EP4240397A1 - Zusammensetzungen und verfahren zur behandlung von krebs mit anti-cd19/cd22-immuntherapie - Google Patents

Zusammensetzungen und verfahren zur behandlung von krebs mit anti-cd19/cd22-immuntherapie

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Publication number
EP4240397A1
EP4240397A1 EP21836263.0A EP21836263A EP4240397A1 EP 4240397 A1 EP4240397 A1 EP 4240397A1 EP 21836263 A EP21836263 A EP 21836263A EP 4240397 A1 EP4240397 A1 EP 4240397A1
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European Patent Office
Prior art keywords
car
cell
domain
cells
nucleic acid
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EP21836263.0A
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English (en)
French (fr)
Inventor
Dina SCHNEIDER
Rimas J. ORENTAS
Boro Dropulic
Peirong Hu
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Lentigen Technology Inc
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Lentigen Technology Inc
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Priority claimed from US17/090,797 external-priority patent/US11242389B2/en
Application filed by Lentigen Technology Inc filed Critical Lentigen Technology Inc
Publication of EP4240397A1 publication Critical patent/EP4240397A1/de
Pending legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464412—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464413—CD22, BL-CAM, siglec-2 or sialic acid binding Ig-related lectin 2
    • 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
    • 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
    • C07K14/70517—CD8
    • 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
    • C07K16/2803—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 immunoglobulin superfamily
    • 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
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/804—Blood cells [leukemia, lymphoma]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/27—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by targeting or presenting multiple antigens
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C07K14/70521—CD28, CD152
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    • 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
    • C07K14/70532—B7 molecules, e.g. CD80, CD86
    • 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
    • C07K14/7056—Lectin superfamily, e.g. CD23, CD72
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    • 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
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
    • 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
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • 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
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • 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

Definitions

  • This application relates to the field of cancer, particularly to CARs targeting CD 19 and CD22 B cell antigens simultaneously, via CD19/CD22 antigen-targeting domains and chimeric antigen receptors (CARs) containing such CD19/CD22 antigen targeting domains and methods of use thereof.
  • CARs chimeric antigen receptors
  • Cancer is one of the most deadly threats to human health. In the U.S. alone, cancer affects nearly 1.3 million new patients each year, and is the second leading cause of death after cardiovascular disease, accounting for approximately 1 in 4 deaths. Solid tumors are responsible for most of those deaths. Although there have been significant advances in the medical treatment of certain cancers, the overall 5 -year survival rate for all cancers has improved only by about 10% in the past 20 years. Cancers, or malignant tumors, metastasize and grow rapidly in an uncontrolled manner, making treatment extremely difficult.
  • CD 19 is a 85-95 kDa transmembrane cell surface glycoprotein receptor.
  • CD 19 is a member of immunoglobulin (Ig) superfamily of proteins, and contains two extracellular Ig-like domains, a transmembrane, and an intracellular signaling domain (Tedder TF, Isaacs, CM, 1989, J Immunol 143:712-171).
  • Ig immunoglobulin
  • CD 19 modifies B cell receptor signaling, lowering the triggering threshold for the B cell receptor for antigen (Carter, RH, and Fearon, DT, 1992, Science, 256: 105- 107) , and co-ordinates with CD81 and CD21 to regulate this essential B cell signaling complex (Bradbury, LE, Kansas GS, Levy S, Evans RL, Tedder TF, 1992, J Immunol, 149:2841-50).
  • B cell ontogeny CD 19 is able to signal at the pro-B, pre-pre-B cell, pre-B, early B cell stages independent of antigen receptor, and is associated with Src family protein tyrosine kinases, is tyrosine phosphorylated, inducing both intracellular calcium mobilization and inositol phospholipid signaling (Uckun FM, Burkhardt AL, Jarvis L, Jun X, Stealy B, Dibirdik I, Myers DE, Tuel-Ahlgren L, Bolen JB, 1983, J Biol Chem 268:21172-84).
  • CD 19 is expressed in a tightly regulated manner on normal B cells, being restricted to early B cell precursors at the stage of IgH gene rearrangement, mature B cells, but not expressed on hematopoietic stem cells, or mature plasma cells (Anderson, KC, Bates, MP, Slaughenhout BL, Pinkus GS, Schlossman SF, Nadler LM, 1984, Blood 63: 1424- 1433).
  • CD22 also known as SIGLEC-2 (sialic acid-binding immunoglobulin-likelectin-2), is 95 kDa transmembrane surface glycoprotein and contains 6 Ig-like C2-type domains and one Ig-like V-type domain (uniprot.org/uniprot/P20273#structure, accessed 07/12/2017).
  • SIGLEC-2 sialic acid-binding immunoglobulin-likelectin-2
  • CD22 is expressed on the B-cell surface starting at the pre-B cell stage, persists on mature B cells and is lost on plasma cells (Nitschke L, 2009, Immunological Reviews, 230:128- 143).
  • CD22 contains intracellular ITIM (immuoreceptor tyrosine-based inhibition motifs) domains which following the engagement of the B cell receptor for antigen serve to down- modulate cellular activation.
  • Antibody binding of CD22 induces co-localization with SHP-1, and intracellular phosphatase that also serves to down-modulate phosorylation-based signal transduction (Lumb S, Fleishcer SJ, Wiedemann A, Daridon C, Maloney A, Shock A, Domer T, 2016, Journal of Cell Communication and Signaling, 10: 143-151).
  • CD22 is expressed in a tightly regulated manner on normal B cells, but not expressed on hematopoietic stem cells, or mature plasma cells, making it a suitable target antigen for B cell leukemias.
  • CD22 on both adult and pediatric (pre-B-ALL) B cell malignancies has led to exploiting this target for both antibody and chimeric antigen receptor (CAR)-T cell-based therapy
  • CAR chimeric antigen receptor
  • Haso W Lee DW, Shah NN, Stetler-Stevenson M, Yuan CM, Pastan IH, Dimitrov DS, Morgan RA, FitzGerlad DJ, Barrett DM, Wayne AS, Mackall CL, Orentas RJ, 2013, Blood, 121 : 1165-1174
  • Inotuzumab Ozogamicin (CMC-544, a humanized version of the murine monoclonal antibody G5/44) is an antibody drug conjugate and is currently being evaluated in clinical trials, either as a single agent or given in combination with chemotherapy (NCT01664910, sponsor: M.D. Anderson Cancer Center) (DiJoseph JF, et al., 2004, Blood, 103: 1807-1814).
  • Epratuzumab is a chimeric protein composed of murine CDRs grafted onto a human antibody framework.
  • binding moi eties for CD22 employed in CAR constructs utilize a domain derived from these murine antibodies and do not effectively activate T cells that target this CD22 domain (such as the HA22 anti-CD22 binder used as the basis for Moxetumomab pasudotox, see James SE, Greenberg PD, Jensen MC, Lin Y, Wang J, Till BG, Raubitschek AA, Forman SJ, Press OW, 2008, Journal of Immunology 180:7028-7038).
  • HA22 anti-CD22 binder used as the basis for Moxetumomab pasudotox
  • the m971 domain was proven effective as a CAR in work supervised by another of the inventors in this application, Dr. Rimas Orentas (Haso W, et al., 2013, Anti-CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia, Blood, 121 : 1165-1174).
  • CD 19 on both adult and pediatric (pre-B-ALL) B cell malignancies has led to exploiting this target for both antibody and chimeric antigen receptor (CAR)-T cell-based therapy
  • CAR chimeric antigen receptor
  • CD22 antigen on lymphomas (DLBCL, FL), and leukemias (CLL) make it an attractive additional target for efficient
  • the present standard of care for B-lineage leukemias may consists of remission induction treatment by high dose of chemotherapy or radiation, followed by consolidation, and may feature stem cell transplantation and additional courses of chemotherapy as needed see the world wide web at cancer.gov).
  • CD 19 on both adult and pediatric (pre-B-ALL) B cell malignancies has led to exploiting this target for both antibody and chimeric antigen receptor (CAR)-T cell-based therapy
  • CAR chimeric antigen receptor
  • bi-specific antibodies that link an anti-CD19 or anti-CD22 binding motif to a T cell binding motif (i.e. Blinatumomab, Blincyto® indicated for the treatment of Philadelphia chromosome-negative relapsed or refractory B-cell precursor acute lymphoblastic leukemia (ALL).
  • Blinatumomab Blincyto® indicated for the treatment of Philadelphia chromosome-negative relapsed or refractory B-cell precursor acute lymphoblastic leukemia (ALL).
  • ALL Philadelphia chromosome-negative relapsed or refractory B-cell precursor acute lymphoblastic leukemia
  • Chimeric Antigen Receptors are hybrid molecules comprising three essential units: (1) an extracellular antigen-binding motif, (2) linking/transmembrane motifs, and (3) intracellular T-cell signaling motifs (Long AH, Haso WM, Orentas RJ. Lessons learned from a highly-active CD22-specific chimeric antigen receptor. Oncoimmunology. 2013; 2 (4):e23621).
  • the antigen-binding motif of a CAR is commonly fashioned after an single chain Fragment variable (ScFv), the minimal binding domain of an immunoglobulin (Ig) molecule.
  • Alternate antigen-binding motifs such as receptor ligands (i.e., IL- 13 has been engineered to bind tumor expressed IL- 13 receptor), intact immune receptors, library-derived peptides, and innate immune system effector molecules (such as NKG2D) also have been engineered.
  • Alternate cell targets for CAR expression such as NK or gamma-delta T cells are also under development (Brown CE et al. Clin Cancer Res. 2012;18(8):2199-209; Lehner M et al. PLoS One. 2012; 7 (2):e31210).
  • the linking motifs of a CAR can be a relatively stable structural domain, such as the constant domain of IgG, or designed to be an extended flexible linker.
  • Structural motifs such as those derived from IgG constant domains, can be used to extend the ScFv binding domain away from the T-cell plasma membrane surface. This may be important for some tumor targets where the binding domain is particularly close to the tumor cell surface membrane (such as for the disialoganglioside GD2; Orentas et al., unpublished observations).
  • the signaling motifs used in CARs always include the CD3-( ⁇ chain because this core motif is the key signal for T cell activation.
  • the first reported second-generation CARs featured CD28 signaling domains and the CD28 transmembrane sequence. This motif was used in third-generation CARs containing CD137 (4-1BB) signaling motifs as well (Zhao Y et al. J Immunol. 2009; 183 (9): 5563-74). With the advent of new technology, the activation of T cells with beads linked to anti-CD3 and anti-CD28 antibody, and the presence of the canonical “signal 2” from CD28 was no longer required to be encoded by the CAR itself.
  • third-generation vectors were found to be not superior to second-generation vectors in in vitro assays, and they provided no clear benefit over second-generation vectors in mouse models of leukemia (Haso W, Lee DW, Shah NN, Stetler-Stevenson M, Yuan CM, Pastan IH, Dimitrov DS, Morgan RA, FitzGerald DJ, Barrett DM, Wayne AS, Mackall CL, Orentas RJ.
  • CD19-specific CARs that are in a second generation CD28/CD3-( ⁇ (Lee DW et al. American Society of Hematology Annual Meeting. New Orleans, LA; December 7-10, 2013) and a CD137/CD3-( ⁇ signaling format (Porter DL et al. N Engl J Med. 2011; 365 (8): 725-33).
  • CD137 other tumor necrosis factor receptor superfamily members such as 0X40 also are able to provide important persistence signals in CAR-transduced T cells (Yvon E et al. Clin Cancer Res. 2009;15(18):5852-60).
  • CD3-( ⁇ and CD28 signal units are split between two different CAR constructs expressed in the same cell; in another, two CARs are expressed in the same T cell, but one has a lower affinity and thus requires the alternate CAR to be engaged first for full activity of the second (Lanitis E et al. Cancer Immunol Res.
  • a second challenge for the generation of a single ScFv-based CAR as an immunotherapeutic agent is tumor cell heterogeneity.
  • At least one group has developed a CAR strategy for glioblastoma whereby the effector cell population targets multiple antigens (HER2, IL-13Ra, EphA2) at the same time in the hope of avoiding the outgrowth of target antigen-negative populations.
  • HER2, IL-13Ra, EphA2 multiple antigens
  • T-cell-based immunotherapy has become a new frontier in synthetic biology; multiple promoters and gene products are envisioned to steer these highly potent cells to the tumor microenvironment, where T cells can both evade negative regulatory signals and mediate effective tumor killing.
  • the elimination of unwanted T cells through the drug-induced dimerization of inducible caspase 9 constructs with chemical -based dimerizers, such as API 903, demonstrates one way in which a powerful switch that can control T-cell populations can be initiated pharmacologically (Di Stasi A et al. N Engl J Med. 2011;365(18): 1673-83).
  • effector T-cell populations that are immune to the negative regulatory effects of transforming growth factor-P by the expression of a decoy receptor further demonstrates the degree to which effector T cells can be engineered for optimal antitumor activity (Foster AE et al. J Immunother. 2008;31(5):500-5).
  • CARs can trigger T-cell activation in a manner similar to an endogenous T-cell receptor, a major impediment to the clinical application of this technology to date has been limited in vivo expansion of CAR+ T cells, rapid disappearance of the cells after infusion, and disappointing clinical activity. This may be due in part to the murine origin of some of the CAR sequences employed.
  • Blinotumomab (bi-specific anti -CD 19 and anti-CD3 antibody) has shown impressive results for the gravely ill patients who have received this therapy. Nevertheless the durable remission rate is less than 40%, and at best only 50% of responders can be salvaged to hematopoietic stem cell transplant (HSCT) (see Gore et al., 2014, NCT01471782 and Von Stackelberg, et al., 2014, NCT01471782, summarized in: Benjamin, JE, Stein AS, 2016, Therapeutic Advances in Hematology 7: 142-156). The requirement of patients who have received either bi-specific antibody or CAR-T therapy to subsequently undergo HSCT in order to maintain durable responses remains an area of active debate.
  • HSCT hematopoietic stem cell transplant
  • the present invention addresses these needs by providing CAR compositions and therapeutic methods that can be used to treat cancers and other diseases and/or conditions.
  • the present invention as disclosed and described herein provides CARs that may be used for the treatment of diseases, disorders or conditions associated with dysregulated expression of CD 19 and/ or CD22 and which CARs contain tandem CD19/CD22 antigen binding domains that exhibit a high surface expression on transduced T cells, exhibit a high degree of cytolysis of CD19-expressing cells, and in which the transduced T cells demonstrate in vivo expansion and persistence.
  • Novel tandem CD 19 and CD22-targeting antibodies or antigen binding domains thereof in which the CD 19 targeting moiety is positioned either before or after the CD22 targeting moiety in the amino acid sequence (hereinafter termed “CD19/CD22”), and chimeric antigen receptors (tandem CARs) that contain such CD 19 and/or CD22 antigen binding domains are provided herein, as well as host cells (e.g., T cells) expressing the receptors, and nucleic acid molecules encoding the receptors.
  • the CARs exhibit a high surface expression on transduced T cells, with a high degree of cytolysis, and with transduced T cell expansion and persistence in vivo.
  • an isolated nucleic acid molecule encoding a tandem CD19/CD22 chimeric antigen receptor (CAR) comprising, from N-terminus to C-terminus, at least one CD19/CD22 antigen binding domain, at least one transmembrane domain, and at least one intracellular signaling domain, wherein the tandem CD19/CD22 CAR comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, and 86.
  • an isolated nucleic acid molecule encoding a tandem CD19/CD22 chimeric antigen receptor (CAR) comprising, from N-terminus to C-terminus, at least one CD19/CD22 antigen binding domain, at least one transmembrane domain, and at least one intracellular signaling domain, wherein the tandem CD19/CD22 CAR encoded by the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 60, 62, 64, 66, 68, 70, 72, 74,
  • tandem CD19/CD22 CAR comprising the amino acid sequence selected from the group consisting of SEQ ID NO. 2, 4, 61, 63, 65, 67, 69, 71, 73, 75,
  • an isolated nucleic acid molecule encoding the CAR is provided wherein the encoded extracellular CD19/CD22 antigen binding domain comprises at least one single chain variable fragment of an antibody that binds to CD19/CD22.
  • an isolated nucleic acid molecule encoding the CAR wherein the encoded extracellular CD19/CD22 antigen binding domain comprises at least one heavy chain variable region of an antibody that binds to CD19/CD22.
  • an isolated nucleic acid molecule encoding the CAR is provided wherein the encoded CAR extracellular CD19/CD22 antigen binding domain further comprises at least one lipocalin-based antigen binding antigen (anticalins) that binds to CD19/CD22.
  • the encoded CAR extracellular CD19/CD22 antigen binding domain further comprises at least one lipocalin-based antigen binding antigen (anticalins) that binds to CD19/CD22.
  • an isolated nucleic acid molecule wherein the encoded extracellular CD19/CD22 antigen binding domain is connected to the transmembrane domain by a linker domain.
  • an isolated nucleic acid molecule encoding the CAR is provided wherein the encoded CD19/CD22 extracellular antigen binding domain is preceded by a sequence encoding a leader or signal peptide.
  • an isolated nucleic acid molecule encoding the CAR comprising at least one CD19/CD22 antigen binding domain encoded by a nucleotide sequence comprising a CD19/CD22 nucleotide sequence contained within SEQ ID Nos: 1, 3, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, and 86, respectively, and wherein the CAR additionally encodes an extracellular antigen binding domain targets an antigen that includes, but is not limited to, CD22, ROR1, mesothelin, CD33, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, Glycolipid F77, EGFRvIII, GD-2, TSLPR, NY- ESO-1 TCR, MAGE A3 TCR, or any combination thereof.
  • the CAR construct is comprised of two CAR chains co-expressed in the same cell via a 2A ribosomal skip element, one CAR chain comprises a targeting domain directed toward CD 19 antigen, and another CAR chain comprises a CAR targeting domain directed toward CD22 antigen. Fused in frame to the targeting domain, each chain comprises a hinge/linker/spacer domain, a transmembrane domain, and a CD3z activation domain. None, one or more co-stimulatory domains may be included in frame in each CAR chain.
  • the CAR chain comprises two co-stimulatory domains linked sequentially (a third generation CAR).
  • an isolated nucleic acid molecule encoding the CAR wherein the additionally encoded extracellular antigen binding domain comprises an anti-CD22 ScFv antigen binding domain, an anti-RORl ScFv antigen binding domain, an anti-mesothelin ScFv antigen binding domain, an anti-CD33 ScFv antigen binding domain, an anti-CD38 ScFv antigen binding domain, an anti-CD123 (IL3RA) ScFv antigen binding domain, an anti-CD138 ScFv antigen binding domain, an anti-BCMA (CD269) ScFv antigen binding domain, an anti- GPC2 ScFv antigen binding domain, an anti-GPC3 ScFv antigen binding domain, an anti-FGFR4 ScFv antigen binding domain, an anti-TSLPR ScFv antigen binding domain an anti-c-Met ScFv antigen binding domain, an anti-PMSA ScFv antigen binding domain, an anti-glycolipid F77 ScFv antigen binding domain, an anti-EGFR
  • the CARs provided herein further comprise a linker or spacer domain.
  • an isolated nucleic acid molecule encoding the CAR is provided wherein the extracellular CD19/CD22 antigen binding domain, the intracellular signaling domain, or both are connected to the transmembrane domain by a linker or spacer domain.
  • an isolated nucleic acid molecule encoding the CAR wherein the encoded linker domain is derived from the extracellular domain of CD8 or CD28, and is linked to a transmembrane domain.
  • an isolated nucleic acid molecule encoding the CAR is provided wherein the encoded CAR further comprises a transmembrane domain that comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137 and CD154, or a combination thereof
  • an isolated nucleic acid molecule encoding the CAR is provided wherein the encoded intracellular signaling domain further comprises a CD3 zeta intracellular domain.
  • an isolated nucleic acid molecule encoding the CAR is provided wherein the encoded intracellular signaling domain is arranged on a C-terminal side relative to the CD3 zeta intracellular domain.
  • an isolated nucleic acid molecule encoding the CAR is provided wherein the encoded at least one intracellular signaling domain comprises a costimulatory domain, a primary signaling domain, or a combination thereof.
  • an isolated nucleic acid molecule encoding the CAR is provided wherein the encoded at least one costimulatory domain comprises a functional signaling domain of 0X40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD1 la/CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or a combination thereof.
  • an isolated nucleic acid molecule encoding the CAR is provided that further contains a leader sequence or signal peptide wherein the leader or signal peptide nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 11.
  • an isolated nucleic acid molecule encoding the CAR is provided wherein the encoded leader sequence comprises the amino acid sequence of SEQ ID NO: 12.
  • a chimeric antigen receptor comprising, from N- terminus to C-terminus, at least one CD19/CD22 antigen binding domain, at least one transmembrane domain, and at least one intracellular signaling domain.
  • a CAR wherein the extracellular CD19/CD22 antigen binding domain comprises at least one single chain variable fragment of an antibody that binds to the antigen, or at least one heavy chain variable region of an antibody that binds to the antigen, or a combination thereof.
  • a CAR wherein the at least one transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, TNFRSF19, or a combination thereof.
  • a protein selected from the group consisting of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, TNFRSF19, or a combination thereof.
  • the CAR is provided wherein CAR additionally encodes an extracellular antigen binding domain comprising CD22, ROR1, mesothelin, CD33, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, TSLPR, c-Met, PSMA, Glycolipid F77, EGFRvIII, GD-2, TSLPR, NY-ESO-1 TCR, MAGE A3 TCR, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, or any combination thereof.
  • CAR additionally encodes an extracellular antigen binding domain comprising CD22, ROR1, mesothelin, CD33, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, TSLPR, c-Met, PSMA, Glycolipid F77, EGFRvIII, GD-2, TSLPR, NY-ESO-1 TCR, MAGE A
  • the CAR is provided wherein the extracellular antigen binding domain comprises an anti-CD22 ScFv antigen binding domain, an anti-RORl ScFv antigen binding domain, an anti-mesothelin ScFv antigen binding domain, an anti-CD33 ScFv antigen binding domain, an anti-CD38 ScFv antigen binding domain, an anti-CD123 (IL3RA) ScFv antigen binding domain, an anti-CD138 ScFv antigen binding domain, an anti -BCMA (CD269) ScFv antigen binding domain, an anti-GPC2 ScFv antigen binding domain, an anti-GPC3 ScFv antigen binding domain, an anti-FGFR4 ScFv antigen binding domain, anti-TSLPR ScFv antigen binding domain, an anti-c-Met ScFv antigen binding domain, an anti-PMSA ScFv antigen binding domain, an anti-glycolipid F77 ScFv antigen binding domain, an anti-EGFRvIII ScFv antigen binding domain, an anti-GD
  • a CAR is provided wherein the at least one intracellular signaling domain comprises a costimulatory domain and a primary signaling domain.
  • a CAR wherein the at least one intracellular signaling domain comprises a costimulatory domain comprising a functional signaling domain of a protein selected from the group consisting of 0X40, CD70, CD27, CD28, CD5, ICAM-1, LFA- 1 (CDl la/CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or a combination thereof.
  • a protein selected from the group consisting of 0X40, CD70, CD27, CD28, CD5, ICAM-1, LFA- 1 (CDl la/CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or a combination thereof.
  • the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 1.
  • the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 2. In another embodiment, the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 3.
  • the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 4.
  • the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 60.
  • the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 61.
  • the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 62.
  • the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 63.
  • the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 64.
  • the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 65.
  • the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 66.
  • the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 67.
  • the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 68.
  • the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 69.
  • the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 70.
  • the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 71.
  • the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 72.
  • the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 73. In one embodiment, the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 74.
  • the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 75.
  • the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 76.
  • the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 77.
  • the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 78.
  • the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 79.
  • the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 80.
  • the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 81.
  • the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 82.
  • the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 83.
  • the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 84.
  • the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 85.
  • the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 86.
  • the nucleic acid sequence encodes a CAR comprising the amino acid sequence of SEQ ID NO: 87.
  • the CARs disclosed herein are modified to express or contain a detectable marker for use in diagnosis, monitoring, and/or predicting the treatment outcome such as progression free survival of cancer patients or for monitoring the progress of such treatment.
  • the nucleic acid molecule encoding the disclosed CARs can be contained in a vector, such as a viral vector.
  • the vector is a DNA vector, an RNA vector, a plasmid vector, a cosmid vector, a herpes virus vector, a measles virus vector, a lentivirus vector, adenoviral vector, or a retrovirus vector, or a combination thereof.
  • the vector further comprises a promoter wherein the promoter is an inducible promoter, a tissue specific promoter, a constitutive promoter, a suicide promoter or any combination thereof.
  • the vector expressing the CAR can be further modified to include one or more operative elements to control the expression of CAR T cells, or to eliminate CAR-T cells by virtue of a suicide switch.
  • the suicide switch can include, for example, an apoptosis inducing signaling cascade or a drug that induces cell death.
  • the vector expressing the CAR can be further modified to express an enzyme such thymidine kinase (TK) or cytosine deaminase (CD).
  • host cells including the nucleic acid molecule encoding the CAR are also provided.
  • the host cell is a T cell, such as a primary T cell obtained from a subject.
  • the host cell is a CD8 + T cell.
  • a pharmaceutical composition comprising an anti-tumor effective amount of a population of human T cells, wherein the T cells comprise a nucleic acid sequence that encodes a chimeric antigen receptor (CAR) comprising the amino acid sequence of SEQ ID NO. 2, 4, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, and 87, wherein the CAR comprises at least one extracellular antigen binding domain comprising a CD19/CD22 antigen binding domain, at least one linker domain, at least one transmembrane domain, and at least one intracellular signaling domain, wherein the T cells are T cells of a human having a cancer.
  • CAR chimeric antigen receptor
  • the cancer includes, inter alia, a hematological cancer such as leukemia (e.g., chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), or chronic myelogenous leukemia (CML), lymphoma (e.g., mantle cell lymphoma, non-Hodgkin's lymphoma or Hodgkin's lymphoma) or multiple myeloma, or a combination thereof.
  • leukemia e.g., chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), or chronic myelogenous leukemia (CML)
  • lymphoma e.g., mantle cell lymphoma, non-Hodgkin's lymphoma or Hodgkin's lymphoma
  • multiple myeloma e.g., multiple myeloma, or a combination thereof.
  • a pharmaceutical composition wherein the at least one transmembrane domain of the CAR contains a transmembrane domain of a protein selected from the group consisting of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, Mesothelin, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF19, or a combination thereof.
  • a protein selected from the group consisting of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, Mesothelin, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF19, or a combination thereof.
  • a pharmaceutical composition wherein the human cancer includes an adult carcinoma comprising oral and pharynx cancer (tongue, mouth, pharynx, head and neck), digestive system cancers (esophagus, stomach, small intestine, colon, rectum, anus, liver, interhepatic bile duct, gallbladder, pancreas), respiratory system cancers (larynx, lung and bronchus), bones and joint cancers, soft tissue cancers, skin cancers (melanoma, basal and squamous cell carcinoma), pediatric tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing’s sarcoma), tumors of the central nervous system (brain, astrocytoma, glioblastoma, glioma), and cancers of the breast, the genital system (uterine cervix, uterine corpus, ovary, vulva, vagina, prostate, testis,
  • a pharmaceutical composition comprising an antitumor effective amount of a population of human T cells of a human having a cancer wherein the cancer is a refractory cancer non-responsive to one or more chemotherapeutic agents.
  • the cancer includes hematopoietic cancer, myelodysplastic syndrome pancreatic cancer, head and neck cancer, cutaneous tumors, minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adult B cell malignancies including, CLL (Chronic lymphocytic leukemia), CML (chronic myelogenous leukemia), non-Hodgkin’s lymphoma (NHL), pediatric B cell malignancies (including B lineage ALL (acute lymphocytic leukemia)), multiple myeloma lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma or other hematological cancer and solid tumors, or any combination thereof.
  • ALL acute lymphoblastic le
  • CAR-T cells methods of making CAR-containing T cells.
  • the methods include transducing a T cell with a vector or nucleic acid molecule encoding a disclosed CAR that specifically binds CD 19 and/or CD22, thereby making the CAR-T cell.
  • a method of generating a population of RNA-engineered cells comprises introducing an in vitro transcribed RNA or synthetic RNA of a nucleic acid molecule encoding a disclosed CAR into a cell of a subject, thereby generating a CAR cell.
  • the disease, disorder or condition associated with the expression of CD 19 is cancer including hematopoietic cancer, myelodysplastic syndrome pancreatic cancer, head and neck cancer, cutaneous tumors, minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adult B cell malignancies including, CLL (Chronic lymphocytic leukemia), CML (chronic myelogenous leukemia), non-Hodgkin’s lymphoma (NHL), pediatric B cell malignancies (including B lineage ALL (acute lymphocytic leukemia)), multiple myeloma lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma or other hematological cancer and solid tumors, or any combination thereof.
  • ALL acute lymphoblastic leukemia
  • AML acute myeloid leukemia
  • NHL chronic myeloid leukemia
  • NHL chronic myeloid leukemia
  • NHL chronic myeloid leukemia
  • a method of blocking T-cell inhibition mediated by a CD 19- and/or CD22 expressing cell and altering the tumor microenvironment to inhibit tumor growth in a mammal comprising administering to the mammal an effective amount of a composition comprising a CAR comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, and 87.
  • the cell is selected from the group consisting of a CD 19 and/or CD22-expressing tumor cell, a tumor-associated macrophage, and any combination thereof.
  • a method of inhibiting, suppressing or preventing immunosuppression of an anti-tumor or anti-cancer immune response in a mammal comprising administering to the mammal an effective amount of a composition comprising a CAR selected from the group consisting of SEQ ID NOs: 2, 4, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, and 87.
  • the CAR inhibits the interaction between a first cell with a T cell, wherein the first cell is selected from the group consisting of a CD 19 and/or CD22- expressing tumor cell, a tumor-associated macrophage, and any combination thereof.
  • a method for inducing an anti-tumor immunity in a mammal comprising administering to the mammal a therapeutically effective amount of a T cell transduced with vector or nucleic acid molecule encoding a disclosed CAR.
  • a method of treating or preventing cancer in a mammal comprising administering to the mammal one or more of the disclosed CARs, in an amount effective to treat or prevent cancer in the mammal.
  • the method includes administering to the subject a therapeutically effective amount of host cells expressing a disclosed CAR that specifically binds CD 19 and/or CD22 and/or one or more of the aforementioned antigens, under conditions sufficient to form an immune complex of the antigen binding domain on the CAR and the extracellular domain of CD 19 and/or CD22 and/or one or more of the aforementioned antigens in the subject.
  • a method for treating a mammal having a disease, disorder or condition associated with an elevated expression of a tumor antigen comprising administering to the subject a pharmaceutical composition comprising an anti -tumor effective amount of a population of T cells, wherein the T cells comprise a nucleic acid sequence that encodes a chimeric antigen receptor (CAR), wherein the CAR includes at least one extracellular CD 19 and/or CD22 antigen binding domain, or any combination thereof, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, and wherein the T cells are T cells of the subject having cancer.
  • CAR chimeric antigen receptor
  • a method for treating cancer in a subject in need thereof comprising administering to the subject a pharmaceutical composition comprising an antitumor effective amount of a population of T cells, wherein the T cells comprise a nucleic acid sequence that encodes a chimeric antigen receptor (CAR), wherein the CAR comprises the amino acid sequence of SEQ ID NOs. 2, 4, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, and 87, or any combination thereof, wherein the T cells are T cells of the subject having cancer.
  • CAR chimeric antigen receptor
  • the at least one transmembrane domain comprises a transmembrane the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD19, CD22, Mesothelin, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF16, TNFRSF19, or a combination thereof
  • a method for generating a persisting population of genetically engineered T cells in a human diagnosed with cancer.
  • the method comprises administering to a human a T cell genetically engineered to express a CAR wherein the CAR comprises the amino acid sequence of SEQ ID NOs.
  • the persisting population of genetically engineered T cells, or the population of progeny of the T cells persists in the human for at least one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, two years, or three years after administration.
  • the progeny T cells in the human comprise a memory T cell.
  • the T cell is an autologous T cell.
  • any of the aforementioned cancers, diseases, disorders or conditions associated with an elevated expression of a tumor antigen may be treated or prevented or ameliorated using one or more of the CARs disclosed herein.
  • kits for making a chimeric antigen receptor T-cell as described supra or for preventing, treating, or ameliorating any of the cancers, diseases, disorders or conditions associated with an elevated expression of a tumor antigen in a subject as described supra, comprising a container comprising any one of the nucleic acid molecules, vectors, host cells, or compositions disclosed supra or any combination thereof, and instructions for using the kit.
  • FIGURES 1A and IB depict the construction of a tandem CARs targeting CD22 and CD19.
  • FIGURE 1A The anti-CD22 and anti-CD19 dual targeting CAR construct (CAR22-19) was generated by linking single chain fragment variable sequence targeting the CD22 antigen membrane distal domain to a single chain fragment variable sequence targeting the CD 19 antigen (membrane proximal) via a flexible glycine-serine linker. The resulting CD22-CD19 dual targeting domain was then connected in frame to CD8 hinge and transmembrane domain, the 4- 1BB (CD137) signaling domain and the CD3 zeta signaling domain.
  • CAR22-19 The anti-CD22 and anti-CD19 dual targeting CAR construct was generated by linking single chain fragment variable sequence targeting the CD22 antigen membrane distal domain to a single chain fragment variable sequence targeting the CD 19 antigen (membrane proximal) via a flexible glycine-serine linker.
  • the resulting CD22-CD19 dual targeting domain was
  • FIGURE IB Tandem targeting constructs CAR19-22 was generated in a similar manner, except that the single chain fragment variable regions of CD 19 (distal to cell membrane) was linked via glycine serine flexible linker to a single chain variable region of CD22 targeting sequence (membrane proximal), followed by CD8, 4- IBB and CD3 zeta domains.
  • Each CAR T construct is capable of activation via binding to either CD 19 or CD22 tumor antigens, or both.
  • FIGURE 2 depicts surface expression of tandem-CAR T constructs LTG 2681 (CAR22- 19) and LTG2791 (C ARI 9-22) in human primary T cells.
  • CAR T expression was determined by flow cytometry. T cells were activated with Miltenyi Biotec TransActTM CD3 CD28 reagent in the presence of IL-2, and transduced with LV as described in Materials and Methods. On culture day 8, viable transduced T cells (7-AAD negative) were assayed for CAR surface expression using one of three staining methods: CD19 Fc followed by anti-Fc-AF647 (top panel), or CD22-his reagent followed by anti-his-PE staining (bottom panel). The LV used in transduction is listed on the top of each column. Percentage of CAR T-positive populations in relation to non-transduced T cell control (UTD) is noted above each histogram. Representative data of three separate donors is shown.
  • FIGURE 3 depicts CAR T cytotoxicity in vitro. Luciferase-based cytotoxicity assays were performed using, Raji CD19+CD22+, REH CD19+CD22+, or CD 19- CD22- cell line 293 T , stably transduced with luciferase. A comparison between CAR 22-19 (LTG2681) and CAR 19-22 (LTG2719), which differ only in the order of antigen targeting domains. Comparator singletargeting CAR19 (pLTG1538) and CAR22 (pLTG2200), and negative control untransduced T cells were included. CAR T cells and target tumor cells were co-incubated overnight at the listed effector to target (E:T) ratios, x-axis. Error bars represent mean values from three technical replicates. One experiment representing three separate experiments in T cells from three donors, is shown.
  • E:T effector to target
  • FIGURES 4A and 4B depict CAR T cytotoxicity in vitro against tumor lines A431 or 293 T transduced to over express one target antigen only, in order to confirm the specificity of each binder domain of the CD22 CD 19 -targeting CAR T cells.
  • Luciferase-based cytotoxicity assays were performed using, 293T cells, 293T CD19+, 293T CD20+, or 293T CD22+ cell lines (FIGURE 4 A), or A431, A431 CD19+, A431 CD22+, A431 CD20+ cell lines stably transduced with luciferase (FIGURE 4B).
  • Data points represent mean values from triplicate determination from one experiment, representing three independent experiments performed with CAR T cells from three separate donors.
  • FIGURE 5 CAR T cytokine release in response to leukemia cell lines. Cytokine production by CAR-T, listed on the x-axis, upon overnight co-culture with the Raji leukemia line at an E:T ratio of 10: 1, was measured using ELISA. Bars represent mean +SD of three replicate samples. Data are representative of three independent experiments performed with CAR T cells from three separate donors.
  • FIGURES 6A-D depict a schematic representation of the various anti-CD22-19 CAR designs with different co-stimulatory domains.
  • a second-generation CAR FIGURE 6A
  • third generation CAR FIGGURE 6B
  • bicistronic CARs combining one second generation CAR chain targeting the CD 19 antigen and another first generation CAR chain targeting the CD22 antigen (FIGURE 6C), or two second generation CAR chains (FIGURE 6D) co-expressed in the same cell are shown.
  • FIGURE 7 depicts the expression of anti-CD22-19 CAR with different transmembrane and co-stimulatory domains as detected by flow cytometry.
  • CAR T cells are stained for the expression of CD 19 scFv and CD22 scFv simultaneously. Construct number and transmembrane and costimulatory domain configuration are noted above each flow diagram. Data are representative of three transduction experiments in T cells form different healthy donors.
  • E effector to target
  • FIGURES 10A-I depict the in vivo functionality of CAR22 19 constructs with Raji animal model.
  • Figure 10A NSG mice were engrafted with Raji-luc tumor cells at 5xl0 5 Raji/mouse. On day 7, 5 x 10 6 CAR T cells per mouse were injected. Tumor burden measured by bioluminescent imaging at indicated time point was shown.
  • Figure 10B Graphic summary of tumor growth kinetics of mice treated with CARs with TNFRSF costimulatory domain.
  • FigurelOD Total Car+ T cell and Car+ CD8 T cell in peripheral blood were measured at day 14, and showed in scatter dot plot. Lines indicated mean of each group.
  • Figure 10E Total Car+ T cell and Car+ CD8 T cell in peripheral blood were measured at day 21, and showed in scatter dot plot. Lines indicated mean of each group.
  • FigurelOF Total Car+ T cell and Car+ CD8 T cell in peripheral blood were measured at day 28, and showed in scatter dot plot. Lines indicated mean of each group.
  • FigurelOG Total Car+ T cell and Car+ CD8 T cell in peripheral blood were measured at day 14, and showed in scatter dot plot. Lines indicated mean of each group.
  • FigurelOH Total Car+ T cell and Car+ CD8 T cell in peripheral blood were measured at day 21, and showed in scatter dot plot. Lines indicated mean of each group.
  • Figure 101 Total Car+ T cell and Car+ CD8 T cell in peripheral blood were measured at day 28, and showed in scatter dot plot. Lines indicated mean of each group.
  • One way ANOVA with Dunnett’s post hoc test was performed to compare with UTD group at each time point. * : P ⁇ 0.05; *** P ⁇ 0.001; **** P ⁇ 0.0001.
  • FIGURES 11A-E depict the characterization of selected CAR22 19 construct in heterogeneous Raji model.
  • Bioluminescence data was log- transformed prior to Two-Way ANOVA test, followed by Tukey’s multiple comparisons.
  • Figure 11C Peripheral blood was harvested on day 29. Genomic DNA was extracted from 1-2 million cells of bone marrow or spleen, or 100 pl whole blood. Fifty ng DNA per sample was used in a real time quantitative PCR to analyze CAR T persistence.
  • Figure 11D Bone marrow and spleens was harvested on day 29. Genomic DNA was extracted from 1-2 million cells of bone marrow or spleen, or 100 pl whole blood. Fifty ng DNA per sample was used in a real time quantitative PCR to analyze CAR T persistence.
  • Figure 1 IE Spleens were harvested on day 29.
  • Genomic DNA was extracted from 1-2 million cells of bone marrow or spleen, or 100 pl whole blood. Fifty ng DNA per sample was used in a real time quantitative PCR to analyze CAR T persistence. N 5-6/group, mean ⁇ SD. Statistical analysis was performed with one way ANOVA followed with Tukey’s multiple comparison. * : P ⁇ 0.05; ** P ⁇ 0.01; *** P ⁇ 0.001; ****
  • FIGURES 12A-P depict the CAR22-19 CAR variants against Raji clones with high and low CD22 surface density in an overnight cytotoxicity assay.
  • Figure 12A Overlaid flow histogram of all the Raji cell lines with different CD22 density was shown. The surface antigen density for CD22high and CD22 low Raji cell line is listed on the top of histogram.
  • Figures 12B-12P CAR22-19s, CAR22, or UTD control T cells were co-cultured with target cells overnight at E:T ratios 20: 1, 10: 1 and 5: 1. Percentage of specific target lysis of each CAR construct was presented from panel B to P. Each data point represents mean ⁇ SEM of CAR T cells generated from different donors in separate experiments.
  • FIGURES 13A-P depict the sensitivity of CAR22-19 variants to Raji clones with CD19 high and low density lines.
  • Figurel3A The flow overlay histogram showed surface CD19 expression level of each cell line, the calculated surface target molecule number is listed above the panel.
  • Figures 13B-13P, CAR22-19s, CAR19, or UTD control T cells were co-cultured with target cells overnight at E:T ratios 20:1, 10: 1 and 5: 1. Percentage of specific target lysis of each construct was presented individually. Solid line represented CD 19 high cell line and dotted line stood for CD19 low cell line.
  • an antigen includes single or plural antigens and can be considered equivalent to the phrase “at least one antigen.”
  • the term “comprises” means “includes.”
  • “comprising an antigen” means “including an antigen” without excluding other elements.
  • the phrase “and/or” means “and” or “or.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated.
  • the present disclosure provides for CD19/CD22 antibodies or fragments thereof as well as chimeric antigen receptors (CARs) having such CD19/CD22 antigen binding domains.
  • the enhancement of the functional activity of the CAR directly relates to the enhancement of functional activity of the CAR-expressing T cell.
  • the CARs exhibit both a high degree of cytokine-induced cytolysis and cell surface expression on transduced T cells, along with an increased level of in vivo T cell expansion and persistence of the transduced CAR-expressing T cell.
  • the CARs of the present disclosure are advantageous in that one CART lentiviral product may be utilized to treat multiple patient populations (i.e. CD19+, CD22+ or double CD19+CD22+ cancer patients), which allows flexibility in circumstances where resources are limited.
  • CARs Chimeric Antigen Receptors
  • the choice of each of these protein domains is a key design feature, as is the way in which they are specifically combined.
  • Each design domain is an essential component that can be used across different CAR platforms to engineer the function of lymphocytes. For example, the choice of the extracellular binding domain can make an otherwise ineffective CAR be effective.
  • the invariable framework components of the immunoglobulin-derived protein sequences used to create the extracellular antigen binding domain of a CAR can either be entirely neutral, or they can self-associate and drive the T cell to a state of metabolic exhaustion, thus making the therapeutic T cell expressing that CAR far less effective. This occurs independently of the antigen binding function of this CAR domain. Furthermore, the choice of the intracellular signaling domain(s) also can govern the activity and the durability of the therapeutic lymphocyte population used for immunotherapy.
  • the CARs disclosed herein are expressed at a high level in a cell.
  • a cell expressing the CAR has a high in vivo proliferation rate, produces large amounts of cytokines, and has a high cytotoxic activity against a cell having, on its surface, a CD19/CD22 antigen to which a CAR binds.
  • the use of an extracellular CD19/CD22 antigen binding domain results in generation of a CAR that functions better in vivo, while avoiding the induction of anti-CAR immunity in the host immune response and the killing of the CAR T cell population.
  • the CARs expressing the extracellular CD19/CD22 ScFv antigen binding domain exhibit superior activities/properties including i) prevention of poor CAR T persistence and function as seen with mouse-derived binding sequences; ii) lack of regional (i.e. intrapleural) delivery of the CAR to be efficacious; and iii) ability to generate CAR T cell designs based both on binders with high and low affinity to CD19/CD22.
  • inventive CARs including a description of their extracellular CD19/CD22 antigen binding domain, the transmembrane domain and the intracellular domain, along with additional description of the CARs, antibodies and antigen binding fragments thereof, conjugates, nucleotides, expression, vectors, and host cells, methods of treatment, compositions, and kits employing the disclosed CARs.
  • the CARs disclosed herein comprise at least one CD19/CD22 antigen binding domain capable of binding to CD19/CD22, at least one transmembrane domain, and at least one intracellular domain.
  • a chimeric antigen receptor is an artificially constructed hybrid protein or polypeptide containing the antigen binding domains of an antibody (e.g., single chain variable fragment (ScFv)) linked to T-cell signaling domains via the transmembrane domain.
  • Characteristics of CARs include their ability to redirect T-cell specificity and reactivity toward a selected target in a non-MHC -restricted manner, and exploiting the antigen-binding properties of monoclonal antibodies.
  • the non-MHC -restricted antigen recognition gives T cells expressing CARs the ability to recognize antigen independent of antigen processing, thus bypassing a major mechanism of tumor escape.
  • CARs advantageously do not dimerize with endogenous T cell receptor (TCR) alpha and beta chains.
  • the intracellular T cell signaling domains of the CARs can include, for example, a T cell receptor signaling domain, a T cell costimulatory signaling domain, or both.
  • the T cell receptor signaling domain refers to a portion of the CAR comprising the intracellular domain of a T cell receptor, such as, for example, and not by way of limitation, the intracellular portion of the CD3 zeta protein.
  • the costimulatory signaling domain refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule, which is a cell surface molecule other than an antigen receptor or their ligands that are required for an efficient response of lymphocytes to antigen.
  • the CAR comprises a target-specific binding element otherwise referred to as an antigen binding domain or moiety.
  • the choice of domain depends upon the type and number of ligands that define the surface of a target cell.
  • the antigen binding domain may be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state.
  • cell surface markers that may act as ligands for the antigen binding domain in the CAR include those associated with viral, bacterial and parasitic infections, autoimmune disease and cancer cells.
  • the CAR can be engineered to target a tumor antigen of interest by way of engineering a desired antigen binding domain that specifically binds to an antigen on a tumor cell.
  • Tumor antigens are proteins that are produced by tumor cells that elicit an immune response, particularly T-cell mediated immune responses. The selection of the antigen binding domain will depend on the particular type of cancer to be treated. Tumor antigens include, for example, a glioma-associated antigen, carcinoembryonic antigen (CEA), .beta.
  • telomere reverse transcriptase RU1, RU2 (AS)
  • intestinal carboxyl esterase mut hsp70- 2, M-CSF
  • PSA prostate-specific antigen
  • PAP PAP
  • NY-ESO-1 LAGE-la
  • p53 prostein
  • PSMA Her2/neu
  • survivin and telomerase prostate-carcinoma tumor antigen-1 (PCTA-1)
  • MAGE ELF2M
  • neutrophil elastase neutrophil elastase
  • ephrinB2 CD22
  • insulin growth factor (IGF)-I IGF-II
  • IGF- I receptor IGF- I receptor
  • the tumor antigen comprises one or more antigenic cancer epitopes associated with a malignant tumor.
  • Malignant tumors express a number of proteins that can serve as target antigens for an immune attack. These molecules include, but are not limited to, tissuespecific antigens such as MART-1, tyrosinase and GP 100 in melanoma and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer.
  • Other target molecules belong to the group of transformation-related molecules such as the oncogene HER-2/Neu/ErbB- 2.
  • Yet another group of target antigens are onco-fetal antigens such as carcinoembryonic antigen (CEA).
  • B-cell lymphoma the tumor-specific idiotype immunoglobulin constitutes a truly tumor-specific immunoglobulin antigen that is unique to the individual tumor.
  • B-cell differentiation antigens such as CD 19, CD22, CD22, BCMA, ROR1, and CD37 are other candidates for target antigens in B-cell lymphoma.
  • Some of these antigens (CEA, HER-2, CD 19, CD22, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies with limited success.
  • the tumor antigens are CD19/CD22 and the tumors associated with expression of CD19/CD22 comprise lung mesothelioma, ovarian, and pancreatic cancers that express high levels of the extracellular proteins CD19/CD22, or any combination thereof.
  • the type of tumor antigen may also be a tumor-specific antigen (TSA) or a tumor- associated antigen (TAA).
  • TSA tumor-specific antigen
  • TAA tumor- associated antigen
  • a TSA is unique to tumor cells and does not occur on other cells in the body.
  • a TAA is not unique to a tumor cell and instead is also expressed on a normal cell under conditions that fail to induce a state of immunologic tolerance to the antigen.
  • the expression of the antigen on the tumor may occur under conditions that enable the immune system to respond to the antigen.
  • TAAs may be antigens that are expressed on normal cells during fetal development when the immune system is immature and unable to respond or they may be antigens that are normally present at extremely low
  • TSAs or TAAs include the following: Differentiation antigens such as MART- 1 /Mel an A (MART -I), gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumorspecific multi-lineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl 5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumorsuppressor genes such as p53, Ras, HER-2/neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7.
  • Differentiation antigens such as MART- 1 /Mel an
  • the antigen binding domain portion of the CAR targets an antigen that includes but is not limited to CD19, CD20, CD22, ROR1, CD33, CD38, CD123, CD138, BCMA, c-Met, PSMA, Glycolipid F77, EGFRvIII, GD-2, FGFR4, TSLPR, NY-ESO- 1 TCR, MAGE A3 TCR, and the like.
  • the antigen binding domain portion of the CAR targets the extracellular CD19/CD22 antigen.
  • the general scheme is set forth in FIGURES 1A and IB and includes, from the N-terminus to the C- terminus, a signal or leader peptide, anti-CD19/CD22 ScFv (where the CD 19 binder is distal to the T cell membrane and the CD22 binder is proximal to the T cell membrane, or where the CD22 binder is distal to the T cell membrane and the CD 19 binder is proximal to the T cell membrane), CD8 extracellular linker, CD8 transmembrane domain, 4- IBB costimulatory domain, CD3 zeta activation domain.
  • the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 1 (Leader-CD22 VH-(GGGGS)-3 CD22 VL (GGGGS)-5 CD 19 VH (GGGGS)-3 CD19 VL CD8 hinge+TM-4-lBB- CD3z (Construct 2219)), and encodes the CAR comprising the amino acid sequence as set forth in SEQ ID NO: 2 (Leader-CD22 VH-(GGGGS)-3 CD22 VL (GGGGS)-5 CD19 VH (GGGGS)-3 CD19 VL CD8 hinge+TM-4-lBB- CD3z (Construct 2219).
  • the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 1, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes the CAR comprising the amino acid sequence as set forth in SEQ ID NO: 2 or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof Leader-CD22 VH- (GGGGS)-3 CD22 VL (GGGGS)-5 CD19 VH (GGGGS)-3 CD19 VL CD8 hinge+TM-4-lBB- CD3z (Construct 2219).
  • the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 3 (Leader-CD19 VH (GGGGS)3 - CD19 VL -(GGGGS)5 -CD22 VL-(GGGGS)3 - CD22 VH CD8 hinge+TM-4-lBB- CD3z (Construct 1922) (FIGURE 2)), and encodes the CAR comprising the amino acid sequence as set forth in SEQ ID NO: 4 [Leader- CD19 VH (GGGGS)3 - CD 19 VL -(GGGGS)5 -CD22 VL-(GGGGS)3 - CD22 VH CD8 hinge+TM-4-lBB- CD3z (Construct 1922)].
  • the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 3 or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes the CAR comprising the amino acid sequence as set forth in SEQ ID NO: 4 or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof (Leader- CD19 VH (GGGGS)3 - CD 19 VL -(GGGGS)5 -CD22 VL-(GGGGS)3 - CD22 VH CD8 hinge+TM-4-lBB- CD3z (Construct 1922)).
  • Example 2 The surface expression of anti-CD19/CD22 CARs incorporating single chain fragment variable (ScFv) sequences reactive with CD19/CD22 antigen, is shown in Example 2 infra.
  • the expression level for each ScFv-containing CAR was determined by flow cytometric analysis of LV-transduced T cells from healthy donors using one of two detection methods: i) CD22 his, followed anti-his-FL; ii) CD19 Fc recombinant protein, followed by anti Fc-FL.
  • the ScFv-based anti-CD19/CD22 CAR constructs CAR22-19 and CAR19-22 were highly expressed in human primary T cells as compared to non-transduced T cell controls.
  • FIGURE 3 Human primary T cells were transduced with LV encoding CAR constructs (CAR 22-19 (LTG2681, D0023), CAR 19-22 (D0024), CAR19 (LTG1538), or CAR22 (LTG2200); see Methods), then incubated for 18 hours with the Raji, REH, K562 or 293T cell lines, stably transduced with firefly luciferase, for luminescence based in vitro killing assays. Raji and Reh leukemia lines express CD 19 and CD22 on their surface, while the negative controls, 293 T do not.
  • 293T-luc lines and A431-luc lines were created that express CD19, or CD20, or CD22 (FIGURES 4A and 4B).
  • 293T-19+ were lysed by the CAR 19 (LTG1538) and tandem CAR 22-19 (LTG 2681), but not by CAR22 LTG 2200 or untransduced T cells control (FIGURE 3).
  • 293T-CD22+ were lysed by the tandem CAR 22-19 (LTG 2681) and the single CAR20 LTG2200, but not by the single CAR19 (LTG 1538), or untransduced control, demonstrating antigen specificity of the tandem CAR.
  • tandem CAR 22-19 will be effective against tumor cells even if one of the two antigens (CD 19 CD22) was downregulated and is no longer expressed. Therefore tandem CARs, in contract to single CARs, can mitigate tumor antigen escape.
  • the capacity of anti-CD19/CD22 CAR T cells for cytokine secretion was then evaluated (FIGURE 5).
  • the CD19+CD22+ Raji tumor cells were co-incubated with the tandem 22-19 CAR T cells (LTG2681) or positive control CAR19 (LTG1538), positive control CAR22 (LTG2200), or negative control untransduced T cells (UTD) at effector to target ratio of 10: 1 overnight, and culture supernatants were analyzed by ELISA for IFN gamma, TNF alpha, and IL-2.
  • tandem CAR 22-19 strongly induced cytokines in response to tumor cells, whereas the negative control (untransduced, UTD.) yielded no appreciable cytokine induction.
  • tandem CAR T-expressing cells LTG2681 showed similar levels of IFN gamma, TNF alpha, IL-2, to CAR22 control (LTG2200), and somewhat higher cytokine response than the single CAR19 (LTG1538) control, demonstrating the high potency of the tandem CAR.
  • CAR 22-19 produced no cytokine secretion in the absence of tumor cells (CART alone group), which further confirms CAR specificity, and indicates a lack of tonic signaling by the tandem car.
  • the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 60 [CD22-19 CD8 BBz (Construct LTG 2737) (FIGURES 6, 7, 8, and 9, respectively)], and encodes the CAR comprising the amino acid sequence as set forth in SEQ ID NO: 61 [CD22-19 CD8 BBz (Construct LTG2737)].
  • the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 60 or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes the CAR comprising the amino acid sequence as set forth in SEQ ID NO: 61 or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof (CD22-19 CD8 BBz (Construct LTG2737)).
  • the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 64 [CD22-19 CD8 ICOSz DNA (Construct D0136) (FIGURES 6, 7, 8, and 9, respectively)), and encodes the CAR comprising the amino acid sequence as set forth in SEQ ID NO: 65 [CD22-19 CD8 ICOSz DNA (Construct D0136)].
  • the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 64 or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes the CAR comprising the amino acid sequence as set forth in SEQ ID NO: 65 or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof (CD22-19 CD8 ICOSz DNA (Construct D0136)).
  • the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 70 [CD22-19 CD28 CD28z (Construct DO 139) ( FIGURES 6, 7, 8, and 9, respectively)], and encodes the CAR comprising the amino acid sequence as set forth in SEQ ID NO: 71 [CD22-19 CD28 CD28z (Construct DO 139)].
  • the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 70 or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes the CAR comprising the amino acid sequence as set forth in SEQ ID NO: 71 or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof (CD22-19 CD28 CD28z (Construct DO 139)).
  • the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 76 [CD19 CD8H&TM ICOS z_CD22 CD8H&TM 3z (Construct D0146) (FIGURES 6, 7, 8, and 9, respectively)], and encodes the CAR comprising the amino acid sequence as set forth in SEQ ID NO: 77 [CD19 CD8H&TM ICOS z_CD22 CD8H&TM 3z (Construct DO 146)] .
  • the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 76 or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes the CAR comprising the amino acid sequence as set forth in SEQ ID NO: 77 or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof (CD19 CD8H&TM ICOS z_CD22 CD8H&TM 3z (Construct D0146)).
  • the nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO: 62, 66, 68, 72, 74, 78, 80, 82, 84, and 86 or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes the CAR comprising the amino acid sequence as set forth in SEQ ID NO: 63, 67, 69, 73, 75, 79, 81, 83, 85, and 87 or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof.
  • dual-targeting CAR constructs comprised of different co-stimulatory domains were designed.
  • CAR constructs are listed in Table 1 of Example 3, infra. Schematic representations of CAR design configurations are provided in FIGURES 6A-C.
  • the tandem CAR antigen-binding domain comprised of anti-CD22 ScFv and anti- CD19 scFv sequences, were linked in tandem, in the following order: anti-CD22 scFv-anti CD 19- scFv-hinge -transmembrane domain - endodomain (FIGURES 6A, 6B).
  • the targeting tandem domain configuration was based on CAR 22-19 (LTG2681) in all cases.
  • Anti-CD 22-19 CAR construct LTG2737 contained the CAR sequence identical to the Anti-CD 22-19 CAR construct LTG2681, but without use of the Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE) during its construction.
  • WP Woodchuck Hepatitis Virus
  • WPRE Posttranscriptional Regulatory Element
  • CAR T constructs were generated by linking the CAR antigen-binding domain in frame to CD8a hinge and transmembrane domains (constructs LTG2737, D0135, D0136, D0137, D0145, D0146, D0147, D0148, and D0149) as described in Example 3, infra.
  • transmembrane domain sequence matching the co- stimulatory domain was utilized: CD28 for D139, D140, 0X40 for D0137, D0147, D0148.
  • the transmembrane domain was linked in frame to a co-stimulatory domain derived from 4- IBB (LTG2737), CD28 (D0135, D0139, D0140), ICOS (D0136, D0146, D0148, D0149), 0X40 (D0137, D0145, D0147, D0148) or CD27 (D0138, D0149). All CAR molecules contained the CD3 zeta signaling domain (CD247, aa 52-163, Ref sequence ID: NP_000725.1).
  • the endodomain of the CAR was comprised of two costimulatory domains, CD28 and 4-1BB, connected in tandem (D0140, FIGURE 6B).
  • two distinct CAR molecules were co-expressed in the same T cell using a 2A ribosomal skip element for bicistronic expression (D146, D147, D148, D149, FIGURE 6C, 6D).
  • each CAR chain contained only one scFv, targeting either the CD 19 or CD22 antigen, and both chains were co-expressed in each transduced T cell via transduction with a single lentiviral vector encoding the bicistronic sequence.
  • no co-stimulatory domain was used, so that the CD3 ⁇ activation domain was linked in frame directly to the transmembrane domain of one of the CAR chains expressed concurrently in the same cell (DO 146, DO 147, FIGURE 6C).
  • CAR constructs sequences were cloned into a third generation lentiviral plasmid backbone under the control of the human EF-la promoter (Lentigen Technology Inc., Gaithersburg, MD).
  • FIGURE 7 The surface expression of anti-CD22-19 CAR incorporating various co-stimulatory domains, is shown in FIGURE 7.
  • the expression level for each ScFv-containing CAR was determined by flow cytometric analysis of LV-transduced T cells from healthy donors using simultaneous staining for the two scFv CAR targeting domains i) CD22-his, followed anti-his-PE; ii) CD19 Fc recombinant protein, followed by anti Fc-A647. All anti-CD22-19 CAR were highly expressed in human primary T cells as compared to non-transduced T cell controls. CAR expression levels ranged from 71%-90% (FIGURE 7).
  • FIGURES 9A-9D high cytolytic activity of the anti-CD22-19 CAR was demonstrated: Human primary T cells were transduced with LV encoding CAR constructs LTG2737, D0135, D0136, D0137, D0138, D0139, D0140, D0145, D0146), then incubated for 18 hours with the Raji, 293T, 293TCD19 or 293TCD22 cell lines, stably transduced with firefly luciferase, for luminescence based in vitro killing assays. Effector to target (ET) ratios of 2.5:1, 5: 1 or 10: 1 were used, as noted in the legend to the right of each plot.
  • E effector to target
  • Raji cells express CD19 and CD22 on their surface, while the negative controls, 293T do not.
  • the 293TCD19 and 293TCD22 target lines were generated to stably express either the CD 19 or CD22 target antigen, respectively, and were used to evaluate the capability of the dual -targeting CAR constructs with different costimulatory domains to accomplish target lysis when triggered by each single target antigen, CD 19 or CD22, independently of the other antigen.
  • the capacity of anti-CD22-19 CAR with various co- stimulatory domains for cytokine secretion was then evaluated (FIGURE 8).
  • the CD19+CD22+ Raji tumor cells were coincubated with the tandem anti-CD22-19 CAR T cells expressing constructs LTG2737, D0135, D0136, D0137, D0138, D0139, D0140, D0145, D0146, or negative control untransduced T cells (UTD) at effector to target ratio of 10: 1 overnight, and culture supernatants were analyzed by ELISA for IFN gamma, TNF alpha, and IL-2 (FIGURE 8).
  • anti-CD22-19 CAR produced little to no cytokine secretion in the absence of tumor cells (CAR T alone group), which further confirms CAR specificity, and indicates a lack of tonic signaling by the tandem anti-CD22-19 CAR with various co-stimulatory domains.
  • the enhanced therapeutic function associated with the exemplary tandem CD22 and CD 19 targeting CARs of the invention include, for example, and not by way of limitation, a) improved lateral movement within the plasma membrane allowing for more efficient signal transduction, b) superior location within plasma membrane microdomains, such as lipid rafts, and greater ability to interact with transmembrane signaling cascades associated with T cell activation, c) superior location within the plasma membrane by preferential movement away from dampening or down-modulatory interactions, such as less proximity to or interaction with phosphatases such as CD45, and d) superior assembly into T cell receptor signaling complexes (i.e. the immune synapse), or e) superior ability to engage with tumor antigen due to two distinct targeting domains present in each CAR molecule, or any combination thereof.
  • CD19/CD22 variable heavy chain only and ScFv antigen binding domains While the disclosure has been illustrated with an exemplary extracellular CD19/CD22 variable heavy chain only and ScFv antigen binding domains, other nucleotide and/or amino acid variants within the CD19/CD22 variable heavy chain only and ScFv antigen binding domains may be used to derive the CD19/CD22 antigen binding domains for use in the CARs described herein.
  • the CAR can be additionally engineered to include the appropriate antigen binding domain that is specific to the desired antigen target.
  • an antibody for CD19/CD22 can be used as the antigen bind domain incorporation into the CAR.
  • the antigen binding domain portion of the CAR additionally targets CD33.
  • the antigen binding domain in the CAR is anti-CD33 ScFv, wherein the nucleic acid sequence of the anti-CD33 ScFv comprises the sequence set forth in SEQ ID NO: 46.
  • the anti-CD33 ScFv comprises the nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 46.
  • the anti- CD19/CD22 ScFv portion of the CAR comprises the amino acid sequence set forth in SEQ ID NO: 47.
  • the antigen binding domain portion of the CAR additionally targets mesothelin.
  • the antigen binding domain in the CAR is anti- mesothelin ScFv, wherein the nucleic acid sequence of the anti-mesothelin ScFv comprises the sequence set forth in SEQ ID NO: 48.
  • the anti-mesothelin ScFv comprises the nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 48.
  • the anti-mesothelin ScFv portion of the CAR comprises the amino acid sequence set forth in SEQ ID NO: 49.
  • a CAR capable of binding to a non-TSA or non-TAA including, for example and not by way of limitation, an antigen derived from Retroviridae (e.g. human immunodeficiency viruses such as HIV-1 and HIV-LP), Picornaviridae (e.g.
  • poliovirus hepatitis A virus, enterovirus, human coxsackievirus, rhinovirus, and echovirus
  • rubella virus coronavirus
  • vesicular stomatitis virus rabies virus
  • ebola virus parainfluenza virus
  • mumps virus measles virus
  • respiratory syncytial virus influenza virus
  • hepatitis B virus parvovirus
  • Adenoviridae Herpesviridae [e.g. type 1 and type 2 herpes simplex virus (HSV), varicella-zoster virus, cytomegalovirus (CMV), and herpes virus]
  • Herpesviridae e.g. type 1 and type 2 herpes simplex virus (HSV), varicella-zoster virus, cytomegalovirus (CMV), and herpes virus
  • Herpesviridae e.g. type 1 and type 2 herpes simplex virus (HSV), varicella-zoster virus
  • a CAR capable of binding to an antigen derived from a bacterial strain of Staphylococci, Streptococcus, Escherichia coli, Pseudomonas, or Salmonella.
  • a CAR capable of binding to an antigen derived from an infectious bacterium, for example, Helicobacter pyloris, Legionella pneumophilia, a bacterial strain of Mycobacteria sps. (e.g. M. tuberculosis, M. avium, M. intracellulare, M. kansaii, or M.
  • the CAR comprises one or more transmembrane domains fused to the extracellular CD19/CD22 antigen binding domain of the CAR.
  • the transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.
  • Transmembrane regions of particular use in the CARs described herein may be derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T- cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF16, or TNFRSF19.
  • the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine.
  • a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.
  • a short oligo- or polypeptide linker preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR.
  • a glycine-serine doublet provides a particularly suitable linker.
  • the transmembrane domain that naturally is associated with one of the domains in the CAR is used in addition to the transmembrane domains described supra.
  • the transmembrane domain can be selected or 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 members of the receptor complex.
  • the transmembrane domain in the CAR of the invention is the CD8 transmembrane domain.
  • the CD8 transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 35.
  • the CD8 transmembrane domain comprises the nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 36.
  • the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 36.
  • the encoded transmembrane domain comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) of an amino acid sequence of SEQ ID NO: 36, or a sequence with 95-99% identity to an amino acid sequence of SEQ ID NO: 36.
  • the transmembrane domain of the CAR comprises the CD8.alpha.hinge domain.
  • the CD8 hinge domain comprises the nucleic acid sequence of SEQ ID NO: 37.
  • the CD8 hinge domain comprises the nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 38.
  • the CD8 hinge domain comprises the amino acid sequence of SEQ ID NO: 38, or a sequence with 95-99% identify thereof.
  • an isolated nucleic acid molecule wherein the encoded linker domain is derived from the extracellular domain of CD8, and is linked to the transmembrane CD8 domain, the transmembrane CD28 domain, or a combination thereof.
  • a spacer domain can be arranged between the extracellular domain and the transmembrane domain, or between the intracellular domain and the transmembrane domain.
  • the spacer domain means any oligopeptide or polypeptide that serves to link the transmembrane domain with the extracellular domain and/or the transmembrane domain with the intracellular domain.
  • the spacer domain comprises up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids.
  • the linker can include a spacer element, which, when present, increases the size of the linker such that the distance between the effector molecule or the detectable marker and the antibody or antigen binding fragment is increased.
  • spacers are known to the person of ordinary skill, and include those listed in U.S. Pat. Nos. 7,964,566, 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149,
  • the spacer domain preferably has a sequence that promotes binding of a CAR with an antigen and enhances signaling into a cell.
  • an amino acid that is expected to promote the binding include cysteine, a charged amino acid, and serine and threonine in a potential glycosylation site, and these amino acids can be used as an amino acid constituting the spacer domain.
  • the spacer domain As the spacer domain, the entire or a part of amino acid numbers 137-206 (SEQ ID NO: 39) which is a hinge region of CD8. alpha. (NCBI RefSeq: NP. sub. —001759.3), amino acid numbers 135 to 195 of CD8.beta. (GenBank: AAA35664.1), amino acid numbers 315 to 396 of CD4 (NCBI RefSeq: NP.sub. -000607.1), or amino acid numbers 137 to 152 of CD28 (NCBI RefSeq: NP.sub.— 006130.1) can be used. Also, as the spacer domain, a part of a constant region of an antibody H chain or L chain can be used. Further, the spacer domain may be an artificially synthesized sequence.
  • a signal peptide sequence can be linked to the N-terminus.
  • the signal peptide sequence exists at the N-terminus of many secretory proteins and membrane proteins, and has a length of 15 to 30 amino acids. Since many of the protein molecules mentioned above as the intracellular domain have signal peptide sequences, the signal peptides can be used as a signal peptide for the CAR.
  • the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 12.
  • Intracellular Domain The cytoplasmic domain or otherwise the intracellular signaling domain of the CAR is responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been placed in.
  • effector function refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines.
  • intracellular signaling domain refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain.
  • intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.
  • intracellular signaling domains for use in the CAR include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.
  • TCR T cell receptor
  • T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequence: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic signaling sequences).
  • primary cytoplasmic signaling sequences those that initiate antigen-dependent primary activation through the TCR
  • secondary cytoplasmic signaling sequences those that act in an antigen-independent manner to provide a secondary or costimulatory signal
  • Primary cytoplasmic signaling sequences regulate primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way.
  • Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or IT AMs.
  • ITAM containing primary cytoplasmic signaling sequences that are of particular use in the CARs disclosed herein include those derived from TCR zeta (CD3 Zeta), FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and
  • CD66d Specific, non-limiting examples, of the ITAM include peptides having sequences of amino acid numbers 51 to 164 of CD3 zeta, (NCBI RefSeq: NP.sub. -932170.1), amino acid numbers 45 to 86 of Fc epsilon RI gamma, (NCBI RefSeq: NP.sub.— 004097.1), amino acid numbers 201 to 244 of Fc epsilon RI beta. (NCBI RefSeq: NP.sub.— 000130.1), amino acid numbers 139 to 182 of CD3 gamma. (NCBI RefSeq: NP. sub.
  • NCBI RefSeq amino acid numbers 128 to 171 of CD3 delta.
  • NCBI RefSeq amino acid numbers 153 to 207 of CD3.
  • epsilon. NCBI RefSeq: NP. sub. —000724.1
  • amino acid numbers 402 to 495 of CD5 NCBI RefSeq: NP.sub. -055022.2
  • amino acid numbers 707 to 847 of 0022 NCBI RefSeq: NP. sub.
  • the cytoplasmic signaling molecule in the CAR comprises a cytoplasmic signaling sequence derived from CD3 zeta.
  • the intracellular domain of the CAR can be designed to comprise the CD3-zeta signaling domain by itself or combined with any other desired cytoplasmic domain(s) useful in the context of the CAR.
  • the intracellular domain of the CAR can comprise a CD3 zeta chain portion and a costimulatory signaling region.
  • the costimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule.
  • a costimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient response of lymphocytes to an antigen.
  • costimulatory molecules examples include CD27, CD28, 4-1BB (CD137), 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, and the like.
  • costimulatory molecules include peptides having sequences of amino acid numbers 236 to 351 of CD2 (NCBI RefSeq: NP.sub.— 001758.2), amino acid numbers 421 to 458 of CD4 (NCBI RefSeq: NP.sub.— 000607.1), amino acid numbers 402 to 495 of CD5 (NCBI RefSeq: NP.sub.— 055022.2), amino acid numbers 207 to 235 of CD8 alpha.
  • NCBI RefSeq NP.sub.— 001759.3
  • amino acid numbers 196 to 210 of CD83 GenBank: AAA35664.1
  • amino acid numbers 181 to 220 of CD28 NCBI RefSeq: NP.sub.
  • the cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR may be linked to each other in a random or specified order.
  • a short oligo- or polypeptide linker preferably between 2 and 10 amino acids in length may form the linkage.
  • a glycine-serine doublet provides a particularly suitable linker.
  • the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28. In another embodiment, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of 4-1BB. In yet another embodiment, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28 and 4-1BB.
  • the intracellular domain in the CAR is designed to comprise the signaling domain of 4- IBB and the signaling domain of CD3-zeta, wherein the signaling domain of 4-1BB comprises the nucleic acid sequence set forth in SEQ ID NO: 40 and the signaling domain of CD3-zeta comprises the nucleic acid sequence set forth in SEQ ID NO: 42.
  • the intracellular domain in the CAR is designed to comprise the signaling domain of 4- IBB and the signaling domain of CD3-zeta, wherein the signaling domain of 4- IBB comprises the nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 41 and the signaling domain of CD3-zeta comprises the nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 43.
  • the intracellular domain in the CAR is designed to comprise the signaling domain of 4- IBB and the signaling domain of CD3-zeta, wherein the signaling domain of 4-1BB comprises the amino acid sequence set forth in SEQ ID NO: 41 and the signaling domain of CD3-zeta comprises the amino acid sequence set forth in SEQ ID NO: 43.
  • the term "functional portion" when used in reference to a CAR refers to any part or fragment of one or more of the CARs disclosed herein, which part or fragment retains the biological activity of the CAR of which it is a part (the parent CAR).
  • Functional portions encompass, for example, those parts of a CAR that retain the ability to recognize target cells, or detect, treat, or prevent a disease, to a similar extent, the same extent, or to a higher extent, as the parent CAR.
  • the functional portion can comprise, for instance, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more, of the parent CAR.
  • the functional portion can comprise additional amino acids at the amino or carboxy terminus of the portion, or at both termini, which additional amino acids are not found in the amino acid sequence of the parent CAR.
  • the additional amino acids do not interfere with the biological function of the functional portion, e.g., recognize target cells, detect cancer, treat or prevent cancer, etc. More desirably, the additional amino acids enhance the biological activity, as compared to the biological activity of the parent CAR.
  • the term "functional variant” as used herein refers to a CAR, polypeptide, or protein having substantial or significant sequence identity or similarity to a parent CAR, which functional variant retains the biological activity of the CAR of which it is a variant.
  • Functional variants encompass, for example, those variants of the CAR described herein (the parent CAR) that retain the ability to recognize target cells to a similar extent, the same extent, or to a higher extent, as the parent CAR.
  • the functional variant can, for instance, be at least about 30%, 50%, 75%, 80%, 90%, 98% or more identical in amino acid sequence to the parent CAR.
  • a functional variant can, for example, comprise the amino acid sequence of the parent CAR with at least one conservative amino acid substitution.
  • the functional variants can comprise the amino acid sequence of the parent CAR with at least one non-conservative amino acid substitution.
  • the non-conservative amino acid substitution may enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the parent CAR.
  • Amino acid substitutions of the CARs are preferably conservative amino acid substitutions.
  • Conservative amino acid substitutions are known in the art, and include amino acid substitutions in which one amino acid having certain physical and/or chemical properties is exchanged for another amino acid that has the same or similar chemical or physical properties.
  • the conservative amino acid substitution can be an acidic/negatively charged polar amino acid substituted for another acidic/negatively charged polar amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain substituted for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Vai, He, Leu, Met, Phe, Pro, Trp, Cys, Vai, etc.), a basic/positively charged polar amino acid substituted for another basic/positively charged polar amino acid (e.g.
  • Lys, His, Arg, etc. an uncharged amino acid with a polar side chain substituted for another uncharged amino acid with a polar side chain (e.g., Asn, Gin, Ser, Thr, Tyr, etc.), an amino acid with a betabranched side-chain substituted for another amino acid with a beta-branched side-chain (e.g., He, Thr, and Vai), an amino acid with an aromatic side-chain substituted for another amino acid with an aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc.
  • a polar side chain substituted for another uncharged amino acid with a polar side chain e.g., Asn, Gin, Ser, Thr, Tyr, etc.
  • an amino acid with a betabranched side-chain substituted for another amino acid with a beta-branched side-chain e.g., He, Thr, and Vai
  • an amino acid with an aromatic side-chain substituted for another amino acid with an aromatic side chain e
  • the CAR can consist essentially of the specified amino acid sequence or sequences described herein, such that other components, e.g., other amino acids, do not materially change the biological activity of the functional variant.
  • the CARs can be of any length, i.e., can comprise any number of amino acids, provided that the CARs (or functional portions or functional variants thereof) retain their biological activity, e.g., the ability to specifically bind to antigen, detect diseased cells in a mammal, or treat or prevent disease in a mammal, etc.
  • the CAR can be about 50 to about 5000 amino acids long, such as 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more amino acids in length.
  • the CARs can comprise synthetic amino acids in place of one or more naturally-occurring amino acids.
  • Such synthetic amino acids are known in the art, and include, for example, aminocyclohexane carboxylic acid, norleucine, -amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4- nitrophenylalanine, 4- chlorophenylalanine, 4-carboxyphenylalanine, P-phenylserine P-hydroxyphenylalanine, phenylglycine, a-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid
  • the CARs can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via, e.g., a disulfide bridge, or converted into an acid addition salt and/or optionally dimerized or polymerized, or conjugated.
  • the CARs can be obtained by methods known in the art.
  • the CARs may be made by any suitable method of making polypeptides or proteins. Suitable methods of de novo synthesizing polypeptides and proteins are described in references, such as Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2000; Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, ed. Westwood et al., Oxford University Press, Oxford, United Kingdom, 2001; and U.S. Patent 5,449,752.
  • polypeptides and proteins can be recombinantly produced using the nucleic acids described herein using standard recombinant methods. See, for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Further, some of the CARs (including functional portions and functional variants thereof) can be isolated and/or purified from a source, such as a plant, a bacterium, an insect, a mammal, e.g., a rat, a human, etc.
  • a source such as a plant, a bacterium, an insect, a mammal, e.g., a rat, a human, etc.
  • the CARs described herein can be commercially synthesized by companies.
  • the CARs can be synthetic, recombinant, isolated, and/or purified.
  • One embodiment further provides a CAR, a T cell expressing a CAR, an antibody, or antigen binding domain or portion thereof, which specifically binds to one or more of the antigens disclosed herein.
  • a “T cell expressing a CAR,” or a “CAR T cell” means a T cell expressing a CAR, and has antigen specificity determined by, for example, the antibody-derived targeting domain of the CAR.
  • antibody can include an antibody and antigen binding fragments thereof.
  • antibody is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multi -specific antibodies e.g., bispecific antibodies), and antigen binding fragments thereof, so long as they exhibit the desired antigen-binding activity.
  • Non-limiting examples of antibodies include, for example, intact immunoglobulins and variants and fragments thereof known in the art that retain binding affinity for the antigen.
  • a “monoclonal antibody” is an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • a monoclonal antibody is an antibody produced by a single clone of B lymphocytes or by a cell into which nucleic acid encoding the light and heavy variable regions of the antibody of a single antibody (or an antigen binding fragment thereof) have been transfected, or a progeny thereof.
  • monoclonal antibodies are isolated from a subject. Monoclonal antibodies can have conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. Exemplary methods of production of monoclonal antibodies are known, for example, see Harlow & Lane, Antibodies, A Laboratory Manual, 2nd ed. Cold Spring Harbor Publications, New York (2013).
  • an immunoglobulin typically has heavy (H) chains and light (L) chains interconnected by disulfide bonds.
  • Immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable domain genes.
  • Each heavy and light chain contains a constant region (or constant domain) and a variable region (or variable domain; see, e.g., Kindt et al. Kuby Immunology, 6 th ed., W.H. Freeman and Co., page 91 (2007).)
  • the heavy and the light chain variable regions combine to specifically bind the antigen.
  • only the heavy chain variable region is required.
  • naturally occurring camelid antibodies consisting of a heavy chain are functional and stable in the absence of light chain (see, e.g., Hamers- Casterman et al., Nature, 363:446-448, 1993; Sheriff et al., Nat. Struct. Biol., 3:733-736, 1996).
  • VH refers to the variable region of an antibody heavy chain, including that of an antigen binding fragment, such as Fv, ScFv, dsFv or Fab.
  • VL refers to the variable domain of an antibody light chain, including that of an Fv, ScFv, dsFv or Fab.
  • Light and heavy chain variable regions contain a “framework” region interrupted by three hypervariable regions, also called “complementarity-determining regions” or “CDRs” (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991).
  • CDRs complementarity-determining regions
  • the sequences of the framework regions of different light or heavy chains are relatively conserved within a species.
  • the framework region of an antibody that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.
  • the CDRs are primarily responsible for binding to an epitope of an antigen.
  • the amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (“Sequences of Proteins of Immunological Interest,” 5 th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991; “Kabat” numbering scheme), Al-Lazikani et al., (JMB 273,927-948, 1997; “Chothia” numbering scheme), and Lefranc et al. (“IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev. Comp.
  • the CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 (from the N-terminus to C-terminus), and are also typically identified by the chain in which the particular CDR is located.
  • a VH CDR3 is the CDR3 from the variable domain of the heavy chain of the antibody in which it is found
  • a VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found.
  • Light chain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3.
  • Heavy chain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3.
  • an “antigen binding fragment” is a portion of a full length antibody that retains the ability to specifically recognize the cognate antigen, as well as various combinations of such portions.
  • antigen binding fragments include Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. ScFv); and multi-specific antibodies formed from antibody fragments.
  • Antibody fragments include antigen binding fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (see, e.g., Kontermann and Dubel (Ed), Antibody Engineering, Vols. 1-2, 2nd Ed., Springer Press, 2010).
  • a single-chain antibody is a genetically engineered molecule containing the VH and VL domains of one or more antibody(ies) linked by a suitable polypeptide linker as a genetically fused single chain molecule (see, for example, Bird et al., Science, 242:423 426, 1988; Huston et al., Proc. Natl. Acad. Sci., 85:5879 5883, 1988; Ahmad et al., Clin. Dev. Immunol., 2012, doi: 10.1155/2012/980250; Marbry, IDrugs, 13:543-549, 2010).
  • Diabodies also are included, which are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see, for example, Holliger et al., Proc. Natl. Acad. Sci., 90:6444 6448, 1993; Poljak e/ a/., Structure, 2: 1121 1123, 1994).
  • Antibodies also include genetically engineered forms such as chimeric antibodies (such as humanized murine antibodies) and heteroconjugate antibodies (such as bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd Ed., W.H. Freeman & Co., New York, 1997.
  • Non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly, or can be obtained, for example, by screening combinatorial libraries consisting of variable heavy chains and variable light chains as described by Huse et al., Science 246: 1275-1281 (1989), which is incorporated herein by reference.
  • These and other methods of making, for example, chimeric, humanized, CDR-grafted, single chain, and bifunctional antibodies are well known to those skilled in the art (Winter and Harris, Immunol.
  • an “antibody that binds to the same epitope” as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more.
  • Antibody competition assays are known, and an exemplary competition assay is provided herein.
  • a “humanized” antibody or antigen binding fragment includes a human framework region and one or more CDRs from a non-human (such as a mouse, rat, or synthetic) antibody or antigen binding fragment.
  • the non-human antibody or antigen binding fragment providing the CDRs is termed a “donor,” and the human antibody or antigen binding fragment providing the framework is termed an “acceptor.”
  • all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they can be substantially identical to human immunoglobulin constant regions, such as at least about 85-90%, such as about 95% or more identical.
  • all parts of a humanized antibody or antigen binding fragment, except possibly the CDRs are substantially identical to corresponding parts of natural human antibody sequences.
  • a “chimeric antibody” is an antibody which includes sequences derived from two different antibodies, which typically are of different species.
  • a chimeric antibody includes one or more CDRs and/or framework regions from one human antibody and CDRs and/or framework regions from another human antibody.
  • a “fully human antibody” or “human antibody” is an antibody which includes sequences from (or derived from) the human genome, and does not include sequence from another species.
  • a human antibody includes CDRs, framework regions, and (if present) an Fc region from (or derived from) the human genome.
  • Human antibodies can be identified and isolated using technologies for creating antibodies based on sequences derived from the human genome, for example by phage display or using transgenic animals (see, e.g., Barbas et al. Phage display: A Laboratory Manuel. 1st Ed. New York: Cold Spring Harbor Laboratory Press, 2004. Print.; Lonberg, Nat. Biotech., 23: 1117-1125, 2005; Lonenberg, Curr. Opin. Immunol., 20:450- 459, 2008).
  • An antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or may be different. For instance, a naturally- occurring immunoglobulin has two identical binding sites, a single-chain antibody or Fab fragment has one binding site, while a bispecific or bifunctional antibody has two different binding sites.
  • Methods of testing antibodies for the ability to bind to any functional portion of the CAR include any antibody-antigen binding assay, such as, for example, radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, and competitive inhibition assays (see, e.g., Janeway et al., infra, U.S. Patent Application Publication No. 2002/0197266 Al, and U.S. Patent No. 7,338,929).
  • RIA radioimmunoassay
  • ELISA ELISA
  • Western blot Western blot
  • immunoprecipitation immunoprecipitation
  • competitive inhibition assays see, e.g., Janeway et al., infra, U.S. Patent Application Publication No. 2002/0197266 Al, and U.S. Patent No. 7,338,929.
  • a CAR, a T cell expressing a CAR, an antibody, or antigen binding portion thereof can be modified to comprise a detectable label, such as, for instance, a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase), and element particles (e.g., gold particles).
  • FITC fluorescein isothiocyanate
  • PE phycoerythrin
  • an enzyme e.g., alkaline phosphatase, horseradish peroxidase
  • element particles e.g., gold particles
  • a CAR, a T cell expressing a CAR, or monoclonal antibodies, or antigen binding fragments thereof, specific for one or more of the antigens disclosed herein can be conjugated to an agent, such as an effector molecule or detectable marker, using any number of means known to those of skill in the art. Both covalent and noncovalent attachment means may be used. Conjugates include, but are not limited to, molecules in which there is a covalent linkage of an effector molecule or a detectable marker to an antibody or antigen binding fragment that specifically binds one or more of the antigens disclosed herein.
  • effector molecules and detectable markers can be used, including (but not limited to) chemotherapeutic agents, anti-angiogenic agents, toxins, radioactive agents such as 125 1, 32 P, 14 C, 3 H and 35 S and other labels, target moieties and ligands, etc.
  • the choice of a particular effector molecule or detectable marker depends on the particular target molecule or cell, and the desired biological effect.
  • the effector molecule can be a cytotoxin that is used to bring about the death of a particular target cell (such as a tumor cell).
  • the procedure for attaching an effector molecule or detectable marker to an antibody or antigen binding fragment varies according to the chemical structure of the effector.
  • Polypeptides typically contain a variety of functional groups; such as carboxylic acid (COOH), free amine (- NH2) or sulfhydryl (-SH) groups, which are available for reaction with a suitable functional group on an antibody to result in the binding of the effector molecule or detectable marker.
  • the antibody or antigen binding fragment is derivatized to expose or attach additional reactive functional groups.
  • the derivatization may involve attachment of any of a number of known linker molecules such as those available from Pierce Chemical Company, Rockford, IL.
  • the linker can be any molecule used to join the antibody or antigen binding fragment to the effector molecule or detectable marker.
  • the linker is capable of forming covalent bonds to both the antibody or antigen binding fragment and to the effector molecule or detectable marker.
  • Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers.
  • the linkers may be joined to the constituent amino acids through their side groups (such as through a disulfide linkage to cysteine) or to the alpha carbon amino and carboxyl groups of the terminal amino acids.
  • the linker can include a spacer element, which, when present, increases the size of the linker such that the distance between the effector molecule or the detectable marker and the antibody or antigen binding fragment is increased.
  • spacers are known to the person of ordinary skill, and include those listed in U.S. Pat. Nos. 7,964,566, 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149,
  • the linker is cleavable under intracellular conditions, such that cleavage of the linker releases the effector molecule or detectable marker from the antibody or antigen binding fragment in the intracellular environment.
  • the linker is not cleavable and the effector molecule or detectable marker is released, for example, by antibody degradation.
  • the linker is cleavable by a cleaving agent that is present in the intracellular environment (for example, within a lysosome or endosome or caveolea).
  • the linker can be, for example, a peptide linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease.
  • the peptide linker is at least two amino acids long or at least three amino acids long.
  • the linker can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids long, such as 1-2, 1-3, 2-5, 3-10, 3-15, 1-5, 1-10, 1-15 amino acids long.
  • Proteases can include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives resulting in the release of active drug inside target cells (see, for example, Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67- 123).
  • a peptide linker that is cleavable by the thiol-dependent protease cathepsin-B can be used (for example, a Phenylalanine -Leucine or a Glycine- Phenylalanine -Leucine-Glycine linker).
  • Other examples of such linkers are described, for example, in U.S. Pat. No. 6,214,345, incorporated herein by reference.
  • the peptide linker cleavable by an intracellular protease is a Valine-Citruline linker or a Phenylalanine-Lysine linker (see, for example, U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin with the Valine- Citruline linker).
  • the cleavable linker is pH-sensitive, z.e., sensitive to hydrolysis at certain pH values.
  • the pH-sensitive linker is hydrolyzable under acidic conditions.
  • an acid-labile linker that is hydrolyzable in the lysosome for example, a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or the like
  • an acid-labile linker that is hydrolyzable in the lysosome for example, a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or the like
  • the hydrolyzable linker is a thioether linker (such as, for example, a thioether attached to the therapeutic agent via an acylhydrazone bond (see, for example, U.S. Pat. No. 5,622,929).
  • the linker is cleavable under reducing conditions (for example, a disulfide linker).
  • a disulfide linker for example, a disulfide linker.
  • disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3- (2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT (N- succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)-, SPDB and SMPT.
  • SATA N-succinimidyl-S-acetylthioacetate
  • SPDP N-succinimidyl-3- (2-pyri
  • the linker is a malonate linker (Johnson et al., 1995, Anticancer Res. 15: 1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1305-12).
  • the linker is not cleavable and the effector molecule or detectable marker is released by antibody degradation. (See U.S. Publication No. 2005/0238649 incorporated by reference herein in its entirety).
  • the linker is resistant to cleavage in an extracellular environment. For example, no more than about 20%, no more than about 15%, no more than about 10%, no more than about 5%, no more than about 3%, or no more than about 1% of the linkers, in a sample of conjugate, are cleaved when the conjugate is present in an extracellular environment (for example, in plasma). Whether or not a linker is resistant to cleavage in an extracellular environment can be determined, for example, by incubating the conjugate containing the linker of interest with plasma for a predetermined time period (for example, 2, 4, 8, 16, or 24 hours) and then quantitating the amount of free effector molecule or detectable marker present in the plasma.
  • a predetermined time period for example, 2, 4, 8, 16, or 24 hours
  • conjugates of a CAR, a T cell expressing a CAR, an antibody, or antigen binding portion thereof, and one or more small molecule toxins such as a calicheamicin, maytansinoids, dolastatins, auristatins, a trichothecene, and CC1065, and the derivatives of these toxins that have toxin activity, are provided.
  • Maytansine compounds suitable for use as maytansinoid toxin moieties are well known in the art, and can be isolated from natural sources according to known methods, produced using genetic engineering techniques (see Yu et al (2002) PNAS 99:7968-7973), or maytansinol and maytansinol analogues prepared synthetically according to known methods.
  • Maytansinoids are mitototic inhibitors which act by inhibiting tubulin polymerization. Maytansine was first isolated from the east African shrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it was discovered that certain microbes also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S.
  • Additional toxins can be employed with a CAR, a T cell expressing a CAR, an antibody, or antigen binding portion thereof.
  • exemplary toxins include Pseudomonas exotoxin (PE), ricin, abrin, diphtheria toxin and subunits thereof, ribotoxin, ribonuclease, saporin, and calicheamicin, as well as botulinum toxins A through F.
  • PE Pseudomonas exotoxin
  • ricin ricin
  • abrin diphtheria toxin and subunits thereof
  • ribotoxin ribonuclease
  • saporin and calicheamicin
  • botulinum toxins A through F as well known in the art and many are readily available from commercial sources (for example, Sigma Chemical Company, St. Louis, MO).
  • Contemplated toxins also include variants of the toxins (see, for example, see, U.S. Patent Nos
  • Saporin is a toxin derived from Saponaria officinalis that disrupts protein synthesis by inactivating the 60S portion of the ribosomal complex (Stirpe et al., Bio/Technology, 10:405-412, 1992).
  • the toxin has no mechanism for specific entry into cells, and therefore requires conjugation to an antibody or antigen binding fragment that recognizes a cell-surface protein that is internalized in order to be efficiently taken up by cells.
  • Diphtheria toxin is isolated from Corynebacterium diphtheriae. Typically, diphtheria toxin for use in immunotoxins is mutated to reduce or to eliminate non-specific toxicity.
  • a mutant known as CRM 107 which has full enzymatic activity but markedly reduced non-specific toxicity, has been known since the 1970’s (Laird and Groman, J. Virol. 19:220, 1976), and has been used in human clinical trials. See, U.S. Patent No. 5,792,458 and U.S. Patent No. 5,208,021.
  • Ricin is the lectin RCA60 from Ricinus communis (Castor bean).
  • Ricinus communis agglutinin occurs in two forms designated RCAeo and RCA120 according to their molecular weights of approximately 65 and 120 kD, respectively (Nicholson & Blaustein, J. Biochim. Biophys. Acta 266:543, 1972). The A chain is responsible for inactivating protein synthesis and killing cells.
  • the B chain binds ricin to cell-surface galactose residues and facilitates transport of the A chain into the cytosol (Olsnes et al., Nature 249:627-631, 1974 and U.S. Patent No. 3,060,165).
  • Ribonucleases have also been conjugated to targeting molecules for use as immunotoxins (see Suzuki et al., Nat. Biotech. 17:265-70, 1999).
  • Exemplary ribotoxins such as a-sarcin and restrictocin are discussed in, for example Rathore et al., Gene 190:31-5, 1997; and Goyal and Batra, Biochem. 345 Pt 2:247-54, 2000.
  • Calicheamicins were first isolated from Micromonospora echinospora and are members of the enediyne antitumor antibiotic family that cause double strand breaks in DNA that lead to apoptosis (see, for example Lee et al., J. Antibiot. 42: 1070-87,1989). The drug is the toxic moiety of an immunotoxin in clinical trials (see, for example, Gillespie et al., Ann. Oncol. 11 :735-41, 2000).
  • Abrin includes toxic lectins from Abrus precatorius.
  • the toxic principles, abrin a, b, c, and d have a molecular weight of from about 63 and 67 kD and are composed of two disulfide- linked polypeptide chains A and B.
  • the A chain inhibits protein synthesis; the B chain (abrin-b) binds to D-galactose residues (see, Funatsu et al., Agr. Biol. Chem. 52: 1095, 1988; and Olsnes, Methods Enzy mol. 50:330-335, 1978).
  • a CAR, a T cell expressing a CAR, monoclonal antibodies, antigen binding fragments thereof, specific for one or more of the antigens disclosed herein, can also be conjugated with a detectable marker; for example, a detectable marker capable of detection by ELISA, spectrophotometry, flow cytometry, microscopy or diagnostic imaging techniques (such as computed tomography (CT), computed axial tomography (CAT) scans, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging NMRI), magnetic resonance tomography (MTR), ultrasound, fiberoptic examination, and laparoscopic examination).
  • CT computed tomography
  • CAT computed axial tomography
  • MRI magnetic resonance imaging
  • NMRI nuclear magnetic resonance imaging
  • MMR magnetic resonance tomography
  • detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioactive isotopes and heavy metals or compounds (for example super paramagnetic iron oxide nanocrystals for detection by MRI).
  • useful detectable markers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5- dimethylamine-l-napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors and the like.
  • Bioluminescent markers are also of use, such as luciferase, Green fluorescent protein (GFP), Yellow fluorescent protein (YFP).
  • a CAR, a T cell expressing a CAR, an antibody, or antigen binding portion thereof can also be conjugated with enzymes that are useful for detection, such as horseradish peroxidase, P-galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like.
  • enzymes that are useful for detection such as horseradish peroxidase, P-galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like.
  • a CAR, a T cell expressing a CAR, an antibody, or antigen binding portion thereof may also be conjugated with biotin, and detected through indirect measurement of avidin or streptavidin binding. It should be noted that the avidin itself can be conjugated with an enzyme or a fluorescent label.
  • a CAR, a T cell expressing a CAR, an antibody, or antigen binding portion thereof, may be conjugated with a paramagnetic agent, such as gadolinium.
  • Paramagnetic agents such as superparamagnetic iron oxide are also of use as labels.
  • Antibodies can also be conjugated with lanthanides (such as europium and dysprosium), and manganese.
  • An antibody or antigen binding fragment may also be labeled with a predetermined polypeptide epitopes recognized by a secondary reporter (such as leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags).
  • a CAR, a T cell expressing a CAR, an antibody, or antigen binding portion thereof, can also be conjugated with a radiolabeled amino acid.
  • the radiolabel may be used for both diagnostic and therapeutic purposes. For instance, the radiolabel may be used to detect one or more of the antigens disclosed herein and antigen expressing cells by x-ray, emission spectra, or other diagnostic techniques. Further, the radiolabel may be used therapeutically as a toxin for treatment of tumors in a subject, for example for treatment of a neuroblastoma.
  • labels for polypeptides include, but are not limited to, the following radioisotopes or radionucleotides: 3 H, 14 C, 15 N, 35 S, 90 Y, "Tc, U1 ln, 125 I, and 131 I.
  • radiolabels may be detected using photographic film or scintillation counters
  • fluorescent markers may be detected using a photodetector to detect emitted illumination.
  • Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.
  • nucleic acid comprising a nucleotide sequence encoding any of the CARs, an antibody, or antigen binding portion thereof, described herein (including functional portions and functional variants thereof).
  • the nucleic acids of the invention may comprise a nucleotide sequence encoding any of the leader sequences, antigen binding domains, transmembrane domains, and/or intracellular T cell signaling domains described herein.
  • the nucleotide sequence may be codon-modified. Without being bound to a particular theory, it is believed that codon optimization of the nucleotide sequence increases the translation efficiency of the mRNA transcripts. Codon optimization of the nucleotide sequence may involve substituting a native codon for another codon that encodes the same amino acid, but can be translated by tRNA that is more readily available within a cell, thus increasing translation efficiency. Optimization of the nucleotide sequence may also reduce secondary mRNA structures that would interfere with translation, thus increasing translation efficiency.
  • the nucleic acid may comprise a codon-modified nucleotide sequence that encodes the antigen binding domain of the inventive CAR. In another embodiment of the invention, the nucleic acid may comprise a codon-modified nucleotide sequence that encodes any of the CARs described herein (including functional portions and functional variants thereof).
  • Nucleic acid as used herein includes “polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” and generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, synthesized or obtained (e.g., isolated and/or purified) from natural sources, which can contain natural, non-natural or altered nucleotides, and which can contain a natural, non-natural or altered internucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide.
  • the nucleic acid does not comprise any insertions, deletions, inversions, and/or substitutions. However, it may be suitable in some instances, as discussed herein, for the nucleic acid to comprise one or more insertions, deletions, inversions, and/or substitutions.
  • a recombinant nucleic acid may be one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques, such as those described in Sambrook et al., supra.
  • the nucleic acids can be constructed based on chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. See, for example, Sambrook et al., supra, and Ausubel et al., supra.
  • a nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides).
  • modified nucleotides that can be used to generate the nucleic acids include, but are not limited to, 5 -fluorouracil, 5-bromouracil, 5- chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1 - methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3 -methylcytosine, 5- methylcytosine, N6-substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxyaminomethyl-2-thiouracil, beta-D-
  • the nucleic acid can comprise any isolated or purified nucleotide sequence which encodes any of the CARs or functional portions or functional variants thereof.
  • the nucleotide sequence can comprise a nucleotide sequence which is degenerate to any of the sequences or a combination of degenerate sequences.
  • An embodiment also provides an isolated or purified nucleic acid comprising a nucleotide sequence which is complementary to the nucleotide sequence of any of the nucleic acids described herein or a nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein.
  • the nucleotide sequence which hybridizes under stringent conditions may hybridize under high stringency conditions.
  • high stringency conditions is meant that the nucleotide sequence specifically hybridizes to a target sequence (the nucleotide sequence of any of the nucleic acids described herein) in an amount that is detectably stronger than non-specific hybridization.
  • High stringency conditions include conditions which would distinguish a polynucleotide with an exact complementary sequence, or one containing only a few scattered mismatches from a random sequence that happened to have a few small regions (e.g., 3-10 bases) that matched the nucleotide sequence. Such small regions of complementarity are more easily melted than a full-length complement of 14-17 or more bases, and high stringency hybridization makes them easily distinguishable.
  • Relatively high stringency conditions would include, for example, low salt and/or high temperature conditions, such as provided by about 0.02-0.1 M NaCl or the equivalent, at temperatures of about 50-70 °C.
  • Such high stringency conditions tolerate little, if any, mismatch between the nucleotide sequence and the template or target strand, and are particularly suitable for detecting expression of any of the inventive CARs. It is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide.
  • nucleic acid comprising a nucleotide sequence that is at least about 70% or more, e.g., about 80%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to any of the nucleic acids described herein.
  • the nucleic acids can be incorporated into a recombinant expression vector.
  • an embodiment provides recombinant expression vectors comprising any of the nucleic acids.
  • the term "recombinant expression vector” means a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, polypeptide, or peptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed within the cell.
  • the vectors are not naturally-occurring as a whole.
  • the recombinant expression vectors can comprise any type of nucleotides, including, but not limited to DNA and RNA, which can be single-stranded or double- stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides.
  • the recombinant expression vectors can comprise naturally-occurring or non-naturally-occurring intemucleotide linkages, or both types of linkages.
  • the non-naturally occurring or altered nucleotides or internucleotide linkages do not hinder the transcription or replication of the vector.
  • the recombinant expression vector can be any suitable recombinant expression vector, and can be used to transform or transfect any suitable host cell.
  • Suitable vectors include those designed for propagation and expansion or for expression or both, such as plasmids and viruses.
  • the vector can be selected from the group consisting of the pUC series (Fermentas Life Sciences, Glen Burnie, MD), the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, CA).
  • Bacteriophage vectors such as ZuTIO, LbTI 1, /.Zap 11 (Stratagene), EMBL4, and XNMI 149, also can be used.
  • plant expression vectors include pBIOl, pBI101.2, pBHOl .3, pBI121 and pBIN19 (Clontech).
  • animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech).
  • the recombinant expression vector may be a viral vector, e.g, a retroviral vector or a lentiviral vector.
  • a lentiviral vector is a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009).
  • Other examples of lentivirus vectors that may be used in the clinic include, for example, and not by way of limitation, the LENTIVECTOR.RTM. gene delivery technology from Oxford BioMedica pic, the LENTIMAX.TM. vector system from Lentigen and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.
  • Transfection methods include calcium phosphate co-precipitation (see, e.g., Graham et al., supra), direct micro injection into cultured cells (see, e.g., Capecchi, Cell, 22: 479-488 (1980)), electroporation (see, e.g., Shigekawa et al., BioTechniques, 6: 742-751 (1988)), liposome mediated gene transfer (see, e.g., Mannino et al., BioTechniques, 6: 682-690 (1988)), lipid mediated transduction (see, e.g., Feigner et al., Proc. Natl. Acad. Sci. USA, 84: 7413-7417 (1987)), and nucleic acid delivery using high velocity microprojectiles (see, e.g., Klein et al, Nature, 327: 70-73 (1987)).
  • the recombinant expression vectors can be prepared using standard recombinant DNA techniques described in, for example, Sambrook et al., supra, and Ausubel et al., supra.
  • Constructs of expression vectors, which are circular or linear, can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell.
  • Replication systems can be derived, e.g., from ColEl, 2 p plasmid, X, SV40, bovine papilloma virus, and the like.
  • the recombinant expression vector may comprise regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host cell (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate, and taking into consideration whether the vector is DNA- or RNA-based.
  • the recombinant expression vector may comprise restriction sites to facilitate cloning.
  • the recombinant expression vector can include one or more marker genes, which allow for selection of transformed or transfected host cells.
  • Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like.
  • Suitable marker genes for the inventive expression vectors include, for instance, neomycin/G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.
  • the recombinant expression vector can comprise a native or nonnative promoter operably linked to the nucleotide sequence encoding the CAR (including functional portions and functional variants thereof), or to the nucleotide sequence which is complementary to or which hybridizes to the nucleotide sequence encoding the CAR.
  • the selection of promoters e.g., strong, weak, inducible, tissue-specific and developmental-specific, is within the ordinary skill of the artisan.
  • the combining of a nucleotide sequence with a promoter is also within the skill of the artisan.
  • the promoter can be a non-viral promoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, or a promoter found in the long-terminal repeat of the murine stem cell virus.
  • CMV cytomegalovirus
  • the recombinant expression vectors can be designed for either transient expression, for stable expression, or for both. Also, the recombinant expression vectors can be made for constitutive expression or for inducible expression.
  • the recombinant expression vectors can be made to include a suicide gene.
  • suicide gene refers to a gene that causes the cell expressing the suicide gene to die.
  • the suicide gene can be a gene that confers sensitivity to an agent, e.g., a drug, upon the cell in which the gene is expressed, and causes the cell to die when the cell is contacted with or exposed to the agent.
  • agent e.g., a drug
  • HSV Herpes Simplex Virus
  • TK thymidine kinase
  • An embodiment further provides a host cell comprising any of the recombinant expression vectors described herein.
  • the term "host cell” refers to any type of cell that can contain the inventive recombinant expression vector.
  • the host cell can be a eukaryotic cell, e.g., plant, animal, fungi, or algae, or can be a prokaryotic cell, e.g., bacteria or protozoa.
  • the host cell can be a cultured cell or a primary cell, /. ⁇ ., isolated directly from an organism, e.g., a human.
  • the host cell can be an adherent cell or a suspended cell, /. ⁇ ., a cell that grows in suspension.
  • Suitable host cells are known in the art and include, for instance, DH5a E. coli cells, Chinese hamster ovarian cells, monkey VERO cells, COS cells, HEK293 cells, and the like.
  • the host cell may be a prokaryotic cell, e.g., a DH5a cell.
  • the host cell may be a mammalian cell.
  • the host cell may be a human cell.
  • the host cell can be of any cell type, can originate from any type of tissue, and can be of any developmental stage, the host cell may be a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC).
  • PBL peripheral blood lymphocyte
  • PBMC peripheral blood mononuclear cell
  • the host cell may be a T cell.
  • the T cell can be any T cell, such as a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupTl, etc., or a T cell obtained from a mammal. If obtained from a mammal, the T cell can be obtained from numerous sources, including but not limited to blood, bone marrow, lymph node, the thymus, or other tissues or fluids. T cells can also be enriched for or purified.
  • the T cell may be a human T cell.
  • the T cell may be a T cell isolated from a human.
  • the T cell can be any type of T cell and can be of any developmental stage, including but not limited to, CD4+/CD8+ double positive T cells, CD4+ helper T cells, e.g., Thl and Th2 cells, CD8+ T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memory T cells, memory stem cells, i.e. Tscm, naive T cells, and the like.
  • the T cell may be a CD8+ T cell or a CD4+ T cell.
  • the CARs as described herein can be used in suitable non-T cells.
  • suitable non-T cells are those with an immune-effector function, such as, for example, NK cells, and T-like cells generated from pluripotent stem cells.
  • the population of cells can be a heterogeneous population comprising the host cell comprising any of the recombinant expression vectors described, in addition to at least one other cell, e.g., a host cell (e.g., a T cell), which does not comprise any of the recombinant expression vectors, or a cell other than a T cell, e.g., a B cell, a macrophage, a neutrophil, an erythrocyte, a hepatocyte, an endothelial cell, an epithelial cell, a muscle cell, a brain cell, etc.
  • a host cell e.g., a T cell
  • a cell other than a T cell e.g., a B cell, a macrophage, a neutrophil, an erythrocyte, a hepatocyte, an endothelial cell, an epithelial cell, a muscle cell, a brain cell, etc.
  • the population of cells can be a substantially homogeneous population, in which the population comprises mainly host cells e.g., consisting essentially of) comprising the recombinant expression vector.
  • the population also can be a clonal population of cells, in which all cells of the population are clones of a single host cell comprising a recombinant expression vector, such that all cells of the population comprise the recombinant expression vector.
  • the population of cells is a clonal population comprising host cells comprising a recombinant expression vector as described herein.
  • CARs including functional portions and variants thereof
  • nucleic acids can be isolated and/or purified.
  • a purified (or isolated) host cell preparation is one in which the host cell is more pure than cells in their natural environment within the body. Such host cells may be produced, for example, by standard purification techniques.
  • a preparation of a host cell is purified such that the host cell represents at least about 50%, for example at least about 70%, of the total cell content of the preparation.
  • the purity can be at least about 50%, can be greater than about 60%, about 70% or about 80%, or can be about 100%.
  • an embodiment provides a method of treating or preventing cancer in a mammal, comprising administering to the mammal the CARs, the nucleic acids, the recombinant expression vectors, the host cells, the population of cells, the antibodies and/or the antigen binding portions thereof, and/or the pharmaceutical compositions in an amount effective to treat or prevent cancer in the mammal.
  • An embodiment further comprises lymphodepleting the mammal prior to administering the CARs disclosed herein.
  • lymphodepletion include, but may not be limited to, nonmyeloablative lymphodepleting chemotherapy, myeloablative lymphodepleting chemotherapy, total body irradiation, etc.
  • the cells can be cells that are allogeneic or autologous to the mammal.
  • the cells are autologous to the mammal.
  • allogeneic means any material derived from a different animal of the same species as the individual to whom the material is introduced.
  • allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically.
  • autologous means any material derived from the same individual to whom it is later to be re-introduced into the individual.
  • the mammal referred to herein can be any mammal.
  • the term "mammal” refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits.
  • the mammals may be from the order Carnivora, including Felines (cats) and Canines (dogs).
  • the mammals may be from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses).
  • the mammals may be of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes).
  • the mammal is a human.
  • the cancer can be any cancer, including any of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder carcinoma), bone cancer, brain cancer (e.g., meduloblastoma), breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, leukemia,
  • bladder cancer
  • the treatment or prevention provided by the method can include treatment or prevention of one or more conditions or symptoms of the disease, e.g., cancer, being treated or prevented.
  • prevention can encompass delaying the onset of the disease, or a symptom or condition thereof.
  • Another embodiment provides a method of detecting the presence of cancer in a mammal, comprising: (a) contacting a sample comprising one or more cells from the mammal with the CARs, the nucleic acids, the recombinant expression vectors, the host cells, the population of cells, the antibodies, and/or the antigen binding portions thereof, or the pharmaceutical compositions, thereby forming a complex, (b) and detecting the complex, wherein detection of the complex is indicative of the presence of cancer in the mammal.
  • the sample may be obtained by any suitable method, e.g., biopsy or necropsy.
  • a biopsy is the removal of tissue and/or cells from an individual. Such removal may be to collect tissue and/or cells from the individual in order to perform experimentation on the removed tissue and/or cells. This experimentation may include experiments to determine if the individual has and/or is suffering from a certain condition or disease-state.
  • the condition or disease may be, e.g., cancer.
  • the sample comprising cells of the mammal can be a sample comprising whole cells, lysates thereof, or a fraction of the whole cell lysates, e.g., a nuclear or cytoplasmic fraction, a whole protein fraction, or a nucleic acid fraction.
  • the cells can be any cells of the mammal, e.g., the cells of any organ or tissue, including blood cells or endothelial cells.
  • the contacting can take place in vitro or in vivo with respect to the mammal.
  • the contacting is in vitro.
  • the CARs disclosed herein, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, populations of cells, or antibodies, or antigen binding portions thereof, described herein can be labeled with a detectable label such as, for instance, a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme e.g., alkaline phosphatase, horseradish peroxidase), and element particles (e.g., gold particles) as disclosed supra.
  • a detectable label such as, for instance, a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme e.g., alkaline phosphatase, horseradish peroxidase), and element particles (e.g., gold particles) as disclosed supra
  • cytokines e.g., interferon-y, granulocyte/monocyte colony stimulating factor (GM-CSF), tumor necrosis factor a (TNF-a) or interleukin 2 (IL-2)
  • cytokines e.g., interferon-y, granulocyte/monocyte colony stimulating factor (GM-CSF), tumor necrosis factor a (TNF-a) or interleukin 2 (IL-2)
  • GM-CSF granulocyte/monocyte colony stimulating factor
  • TNF-a tumor necrosis factor a
  • IL-2 interleukin 2
  • Another embodiment provides for the use of the CARs, nucleic acids, recombinant expression vectors, host cells, populations of cells, antibodies, or antigen binding portions thereof, and/or pharmaceutical compositions of the invention, for the treatment or prevention of a proliferative disorder, e.g., cancer, in a mammal.
  • a proliferative disorder e.g., cancer
  • the cancer may be any of the cancers described herein.
  • any method of administration can be used for the disclosed therapeutic agents, including local and systemic administration.
  • topical, oral, intravascular such as intravenous, intramuscular, intraperitoneal, intranasal, intradermal, intrathecal and subcutaneous administration can be used.
  • the particular mode of administration and the dosage regimen will be selected by the attending clinician, taking into account the particulars of the case (for example the subject, the disease, the disease state involved, and whether the treatment is prophylactic).
  • one or more routes of administration may be used; for example, a chemotherapeutic agent may be administered orally and an antibody or antigen binding fragment or conjugate or composition may be administered intravenously.
  • Methods of administration include injection for which the CAR, CAR T Cell, conjugates, antibodies, antigen binding fragments, or compositions are provided in a nontoxic pharmaceutically acceptable carrier such as water, saline, Ringer's solution, dextrose solution, 5% human serum albumin, fixed oils, ethyl oleate, or liposomes.
  • a nontoxic pharmaceutically acceptable carrier such as water, saline, Ringer's solution, dextrose solution, 5% human serum albumin, fixed oils, ethyl oleate, or liposomes.
  • local administration of the disclosed compounds can be used, for instance by applying the antibody or antigen binding fragment to a region of tissue from which a tumor has been removed, or a region suspected of being prone to tumor development.
  • sustained intra-tumoral (or near-tumoral) release of the pharmaceutical preparation that includes a therapeutically effective amount of the antibody or antigen binding fragment may be beneficial.
  • the conjugate is applied as an eye drop topically to the cornea, or intravitreally into the eye.
  • the disclosed therapeutic agents can be formulated in unit dosage form suitable for individual administration of precise dosages.
  • the disclosed therapeutic agents may be administered in a single dose or in a multiple dose schedule.
  • a multiple dose schedule is one in which a primary course of treatment may be with more than one separate dose, for instance 1-10 doses, followed by other doses given at subsequent time intervals as needed to maintain or reinforce the action of the compositions.
  • Treatment can involve daily or multi-daily doses of compound(s) over a period of a few days to months, or even years.
  • the dosage regime will also, at least in part, be determined based on the particular needs of the subject to be treated and will be dependent upon the judgment of the administering practitioner.
  • Typical dosages of the antibodies or conjugates can range from about 0.01 to about 30 mg/kg, such as from about 0.1 to about 10 mg/kg.
  • the subject is administered a therapeutic composition that includes one or more of the conjugates, antibodies, compositions, CARs, CAR T cells or additional agents, on a multiple daily dosing schedule, such as at least two consecutive days, 10 consecutive days, and so forth, for example for a period of weeks, months, or years.
  • the subject is administered the conjugates, antibodies, compositions or additional agents for a period of at least 30 days, such as at least 2 months, at least 4 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months.
  • the disclosed methods include providing surgery, radiation therapy, and/or chemotherapeutics to the subject in combination with a disclosed antibody, antigen binding fragment, conjugate, CAR or T cell expressing a CAR (for example, sequentially, substantially simultaneously, or simultaneously).
  • chemotherapeutics for example, sequentially, substantially simultaneously, or simultaneously.
  • Methods and therapeutic dosages of such agents and treatments are known to those skilled in the art, and can be determined by a skilled clinician.
  • Preparation and dosing schedules for the additional agent may be used according to manufacturer's instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service, (1992) Ed., M. C. Perry, Williams & Wilkins, Baltimore, Md.
  • the combination therapy can include administration of a therapeutically effective amount of an additional cancer inhibitor to a subject.
  • additional therapeutic agents that can be used with the combination therapy include microtubule binding agents, DNA intercalators or cross-linkers, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors. These agents (which are administered at a therapeutically effective amount) and treatments can be used alone or in combination.
  • any suitable anticancer or anti-angiogenic agent can be administered in combination with the CARS, CAR- T cells, antibodies, antigen binding fragment, or conjugates disclosed herein. Methods and therapeutic dosages of such agents are known to those skilled in the art, and can be determined by a skilled clinician.
  • Additional chemotherapeutic agents include, but are not limited to alkylating agents, such as nitrogen mustards (for example, chlorambucil, chlormethine, cyclophosphamide, ifosfamide, and melphalan), nitrosoureas (for example, carmustine, fotemustine, lomustine, and streptozocin), platinum compounds (for example, carboplatin, cisplatin, oxaliplatin, and BBR3464), busulfan, dacarbazine, mechlorethamine, procarbazine, temozolomide, thiotepa, and uramustine; antimetabolites, such as folic acid (for example, methotrexate, pemetrexed, and raltitrexed), purine (for example, cladribine, clofarabine, fludarabine, mercaptopurine, and tioguanine), pyrimidine (for example, capecitabine),
  • the combination therapy may provide synergy and prove synergistic, that is, the effect achieved when the active ingredients used together is greater than the sum of the effects that results from using the compounds separately.
  • a synergistic effect may be attained when the active ingredients are: (1) co-formulated and administered or delivered simultaneously in a combined, unit dosage formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen.
  • a synergistic effect may be attained when the compounds are administered or delivered sequentially, for example by different injections in separate syringes.
  • an effective dosage of each active ingredient is administered sequentially, i.e. serially
  • combination therapy effective dosages of two or more active ingredients are administered together.
  • an effective amount of an antibody or antigen binding fragment that specifically binds to one or more of the antigens disclosed herein or a conjugate thereof is administered to a subject having a tumor following anti-cancer treatment. After a sufficient amount of time has elapsed to allow for the administered antibody or antigen binding fragment or conjugate to form an immune complex with the antigen expressed on the respective cancer cell, the immune complex is detected. The presence (or absence) of the immune complex indicates the effectiveness of the treatment. For example, an increase in the immune complex compared to a control taken prior to the treatment indicates that the treatment is not effective, whereas a decrease in the immune complex compared to a control taken prior to the treatment indicates that the treatment is effective.
  • compositions are provided herein for use in gene therapy, immunotherapy and/or cell therapy that include one or more of the disclosed CARs, or T cells expressing a CAR, antibodies, antigen binding fragments, conjugates, CARs, or T cells expressing a CAR that specifically bind to one or more antigens disclosed herein, in a carrier (such as a pharmaceutically acceptable carrier).
  • a carrier such as a pharmaceutically acceptable carrier.
  • the compositions can be prepared in unit dosage forms for administration to a subject. The amount and timing of administration are at the discretion of the treating clinician to achieve the desired outcome.
  • the compositions can be formulated for systemic (such as intravenus) or local (such as intra-tumor) administration.
  • a disclosed CARs, or T cells expressing a CAR, antibody, antigen binding fragment, conjugate is formulated for parenteral administration, such as intravenous administration.
  • Compositions including a CAR, or T cell expressing a CAR, a conjugate, antibody or antigen binding fragment as disclosed herein are of use, for example, for the treatment and detection of a tumor, for example, and not by way of limitation, a neuroblastoma.
  • the compositions are useful for the treatment or detection of a carcinoma.
  • the compositions including a CAR, or T cell expressing a CAR, a conjugate, antibody or antigen binding fragment as disclosed herein are also of use, for example, for the detection of pathological angiogenesis.
  • compositions for administration can include a solution of the CAR, or T cell expressing a CAR, conjugate, antibody or antigen binding fragment dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier.
  • a pharmaceutically acceptable carrier such as an aqueous carrier.
  • aqueous carriers can be used, for example, buffered saline and the like. These solutions are sterile and generally free of undesirable matter.
  • These compositions may be sterilized by conventional, well known sterilization techniques.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, adjuvant agents, and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
  • concentration of a CAR, or T cell expressing a CAR, antibody or antigen binding fragment or conjugate in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the subject’s needs. Actual methods of preparing such dosage forms for use in in gene therapy, immunotherapy and/or cell therapy are known, or will be apparent, to those skilled in the art.
  • a typical composition for intravenous administration includes about 0.01 to about 30 mg/kg of antibody or antigen binding fragment or conjugate per subject per day (or the corresponding dose of a CAR, or T cell expressing a CAR, conjugate including the antibody or antigen binding fragment).
  • Actual methods for preparing administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA (1995).
  • a CAR, or T cell expressing a CAR, antibodies, antigen binding fragments, or conjugates may be provided in lyophilized form and rehydrated with sterile water before administration, although they are also provided in sterile solutions of known concentration.
  • the CARs, or T cells expressing a CAR, antibody or antigen binding fragment or conjugate solution is then added to an infusion bag containing 0.9% sodium chloride, USP, and in some cases administered at a dosage of from 0.5 to 15 mg/kg of body weight.
  • a CAR, or T cell expressing a CAR, antibodies, antigen binding fragments and conjugates thereof can be administered by slow infusion, rather than in an intravenous push or bolus.
  • a higher loading dose is administered, with subsequent, maintenance doses being administered at a lower level.
  • an initial loading dose of 4 mg/kg antibody or antigen binding fragment (or the corresponding dose of a conjugate including the antibody or antigen binding fragment) may be infused over a period of some 90 minutes, followed by weekly maintenance doses for 4-8 weeks of 2 mg/kg infused over a 30 minute period if the previous dose was well tolerated.
  • Controlled release parenteral formulations can be made as implants, oily injections, or as particulate systems.
  • Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles.
  • Microcapsules contain the therapeutic protein, such as a cytotoxin or a drug, as a central core. In microspheres, the therapeutic is dispersed throughout the particle.
  • Particles, microspheres, and microcapsules smaller than about 1 pm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively.
  • Capillaries have a diameter of approximately 5 pm so that only nanoparticles are administered intravenously.
  • Microparticles are typically around 100 pm in diameter and are administered subcutaneously or intramuscularly. See, for example, Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219- 342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339, (1992).
  • Polymers can be used for ion-controlled release of the CARs, or T cells expressing a CAR, antibody or antigen binding fragment or conjugate compositions disclosed herein.
  • Various degradable and nondegradable polymeric matrices for use in controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26:537-542, 1993).
  • the block copolymer, polaxamer 407 exists as a viscous yet mobile liquid at low temperatures but forms a semisolid gel at body temperature. It has been shown to be an effective vehicle for formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res.
  • hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema et al., Int. J. Pharm. 112:215-224, 1994).
  • liposomes are used for controlled release as well as drug targeting of the lipid-capsulated drug (Betageri et al, Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Numerous additional systems for controlled delivery of therapeutic proteins are known (see U.S. Patent No. 5,055,303; U.S. Patent No. 5,188,837; U.S.
  • kits employing the CARs disclosed herein are also provided.
  • kits for treating a tumor in a subject, or making a CAR T cell that expresses one or more of the CARs disclosed herein will typically include a disclosed antibody, antigen binding fragment, conjugate, nucleic acid molecule, CAR or T cell expressing a CAR as disclosed herein. More than one of the disclosed antibodies, antigen binding fragments, conjugates, nucleic acid molecules, CARs or T cells expressing a CAR can be included in the kit.
  • the kit can include a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container typically holds a composition including one or more of the disclosed antibodies, antigen binding fragments, conjugates, nucleic acid molecules, CARs or T cells expressing a CAR.
  • the container may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • a label or package insert indicates that the composition is used for treating the particular condition.
  • the label or package insert typically will further include instructions for use of a disclosed antibodies, antigen binding fragments, conjugates, nucleic acid molecules, CARs or T cells expressing a CAR, for example, in a method of treating or preventing a tumor or of making a CAR T cell.
  • the package insert typically includes instructions customarily included in commercial packages of therapeutic products that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.
  • the instructional materials may be written, in an electronic form (such as a computer diskette or compact disk) or may be visual (such as video files).
  • the kits may also include additional components to facilitate the particular application for which the kit is designed.
  • the kit may additionally contain means of detecting a label (such as enzyme substrates for enzymatic labels, filter sets to detect fluorescent labels, appropriate secondary labels such as a secondary antibody, or the like).
  • the kits may additionally include buffers and other reagents routinely used for the practice of a particular method. Such kits and appropriate contents are well known to those of skill in the art.
  • a naive human ScFv (recombinant single chain fragment variable of immunoglobulin) phage display library (approximate diversity, IO 10 unique specificities), constructed from peripheral blood B cells of 50 healthy donors (Z. Y. Zhu and D. S. Dimitrov, unpublished data), were used for selection of ScFvs for recombinant human CD 19 protein (Miltenyi Biotec, unpublished).
  • Amplified libraries of 10 12 phage-displayed ScFv were incubated with 5, 3, and 1, pg of coated CD19 in a 5xl00-pl volume, distributed equally in 5 wells of a 96-well plate for 2 h at room temperature during the first, second and third rounds of biopanning, respectively.
  • the wells were washed 5 times for the first round and 10 times for the later rounds with phosphate-buffered saline containing 0.05% Tween 20 (PBST) to remove nonspecifically bound phage, the bound phage were mixed with TGI competent cells for 1 hour at 37°C, and the phage was amplified from the infected cells and used in the next round of biopanning.
  • PBST phosphate-buffered saline containing 0.05% Tween 20
  • 380 clones were randomly picked from the infected TGI cells and each inoculated into 150 pl 2YT medium containing 100 pg/ml carbenicillin and 0.2% glucose in 96-well plates by using the automated BioRobotics BioPick colony picking system (Genomic Solutions, Ann Arbor, MI). After the bacterial cultures reached an optical density at 600 nm (OD600) of 0.5, helper phage M13K07 at a multiplicity of infection (MOI) of 10 and kanamycin at 50 pg/ml (final concentration) were added to the medium, and the plates were further incubated at 30°C overnight in a shaker at 250 rpm.
  • MOI multiplicity of infection
  • the phage supernatants were mixed with 3% nonfat milk in PBS at a 4: 1 volume ratio and used for enzyme-linked immunosorbent assay (ELISA) to identify clones of phage displaying ScFvs or VHs with high CD 19 binding affinity.
  • the supernatants were incubated for 2 h at room temperature with recombinant human CD 19 coated at 50 ng per well in 96-well plates and washed five times with PBST, (after overnight incubation at 4°C it was blocked with 3% nonfat milk in PBS and washed three times with PBS containing 0.05% Tween 20.) CD19-bound phage were detected using horseradish peroxidase-conjugated goat anti-M13 antibody.
  • VH and VL of the selected clones were DNA sequenced, and the ScFvs encoded by clones with unique sequences were expressed and purified as described below. Plasmids extracted from these clones were used for transformation of HB2151 cells. A single colony was picked from the plate containing freshly transformed cells, inoculated into 200 ml 2YT medium containing 100 pg/ml ampicillin and 0.2% glucose, and incubated at 37°C with shaking at 250 rpm. When the culture OD at 600 nm reached 0.90, isopropyl-P-d-thiogalactopyranoside at a 0.5 mM final concentration was added, and the culture was further incubated overnight at 30°C.
  • the bacterial pellet was collected after centrifugation at 8,000 x g for 20 min and resuspended in PBS buffer containing 0.5 mU polymixin B (Sigma-Aldrich, St. Louis, MO). After 30 min incubation with rotation at 50 rpm at room temperature, the resuspended pellet was centrifuged at 25,000 x g for 25 min at 4°C, and the supernatant was used for ScFv purification using the Ni-NTA resin following vendor protocol (Qiagen).
  • the same ScFv starting material as for phage display was also incorporated into a yeast ScFv display system.
  • yeast libraries expressing the human scFv library were also screened.
  • cell panning on CHOK1 transfected with CD19 cells was performed. For the first round of panning on the cell surface, two days prior to panning, the CHOK1-CD19 cells were seeded into 6-well plates and grown to 50% confluency in F12 K medium.
  • the cell mix containing both the yeast and CHOK1 cells were then inoculated into 10 mL SDCAA medium and amplified overnight at 30°C and then induced in SGCAA medium at 30°C for 16 hours.
  • SDCAA medium For the second round of cell panning, a similar protocol as above was performed, but more stringent wash conditions were used. This method of panning yielded the ml9217 binder. Further characterization of this binder as well as others from phage display indicated that affinity maturation was required, as the biological characteristics of the CAR created from this hit were still not optimal.
  • a yeast-display ml9217 mutant scFv library was created by using error-prone PCR to create random point mutations in scFv gene sequences. After electroporation, the resulting mutant library was then grown overnight at 30°C for 16 hours in SDCAA medium and then switched into SGCAA medium at 30°C for another 16 hours. The mutant library was then sorted through MACS (immunomagentic column, Miltenyi Biotec) with CD19-Fc as the capture antigen to downsize the library and to increase the population of mutants that could bind to CD19-Fc.
  • MACS microsomal analysis
  • the strongest binders were then selected by double staining the pools with Anti-c-Myc-Alexa 488 and CD19-Fc/Anti-Hu-Fc and selecting for the binders that had the highest binding affinities as well as c-Myc expression levels. This process was then repeated two more times, until flow cytometry of yeast particles with fluorescently tagged antigen yielded average binding affinities of the mutant pools that were increased over the starting construct. Binding affinities were estimated by flow cytometry of yeast pools using decreasing amounts of labeled CD 19.
  • phage display candidates did not yield biologically functional CAR constructs and thus ScFv identification that yielded biologically active binders were generated by yeast display.
  • human anti-CD19 ScFv binders M19217 (LTG2050, founder clone, EC50 of 0.5 ug/ml), and the following affinity matured binders (EC50 ⁇ 0.01 ug/ml): M19217-1 (LTG2065), Ml 9217-2 (LTG2066), Ml 9217-7 (LTG2067), Ml 9217-23 (LTG2068), Ml 9217-29 (LTG2069), M19217-38 (LTG2070), and M19217-40 (LTG2071) respectively.
  • a naive human ScFv (recombinant single chain fragment variable of immunoglobulin) phage display library (approximate diversity, 10 10 unique specificities), constructed from peripheral blood B cells of 50 healthy donors (Z. Y. Zhu and D. S. Dimitrov, unpublished data), were used for selection of ScFvs for recombinant human CD 19 protein (Miltenyi Biotec, unpublished).
  • Amplified libraries of 10 12 phage-displayed ScFv were incubated with 5, 3, and 1, pg of coated CD22 in a 5xl00-pl volume, distributed equally in 5 wells of a 96-well plate for 2 h at room temperature during the first, second and third rounds of biopanning, respectively.
  • the wells were washed 5 times for the first round and 10 times for the later rounds with phosphate-buffered saline containing 0.05% Tween 20 (PBST) to remove nonspecifically bound phage, the bound phage were mixed with TGI competent cells for 1 hour at 37°C, and the phage was amplified from the infected cells and used in the next round of biopanning.
  • PBST phosphate-buffered saline containing 0.05% Tween 20
  • 380 clones were randomly picked from the infected TGI cells and each inoculated into 150 pl 2YT medium containing 100 pg/ml carbenicillin and 0.2% glucose in 96-well plates by using the automated BioRobotics BioPick colony picking system (Genomic Solutions, Ann Arbor, MI). After the bacterial cultures reached an optical density at 600 nm (OD600) of 0.5, helper phage M13K07 at a multiplicity of infection (MOI) of 10 and kanamycin at 50 pg/ml (final concentration) were added to the medium, and the plates were further incubated at 30°C overnight in a shaker at 250 rpm.
  • MOI multiplicity of infection
  • the phage supernatants were mixed with 3% nonfat milk in PBS at a 4: 1 volume ratio and used for enzyme-linked immunosorbent assay (ELISA) to identify clones of phage displaying ScFvs or VHs with high CD22 binding affinity.
  • the supernatants were incubated for 2 h at room temperature with recombinant human CD22 coated at 50 ng per well in 96-well plates and washed five times with PBST, (after overnight incubation at 4°C it was blocked with 3% nonfat milk in PBS and washed three times with PBS containing 0.05% Tween 20.) CD22-bound phage were detected using horseradish peroxidase-conjugated goat anti-M13 antibody.
  • VH and VL of the selected clones were DNA sequenced, and the ScFvs encoded by clones with unique sequences were expressed and purified as described below. Plasmids extracted from these clones were used for transformation of HB2151 cells. A single colony was picked from the plate containing freshly transformed cells, inoculated into 200 ml 2YT medium containing 100 pg/ml ampicillin and 0.2% glucose, and incubated at 37°C with shaking at 250 rpm. When the culture OD at 600 nm reached 0.90, isopropyl-P-d-thiogalactopyranoside at a 0.5 mM final concentration was added, and the culture was further incubated overnight at 30°C.
  • the bacterial pellet was collected after centrifugation at 8,000 x g for 20 min and resuspended in PBS buffer containing 0.5 mU polymixin B (Sigma-Aldrich, St. Louis, MO). After 30 min incubation with rotation at 50 rpm at room temperature, the resuspended pellet was centrifuged at 25,000 x g for 25 min at 4°C, and the supernatant was used for ScFv purification using the Ni-NTA resin following vendor protocol (Qiagen). c) ELISA binding assay
  • the same ScFv starting material as for phage display was also incorporated into a yeast ScFv display system.
  • yeast libraries expressing the human scFv library were also screened.
  • cell panning on CHOK1 transfected with CD22 cells was performed. For the first round of panning on the cell surface, two days prior to panning, the CHOK1-CD22 cells were seeded into 6-well plates and grown to 50% confluency in F12 K medium.
  • the cell mix containing both the yeast and CHOK1 cells were then inoculated into 10 mL SDCAA medium and amplified overnight at 30°C and then induced in SGCAA medium at 30°C for 16 hours.
  • a similar protocol as above was performed, but more stringent wash conditions were used.
  • This method of panning yielded the 16P, 24P, 25P, 1 IS and 12S binders. Binder sequences were incorporated into CART constructs as described in Example 2, infra, in a series of in vitro CART functional assays. Characterization of these binders from phage display in CART format revealed that only 16P binder had specific tumor-lytic activity in vitro, but it was low as compared to CAR positive control.
  • a yeast-display mutant scFv library was created by using error-prone PCR to create random point mutations in scFv gene sequences. After electroporation, the resulting mutant library was then grown overnight at 30°C for 16 hours in SDCAA medium and then switched into SGCAA medium at 30°C for another 16 hours. The mutant library was then sorted through MACS (immunomagnetic column, Miltenyi Biotec) with CD22-Fc as the capture antigen to downsize the library and to increase the population of mutants that could bind to CD22-Fc.
  • MACS microsomal analysis
  • the strongest binders were then selected by double staining the pools with Anti-c- Myc-Alexa 488 and CD19-Fc/Anti-Hu-Fc and selecting for the binders that had the highest binding affinities as well as c-Myc expression levels. This process was then repeated two more times, until flow cytometry of yeast particles with fluorescently tagged antigen yielded average binding affinities of the mutant pools that were increased over the starting construct. Binding affinities were estimated by flow cytometry of yeast pools using decreasing amounts of labeled CD22.
  • ScFv for biologically active and highly specific binders were generated by yeast display. Based upon flow cytometry analysis of yeast-displayed ScFv, thirteen ScFv clones specific for recombinant human CD22 were identified and labeled as human anti-CD22 ScFv binders 16P (LTG2202, founder clone, EC50 of 0.5 ug/ml), and the following affinity matured binders (EC50 ⁇ 0.01 ug/ml): 16P1, 16P2, 16P3, 16P3v2, 16P6, 16P8, 16P10, 16P13, 16P15, 16P17, 16P20, and 16P20v2 respectively.
  • the generation of a tandem CAR incorporating the anti-CD22 scFv 16P17 sequence is outlined in EXAMPLE 2, infra.
  • This example discusses the creation of a dual-targeting CAR, which targets tumor antigens CD19 and CD22 simultaneously.
  • This approach has been postulated to help mitigate tumor antigen escape, which accounts for a significant portion of CAR therapy failures using single CAR approaches, namely anti-CD19 CAR, or anti-CD22 CAR therapy (Sotillo, Maria, et al. "Convergence of acquired mutations and alternative splicing of CD 19 enables resistance to CART-19 immunotherapy.” Cancer discovery (2015). Gardner, Rebecca, et al.
  • CD 19 is a 85-95 kDa transmembrane cell surface glycoprotein receptor.
  • CD 19 is a member of immunoglobulin (Ig) superfamily of proteins, and contains two extracellular Ig-like domains, a transmembrane, and an intracellular signaling domain (Tedder TF, Isaacs, CM, 1989, J Immunol 143:712-171).
  • Ig immunoglobulin
  • CD 19 modifies B cell receptor signaling, lowering the triggering threshold for the B cell receptor for antigen (Carter, RH, and Fearon, DT, 1992, Science, 256: 105- 107) , and co-ordinates with CD81 and CD21 to regulate this essential B cell signaling complex (Bradbury, LE, Kansas GS, Levy S, Evans RL, Tedder TF, 1992, J Immunol, 149:2841-50).
  • B cell ontogeny CD 19 is able to signal at the pro-B, pre-pre-B cell, pre-B, early B cell stages independent of antigen receptor, and is associated with Src family protein tyrosine kinases, is tyrosine phosphorylated, inducing both intracellular calcium mobilization and inositol phospholipid signaling (Uckun FM, Burkhardt AL, Jarvis L, Jun X, Stealy B, Dibirdik I, Myers DE, Tuel-Ahlgren L, Bolen JB, 1983, J Biol Chem 268:21172-84).
  • CD 19 is expressed in a tightly regulated manner on normal B cells, being restricted to early B cell precursors at the stage of IgH gene rearrangement, mature B cells, but not expressed on hematopoietic stem cells, or mature plasma cells (Anderson, KC, Bates, MP, Slaughenhout BL, Pinkus GS, Schlossman SF, Nadler LM, 1984, Blood 63: 1424- 1433).
  • Homo sapiens CD22 (SIGLEC-2, Leul4) is a well-investigated cell surface glycoprotein expressed on B cell leukemias and lymphomas. At least two anti-CD22 antibody drug (Inotuzumab Ozogamicin) or immunotoxin conjugates (Moxetumomab Pasudotox) have been the subject of clinical trials (NCT02981628, NCT00659425).
  • CAR Construct LTG1538 utilizing the mouse hybridoma FMC63-derived scFv is an activity control. This mouse-derived sequence is the current binder employed in commercial development (See KTE-C19, Kite Pharma, and CTL019, Novartis).
  • the m971 CAR LTG2200 is equivalent to the CAR being evaluated at the NCI, and is used as an anti- CD22 CAR control.
  • the Burkitt lymphoma cell line Raji, and the chronic myelogenous leukemia line K562 were purchased from American Tissue Culture Collection (ATCC, Manassass, VA).
  • the REH leukemia line was purchased from DSMZ (Leibniz Institute DSMZ, Braunschwieg, Germany). Cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT) and 2mM L-Glutamax (Thermo Fisher Scientific, Grand Island, NY).
  • Human Embryonic kidney line 293T was purchased from ATCC (Gibco/Thermo Fisher Scientific, Grand Island, NY).
  • Single-cell clones of luciferase-expressing cell lines were generated by stably transducing wild-type tumor lines with lentiviral vector encoding firefly luciferase (Lentigen Technology, Inc., Gaithersburg, MD), followed by cloning and selection of luciferase-positive clones.
  • the Raji clone was generated by passaging luciferase - transduced Raji cells in the mice and was selected for its proliferative capacity.
  • Whole blood was collected from healthy volunteers at Oklahoma Blood Institute (OBI) with donors’ written consent. Processed buffy coats were purchased from OBI (Oklahoma City, OK).
  • the CD4-positive and CD8-positive human T cells were purified from buffy coats via positive selection using a 1 : 1 mixture of CD4- and CD8- MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to manufacturer’s protocol.
  • CAR antigen-binding domains were derived from human anti-CD22 ScFv or heavy chain variable fragments.
  • CAR T constructs were generated by linking the binder sequence in frame to CD8a linking and transmembrane domains (aa 123-191, Ref sequence ID NP_001759.3), and then to 4-1BB (CD137, aa 214-255, UniProt sequence ID Q07011) signaling domain and CD3 zeta signaling domain (CD247, aa 52-163, Ref sequence ID: NP 000725.1).
  • CAR constructs sequences were cloned into a third generation lentiviral plasmid backbone (Lentigen Technology Inc., Gaithersburg, MD).
  • Lentiviral vector (LV) containing supernatants were generated by transient transfection of HEK 293T cells and vector pelleted by centrifugation of lentiviral vector-containing supernatants, and stored at -80°C.
  • T cells Human primary T cells from healthy volunteers were purified from whole blood or buffy coats (purchased from commercial provider with donor’s written consent) using immunomagnetic bead selection of CD4 + and CD8 + cells according to manufacturer’s protocol (Miltenyi Biotec, Bergisch Gladbach, Germany). T cells were cultivated in TexMACS medium supplemented with 200 lU/ml IL-2 at a density of 0.3 to 2 x 10 6 cells/ml, activated with CD3/CD28 MACS® GMP T Cell TransAct reagent (Miltenyi Biotec) and transduced on day 2 with lentiviral vectors encoding CAR constructs in the presence of 10 ug/ml protamine sulfate (Sigma- Aldrich, St. Louis, MO) overnight, and media exchanged on day 3. Cultures were propagated in TexMACS medium supplemented with 200 lU/ml IL-2 until harvest on day 8-13.
  • CTL assay To determine cell-mediated cytotoxicity (CTL assay), 5,000 target cells stably transduced with firefly luciferase were combined with CAR T cells at various effector to target ratios and incubated overnight. SteadyGlo reagent (Promega, Madison WI) was added to each well and the resulting luminescence quantified as counts per second (sample CPS). Target only wells (max CPS) and target only wells plus 1% Tween-20 (min CPS) were used to determine assay range. Percent specific lysis was calculated as: (1 -(sample CPS-min CPS)/(max CPS-min CPS)). Supernatants from co-cultures at E:T ratio of 10:1 were removed and analyzed by ELISA (eBioscience, San Diego, CA) for IFNy, TNFa and IL-2 concentration.
  • ELISA eBioscience, San Diego, CA
  • CAR T transduced cells were harvested from culture, washed two times in cold AutoMACS buffer supplemented with 0.5% bovine serum albumin (Miltenyi Biotec), and CAR surface expression detected by staining with CD22-Fc peptide followed by anti Fc-PE conjugate (Jackson ImmunoResearch, West Grove, PA).
  • Anti-CD4 antibody conjugated to VioBlue fluorophore was used where indicated, as per vendors’ protocol.
  • Nontransduced cells were used as negative controls. Dead cells in all studies were excluded by 7AAD staining (BD Biosciences, San Jose, CA).
  • Tandem CAR constructs for dual targeting of CD22 and CD 19 tumor antigens were developed in order to overcome tumor antigen escape. Shema of tandem CAR design is shown in FIGURES 1A and IB. Fully human scFv binders targeting CD19 and CD22 were connected in tandem by a flexible linker, in either 22-19 or 19-22 orientation, noted membrane distal to proximal. Then, the resulting CAR binding segment was linked in frame to CD8 hinge and transmembrane domain, 4- IBB costimulatory domain and CD3 zeta activation domain to generate CAR 22-19 (LTG2681, FIGURE 1A), or CAR19-22 (LTG2719, FIGURE IB). CAR sequences were incorporated into a 3rd generation lentiviral vectors and applied to primary human T cells for transduction.
  • CAR Construct LTG1538 utilizing the mouse hybridoma FMC63-derived scFv is an activity control. This mouse-derived sequence is the current binder employed in commercial development (See KTE-C19, Kite Pharma, and CTL019, Novartis).
  • the m971 CAR LTG2200 is equivalent to the CAR being evaluated at the NCI, and is used as an anti- CD22 CAR control.
  • FIGURE 2 The surface expression of anti-CD19/CD22 CARs incorporating single chain fragment variable (ScFv) sequences reactive with CD19/CD22 antigen, is shown in FIGURE 2.
  • the expression level for each ScFv-containing CAR was determined by flow cytometric analysis of LV-transduced T cells from healthy donors using one of two detection methods: i) CD22-his, followed anti-his-PE; ii) CD19 Fc recombinant protein, followed by anti Fc-A647.
  • tandem CAR 22-19 and CAR19-22 were functional in tumor lines co-expressing the CD 19 and CD22 antigens, and had no spontaneous killing activity against CD19-CD22- cell line 293T, underscoring target specificity of these constructs.
  • each scFv in a tandem CAR in isolation was tested by using tumor lines A431 or 293T, engineered to express only a single target antigen (CD19 or CD22, or irrelevant antigen CD20).
  • 293T-luc lines were created that express CD19 (293 T luc CD-19+), or CD22 (293 T luc -20+), and line CD20+ (293 T luc CD20) was used as an irrelevant target control (FIGURE 4A).
  • 293T-19+ were lysed by the CAR 19 (LTG1538) and tandem CAR 22-19 (LTG 2681), but not by CAR22 LTG 2200 or untransduced T cells control (FIGURE 3).
  • 293T-CD22+ were lysed by the tandem CAR 22-19 (LTG 2681) and the single CAR20 LTG2200, but not by the single CAR19 (LTG 1538), or untransduced control, demonstrating antigen specificity of the tandem CAR. Finally, no CAR construct lysed the 293 T luc CD20 cells, since this antigen was not targeted (FIGURE 4A) .
  • tandem CAR T cells were tested in an overnight killing assay against A431 clones expressing only CD 19 antigen (A431 luc CD 19), or only CD22 antigen (A431 luc-CD22), or an irrelevant target control line A431 luc -CD20, expressing the antigen CD20, which the tandem CARs 19-22 and 22-19 are not intended to recognize (FIGURE 4B).
  • A431 luc CD 19 line was lysed only by the CAR 19 (LTG1538) and tandem CAR 22-19 (LTG 2681), but not by CAR22 LTG 2200 or untransduced T cells control, whereas line A431 luc CD22 was lysed by the tandem CAR 22-19 (LTG 2681) and the single CAR20 LTG2200, but not by the single CAR19 (LTG 1538), or untransduced control, demonstrating antigen specificity of the tandem CAR. Moreover, no CAR construct lysed A431 luc CD20 cells, since this antigen was not targeted (FIGURE 4B). This results underscores the independent functionality and specificity of each targeting domain of the tandem CAR22-19 (FIGURES 4A-4B), and the potential of tandem CARs targeting the CD 19 and CD22 antigens to mitigate tumor antigen escape.
  • the capacity of anti-CD19/CD22 CAR T cells for cytokine secretion was then evaluated (FIGURE 5).
  • the CD19+CD22+ Raji tumor cells were co-incubated with the tandem 22-19 CAR T cells (LTG2681) or positive control CAR19 (LTG1538), positive control CAR22 (LTG2200), or negative control untransduced T cells (UTD) at effector to target ratio of 10: 1 overnight, and culture supernatants were analyzed by ELISA for IFN gamma, TNF alpha, and IL-2.
  • tandem CAR 22-19 strongly induced cytokines in response to tumor cells, whereas the negative control (untransduced, UTD.) yielded no appreciable cytokine induction.
  • tandem CAR T-expressing cells LTG2681 showed similar levels of IFN gamma, TNF alpha, IL-2, to CAR22 control (LTG2200), and somewhat higher cytokine response than the single CAR19 (LTG1538) control, demonstrating the high potency of the tandem CAR.
  • CAR 22-19 produced no cytokine secretion in the absence of tumor cells (CART alone group), which further confirms CAR specificity, and indicates a lack of tonic signaling by the tandem car.
  • Dual-targeting CAR 22-19 constructs incorporating various co-stimulatory domains demonstrate robust anti-tumor function
  • This Example discusses tandem CD22- and CD 19- dual targeting CAR constructs, which are incorporating various co- stimulatory domains.
  • the co-stimulatory domains utilized in CAR 22-19 design in this example included ICOS, 0X40, CD27, CD137/4-1BB, and CD28.
  • These costimulatory domain sequences are derived from T cell surface molecules known to be involved in positive regulation of T cell function, including T cell activation, expansion, persistence, phenotypic differentiation, memory formation, and anti-tumor responses (Zhao Z, Condomines
  • Concurrent dual targeting of CD 19 and CD22 antigens is designed to mitigate tumor antigen escape, which has hindered therapeutic benefit in a sub-population of patients who have received either CD 19 or CD22-targeted single CAR therapy (Sotillo, Maria, et al. "Convergence of acquired mutations and alternative splicing of CD 19 enables resistance to CART- 19 immunotherapy.” Cancer discovery (2015). Gardner, Rebecca, et al. "Acquisition of a CD 19 negative myeloid phenotype allows immune escape of MLL-rearranged B-ALL from CD 19 CAR- T cell therapy.” Blood (2016): blood-2015., Fry, Terry J., et al. "CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19-targeted CAR immunotherapy.” Nature medicine 24.1 (2016): 20.).
  • the Burkitt lymphoma cell line Raji was purchased from American Tissue Culture Collection (ATCC, Manassass, VA). Cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT) and 2mM L-Glutamax (Thermo Fisher Scientific, Grand Island, NY). Human Embryonic kidney line 293T was purchased from ATCC (Gibco/Thermo Fisher Scientific, Grand Island, NY).
  • Single-cell clones of luciferaseexpressing cell lines were generated by stably transducing wild-type tumor lines with lentiviral vector encoding firefly luciferase (Lentigen Technology, Inc., Gaithersburg, MD), followed by cloning and selection of luciferase-positive clones.
  • CD19 and CD22 expressing 293T cell line clones designated 293TCD19 and 293TCD22, respectively, were generated by lentiviral transduction of human CD 19 protein and human CD22 proteins into the parental 293T-luciferase expressing clone, following by single-cell cloning, selection and expansion of CD 19- or CD22- positive target cell clones, as appropriate.
  • Stable luciferase-expressing Raji clone was generated by passaging luciferase - transduced Raji cells in the mice and was selected for its proliferative capacity.
  • Whole blood was collected from healthy volunteers at Oklahoma Blood Institute (OBI) with donors’ written consent.
  • Processed buffy coats were purchased from OBI (Oklahoma City, OK).
  • the CD4-positive and CD8-positive human T cells were purified from buffy coats via positive selection using a 1 : 1 mixture of CD4- and CD8- MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to manufacturer’s protocol.
  • the tandem CAR antigen-binding domain was derived from a human anti-CD22 ScFv and human anti-CD19 scFv sequences, linked in tandem, in configuration anti-CD22 scFv-anti CD 19- scFv-hinge -transmembrane domain - endodomain.
  • CAR T constructs were generated by linking the CAR antigen-binding domain in frame to CD8a hinge and transmembrane domains (aa 123-191, Ref sequence ID NP 001759.3), and then to 4-1BB (CD137, aa 214-255, UniProt sequence ID Q07011) signaling domain and CD3 zeta signaling domain (CD247, aa 52-163, Ref sequence ID: NP 000725.1).
  • 4-1BB co-stimulatory domain was substituted for the human ICOS, CD27, CD28, OX-40 co-stimulatory domain, using the full signaling domain sequence of each molecule.
  • the CD8 transmembrane domain was substituted by transmembrane sequence derived from same protein as the costimulatory domain.
  • the endodomain of the CAR comprised two costimulatory domains connected in tandem.
  • no co-stimulatory domain was used, and the CD3( ⁇ activation domain was linked in frame directly to the transmembrane domain.
  • two CAR chains were encoded in the same bicistronic expression cassette, separated by 2A ribosomal skip element, thus enabling co-expression of the two CAR chains in each T cell transduced with the lentiviral construct encoding the bicistronic CAR.
  • Lentiviral vector (LV) containing supernatants were generated by transient transfection of HEK 293T cells and vector pelleted by centrifugation of lentiviral vector-containing supernatants, and stored at - 80°C.
  • T cells Human primary T cells from healthy volunteers were purified from whole blood or buffy coats (purchased from commercial provider with donor’s written consent) using immunomagnetic bead selection of CD4 + and CD8 + cells according to manufacturer’s protocol (Miltenyi Biotec, Bergisch Gladbach, Germany). T cells were cultivated in TexMACS medium supplemented with 200 lU/ml IL-2 at a density of 0.3 to 2 x 10 6 cells/ml, activated with CD3/CD28 MACS® GMP T Cell TransAct reagent (Miltenyi Biotec) and transduced on day 2 with lentiviral vectors encoding CAR constructs in the presence of 10 ug/ml protamine sulfate (Sigma- Aldrich, St. Louis, MO) overnight, and media exchanged on day 3. Cultures were propagated in TexMACS medium supplemented with 200 lU/ml IL-2 until harvest on day 8-13.
  • CTL assay To determine cell-mediated cytotoxicity (CTL assay), 5,000 target cells stably transduced with firefly luciferase were combined with CAR T cells at various effector to target ratios and incubated overnight. SteadyGlo reagent (Promega, Madison WI) was added to each well and the resulting luminescence quantified as counts per second (sample CPS). Target only wells (max CPS) and target only wells plus 1% Tween-20 (min CPS) were used to determine assay range. Percent specific lysis was calculated as: (1 -(sample CPS-min CPS)/(max CPS-min CPS)). Supernatants from co-cultures at E:T ratio of 10:1 were removed and analyzed by ELISA (eBioscience, San Diego, CA) for IFNy, TNFa and IL-2 concentration. (e) Flow Cytometric Analysis
  • CAR T transduced cells were harvested from culture, washed two times in cold AutoMACS buffer supplemented with 0.5% bovine serum albumin (Miltenyi Biotec), and CAR surface expression detected by staining with CD22-His peptide followed by anti-His secondary detection reagent, simultaneously with CD19 Fc peptide followed by anti-Fc conjugate (the secondary detection reagents were purchased form Jackson ImmunoResearch, West Grove, PA).
  • Anti-CD4 antibody conjugated to VioBlue fluorophore was used where indicated, as per vendors’ protocol. Non-transduced cells were used as negative controls.
  • Dual-targeting CAR constructs comprised of different co-stimulatory domains were designed.
  • CAR constructs are listed in Table 1. Schematic representation of CAR design configurations is provided in FIGURES 6A-D.
  • the tandem CAR antigen-binding domain comprised of anti-CD22 ScFv and anti-CD19 scFv sequences, were linked in tandem, in the following order: anti-CD22 scFv-anti CD19-scFv-hinge -transmembrane domain - endodomain (FIGURES 6A and 6B).
  • the targeting tandem domain configuration was based on CAR 22-19 (LTG2681) in all cases.
  • Anti-CD 22-19 CAR construct LTG2737 contained the CAR sequence identical to the Anti-CD 22-19 CAR construct LTG2681, but without use of the Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE) during its construction.
  • WP Woodchuck Hepatitis Virus
  • WPRE Posttranscriptional Regulatory Element
  • CAR T constructs were generated by linking the CAR antigen-binding domain in frame to CD8a hinge and transmembrane domains (constructs LTG2737, D0135, D0136, D0137, D0145, D0146, D0147, D0148, and D0149).
  • a transmembrane domain sequence matching the co-stimulatory domain was utilized: CD28 for D139, D140, 0X40 for D0137, DO 147, DO 148.
  • the transmembrane domain was linked in frame to a co-stimulatory domain derived from 4-1BB (LTG2737), CD28 (D0135, D0139, D0140), ICOS (D136, D146, D148, D149), 0X40 (D0137, D0145, D0147, D0148) or CD27 (D0138, D0149).
  • All CAR molecules contained the CD3 zeta signaling domain (CD247, aa 52-163, Ref sequence ID: NP 000725.1).
  • the endodomain of the CAR was comprised of two co-stimulatory domains, CD28 and 4-1BB, connected in tandem (D0140, FIGURE 6B).
  • two distinct CAR molecules were co-expressed in the same T cell using a 2A ribosomal skip element for bicistronic expression (D146, D147, D148, D149, FIGURE 6C, 6D).
  • each CAR chain contained only one scFv, targeting either the CD 19 or CD22 antigen, and both chains were co-expressed in each transduced T cell via transduction with a single lentiviral vector encoding the bicistronic sequence.
  • no co-stimulatory domain was used, so that the CD3( ⁇ activation domain was linked in frame directly to the transmembrane domain of one of the CAR chains expressed concurrently in the same cell (DO 146, DO 147, FIGURE 6C).
  • CAR constructs sequences were cloned into a third generation lentiviral plasmid backbone under the control of the human EF-la promoter (Lentigen Technology Inc., Gaithersburg, MD).
  • FIGURE 7 The surface expression of anti-CD22-19 CAR incorporating various co-stimulatory domains, is shown in FIGURE 7.
  • the expression level for each ScFv-containing CAR was determined by flow cytometric analysis of LV-transduced T cells from healthy donors using simultaneous staining for the two scFv CAR targeting domains i) CD22-his, followed anti-his-PE; ii) CD19 Fc recombinant protein, followed by anti Fc-A647. All anti-CD22-19 CAR were highly expressed in human primary T cells as compared to non-transduced T cell controls. CAR expression levels ranged from 71%-90% (FIGURE 7).
  • FIGURES 9A-D high cytolytic activity of the anti-CD22-19 CAR was demonstrated: Human primary T cells were transduced with LV encoding CAR constructs LTG2737, D0135, D0136, D0137, D0138, D0139, D0140, D0145, D0146), then incubated for 18 hours with the Raji, 293 T, 293TCD19 or 293TCD22 cell lines, stably transduced with firefly luciferase, for luminescence based in vitro killing assays. Effector to target (ET) ratios of 2.5: 1, 5: 1 or 10:1 were used, as noted in the legend to the right of each plot.
  • E effector to target
  • Raji cells express CD19 and CD22 on their surface, while the negative controls, 293T do not.
  • the 293TCD19 and 293TCD22 target lines were generated to stably express either the CD 19 or CD22 target antigen, respectively, and were used to evaluate the capability of the dual-targeting CAR constructs with different costimulatory domains to accomplish target lysis when triggered by each single target antigen, CD 19 or CD22, independently of the other antigen.
  • the capacity of anti-CD22-19 CAR with various co- stimulatory domains for cytokine secretion was then evaluated (FIGURE 8).
  • the CD19+CD22+ Raji tumor cells were coincubated with the tandem anti-CD22-19 CAR T cells expressing constructs LTG2737, D0135, D0136, D0137, D0138, D0139, D0140, D0145, D0146, or negative control untransduced T cells (UTD) at effector to target ratio of 10: 1 overnight, and culture supernatants were analyzed by ELISA for IFN gamma, TNF alpha, and IL-2 (FIGURE 8).
  • anti-CD22-19 CAR produced little to no cytokine secretion in the absence of tumor cells (CAR T alone group), which further confirms CAR specificity, and indicates a lack of tonic signaling by the tandem anti-CD22-19 CAR with various co-stimulatory domains.
  • the Burkitt lymphoma cell line Raji was cultured in Corning Glutgro RPMI-1640 (Coming, NY) and 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT). Raji cells were transduced with firefly luciferase gene. The Raji clone used herein was generated by transplanting luciferase expressing Raji cells in the mice and was selected for its proliferative capacity.
  • CD 19 knockout (KO), CD22KO and CD19CD22 double KO lines were generated based on luciferase-expressing Raji using CRISPR Cas9 gene editing approach.
  • Rajil9KO line LentiCRISPR v2 plasmids carrying human single guide RNA (sgRNA, AAGCGGGGACTCCCGAGACC, Genscript, Piscataway, NJ), were electroporated into Raji cells with Lonza 4D- Nuclefector X unit.
  • Alt-R CRISPR-Cas9 crRNA RNP complex (guide RNA: CCCGAGTGCTGGACCTTCAC, PAM CGQ IDT, Coralville, Iowa) was generated and introduced into Raji cell lines via electroporation as vender instruction.
  • guide RNA CCCGAGTGCTGGACCTTCAC, PAM CGQ IDT, Coralville, Iowa
  • CD19 or CD22 microbeads Single cell cloning was achieved by limiting dilution (0.5 cell per 96 well). All the knock out cell lines were verified with surface protein staining by flow cytometry and DNA sequencing.
  • CD19CD22 double KO line was generated based on CD22KO line followed same procedure as aforementioned CD 19 knockout strategy.
  • CD19CD22 double KO Raji cell line was stably transduced with lentiviral vector encoding CD 19 or CD22 fused with puromycin by F2A peptide.
  • F2A peptide F2A peptide
  • Cytotoxicity overnight killing assay was performed to evaluate functionality of different CAR constructs as described elsewhere (73). Briefly, CAR T cells were co-cultured with 5,000 target cells carrying firefly luciferase activity for 16-18 hours at various effector to target ratios. SteadyGlo reagent (Promega, Madison WI) was added to each well, the resulting luminescence was quantified by GloMax Luminometer and used to calculate specific percentage of lysis.
  • Flow cytometric analysis was performed to detect the cell surface molecule expression.
  • Cells were washed and stained with cold AutoMACS buffer supplemented with 0.5% bovine serum albumin (Miltenyi Biotec), and resuspended in 200 ul Running Buffer before acquired by MACSQuant®10 Analyzer (Miltenyi Biotec).
  • CD22-his peptide(Abcam, Cambridge, MA) and CD19Fc peptide R&D System, Minneapolis, MN
  • anti His-PE or anti Fc-AF647 Jackson ImmunoResearch, West Grove, PA.
  • T cell subtypes was further identified with anti-CD4 antibody conjugated to VioBlue fluorophore.
  • ABS antibody binding capacity assay was performed as per manufacturer’s protocol using BD Bioscience ( San Jose, CA) PE Fluorescence Quantitation Kit and BD anti human CD19-PE(SJ25C1) and anti-human CD22-PE(S-HCL-1).A11 the antibodies and staining reagents listed were from Miltenyi Biotec unless stated otherwise. Flow data were analyzed with FlowJo software (Ashland, OR).
  • NSG NOD.Cg-/ J /'A/c' c ' f/ mice were injected with
  • Tumor burden was measured using bioluminescent imaging by Xenogen IVIS-200 instrument (Perkin Elmer, Shelton, Connecticut). Mice with comparable tumor burden were randomly distributed into each group for CAR T administration at day seven. Kinetics of tumor development were measured by living animal imaging at indicated time point. Tissue samples, including blood, bone marrow and spleen were harvested upon request.
  • Mouse blood, bone marrow and spleen were harvested at the endpoint of animal study.
  • Tissue samples were extracted with DNeasy Blood & Tissue Kit (Qiagen, Germantown, MD) as manual instructed, and genomic DNA was quantified on Nanodrop (ThermoFisher, Carlsbad, CA).
  • Nanodrop ThermoFisher, Carlsbad, CA
  • Fifty ng DNA per sample was used per reaction in realtime PCR with TaqMan Fast Advanced Master (ThermoFisher, Carlsbad, CA) on BioRad CFX qPCR instrument. Primer and probes were synthesized by IDT Technology.
  • Thermal cycling conditions is 50 °C 2 minutes, followed by 95 °C 20 seconds, 40 repeats of 95°C 5 seconds, 56 °C 20 seconds and 65 °C 20 seconds 3 -step amplification cycle.
  • Viral copy number (VCN) of each sample was calculated based on standard curve.
  • Tan For tandem CARs, Tan For: 5’- GAGCGACATAGGCAACAAGA-3’,
  • Tan probe 5 ACAGAAACCAGGTCAAGCACCTGT-3’.
  • Dual-targeting probe 5’-ACTACGCCGTGTCCGTGAAGAATC-3’
  • a series of CAR constructs capable of simultaneous targeting of CD 19 and CD22 B-cell antigens were constructed. All constructs were fully human, and utilized human scFv sequences against CD19 (ml9217-l), and CD22 (16P17). Second generation CARs, third generation CARs and bicistronic dual-targeting CARs to CD19 and CD22 were constructed (Table 2).
  • CAR D0145 was the least effective CAR in vivo (Fig. 10A and 10B), despite showing greater expression and functionality than D0137 in vitro ( Figures 9A-9D).
  • CAR D0135 performed better than D0139 in vivo, despite similar in vitro function (Fig. 10A, IOC, Figures 9A-9D).
  • the expansion and persistence of the 22-19 CAR variants in vivo was measured in peripheral blood on study days 14, 21 and 28 by flow cytometric enumeration of total human T cells (Fig.
  • the third generation CAR 22-19 with the 28-BB co-stimulation was able to maintain the longest persistence of the total T cells as well as the CD8 + T compartment, which through differentiation to CTLs (cytotoxic T cell lymphocytes, an effector subset capable of killing tumor cells), is thought to contribute greatly to CAR overall anti-tumor function.
  • CTLs cytotoxic T cell lymphocytes, an effector subset capable of killing tumor cells
  • early CAR expansion on day 14 was necessary for effective tumor clearance, even in the absence of long-term blood persistence, as seen for CARs D0146, D0135, D0136, D0137 and D0138.
  • long-term persistence in the absence of early CAR T expansion was insufficient for tumor clearance, as seen for the second generation 22-19 BB tandem CAR LTG2737, and CAR DO 145.
  • CAR constructs that mediated strong tumor regression in the wild-type Raji xenograft were further investigated in the Raji antigen- heterogeneous model.
  • Mice were implanted with an equal ratio mixture of Raji 19KO, Raji22KO, and the wild-type Raji tumor cells, all stably expressing firefly luciferase, to facilitate detection of tumor progression.
  • tandem CAR 22-19 BB( ⁇ (LTG2737) anti-tumor function in vivo can be modulated, and dramatically improved, by the utilization of alternative costimulatory domains.
  • the optimization of CAR 22-19 co-stimulatory sequences, hinge and transmembrane domains, as well as implementation of the third generation CAR architecture yielded tandem 22-19 CAR candidates with superior anti-tumor functionality. It is therefore important to determine whether these improved CAR constructs are better suited for detecting low- antigen density tumor cell variants, which remain a daunting obstacle for CAR clinical efficacy.
  • Alternative co-stimulatory domains improved 22-19BBz CAR T cell response towards tumor targets with low antigen density
  • Tandem CAR constructs and single-targeting CAR22 control were coincubated with Raji 111 , or Raji low targets at E:T ratio of 5: 1, 10:1 or 20: 1 overnight.
  • the reduction of lysis potency with reduced CD22 surface density was most prominent for the LTG2737 tandem 22- 19 CAR construct with BB co-stimulatory domain, and especially dramatic loss of cytolytic ability was seen against the CD22 low target density line (Fig. 12B).
  • nucleic and amino acid sequences listed below are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing:
  • SEQ ID NO: 1 nucleotide sequence of LTG2681 D0023 Leader-CD22 VH-(GGGGS)-3 CD22 VL (GGGGS)-5 CD 19 VH (GGGGS)-3 CD 19 VL CD8 hinge+TM-4-lBB- CD3z (Construct CAR 2219)
  • SEQ ID NO: 3 nucleotide sequence of LTG2791 D0024 Leader-CD19 VH (GGGGS)3 - CD 19 VL -(GGGGS)5 -CD22 VH (GGGGS)3 - CD22 VH CD8 hinge+TM-4-lBB- CD3z (Construct CAR 1922)
  • SEQ ID NO: 5 nucleotide sequence of fully human CAR19 LTG2065 (M19217-1-CD8 TM- 4- IBB zeta)
  • SEQ ID NO: 7 nucleotide sequence of mouse scFv CAR19 LTG1538
  • SEQ ID NO: 11 nucleotide sequence of leader/signal peptide sequence (LP) atgctgctgctggtgaccagcctgctgctgtgcgaactgccgcatccggcgtttctgctgattccg
  • SEQ ID NO: 35 nucleotide sequence of DNA CD8 transmembrane domain atttgggccccgctggccacttgcggcgtgctcctgctgtcgctggtcatcaccctt tactgc
  • SEQ ID NO: 37 nucleotide sequence of DNA CD8 hinge domain actaccacccctgcccctcggccgccgactccggccccaaccatcgcaagccaacccctc tccttgcgccccgaagcttgccgcccggccgcgggtggagccgtgcatacccgggggctg gactttgcctgcgatatctac
  • SEQ ID NO: 39 amino acid sequence of amino acid numbers 137 to 206 hinge and transmembrane region of CD8. alpha. (NCBI RefSeq: NP. sub. —001759.3)
  • SEQ ID NO: 40 nucleotide sequence of DNA signaling domain of 4- IBB aagaggggccggaagaagctgctttacatcttcaagcagccgttcatgcggcccgtgcag acgactcaggaagaggacggatgctcgtgcagattccctgaggaggaagaggggggatgc gaactg
  • SEQ ID NO: 42 nucleotide sequence of DNA signaling domain of CD3-zeta cgcgtcaagttctcacggtccgccgacgccccgcatatcaacagggccagaatcagctc tacaacgagctgaacctgggaaggagagaggagtacgacgtgctggacaagcgacgcgga cgcgacccggagatgggggggggaaaccacggcggaaaaccctcaggaaggactgtacaac gaactccagaaagacaagatggcggaagcctactcagaaatcgggatgaagggagagcgg aggaggagagcgg aggaggaggaggagagcgg aggaggaggaggaggaggtcacgacgggctgtaccagggactgagggaccg
  • SEQ ID NO: 43 amino acid sequence of CD3zeta Arg Vai Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gin Gin Gly Gin Asn Gin Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Vai Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gin Glu Gly Leu Tyr Asn Glu Leu Gin Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu He Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gin Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gin Ala Leu Pro Pro Arg
  • SEQ ID NO: 46 nucleotide sequence of anti-CD33 CAR (LTG1936)
  • SEQ ID NO: 48 nucleotide sequence of anti-mesothelin CAR (LTG1904)
  • SEQ ID NO: 50 nucleotide sequence of heavy chain scFv 16P17
  • SEQ ID NO: 52 nucleotide sequence of light chain scFv 16P17
  • SEQ ID NO: 54 nucleotide sequence of heavy chain scFv Ml 9217-1
  • SEQ ID NO: 55 nucleotide sequence of heavy chain scFv Ml 9217-1
  • SEQ ID NO: 56 nucleotide sequence of light chain scFv M19217-1
  • SEQ ID NO: 58 nucleotide sequence of CAR22 LTG2200 (M971-CD8TM-4-lBB-zeta)
  • SEQ ID NO: 60 nucleotide sequence of CAR LTG2737 (CD22-19 CD8 BBz)
  • TTCCCAA ACCCTTTCTCTCTCACGTGTGCAATTAGCGGCGATAGTGTATCCTCTAA
  • SEQ ID NO: 68 nucleotide sequence of CARD0138 CD22-19 CD8 CD27z ATGCTCTTGCTCGTGACTTCTTTGCTTTTGTGCGAACTTCCGCACCCAGCCTTC
  • TTCCCAA ACCCTTTCTCTCTCACGTGTGCAATTAGCGGCGATAGTGTATCCTCTAA
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