(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY(PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`3 October 2002 (03.10.2002)
`
`
`
`(10) International Publication Number
`WO 02/077029 A2
`
`(51) International Patent Classification’:
`
`(US). FORMAN,Stephen [US/US]; 2580 Oak Knoll Av-
`enue, San Marino, CA 91108 (US). RAUBITSCHEK,An-
`drew [US/US]; 1691 El Molino, San Marino, CA 91108
`(21) International Application Number:=PCT/US01/42997
`(US).
`
`C07K 14/705
`
`(22) International Filing Date:
`7 November 2001 (07.11.2001)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`60/246,117
`
`7 November 2000 (07.11.2000)
`
`US
`
`(74) Agents: FIGG, E., Anthonyet al.; Rothwell, Figg, Ernst
`& Manbeck, P.C., Suite 701-E, 555 13th Street, N.W, Wash-
`ington, DC 20004 (US).
`
`(81) Designated States (national): AU, CA, JP, US.
`
`(84) Designated States (regional): European patent (AT, BE,
`CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC,
`NL,PT, SE, TR).
`
`(71) Applicant: CITY OF HOPE [US/US]; 1500 East Duarte
`Road, Duarte, CA 91010-3000 (US).
`
`without international search report and to be republished
`upon receipt of that report
`
`Published:
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): JENSEN, Michael,
`C. [US/US]; 2305 Woodlyn Road, Pasadena, CA 91104
`
`For two-letter codes and other abbreviations, refer to the "Guid-
`ance Notes on Codes andAbbreviations" appearing at the begin-
`ning ofeach regular issue ofthe PCT Gazette.
`
`(54) Title: CD19-SPECIFIC REDIRECTED IMMUNE CELLS
`
`oGh pAn
`
`On COlE1
`
` Pacli751)
`
`
`
`~ Span
`CMV Promoter
`
`
`A
`
`WO02/077029A2
`
`Intron A
`
`CD19R scFvFc:Zeta
`
`chimeric T cell receptor by electroportion using naked DNA encodingthe receptor.
`
`engineered,
`Genetically
`(57) Abstract:
`redirected
`immune
`cells
`CD19-specific
`expressing a cell surface protein having an
`extracellular domain comprising a receptor
`which is specific for CD19, an intracellular
`signaling domain,
`and a transmembrane
`domain.
`Use of such cells for cellular
`
`immunotherapy of CD19* malignancies
`and for abrogating any untoward B cell
`function.
`In one embodiment, the immune
`cell is a T cell and the cell surface protein is
`a single chain svFvFc:€
`receptor where scFc
`designates the Vy and V, chains of a single
`chain monoclonal antibody to CD19, Fe
`represents at least part of a constant region
`of an IgG, and €
`represents the intracellular
`signaling domain of the zeta chain of human
`CD3.
`The extracellular domain scFvFc
`
`and the intracellular domain ¢ are linked
`by a transmembrane domain such as the
`transmembrane domain of CD4. A method
`
`of making a redirected T cell expressing a
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`PCT/US01/42997
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`CD19-SPECIFIC REDIRECTED IMMUNE CELLS
`
`Cross-reference to Related Application:
`
`[0001]
`
`This application claims priority to Provisional Application Serial No.
`
`60/246,117, filed November 7, 2000, the disclosure of which is incorporated by reference.
`
`BACKGROUND OF THE INVENTION
`
`[0002]
`
`This invention relates to the field of genetically engineered, redirected
`
`immunecells andto the field of cellular immunotherapy of B-cell malignancies, B-cell
`
`lymphoproliferative syndromes and B-cell mediated autoimmunediseases.
`
`The publications and other materials used herein to illuminate the background ofthe
`
`invention or provide additionaldetails respecting the practice are incorporated by
`
`reference.
`
`[0003]
`
`Approximately half of all hematopoietic stem cell transplantation (HSC)
`
`procedures performed inthe United States are for the treatment of hematologic
`
`malignancy [1]. Theinitial obstacles for successful HSC transplantation were in large part
`
`due to inadequate treatment modalities for ameliorating regimen-related toxicities and for
`
`controlling opportunistic infections and graft-versus-host disease (GVHD)[2-5]. As
`
`supportive care measures have improved overthe last decade, post-transplant disease
`
`relapse has emerged as the major impediment to improving the outcomeofthis patient
`population [6-10]. The inability of maximally intensive preparative regimens combined
`with immunologic graft-versus-tumorreactivity to eradicate minimal residual disease is
`
`the mechanism oftreatment failure in allogeneic transplantation while, in the autologous
`
`setting, tumor contamination of the stem cell graft can also contribute to post-transplant
`
`relapse [11]. Targeting minimalresidual disease early after transplantation is one strategy
`to consolidate the tumor cytoreduction achieved with myeloablative preparative regimens
`
`and purge, in vivo, malignant cells transferred with autologous stem cell grafts. The utility
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`of therapeutic modalities for targeting minimal residual disease shortly following stem
`
`cell rescue is dependent on both a limited spectrum of toxicity and the susceptibility of
`
`residual tumorcells to the modality's antitumor effector mechanism(s). The successful
`elimination of persistent minimalresidual disease should not only have a major impact on
`the outcomeoftransplantation for hematologic malignancy utilizing current
`
`myeloablative preparative regimens but may also provide opportunities to decrease the
`
`intensity of these regimens andtheir attendanttoxicities.
`
`[0004]
`
`The prognosis for patients with bcr-abl positive Acute Lymphoblastic
`
`Leukemia (ALL) treated with chemotherapy is poor and allogeneic transplantation has
`
`offered a curative option for many patients when an appropriate donor was available. For
`example at the City of Hope, 76 patients with bcr-abl positive ALL were treated with
`allogeneic Bone Marrow Transplantation (BMT) from a HLA matched donor. Of these
`patients, 26 were in first remission, 35 were transplantedafter first remission. The two
`year probability of disease free survival was 68% with a 10% relapserate in those patients
`transplanted in first remission whereas for those patients transplantedafter first remission,
`the disease-free survival and relapse rate were 36% and 38%,respectively [12]. Post-
`transplant Polymerase Chain Reaction (PCR) screening of blood and marrow for ber-abl
`transcript is under evaluation as a molecular screening tool for identifying early those
`transplant recipients at high risk for later developmentof overt relapse [13,14]. Patients
`
`for whom detectable p190 transcript was detected following BMThad a 6.7 higher
`
`incidence of overt relapse than PCR negative patients. The median time from the
`
`development ofa positive signal to morphologic relapse was 80-90 days in these patients.
`The identification of patients in the earliest phases of post-transplant relapse affords the
`
`opportunity for making therapeutic interventions when tumor burden is low and
`potentially most amenableto salvage therapy.
`[0005]
`Recent advancesin the field of immunology have elucidated many of the
`
`molecular underpinnings of immune system regulation and have provided novel
`
`opportunities for therapeutic immune system manipulation, including tumor
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`immunotherapy. Evidence supporting the potential of immune-mediated eradication of
`
`residual tumorcells following allogeneic transplantation can be inferred by comparing the
`
`disparate relapse rates between recipients of syngeneic and non-T cell depleted matched
`
`sibling transplants. Patients with chronic myelogenous leukemia in chronic phase (CML-
`CP), acute myelogenousleukemia in first complete remission (1* CR), and acute
`
`lymphoblastic leukemia in 1 CR who received a marrow transplant from a syngeneic
`
`donorhad an actuarial probability of relapse at 3 years of 45%, 49%, and 41%,
`
`respectively, whereas the rates for recipients of a non-T depleted marrow transplant from
`
`an HLA identical sibling for the same diseases were 12%, 20%, and 24%, respectively
`
`[15-17]. The reduction of relapse rates following allogeneic bone marrow transplantation
`
`has been most significant in patients who develop acute and/or chronic GVHD.Currently,
`efforts are focused on developing strategies to selectively augmentthe graft-versus-
`leukemia (GVL) responsein order to reduce post-transplant relapse rates without the
`
`attendant toxicities of augmented GVHD.
`
`[0006]
`
`Studies in animal models have established that donor MHC-restricted CD8*
`
`and CD4* a/f* T cells specific for minor histocompatibility antigens encoded by
`
`polymorphic genes that differ between the donor andrecipient are the principle mediators
`
`of acute GVHD and GVL [18-21]. Recently, patients with CML in chronic phase who
`
`relapse after allogeneic BMT havebeenidentified as a patient population for whom the
`
`infusion of donor lymphocytes (DLI) successfully promotes a GVL effect [22,23].
`
`Complete response rates of approximately 75% are achieved with DLIcell doses in the
`range of 0.25-12.3x10° mononuclear cells/kg [24]. Although the antitumoractivity of
`
`donor lymphocyte infusion underscores the potential of cellular immunotherapy for CML,
`
`the clinical benefit of DLI has not been generalizable to all forms of hematologic
`
`malignancy. Relapsed ALL is muchless responsive to DLI with a reported CR rate of
`
`less than 20%; when tumor responses are observed, they are typically associated with
`
`significant GVHD morbidity and mortality [25]. In order to increase the therapeutic ratio
`
`of DLI, genetic modification of donor lymphocytes to express a suicide gene is being
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`evaluated as a strategy to permit the in vivo ablation of donor lymphocytes should toxicity
`
`from GVHDwarrant this maneuver [26,27]. Alternately, efforts are underwayto identify
`
`genes encoding minor histocompatibility antigens (mHA’s) with restricted hematopoietic
`expression thatelicit donor antigen-specific T cell responses. The isolation, ex vivo
`expansion, andre-infusion of donor-derived clones specific for these mHA’s hasthe
`potential of selectively augmenting GVL following allogeneic bone marrow
`
`transplantation [28-30].
`[0007]
`Non-transformed B-cells and malignant B-cells express an array ofcell-
`surface moleculesthat define their lineage commitment and stage of maturation. These
`
`were identified initially by murine monoclonal antibodies and more recently by molecular
`genetic techniques. Expression of several of these cell-surface molecules is highly
`restricted to B-cells and their malignant counterparts. CD20 is a clinically useful cell-
`
`surface target for B-cell lymphoma immunotherapy with anti-CD20 monoclonal
`antibodies. This 33-kDaprotein hasstructural features consistent with its ability to
`
`function as a calcium ion channel and is expressed on normal pre-B and mature B cells,
`
`but not hematopoietic stem cells nor plasmacells [31-33]. CD20 does not modulate nor
`does it shed from the cell surface [34]. Jn vitro studies have demonstrated that CD20
`
`crosslinking by anti-CD20 monoclonalantibodies can trigger apoptosis of lymphomacells
`[35,36]. -Clinical trials evaluating the antitumoractivity of chimeric anti-CD20 antibody
`IDEC-C2B8 (Rituximab) in patients with relapsed follicular lymphoma have documented
`tumor responsesin nearly half the patients treated, although the clinical effect is usually
`transient [37-40]. Despite the prolonged ablation of normal CD20* B-cells, patients
`receiving Rituximab have not manifested complications attributable to B-cell
`lymphopenia [41]. Radioimmunotherapy with *I-conjugated and °°Y-conjugated anti-
`CD20antibodies also has shown promisingclinical activity in patients with
`
`relapsed/refractory high-grade Non-Hodgkins Lymphomabut hematopoietic toxicities
`from radiation have been significant, often requiring stem cell support [42].
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`[0008]
`
`Unlike CD20, CD19 is expressed on all human B-cells beginning from the
`
`initial commitmentof stem cells to the B lineage andpersisting until terminal
`
`differentiation into plasmacells [43]. CD19 is a type I transmembrane protein that
`
`associates with the complement 2 (CD21), TAPA-1, and Leu13 antigens forming a B-cell
`
`signal transduction complex. This complex participates in the regulation of B-cell
`proliferation [44]. Although CD19 does not shed from thecell surface, it does internalize
`[45]. Accordingly, targeting CD19 with monoclonal antibodies conjugated with toxin
`molecules is currently being investigated as a strategy to specifically deliver cytotoxic
`
`agents to the intracellular compartment of malignant B-cells [46-48]. Anti-CD19
`antibody conjugatedto blocked ricin and poke-weedantiviral protein (PAP) dramatically
`
`increase specificity and potency of leukemiacell killing both in ex vivo bone marrow
`purging procedures and when administered to NOD-SCIDanimals inoculated with CD19*
`leukemiacells [49]. Jn vitro leukemia progenitor cell assays have provided evidence that
`
`the small percentage of leukemic blasts with the capacity for self-renewal express CD19
`
`on their cell surface. This conclusion was derived from the observations that leukemic
`
`progenitor activity is observed exclusively in fresh marrow samples sorted for CD19
`positive cells and is not observed in the CD19 negative cell population [50]. Additionally
`B43-PAPtreatmentof relapsed leukemic marrow specimensablates progenitorcell
`
`activity while a PAP conjugated antibody with an irrelevant specificity had no such
`
`activity [51]. Systemic administration of the CD19-specific immunotoxin B43-PAP is
`
`currently undergoing investigation in phase I/II clinicaltrials in patients with high risk
`
`pre-B ALL[52].
`
`[0009]
`
`Despite the antitumoractivity of monoclonal anti-CD20 and anti-CD19
`
`antibody therapy observed in clinicaltrials, the high rate of relapse in these patients
`
`underscores the limited capacity of current antibody-based immunotherapyto eliminateall
`
`tumorcells [53]. In contrast, the adoptive transfer of tumor-specific T cells can result in
`complete tumoreradication in animal models and a limited numberofclinical settings
`
`[54,55]. Theability of transferred T cells to directly recognize and lyse tumortargets,
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`produce cytokines that recruit and activate antigen non-specific antitumor effector cells,
`migrate into tumor masses, and proliferate following tumor recognition all contributeto
`
`the immunologic clearance of tumorby T cells [56]. Expression-cloning technologies
`have recently permitted the genetic identification of a growing number of genes expressed
`by human tumors to which T cell responses have beenisolated [57,58]. To date leukemia
`and lymphoma-specific antigens have not been identified that are both broadly expressed
`by malignant B-cells and elicit T cell responses. Consequently, preclinical and clinical
`investigation has focused on combining antibody targeting of tumors with T cell effector
`
`mechanisms by constructing bispecific antibodies consisting of CD20 or CD19 binding
`
`sites and a bindingsite for a cell-surface CD3 complex epitope. Such bispecific
`antibodies can co-localize leukemia and lymphomatargets with activated T cells resulting
`
`in target cell lysis in vitro [59-61]. The in vivo antitumoractivity of such bispecific
`antibodies has been limited, however, both in animal models as well as in clinical practice
`
`[62]. The discrepancy betweenin vitro activity and in vivo effect likely reflects the
`inherent limitations in antibody immunotherapy compoundedbythe obstacles associated
`
`with engaging T cells and tumor cells via a soluble linker in a mannerthat yields a
`
`persistent and functional cellular immuneresponse [63].
`
`[00010]
`
`The safety of adoptively transferring antigen-specific CTL clones in
`
`humanswasoriginally examined in bone marrow transplant patients who received donor-
`
`derived CMV-specific T cells [56]. Previous studies have demonstrated that the
`
`reconstitution of endogenous CMV-specific T cell responses following allogeneic bone
`
`marrow transplantation (BMT)correlates with protection from the development of severe
`
`CMVdisease [64]. In an effort to reconstitute deficient CMV immunity following BMT,
`
`CD8* CMV-specific CTL clones were generated from CMV seropositive HLA-matched
`
`sibling donors, expanded, and infused into sibling BMTrecipients at risk for developing
`CMVdisease. Fourteen patients were treated with four weekly escalating doses of these
`CMV-specific CTL clones to a maximum cell dose of 10° cells/m? without any attendant
`toxicity [65]. Peripheral blood samples obtained from recipients of adoptively transferred
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`T cell clones were evaluated for in vivo persistence of transferred cells. The recoverable
`CMV-specific CTL activity increased after each successive infusion of CTL clones, and
`persisted at least 12 weeks after the last infusion. However, long term persistence of
`CD8*clones without a concurrent CD4* helper response was not observed. Nopatients
`developed CMV viremia or disease. These results demonstrate that ex-vivo expanded
`CMV-specific CTL clones can be safely transferred to BMTrecipients and can persist in
`vivo as functional effector cells that may provide protection from the development of
`
`CMVdisease.
`
`A complication of bone marrow transplantation, particularly when marrow
`[00011]
`is depleted of T cells, is the development of EBV-associated lymphoproliferative disease
`[66]. This rapidly progressiveproliferation ofEBV-transformed B-cells mimics
`immunoblastic lymphomaand is a consequence of deficient EBV-specific T cell
`immunity in individuals harboring latent virus or immunologically naive individuals
`receiving a virus inoculum with their marrow graft. Clinicaltrials by Rooneyetal. have
`demonstrated that adoptively transferred ex-vivo expanded donor-derived EBV-specific T
`cell lines can protect patients at high risk for developmentof this complication as well as
`mediate the eradication of clinically evident EBV-transformed B cells [54]. No
`significant toxicities were observed in the forty-one children treated with cell doses in the
`range of 4x10’ to 1.2x10° cells/m’.
`[00012]
`Genetic modification of T cells used in clinical trials has been utilized to
`
`markcells for in vivo tracking and to endow T cells with novel functional properties.
`
`Retroviral vectors have been used most extensively for this purpose duetotheir relatively
`
`high transduction efficiency and lowin vitro toxicity to T cells [67]. These vectors,
`however, are time consuming and expensive to prepare as clinical grade material and
`
`must be meticulously screened for the absence of replication competent viral mutants
`
`[68]. Rooneyet al. transduced EBV-reactive T cell lines with the NeoR geneto facilitate
`assessmentofcell persistence in vivo by PCR specific for this marker gene [69]. Riddell
`et al. have conducted a PhaseI trial to augment HIV-specific immunity in HIV
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`seropositive individuals by adoptive transfer using HIV-specific CD8* CTL clones[70].
`
`These clones were transduced with the retroviral vector tgLS'HyTK which directs the
`
`synthesis of a bifunctional fusion protein incorporating hygromycin phosphotransferase
`
`and herpes virus thymidine kinase (HSV-TK)permitting in vitro selection with
`
`hygromycin and potential in vivo ablation of transferred cells with gancyclovir. Six HIV
`
`infected patients were treated with a series of four escalating cell dose infusions without
`
`toxicities, with a maximum cell dose of 5x10 cells/m? [70].
`
`[00013]
`
`Asan alternate to viral gene therapy vectors, Nabel et al. used plasmid
`
`DNAencoding an expression cassette for an anti-HIV gene in a PhaseI clinicaltrial.
`
`Plasmid DNA wasintroduced into T cells by particle bombardment with a gene gun [71].
`Genetically modified T cells were expandedand infused back into HIV-infected study
`subjects. Although this study demonstrated the feasibility of using a non-viral genetic
`
`modification strategy for primary human T cells, one limitation of this approach is the
`
`episomal propagation of the plasmid vector in T cells. Unlike chromosomally integrated
`
`transferred DNA, episomal propagation of plasmid DNAcarriesthe risk of loss of
`
`transferred genetic material with cell replication and of repetitive random chromosomal
`
`integration events.
`
`[00014]
`
`Chimeric antigen receptors engineered to consist of an extracellular single
`
`chain antibody (scFvFc) fused to the intracellular signaling domain of the T cell antigen
`
`receptor complex zeta chain (€) have the ability, when expressed in T cells, to redirect
`antigen recognition based on the monoclonal antibody’s specificity [72]. The design of
`
`scFvFc:¢ receptors with target specificities for tumorcell-surface epitopesis a
`
`conceptually attractive strategy to generate antitumor immuneeffector cells for adoptive
`
`therapy as it does not rely on pre-existing anti-tumor immunity. These receptors are
`
`“universal” in that they bind antigen in a MHC independentfashion, thus, one receptor
`
`construct can be used to treat a population of patients with antigen positive tumors.
`
`Several constructs for targeting human tumors have been described in the literature
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`including receptors with specificities for Her2/Neu, CEA, ERRB-2, CD44v6,and
`
`epitopes selectively expressed on renal cell carcinoma [73-77]. These epitopesall share
`
`the common characteristic of being cell-surface moieties accessible to scFv binding by the
`
`chimeric T cell receptor. Jn vitro studies have demonstrated that both CD4* and CD8* T
`
`cell effector functions canbe triggered via these receptors. Moreover, animal models
`
`have demonstrated the capacity of adoptively transferred scFvFc:¢ expressing T cells to
`
`eradicate established tumors [78]. The function of primary human T cells expressing
`
`tumor-specific scFvFc:C receptors have been evaluated in vitro; these cells specifically
`
`lyse tumortargets and secrete an array of pro-inflammatory cytokines including IL-2,
`
`TNF, IFN-y, and GM-CSF [79]. Phase I pilot adoptive therapy studies are underway
`
`utilizing autologous scFvFc:C-expressing T cells specific for HIV gp120 in HIV infected
`
`individuals and autologous scFvFc:C-expressing T cells with specificity for TAG-72
`expressed on a variety of adenocarcinomasincluding breast and colorectal
`
`adenocarcinoma.
`
`[00015]
`
`Investigators at City of Hope have engineered a CD20-specific scFvFc:¢
`
`receptor constructfor the purposeof targeting CD20+ B-cell malignancy [80]. Preclinical
`
`laboratory studies have demonstrated thefeasibility of isolating and expanding from
`
`healthy individuals and lymphoma patients CD8+ CTL clonesthat contain a single copy
`
`of unrearranged chromosomally integrated vector DNA and express the CD20-specific
`
`scFvFc:z6 receptor [81]. To accomplish this, purified linear plasmid DNA containing the
`
`chimeric receptor sequence underthetranscriptional control of the CMV immediate/early
`
`promoter and the NeoR gene under the transcriptional control of the SV40 early promoter
`was introduced into activated human peripheral blood mononuclear cells by exposure of
`
`cells and DNAto a brief electrical current, a procedure called electroporation [82].
`
`Utilizing selection, cloning, and expansion methods currently employed in FDA-approved
`
`clinical trials at the FHCRC, gene modified CD8+ CTL clones with CD20-specific
`
`cytolytic activity have been generated from each of six healthy volunteers in 15 separate
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`electroporation procedures [81]. These clones when co-cultured with a panel of human
`
`CD20+ lymphomacell lines proliferate, specifically lyse target cells, and are stimulated to
`
`produce cytokines.
`[00016]
`| It is desired to develop additional redirected immunecells and, in a
`preferred embodiment, redirectedTcells, for treating B-cell malignancies and B-cell
`
`mediated autoimmunedisease.
`
`SUMMARYOF THE INVENTION
`
`[00017]
`
`In one aspect, the present invention provides genetically engineered T cells
`
`which express and bear onthe cell surface membrane a CD19-specific chimeric T cell
`
`receptor(referred to herein as “CD19R”) having an intracellular signaling domain, a
`
`transmembrane domain (TM) and a CD19-specific extracellular domain (also referred to
`
`herein as “CD19-specific T cells”). The present invention also provides the CD19-
`
`specific chimeric T cell receptors, DNA constructs encoding the receptors, and plasmid
`
`expression vectors containing the constructs in proper orientation for expression.
`
`[00018]
`
`In a second aspect, the present invention provides a methodoftreating a
`
`CD19* malignancy in a mammal which comprises administering CD19-specific T cells to
`the mammalin a therapeutically effective amount. In one embodiment, CD8* CD19-
`specific T cells are administered, preferably with CD4* CD19-specific T cells. Ina
`
`second embodiment, CD4* CD19-specific T cells are administered to a mammal,
`
`preferably with CD8* cytotoxic lymphocytes which do not express the CD19-specific
`chimeric receptor of the invention, optionally in combination with CD8* CD19-specific
`
`redirected T cells.
`
`In another aspect, the present invention provides a methodof abrogating
`[00019]
`any untoward B cell function in a mammal which comprises administering to the mammal
`CD19-specific redirected T cells in a therapeutically effective amount. These untoward B
`
`cell functions can include B-cell mediated autoimmunedisease (e.g., lupus or rheumatoid
`
`arthritis) as well as any unwanted specific immune responseto a given antigen.
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`WO02/077029
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`PCT/US01/42997
`
`[00020]
`
`In another aspect, the present invention provides a method of making and
`
`expanding the CD19-specific redirected T cells which comprises transfecting T cells with
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`an expression vector containing a DNA construct encoding the CD19-specific chimeric
`receptor, then stimulating the cells with CD19" cells, recombinant CD19,or an antibody
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`to the receptor to cause the cells to proliferate. In one embodiment, the redirected T cells
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`are prepared by electroporation. In a second embodiment,the redirected T cells are
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`prepared by using viral vectors.
`
`[00021]
`
`In another aspect, the present invention provides a method oftargeting
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`Natural Killer (NK) cells which express and bear onthe cell surface membrane a CD19-
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`specific chimeric immunereceptor having an intracellular signaling domain, a
`
`transmembrane domain (TM)and a CD19-specific extracellular domain.
`- [00022]
`In another aspect, the present invention provides a method oftargeting
`macrophagecells which express and bear onthe cell surface membrane a CD19-specific
`
`chimeric immunereceptor having an intracellular signaling domain, a transmembrane
`
`domain (TM) and a CD19-specific extracellular domain.
`
`[00023]
`
`In another aspect, the present invention provides a method oftargeting
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`neutrophils cells which express and bear on the cell surface membrane a CD19-specific
`
`chimeric immunereceptor having an intracellular signaling domain, a transmembrane
`
`domain (TM)and a CD19-specific extracellular domain.
`
`[00024]
`
`In another aspect, the present invention provides a method oftargeting
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`stem cells which express and bear on the cell surface membrane a CD19-specific chimeric
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`immunereceptor having an intracellular signaling domain, a transmembrane domain
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`(TM) and a CD19-specific extracellular domain.
`
`[00025]
`
`In another aspect, the invention provides a CD-19-specific chimeric T-cell
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`receptor comprising an intracellular signalling domain, a transmembrane domain and a
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`CD19-specific extracellular domain.
`
`[00026]
`
`In one embodiment, the CD19-specific chimeric T cell receptor of the
`
`invention comprises scFvFc:¢, where scFvFc represents the extracellular domain, scFv
`
`11
`
`UPenn Ex. 2028
`
`Miltenyi v. UPenn
`IPR2022-00853
`
`UPenn Ex. 2028
`Miltenyi v. UPenn
`IPR2022-00853
`
`

`

`WO02/077029
`
`PCT/US01/42997
`
`designates the V,, and V, chains ofa single chain monoclonal antibody to CD19, Fe
`represents at least part of a constant region of an IgG,, and ¢ represents the intracellular
`
`signaling domain of the zeta chain of human CD3.
`
`[00027]
`
`In another embodiment, the CD19-specific chimeric T cell receptor of the
`
`invention comprises the scFvFc extracellular domain and the ¢ intracellular domain are
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`linked by the transmembrane domain of human CD4.
`
`[00028]
`
`In another embodiment, the CD19-specific chimeric T cell receptor of the
`
`invention comprises amino acids 23-634 of SEQ ID NO:2.
`
`[00029]
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`In another aspect, the invention provides a plasmid expression vector
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`containing a DNA construct encoding a chimeric T-cell receptor of the invention in
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`properorientation for expression.
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`BRIEF DESCRIPTION OF THE FIGURES
`
`[00030]
`
`Figures 1A-1C show the double-stranded DNA sequence and amino acid
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`sequence for the CD19:zeta chimeric immunoreceptor of the present invention, SEQ ID
`
`NO.1 and show the source of the DNA segments found in the chimeric immunoreceptor.
`
`[00031]
`
`[00032]
`
`Figure 2 is a schematic representation of the plasmid pwG-CD19R/HyTK.
`
`Figure 3 shows Western blot analyses which demonstrate the expression of
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`the CD19R/scFvFe:¢ chimeric receptor.
`
`[00033]
`
`Figure 4 is a graphical representation showing the antigen-specific cytolytic
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`activity of T-cells expressing the CD19R/scFvFe:¢ chimeric receptor.
`
`[00034]
`
`Figure 5 is a graphical representation of the production of interferon-y by T
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`cells expressing the CD19R/scFvFc:¢ chimeric receptor that are incubatedin the presence
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`of variouscell lines expressing CD-19.
`
`[00035]
`
`Figure 6 A-E are graphical representations showing the antigen-specific
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`cytolytic activity of CD19R/scFvFc:¢ chimeric receptor redirected T-cell clones.
`
`12
`
`UPenn Ex. 2028
`
`Miltenyi v. UPenn
`IPR2022-00853
`
`UPenn Ex. 2028
`Miltenyi v. UPenn
`IPR2022-00853
`
`

`

`WO02/077029
`
`PCT/US01/42997
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`[00036]
`
`The present invention is directed to genetically engineered, redirected T
`
`cells and to their use for cellular immunotherapy of B-cell malignancies, Epstein Barr
`
`Virus-related lymphoproliferative disorders, and B-cell mediated autoimmunediseases.
`[00037]
`In one aspect, the present invention provides genetically engineered T cells
`which express and bear on the cell surface membrane a CD19-specific chimeric T cell
`
`receptor having an intracellular signaling domain, a transmembrane domain and a CD19-
`
`specific extracellular domain (referred to herein as CD19-specific T cells). The
`
`extracellular domain comprises a CD19-specific receptor. Individual T cells of the
`invention may be CD4*/CD8, CD4/CD8*, CD4/CD8’ or CD4*/CD8". The T cells may be
`a mixed population of CD4*/CD8and CD4/CD8*'cells or a population of a single clone.
`CD4*Tcells of the invention produce IL-2 when co-cultured in vitro with CD19"
`lymphomacells. CD8* T cells of the invention lyse CD19* human lymphomatarget cells
`
`whenco-cultured in vitro with the target cells. The invention further provides the CD19-
`
`specific chimeric T cell receptors, DNA constructs encoding the receptors, and plasmid
`
`expression vectors containing the constructs in proper orientation for expression.
`
`[00038}
`
`In a preferred embodiment, CD19-specific redirected T cells express CD19-
`
`specific chimeric receptor scFvFe:¢, where scFv designates the Vy and V, chains of a
`single chain monoclonal antibody to CD19,Fc represents at least part of a constant region
`
`of a human IgG,, and ¢ represents the intracellular signaling domain of the zeta chain of
`
`human CD3. The extracellular domain scFvFc andthe intracellular domain ¢ are linked
`
`by a transmembrane domain such as the transmembrane domain of CD4. In other
`
`embodiments, the human Fc constant region may be provided by other species of antibody
`
`such as IgG, for example.
`
`[00039]
`
`In a specific preferred embodiment, a full length scFvFc:¢ cDNA,
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`designated SEQ ID NO.1 or “CD19R:zeta,” comprises the