8224035
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`UNITED STATES DEPARTMENT OF COJV[M:ERCE
`
`United Stntcs Pntcnt nnd Trndcmnrk Office
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`March 11, 2022
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`THIS IS TO CERTIFY THAT ANNEXED IS A TRUE COPY FROM THE
`RECORDS OF THIS OFFICE OF THE FILE WRAPPER AND CONTENTS
`OF:
`
`APPLICATION NUMBER: 61/421,470
`FILING DATE: December 09, 2010
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`Certified by
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`Under Secretary of Commerce
`for Intellectual Property
`and Director of the United States
`Patent and Trademark Office
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`Miltenyi Ex. 1020 Page 1
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`046483-6001-Pl-US (600640)
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`TITLE OF THE INVENTION
`Compositions and Methods for Treatment of Chronic Lymphocytic Leukemia
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`STATEMENT REGARDING FEDERALL YSPONSORED RESEARCH OR
`DEVELOPMENT
`This invention was made with U.S. government support under Grant Nos.
`1RO1CA105216, RO1AI057838 and ROl 1113482 awarded by the National Institutes of
`Health (NIH). The government has certain rights in the invention.
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`BACKGROUND OF THE INVENTION
`The large majority of patients having B-cell malignancies, including chronic
`lymphocytic leukemia (CLL) will die from their disease. One approach to treating these
`patients is to genetically modify T cells to target antigens expressed on tumor cells
`through the expression of chimeric antigen receptors (CARs), CARs are antigen
`receptors that are designed to recognize cell surface antigens in a human leukocyte
`antigen-independent manner. Attempts in using genetically modified cells expressing
`CARs to treat these types of patients have met with very limited success. See for
`example, Brentjens et al., 2010, Molecular Therapy, 18:4, 666-668; Morgan et al., 2010,
`20 Molecular Therapy, published online Febrnary 23, 2010, pages 1-9; and, Till et al., 2008,
`Blood, 112:2261-2271.
`, Thus, there is an urgent need in the art for compositions and methods for
`treatment of CLL, The present invention addresses this need.
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`DETAILED DESCRIPTION
`The present invention provides compositions and methods for treatment of CLL.
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`Compositions
`As exemplified elsewhere herein, the present invention includes a vector
`comprising an crCD19 CAR transgene, Preferably, the vector is a retroviral vector. More
`preferably, the vectm· is a self-inactivating lentiviral vector as described elsewhere herein.
`The invention also includes autologous cells that are transfected with the vector of
`the invention. Preferably, the cells are T cells and more preferably, they are autologous T
`cells.
`
`Methods of Treating
`As described in detail elsewhere herein, the invention includes a method of
`treating an patient with CLL, wherein the method comprises administering to the patient
`autologous T cells that have been transfected so as to express a CD 19 CAR antigen.
`Preferably, the cells are administered to the patient by infusion.
`The precise protocols used to treat patients are described elsewhere herein and can
`also be found at clinicaltrials.dot.gov under trial no NCT01029366. Interim results from
`this study, where three patients were treated, establish that successful expansion and
`.transduction of T cells was accomplished in all three patients. The actual manufacturing
`of final product was more difficult in CLL patients than in previous trials, CARs with 4-
`1 BB:z signaling domains have massive expansion in vivo in two of three patients with
`advanced CLL. The cells persisted in blood and migrated to bone marrow for at least
`sixty days in substantial numbers post-infusion. CART cells expanded in vivo compared
`to the infused amount. Anti-tumor effects were observed: patient 1 CR, patient 2 PR,
`patient 3 CRn, And) the treatment was promising in chemotherapy refractory patients.
`
`Definitions
`Unless defined otherwise, all technical and scientific tem1s used herein have the
`same meaning as conunonly understood by one of ordinary skill in the at1 to which this
`invention belongs. Although any methods and materials similar or equivalent to those
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`described herein can be used in the practice or testing of the present invention, the
`preferred methods and materials are described.
`As used herein, each of the following terms has the meaning associated with it in
`this section.
`The articles "a" and "an" are used herein to refer to one or to more than one (i.e.,
`to at least one) of the grammatical object of the article. By way of example, "an element"
`means one element or more than one element.
`'~About" as used herein when referring to a measurable value such as an amount, a
`temporal duration, and the like, is meant to encompass variations of ±20% or ±10%,
`10 more preferably ±5%, even more preferably ±1 %, and still more preferably ±0.1 % from
`the specified value, as such variations are appropriate to perform the disclosed methods,
`The term "antibody" as used herein, refers to an immunoglobulin molecule, which
`is able to specifically bind to a specific epitope on an antigen, Antibodies can be intact
`immunoglobulins derived from natural sources or from recombinant sources and can be
`immunoactive portions of intact immunoglobulins. Antibodies are typically tetramers of
`immunoglobulin molecules. The antibodies in the present invention may exist in a
`variety of forms including, for example, polyclonal antibodies, monoclonal antibodies,
`Fv, Fab and F(ab)2, as well as single chain antibodies and humanized antibodies.
`The term "antigen" or "Ag" as used herein is defined as a molecule that provokes
`an immune response. This immune response may involve either antibody production, or
`the activation of specific immunologically-competent cells, or both, The skilled artisan
`will understand that any macromolecule, including vittually all proteins or peptides, can
`serve as an antigen. Fmihermore, antigens can be derived from recombinant or genomic
`DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide
`sequences or a partial nucleotide sequence encoding a protein that elicits an immune
`response can encode an ''antigen" as that term is used herein. Furthermore, one skilled in
`tl}e art will understand that an antigen need not be encoded solely by a full length
`nucleotide sequence of a gene, It is readily apparent that the present invention includes,
`but is not limited to, the use of partial nucleotide sequences of more than one gene and
`that these nucleotide sequences can be arranged in various combinations to elicit the
`desired immune response. Moreover, a skilled artisan will understand that an antigen
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`need not be encoded by a "gene" at all. It is readily apparent that an antigen can be
`generated synthesized or can be derived from a biological sample. Such a biological
`sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a
`biological fluid,
`A "coding region" of a gene consists of the nucleotide residues of the coding
`strand of the gene and the nucleotides of the non-coding strand of the gene which are
`homologous with or complementary to, respectively, the coding region of an mRNA
`molecule which is produced by transcription of the gene.
`A "coding region'' of an mRNA molecule also consists of the nucleotide residues
`of the mRN A molecule which are matched with an anti-codon region of a transfer RNA
`molecule during translation of the mRNA molecule or which encode a stop codon. The
`coding region may thus include nucleotide residues corresponding to amino acid residues
`which are not present in the mature protein encoded by the mRNA molecule (e.g., amino
`acid residues in a protein expo1t signal sequence).
`"Encoding" refers to the inherent property of specific sequences of nucleotides in
`a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for
`synthesis of other polymers and macromolecules in biological processes having either a
`defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of
`amino acids and the biological properties resulting therefrom. Thus, a gene encodes a
`protein if transcdption and translation of mRN A corresponding to that gene produces the
`protein in a cell or other biological system. Both the coding strand, the nucleotide
`sequenc~ of which is identical to the mRNA sequence and is usually provided in
`sequence listings, and the non-coding strand, used as the template for transcription of a
`gene or cDNA, can be referred to as encoding the protein or other product of that gene or
`cDNA.
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`Unless otherwise specified; a "nucleotide sequence encoding an amino acid
`sequence" includes all nucleotide sequences that are degenerate versions of each other
`and that encode the same amino acid sequence. Nucleotide sequences that encode
`proteins and RNA may include introns.
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`A "disease" is a state of health of an animal, preferably a mammal and more
`preferably, a human, wherein the animal cannot maintain homeostasis, and wherein if the
`disease is not ameliorated then the animal's health continues to deteriorate,
`In contrast, a "disorder" in· an animal is a state of health in which the animal is
`able to maintain homeostasis, but in which the animal's state of health is less favorable
`than it would be in the absence of the disorder. Left untreated, a disorder does not
`necessarily cause a further decrease in the animal's state of health,
`A disease or disorder is "alleviated" if the severity of a symptom of the disease or
`disorder, the frequency with which such a symptom is experienced by a patient, or both,
`is reduced,
`An "effective amount" or "therapeutically effective amount" of a compound is
`that amount of compound which is sufficient to provide a beneficial effect to the subject
`to which the compound is administered. An "effective amount" of a delivery vehicle is
`that amount sufficient to effectively bind or deliver a compound.
`As used herein, the term "fragment," as applied to a nucleic acid, refers to a
`subsequence of a larger nucleic acid, A "fragment" of a nucleic acid can be at least about
`15 nucleotides in length; for example, at least about 50 nucleotides to about 100
`nucleotides; at least about 100 to about 500 nucleotides, at least about 500 to about 1000
`nucleotides, at least about 1000 nucleotides to about 1500 nucleotides; or about 1500
`nucleotides to about 2500 nucleotides; or about 2500 nucleotides (and any integer value
`in between).
`As used herein, the term Hfragment," as applied to a protein or peptide, refers to a
`subsequence of a larger protein or peptide, A "fragment" of a protein or peptide can be at
`least about 20 amino acids in length; for example at least about 50 amino acids in length;
`at least about 100 amino acids in length, at least about 200 amino acids in length, at least
`about 300 amino acids in length, and at least about 400 amino acids in length (and any
`integer value in between).
`The term "inununoglobulin" or "lg", as used herein is defined as a class of
`proteins, which function as antibodies. The five members included in this class of
`proteins are IgA, lgG, IgM, lgD, and IgE. IgA is the primary antibody that is present in
`body secretions, such as saliva, tears, breast milk, gastrointestinal secretions and mucus
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`secretions of the respiratory and genitourinary tracts. IgG is the most common
`circulating antibody. IgM is the main immunoglobulin produced in the primary immune
`response in most mammals. It is the most efficient immunoglobulin in agglutination,
`complement fixation, and other antibody responses, and is important in defense against
`bacteria and viruses. IgD is the immunoglobulin that has no known antibody function,
`but may serve as an antigen receptor. IgE is the immunoglobulin that mediates
`immediate hypersensitivity by causing release of mediators from mast cells and basophils
`upon exposure to allergen.
`"Isolated" means altered or removed from the natural state. For example, a
`nucleic acid or a peptide naturally present in a living animal is not '~isolated," but the
`same nucleic acid or peptide partially or completely separated from the coexisting
`materials of its natural state is '{isolated." An isolated nucleic acid or protein can exist in
`substantially purified form, or can exist in a non-native environment such as, for
`example, a host cell.
`"Naturally occurring" as used herein describes a composition that can be found in
`nature as distinct from being mtificially produced. For example, a nucleotide sequence .
`present in an organism, which can be isolated from a source in nature and which has not
`been intentionally modified by a person in the laboratory, is naturally occurring.
`Unless otherwise specified, a "nucleotide sequence encoding an amino acid
`sequence" includes all nucleotide sequences that are degenerate versions of each other
`and that encode the same amino acid sequence. The phrase nucleotide sequence that
`encodes a protein or an RNA may also include introns to the extent that the nucleotide
`sequence encoding the protein may in some version contain an intrnn(s).
`The term "polynucleotide" as used herein is defined as a chain of nucleotides.
`Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and
`polynucleotides as used herein are interchangeable. One skilled in the art has the general
`knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the
`monomeric "nucleotides." The monomeric nucleotides can be hydrolyzed into
`nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic
`acid sequences which are obtained by any means available in the art, including, without
`limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a
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`recombinant library or a cell genome, using ordinary cloning technology and PCRTri\ and
`the like, and by synthetic means.
`As used herein, the terms "peptide," "polypeptide," and "protein" are used
`interchangeably, and refer to a compound comprised of amino acid residues covalently
`linked by peptide bonds. A protein or peptide must contain at least two amino acids, and
`no limitation is placed on the maximum number of amino acids that can comprise a
`protein's or peptide's sequence. Polypeptides include any peptide or protein comprising
`two or more amino acids joined to each other by peptide bonds. As used herein, the term
`refers to both short chains, which also commonly are referred to in the art as peptides,
`oligopeptides and oligomers, for example, and to longer chains, which generally are
`referred to in the art as proteins, of which there are many types. "Polypeptides" include,
`for example, biologically active fragments, substantially homologous polypeptides,
`oligopeptides, homodimers, heterodimers, variants of polypeptides, modified
`polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides
`include natural peptides, recombinant peptides, synthetic peptides, or a combination
`thereof.
`The terms "patientt "subject," "individualt and the like are used interchangeably
`herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to
`the methods described herein. Preferably, the patient, subject or individual is a mammal,
`and more preferable, a human.
`The term "treatmenf' as used within the context of the present invention is meant
`to include therapeutic treatment as well as prophylactic, or suppressive measures for the
`disease or disorder. Thus, for example, the term treatment includes the administration of
`an agent prior to or following the onset of a disease or disorder thereby preventing or
`removing all signs of the disease or disorder. As another example, administration of the
`agent after clinical manifestation of the disease to combat the symptoms of the disease
`comprises "treatment" of the disease.
`A "therapeutic" treatment is a treatment administered to a subject who exhibits
`signs of pathology, for the purpose of diminishing or eliminating those signs.
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`As used herein, "treating a disease or disorder" means reducing the frequency
`with which a symptom of the disease or disorder is experienced by a patient. Disease and
`disorder are used interchangeably herein.
`"Variant" as the term is used herein, is a nucleic acid sequence or a peptide
`sequence that differs in sequence from a reference nucleic acid sequence or peptide
`sequence respectively, but retains essential propet1ies of the reference molecule.
`Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence
`of a peptide encoded by the reference nucleic acid, or may result in amino acid
`substitutions, additions, deletions, fusions and truncations. Changes in the sequence of
`peptide variants are typically limited or conservative, so that the sequences of the
`reference peptide and the variant are closely similar overall and, in many regions,
`identical. A variant and reference peptide can differ in amino acid sequence by one or
`more substitutions, additions, deletions in any combination. A variant of a nucleic acid
`or peptide can be a naturally occurring such as an allelic variant, or can be a variant that
`is not known to occur naturally. Non-naturally occurring variants of nucleic acids and
`peptides may be made by mutagenesis techniques or by direct synthesis.
`For sequence comparison, typically one sequence acts as a reference sequence, to
`which test sequences may be compared. When using a sequence comparison algorithm,
`test and reference sequences are input into a computer, subsequent coordinates are
`designated, if necessary, and sequence algorithm program parameters are designated. The
`sequence comparison algorithm then calculates the percentage sequence identity for the
`test sequence(s) relative to the reference sequence, based on the designated program
`parameters. ·
`As used herein, "vaccination" is intended for prophylactic or therapeutical
`vaccination.
`Ranges: throughout this disclosure, various aspects of the invention can be
`presented in a range format. It should be understood that the description in range format
`is merely for convenience and brevity and should not be construed as an inflexible
`limitation on the scope of the invention. Accordingly, the description of a range should be
`considered to have specifically disclosed all the possible subranges as well as individual
`numerical values within that range. For example, description of a range such as from 1 to
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`6 should be considered to have specifically disclosed subranges such as from 1 to 3, from
`1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual
`numbers within that range, for example, 1, 2 1 2.7 1 3 1 4, 5, 5.3, and 6. This applies
`regardless of the breadth of the range.
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`EXPERIMENT AL EXAMPLES
`The invention is further described in detail by reference to the following
`experimental examples. These examples are provided for purposes of illustration only,
`and are not intended to be limiting unless otherwise specified. Thus, the invention should
`in no way be construed as being limited to the following examples, but rather, should be
`construed to encompass any and all variations which become evident as a result of the
`teaching provided herein.
`Without fmiher description, it is believed that one of ordinary skill in the
`ai1 can, using the preceding description and the following illustrative examples, make and
`utilize the compounds of the present invention and practice the claimed methods. The
`following working examples therefore, specifically point out the preferred embodiments
`of the present invention, and are not to be construed as limiting in any way the remainder
`of the disclosure.
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`Example I: Chimeric Receptors Containing CD 13 7 Signal Transduction Domains
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`Mediate Enhanced Survival of T Cells and Increased Anti"Leukemic Efficacy In Vivo
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`Abstract
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`Persistence of T cells engineered with chimeric antigen receptors (CARs) has been a
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`major barrier to use of these cells for molecularly targeted adoptive immunotherapy. To
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`address this issue, we created a series of CARs that contain the TCR-s signal
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`transduction domain with the CD28 and/or CD137 (4-lBB) intracellular domains in
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`tandem. After short-term expansion, primary human T cells were subjected to lentiviral
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`gene transfer, resulting in large numbers of cells with >85% CAR expression. In an
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`immunodeficient mouse xenograft model of primary human pre-B-cell acute
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`lymphoblastic leukemia, human T cells expressing anti-CDI9 CARs containing CD137
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`exhibited the greatest anti-leukemic efficacy and prolonged (>6 months) survival in vivo,
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`and were significantly more effective than cells expressing CARs containing TCR-l;
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`alone or CD28-s signaling receptors, We uncovered a previously unrecognized, antigen(cid:173)
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`independent effect of CARs expressing the .CD 13 7 cytoplasmic domain that likely _
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`contributes to the enh_anced antileukemic efficacy and survival in tumor bearing mice.
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`Furthermore, our studies revealed significant discrepancies between in vitro and in vivo
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`surrogate measures of CAR efficacy. Together these results suggest that incorporation of
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`the CD 137 signaling domain in CARs should improve the persistence of CAR.s in the
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`tumor microenvironment and hence maximize their anti tumor activity.
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`With the advent of efficient gene transfer technologies, such as murine retroviral
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`and HIV-derived lentiviral vectors, it has become feasible to confer novel antigenic
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`specificity to T cells by transfer of chimeric antigen receptors (CARs) with stable, long(cid:173)
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`tenn expression. This technology has been used to generate T cells specific for HIV and
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`several human tumor antigens, and some of these engineered T cells have been tested in
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`Phase I/II studies in humans demonstrating the feasibility and relative safety of this
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`approach [1-3]. One study has demonstrated anti-tumor activity in patients with
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`neuroblastoma given a single CAR infusion [4].
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`CARs combine the antigen recognition domain of antibody ~th the intracellular
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`· domain of the TCR-t;, chain or FcyRI protein into a single chimeric protein that are
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`capable of triggering T-cell activation in a manner very similar to that of the endogenous
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`TCR [5, 6]. Several studies de1nonstrate that the addition of co-stimulatory domains,
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`particularly the intracellular domain of CD28 can significantly augment the ability of
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`these receptors to stimulate cytokine secretion and enhance antitumor•efficacy in pre(cid:173)
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`clinical animal models using both solid tumors and leukemia that lack the expression of
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`the CD28 receptor ligands CD80 and CD86 [7-9]. Inclusion of domains from receptors
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`such as the TNF receptor family members, CD134(OX-40) and CDI37 (4-lBB) into
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`CARs has also been shown to augment CAR-mediated T-cell responses [10,11]. Gene
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`transfer approaches using these engineered CARs may therefore provide significant
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`improvements over current adoptive immunotherapy strategies that must rely upon the
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`endogenous TCR specificities, for which significant issues of TCR repertoire limitation
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`and impaired tumor MHC class I expression may exist.
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`In this study, we have addressed the issue of limited in vivo persistence of CARs
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`by defining the relative contributions ofTCR-s, CD137 and CD28 signaling domains in
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`mice engrafted with hematopoietic malignancies. We chose the human CD 19 antigen as
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`our initial target for several reasons: 1) CD19 displays a pattern of expression that is
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`highly restricted to B-cells and B-cell progenitor cells [12], 2) CD 19 does not appear to
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`be expressed by hematopoietic stem cells permitting the targeting of the B-cell lineage
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`without affecting other hematopoietic lineages (13], and 3) CD 19 is widely expressed by
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`malignant cells that are derived from the B cell lineage including most lymphomas and
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`lymphocytic leukemias (14]. After optimizing the generation of CARs with an efficient
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`T-cell culture process, in vitro studies indicate that inc01poration of either CD28 or 4-
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`IBB signaling domains enhances activity over TCR--s, confirming previous studies. In
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`contrast, compared to CARs that contain CD28, our in vivo studies indicate that CARs
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`containing CD 137 have superior antileukemic efficacy and improved persistence in a
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`primary human acute lymphoblastic leukemia xenograft model. Furthermore, we also
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`find that CARs expressing CD137 signaling domains can provide significant activity that
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`appears to be antigen independent and may contribute to the efficacy of CARs in vivo.
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`Results
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`E/ficie11t generation CAR. T cells using artificial bead-based APCs and lentivira/ gene .
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`transfer
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`Lentiviral vectors can transfer genes with into activated CD4+ and CDS+ human
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`T cells with high efficiency but_ expression of the vector-encoded transgene depends on
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`the internal promoter that drives its transcription. Therefore, successful CAR expression
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`and gene therapies with CAR-expressing T cells rely on the ability ofT cells to maintain
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`adequate receptor expression over long periods of time, We tested several promoters to
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`identify the one with the highest stable expression in both primary CD4+ and CD8+ T
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`cells. Transduction was perfonned at limiting dilution to ensure that the cells have a
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`single integrated vector per cell (data not shown). While the CMV promoter exhibited
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`high levels of expression of a GFP transgene early after transduction, expression .
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`decreased to < 25% of the initial expression after 10 days of culture (Fig. 1 b ). The
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`distribution of CMV-driven GFP expression was also quite variable compared with the
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`other promoters tested (Suppl. Fig. S1). In contrast, the EF-la promoter not only
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`induced the highest level of GFP expression but also optimally maintained it in both CD4
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`cells and CD8 cells (Fig. 1b). These findings confirmed and extended other studies in
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`primary human T cells [15]. The EF-la promoter was therefore selected for all future
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`studies using CARs. By using lentiviral vectors and transductions at an MOI of 5, the
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`different CARs could be expressed high expression in > 85% primary human T cells
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`(Fig. le). Western blotting under both reducing and non-reducing conditions
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`demonstrated that the CARs are present as both covalent dimers and monomers within T
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`cells (Suppl. Fig. S2). Using the artificial bead-based APC system previously described
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`by our laboratory [16], > 50-fold expansion of CAR+ T cells could be achieved over the
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`course of transduction and growth in~ 10 days (Fig. ld).
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`Fu11ctio11al characterization of anti-CD19 CAR-expressing primary ltuma11 T cells
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`To enhance the functionality of the immunoreceptor, we introduce the signal
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`transduction domains of CD28 or CD 13 7 in the TCR-l; containing CAR (Fig. 1 a).
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`Similar to data reported by other groups [11,17], the introduction of costimulatory
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`domains into CARs does not improve the antigen-specific cytotoxicity triggered by these
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`receptors (Fig. 2b). Lytic activity of transduced T cells against K562 target cells
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`expressing CD 19 correlated with the transduction efficiency of the T cells ( data not
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`shown). CAR-triggered cytotoxicity is antigen-specific with only negligible lysis ofwild(cid:173)
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`type K562 cells that lack expression of the CD19 antigen (Fig. 2a). CAR+ T cells are
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`also able to efficiently kill primary pre-BALL cells that express physiologic levels of
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`CD19 (Fig. 2b). Of note, these primary ALL cells lack expression of endogenous CO80
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`or CD86 ( data not shown).
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`Following CAR activation with CD19+ K562 cells, CD4+ T cells expressing
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`CARs produced abundant quantitiesofIL-2 and IFN-y (Fig. 3) comparable to cells
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`stimulated via the endogenous TCR and CD28 receptors (data not shown). T cells
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`expressing CD28 and CD 13 7 domain containing CARs produced greater quantities of IL-
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`2 when compared with cells expressing the aCD19-<'.; receptor (Fig. 3). The production of
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`the type 2 cytokines, IL-4 and IL- I 0, by CD4+ T cells was also stimulated by all of the
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`CARs tested; however, the levels of these cytokines were much.lower, consistent with the
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`Th I-like phenotype ofT cells generated by anti-CD3 and CD28 stimulatory beads [16). It
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`was notable that the incorporation of the CD 13 7 domain into CARs decreased the
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`production of these type 2 cytokines, consistent with previous repo1is of the 4~ lBB
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`signaling pathway .in natural T cells [18]. All CARs stimulated IFN-y production by
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`CDS+ T cells. These findings confirm that the addition of co-stimulatory domains into
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`CARs modulates cytokine secretion in a manner that is dependent on the type of
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`costimulatory domain [7,S,17,19]. However, it is less well appreciated that the pattern of
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`cytokine expression is altered by incorporation of different signal transduction domains
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`into the CARs. These differences may have imp011ant consequences for the functionality
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`ofT cells engineered to express CARs.
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`The effects of costimulatory domains on CAR-driven T cell proliferation
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`The generation of a robust and sustained anti-twnor immune response requires not
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`only triggering of cytotoxicity and cytokine production but also stimulation of T-cell
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`proliferation. To assess the relative contribution of different costimulatory domains to
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`proliferative signals delivered by CARs~ we engineered primary human T cells to express
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`CARs in conjunction with GFP to permit evaluation of both CAR+ and CAR"T cells in
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`the same culture. Following T-cell re-stimulation with CDI9+ K562 (K562-CD19 cells))
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`T cells expressing the a CD 19-28-l; receptor exhibited proliferation comparable to that
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`obtained with full stimulation of the endogenous TCR complex with K562 cells loaded
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`with anti-CD3 and CD28 antibodies~ a condition shown previously to support long-term
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`expansion of primary human T cells (KT32-BBL) [20] (Fig, 4a[v]). The aCD19-28-BB(cid:173)
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`S triple receptor also stimulated CD19 driven proliferation (Fig. 4a[iv]), but to a lesser
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`extent that the aCD19-28-l; double costimulatory receptor. No significant proliferation
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`was observed when these same T cells. were stimulated with wild-type K562 cells lacking
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`the CD19 antigen (K.562 wt). As previously shown by other investigators [17,19)21)22),
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`T cells expressing the a CD 19...t;, receptor showed little proliferation upon exposure to the.
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`surrogate CD 19 antigen (Fig. 4a[ii]), demonstrating the dependence of CAR-driven
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`proliferation on co-stimulatory signals.
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`Unexpectedly, T cells containing the aCD 19-BB-s double costimulatocy domain
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`CAR had significantly increased proliferative capacity during in vitro expansion
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`Miltenyi Ex. 1020 Page 17
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`independently of receptor ligation with the surrogate CD19 antigen (Fig. 4a[iii] and 4b).
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`This increased proliferation was obse1ved in both CD4+ and CD8+ T cells (data not
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`shown), and it was associated with a prolonged blast phase after the initial stimulation
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`and transduction, as revealed by a longer maintenance of an elevated mean cellular
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`volume (Fig. 4c), a parameter that correlates well with log phase proliferation ofT cells
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`[16]. These findings suggest that incorporation of the CD137 intracellular domain
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`mediates antigen-independent activity that is similar to that provided by the natural 4-
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`lBB receptor in T cells following ligation [23]. As a result of the enhanced proliferation
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`obse1ved following the initial activation of T cells via aCD3/aCD28-coated beads used
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`to enhance T-cell transduction [24,25] (Fig 4b), CAR+ T cells expressing the aCD19-
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`BB-½ receptor had relatively low CAR-driven proliferation (Fig. 4a[iii]).
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`Evaluating anti-tumor responses of CAR+ human primary T cells in vivo
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`Other than the antigen-in.dependent proliferation of the 4-lBB containing CAR,
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`the above in vitro findings, in aggregate, suggested that the aCD19-28-l; CAR would be
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`the most effective receptor for generating a sustained anti-leukemic T_cell response in
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`vivo. We evaluated the in vivo efficacy of aCD19 CARs in vivo model of ALL (Fig. 5a)
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`in which primary human pre-BALL cel