Costimulatory Chimeric Antigen Receptor T Cells Targeting IL13R-alpha-2
20180265844 ยท 2018-09-20
Inventors
Cpc classification
A61K39/4611
HUMAN NECESSITIES
C07K2319/75
CHEMISTRY; METALLURGY
C12N2740/16043
CHEMISTRY; METALLURGY
C07K2319/33
CHEMISTRY; METALLURGY
C07K2319/30
CHEMISTRY; METALLURGY
C07K14/70578
CHEMISTRY; METALLURGY
C07K2319/74
CHEMISTRY; METALLURGY
A61K2239/38
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
C07K14/705
CHEMISTRY; METALLURGY
C07K14/715
CHEMISTRY; METALLURGY
Abstract
Chimeric transmembrane immunoreceptors (CAR) which include an extracellular domain that includes IL-13 or a variant thereof that binds interleukin-13R?2 (IL13R?2), a transmembrane region, a costimulatory domain and an intracellular signaling domain are described.
Claims
1. A population of human T cells transduced by a vector comprising an expression cassette encoding a chimeric antigen receptor, wherein chimeric antigen receptor comprises: a human IL-13 variant comprising the amino acid sequence of SEQ ID NO: 3 with up to 5 single amino acid substitutions, provided that the amino acid at position 11 of SEQ ID NO: 3 other than E; a transmembrane domain selected from: a CD4 transmembrane domain or variant thereof having 1-5 amino acid substations, a CD8 transmembrane domain or variant thereof having 1-5 amino acid substitutions, a CD28 transmembrane domain or a variant thereof having 1-5 amino acid substations, and a CD3? transmembrane domain or a variant thereof having 1-5 amino acid substitutions; a costimulatory domain selected from the group consisting of: a CD28 costimulatory domain or a variant thereof having 1-5 amino acid substitutions, a 4IBB costimulatory domain or a variant thereof having 1-5 amino acid substitutions and an OX40 costimulatory domain or a variant thereof having 1-5 amino acid substitutions; and CD3 ? signaling domain of a variant thereof having 1-5 amino acid substitutions.
2. The population of human T cells of claim 1, wherein the chimeric antigen receptor comprising an amino acid sequence selected from SEQ ID NOs: 10, 31-48 and 52.
3. The population of human T cells of claim 1, wherein the chimeric antigen receptor comprises SEQ ID NO: 10.
4. A composition comprising T cells harboring a nucleic acid molecule comprising a nucleotide sequence encoding a chimeric antigen receptor molecule comprising the amino acid sequence of SEQ ID NO: 10.
5. A composition of comprising T cells harboring an expression vector comprising a nucleotide sequence encoding a chimeric antigen receptor molecule comprising the amino acid sequence of SEQ ID NO: 10.
6. The composition of claim 5, wherein the expression vector is a lentiviral vector.
7. The composition of claim 4, wherein the nucleic acid molecule further comprises a nucleotide sequence encoding a GMSCFRa signal sequence preceding the nucleotide sequence encoding the chimeric antigen receptor.
8. The composition of claim 7, wherein the GMSCFRa signal sequence comprises the amino acid sequence of SEQ ID NO:2.
9. The composition of claim 4, wherein the nucleic acid molecule further comprises a nucleotide sequence encoding a T2A ribosome skip sequence following the nucleotide sequence encoding the chimeric antigen receptor.
10. The composition of claim 9, wherein T2A ribosome skip sequence comprises the amino acid sequence of SEQ ID NO:8.
11. The composition of claim 9, wherein the nucleic acid molecule further comprises a nucleotide sequence encoding a truncated CD19 following the nucleotide sequence encoding the T2A ribosome skip sequence.
12. The composition of claim 10, wherein the truncated CD19 comprises the amino acid sequence of SEQ ID NO:9.
13. The composition of claim 5, wherein the T cells comprise central memory T cells.
Description
DESCRIPTION OF DRAWINGS
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DETAILED DESCRIPTION
[0052] Described below is the structure, construction and characterization of various IL13R?2-specific chimeric antigen receptors. A chimeric antigen (CAR) is a recombinant biomolecule that contains, at a minimum, an extracellular recognition domain, a transmembrane region, and an intracellular signaling domain. The term antigen, therefore, is not limited to molecules that bind antibodies, but to any molecule that can bind specifically to a target. For example, a CAR can include a ligand that specifically binds a cell surface receptor. The extracellular recognition domain (also referred to as the extracellular domain or simply by the recognition element which it contains) comprises a recognition element that specifically binds to a molecule present on the cell surface of a target cell. The transmembrane region anchors the CAR in the membrane. The intracellular signaling domain comprises the signaling domain from the zeta chain of the human CD3 complex and optionally comprises one or more costimulatory signaling domains. CARs can both to bind antigen and transduce T cell activation, independent of MHC restriction. Thus, CARs are universal immunoreceptors which can treat a population of patients with antigen-positive tumors irrespective of their HLA genotype. Adoptive immunotherapy using T lymphocytes that express a tumor-specific CAR can be a powerful therapeutic strategy for the treatment of cancer.
[0053] One IL13R?2-specific CAR described herein is referred to as IL13(EQ)BB?. This CAR includes a variety of important features including: a IL13?2 ligand having an amino acid change that improves specificity of biding to IL13?2; the domain of CD137 (4-1BB) in series with CD3? to provide beneficial costimulation; and an IgG4 Fc region that is mutated at two sites within the CH2 region (L235E; N297Q) in a manner that reduces binding by Fc receptors (FcRs). Other CAR described herein contain a second costimulatory domain.
[0054] In some cases the CAR described herein, including the IL13(EQ)BB? CAR can be produced using a vector in which the CAR open reading frame is followed by a T2A ribosome skip sequence and a truncated CD19 (CD19t), which lacks the cytoplasmic signaling tail (truncated at amino acid 323). In this arrangement, co-expression of CD19t provides an inert, non-immunogenic surface marker that allows for accurate measurement of gene modified cells, and enables positive selection of gene-modified cells, as well as efficient cell tracking and/or imaging of the therapeutic T cells in vivo following adoptive transfer. Co-expression of CD19t provides a marker for immunological targeting of the transduced cells in vivo using clinically available antibodies and/or immunotoxin reagents to selectively delete the therapeutic cells, and thereby functioning as a suicide switch.
[0055] Gliomas, express IL13 receptors, and in particular, high-affinity IL13 receptors. However, unlike the IL13 receptor, glioma cells overexpress a unique IL13R?2 chain capable of binding IL13 independently of the requirement for IL4R? or ?c44. Like its homolog IL4, IL13 has pleotropic immunoregulatory activity outside the CNS. Both IL13 and IL4 stimulate IgE production by B lymphocytes and suppress pro-inflammatory cytokine production by macrophages.
[0056] Detailed studies using autoradiography with radiolabeled IL13 have demonstrated abundant IL13 binding on nearly all malignant glioma tissues studied. This binding is highly homogeneous within tumor sections and in single cell analysis. However, molecular probe analysis specific for IL13R?2 mRNA did not detect expression of the glioma-specific receptor by normal brain elements and autoradiography with radiolabeled IL13 also could not detect specific IL13 binding in the normal CNS. These studies suggest that the shared IL13R?1/IL4?/?c receptor is not expressed detectably in the normal CNS. Therefore, IL13R?2 is a very specific cell-surface target for glioma and is a suitable target for a CAR designed for treatment of a glioma.
[0057] Binding of IL13-based therapeutic molecules to the broadly expressed IL13R?1/IL4?/?c receptor complex, however, has the potential of mediating undesired toxicities to normal tissues outside the CNS, and thus limits the systemic administration of these agents. An amino acid substitution in the IL13 alpha helix A at amino acid 13 of tyrosine for the native glutamic acid selectively reduces the affinity of IL13 to the IL13R?1/IL4?/?c receptor. Binding of this mutant (termed IL13(E13Y)) to IL13R?2, however, was increased relative to wild-type IL13. Thus, this minimally altered IL13 analog simultaneously increases IL13's specificity and affinity for glioma cells. Therefore, CAR described herein include an IL13 containing a mutation (E to Y or E to some other amino acid such as K or R or L or V) at amino acid 13 (according to the numbering of Debinski et al. 1999 Clin Cancer Res 5:3143s). IL13 having the natural sequence also may be used, however, and can be useful, particularly in situations where the modified T cells are to be locally administered, such as by injection directly into a tumor mass.
[0058] The CAR described herein can be produced by any means known in the art, though preferably it is produced using recombinant DNA techniques. Nucleic acids encoding the several regions of the chimeric receptor can be prepared and assembled into a complete coding sequence by standard techniques of molecular cloning known in the art (genomic library screening, PCR, primer-assisted ligation, site-directed mutagenesis, etc.) as is convenient. The resulting coding region is preferably inserted into an expression vector and used to transform a suitable expression host cell line, preferably a T lymphocyte cell line, and most preferably an autologous T lymphocyte cell line.
[0059] Various T cell subsets isolated from the patient, including unselected PBMC or enriched CD3 T cells or enriched CD3 or memory T cell subsets, can be transduced with a vector for CAR expression. Central memory T cells are one useful T cell subset. Central memory T cell can be isolated from peripheral blood mononuclear cells (PBMC) by selecting for CD45RO+/CD62L+ cells, using, for example, the CliniMACS? device to immunomagnetically select cells expressing the desired receptors. The cells enriched for central memory T cells can be activated with anti-CD3/CD28, transduced with, for example, a SIN lentiviral vector that directs the expression of an IL13R?2-specific CAR (e.g., IL13(EQ)BB?) as well as a truncated human CD19 (CD19t), a non-immunogenic surface marker for both in vivo detection and potential ex vivo selection. The activated/genetically modified central memory T cells can be expanded in vitro with IL-2/IL-15 and then cryopreserved.
Example 1: Construction and Structure of an IL13R?2-Specific CAR
[0060] The structure of a useful IL13R?2-specific CAR is described below. The codon optimized CAR sequence contains a membrane-tethered IL-13 ligand mutated at a single site (E13Y) to reduce potential binding to IL13R?1, an IgG4 Fc spacer containing two mutations (L235E; N297Q) that greatly reduce Fc receptor-mediated recognition models, a CD4 transmembrane domain, a costimulatory 4-1BB cytoplasmic signaling domain, and a CD3? cytoplasmic signaling domain. A T2A ribosome skip sequence separates this IL13(EQ)BB? CAR sequence from CD19t, an inert, non-immunogenic cell surface detection/selection marker. This T2A linkage results in the coordinate expression of both IL13(EQ)BB? and CD19t from a single transcript.
[0061] The IL13(EQ)BBZ sequence was generated by fusion of the human GM-CSF receptor alpha leader peptide with IL13(E13Y) ligand 5 L235E/N297Q-modified IgG4 Fc hinge (where the double mutation interferes with FcR recognition), CD4 transmembrane, 4-1BB cytoplasmic signaling domain, and CD3? cytoplasmic signaling domain sequences. This sequence was synthesized de novo after codon optimization. The T2A sequence was obtained from digestion of a T2A-containing plasmid. The CD19t sequence was obtained from that spanning the leader peptide sequence to the transmembrane components (i.e., basepairs 1-972) of a CD19-containing plasmid. All three fragments, 1) IL13(EQ)BBZ, 2) T2A, and 3) CD19t, were cloned into the multiple cloning site of the epHIV7 lentiviral vector. When transfected into appropriate cells, the vector integrates the sequence depicted schematically in
[0062] As shown schematically in
Example 2: Construction and Structure of epHIV7 Used for Expression of an IL13R?2-Specific CAR
[0063] The pHIV7 plasmid is the parent plasmid from which the clinical vector IL13(EQ)BBZ-T2A-CD19t_epHIV7 was derived in the T cell Therapeutics Research Laboratory (TCTRL) at City of Hope (COH). The epHIV7 vector used for expression of the CAR was produced from pHIV7 vector. Importantly, this vector uses the human EF1 promoter to drive expression of the CAR. Both the 5 and 3 sequences of the vector were derived from pv653RSN as previously derived from the HXBc2 provirus. The polypurine tract DNA flap sequences (cPPT) were derived from HIV-1 strain pNL4-3 from the NIH AIDS Reagent Repository. The woodchuck post-transcriptional regulatory element (WPRE) sequence was previously described.
[0064] Construction of pHIV7 is schematically depicted in
[0065] A packaging signal, psi w, is required for efficient packaging of viral genome into the vector. The RRE and WPRE enhance the RNA transcript transport and expression of the transgene. The flap sequence, in combination with WPRE, has been demonstrated to enhance the transduction efficiency of lentiviral vector in mammalian cells.
[0066] The helper functions, required for production of the viral vector), are divided into three separate plasmids to reduce the probability of generation of replication competent lentivirus via recombination: 1) pCgp encodes the gag/pol protein required for viral vector assembly; 2) pCMV-Rev2 encodes the Rev protein, which acts on the RRE sequence to assist in the transportation of the viral genome for efficient packaging; and 3) pCMV-G encodes the glycoprotein of the vesiculo-stomatitis virus (VSV), which is required for infectivity of the viral vector.
[0067] There is minimal DNA sequence homology between the pHIV7 encoded vector genome and the helper plasmids. The regions of homology include a packaging signal region of approximately 600 nucleotides, located in the gag/pol sequence of the pCgp helper plasmid; a CMV promoter sequence in all three helper plasmids; and a RRE sequence in the helper plasmid pCgp. It is highly improbable that replication competent recombinant virus could be generated due to the homology in these regions, as it would require multiple recombination events. Additionally, any resulting recombinants would be missing the functional LTR and tat sequences required for lentiviral replication.
[0068] The CMV promoter was replaced by the EF1?-HTLV promoter (EF1p), and the new plasmid was named epHIV7 (
[0069] The lentiviral genome, excluding gag/pol and rev that are necessary for the pathogenicity of the wild-type virus and are required for productive infection of target cells, has been removed from this system. In addition, the IL13(EQ)BBZ-T2ACD19t_epHIV7 vector construct does not contain an intact 3LTR promoter, so the resulting expressed and reverse transcribed DNA proviral genome in targeted cells will have inactive LTRs. As a result of this design, no HIV-I derived sequences will be transcribed from the provirus and only the therapeutic sequences will be expressed from their respective promoters. The removal of the LTR promoter activity in the SIN vector is expected to significantly reduce the possibility of unintentional activation of host genes (56). Table 4 summarizes the various regulator elements present in IL13(EQ)BBZ-T2ACD19t_epHIV7.
TABLE-US-00004 TABLE 4 Functional elements of IL13(EQ)41BBZ-T2A-CD19t_epHIV7 Location Regulatory Elements (Nucleotide and Genes Numbers) Comments U5 87-171 5 Unique sequence psi 233-345 Packaging signal RRE 957-1289 Rev-responsive element flap 1290-1466 Contains polypurine track sequence and central termination sequence to facilitate nuclear import of pre-integration complex EF1p Promoter 1524-2067 EF1-alpha Eukaryotic Promoter sequence driving expression of CD19Rop IL13-IgG4 (EQ)- 2084-4753 Therapeutic insert 41BB-Zeta-T2A- CD19t WPRE 4790-5390 Woodchuck hepatitis virus derived regulatory element to enhance viral RNA transportation delU3 5405-5509 3 U3 with deletion to generate SIN vector R 5510-5590 Repeat sequence within LTR U5 5591-5704 3 U5 sequence in LTR Amp.sup.R 6540-7398 Ampicillin-resistance gene CoE1 ori 7461-8342 Replication origin of plasmid SV40 ori 8639-8838 Replication origin of SV40 CMV promoter 8852-9451 CMV promoter to generate viral genome RNA R 9507-86 Repeat sequence within LTR
Example 3: Production of Vectors for Transduction of Patient T Cells
[0070] For each plasmid (IL13(EQ)BBZ-T2A-CD19t_epHIV7; pCgp; pCMV-G; and pCMV-Rev2), a seed bank is generated, which is used to inoculate the fermenter to produce sufficient quantities of plasmid DNA. The plasmid DNA is tested for identity, sterility and endotoxin prior to its use in producing lentiviral vector.
[0071] Briefly, cells were expanded from the 293T working cell (WCB), which has been tested to confirm sterility and the absence of viral contamination. A vial of 293T cells from the 293T WCB was thawed. Cells were grown and expanded until sufficient numbers of cells existed to plate an appropriate number of 10 layer cell factories (CFs) for vector production and cell train maintenance. A single train of cells can be used for production.
[0072] The lentiviral vector was produced in sub-batches of up to 10 CFs. Two sub-batches can be produced in the same week leading to the production of approximately 20 L of lentiviral supernatant/week. The material produced from all sub-batches were pooled during the downstream processing phase, in order to produce one lot of product. 293T cells were plated in CFs in 293T medium (DMEM with 10% FBS). Factories were placed in a 37? C. incubator and horizontally leveled in order to get an even distribution of the cells on all the layers of the CF. Two days later, cells were transfected with the four lentiviral plasmids described above using the CaPO4 method, which involves a mixture of Tris:EDTA, 2M CaCl2, 2?HBS, and the four DNA plasmids. Day 3 after transfection, the supernatant containing secreted lentiviral vectors was collected, purified and concentrated. After the supernatant was removed from the CFs, End-of-Production Cells were collected from each CF. Cells were trypsinized from each factory and collected by centrifugation. Cells were resuspended in freezing medium and cryopreserved. These cells were later used for replication-competent lentivirus (RCL) testing.
[0073] To purify and formulate vectors crude supernatant was clarified by membrane filtration to remove the cell debris. The host cell DNA and residual plasmid DNA were degraded by endonuclease digestion (Benzonase?). The viral supernatant was clarified of cellular debris using a 0.45 ?m filter. The clarified supernatant was collected into a pre-weighed container into which the Benzonase? is added (final concentration 50 U/mL). The endonuclease digestion for residual plasmid DNA and host genomic DNA as performed at 37? C. for 6 h. The initial tangential flow ultrafiltration (TFF) concentration of the endonuclease-treated supernatant was used to remove residual low molecular weight components from the crude supernatant, while concentrating the virus ?20 fold. The clarified endonuclease-treated viral supernatant was circulated through a hollow fiber cartridge with a NMWCO of 500 kD at a flow rate designed to maintain the shear rate at 4,000 sec-1 or less, while maximizing the flux rate. Diafiltration of the nuclease-treated supernatant was initiated during the concentration process to sustain the cartridge performance. An 80% permeate replacement rate was established, using 4% lactose in PBS as the diafiltration buffer. The viral supernatant was brought to the target volume, representing a 20-fold concentration of the crude supernatant, and the diafiltration was continued for 4 additional exchange volumes, with the permeate replacement rate at 100%.
[0074] Further concentration of the viral product was accomplished by using a high speed centrifugation technique. Each sub-batch of the lentivirus was pelleted using a Sorvall RC-26 plus centrifuge at 6000 RPM (6,088 RCF) at 6? C. for 16-20 h. The viral pellet from each sub-batch was then reconstituted in a 50 mL volume with 4% lactose in PBS. The reconstituted pellet in this buffer represents the final formulation for the virus preparation. The entire vector concentration process resulted in a 200-fold volume reduction, approximately. Following the completion of all of the sub-batches, the material was then placed at ?80? C., while samples from each sub-batch were tested for sterility. Following confirmation of sample sterility, the sub-batches were rapidly thawed at 37? C. with frequent agitation. The material was then pooled and manually aliquoted in the Class II Type A/B3 biosafety cabinet in the viral vector suite. A fill configuration of 1 mL of the concentrated lentivirus in sterile USP class 6, externally threaded O-ring cryovials was used. Center for Applied Technology Development (CATD)'s Quality Systems (QS) at COH released all materials according to the Policies and Standard Operating Procedures for the CBG and in compliance with current Good Manufacturing Practices (cGMPs).
[0075] To ensure the purity of the lentiviral vector preparation, it was tested for residual host DNA contaminants, and the transfer of residual host and plasmid DNA. Among other tests, vector identity was evaluated by RT-PCR to ensure that the correct vector is present. All release criteria were met for the vector intended for use in this study.
Example 4: Preparation of T Cells Suitable for Use in ACT
[0076] T lymphocytes are obtained from a patient by leukopheresis, and the appropriate allogenic or autologous T cell subset, for example, Central Memory T cells (T.sub.CM), are genetically altered to express the CAR, then administered back to the patient by any clinically acceptable means, to achieve anti-cancer therapy.
[0077] An outline of the manufacturing strategy for T.sub.CM is depicted in
[0078] Following enrichment, T.sub.CM cells are formulated in complete X-Vivo15 plus 50 IU/mL IL-2 and 0.5 ng/mL IL-15 and transferred to a Teflon cell culture bag, where they are stimulated with Dynal ClinEx? Vivo CD3/CD28 beads. Up to five days after stimulation, cells are transduced with IL13(EQ)BBZ-T2A-CD19t_epHIV7 lentiviral vector at a multiplicity of infection (MOI) of 1.0 to 0.3. Cultures are maintained for up to 42 days with addition of complete X-Vivo15 and IL-2 and IL-15 cytokine as required for cell expansion (keeping cell density between 3?10.sup.5 and 2?10.sup.6 viable cells/mL, and cytokine supplementation every Monday, Wednesday and Friday of culture). Cells typically expand to approximately 10.sup.9 cells under these conditions within 21 days. At the end of the culture period cells are harvested, washed twice and formulated in clinical grade cryopreservation medium (Cryostore CS5, BioLife Solutions).
[0079] On the day(s) of T cell infusion, the cryopreserved and released product is thawed, washed and formulated for re-infusion. The cryopreserved vials containing the released cell product are removed from liquid nitrogen storage, thawed, cooled and washed with a PBS/2% human serum albumin (HSA) Wash Buffer. After centrifugation, the supernatant is removed and the cells resuspended in a Preservative-Free Normal Saline (PFNS)/2% HSA infusion diluent. Samples are removed for quality control testing.
[0080] Two qualification runs on cells procured from healthy donors were performed using the manufacturing platform described above. Each preclinical qualification run product was assigned a human donor (HD) numberHD006.5 and HD187.1. Importantly, as shown in Table 5, these qualification runs expanded >80 fold within 28 days and the expanded cells expressed the IL13(EQ)BB?/CD19t transgenes.
TABLE-US-00005 TABLE 5 Summary of Expression Data from Pre-clinical Qualification Run Product Cell Product CAR CD19 CD4+ CD8+ Fold Expansion HD006.5 20% 22% 24% 76% 84-fold (28 days) Hd187.1 18% 25% 37% 63% 259-fold (28 days)
Example 5: Flow Cytometric Analysis of Surface Transgene and T Cell Marker Expression in IL13(EQ)BB?/CD19t+ T.SUB.CM
[0081] The two preclinical qualification run products described in Example 4 were used in pre-clinical studies to as described below.
Example 6: Effector Activity of IL13(EQ)BB?/CD19t+ T.SUB.CM
[0082] The effector activity of IL13(EQ)BB?/CD19t+ T.sub.CM was assessed and the results of this analysis are depicted in
Example 7: In Vivo Anti-Tumor Activity of IL13(EQ)BB?/CD19t+ T.SUB.CM
[0083] The studies described below demonstrate that IL13(EQ)BB?/CD19t+ T.sub.CM exhibit anti-tumor efficacy in in vivo mouse models. Specifically, we have evaluated the anti-tumor potency of IL13(EQ)BB?/CD19t+ T.sub.CM against the IL13R?2+ primary low-passage glioblastoma tumor sphere line PBT030-2, which has been engineered to express both EGFP and firefly luciferase (ffLuc) reporter genes (PBT030-2 EGFP:ffLuc) A panel of primary lines (PBT) from patient glioblastoma specimens grown as tumor spheres (TSs) in serum-free media. These expanded TS lines exhibit stem cell-like characteristics, including expression of stem cell markers, multilineage differentiation and capacity to initiate orthotopic tumors in immunocompromised mice (NSG) at low cell numbers. The PBT030-2 EGFP:ffLuc TS-initiated xenograft model (0.1?10.sup.6 cells; 5 day engraftment) has been previously used to evaluate in vivo anti-tumor activity in NSG mice of IL13R?2-specific CAR expressing T cells, whereby three injections of 2?10.sup.6 cytolytic T lymphocytes (CTLs) over a course of 2 weeks were shown to reduce tumor growth. However, in those experiments the majority of the PBT030-2 tumors eventually recurred. By comparison, a single injection of IL13(EQ)BB?/CD19t+ T.sub.CM (1.1?10.sup.6 CAR+T.sub.CM; 2?10.sup.6 total T.sub.CM) exhibited robust anti-tumor activity against PBT030-2 EGFP:ffLuc TS-initiated tumors (0.1?10.sup.6 cells; 5 day engraftment) as shown in
[0084] Briefly, EGFP-ffLuc+ PBT030-2 tumor cells (1?10.sup.5) were stereotactically implanted into the right forebrain of NSG mice. On day 5, mice received either 2?10.sup.6 IL13(EQ)BB?/CD19t+ T.sub.CM (1.1?106 CAR+; n=6), 2?10.sup.6 mock T.sub.CM (no CAR; n=6) or PBS (n=6).
Example 8: Comparison of IL13(EQ)BB?+T.SUB.CM .and Non-T.SUB.CM .IL13-Zetakine CD8+ CTL Clones in Antitumor Efficacy and T Cell Persistence
[0085] The studies described below compare IL13(EQ)BB?+T.sub.CM and a previously created IL13R?2-specific human CD8+ CTLs (IL13-zetakine CD8+ CTL (described in Brown et al. 2012 Clin Cancer Res 18:2199 and Kahlon et al. 2004 Cancer Res 64:9160). The IL13-zetakine uses a CD3? stimulatory domain, lacks a co-stimulatory domain and uses the same IL13 variant as IL13(EQ)BB?+.
[0086] A panel of primary lines (PBT) from patient glioblastoma specimens grown as tumor spheres (TSs) in serum-free media was generated (Brown et al. 2012 Clin Cancer Res 18:2199; Brown et al. 2009 Cancer Res 69:8886). These expanded TS lines exhibit stem cell-like characteristics, including expression of stem cell markers, multi-lineage differentiation and capacity to initiate orthotopic tumors in immunocompromised mice (NSG) at low cell numbers. The IL13R?2+ primary low-passage glioblastoma TS line PBT030-2, which has been engineered to express both EGFP and firefly luciferase (ffLuc) reporter genes (PBT030-2 EGFP:ffLuc) (Brown et al. 2012 Clin Cancer Res 18:2199) was used for the experiments outlined below.
[0087] First, a single dose (1?10.sup.6 CAR T cells) of IL13(EQ)BB?+T.sub.CM product was compared to IL13-zetakine CD8+ CTL clones evaluated against day 8 PBT030-2 EGFP:ffuc TS-initiated xenografts (0.1?10.sup.6 cells). While both IL13R?2-specific CAR T cells (IL13-zetakine CTL and IL13(EQ)BB? T.sub.CM) demonstrated antitumor activity against established PBT030-2 tumors as compared to untreated and mock T.sub.CM (CAR-negative) controls (
[0088] To further compare the therapeutic effectiveness of these two IL13R?2-CAR T cell products, a dose titration of 1.0, 0.3 and 0.1?10.sup.6 CART cells against day 8 PBT030-2 EGFP:ffuc TS-initiated tumors was performed (
Example 9: Comparison of CAR T Cell Delivery Route for Treatment of Large TS-Initiated PBT Tumors
[0089] Described below are studies that compare the route of delivery, intraveneous (i.v.) or intracranial (i.c.), on antitumor activity against invasive primary PBT lines. In pilot studies (data not shown), it was unexpectedly observed that i.v. administered IL13(EQ)BB?+ Tcm provided no therapeutic benefit as compared to PBS for the treatment of small (day 5) PBT030-2 EGFP:ffLuc tumors. This is in contrast to the robust therapeutic efficacy observed with i.c. administered CAR+ T cells. Reasoning that day 5 PBT030-2 tumors may have been too small to recruit therapeutic T cells from the periphery, a comparison was made of i.v. versus i.c. delivery against larger day 19 PBT030-2 EGFP:ffLuc tumors. For these studies, PBT030-2 engrafted mice were treated with either two i.v. infusions (5?10.sup.6 CAR+ Tcm; days 19 and 26) or four i.c. infusions (1?10.sup.6 CAR+ Tcm; days 19, 22, 26 and 29) of IL13(EQ)BBZ+ Tcm, or mock Tcm (no CAR). Here too no therapeutic benefit as monitored by Xenogen imaging or Kaplan-Meier survival analysis for i.v. administered CAR+ T cells (
[0090] Turnor derived cytokines, particularly MCP-1/CCL2, are important in recruiting T cells to the tumor. Thus, PBT030-2 tumor cells were evaluated and it was found that this line produces high levels of MCP-1/CCL2 comparable to U251T cells (data not shown), a glioma line previously shown to attract i.v. administered effector CD8+ T cells to i.c. engrafted tumors. Malignant gliomas are highly invasive tumors and are often multi-focal in presentation. The studies described above establish that IL13BBZ T.sub.CM can eliminate infiltrated tumors such as PBT030-2, and mediate long-term durable antitumor activity. The capacity of intracranially delivered CAR T cells to traffic to multifocal disease was also examined. For this study PBT030-2 EGFP:ffLuc TSs were implanted in both the left and right hemispheres (
Example 10: Comparison of Costimulatory Domains
[0091] A series of studies were conducted to evaluate various costimulatory domains. The various CAR evaluated are depicted schematically in
[0092] CD4 and CD8 T.sub.CM were lentivirally transduced and CAR-expressing T cells were immunomagnetically enriched via anti-CD19. CD19 and IL13 (i.e., CAR) expression levels as measured by flow cytometry. The results are shown in
[0093] The ability of the indicated mock-transduced or CAR-expressing T cells to kill IL13R?2-expressing PBT030-2 tumor cell targets was determined in a 4-hour .sup.51Cr-release assay at the indicated effector:target ratios. The results of this study are in
[0094] The in vivo efficacy of the various CAR was examined as follows. Briefly, NSG mice received an intracranial injection of ffLuc+ PBT030-2 tumor cells on day 0, and were randomized into 6 groups (n=9-10 mice per group) for i.c. treatment with either PBS (Tumor Only), mock-transduced T cells or T cells expressing the indicated IL13R?2-specific CAR on day 8. Quantitative bioluminescence imaging was then carried out to monitor tumor growth over time. Bioluminescence images for representative mice in each group (
Example 11: Amino Acid Sequence of IL13(EQ)BB?/CD19t
[0095] The complete amino acid sequence of IL13(EQ)BB?/CD19t is depicted in
[0096] The mature chimeric antigen receptor sequence (SEQ ID NO:10) includes: a 112 amino acid IL-13 sequence (SEQ ID NO:3; amino acid substitution E13Y shown in bold); a 229 amino acid IgG4 sequence (SEQ ID NO:4; with amino acid substitutions L235E and N297Q shown in bold); at 22 amino acid CD4 sequence (SEQ ID NO:5); a 42 amino acid 4-1BB sequence (SEQ ID NO:6); a 3 amino acid Gly linker; and a 112 amino acid CD3? sequence (SEQ ID NO:7). Within this CAR sequence (SEQ ID NO:10) is the IL-13/IgG4/CD4t/41-BB sequence (SEQ ID NO:11), which includes: a 112 amino acid IL-13 sequence (SEQ ID NO:3; amino acid substitution E13Y shown in bold); a 229 amino acid IgG4 sequence (SEQ ID NO:4; with amino acid substitutions L235E and N297Q shown in bold); at 22 amino acid CD4 sequence (SEQ ID NO:5); and a 42 amino acid 4-1BB sequence (SEQ ID NO:6). The IL13/IgG4/CD4t/4-1BB sequence (SEQ ID NO:11) can be joined to the 112 amino acid CD3? sequence (SEQ ID NO:7) by a linker such as a Gly Gly Gly linker. The CAR sequence (SEQ ID NO:10) can be preceded by a 22 amino acid GMCSF signal peptide (SEQ ID NO:2).
[0097]
Example 12: Amino Acid Sequence of IL13(EQ)BB?/CD19t
[0098]
[0099] A panel of CAR comprising human IL13(E13Y) domain, a CD28 tm domain, a CD28gg costimulatory domain, a 4-1BB costimulatory domain, and a CD3? domain CAR backbone and including either a HL (22 amino acids) spacer, a CD8 hinge (48 amino acids) spacer, IgG4-HL-CH3 (129 amino acids) spacer or a IgG4(EQ) (229 amino acids) spacer were tested for their ability to mediate IL13R?2-specific killing as evaluated in a 72-hour co-culture assay. With the exception of HL (22 amino acids) which appeared to have poor CAR expression in this system, all were active.