224] A "tumor" can be liquid or solid depending on the cell origin. A solid tumor is a swelling or lesion formed by an abnormal growth of cells (called neoplastic cells or tumor cells). A "tumor cell" is an abnormal cell that grows by a rapid, uncontrolled cellular proliferation and continues to grow after the stimuli that initiated the new growth cease and can be considered a solid tumor or liquid tumor in the art depending on the cell origin. Tumors show partial or complete lack of structural organization and functional coordination with the normal tissue, and usually form a distinct mass of tissue, which may be benign, pre-malignant or malignant. Liquid tumors also refer to the total mass of circulating neoplastic cells, for examples in hematopoietic malignancies such as leukemia.
[00225] Particular embodiments of the disclosure are used to treat AML, BPDCN, MDS, natural killer cell lymphomas, hairy cell leukemia, ALL, CML, other leukemias, hematological cancers or tumors, (HL, B-cell HL, NHL, MCL, T cell lymphoma, multiple myeloma (refractory, relapsed, etc.), SM, HES, myelofibrosis, anemia, SLE, psoriasis, and scleroderma.
[00226] For administration, therapeutically effective amounts (also referred to herein as doses) can be initially estimated based on results from in vitro assays and/or animal model studies. Such information can be used to more accurately determine useful doses in subjects of interest. The actual dose amount administered to a particular subject can be determined by a physician, veterinarian or researcher taking into account parameters such as physical and physiological factors including target, body weight, severity of condition, type of cancer, stage of cancer, previous or concurrent therapeutic interventions, idiopathy of the subject and route of administration.
[00227] Therapeutically effective amounts to administer can include greater than 102 cells, greater than 103 cells, greater than 104 cells, greater than 105 cells, greater than 106 cells, greater than 107 cells, greater than 108 cells, greater than 109 cells, greater than 1010 cells, or greater than 1011.
[00228] Useful doses can range from 0.1 to 5 pg/kg or from 0.5 to 1 pg /kg. In other examples, a dose can include 1 pg /kg, 15 pg /kg, 30 pg /kg, 50 pg/kg, 55 pg/kg, 70 pg/kg, 90 pg/kg, 150 pg/kg, 350 pg/kg, 500 pg/kg, 750 pg/kg, 1000 pg/kg, 0.1 to 5 mg/kg or from 0.5 to 1 mg/kg. In other examples, a dose can include 1 mg/kg, 10 mg/kg, 30 mg/kg, 50 mg/kg, 70 mg/kg, 100 mg/kg, 300 mg/kg, 500 mg/kg, 700 mg/kg, 1000 mg/kg or more.
[00229] Therapeutically effective amounts can be achieved by administering single or multiple doses during the course of a treatment regimen (e.g., daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months or yearly). [00230] As indicated, the compositions and formulations disclosed herein can be administered by, e.g., injection, infusion, perfusion, or lavage and can more particularly include administration through one or more bone marrow, intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, and/or subcutaneous infusions and/or bolus injections.
[00231] In certain embodiments, cells or nanoparticle-based formulations are administered to a patient in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities. In particular embodiments, cells or nanoparticle-based formulations may be used in combination with chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as alemtuzumab, anti-CD3 antibodies or other antibody therapies, cytoxin, fludarabine, cyclophosphamide, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. In particular embodiments, cells or nanoparticle- based formulations may be used in conjunction with cyclophosphamide. In particular embodiments, cells or nanoparticle-based formulations may be used in conjunction with fludarabine. In particular embodiments, cells or nanoparticle-based formulations may be used in conjunction with cyclophosphamide and fludarabine.
[00232] FIG. 25 illustrates three exemplary clinical applications of treatment strategies disclosed herein. An initial clinical trial could include patients having R/R AML who have a suitable allogenic donor identified. As shown in the top panel, chemotherapy can be administered to a patient having R/R AML for a first time period. Then, chemotherapy can be reduced and the patient can be infused with anti-CLL-1/anti-CD123 CAR-T cells for a second time period. Upon myeloid recovery, chemotherapy can then be administered to the patient for a third time period.
[00233] As shown in the middle panel, chemotherapy can be administered to a patient having R/R AML for a first time period. Then, chemotherapy can be reduced and the patient can be infused with CLL-1/CD123 CAR-T cells for a second time period. Upon myeloid recovery, a control feature (tag cassette, transduction marker and/or suicide switch) can be activated in the CLL- 1/CD123 CAR-T cells and chemotherapy can then be administered to the patient for a third time period.
[00234] As shown in the lower panel, chemotherapy can be administered to a patient having R/R AML for a first time period. Then, chemotherapy can be reduced and the patient can be infused with CLL-1/CD123 CAR-T cells for a second time period. A control feature can be activated in the CLL-1/CD123 CAR-T cells, the patient can then undergo myeloablation followed by a bone marrow transplant (BMT). Other clinical applications of the CLL-1/CD123 CAR-T cells are also within the scope of this disclosure.
[00235] (xii) Exemplary Embodiments.
1. A chimeric antigen receptor (CAR) system including a cell genetically modified to express at least two CAR constructs wherein each expressed CAR construct includes
an extracellular component linked to an intracellular component through a transmembrane domain;
wherein the extracellular component of at least one CAR construct expressed by the cell includes a binding domain that specifically binds CLL-1 ; and wherein the extracellular component of a second CAR construct expressed by the cell includes a binding domain that specifically binds CD123;
and wherein the intracellular components of the CAR constructs include an effector domain.
2. The CAR system of embodiment 1 , wherein the binding domain that specifically binds CLL- 1 is a single chain variable fragment (sFv) including the variable light chain and the variable heavy chain complementarity determining regions (CDRs) of SC02357 as set forth in SEC ID NO: 261-266, according to Kabat numbering.
3. The CAR system of embodiment 2, wherein the linker of the scFv is selected from SEC ID NO: 212 and SEC ID NO: 213.
4. The CAR system of any embodiments 1-3, wherein the binding domain that specifically binds CD123 is a single chain variable fragment (scFv) including the variable light chain and the variable heavy chain of KLON120, as set forth in SEC ID NO: 19 and SEC ID NO: 18.
5. The CAR system of embodiment 4, wherein the variable heavy chain of KLON120 and the variable light chain of KLON120 are linked through a Gly-Ser linker including SEC ID NO: 212.
6. A chimeric antigen receptor (CAR) system of any of embodiments 1-5, including a cell genetically modified to express a CAR construct including an extracellular component linked to an intracellular component through a transmembrane domain;
wherein the extracellular component includes a binding domain that specifically binds CLL-1 and a binding domain that specifically binds CD123
and wherein the intracellular components of the CAR construct include an effector domain.
7. The CAR system of any of embodiments 1 -6, wherein at least one CAR construct within the system includes a multimerization domain selected from SEC ID NO: 12, SEC ID NO: 15, SEC ID NO: 16, SEC ID NO: 17, SEC ID NO: 20, SEC ID NO: 24, and SEC ID NO: 28. The CAR system of embodiment 7, wherein the multimerization domain is between the binding domain and the transmembrane domain.
The CAR system of any of embodiments 1-8, wherein the cell is selected from a T cell, natural killer cell, monocyte/macrophage, hematopoietic stem cell or hematopoietic progenitor cell.
The CAR system of embodiment 9, wherein the cell is a T cell selected from a CD3 T cell, a CD4 T cell, a CD8 T cell, a central memory T cell, an effector memory T cell, and/or a naive T cell.
The CAR system of any of embodiments 1-10, including CD4 T cells and CD8 T cells. The CAR system of embodiment 11 , including a 1 :1 ratio of CD4 T cells to CD8 T cells. The CAR system of any of embodiments 1-12, including at least two cell types genetically modified ex vivo to express a CAR construct of the system.
The CAR system of embodiment 13, wherein the at least two cell types include T cells and natural killer cells, T cells and monocyte/macrophages, T cells and hematopoietic stem cells, T cells and hematopoietic progenitor cells, natural killer cells and monocyte/macrophages, natural killer cells and hematopoietic stem cells, natural killer cells and hematopoietic progenitor cells, monocyte/macrophages and hematopoietic stem cells, monocyte/macrophages and hematopoietic progenitor cells, or hematopoietic stem cells and hematopoietic progenitor cells.
A chimeric antigen receptor (CAR) system including cells genetically modified to express at least one CAR, wherein binding domains of CAR within the system specifically bind CLL-1 or CD123, and wherein each CAR in the system:
includes:
an extracellular component linked to an intracellular component through a transmembrane domain;
wherein extracellular components within the system include:
a binding domain that specifically binds CLL-1 ;
a binding domain that specifically binds CD123; or
a binding domain that specifically binds CLL-1 and a binding domain that specifically binds CD123 (FIG. 1 , tandem);
and wherein the intracellular component includes an effector domain.
The CAR system of embodiment 15, wherein at least two CAR within the system are expressed by a same genetically modified cell.
The CAR system of embodiments 15 or 16, wherein the extracellular component and/or the intracellular component and/or the transmembrane domain of the expressed CAR include a multimerization domain that results in multimerization of the expressed CAR following expression by a genetically modified cell (FIG. 1 , dimerized CAR).
The CAR system of any of embodiments 15-17, wherein at least two CAR within the system are expressed by a same genetically modified cell and wherein the first CAR includes a binding domain that specifically binds CLL-1 and the second CAR includes a binding domain that specifically binds CD123 (FIG. 1 , dual CAR).
The CAR system of any of embodiments 15-17, wherein at least two CAR within the system are expressed by a same genetically modified cell and wherein the first CAR includes a binding domain that specifically binds CLL-1 and the second CAR includes a binding domain that specifically binds CLL-1.
The CAR system of any of embodiments 15-17, wherein at least two CAR within the system are expressed by a same genetically modified cell and wherein the first CAR includes a binding domain that specifically binds CD123 and the second CAR includes a binding domain that specifically binds CD123.
The CAR system of embodiment 15, wherein CAR within the system including a binding domain that specifically binds CLL-1 are expressed by different genetically-modified cells than CAR within the system including a binding domain that specifically binds CD123 (FIG. 1 , dual population).
The CAR system of any of embodiments 15-21 , wherein the binding domain of at least one CAR within the system is derived from a binding domain that specifically binds CLL-1 and the binding domain of at least one CAR within the system is derived from a binding domain that specifically binds CD123.
The CAR system of any of embodiments 15-22, wherein the binding domain that specifically binds CLL-1 is a single chain variable fragment (sFv) including the variable light chain and the variable heavy chain CDRs of SC02357 as set forth in SEQ ID NO: 261-266, according to Kabat numbering.
The CAR system of embodiment 23, wherein the linker of the scFv is selected from SEQ ID NO: 212 and SEQ ID NO: 213.
The CAR system of any of embodiments 15-24, wherein the binding domain that specifically binds CD123 is a single chain variable fragment (scFv) including the variable light chain and the variable heavy chain of KLON120, as set forth in SEQ ID NO: 19 and SEQ ID NO: 18.
The CAR system of embodiment 25, wherein the KLQN120 variable heavy chain and the KLON120 variable light chain are linked through a Gly-Ser linker including SEQ ID NO: 212. The CAR system of any of embodiments 15-26, wherein the binding domain of at least one CAR within the system is selected from SEC ID NO: 139 and SEC ID NO: 267.
The CAR system of any of embodiments 15-27, wherein all binding domains within the system that specifically bind CLL-1 are selected from SEQ ID NO: 139 and SEQ ID NO: 267.
The CAR system of any of embodiments 15-28, wherein the binding domain of at least one CAR within the system includes SEQ ID NO: 138.
The CAR system of any of embodiments 15-29, wherein all binding domains within the system that specifically bind CD123 include SEQ ID NO: 138.
The CAR system of any of embodiments 15-30, wherein the binding domains include a binding domain as set forth in SEQ ID NO: 138 and a binding domain selected from SEQ ID NO 139 and SEQ ID NO: 267.
The CAR system of any of embodiments 15-31 , wherein a binding domain within the system that specifically binds CLL-1 is derived from the CDRs of M26, M31 , G4, M22, M29, M2, M5, or G12 according to Kabat numbering.
The CAR system of any of embodiments 15-32, wherein a binding domain within the system that specifically binds CD123 is derived from the CDRs of IMGN632, ADAMTS2, 7G3, 32716, 32701 , 32703, or 26292 according to Kabat numbering.
The CAR system of any of embodiments 15-33, wherein a CAR within the system includes SC02357-lgG4 hinge-28TM-41 BB-3Z; KLON120-lgG4 hinge-28TM-41 BB-3Z; anti- CD123_scFV-hsCD28_TM-hs4-1 BB_CPD-hsCD3^CPD-T2A-hsEGFRt and/or anti- 0ίί_30Rn-Iΐ30028_TM-ΐΊ34-1 BB_0R0-ΐΊ3003z_0R0-T2A-ΐΊ3E0RRΐ.
The CAR system of embodiment 34, wherein SC02357 includes a single chain variable fragment that specifically binds CLL-1 including the variable light chain and the variable heavy chain CDRs of SC02357 as set forth in SEQ ID NO: 261-266, according to Kabat numbering.
The CAR system of embodiment 35, wherein the linker of the scFv is selected from SEQ ID NO: 212 and SEQ ID NO: 213.
The CAR system of any of embodiments 34-35, wherein KLON120 includes a single chain variable fragment that specifically binds CD123 including the variable heavy chain and the variable light chain of KLON 120 as set forth in SEQ ID NO: 19 and SEQ ID NO: 18.
The CAR system of embodiment 37, wherein the variable heavy chain of KLON120 and the variable light chain of KLON120 are linked through a Gly-Ser linker including SEQ ID NO: 212.
The CAR system of any of embodiments 34-38, wherein the lgG4 hinge can be selected from SEQ ID NO: 38 or SEQ ID NO: 257.
The CAR system of any of embodiments 15-39, wherein at least two CAR constructs within the system include a multimerization domain that results in multimerization of the CAR following expression.
The CAR system of embodiment 40, wherein the multimerization domains include a PRKAR1A dimerization domain, PRKAR1 E (SEQ ID NO: 53), and/or a PRKAR1 R dimerization domain.
The CAR system of embodiment 41 , wherein the PRKAR1 A dimerization domain is selected from SEQ ID NO: 48 and SEQ ID NO: 50.
The CAR system of embodiments 41 or 42, wherein the PRKAR1 R dimerization domain is selected from SEQ ID NO: 49 and SEQ ID NO: 55.
The CAR system of any of embodiments 40, 41 , or 43, wherein the multimerization domains include a PRKAR1 R dimerization domain selected from SEQ ID NO: 49 and SEQ ID NO: 55 and the PRKAR1 E dimerization domain (SEQ ID NO: 53).
The CAR system of any of embodiments 40-44, wherein the multimerization domains are selected from the leucine zipper domain of Jun (SEQ ID NO: 74), the dimerization domain of Fos (SEQ ID NO: 75), a consensus sequence for a WW motif (SEQ ID NO: 76), the dimerization domain of the SH2B adapter protein (SEQ ID NO: 77), the SH3 domain of IB1 (SEQ ID NO: 78), the PTB domain of human DOK-7 (SEQ ID NO: 79), the PDZ-like domain of SATB1 (SEQ ID NO: 80), the WD40 repeats of APAF (SEQ ID NO: 81), the PAS motif of the dioxin receptor (SEQ ID NO: 82), the EF hand motif of parvalbumin (SEQ ID NO: 83), and a C4b multimerization domain selected from SEQ ID NOs: 84 - 116 and/or ferritin. The CAR system of any of embodiments 40, 41 , 42, or 45, wherein the multimerized CAR form a complete homomultimer following multimerization.
The CAR system of any of embodiments 40-42, or 45, wherein the multimerized CAR form a partial homomultimer following multimerization.
The CAR system of any of embodiments 40, 41 , 43-45, wherein the multimerized CAR form a heteromultimer following multimerization.
The CAR system of any of embodiments 40-42, 45, or 46, wherein the multimerized CAR form a complete homodimer following multimerization.
The CAR system of any of embodiments 40-42, 45, or 47, wherein the multimerized CAR form a partial homodimer following multimerization. The CAR system of any of embodiments 40, 41 , 43-45, or48, wherein the multimerized CAR form a heterodimer following multimerization.
The CAR system of any of embodiments 40-51 , wherein a CAR within the system includes
SC02357-PRKAR1 A-28TM-41 BB-3Z; SC02357-PRKAR1AE-28TM-41 BB-3Z; SC02357- PRKAR1 AR-28TM-41 BB-3Z; KLON120-PRKAR1A-28TM-41 BB-3Z; KLON120-
PRKAR1 AR-28TM-41 BB-3Z ; KLON120-PRKAR1 AE-28TM-41 BB-3Z ; MDT-000828 (SEQ ID NO: 17); MDT-000830 (SEQ ID NO: 20); MDT-000831 (SEQ ID NO: 24); and/or MDT- 000832 (SEQ ID NO: 28).
The CAR system of any of embodiments 52, wherein PRKAR1A is selected from SEQ ID NO: 48 and SEQ ID NO: 50.
The CAR system of embodiment 52, wherein PRKAR1AE is SEQ ID NO: 53.
The CAR system of embodiment 52, wherein PRKAR1AR is selected from SEQ ID NO: 49 and SEQ ID NO: 55.
The CAR system of any of embodiments 15-55, further including a spacer region between the binding domain and the transmembrane domain.
The CAR system of any of embodiments 15-56, wherein the transmembrane domain of at least one CAR within the system is a CD28 transmembrane domain.
The CAR system of any of embodiments 15-57, wherein the effector domain of at least one CAR within the system is selected from a 4-1 BB effector domain and a ΰϋ3z effector domain.
The CAR system of any of embodiments 15-58, further including a control feature selected from a tag cassette, a transduction marker, and/or a suicide switch.
The CAR system of any of embodiments 15-59, wherein the genetically-modified cells are T cells, natural killer cells, monocytes/macrophages, hematopoietic stem cells or hematopoietic progenitor cells.
The CAR system of embodiment 60, wherein the T cells are selected from CD3 T cells, CD4 T cell, CD8 T cells, central memory T cells, effector memory T cells, and/or naive T cells.
The CAR system of embodiment 61 , including CD4 T cells and CD8 T cells.
The CAR system of embodiment 62, including a 1 : 1 ratio of CD4 T cells to CD8 T cells. The CAR system of any of embodiments 15-63, wherein the genetically-modified cells include at least two cell types genetically modified ex vivo to express a CAR construct of the system.
The CAR system of embodiment 64, wherein the at least two cell types include T cells and natural killer cells, T cells and monocyte/macrophages, T cells and hematopoietic stem cells, T cells and hematopoietic progenitor cells, natural killer cells and monocyte/macrophages, natural killer cells and hematopoietic stem cells, natural killer cells and hematopoietic progenitor cells, monocyte/macrophages and hematopoietic stem cells, monocyte/macrophages and hematopoietic progenitor cells, or hematopoietic stem cells and hematopoietic progenitor cells.
The CAR system of any of embodiments 1-65, wherein the genetically-modified cells are ex vivo or in vivo.
A cell genetically modified to express a CAR of a system of any of embodiments 1-66. The cell of embodiment 67, wherein the cell is ex vivo or in vivo.
The cell of embodiment 67 or 68, wherein the cell is a T cell, natural killer cell, monocyte/macrophage, hematopoietic stem cell or a hematopoietic progenitor cell.
The cell of embodiment 69, wherein the T cell is selected from a CD3 T cell, a CD4 T cell, a CD8 T cell, a central memory T cell, an effector memory T cell, or a naive T cell.
The cell of embodiment 70, wherein the cell is a CD4 T cell or a CD8 T cell.
A composition including cells genetically modified to express the CAR system of any of embodiments 15-66 and/or nanoparticles that that result in in vivo genetic modification of cells to express the CAR system.
The composition of embodiment 72, wherein the cells are T cells, natural killer cells, monocyte/macrophages, hematopoietic stem cells or hematopoietic progenitor cells.
The composition of embodiment 73, wherein the T cells are selected from CD3 T cells, CD4 T cells, CD8 T cells, central memory T cells, effector memory T cells, and/or naive T cells. The composition of embodiment 74, wherein the T cells are CD4 T cells and CD8 T cells. The composition of embodiment 75, including a 1 : 1 ratio of CD4 T cells and CD8 T cells. The composition of any of embodiments 72-76, wherein the cells include a formulation of at least two cell types genetically modified ex vivo to express the CAR system.
The composition of embodiment 77, wherein the at least two cell types include T cells and natural killer cells, T cells and monocyte/macrophages, T cells and hematopoietic stem cells, T cells and hematopoietic progenitor cells, natural killer cells and monocyte/macrophages, natural killer cells and hematopoietic stem cells, natural killer cells and hematopoietic progenitor cells, monocyte/macrophages and hematopoietic stem cells, monocyte/macrophages and hematopoietic progenitor cells, or hematopoietic stem cells and hematopoietic progenitor cells.
A method of treating a subject in need thereof including administering a therapeutically effective amount of a composition of any of embodiments 72-78 to the subject wherein the therapeutically effective amount results in in vivo expression of the CAR system of any of embodiments 1-66 within the subject thereby treating the subject in need thereof.
80. The method of embodiment 79, wherein the treating provides an anti-cancer effect.
81. The method of embodiment 80, wherein the anti-cancer effect is against acute myeloid leukemia.
[00236] (xiii) Experimental Examples. The following examples are illustrative of disclosed methods and compositions. In light of this disclosure, those of skill in the art will recognize that variations of these examples and other examples of the disclosed compositions and methods would be possible without undue experimentation.
[00237] Example 1. CAR Engineering, Lentivirus Production, T Cell Transduction, and Readout Assays. Patients were treated using a CD19 CAR including a short lgG4-hinge only spacer, CD28 transmembrane (TM) domain, 4-1 BB co-stimulatory domain, and a ΰϋ3z signaling domain that is separated by a T2A ribosomal skip sequence from a truncated human EGFR (tEGFR), which serves as a marker of transgene expression (see Turtle et al., Sci Transl Med. 2016;8(355):355ra1 16-355ra1 16; Turtle et al., J Clin Invest. 2016; 126(6):2123-2138; and Hudecek et al., Cancer Immunol Res. 2015;3(2): 125—135). The epHIV7 lentiviral backbone used in the CD19 CAR was employed to design novel CAR constructs that when introduced into primary T cells redirect the specificity of the T cells to either CLL-1 or CD123.
[00238] Figure 1 of International Application No. PCT/US2013/055862, filed August 20, 2013, the entire contents of which are particularly incorporated by reference herein, depicts vector maps of exemplary CAR. Herein, CLL-1 -CAR were generated that include a human CLL-1 scFv (SC02357) derived from a phage display library. CD123-CAR were generated that include a mouse scFv (Klon120) derived from a phage display library from mouse spleen RNA.
[00239] CLL-1- and CD123- CAR including the following in their extracellular domain were also generated: i) an lgG4 hinge“short” spacer (12 amino acids); ii) an lgG4 hinge and CH3 domain containing an“intermediate” spacer (1 19 amino acids); and iii) an lgG4 hinge, CH2, and CH3 domain containing a“long” spacer (229 amino acids).
[00240] FIGs. 6A, 6B depict the results of the CLL-1-CAR spacer analysis. As shown, it was found that the CLL-1 scFv proliferates and produces cytokines optimally on a short spacer. More particularly, FIG. 6A demonstrates that CLL1-short, CLL1-int, and CLL1-long CD8 CAR-T cells are effective at specifically lysing K562 cells transduced with CLL1 (K-CLL1) but not CD19 (K-19) at varying effector to target ratios. It can be seen that CFSE labelled CLL1 -short, CLL1 -int, and CLL1-long CD8 CAR-T cells have specific proliferation in response to K-CLL1 but not K-19 cells. In both of these experiments, CD19-short CAR activity is shown by way of comparison. FIG. 6B demonstrates the specific production of the cytokines IL-2, TNF-a, and IFN-g by CLL1 -short, CLL1 -int, and CLL1-long CD8 CAR-T cells when co-cultured with K-CLL1 , with minimal cytokine production when co-cultured with K-19 cells.
[00241] FIGs. 7A, 7B depict the results of the CD123-CAR spacer analysis. As shown, it was found that the CD123 scFv proliferates and produces cytokines optimally on a short spacer. More particularly, FIG. 7A demonstrates that CD123-short, CD123-int, and CD123-long CD8 CAR-T cells are effective at specifically lysing K562 cells transduced with CD123 (K-123) but not CD19 (K-19) at varying effector to target ratios. It can be seen that CFSE labelled CD123-short, CD123- int, and CD123-long CD8 CAR-T cells have specific proliferation in response to K-123 but not K- 19 cells. In both of these experiments, CD 19-short CAR activity is shown by way of comparison. FIG. 7B demonstrates the specific production of the cytokines IL-2, TNF-a, and IFN-g by CD123- short, CD123-int, and CD123-long CD8 CAR-T cells when co-cultured with K-123, with minimal cytokine production when co-cultured with K-19 cells. In all assays, CD19-short CAR activity is shown by way of comparison. Collectively, these demonstrate the specificity of the CAR constructs provided herein for CD123 expressing target cells and its activity.
[00242] Example 2. Design and Analysis of Tandem CLL-1/CD123 CAR. Tandem CLL-1/CD123 CAR including the following extracellular domains were generated: CLL1-G4Sx1-CD123, CD123- G4Sx1-CLL1 , CD123-G4Sx3-CLL1 , CLL1-G4Sx4-CD123, CD123-G4Sx4-CLL1 , CLL1-
EAAAKx3-CD123, and CD123-EAAAKx3-CLL1. That is, each tandem CLL-1/CD123 CAR includes a different order of its CLL-1 and CD123 scFvs and/or different linker modules between its CLL-1 and CD123 scFvs.
[00243] The CD123-G4Sx3-CLL1 and CLL1-G4Sx4-CD123 tandem CAR were analyzed. FIG. 8A demonstrates that CD123-G4Sx3-CLL1 and CLL1-CLL1-G4Sx4-CD123 tandem CD8 CAR-T cells are effective at specifically lysing K562 cells transduced with CLL1 (K-CLL1), CD123 (K- 123), or both CLL1 and CD123 (K-123CLL1) but not CD19 (K-19) at varying effector to target ratios. FIG. 8B demonstrates that CFSE labelled CD123-G4Sx3-CLL1 and CLL1-CLL1-G4Sx4- CD123 tandem CD8 CAR-T cells have specific proliferation in response to K-CLL1 , K-123, and K-123CLL1 cells but not K-19 cells.
[00244] Example 3. CD8+ T cells can be enriched by immunomagnetic positive selection using CD8+ microbeads (MILTENYI BIOTEC™) and cryopreserved. Thawed CD8+ T cells can be activated on day zero with anti-CD3/CD28 paramagnetic beads (LIFE SCIENCES™) in medium with IL-15 1 ng/mL. On day one, cultures can be transduced with the CAR lentiviral supernatant. Paramagnetic beads can be removed on day five, and on day seven, tEGFR+ transduced T cells can be flow sorted and expanded for seven additional days in IL-15.
[00245] On day 14, CAR-T cells can be assayed for their proliferative capacity and ability to induce target cell cytotoxicity after stimulation with antigen-positive (K562/CLL-1) or antigen negative control (K562) target cells. After K562/CLL-1 stimulation, the percentage of proliferating CAR-T cells can be assessed by CFSE dilution and the percentage of K562/CLL-1 target cells lysed can be assessed by flow cytometry. These experiments can be performed using T cells from five healthy donors. In each assay, this will provide power of 0.8 to identify a difference of 20% in function between different CAR-T cell populations, assuming an a error of 0.05 and a standard deviation of 15%.
[00246] Example 4. The capacity of humanized MISTRG/IL6 mice to allow engraftment of primary AML blasts and human hematopoiesis can be harnessed, and the capacity of CLL-1- and CD123- specific Tandem CAR-, Dual CAR-, Dual Population CAR, and multimerizing CAR cells (developed above) that are manufactured from AML patients to eliminate autologous AML blasts can be compared.
[00247] Antibodies and antibody fragments targeting specific antigens are referred to, herein, as the antibody name, antibody fragment name, antigen target, and“anti-“ the antigen target. For example, molecules targeting CD123 are referred to as KLON120, anti-CD123, CD123, scFv targeting CD123, CD123_VH and CD123_VL, etc.
[00248] (xiv) Closing Paragraphs. Variants of the sequences disclosed and referenced herein are also included. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological activity can be found using computer programs well known in the art, such as DNASTAR™ (Madison, Wisconsin) software. Preferably, amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e. , substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains.
[00249] In a peptide or protein, suitable conservative substitutions of amino acids are known to those of skill in this art and generally can be made without altering a biological activity of a resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub. Co., p. 224). Naturally occurring amino acids are generally divided into conservative substitution families as follows: Group 1 : Alanine (Ala), Glycine (Gly), Serine (Ser), and Threonine (Thr); Group 2: (acidic): Aspartic acid (Asp), and Glutamic acid (Glu); Group 3: (acidic; also classified as polar, negatively charged residues and their amides): Asparagine (Asn), Glutamine (Gin), Asp, and Glu; Group 4: Gin and Asn; Group 5: (basic; also classified as polar, positively charged residues): Arginine (Arg), Lysine (Lys), and Histidine (His); Group 6 (large aliphatic, nonpolar residues): Isoleucine (lie), Leucine (Leu), Methionine (Met), Valine (Val) and Cysteine (Cys); Group 7 (uncharged polar): Tyrosine (Tyr), Gly, Asn, Gin, Cys, Ser, and Thr; Group 8 (large aromatic residues): Phenylalanine (Phe), Tryptophan (Trp), and Tyr; Group 9 (non polar): Proline (Pro), Ala, Val, Leu, lie, Phe, Met, and Trp; Group 11 (aliphatic): Gly, Ala, Val, Leu, and lie; Group 10 (small aliphatic, nonpolar or slightly polar residues): Ala, Ser, Thr, Pro, and Gly; and Group 12 (sulfur-containing): Met and Cys. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.
[00250] In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1), 105-32). Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values are: lie (+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); Glutamate (-3.5); Gin (-3.5); aspartate (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5).
[00251] It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e. , still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity.
[00252] As detailed in US 4,554, 101 , the following hydrophilicity values have been assigned to amino acid residues: Arg (+3.0); Lys (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); Ser (+0.3); Asn (+0.2); Gin (+0.2); Gly (0); Thr (-0.4); Pro (-0.5±1); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Val (-1.5); Leu (-1.8); lie (-1.8); Tyr (-2.3); Phe (-2.5); Trp (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.
[00253] As outlined above, amino acid substitutions may be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
[00254] As indicated elsewhere, variants of gene sequences can include codon optimized variants, sequence polymorphisms, splice variants, and/or mutations that do not affect the function of an encoded product to a statistically-significant degree.
[00255] Variants of the protein, nucleic acid, and gene sequences disclosed herein also include sequences with at least 70% sequence identity, 80% sequence identity, 85% sequence, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity to the protein, nucleic acid, or gene sequences disclosed herein.
[00256]“% sequence identity” refers to a relationship between two or more sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between protein, nucleic acid, or gene sequences as determined by the match between strings of such sequences. "Identity" (often referred to as "similarity") can be readily calculated by known methods, including those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Oxford University Press, NY (1992). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin). Multiple alignment of the sequences can also be performed using the Clustal method of alignment (Higgins and Sharp CABIOS, 5, 151-153 (1989) with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Relevant programs also include the GCG suite of programs (Wsconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wsconsin); BLASTP, BLASTN, BLASTX (Altschul, et al., J. Mol. Biol. 215:403-410 (1990); DNASTAR (DNASTAR, Inc., Madison, Wsconsin); and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 1 11-20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, N.Y.. Wthin the context of this disclosure it will be understood that where sequence analysis software is used for analysis, the results of the analysis are based on the "default values" of the program referenced. As used herein "default values" will mean any set of values or parameters, which originally load with the software when first initialized.
[00257] Variants also include nucleic acid molecules that hybridizes under stringent hybridization conditions to a sequence disclosed herein and provide the same function as the reference sequence. Exemplary stringent hybridization conditions include an overnight incubation at 42 °C in a solution including 50% formamide, 5XSSC (750 mM NaCI, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5XDenhardt's solution, 10% dextran sulfate, and 20 pg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1XSSC at 50 °C. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, moderately high stringency conditions include an overnight incubation at 37°C in a solution including 6XSSPE (20XSSPE=3M NaCI; 0.2M NaH2P04; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 pg/ml salmon sperm blocking DNA; followed by washes at 50 °C with 1XSSPE, 0.1 % SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5XSSC). Variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
[00258] "Specifically binds" refers to an association of a binding domain (of, for example, a CAR binding domain or a nanoparticle selected cell targeting ligand) to its cognate binding molecule with an affinity or Ka (i.e., an equilibrium association constant of a particular binding interaction with units of 1/M) equal to or greater than 105 M-1 , while not significantly associating with any other molecules or components in a relevant environment sample.“Specifically binds” is also referred to as“binds” herein. Binding domains may be classified as "high affinity" or "low affinity". In particular embodiments, "high affinity" binding domains refer to those binding domains with a Ka of at least 107 M 1 , at least 108 M 1 , at least 109 M 1, at least 1010 M 1, at least 1011 M 1 , at least 1012 M 1, or at least 1013 M 1. In particular embodiments, "low affinity" binding domains refer to those binding domains with a Ka of up to 107 M 1 , up to 106 M 1, up to 105 M 1. Alternatively, affinity may be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 105 M to 10 13 M). In certain embodiments, a binding domain may have "enhanced affinity," which refers to a selected or engineered binding domains with stronger binding to a cognate binding molecule than a wild type (or parent) binding domain. For example, enhanced affinity may be due to a Ka (equilibrium association constant) for the cognate binding molecule that is higher than the reference binding domain or due to a Kd (dissociation constant) for the cognate binding molecule that is less than that of the reference binding domain, or due to an off- rate (K0ff) for the cognate binding molecule that is less than that of the reference binding domain. A variety of assays are known for detecting binding domains that specifically bind a particular cognate binding molecule as well as determining binding affinities, such as Western blot, ELISA, and BIACORE® analysis (see also, e.g., Scatchard, et al., 1949, Ann. N. Y. Acad. Sci. 51:660; and US 5,283, 173, US 5,468,614, or the equivalent).
[00259] Unless otherwise indicated, the practice of the present disclosure can employ conventional techniques of immunology, molecular biology, microbiology, cell biology and recombinant DNA. These methods are described in the following publications. See, e.g., Sambrook, et al. Molecular Cloning: A Laboratory Manual, 2nd Edition (1989); F. M. Ausubel, et al. eds., Current Protocols in Molecular Biology, (1987); the series Methods IN Enzymology (Academic Press, Inc.); M. MacPherson, et al., PCR: A Practical Approach, IRL Press at Oxford University Press (1991); MacPherson et al., eds. PCR 2: Practical Approach, (1995); Harlow and Lane, eds. Antibodies, A Laboratory Manual, (1988); and R. I. Freshney, ed. Animal Cell Culture (1987).
[00260] As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component. Thus, the terms“include” or“including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term“comprise” or“comprises” means includes, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of’ excludes any element, step, ingredient or component not specified. The transition phrase “consisting essentially of” limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment. A material effect would cause a statistically significant reduction in the dimerization of CAR expressed by a genetically modified cell.
[00261] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term“about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±1 1 % of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1 % of the stated value.
[00262] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
[00263] The terms“a,”“an,”“the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
[00264] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
[00265] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
[00266] Furthermore, numerous references have been made to patents, printed publications, journal articles and other written text throughout this specification (referenced materials herein). Each of the referenced materials are individually incorporated herein by reference in their entirety for their referenced teaching.
[00267] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.
[00268] The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and/or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.
[00269] Definitions and explanations used in the present disclosure are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the examples or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 3rd Edition or a dictionary known to those of ordinary skill in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology (Eds. Attwood T et al. , Oxford University Press, Oxford, 2006).

Claims

What is claimed is:
1. A chimeric antigen receptor (CAR) system comprising a cell genetically modified to express at least two CAR constructs wherein each expressed CAR construct comprises an extracellular component linked to an intracellular component through a transmembrane domain;
wherein the extracellular component of at least one CAR construct expressed by the cell comprises a binding domain that specifically binds CLL-1 ; and wherein the extracellular component of a second CAR construct expressed by the cell comprises a binding domain that specifically binds CD123;
and wherein the intracellular components of the CAR constructs comprise an effector domain.
2. The CAR system of claim 1 , wherein the binding domain that specifically binds CLL-1 is a single chain variable fragment (sFv) comprising the variable light chain and the variable heavy chain complementarity determining regions (CDRs) of SC02357 as set forth in SEQ ID NO: 261-266, according to Kabat numbering.
3. The CAR system of claim 2, wherein the linker of the scFv is selected from SEQ ID NO:
212 and SEQ ID NO: 213.
4. The CAR system of claim 1 , wherein the binding domain that specifically binds CD123 is a single chain variable fragment (scFv) comprising the variable light chain and the variable heavy chain of KLON120, as set forth in SEQ ID NO: 19 and SEQ ID NO: 18.
5. The CAR system of claim 4, wherein the variable heavy chain of KLON120 and the variable light chain of KLON120 are linked through a Gly-Ser linker comprising SEQ ID NO: 212.
6. A chimeric antigen receptor (CAR) system of claim 1 , comprising a cell genetically modified to express a CAR construct comprising an extracellular component linked to an intracellular component through a transmembrane domain;
wherein the extracellular component comprises a binding domain that specifically binds CLL-1 and a binding domain that specifically binds CD123
and wherein the intracellular components of the CAR construct comprise an effector domain.
7. The CAR system of claim 1 , wherein at least one CAR construct within the system comprises a multimerization domain selected from SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 24, and SEQ ID NO: 28.
8. The CAR system of claim 7, wherein the multimerization domain is between the binding domain and the transmembrane domain.
9. The CAR system of claim 1 , wherein the cell is selected from a T cell, natural killer cell, monocyte/macrophage, hematopoietic stem cell or hematopoietic progenitor cell.
10. The CAR system of claim 9, wherein the cell is a T cell selected from a CD3 T cell, a CD4 T cell, a CD8 T cell, a central memory T cell, an effector memory T cell, and/or a naive T cell.
11. The CAR system of claim 10, comprising CD4 T cells and CD8 T cells.
12. The CAR system of claim 1 1 , comprising a 1 : 1 ratio of CD4 T cells to CD8 T cells.
13. The CAR system of claim 1 , comprising at least two cell types genetically modified ex vivo to express a CAR construct of the system.
14. The CAR system of claim 13, wherein the at least two cell types comprise T cells and natural killer cells, T cells and monocyte/macrophages, T cells and hematopoietic stem cells, T cells and hematopoietic progenitor cells, natural killer cells and monocyte/macrophages, natural killer cells and hematopoietic stem cells, natural killer cells and hematopoietic progenitor cells, monocyte/macrophages and hematopoietic stem cells, monocyte/macrophages and hematopoietic progenitor cells, or hematopoietic stem cells and hematopoietic progenitor cells.
15. A chimeric antigen receptor (CAR) system comprising cells genetically modified to express at least one CAR, wherein binding domains of CAR within the system specifically bind CLL- 1 or CD123, and wherein each CAR in the system:
comprises:
an extracellular component linked to an intracellular component through a transmembrane domain;
wherein extracellular components within the system comprise:
a binding domain that specifically binds CLL-1 ;
a binding domain that specifically binds CD123; or
a binding domain that specifically binds CLL-1 and a binding domain that specifically binds CD123 (FIG. 1 , tandem);
and wherein the intracellular component comprises an effector domain.
16. The CAR system of claim 15, wherein at least two CAR within the system are expressed by a same genetically modified cell.
17. The CAR system of claim 15, wherein the extracellular component and/or the intracellular component and/or the transmembrane domain of the expressed CAR comprise a multimerization domain that results in multimerization of the expressed CAR following expression by a genetically modified cell (FIG. 1 , dimerized CAR).
18. The CAR system of claim 15, wherein at least two CAR within the system are expressed by a same genetically modified cell and wherein the first CAR comprises a binding domain that specifically binds CLL-1 and the second CAR comprises a binding domain that specifically binds CD123 (FIG. 1 , dual CAR).
19. The CAR system of claim 15, wherein at least two CAR within the system are expressed by a same genetically modified cell and wherein the first CAR comprises a binding domain that specifically binds CLL-1 and the second CAR comprises a binding domain that specifically binds CLL-1.
20. The CAR system of claim 15, wherein at least two CAR within the system are expressed by a same genetically modified cell and wherein the first CAR comprises a binding domain that specifically binds CD123 and the second CAR comprises a binding domain that specifically binds CD123.
21. The CAR system of claim 15, wherein CAR within the system comprising a binding domain that specifically binds CLL-1 are expressed by different genetically-modified cells than CAR within the system comprising a binding domain that specifically binds CD123 (FIG. 1 , dual population).
22. The CAR system of claim 15, wherein the binding domain of at least one CAR within the system is derived from a binding domain that specifically binds CLL-1 and the binding domain of at least one CAR within the system is derived from a binding domain that specifically binds CD123.
23. The CAR system of claim 15, wherein the binding domain that specifically binds CLL-1 is a single chain variable fragment (sFv) comprising the variable light chain and the variable heavy chain CDRs of SC02357 as set forth in SEQ ID NO: 261-266, according to Kabat numbering.
24. The CAR system of claim 23, wherein the linker of the scFv is selected from SEQ ID NO:
212 and SEQ ID NO: 213.
25. The CAR system of claim 15, wherein the binding domain that specifically binds CD123 is a single chain variable fragment (scFv) comprising the variable light chain and the variable heavy chain of KLON120, as set forth in SEQ ID NO: 19 and SEQ ID NO: 18.
26. The CAR system of claim 25, wherein the KLON120 variable heavy chain and the KLON120 variable light chain are linked through a Gly-Ser linker comprising SEQ ID NO: 212.
27. The CAR system of claim 15, wherein the binding domain of at least one CAR within the system is selected from SEQ ID NO: 139 and SEQ ID NO: 267.
28. The CAR system of claim 15, wherein all binding domains within the system that specifically bind CLL-1 are selected from SEQ ID NO: 139 and SEQ ID NO: 267.
29. The CAR system of claim 15, wherein the binding domain of at least one CAR within the system comprises SEQ ID NO: 138.
30. The CAR system of claim 15, wherein all binding domains within the system that specifically bind CD123 comprise SEQ ID NO: 138.
31. The CAR system of claim 15, wherein the binding domains comprise a binding domain as set forth in SEQ ID NO: 138 and a binding domain selected from SEQ ID NO 139 and SEQ ID NO: 267.
32. The CAR system of claim 15, wherein a binding domain within the system that specifically binds CLL-1 is derived from the CDRs of M26, M31 , G4, M22, M29, M2, M5, or G12 according to Kabat numbering.
33. The CAR system of claim 15, wherein a binding domain within the system that specifically binds CD123 is derived from the CDRs of IMGN632, ADAMTS2, 7G3, 32716, 32701 , 32703, or 26292 according to Kabat numbering.
34. The CAR system of claim 15, wherein a CAR within the system comprises SC02357-lgG4 hinge-28TM-41 BB-3Z; KLON120-lgG4 hinge-28TM-41 BB-3Z; anti-CD123_scFV- hsCD28_TM-hs4-1 BB_CPD-hsCD3^CPD-T2A-hsEGFRt and/or anti-CLL_scFV- hsCD28_TM-hs4-1 BB_CPD-hsCD3^CPD-T2A-hsEGFRt.
35. The CAR system of claim 34, wherein SC02357 comprises a single chain variable fragment that specifically binds CLL-1 comprising the variable light chain and the variable heavy chain CDRs of SC02357 as set forth in SEQ ID NO: 261-266, according to Kabat numbering.
36. The CAR system of claim 35, wherein the linker of the scFv is selected from SEQ ID NO:
212 and SEQ ID NO: 213.
37. The CAR system of claim 34, wherein KLON120 comprises a single chain variable fragment that specifically binds CD123 comprising the variable heavy chain and the variable light chain of KLON120 as set forth in SEQ ID NO: 19 and SEQ ID NO: 18.
38. The CAR system of claim 37, wherein the variable heavy chain of KLON120 and the variable light chain of KLON120 are linked through a Gly-Ser linker comprising SEQ ID NO: 212.
39. The CAR system of claim 34, wherein the lgG4 hinge can be selected from SEQ ID NO: 38 or SEQ ID NO: 257.
40. The CAR system of claim 15, wherein at least two CAR constructs within the system comprise a multimerization domain that results in multimerization of the CAR following expression.
41. The CAR system of claim 40, wherein the multimerization domains comprise a PRKAR1A dimerization domain, PRKAR1 E (SEQ ID NO: 53), and/or a PRKAR1 R dimerization domain.
42. The CAR system of claim 41 , wherein the PRKAR1 A dimerization domain is selected from SEQ ID NO: 48 and SEQ ID NO: 50.
43. The CAR system of claim 41 , wherein the PRKAR1 R dimerization domain is selected from SEQ ID NO: 49 and SEQ ID NO: 55.
44. The CAR system of claim 40, wherein the multimerization domains comprise a PRKAR1 R dimerization domain selected from SEQ ID NO: 49 and SEQ ID NO: 55 and the PRKAR1 E dimerization domain (SEQ ID NO: 53).
45. The CAR system of claim 40, wherein the multimerization domains are selected from the leucine zipper domain of Jun (SEQ ID NO: 74), the dimerization domain of Fos (SEQ ID NO: 75), a consensus sequence for a WW motif (SEQ ID NO: 76), the dimerization domain of the SH2B adapter protein (SEQ ID NO: 77), the SH3 domain of IB1 (SEQ ID NO: 78), the PTB domain of human DOK-7 (SEQ ID NO: 79), the PDZ-like domain of SATB1 (SEQ ID NO: 80), the WD40 repeats of APAF (SEQ ID NO: 81), the PAS motif of the dioxin receptor (SEQ ID NO: 82), the EF hand motif of parvalbumin (SEQ ID NO: 83), and a C4b multimerization domain selected from SEQ ID NOs. 84 - 1 16 and/or ferritin.
46. The CAR system of claim 40, wherein the multimerized CAR form a complete homomultimer following multimerization.
47. The CAR system of claim 40, wherein the multimerized CAR form a partial homomultimer following multimerization.
48. The CAR system of claim 40, wherein the multimerized CAR form a heteromultimer following multimerization.
49. The CAR system of claim 40, wherein the multimerized CAR form a complete homodimer following multimerization.
50. The CAR system of claim 40, wherein the multimerized CAR form a partial homodimer following multimerization.
51. The CAR system of claim 40, wherein the multimerized CAR form a heterodimer following multimerization.
52. The CAR system of claim 40, wherein a CAR within the system comprises SC02357-
PRKAR1 A-28TM-41 BB-3Z; SC02357-PRKAR1AE-28TM-41 BB-3Z; SC02357-
PRKAR1 AR-28TM-41 BB-3Z; KLON120-PRKAR1A-28TM-41 BB-3Z; KLON120-
PRKAR1 AR-28TM-41 BB-3Z ; KLON120-PRKAR1 AE-28TM-41 BB-3Z ; MDT-000828 (SEQ ID NO: 17); MDT-000830 (SEQ ID NO: 20); MDT-000831 (SEQ ID NO: 24); and/or MDT- 000832 (SEQ ID NO: 28).
53. The CAR system of claim 52, wherein PRKAR1 A is selected from SEQ ID NO: 48 and SEQ ID NO: 50.
54. The CAR system of claim 52, wherein PRKAR1AE is SEQ ID NO: 53.
55. The CAR system of claim 52, wherein PRKAR1AR is selected from SEQ ID NO: 49 and SEQ ID NO: 55.
56. The CAR system of claim 15, further comprising a spacer region between the binding domain and the transmembrane domain.
57. The CAR system of claim 15, wherein the transmembrane domain of at least one CAR within the system is a CD28 transmembrane domain.
58. The CAR system of claim 15, wherein the effector domain of at least one CAR within the system is selected from a 4-1 BB effector domain and a Oϋ3z effector domain.
59. The CAR system of claim 15, further comprising a control feature selected from a tag cassette, a transduction marker, and/or a suicide switch.
60. The CAR system of claim 15, wherein the genetically-modified cells are T cells, natural killer cells, monocytes/macrophages, hematopoietic stem cells or hematopoietic progenitor cells.
61. The CAR system of claim 60, wherein the T cells are selected from CD3 T cells, CD4 T cell, CD8 T cells, central memory T cells, effector memory T cells, and/or naive T cells.
62. The CAR system of claim 61 , comprising CD4 T cells and CD8 T cells.
63. The CAR system of claim 62, comprising a 1 : 1 ratio of CD4 T cells to CD8 T cells.
64. The CAR system of claim 15, wherein the genetically-modified cells comprise at least two cell types genetically modified ex vivo to express a CAR construct of the system.
65. The CAR system of claim 64, wherein the at least two cell types comprise T cells and natural killer cells, T cells and monocyte/macrophages, T cells and hematopoietic stem cells, T cells and hematopoietic progenitor cells, natural killer cells and monocyte/macrophages, natural killer cells and hematopoietic stem cells, natural killer cells and hematopoietic progenitor cells, monocyte/macrophages and hematopoietic stem cells, monocyte/macrophages and hematopoietic progenitor cells, or hematopoietic stem cells and hematopoietic progenitor cells.
66. The CAR system of claim 15, wherein the genetically-modified cells are ex vivo or in vivo.
67. A cell genetically modified to express a CAR of a system of claim 15.
68. The cell of claim 67, wherein the cell is ex vivo or in vivo.
69. The cell of claim 67, wherein the cell is a T cell, natural killer cell, monocyte/macrophage, hematopoietic stem cell or a hematopoietic progenitor cell.
70. The cell of claim 69, wherein the T cell is selected from a CD3 T cell, a CD4 T cell, a CD8 T cell, a central memory T cell, an effector memory T cell, or a naive T cell.
71. The cell of claim 70, wherein the cell is a CD4 T cell or a CD8 T cell.
72. A composition comprising cells genetically modified to express the CAR system of claim 15 and/or nanoparticles that that result in in vivo genetic modification of cells to express the CAR system.
73. The composition of claim 72, wherein the cells are T cells, natural killer cells, monocyte/macrophages, hematopoietic stem cells or hematopoietic progenitor cells.
74. The composition of claim 73, wherein the T cells are selected from CD3 T cells, CD4 T cells, CD8 T cells, central memory T cells, effector memory T cells, and/or naive T cells.
75. The composition of claim 74, wherein the T cells are CD4 T cells and CD8 T cells.
76. The composition of claim 75, comprising a 1 : 1 ratio of CD4 T cells and CD8 T cells.
77. The composition of claim 72, wherein the cells comprise a formulation of at least two cell types genetically modified ex vivo to express the CAR system.
78. The composition of claim 77, wherein the at least two cell types comprise T cells and natural killer cells, T cells and monocyte/macrophages, T cells and hematopoietic stem cells, T cells and hematopoietic progenitor cells, natural killer cells and monocyte/macrophages, natural killer cells and hematopoietic stem cells, natural killer cells and hematopoietic progenitor cells, monocyte/macrophages and hematopoietic stem cells, monocyte/macrophages and hematopoietic progenitor cells, or hematopoietic stem cells and hematopoietic progenitor cells.
79. A method of treating a subject in need thereof comprising administering a therapeutically effective amount of a composition of claim 72 to the subject wherein the therapeutically effective amount results in in vivo expression of the CAR system of claim 15 within the subject thereby treating the subject in need thereof.
80. The method of claim 79, wherein the treating provides an anti-cancer effect.
81. The method of claim 80, wherein the anti-cancer effect is against acute myeloid leukemia.
PCT/US2019/038596 2018-06-22 2019-06-21 Compositions and methods to target cll-1 and cd123 for the treatment of acute myeloid leukemia and related disorders WO2019246593A2 (en)

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WO2021202793A3 (en) * 2020-03-31 2021-11-11 Fred Hutchinson Cancer Research Center Chimeric antigen receptors targeting cd33
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WO2023154890A3 (en) * 2022-02-11 2023-10-26 Fred Hutchinson Cancer Center Chimeric antigen receptors binding steap1

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