NUCLEIC ACID MOLECULE AND EXPRESSION CASSETTE COMPRISING SUCH MOLECULE

专利摘要:
nucleic acid molecule, expression cassette and use of a population of t cells a family of chimeric antigen receptors (cars) containing a cd123-specific scfv region was developed to target different epitopes on cd123. in some embodiments, such chimeric cd123 antigen receptor gene (cd123car) includes an anti-cd123 scfv region fused in frame to a modified igg4 hinge region comprising an s228p substitution, an l235e substitution, and optionally an n297q substitution; a costimulatory signaling domain; and a t-cell receptor (tcr) zeta chain signaling domain. when expressed on t cells from a healthy donor (cd4/cd8), cd123cars redirect t cell specificity and mediate potent effector activity against cd123+ cell lines and against samples from patients with primary lma. T cells obtained from patients with active lma can be modified to express cd123cdr genes and be able to lyse autologous lma blasts in vitro. finally, a single 5.0 x 10 6 dose of car123t cells causes a significant delay in leukemic progression in mice. these results suggest that transduced cd123car t cells can be used as an immunotherapy for the treatment of high-risk lma.
公开号:BR112015023701B1
申请号:R112015023701-0
申请日:2014-03-14
公开日:2022-01-11
发明作者:Stephen Forman；Armen Mardiros
申请人:City Of Hope；
IPC主号:

专利说明:

Priority in claim
[001] This application claims priority to U.S. Patent Application No. 13/844,048, filed March 15, 2013, which is incorporated into the present invention in its entirety, including the figures. government interest
[002] The present invention was realized with government support with the grants of NIH P50 CA107399, P01 CA030206 and M01 RR0004. The government has certain rights in the present invention. Background of the invention
[003] Acute myeloid leukemia (AML) is a disease characterized by the rapid proliferation of immature myeloid cells in the bone marrow, causing dysfunctional hematopoiesis [1]. First-line treatments for acute myeloid leukemia (AML) have remained unchanged for nearly 50 years, and AML remains a disease with a poor prognosis. Although standard induction chemotherapy can induce complete remissions, many patients relapse and succumb to the disease [2]. Therefore, the development of new therapeutic agents for AML is crucial.
[004] Allogeneic hematopoietic cell transplantation can achieve disease cure in selected patients and underscores the susceptibility of AML to donor-derived immunotherapy. Furthermore, the interleukin 3 receptor alpha chain (CD123) has been identified as a potential immunotherapeutic target as it is overexpressed in AML compared to normal hematopoietic embryonic cells.
[005] Recent advances in AML cell immunophenotyping have revealed several AML-associated cell surface antigens that may act as targets for future therapies [3]. In addition, preclinical investigations using antibodies targeting CD44, CD47, T cell immunoglobulin mucin-3 (TIM-3) and the interleukin 3 receptor alpha chain (IL-3Rα; CD-123) for the treatment of AML have been described and demonstrated promising antileukemic activity in murine models [3,4]. CD123 is expressed in several malignancies including acute and chronic myeloid leukemia, hairy cell leukemia, B-cell precursor acute lymphoblastic leukemia, and blastic plasmacytoid dendritic cell neoplasm. Furthermore, CD123 is not generally expressed on normal hematopoietic embryonic cells, making it an ideal immunotherapeutic target. In addition, two phase I trials for specific CD123 therapeutics have been completed with both drugs offering good safety profiles (Clinical Trials.gov ID: NCT00401739 and NCT00397579). Unfortunately, these CD123-targeted drugs have limited efficacy suggesting that other alternative therapies are needed to observe antileukemic activity.
[006] A possibly more potent alternative therapy for treating AML is the use of T cells expressing chimeric antigen receptors (CARs) that redirect T cell specificity across cell surface antigen-associated tumors (TAAs) in a MHC-independent [5]. In most cases, CARs include a single-chain variable fragment (scFv) of a monoclonal antibody fused to the signaling domain of CD3Z and may contain a costimulatory endodomain [5]. Several groups have developed CARs targeting multiple antigens for the treatment of B-cell malignancies [6-10] and many have undergone evaluation of CAR-expressing T cells in phase 1 clinical trials [11-15]. In contrast, T cells expressing CAR for the treatment of AML remain rare [16,17].
[007] While treatment regimens for AML may achieve complete responses in selected patients, they may not evidence the need for new therapeutic agents that may lead to more durable responses. Several immunotherapies for AML including antigen-specific cytotoxic T lymphocytes, alloreactive natural killer cells, and dendritic cell vaccines are currently being developed. For example, Oka et al. demonstrated that Wilms tumor peptide 1 vaccination can lead to clinical and immune responses in AML patients [33]. However, these targeted therapies are HLA-dependent. For this purpose, it may be desirable to devise a therapeutic target, such as a CAR, that can redirect T cell specificity to select target AML cells in an HLA-independent manner. Summary
[008] A family of chimeric antigen receptors (CARs) containing a specific scFv CD123 region was developed to target different epitopes on CD123. In some embodiments, the CD123 chimeric antigen receptor (CD123CAR) gene includes an anti-CD123 scFv region fused in frame to a modified IgG4 hinge region comprising an alteration of a modified IgG4 spacer region including an S228P substitution, a L235E and optionally a replacement N297Q. The CD123CAR gene also includes at least one costimulatory signaling domain; and a T cell receptor (TCR) zeta chain signaling domain. In some embodiments, the CD123CAR gene includes a nucleotide sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. In other embodiments, the CD123CAR gene encodes an amino acid sequence that includes SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12.
[009] In accordance with the embodiments described below, the CD123CAR genes can be part of an expression cassette that is inserted into a vector (eg, a viral vector). As such, a population of human T cells can be transduced by the vector, resulting in the expression of CD123CAR genes by T cells. When expressed on T cells from healthy donors (CD4/CD8), CD123CARs genes redirect cell specificity and potent effector activity. mediated against CD123+ cell lines, as well as samples from patients with primary AML. CD123CAR T cells do not significantly alter granulocyte/macrophage and erythroid colony formation in vitro, suggesting a differential effect on AML cells as opposed to immune cells.
[0010] T cells obtained from patients with active AML can also be modified to express the CD123CAR genes and are able to lyse autologous AML blasts in vitro. These results suggest that transduced CD123CAR T cells can be used as an immunotherapy for the treatment of high-risk AML. Thus, in accordance with some embodiments, methods for treating AML in a subject are provided, wherein such methods include a step of administering a first population of T cells transduced with a first CD123CAR gene to the subject. The methods may also comprise an additional step of administering the first population of T cells transfected with the first CD123CAR gene combined with a second population of T cells transduced with a second CD123CAR gene to the subject. In some embodiments, the first CD123CAR gene includes a nucleotide sequence selected from SEQ ID NO:3 or SEQ ID NO:4. The second CD123CAR gene may also include a nucleotide sequence selected from SEQ ID NO:3 or SEQ ID NO:4, however, the nucleotide sequence of the second CD123CAR gene may not be the same as that selected for the first CD123CAR gene. This results in a combined treatment of AML using two or more different populations of CD123CAR-transduced T cells, which may have a synergistic effect when compared to using a single population of CD123CAR-transduced T cells. Brief description of figures
[0011] Figure 1 describes that CD123-specific CARs can be expressed on human T cells from healthy donors. (A) Schematic diagram of the CAR containing a modified IgG4 hinge, a modified transmembrane, the CD28 intracellular signaling domain, and the CD3Z signaling domain. The T2A ribosomal skip sequence and the EGFR truncated transduction marker (EGFRt) are also indicated. (B) Representative phenotype of Mock and lenti-transduced T cells from a healthy single donor. After immunomagnetic selection and a cycle of expansion, the modified CAR T cells were stained with biotinylated anti-Fc or biotinylated anti-EGFR, followed by PE-conjugated streptavidin and anti-TCRα/β, anti-CD4, or anti-CD8, and analyzed by flow cytometry. Quadrant placement is based on staining with control isotypes, and the percentage of cells in each quadrant is indicated. (C) Expression of the indicated cell surface markers from three different T cell lines from healthy donors after immunomagnetic selection and an expansion cycle. Data represent mean values ± SEM.
[0012] Figure 2 demonstrates that T cells that express CD123-specific CAR lyse tumor cell lines that express CD123. (A) Kilometric analysis of the flow of 293T cells transfected to express CD123 (top, black line) or CD19 (bottom, black line). Transduced parental Mock 293T cells were stained with anti-CD123 or anti-CD19 antibodies (gray fill, top and bottom) to determine background expression levels. (B) Specific cytotoxicity of CD123-CAR expressing T cells (26292 and 32716) against 293T cells expressing either CD123 (293T-CD123) or CD19 (293T-CD19) by chromium release assay. Data represent mean values from triplicate wells + SD. (C) Kilometric analysis of CD123 flux in the kG1a AML cell line, the EBV-transformed LCL cell line, and the K562 CML cell line. The percentage of positive cells for CD123 staining (black line) relative to isotype controls (gray fill) is indicated in each histogram. (D) Specific cytotoxicity of CD123-CAR T cells (26292 and 32716) against the CD19+ CD123+ LCL cell line and the CD19-CD123+ KG1a cell line by the chromium release assay. LCL expressing OKT3 (LCL-OKT3) and CD-19 - CD123 - K562 cell lines were used as positive control and negative control cell lines respectively. Data represent mean values from triplicate wells ± D.P.
[0013] Figure 3 describes that CD123-specific T cells release INF-y and TNF-a and proliferate in response to target cells that express CD123. CD123 CAR T cells or paired control T cells from three healthy donors were co-cultured with the indicated cell lines for 24 h, with an E:T ratio of 10:1, and the release of IFN-γ and TNF-a was quantified by Luminex multiplex bead technology. (B) Pair-matched CFSE-labeled specific CD19 or CD123 T cells were co-cultured with the cell lines with the indicated stimulator for 96 hours, with an E:T ratio of 2:1, and analyzed by flow cytometry for dilution. of the CFSE. Unstimulated T cells (filled histograms) were used as basal T cell proliferation controls.
[0014] Figure 4 depicts the activation of multiple effector functions of CD4 and CD8 T cells by specific CD123 CARs after co-culture with primary AML samples. T cells made with CAR pair-matching were co-cultured for six hours with three different samples from AML patients (AML 179, 373 and 605), and analyzed for surface CD107a expression and intracellular production of IFN-y and TNF- α. (A, bar graph). Percentage of DAPI-CD3+CD8+EGFRt+ cells expressing CD107a. Data represent mean values + S.D. (A, pie charts). The fractions of CD3+CD8+EGFRt+ cells, which were subjected to degranulation and produced IFN-y and TNF-a, are shown in pie charts. (B) DAPI-CD3+CD4+EGFRt+ population data from the same experiment as described in A and B. (C) Paired specific CD19 or CD123 T cells were co-cultured with the indicated stimulator cells for 72 h and an E:T ratio of 2 :1, and analyzed by flow cytometry for dilution of CFSE in the DAPICD3+ EGFRt+ population. The LCL and K562 cell lines act as negative and positive controls, respectively. Pre B-ALL 802 is a double positive primary patient sample for CD19 and CD123. Quadrant placement is based on unstimulated T cells.
[0015] Figure 5 demonstrates that primary AML cells are chosen as targets by specific CD123 T cells. (A) Paired CFSE-labeled specific CD19 or CD123 T cells were co-cultured for 4 hours with 51 Cr-labeled primary CD34+ LMA samples with an E:T ratio of 25:1. The LCL and K562 cell lines act as positive and negative controls, respectively. Pre B-ALL 802 is a double positive primary patient sample for CD19 and CD123. Data represent mean values from triplicate wells + D.P. (B) Specific lysis of AML blasts from three primary AML patient samples in (A). Data represent mean values ± SEM. * p<0.05 and ** p<0.0005 using Student's unpaired test comparing 26292 and 32716 to CD19R.
[0016] Figure 6 shows the effect of T cells expressing CD123 on leukemic and normal progenitor cells in vitro. (A and B) CD34+ umbilical cord blood (CB) cells (n=3) were immunomagnetically selected for CD34 and co-cultured with specific CD19 or CD123 paired T cells or in a single (untreated) medium for 4 hours with an E: T of 25:1. The foramen cells were plated in semi-solid methylcellulose progenitor culture for 14-18 days and evaluated for the presence of macrophage-granulocyte colony forming unit (CFU-GM, A) and erythoid burst forming units (BFU-E, B ). The percentages are normalized to specific CD19 T cell controls. Data represent mean values ± SEM for three different CB samples. (C) Samples from patients with CD34+ primary AML (AML 493, 519 or 545) were immunomagnetically selected and co-cultured with either specific CD19 or CD123 paired T cells or with medium alone (untreated) for 4 hours with an E:T ratio of 25:1. Cells were then plated in methylcellulose progenitor culture for 14-18 days and evaluated for the presence of leukemia colony forming units (CFU-L). The percentages are normalized to specific CD19 T cell controls. Data represent mean values ± SEM for three different samples of patients with primary AML.*, p < 0.05 using unpaired Student's t test comparing 26292 and 32716 at CD19R. (D) Combined colony formation of CB with cells from (A) or AML cells (C) treated with the CD123 target CAR structure (26292 or 32716) normalized to CD19R. p < 0.05 using unpaired Student's t test. Figure 7 demonstrates that redirected CD 123 CAR T cells derived from AML patients specifically lyse autologous blasts in vitro. (A) T cells from three AML patients were transduced by lentiviruses to express the CD19R, 26292, or 32716 CARs. T cell lines were demonstrated from AML 722 19 days after transduction. (B) Expression of CD123 on target cells used in the 51Cr release assay. The percentage of CD123+ cells and the relative fluorescence index (RFI) of each sample are indicated. (C) Results of 4-hour autologous death assays using T cells engineered from three AML patients as effectors, and 51 Cr-labeled autologous CD34-enriched blasts as target cells. Data represent mean values from triplicate wells + D.P.
[0017] Figure 8 demonstrates changes in tumor size as demonstrated by bioluminescent imaging of NSG mice that were treated five days after injection of the LMA cell line KG1a modified to express firefly luciferase (day 5) with transduced CD123CAR T cells ( 26292) containing the S228P+L235E mutations or the S228P+L235E+N297Q mutations.
[0018] Figure 9 presents a schematic diagram of a chimeric antigen receptor (CAR) that has an antigen-specific single chain Fv, a hinge region, a costimulatory signaling domain, and a zeta chain signaling domain of the receptor. of the T cell according to some modalities (Image by Urba WJ and Longo DL N Engl J Med 2011; 365:754-757).
[0019] Figure 10 shows a schematic diagram of structure 32716CAR showing an L235E mutation and an S228P mutation ("32716CAR (S228P+L235E)") along with the nucleotide sequence of structure 32716CAR(S228P+L235E) (SEQ ID NO:1 — antisense strand (top numbered strand); SEQ ID NO:5 - sense strand (bottom unnumbered strand)) and the amino acid sequence of structure 32716CAR (S228P+L235E) (SEQ ID NO: 9) according to some embodiments. Mutations are shown in bold.
[0020] Figure 11 demonstrates a schematic diagram of the construction of 26292CAR showing an L235E mutation and an S228P mutation ("26292CAR(S228P+L235E)") along with the nucleotide sequence of the construct 26292CAR(S228P+L235E) (SEQ ID NO:2 - antisense strand (top numbered strand); SEQ ID NO:6 - sense strand (bottom unnumbered strand)) and the amino acid sequence of construct 26292CAR(S228P+L235E) (SEQ ID NO:10) according to some embodiments. Mutations are shown in bold.
[0021] Figure 12 shows a schematic diagram of construct 32716CAR showing an L235E mutation, an S228P mutation and an N297Q mutation(“32716CAR(S228P+L235E+N297Q)”) along with the nucleotide sequence of construct 32716CAR(S228P+L235E +N297Q) (SEQ ID NO:3 - antisense strand (top numbered strand); SEQ ID NO:7 - sense strand (bottom unnumbered strand)) and the amino acid sequence of construct 32716CAR(S228P+L235E+N297Q)(SEQ ID NO:11), according to some modalities. Mutations are shown in bold and underlined. The IUPAC base code R corresponds to an A or G, and the IUPAC base code Y corresponds to a T or C.
[0022] Figure 13 shows a schematic diagram of construct 26292CAR showing an L235E mutation, an S228P mutation and an N297Q mutation(“26292CAR(S228P+L235E+N297Q)”) along with the nucleotide sequence of the construct 26292CAR(S228P+L235E +N297Q) (SEQ ID NO:4 - antisense strand (top numbered strand); SEQ ID NO:8 - sense strand (bottom unnumbered strand)) and the amino acid sequence of construct 26292CAR(S228P+L235E+N297Q) (SEQ ID NO:12) according to some arrangements. Mutations are shown in bold. The IUPAC base code R corresponds to an A or G, and the IUPAC base code Y corresponds to a T or C.
[0023] Figure 14 shows the expression of CD123 in primary AML and umbilical cord blood samples. (A) Representative examples of CD123 expression on primary AML cells. Cells were passaged in the DAPI-lineage-CD34+ population and evaluated for CD123 expression (black - isotype control, red - anti-CD123). (B) Percentage of CD123 positive cells in the DAPI-lineage-CD34+ population. Each point represents an individual sample. (C) Relative fluorescence index of CD123 (RFI) in the DAPI-lineage-CD34+ population. RFI is calculated by dividing the average of anti-CD123 cells by the average of cells stained by the control isotype. (D) Histogram of overlapping CD123 expression with LMA 605 (red), LMA 722 (blue, and a sample of umbilical cord blood (gray).Control isotype is shown in black.
[0024] Figure 15 illustrates a gating strategy used to investigate activation of multiple effector functions by CD123-specific cells in response to incubation with samples from patients with primary AML. The gating strategy for polychromatic flow cytometry for identification of T cell effector functions is demonstrated for CD123 CAR cells (based 26292) after co-culture with LMA 373. (A) An initial passage is performed on D3+ cells. (B) A second strategy, using a fluorescence minus a control, is established in EGFRt+ cells. (C) A tertiary strategy is established for CD4+ and CD8+ populations. (D) A final passage is established on CD107a+ cells. (E) Production of IFN-Y and TNF-α within CD107a+ populations. Quadrants were created using samples stained with isotope control. The percentages in each quadrant were observed.
[0025] Figure 16 demonstrates CFSE that is diluted in the CD4 and CD8 populations of CAR expressing T cells. The CD4 (A) and CD8 (B) subpopulations of the cells shown in Figure 5C are demonstrated. After an initial passage in DAPI-CD3+EGFRt+ cells, CD4 and CD8 cells were analyzed for dilution with CFSE after co-culture with samples from patients with primary AML. Quadrant placement is based on unstimulated T cells.Detailed Description
[0026] Certain embodiments of the invention are described in detail, using specific examples, sequences and figures. The enumerated embodiments are not intended to limit the invention, as the invention encompasses all alternatives, modifications and equivalents that may be included within the scope of the present invention as defined by the claims. One skilled in the art will recognize many methods and materials similar or equivalent to those described in the present invention that could be used in the practice of this invention.
[0027] In some embodiments, a gene encoding a target chimeric antigen receptor (CAR) is provided. In accordance with certain embodiments, the gene encodes a specific CD123-CAR (CD123CAR). A CD123CAR gene includes an anti-CD123 single-chain Fv (ScFv) region and one or more of the following domains: a hinge region, a costimulatory signaling domain, an intracellular signaling domain, or a combination thereof.
[0028] In some embodiments, a CD123CAR gene may include, but is not limited to, an anti-CD123 single-chain Fv (scFv) region, a hinge region, and, optionally, a costimulatory signaling domain, and, optionally, a intracellular signaling domain.
[0029] In certain embodiments, a CD123CAR gene may include, but is not limited to, an anti-CD123 single-chain Fv (scFv) region, a hinge region, and an intracellular signaling domain (Figure 9).
[0030] The anti-CD123 sFv region can include a nucleotide sequence which, when expressed, can bind an epitope of CD123. In some embodiments, the anti-CD123 Fv region (scFv) includes a nucleotide encoding a VH and VL domain of recombinant immunotoxins (RITs) 26292 and 32716 [18]. A CD123CAR gene that targets 26292 or 32716 is also called 26292CAR or 32716CAR, respectively. In certain embodiments, an anti-CD123 scFv region includes a nucleotide sequence selected from the following: nucleotides 82-814 of SEQ ID NO:1 or SEQ ID NO:3 to a 32716CAR nucleotides 82-792 of SEQ ID NO:2 or SEQ ID NO :4 for a 26292CAR; or Said nucleotide sequences encode amino acid sequences selected from the following: residues 23-266 of SEQ ID NO:9 or SEQ ID NO:11 when used in a 32716CAR; or residues 23-259 of SEQ ID NO:10 or SEQ ID NO:12 when used in a 26292CAR.
[0031] In certain embodiments, the anti-CD123 scFv region can be modified to enhance binding or to reduce immunogenicity. For example, in one aspect, the anti-CD123 scFv region may be a humanized anti-CD123 scFv region.
[0032] The hinge region may include at least a portion of an immunoglobulin (eg IgG1, IgG2, IgG3, IgG4) remaining between the CH2-CH3 domains. In some embodiments, the hinge region is modified. The modified hinge may have one or more amino acid substitutions or modifications that contribute to reducing the off-target effects of CD123CAR, thereby increasing its specificity and effectiveness. An "amino acid modification" or an "amino acid substitution" or a "substitution" as used in the present invention means an amino acid substitution, insertion and/or deletion in a protein or peptide sequence. An "amino acid substitution" or "substitution" as used in the present invention means a substitution of an amino acid at a particular position in a parent peptide or protein sequence with another amino acid. For example, the S228P substitution refers to a variant protein or peptide where the serine at position 228 is replaced with proline.
[0033] Amino acid substitutions can be made by mutation such that a particular codon in the nucleic acid sequence encoding the protein or peptide is changed to a codon encoding a different amino acid. Such a mutation is produced by making possible a minimal change in nucleotides. Such a substitution mutation can be made to change an amino acid in the resulting protein in a non-conservative manner (i.e., changing the codon of an amino acid belonging to a group of amino acids, which has a particular size or characteristic, to an amino acid belonging to another group) or conservatively (i.e., changing the codon of an amino acid belonging to a group of amino acids having a particular size or characteristic to an amino acid belonging to the same group). Such a conservative change generally produces less change in the structure and function of the resulting protein.
[0034] Examples of various groups of amino acids are listed below:• Amino acids with non-polar R groups: Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, Tryptophan, Methionine• Amino acids with uncharged polar R groups: Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine, Glutamine• Amino acids with polar charged R groups (negatively charged at pH 6.0): Aspartic acid, glutamic acid Basic amino acids (positively charged at pH 6.0): Lysine, Arginine, Histidine (at pH 6.0) ,0)
[0035] Another group comprises the amino acids with phenyl groups: Phenylalanine, tryptophan, tyrosine.
[0036] Another group may be in agreement with the molecular weight (i.e. the size of the R groups), as shown below:

[0037] In certain embodiments, the modified hinge is derived from an IgG1, IgG2, IgG3, or IgG4 that includes one or more amino acid residues substituted with an amino acid residue different from that present in an unmodified hinge. One or more substituted amino acid residues are selected, but not limited to, one or more amino acid residues at positions 220, 226, 228, 229, 230, 233, 234, 235, 234, 237, 238, 239, 243, 247, 267 , 268, 280, 290, 292, 297, 298, 299, 300, 305, 309, 218, 326, 330, 331, 332, 333, 334, 336, 339, or a combination thereof.
[0038] In some embodiments, the modified hinge is derived from an IgG1, IgG2, IgG3, or IgG4 that includes, but is not limited to, one or more of the following amino acid residue substitutions: C220S, C226S, S228P, C229S, a combination of these (50).
[0039] In some embodiments, the modified hinge is derived from an IgG4 hinge having the following amino acid sequence: Pos. 219 ESKYGPPCPS CPAPEFLGGP SVFLFPPKPK DTLMISRTPEVTCVVVDVSQ EDPEVQFNWYPos. 279 VDGVEVHNAK TKPREEQFNS TYRVVSVLTV LHQDWLNGKEYKCKVSNKGL PSSIEKTISKPos. 339 AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIAVEWESNGQPE NNYKTTPPVL Pos. 399 DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK (SEQ ID NO:13)
[0040] In certain embodiments, the modified hinge is derived from IgG4 which includes one or more amino acid residues substituted with an amino acid residue different from that present in an unmodified hinge. One or more amino acid residues are selected, but not limited to, one or more amino acid residues at positions 220, 226, 228, 229, 230, 233, 234, 235, 234, 237, 238, 239,243, 247, 267, 268, 280, 290, 292, 297, 298, 299, 300, 305,309, 218, 326, 330, 331, 332, 333, 334, 336, 339, or a combination thereof.
[0041] In some embodiments, the modified hinge is derived from an IgG4 that includes, but is not limited to, one or more substitutions below amino acid residues: 220S, 226S, 228P, 229S, 230S, 233P, 234A, 234V, 234F, 234A The , 318A, 326A, 326W, 326E, 328F,330L, 330S, 331S, 331S, 332E, 333A, 333S, 333S, 334A, 339D,339Q, 396L, or a combination thereof, where the unmodified hinge amino acid is substituted with the amino acids identified above at the indicated position.
[0042] In some embodiments, the modified IgG4 hinge includes, but is not limited to, a proline (P) substitution in place of serine (S) at position 228 (S228P), a leucine (L) substitution in place of glutamic acid (E) at position 235 (L235E), a substitution of asparagine(N) in place of glutamine (Q) at position 297 (N297Q). In certain embodiments, a modified IgG4 hinge region includes a nucleotide sequence from: nucleotides 814-1500 of SEQ ID NO:1 or SEQ ID NO:3 for a 32716CAR; or nucleotides 793-1479 of SEQ ID NO:2 or SEQ ID NO:4 for a 26292CAR. Said nucleotide sequences encode amino acid sequences selected from: residues 267-495 of SEQ ID NO:1 or SEQ ID NO:3 when used in a 32716CAR; or residues 260-488 of SEQ ID NO:2 or SEQ ID NO:4 when used in a 26292CAR.
[0043] In one embodiment, the hinge region of the modified IgG4 includes an S228P replacement and an L235E ("S228P+L235E") (see Figures 10 and 11). In another embodiment, the hinge region of IgG4 includes an S228P substitution, an L235E substitution, and an N297Q substitution ("S228P+L235e+N297Q") (see Figures 12 and 13 ).
[0044] In some embodiments, the hinge may be modified to replace the Fc spacer region on the C123CAR for a region that lacks Fc binding, such as the hinge region of CD8a. Alternately, the spacer region Fc of the hinge can be deleted. Such substitutions can reduce or eliminate Fc binding.
[0045] The term "position", as used in the present invention, is a location in the sequence of a protein. Positions can be numbered sequentially, or according to an established format, for example a Kabat position or an EU position or EU index as in Kabat. For all positions discussed in the present invention, the numbering is in accordance with the EU index or EU numbering scheme (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition, United States Public Health Service, National Institutes of Health, Bethesda, incorporated herein by reference). The EU index or EU index as in Kabat or numbering scheme refers to the EU antibody numbering (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85 , which is incorporated herein by reference. of Kabat, although well known in the art, can vary from position EU to a particular position. For example, substitutions S228P and L235E described above refer to position EU. However, these substitutions can also correspond to positions Kabat 241 (S241P) and 248 (L248E) [21].
[0046] The costimulatory signaling domain may include any suitable domain including, but not limited to, a 4-1BB costimulatory domain, an OX-40 costimulatory domain, a CD27 costimulatory domain, or a CD28 costimulatory domain. In accordance with the embodiments described in the present invention, a CD123CAR can include at least one costimulatory signaling domain. In one aspect, CD123CAR has a single costimulatory signaling domain, or may include two or more costimulatory signaling domains, such as those described above. In another aspect, the costimulatory domain can be produced from a single costimulatory domain, such as those described above, or alternatively, can be produced from two or more portions of two or more costimulatory domains. Alternately, in some embodiments, CD123CAR does not include a costimulatory signaling domain.
[0047] In one embodiment, the CD123CAR includes a costimulatory signaling domain which is a CD28 costimulatory domain. The CD28 signaling domain may include a modified CD28 transmembrane domain. In one embodiment, such a modified CD28 transmembrane domain has one or more amino acid substitutions or modifications including, but not limited to, a leucine-leucine (LL) to glycine-glycine (GG) substitution at amino acid residues 530-531 of the SEQ ID NO:10 or SEQ ID NO:12 or residues 523-524 of SEQ ID NO:11 or SEQ ID NO:13 (e.g., RLLH ^ RGGH [22]). In certain embodiments, a modified costimulatory signaling domain may include a nucleotide sequence selected from the following: residues 498-564 of SEQ ID NO:1 or SEQ ID NO:3 when used in a 32716CAR; or residues 489-557 of SEQ ID NO:2 or SEQ ID NO:4 when used in a 26292CAR.
[0048] The intracellular signaling domain may include any suitable T cell receptor (TCR) complex, signaling a portion of the domain thereof. In some embodiments, the intracellular signaling domain is a Z-chain signaling domain that may include a nucleotide sequence selected from: nucleotides 1717-2052 of SEQ ID NO:1 or SEQ ID NO:3 for a 32716CAR; or nucleotides 1696-2031 of SEQ ID NO:2 or SEQ ID NO:4 for a 26292CAR.
[0049] Said nucleotide sequences encode amino acid sequences selected from the following: residues 568-679 of SEQ ID NO:1 or SEQ ID NO:3 when used in a 32716CAR; residues 561-672 of SEQ ID NO:2 or SEQ ID NO:4 when used in a 26292CAR.
[0050] Thus, in accordance with the embodiments described above, the CD123CAR gene may include a nucleotide sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4. In other embodiments, the CD123CAR gene can encode an amino acid sequence selected from SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12. (Figures 10, 11, 12, 13). CD123CAR gene expression and T cell transduction
[0051] In some embodiments, the CD123CAR gene is part of an expression cassette. In some embodiments, the expression cassette may, in addition to the CD123CAR gene, also include an accessory gene. When expressed by a T cell, the accessory gene can serve as a marker of T cell selection, an in vivo screening marker, or a suicide gene for transduced T cells.
[0052] In some embodiments, the accessory gene is a truncated EGFR gene (EGFRt). An EGFRt can be used as a non-immunogenic selection tool (e.g., immunomagnetic selection using bionitrated cetumixab combined with anti-biotin microspheres to enrich T cells that have been lentivirally transduced with EGFRt-containing constructs), tracer marker (e.g., flow cytometric analysis for T cell engraftment screening), and suicide gene (eg, by dependent cell cytotoxicity (ADCC) mediated via Cetuximab/Erbitux®). An example of an EGFR gene (EGFRt), which can be used in accordance with embodiments of the present invention, is described in International Application No. PCT/US2010/055329, the contents of which are incorporated by reference in its entirety. In other embodiments, the accessory gene is a truncated CD19 gene (CD19t).
[0053] In another embodiment, the accessory gene is an induced suicide gene. A suicide gene is a recombinant gene that subjects the cell, in which it is expressed, to programmed cell death or antibody-mediated clearance at a desired time. In one embodiment, an induced suicide gene that can be used as an induced caspase 9 gene (see, Staathof et al (2005) An inducible caspase 9 safety switch for T-cell therapy. Blood. Jun 1; 105 (11) 4247-4254, the contents of which are incorporated by reference in their entirety.
[0054] In some embodiments, the expression cassette, which includes a CD123CAR gene described above, can be inserted into a vector for delivery - via transduction or transfection of a target cell. Any suitable vector can be used, for example a bacterial vector, a viral vector, or a plasmid. In some embodiments, the viral vector is selected from a retroviral or lentiviral, a poxvirus, an adenoviral, or an adeno-associated viral. In some embodiments, the vector may transfer a population of healthy T cells. Successfully transduced or transfected target cells express one or more genes that are part of the expression cassette.
[0055] As such, one or more populations of T cells can be transduced with a CD123CAR gene. In some embodiments, the CD123CAR gene includes a nucleotide sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. Thus, in some embodiments, the transduced T cells express a CD123CAR gene encoding an amino acid sequence selected from SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12 (Figures 10, 11, 12, 13). The transduced T cells can be from a donor, or they can be from an individual who has AML and is in need of treatment for AML. In some embodiments, transduced T cells are used in an immunotherapy treatment for the treatment of AML.
[0056] One or more populations of T cells may also be part of a pharmaceutically acceptable composition for administration to an individual. In addition to the transduced CD123CAR T cells, the pharmaceutically effective composition may include one or more pharmaceutically effective carriers. A "pharmaceutically acceptable carrier" as used in the present invention refers to a material, composition, or vehicle that is involved in transporting a treatment of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. Such a vehicle may comprise, for example, a liquid, solid or semi-solid filler, solvent, surfactant, diluent, excipient, auxiliary, binder, buffer, dissolution aid, encapsulating material, sequestering agent, dispersing agent, preservative, lubricant, disintegrant, thickener. , emulsifier, microbial agent, antioxidant, stabilizing agent, coloring agent, or combination thereof.
[0057] Each component of the vehicle is “pharmaceutically acceptable”, that is, it is compatible with other components of the composition and must be suitable for contact with any tissue, organ, or portion of the body, without presenting a risk of toxicity, irritation, allergic response , immunogenicity or any other complication that outweighs its therapeutic benefits.
[0058] Some examples of materials that act as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) natural polymers such as gelatin, collagen, fibrin, fibrinogen, laminin, decorin, hyaluronan, alginate and chitosan; (7) talc; (8) excipients such as cocoa butter, suppository paraffins; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters such as trimethylene carbonate, ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid (or alginate); (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) alcohol, such as ethyl alcohol and propanol; (20) phosphate buffer solutions; (21) thermoplastics, such as polylactic acid, polyglycolic acid; (22) polyesters, such as polycaprolactone; (23) self-assembled peptides; and (24) other compatible non-toxic substances used in pharmaceutical formulations, such as acetone.
[0059] Pharmaceutical compositions may contain acceptable auxiliary substances as needed for approximate physiological conditions such as pH adjustment and buffers, toxicity adjusting agent and the like, e.g. sodium acetate, sodium chloride, potassium chloride, chloride calcium, sodium lactate and the like.
[0060] In one embodiment, the pharmaceutically acceptable carrier is an aqueous carrier, for example, buffered saline and the like. In certain embodiments, the pharmaceutically acceptable carrier is a polar solvent, for example, acetone and alcohol.
[0061] The concentration of CD123CAR-transduced T cells in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, organ size, body weight and the like according to the particular form of administration selected and system needs. biological.
[0062] In certain embodiments, populations of T cells transduced with the CD124CAR gene (i.e., CD124CAR-transduced T cells), such as those described in the present invention, used in methods to target and kill AML cells, can grow in a cell culture. In certain aspects of this modality, the method can be used in a research setting or in vitro to assess the role of CD123 in the etiology of AML, or to assess the targeting abilities of new CD123CAR constructs.
[0063] Treatment of AML with CD123CAR-transduced T cells
[0064] In accordance with some embodiments, CD123CAR genes and T cell populations that are transduced with CD123CAR genes such as those described above can be used in methods for treating AML in an individual. Such methods may include a step of administering a therapeutically effective amount of at least one population of T cells transduced with at least one CD123CAR gene to the subject. In these embodiments, the population of transduced CD123CAR T cells expresses one or more CD123CAR genes, such as those described above. In certain embodiments, T cells are transduced and express a 32716CAR gene construct (SS228P+L235E+N297Q) (Figure 12) or a 26292CAR gene construct (S228P+L235E+N297Q) (Figure 13). When such cells are administered via an adoptive immunotherapy treatment, the transduced T cells specifically target and lyse CD123 cells (i.e., AML cells) in vivo, providing their cancer cell killing therapeutic effect. As described in the examples below, the CD123CSR gene constructs carrying the S228P and L235E mutations in the hinge band offer sufficient protection from off-target effects to produce a sufficient response in in vitro cultured cells. However, these data should not be extrapolated for these effects in in vivo constructs. Researchers always value in vitro data over its ability to transfer a treatment effect to in vivo data. Sometimes in vitro data do not match in vivo data. However, this correlation is unpredictable, because according to Figure 8, the CD123CAR (S228P+L235E) gene constructs (Figures 10-11), which showed a highly effective antitumor cellular effect in vitro, did not show the same effects in vivo. Consequently, an additional mutation was performed in the hinge region (N297Q) to produce CD123CAR constructs (S228P+L235E+N297Q). Unlike the administration of the CD123CAR gene constructs (S228P+L235E), the administration of these constructs results in a significant reduction in the leukemic load.
[0065] The population or populations of T cells bearing the CD123CAR gene or genes that can be used in accordance with the methods described in the present invention can be administered by any suitable route, alone or as part of a pharmaceutical composition. A route of administration refers to any route known in the art, including but not limited to intracranial, parenteral or transdermal. "Parenteral" refers to a route of administration that is generally associated with injection, including infraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intratumoral, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal or transtracheal. In certain embodiments, transduced T cells are administered intravenously or intrathecally.
[0066] The term "effective amount", as used in the present invention, refers to an amount of an agent, compound, treatment or therapy that produces a desired effect. For example, a population of cells may be contacted with an effective amount of an agent, compound, treatment or therapy to study its effect in vitro (e.g., cell culture) or to produce a desired therapeutic effect ex vivo or in vitro. An effective amount of an agent, compound, treatment or therapy can be used to produce a therapeutic effect in a subject, such as preventing or treating a target condition, ameliorating symptoms associated with the condition or producing a desired physiological effect. In such a case, the effective amount of a compound is a "therapeutically effective amount", "therapeutically effective concentration", or "therapeutically effective dose". The precise effective or therapeutically effective amount is an amount of the composition that will produce the best results in terms of treatment efficacy in a given individual or cell population. This amount will vary depending on a number of factors, including, but not limited to, the characteristics of the compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the individual (including age, sex, disease type and stage, general physical condition). , responsiveness to a particular dosage, and type of medication) or cells, the nature of the pharmaceutically acceptable vehicle or vehicles in the formulation, and the route of administration. An effective or therapeutically effective amount can vary depending on whether the compound is administered alone or combined with another compound, drug, therapy, or other therapeutic method or modality. One skilled in the clinical or pharmacological field will be able to determine an effective or therapeutically effective amount through routine experimentation, characterized by monitoring the individual or cellular response to administration of a compound and adjusting the dosage. For additional guidance, see Remington: TheScience and Practice of Pharmacy, 21st edition, Univ. of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, PA, 2005, incorporated herein by reference. Agents, compounds, treatments or therapies that can be used in an effective or therapeutically effective amount to produce a desired effect in accordance with the embodiments described in the present invention can include, but are not limited to, a CD123CAR gene, an expression cassette that includes a CD123CAR gene, a vector that provides an expression cassette that includes a CD123CAR gene for a target cell, such as a T cell, and a population of T cells that are transduced with a CD123CAR gene.
[0067] The terms “treating” or “treatment” of a condition refer to preventing the condition, reducing the onset or rate of development of the condition, reducing the risk of developing the condition, preventing or delaying the development of symptoms associated with the condition, reducing or termination of symptoms associated with the condition, producing a partial or total regression of the condition, or combinations thereof.
[0068] The term "subject" as used in the present invention refers to man or an animal, including all mammals, such as primates (particularly the higher primates), sheep, dogs, rodents (e.g., mouse or rat). ), guinea pigs, goats, pigs, cats, rabbits and cows. In some embodiments, the individual is the man.
[0069] In certain embodiments, methods for treating AML may include a step of administering a therapeutically effective amount of a first population of T cells transduced with a first CD123CAR gene combined with a therapeutically effective amount of a second population of T cells. transduced with a second CD123CAR gene.
[0070] In other embodiments, the induced CD123CAR T cells can be administered combined with one or more additional anti-cancer therapies. "In combination" or "combined with", as used in the present invention, means the treatment of cancer in the same individual using two or more agents, drugs, therapeutics, procedures, treatment regimens, treatment modalities, or a combination thereof, in any order. This includes simultaneous administration, and also with a time spacing of up to several days. Such combined treatment may also include more than one administration of any one or more agents, drugs, therapeutics, procedures, regimens, and treatment modalities. The administration of two or more agents, drugs, therapeutics, procedures, regimens and treatment modalities, or a combination thereof, may utilize the same or different routes of administration.
[0071] Additional anti-cancer therapies, which may be used in accordance with the methods described in the present invention, may include one or more anti-cancer procedures, treatment modalities, anti-cancer therapeutic agents, or a combination thereof. In some embodiments, CD123CAR-transduced T cells may be administered in combination with one or more anticancer procedures or treatment modalities including, but not limited to, stem cell transplantation (e.g., bone marrow or peripheral blood stem cell transplantation using allogeneic, autologous cells; or a non-myeloablative transplant), radiation, or surgical resection. In other embodiments, CD123CAR-transduced T cells can be administered in combination with one or more anticancer therapeutic agents or drugs that can be used to treat AML, including, but not limited to, chemotherapeutics and other anticancer drugs, immunotherapeutics, targeted therapeutics, or a combination thereof. .
[0072] Chemotherapeutics and other anti-cancer drugs that can be administered in combination with CD123CAR-transduced T cells in accordance with the embodiments described in the present invention include, but are not limited to, total transretinoic acid (ATRA), arsenic trioxide, anthracycline antibiotics, and pharmaceutically acceptable salts thereof (e.g. doxorubicin hydrochloride, daunorubicin hydrochloride, idarubicin, mitoxantrone), alkylating agents (e.g. cyclophosphamide, laromustine), antimetabolite analogues (cytarabine, 6-thioguanine, 6-mercaptopurine, methotrexate), demethylating agents (e.g. decitabine, 5-azacytidine), nucleic acid synthesis inhibitors (e.g. vincristine sulfate) or a combination thereof (e.g. “ADE”, which is a combination treatment that includes a combination of cytarabine (Ara -C), daunorubicin hydrochloride and etoposide).
[0073] Immunotherapeutics that can be administered in combination with the CD123CAR-transduced T cells, in accordance with the embodiments described in the present invention, include, but are not limited to, immunomodulatory reagents (e.g., STAT3 inhibitors, lenalidomide) and therapeutic monoclonal antibodies. Therapeutic monoclonal antibodies can be designed (i) to target one or more AML antigens including, but not limited to, CD33 (eg, gemtuzumab, lintuzumab), MUC1 (eg, cantuzumab, ravtansine, clivatuzumab, tetraxetane, pemtumomab); (ii) a B cell antigen (eg, alacizumab pegol, bevacizumab, icrucumab, ramucirumab, ranibizumab).
[0074] Targeted therapeutic agents that can be administered in combination with CD123CAR-transduced T cells in accordance with the embodiments described in the present invention include, but are not limited to, tyrosine kinase inhibitors (imatinib, dasatinib, nilotinib, sunitinib), farnesyl transferase inhibitors (e.g. tipifarnib), FLT inhibitors and c-kit (or CD117) inhibitors (imatinib, dasatinib, nilotinib).
[0075] Example 1: CD123CAR-transduced T cells exhibit potent cytolytic activity and multiple effector functions against AML in vitro Materials and methods
[0076] Cell lines. Unless otherwise specified, all cell lines were maintained in RPMI 1640 medium (Irvine Scientific) supplemented with 2 mM L-glutamine, 25 mM HEPES, and 10% heat-inactivated FCS (Hyclone), henceforth referred to as medium. complete (CM). Peripheral blood mononuclear cells (PBMCs) were transformed with Epstein-Barr virus to produce lymphoblastoid cell lines (LCL) as described previously [19]. LCL-OKT3 cells express membrane-bound OKT3 and are grown in CM supplemented with 0.4 mg/mL hydromycin [20]. KG1a cells (kindly provided by Dr. Ravi Bhatia) were maintained in IMDM (Irvine Scientific) with 25 mM HEPES, 4 mM L-glutamine (Irvine Scientific) and 20% FCS. 293T cells (kindly provided by the Center for Biomedicine and Genetics at City of Hope) were maintained in 10% heat-inactivated DMEM+FCS.
[0077] Primary LMA samples. Primary LMA samples were obtained from the peripheral blood of patients (referred to in the present invention as LMA sample ID Nos. 179, 373, 493, 519, 545, 559, 605, 722 and 813). The characteristics of the samples are summarized in table 1 below. Confidential Sample ID LMA Age/sex Cytogenetics Mutational status Flt3 Clinical status Sample type CD123 (RFI)a positive % of CD123179 7 4/M Intermediate riskt(1;7), t(14;15) ND Recurrence PB 428.32 99 .22373 47/M Low risk Complex of abnormalities in the 3 cell lines ND Recurrence PB 1052.83 99.66493 46/F Intermediate risk Trisomy 8 ND Recurrence PB 23.98 76.80519 44/F del (17p), dic (11; 7), clonal Loss of TP53/17p13.1 ND Relapse PB 63.18 97.40545 5 8/M Intermediate risk T(3;6), del(7) ND Failure in PB induction 52.73 99.32559 59/M Complex of abnormalities, marked hyperdiploidia Negative Recurrence Apheresis 9.30 45.0605 55/M Normal Negative Persistent PB 58.48 99.91722 22/M Intermediate risk t(14;21), del(9q) Negative Untreated PB 33, 53 92.74813 48/F Complex of abnormalities, trisomy 8, trisomy 21, add (17) ND Untreated BP 37.19 90.93Table 1. Characteristics of primary AML samples

[0078] Flow cytometry. Fluorochrome-conjugated isotype control, anti-CD4, anti-CD8, anti-T cell receptor-αβ (TCR-αβ), anti-CD123 (9F5), anti-CD34 (8G12), and anti-CD38 (HIT2) were obtained from BD Biosciences. Biotinylated Anti-Fc was obtained from Jackson ImmunoResearch Laboratories. Biotinylated cetuximab (Erbitux) was obtained from the COH pharmacy and has been previously described[20]. Biotinylated anti-CD2, anti-CD3, anti-CD7, anti-CD10, anti-CD11b, anti-CD19, anti-CD33, and anti-CD235A were obtained from eBioscience. Data collection was performed on a FACSCalibur, LSRII (BD Biosciences), or MACSQuant Analyzer (Miltenyi Biotec), and analyzed using FCS Express, Version 3 (De Novo Software).
[0079] Transfection of 293T cells with CD123. CD123cDNA was amplified from CD123-pMD18-T (Sino Biological Inc.) using polymerase chain reaction and primers (CD123-F: 5'-ATAAGGCCTGCCGCCACCATGGTCCTCCTTTGGCTCACG-3' and CD123-R 5'-ATAGCTAGCTCAAGTTTTCTGCACGACCTGTACTTC-3'). The PCR product was cloned into pMGPac using the StuI and NheI sites. 293T cells were transfected using Lipofectamine 2000 (Life Technologies) according to the manufacturer's instructions. Twenty-four hours post-transfection, CD123 expression was confirmed by flow cytometry.
[0080] Generation of lentiviral vectors. To produce the CAR constructs used in this study, codon-optimized DNA sequences encoding VH and VL chains, IgG4 hinge region, and a modified CD28 transmembrane domain (RLLH^RGGH[22]) were synthesized (GENEART) and cloned into CD19RCAR-T2AEGFRt_epHIV7 [20] using the NheI and RsrII sites to replace CD19RCAR. Lentivirus was produced by transfecting 293T cells with a lentiviral vector and the pCMV-Rev2, pCHGP-2, and pCMV-G vectors using the CalPhosTM mammalian cell transfection kit (Clontech). These constructs 26292 and 32716 CAR are also named as 26292CAR(S228P+L235E) or 26292CAR(S228P+L235E+N297Q) (Figures 11 and 13) and 32716CAR(S228P+L235E) or 32716CAR(S228P+L235E+N297Q) (Figures 1970) and 12). Lentiviral supernatants were collected at 24, 48 and 72 hours post-infection and concentrated by ultracentrifugation.
[0081] Transduction of healthy donors and PBMCs from patients with AML. Unidentified PBMCs were obtained from healthy donors and patients in approved institutional protocols. For healthy donors, T cells were activated using OKT3 (30ng/ml) in CM supplemented 3 times a week with IL-2 25 U/ml and IL-15 0.5 ng/ml (called in the present invention as cell medium). T). Seventy-two (72) hours post-activation, T cells were inoculated with lentiviruses with MOI=3 by centrifugation for 30 minutes at 800g and 32°C. CAR expression was analyzed by flow cytometry 12-14 days post lentiviral transduction. T cells expressing EGFRt were enriched as described previously [20]. T cells were expanded in T cell medium by the rapid expansion method [23].
[0082] For genetic modification of T cells from AML patients, thawed peripheral blood or apheresis product were stimulated using Dynabeads® T-human CD3/CD28 expander (Life Technologies) with a ratio of CD3+ cells:beads of 3:1 in T cell medium. Seventy-two (72) hours post-bead challenge, cells were inoculated with lentiviruses with MOI=3. Beads were removed 9-14 days after initiation of stimulation using a DynaMagTM 50 magnet (Life Technologies) and T cells were maintained in the medium. T cell lines derived from AML patients that express CAR were not immunomagnetically selected prior to use in cell death assays.
[0083] CFSE Proliferation Assay. T cells were labeled with 0.5 µM carboxyfluoroscein succinimidyl ester (CFSE; molecular probes) per manufacturer's instructions. Labeled T cells were co-cultured with or without stimulator cells with an E:T ratio of 2:1 in CM supplemented with 10 U/ml IL-2. After 72-96 hours, cells were harvested and stained with cetuximab as well as propidium iodide or DAPI to exclude dead cells from analysis. Samples were analyzed by flow cytometry to assess the proliferation of live EGFRt-positive cells by CFSE dilution.
[0084] Chromium release and cytokine secretion assay. Target cells were labeled for 1 hour with 51 Cr (PerkinElmer), washed five times and divided in triplicate at 5 x 10 3 cells/well with effector cells in various cell-to-target ratios (E:T). After a 4 hour co-culture, supernatants were collected and radioactivity was measured using a gamma counter or a Topcount (PerkinElmer). The specific percentage of lysis was calculated as described above [24]. Cytokine production after a 24-hour co-culture with an E:T ratio of 10:1 was measured as described previously [25].
[0085] CD107a degranulation and intracellular cytokine production. T cells were co-cultured with target cells with an E:T ratio of 2:1 for six hours at 37°C in the presence of GolgiStopTM (BD Biosciences) and anti-CD107a clone H4A3 or antibody according to isotype control. At the end of the 6 hour incubation, cells were harvested, washed and stained with anti-CD3, CD4, CD8 and biotinylated cetuximab followed by a secondary staining using PE-conjugated streptavidin. Cells were then fixed and permeabilized (Cytofix/CytopermTM BD Biosciences) according to the manufacturer's instructions and stained with anti-IFN-γ (BD Bioscience clone B27) and anti-TNF-α (BD Biosciences clone MAb11). Data acquisition was performed using the MACSQuant analyzer (Miltenyi Biotec) and analysis was performed using FCS Express Version 3 (From New Software).
[0086] Colony forming cells assay. CD34+ cells from umbilical cord blood (BC) mononuclear cells or primary AML samples were selected using immunomagnetic column sorting (Miltenyi Biotech). One hundred and three (103) CD34+ + CB cells were co-cultured with 25 x 10 3 effector cells for 4 hours before plating on semisolid methylcellulose progenitor culture in duplicate wells [26]. Fourteen to eighteen (14 to 18) days later, granulocyte-macrophage colony forming unit (CFU-GM) and erythroid colony charge forming unit (BFUE) were enumerated. For all AML samples, 5 x 10 3 CD34+ AML cells were co-cultured with 125 x 10 3 effector cells for 4 hours prior to plating in semi-solid methylcellulose progenitor culture in duplicate wells.
[0087] Statistical analysis. Analyzes were performed using Graphpad Prism v5.04. Unpaired Student's t-test was used to identify significant differences between treatment groups. ResultsProduction of T cells with CD123CAR expression
[0088] To redirect T cell specificity, lentiviral vectors that encode CD123CRs have been developed. Each of the CARs includes codon-optimized sequences that encode one of two CD123-specific scFvs regions, 26292 and 32716 [18], respectively. The scFvs are fused in structure to the Fc region of human IgG4, a CD28 costimulatory domain and a CD3Z signaling domain. Downstream of the CAR sequence is a T2A ribosomal skip sequence and a truncated human EGFR transduction marker (EGFRt) (Figure 1A). OKT3-stimulated PBMCs from healthy donors were lentitransduced and T cells expressing CAR were isolated by immunomagnetic selection using a biotinylated Erbitux antibody, followed by secondary staining with anti-biotin magnetic beads. After one REM cycle, isolated cells were analyzed by flow cytometry for CAR surface expression and T cell phenotype. Fc and EGFRt expression was greater than 90% in T cell lines produced from three healthy donors, and the final T cell products consisted of a mixture of CD4 and CD8 positive T cells (Figure 1B, 1C). CD123CAR T cells with specific target on tumor cell lines that express CD123
[0089] To confirm the specificity of CD123CAR T cells, the ability of genetically modified T cells to lyse provisionally transfected 293T cells to express CD123 was examined (293T-CD123; Figure 2A). CD123CAR T cells produced efficiently lysed 293T-CD123, but not 293T cells transiently transfected to express CD19, demonstrating CD123-specific recognition (Figure 2B). Next, the in vitro cytolytic capacity of CD123-specific T cells was investigated in relation to tumor cell lines that express CD123 endogenously. CD123 expression in LCL and KG1a cell lines was confirmed by flow cytometry (Figure 2C). CD123-specific T cell lines efficiently lyse the LCL and KG1a target lines, but not the CD123-K562 cell line (Figure 2C). Paired CD-19-specific T cells lysed CD19+LCL targets, but not CD19-KG1a or K562 targets (Figure 2D). The transduced parental Mock cells lysed only the positive control LCL-OKT3 cell lines (Figure 2D). CD123CAR T cells activate multiple effector functions when co-cultured with CD123-positive target cells
[0090] To assess the effector function of CD123-specific T cells, the secretion of IFN-y and TNF-a was measured after co-culture with various tumor cell lines. T cell products expressing CD123CAR produced IFN-y and TNF-a when co-cultured with CD123+ target cells, whereas paired CD19-specific T cells secreted these cytokines only when cultured with the CD19+ LCL or LCL-OKT3 cell line (Figure 3A) . In addition, CD123-specific T cell lines proliferated when co-cultured with CD123+ LCL, LCL-OKT3, or KG1a cell lines, but not with the CD123-K562 cell line (Figure 3B). In contrast, T cells expressing CD19- Paired CAR only proliferate when co-cultured with LCL or LCL-OKT3 (Figure 3B). CD123CAR T cells activate multiple effector functions when co-cultured with primary LMA samples
[0091] The overexpression of CD123 in primary AML samples is well documented [27-29] and confirmed in this study (figure 14). Multifaceted T cell responses are critical for strong immune responses to infections and vaccines, and they also play a role in the antitumor activity of retargeted CAR T cells [30]. To investigate the ability of CD123 CAR T cells to activate multiple effector pathways against primary AML samples, the T cells produced were co-cultured with three samples from AML patients (179, 373 and 605) for 6 hours and evaluated for upregulation of CD107a and production of IGN-y and TNF-a using polychromatic flow cytometry (gating strategy demonstrated in Figure 15). CD107a cell surface mobilization was observed in the CD4 and CD8 compartments of CD123-specific T cells, while in paired CD19R T cells no degranulation was observed relative to primary AML samples (Figure 4A, bar graphs). Subpopulations of CD107a+ CD123CAR T cells are also produced by the cytokines IFN-γ and TNF-α, or by both (Figure 4A, pie charts). This multifunctional response was observed in the CD4 and CD8 populations (Figure 4A and 4B). Additionally, the ability of produced CAR T cells to proliferate in response to co-culture with primary AML samples was examined. Both CD123-specific T cell lines were able to proliferate after co-culture with LMA 813 and pre B-ALL 802 samples (Figure 4C). Proliferation was observed in both CD4 and CD8 populations (figure 16). Paired CD19-specific T cells proliferated when co-cultured with CD19+ pre B-ALL 802, but did not proliferate when co-cultured with LMA 813. T cells expressing CD123CAR targeted on primary LMA cells in vitro Specific CD123 T cells do not eliminate colony formation by umbilical cord cells in vitro
[0092] Established that CD123 is expressed on common myeloid progenitors (CMPs) [31], the effect of T cells produced on the colony-forming ability of CD34-enriched normal cord blood (CB) samples was investigated. The formation of myeloid and erythroid colonies by CB samples was not significantly reduced after a 4-hour co-culture with CD123-CAR expressing T cells, with an E;T ratio of 25:1, when compared to paired CD19R CAR T cells ( figure 6A&B). Next, the ability of CD123-specific T cells to inhibit the growth of clonogenic AML cells was evaluated in vitro. Both CD123 CAR T cell lines significantly reduced leukemic colony formation compared to paired CD19R T cells (Figure 6C). Notably, specific T cells had a greater impact on leukemic colony formation compared to normal myeloid colony formation (Figure 6D, 69% reduction versus 31% reduction, respectively). be genetically modified to express CD123 CARs and specifically target autologous tumor cells like albo
[0093] T cells derived from AML patients are known to poorly repolize actin and form faulty immune synapses with autologous blasts [32]. In addition, CAR-expressing T cells derived from AML patients have yet to be described. Therefore, it was determined whether T cells from AML patients could be genetically modified to express CD123 CARs. Cryopreserved PBMCs (LMA 605 and LMA 722) or apheresis product (LMA 559) were stimulated by CD3/CD28 beads and lentivirally transduced to express CD123 CARs or a CD19R CAR control. All T cells derived from samples from three patients expressed 26292 CAR (40-65% transduction efficiency), 32716 CAR (46-70% transduction efficiency), and CD19R CAR (to assess the ability of CD123-specific cells to kill primary AML cells), paired CD19R CAR or T cells expressing CD123 CAR were co-cultured with CD34-enriched primary AML patient samples in a 4-hour 51Cr release assay. Unlike CD19R T cells, both CD123 CAR T cell lines intensely lysed all primary AML patient samples tested (Figure 5A). Additionally, although no statistical difference was observed between the cytolytic capacity of CD123CAR-expressing T cells, CD123-specific T cells demonstrated significantly increased cytotoxicity when compared to paired CD19R-CAR T cells (Figure 5B). (23-37% transduction efficiency). A representative example of the AML patient phenotype CAR-derived T cells is demonstrated in Figure 7A. Next, the patient's cytolytic potential with AML patient-derived CAR T cells relative to CD34-enriched autologous target cells was evaluated in a 4-hour 51Cr release assay. All of the CD34-enriched autologous cells expressed CD123, albeit with varying percentages and intensities (Figure 7D). LMA 605 and 722-derived T cells efficiently lysed autologous blasts, while LMA 559-derived T cells showed low levels of autologous blast lysis likely due to low and heterogeneous expression of CD123 on LMA 559 blasts (Figure 7C). Discussion
[0094] The modalities described in the present invention include the formation of two new CD123-targeted CARs using scFvs from recombinant immunotoxins (RITs), 26292 and 32716, which bind to distinct epitopes and have similar binding affinities for CD123 [18] . When expressed by a population of T cells, these CD123-targeted CARs redirect T cell specificity against cells that express CD123. Using a 4-hour chromium-51 (51Cr) release assay, T cells from healthy donors, which were engineered to express CD123CRs, lysed CD123+ cell lines and samples from primary AML patients. Additionally, CD123CAR T cells activated multiple effector functions after co-culture with CD123+ cell lines and with samples from patients with primary AML. In addition, CD123-targeted T cells did not significantly reduce the number of granulocyte-macrophage colony-forming units (CFU-GM), or erythroid charge-forming unit (BFU-E) of umbilical cord blood (CB) when compared to CD19 CAR T cells. Notably, although CD19-specific T cells have little impact on leukemic colony formation from primary AML samples, CD123-targeted T cells significantly reduce leukemic colony formation in vitro. It has also been shown that T cells derived from AML patients can express CD123 CARs and lyse autologous blasts in vitro.
[0095] T cells expressing the two CD123-specific CARs can specifically lyse CD123-expressing cell lines and AML patient samples, and activate multiple effector functions in an antigen-specific manner in vitro, demonstrating that both epitopes are potential targets for treatment. No differences were observed between the produced CD123 CAR T cell lines with respect to target cell death, cytokine secretion or proliferation when co-cultured with CD123+ cells. One possible explanation for this is that the binding affinities of the CD123-specific scFvs used in CD123-CARs are in the nanomolar range and differ by less than 3-fold, and therefore do not offer a significant advantage in binding the target antigen and are conferred by the scFv [18]. Expression of multiple surface antigens has been well documented [4,27,34]. Targeting some of these antigens through CAR-expressing T cells may not be feasible. For example, the AML-associated TIM-3 antigen is expressed on a subset of depleted T cells [35,36], and targeting the TIM-3 antigen using CAR-expressing T cells can lead to autolysis of genetically modified cells. Furthermore, CD47 is generally expressed [37], and therefore unlikely to be targeted by the CAR cells produced. The CD33 differentiation antigen is predominantly expressed on myeloid cells, and CD33-targeted immunotherapies such as gemtuzumab ozogamicin, antibodies with bispecific CD33/CD3 T cells, and a CD33 CAR are currently used in clinical and preclinical trials [17, 38, 39]. Like TIM-3, CD33 is expressed on a subset of T cells making it a non-ideal target for CAR-based therapy [40]. Additionally, the antileukemic activity of CD33-targeted therapies was accompanied with a slow recovery of hematopoiesis and cytopenias, as well as the result of CD33 expression in the long-term self-renewal of hematopoietic stem cells (HSCs) [41]. Hepatotoxicities are also a common side effect of CD33-targeted treatments and occur due to the undesired targeting of CD33+ Kupffer cells [42].
[0096] CD123 expression is absent in T cells, restricted predominantly to cells of myeloid lineage [43], and largely absent in HSCs [27]. These observations together make CD123 an attractive target for CAR-mediated T cell therapy. CD123-specific therapeutics showed favorable safety profiles in phase I trials (clinical trials gov ID: NCT00401739 and NCT00397579). Unfortunately, these therapies failed to induce responses in most treated patients. The produced CD123-CAR expressing T cells offer potent cytolytic capacity in vitro against CD123+ cell lines and primary AML samples. The studies described below demonstrate that primary samples from low-risk AML patients were susceptible to CD123 CAR T cell-mediated cytotoxicity. In the small group of primary samples used in the short-term cytotoxicity assays, samples from AML patients, who exhibited high-risk aspects in diagnosis and/or chemoresistance, were sensitive to CD123 CAR killing, similarly to what was observed in experiments using CD123+ cell lines.
[0097] Multifunctional T cell responses are related to viral infection control and may be important in the antitumor CAR T cell response [44]. In addition, patients responding to CD19 CAR T cell therapy showed detectable T cell responses (ie, degranulation, cytokine secretion, or proliferation) post-therapy in response to ex vivo CD19+ targets [11, 12,14] . In the examples below, the functionality of CD123-CAR expressing T cells was demonstrated by analyzing CD107a upregulation, production of inflammatory cytokines, and proliferation of CD123-specific T cells in response to CD123+ cell lines and primary AML samples. . Furthermore, multifunctionality has been observed in both CD4+ and CD8+ compartments, which can promote both constant and increased antileukemic activity within the tumor milieu [45, 46]. The inclusion of other co-stimulatory domains such as 4-1BB, and the use of “younger” less differentiated T cells may also enhance CD123 CAR responses, and are in the area of active research [9,47].
[0098] CD123-specific T cells also do not inhibit normal progenitor colony formation, even with an E:T ratio of 25:1. CD123 expression on CD34+CD38 lineage cells is a hallmark of the common myeloid progenitor cell and therefore a likely target of CD123 CAR T cells[31]. Although a reduction in the relative percentage of myeloid-derived colonies was observed when CB cells were incubated with CD123-specific T cells, the reduction was not significantly less than the paired CD19 CAR T cells. It is possible that the limited sample size is attributed to this result and also experimentation may reveal a significant reduction in the formation of CD123CAR T cell treated umbilical cord blood samples. Additionally, 4-hour co-culture of CAR T cells and CB cells prior to plating may not have a long enough time period to observe an effect on normal myeloid progenitor colony formation, and a long incubation time may reduce the number of myeloid colonies. observed derivatives. However, using the same methodology used for CB cells, a substantial reduction in the number of leukemic colonies formed was observed when samples from CD34-enriched AML patients were incubated with CD123 CAR T cells, suggesting that the 4-hour incubation time is enough to observe an effect between normal and leukemic colony formation. Alternatively, the relatively lower expression of CD123 on CB cells compared to AML cells may in part cause the inability of CD123 CAR T cells to alter myeloid-derived colony formation in vitro. Although other trials have shown that CD123 is expressed only on a small fraction of the CD34+CD38-HSC lineage, and 2 phase I trials using CD123-targeting agents did not reveal long-term myelosuppression, further studies are needed to assess the effect of CD123 CAR T cell therapy on hematopoiesis. To control unwanted toxicities, EGFRt was included in the lentiviral construct to allow ablation of CAR-expressing T cells. Other strategies to modulate CAR T cell activity, such as induction of caspase 9 apoptosis [48] or electroporation of CAR mRNA [49] are also of high interest because of the potential to kill normal cells expressing CD123.
[0099] It has also been shown that cryopreserved PBMCs from AML patients with active disease can be genetically engineered to express CD123 CARs and exhibit potent cytolytic activity against autologous leukemic blasts in 2/3 of the samples. Although T cells expressing CD123 CAR from LMA 559 did not show the potential to lyse autologous blasts with low levels of CD123, these CAR T cells did not lyse CD123+ LC and KG1a cell lines (data not shown), suggesting that T cells in general have the potential to target CD123-expressing target cells. To the best of our knowledge, this is the first demonstration that T cells derived from AML patients can be engineered to express a CAR and exhibit redirected antigen-specific cytotoxicity against autologous blasts.
[00100] In summary, the results of the studies found that the examples below demonstrate that CD123 CAR T cells can distinguish between CD123+ and CD123- cells, and can activate multiple T cell effector functions against a panel of AML patient samples low-risk primary. Notably, CD123-specific T cells do not significantly alter the formation of normal progenitor colonies, but considerably reduce the growth of myeloidclonogenic leukemic progenitors in vitro. It has also been shown that T cells derived from AML patients can be genetically modified to express CD123-specific CARs and lyse autologous blasts in vitro. Therefore, CD123 CAR T cells are promising candidates for AML immunotherapy.Example 2: CAR-transduced CD123 T cells slow leukemic progression in vivo
[00101] CD123CAR Constructs. Constructs 26292CAR (S228P+L235E) and 32716CAR (S228P+L235E) were generated as described in example 1 above. Two additional CD123CAR constructs were also produced including an additional mutation in the IgG4 hinge region at position 297 (N297Q) for each scFv ("26292CAR(S228P+L235E+N297Q") and "32716CAR (S228P+L235E+N297Q)") ( figures 12 and 13, mutations in bold and underlined).
[00102] NSG mice were implanted with AML tumor cells (day 0), and treated with 5.0 x 10 6 CAR+ T cells expressing 26292CAR(S228P+L235E) or 26292CAR (S228P+L235E+N297Q) on day 5, and leukemic progression was monitored by bioluminescent imaging. As shown in Figure 8, the leukemic load increased on day 8 compared to day of treatment in mice treated with T cells transduced with 26292CAR (S228P+L235E), indicating that cells transduced with the CD123CAR construct had mutations in the hinge region in the positions S228P and L235E had no effect in vivo. In contrast, mice treated with T cells transduced with 26292CAR (S228P+L235E+N297Q) demonstrated a reduction in tumor size compared to the day of treatment, indicating that the addition of a mutation in the hinge region at position 297 (N297Q ) produces a CD123CAR construct that is capable of slowing leukemic progression in vivo.References
[00103] The references, patents and published patent applications listed below, and all references cited in the above specification are incorporated by reference in their entirety in the present invention.1. Eaves, C.J. and R.K. Humphries, Acute myeloid leukemia and the Wnt pathway. N Engl J Med, 2010. 362(24): p. 23267.2. Dohner, H., et al., Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European LeukemiaNet. Blood, 2010. 115(3): p. 453-74.3. Majeti, R., Monoclonal antibody therapy directed against human acute myeloid leukemia stem cells. Oncogene, 2011. 30(9): p. 1009-19.4. Kikushige, Y., et al., TIM-3 is a promising target to selectively kill acute myeloid leukemia stem cells. Cell Stem Cell, 2010. 7(6): p. 708-17. 5. Jena, B., G. Dotti, and L.J. Cooper, Redirecting T-cell specificity by introducing a tumor-specific chimeric antigen receptor. Blood, 2010. 116(7): p. 1035-44.6. 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Peinert, S., et al., Gene-modified T cells as immunotherapy for multiple myeloma and acute myeloid leukemia expressing the Lewis Y antigen. Gene Ther, 2010. 17(5): p. 678-86.17. Dutour, A., et al., In Vitro and In Vivo Antitumor Effect of Anti-CD33 Chimeric Receptor-Expressing EBV-CTL against CD33 Acute Myeloid Leukemia. Adv Hematol, 2012. 2012: p. 683065.18. Du, X., M. Ho, and I. Pastan, New immunotoxins targeting CD123, a stem cell antigen on acute myeloid leukemia cells. J Immunother, 2007. 30(6): p. 607-13.19. Pelloquin, F., J.P. Lamelin, and G.M. Lenoir, Human B lymphocytes mortality by Epstein-Barr virus in the presence of cyclosporin A. In Vitro Cell Dev Biol, 1986. 22(12): p. 689-94.20. Wang, X., et al., A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells. Blood, 2011. 118(5): p. 1255-63. 21. Reddy, M.P., et al., Elimination of Fc receptor-dependent effector functions of a modified IgG4 monoclonal antibody to human CD4. J Immunol, 2000. 164(4): p. 1925-33.22. Nguyen, P., I. Moisini, and T.L. Geiger, Identification of a murine CD28 dileucine motif that suppresses singlechain chimeric T-cell receptor expression and function. Blood, 2003. 102(13): p. 4320-5.23. Jensen, M.C., et al., Human T lymphocyte genetic modification with naked DNA. Mol Ther, 2000. 1(1): p. 49-55.24. Riddell, S.R. and P.D. Greenberg, The use of anti-CD3 and anti-CD28 monoclonal antibodies to clone and expand human antigen-specific T cells. J Immunol Methods, 1990. 128(2): p. 189-201.25. Brown, C.E., et al., Recognition and killing of brain tumor stem-like initiating cells by CD8+ cytolytic T cells. Cancer Res, 2009. 69(23): p. 8886-93.26. Bhatia, R., et al., Abnormal function of the bone marrow microenvironment in chronic myelogenous leukemia: role of malignant stromal macrophages. Blood, 1995. 85(12): p. 363645.27. Jordan, C.T., et al., The interleukin-3 receptor alpha chain is a unique marker for human acute myelogenous leukemia stem cells. Leukemia, 2000. 14(10): p. 1777-84.28. Jin, L., et al., Monoclonal antibody-mediated targeting of CD123, IL-3 receptor alpha chain, eliminates human acute myeloid leukemic stem cells. Cell Stem Cell, 2009. 5(1): p. 31-42.29. Munoz, L., et al., Interleukin-3 receptor alpha chain (CD123) is widely expressed in hematologic malignancies. Haematologica, 2001. 86(12): p. 1261-9. 30. Seder, R.A., P.A. Darrah, and M. Roederer, T-cell quality in memory and protection: implications for vaccine design. Nat Rev Immunol, 2008. 8(4): p. 247-58.31. Manz, M.G., et al., Prospective isolation of human clonogenic common myeloid progenitors. Proc Natl Acad Sci US A, 2002. 99(18): p. 11872-7.32. 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Frazier, Integrin-associated protein (CD47) and its ligands. Trends Cell Biol, 2001. 11(3): p. 130-5.38. Walter, R.B., et al., Acute myeloid leukemia stem cells and CD33-targeted immunotherapy. Blood, 2012. 119(26): p. 6198-208. 39. Aigner, M., et al., T lymphocytes can be effectively recruited for ex vivo and in vivo lysis of LMA blasts by a novel CD33/CD3-bispecific BiTE((R)) antibody construct. Leukemia, 2012.40. Hernandez-Caselles, T., et al., A study of CD33 (SIGLEC-3) antigen expression and function on activated human T and NK cells: two isoforms of CD33 are generated by alternative splicing. J Leukoc Biol, 2006. 79(1): p. 46-58.41. Sievers, E.L., et al., Efficacy and safety of gemtuzumab ozogamicin in patients with CD33-positive acute myeloid leukemia in first relapse. J Clin Oncol, 2001. 19(13): p. 3244-54.42. Tsimberidou, A.M., et al., The role of gemtuzumab ozogamicin in acute leukaemia therapy. Br J Haematol, 2006. 132(4): p. 398-409.43. 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权利要求:
Claims (17)
[0001]
1. Nucleic acid molecule CHARACTERIZED in that it encodes a chimeric antigen receptor comprising, from the amino terminus to the carboxy terminus: an anti-CD123 scFv region, an IgG4 hinge region comprising SEQ ID No: 13 showing a substitution of N amino acids for Q at position 79 and an L to E amino acid substitution at position 17 and, optionally, an S to P amino acid substitution at position 10, a transmembrane domain; a co-stimulatory signaling domain selected from the group consisting of: a CD27 co-stimulatory signaling domain, a CD28 co-stimulatory signaling domain, a 4-1BB co-stimulatory signaling domain, and an OX40 co-stimulatory signaling domain; and a T cell receptor zeta chain signaling domain.
[0002]
2. Nucleic acid molecule, according to claim 1, CHARACTERIZED by the fact that the hinge region of IgG4 comprising SEQ ID No: 13 has an amino acid substitution S to P at position 10.
[0003]
3. Nucleic acid molecule, according to claim 1, CHARACTERIZED in that the costimulatory signaling domain is selected from the group consisting of: a CD28 costimulatory signaling domain and a 4-1BB costimulatory signaling domain .
[0004]
4. Nucleic acid molecule, according to claim 1, CHARACTERIZED by the fact that the domain of the anti-CD123 scFv region comprises: the VL and VH domain of the recombinant immunotoxin 26292 or the VL and VH domain of the recombinant immunotoxin 32716.
[0005]
5. A nucleic acid molecule according to claim 1, CHARACTERIZED in that the chimeric antigen receptor comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 11 and SEQ ID NO: 12.
[0006]
A nucleic acid molecule according to claim 1, CHARACTERIZED in that it comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 .
[0007]
7. Nucleic acid molecule, according to claim 1, CHARACTERIZED by the fact that the transmembrane domain is a CD28 transmembrane domain.
[0008]
8. Nucleic acid molecule, according to claim 1, CHARACTERIZED in that the anti-CD123 scFv region is a humanized anti-CD123 scFv region.
[0009]
9. The nucleic acid molecule of claim 1, CHARACTERIZED in that the anti-CD123 scFv region comprises amino acids 23-266 of SEQ ID NO: 9.
[0010]
10. Nucleic acid molecule, according to claim 1, CHARACTERIZED in that the anti-CD123 scFv region comprises amino acids 23-259 of SEQ ID NO: 10.
[0011]
11. A nucleic acid molecule according to claim 1, CHARACTERIZED in that the IgG4 hinge region comprises amino acids 267-495 of SEQ ID NO: 9.
[0012]
12. Nucleic acid molecule, according to claim 1, CHARACTERIZED by the fact that the costimulatory signaling domain is a 4-1BB costimulatory signaling domain.
[0013]
13. Nucleic acid molecule, according to claim 1, CHARACTERIZED by the fact that the costimulatory signaling domain is a CD28 costimulatory signaling domain.
[0014]
14. A nucleic acid molecule according to claim 13, CHARACTERIZED in that the CD28 costimulatory domain comprises amino acids 498-564 of SEQ ID NO: 9.
[0015]
15. A nucleic acid molecule according to claim 13, CHARACTERIZED in that the CD28 costimulatory domain comprises amino acids 489-557 of SEQ ID NO: 10.
[0016]
16. Nucleic acid molecule, according to claim 13, CHARACTERIZED in that the signaling domain of the T cell receptor (TCR) Zeta chain comprises amino acids 568-679 of SEQ ID NO: 9.
[0017]
17. Expression cassette CHARACTERIZED in that it comprises a nucleotide sequence, as defined in claim 1.

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法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2019-10-15| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2019-12-31| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-07-20| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2021-11-03| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2022-01-11| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 14/03/2014, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US13/844,048|2013-03-15|

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US13/844,048|US9657105B2|2013-03-15|2013-03-15|CD123-specific chimeric antigen receptor redirected T cells and methods of their use|

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PCT/US2014/029109|WO2014144622A2|2013-03-15|2014-03-14|Cd123-specific chimeric antigen receptor redirected t cells and methods of their use|

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