Compositions and methods for treating cancer with anti-CD19 / CD20 immunotherapy

Abstract

This invention provides a chimeric antigen receptor that can be used to treat diseases, disorders, or conditions associated with dysregulation of CD19 and / or CD20 expression. [Solution] An isolated nucleic acid molecule is provided that encodes a CD19 / CD20 tandem chimeric antigen receptor (CAR) comprising at least one extracellular antigen-binding domain including a CD19 / CD20 antigen-binding domain, at least one transmembrane domain, and at least one intracellular signaling domain.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 539,483, filed July 31, 2017, which is incorporated herein by reference in its entirety under Section 119(e) of the U.S. Patent Act.

[0002] Sequence List This application includes a sequence listing, submitted electronically in ASCII format, which is incorporated herein by reference throughout. The ASCII copy, created on July 31, 2018, is named Sequence_Listing.txt and is 64 kilobytes in size.

[0003] Areas of this disclosure This application relates to the field of cancer, in particular to CD19 / CD20 antigen-binding domains, chimeric antigen receptors (CARs) containing such CD19 / CD20 antigen-binding domains, and methods of using the same. [Background technology]

[0004] background Cancer is one of the most deadly threats to human health. In the United States alone, nearly 1.3 million people are diagnosed with cancer each year, making it the second leading cause of death after cardiovascular disease and accounting for about a quarter of all deaths. Solid tumors account for the vast majority of these deaths. Despite significant progress in the medical treatment of certain types of cancer, the five-year overall survival rate for all cancers has improved by only about 10% in the last 20 years. Cancer, or malignant tumors, are extremely difficult to treat because they metastasize and grow rapidly without being controlled.

[0005] CD19 is an 85-95 kDa transmembrane cell surface glycoprotein receptor. CD19 is a member of the immunoglobulin (Ig) superfamily and contains two extracellular Ig-like domains, transmembrane and intracellular signaling domains (Tedder TF, Isaacs, CM, 1989, J Immunol vol. 143: pp. 712-171). CD19 modifies B cell receptor signaling, lowering the B cell receptor's trigger threshold for antigens (Carter, RH and Fearon, DT, 1992, Science, vol. 256: pp. 105-107), and, in coordination with CD81 and CD21, regulates this essential B cell signaling complex (Bradbury, LE, Kansas GS, Levy S, Evans RL, Tedder TF, 1992, J Immunol vol. 149: pp. 2841-2850). During B cell development, CD19 can signal independently of antigen receptors at the pro-B, pre-pre-B, pre-B, and early B cell stages, associating with Src family protein tyrosine kinases, undergoing tyrosine phosphorylation, and inducing both intracellular calcium mobilization and inositol phospholipid signaling (Uckun FM, Burkhardt AL, Jarvis L, Jun X, Stealy B, Dibirdik I, Myers DE, Tuel-Ahlgren L, Bolen JB, 1983, J Biol Chem vol. 268: pp. 21172-84). An important point relevant to the treatment of B cell malignancies is that CD19 is expressed on normal B cells in a tightly regulated manner, limited to early B cell precursors and mature B cells at the IgH gene rearrangement stage, but not on hematopoietic stem cells or mature plasma cells (Anderson, KC, Bates, MP, Slaughenho ut BL, Pinkus GS, Schlossman SF, Nadler LM, 1984, Blood Vol. 63: pp. 1424-1433).

[0006] CD20 (also known as LEU-16 or MS4A1) is a transmembrane 4A family protein expressed on the surface of B cells from the pro-B phase to the mature B-cell phase, playing a role in B cell development and differentiation. The CD20 antigen is also expressed on various hematological malignancies, and various monoclonal anti-CD20 antibodies have been used for many years to treat CD20-positive malignancies (summarized in Lim, Sean H. et al., "Anti-CD20 Monoclonal Antibodies: Historical and Future Perspectives," Haematologica 95.1 (2010): pp. 135-143, PMC.Web, July 31, 2017). The anti-CD20 monoclonal antibody rituximab (Rituxan®) is widely used in the treatment of B-cell lymphomas, such as follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL), as well as chronic lymphocytic leukemia (CLL) (Rituxan prescribing information).

[0007] Traditional treatment approaches for lineage B leukemia and lymphoma may involve chemotherapy, radiotherapy, and stem cell transplantation (see mayclinic.org on the World Wide Web). The high toxicity associated with these treatments, and the risk of complications such as relapse, secondary malignancies, or GVHD, necessitate the search for better therapeutic alternatives. Since CD19 is expressed in both adult and pediatric (pre-B-ALL) B-cell malignancies, this target has been utilized for both antibody and chimeric antigen receptor (CAR)-T cell-based therapies (Kochenderfer JN, Wilson WH, Janik JE, Dudley ME, Stetler-Stevenson M, Feldman SA, Maric I, Raffeld M, Nathan DA, Lanier BJ, Morgan RA, Rosenberg SA, 2010, Blood vol. 116: pp. 4099-102; Lee DW, Kochenderfer JN, Stetler-Stevenson M, Cui YK, Delbrook C, Feldman SA, Orentas R, Sabatino M, Shah NN, Steinberg SM, Stroncek D, Tschernia N, Yuan C, Zhang H, Zhang L, Rosenberg SA, Wayne AS, Mackall). CL, 2015, Lancet Vol. 385: pp. 517-518). Furthermore, due to the presence of the CD20 antigen in lymphoma (DLBCL, FL) and leukemia (CLL), the CD20 antigen is an attractive further target for efficient tumor elimination and prevention of tumor antigen escape.

[0008] The current standard of care for lineage B leukemia may consist of induction treatment with high-dose chemotherapy or high-dose radiation, followed by consolidation, and may be characterized by a process of stem cell transplantation and further chemotherapy as needed (see cancer.gov on the World Wide Web). The high toxicity associated with these treatments, and the risk of complications such as relapse, secondary malignancies, or GVHD, prompt the search for better therapeutic alternatives. CD19 expression in both adult and pediatric (pre-B-ALL) B-cell malignancies has led to the utilization of this target for both antibody and chimeric antigen receptor (CAR)-T cell-based therapies (Kochenderfer JN, Wilson WH, Janik JE, Dudley ME, Stetler-Stevenson M, Feldman SA, Maric I, Raffeld M, Nathan DA, Lanier BJ, Morgan RA, Rosenberg SA, 2010, Blood vol. 116: pp. 4099-102; Lee DW, Kochenderfer JN, Stetler-Stevenson M, Cui YK, Delbrook C, Feldman SA, Orentas R, Sabatino M, Shah NN, Steinberg SM, Stroncek D, Tschernia N, Yuan C, Zhang H, Zhang L, Rosenberg SA, Wayne AS, Mackall CL, 2015, Lancet 385:517-28).

[0009] Several novel approaches have been developed to treat B-cell leukemias and lymphomas, including bispecific antibodies that link anti-CD19 or anti-CD20 binding motifs to T-cell binding motifs (i.e., blinatumomab, Blincyto®, indicated for the treatment of Philadelphia chromosome-negative relapsed or refractory B-cell precursor acute lymphoblastic leukemia (ALL)). To date, many of the CD19 or CD20 binding portions used in CAR constructs utilize domains derived from mouse antibodies. Several of these products, including those developed by Novartis and Kite Pharmaceuticals, are currently under review for approval. In April 2017, Novartis announced that CTL019 (tisagenlecleucel) had received FDA Breakthrough Therapy designation for the treatment of adult patients with refractory or relapsed (r / r) DLBCL (diffuse large B-cell lymphoma) that had failed two or more prior therapies, and that this designation was extended to the treatment of r / r B-cell acute lymphoblastic leukemia (ALL). These indications are based on the Phase II JULIET study (NCT02445248) and the ELIANA study (NCT02435849), respectively. The JULIET trial showed an objective response rate (ORR) of 45% at 3 months, with 37% complete response (CR) and 8% partial response (PR). In the ELIANA study, 82% of patients who received the product achieved CR or CR with incomplete recovery, and the relapse-free survival rate at 6 months was 60%. The CAR-T product from Kite Pharmaceuticals (KTE-C19, axicapbutagen siloleucel) has been approved for Breakthrough Therapy designation for diffuse large B-cell lymphoma (DLBCL), transformed follicular lymphoma (TFL), and primary mediastinal B-cell lymphoma (PMBCL). In the Kite ZUMA-3 Phase II trial for KTE-C19 in r / r ALL, a 73% complete response (CR) was reported (at 2 months or more). A significant number of patients still remain unsalvaged by these therapies, regardless of whether antibodies to CAR-T therapy are available, indicating considerable room for improvement in therapeutic approaches.

[0010] Chimeric antigen receptors (CARs) are hybrid molecules containing three essential units: (1) an extracellular antigen-binding motif, (2) a ligation / transmembrane motif, and (3) an intracellular T cell signaling motif (Long AH, Haso WM, Orentas RJ. Lessons learned from a highly-active CD2-specific chimeric antigen receptor. Oncoimmunology. 2013; Vol. 2 (No. 4): e23621). The antigen-binding motif of CARs is generally constructed based on the single-chain fragment variable (ScFv), the smallest binding domain of an immunoglobulin (Ig) molecule. Alternative antigen-binding motifs have also been engineered, for example, receptor ligands (i.e., IL-13 is engineered to bind to the IL-13 receptor expressed by tumors), intact immune receptors, library-derived peptides, and innate immune system effector molecules (e.g., NKG2D). Alternative cell targets for CAR expression (e.g., NK or gamma-delta T cells) are also under development (Brown CE et al. Clin Cancer Res. 2012; Vol. 18 (No. 8): pp. 2199-209; Lehner M et al. PLoS One. 2012; Vol. 7 (No. 2): e31210). Considerable effort is still required to define the most active T cell population for transduction of CAR vectors, determine optimal culture and proliferation techniques, and define the molecular details of the CAR protein structure itself.

[0011] The linking motif of the CAR can be designed to be a relatively stable structural domain, such as the constant domain of IgG, or to be an extended, flexible linker. Using a structural motif such as one derived from the constant domain of IgG, the ScFv-binding domain can be T The binding domain can be extended away from the cell plasma membrane surface. This may be important for some tumor targets where the binding domain is particularly close to the tumor cell surface membrane (e.g., for the disiaroganglioside GD2; Orentas et al., unpublished observation). To date, the signaling motif used in CARs has always included the CD3-ζ chain because this core motif is a crucial signal for T cell activation. The first reported second-generation CARs featured a CD28 signaling domain and a CD28 transmembrane sequence. This motif was similarly used in third-generation CARs containing the CD137(4-1BB) signaling motif (Zhao Y et al. J Immunol. 2009; Vol. 183 (No. 9): pp. 5563-74). With advances in new technologies, it is no longer necessary for T cell activation by beads linked to anti-CD3 and anti-CD28 antibodies, as well as the presence of a canonical "signal 2" derived from CD28, to be encoded by the CAR itself. Using bead activation, third-generation vectors were found not to be superior to second-generation vectors in in vitro assays, and no clear advantage over second-generation vectors was obtained in a mouse model of leukemia (Haso W, Lee DW, Shah NN, Stetler-Stevenson M, Yuan CM, Pastan IH, Dimitrov DS, Morgan RA, FitzGerald DJ, Barrett DM, Wayne AS, Mackall CL, Orentas RJ. Anti-CD22-chimeric antigen receptors targeting B cell precursor acute lymphoblastic leukemia, Blood. 2013; Vol. 121 (No. 7): pp. 1165-1174; Kochenderfer JN et al. Blood. 2012; Vol. 119 (No. 12): pp. 2709-2720). This is the second-generation CD28 / CD3-ζ (Lee DW et al., American Society of Hematology Annual Meeting, New Orleans, LA; December 7-10, 2013) and CD137 / CD3-ζ signaling pathway (Porter). This is supported by the clinical success of CD19-specific CARs described by DL et al. (N Engl J Med. 2011; vol. 365 (no. 8): pp. 725-733). In addition to CD137, other tumor necrosis factor receptor superfamily members such as OX40 can also provide important sustained signaling in CAR-transduced T cells (Yvon E et al.). Clin Cancer Res. 2009; Vol. 15 (No. 18): pp. 5852-5860). It is equally important to include the culture conditions under which the CAR T cell population was cultured, such as the cytokines IL-2, IL-7, and / or IL-15 (Kaiser AD et al.). Cancer Gene Ther. 2015; Vol. 22 (Issue 2): pp. 72-78).

[0012] A current challenge in the broader and more effective application of CAR therapy for cancer concerns the lack of compelling targets. While creating binding factors to cell surface antigens is now readily achievable, discovering tumor-specific cell surface antigens while overlooking normal tissue remains a challenging task. One potential method for conferring higher target cell specificity to CAR-expressing T cells is to use a combinatorial CAR approach. In one system, the CD3-ζ and CD28 signaling units are split between two different CAR constructs expressed in the same cell; in another system, two CARs are expressed in the same T cell, but one has lower affinity and therefore requires the alternative CAR to bind first for the full activity of the second CAR (Lanitis). (E et al., Cancer Immunol Res. 2013;1(1):43 - 53; Kloss CC et al., Nat Biotechnol. 2013;31(1):71 - 5). A second challenge for the generation of a single ScFv - based CAR as an immunotherapeutic agent is the heterogeneity of tumor cells. At least one group has developed a CAR strategy for glioblastoma in which the effector cell population targets multiple antigens (HER2, IL - 13Ra, EphA2) simultaneously in anticipation of avoiding the growth of target - antigen - negative populations (Hegde M et al., Mol Ther. 2013;21(11):20 87 - 101).

[0013] T - cell - based immunotherapy is a new area in synthetic biology; multiple promoters and gene products are envisioned to direct these highly potent cells to the tumor microenvironment, where T cells can evade negative regulatory signals and mediate effective tumor killing. Chemical - based dimerization of an inducible caspase 9 construct, for example, with AP1903, has demonstrated one way in which unwanted T - cell elimination can be pharmacologically initiated, providing a powerful switch to control the T - cell population (Di Stasi A et al., N Engl J Med. 2011;365(18):1673 - 8). The creation of an effector T - cell population that is immune to the negative regulatory effects of transforming growth factor - β by the expression of a decoy receptor further demonstrates the extent to which effector T cells can be engineered for optimal antitumor activity (Foster AE et al., J Immunother. 2008;31(5):500 - 5). Thus, CARs appear to be able to induce T - cell activation in a manner similar to the endogenous T - cell receptor, but the major impediments to the clinical application of this technology to date are the limited in vivo proliferation of CAR + T cells, the rapid disappearance of cells after injection, and off - target clinical activity. This may be due in part to the murine origin of some of the CAR sequences used.

[0014] The use of blinatumomab (bispecific anti-CD19 and anti-CD3 antibody) has shown excellent results for severely ill patients who received this therapy. Nevertheless, the long-term remission rate is less than 40%, and at most only 50% of the responders can be salvaged to hematopoietic stem cell transplantation (HSCT) (see Gore et al., 2014, NCT01471782 and Von Stackelberg et al., 2014, NCT01471782, summarized in Benjamin, JE, Stein AS, 2016, Therapeutic Advances in Hematology 7:142-156). The need for patients who received either bispecific antibody or CAR-T therapy to subsequently undergo HSCT to maintain long-term responses is still actively debated. High responses have been reported for CD19 CAR-T trials, some even exceeding 90%, but the numbers can be close to 70% if the trials are changed to "intent-to-treat" trials (Davis KL, Mackall CL, 2016, Blood Advances 1:265-268). The best results reported 12 months after CAR19 treatment are a 55% RFS and 79% OS in patients who were able to receive the T cell product at the University of Pennsylvania (Maude SL, Teachey DT, Rheingold SR, Shaw PA, Aplenc R, Barrett DM, Barker CS, Callahan C, Frey NV, Farzana N, Lacey SF, Zheng A, Levine B, Melenhorst JJ, Motley L, Prter DL, June CH, Grupp SA, 2016, J Clin Oncol 34, No. 15 Suppl (May 2016) 3011-3011).

[0015] Therefore, there is an urgent and long-standing need in the art to discover new compositions and methods for the treatment of B-ALL and other CD19 and / or CD20-expressing B cell malignancies that use approaches that can exhibit specific and effective anti-tumor effects without the above-mentioned drawbacks. SUMMARY OF THE INVENTION [Problems that the invention aims to solve]

[0016] The present invention addresses these needs by providing CAR compositions and therapeutic methods that can be used to treat cancer and other diseases and / or conditions. In particular, the present invention disclosed and described herein provides CARs that can be used to treat diseases, disorders or conditions associated with dysregulation of CD19 and / or CD20 expression, where C AR contains a tandem CD19 / CD20 antigen-binding domain that exhibits high surface expression in transduced T cells, high cytolysis of CD19-expressing cells, and demonstrates in vivo proliferation and persistence of transduced T cells. [Means for solving the problem]

[0017] overview Novel tandem CD19 and CD20 targeted antibodies or their antigen-binding domains (hereinafter referred to herein as "CD19 / CD20") in which the CD19 targeting portion of the amino acid sequence is located either before or after the CD20 targeting portion, and chimeric antigen receptors (tandem CARs) containing such CD19 and / or CD20 antigen-binding domains, as well as host cells expressing the receptor (e.g., T cells), and nucleic acid molecules encoding the receptor are provided herein. The CARs exhibit high surface expression in transduced T cells, high levels of cytolysis, and in vivo proliferation and persistence of transduced T cells. Methods using the disclosed CARs, host cells, and nucleic acid molecules for treating cancer in a subject are also provided.

[0018] In one embodiment, an isolated nucleic acid molecule is provided encoding a tandem CD19 / CD20 chimeric antigen receptor (CAR) comprising at least one CD19 / CD20 antigen-binding domain, at least one transmembrane domain, and at least one intracellular signaling domain from the N-terminus to the C-terminus, wherein the tandem CD19 / CD20 CAR comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 and 3.

[0019] In one embodiment, an isolated nucleic acid molecule is provided encoding a tandem CD19 / CD20 chimeric antigen receptor (CAR) comprising at least one CD19 / CD20 antigen-binding domain, at least one transmembrane domain, and at least one intracellular signaling domain from the N-terminus to the C-terminus, wherein a tandem CD19 / CD20 CAR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 and 3 encodes a tandem CD19 / CD20 CAR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 4.

[0020] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded extracellular CD19 / CD20 antigen-binding domain contains at least one single-chain variable fragment of an antibody that binds to CD19 / CD20.

[0021] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded extracellular CD19 / CD20 antigen-binding domain includes at least one heavy chain variable region of an antibody that binds to CD19 / CD20.

[0022] In yet another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded CAR extracellular CD19 / CD20 antigen-binding domain further comprises at least one lipocalin-based antigen-binding antigen (anticalin) that binds to CD19 / CD20.

[0023] In one embodiment, an isolated nucleic acid molecule is provided in which an encoded extracellular CD19 / CD20 antigen-binding domain is connected to a transmembrane domain by a linker domain.

[0024] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded CD19 / CD20 extracellular antigen-binding domain is preceded by a sequence encoding a leader or signal peptide.

[0025] In yet another embodiment, an isolated nucleic acid molecule is provided encoding a CAR comprising at least one CD19 / CD20 antigen-binding domain encoded by a nucleotide sequence comprising the CD19 / CD20 nucleotide sequences contained in SEQ ID NOs: 1 and 3, respectively, wherein the CAR further encodes an extracellular antigen-binding domain that targets antigens including, but not limited to, CD22, ROR1, mesoserine, CD33, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, TSLPR, NY-ESO-1 TCR, MAGE A3 TCR, or any combination thereof.

[0026] In certain embodiments, further encoded extracellular antigen-binding domains include: anti-CD22 ScFv antigen-binding domain, anti-ROR1 ScFv antigen-binding domain, anti-mesoserine ScFv antigen-binding domain, anti-CD33 ScFv antigen-binding domain, anti-CD38 ScFv antigen-binding domain, anti-CD123 (IL3RA) ScFv antigen-binding domain, anti-CD138 ScFv antigen-binding domain, anti-BCMA (CD269) ScFv antigen-binding domain, anti-GPC2 ScFv antigen-binding domain, anti-GPC3 ScFv antigen-binding domain, anti-FGFR4 ScFv antigen-binding domain, anti-TSLPR ScFv antigen-binding domain, anti-c-Met ScFv antigen-binding domain, anti-PMSA ScFv antigen-binding domain, anti-glycolipid F77 ScFv antigen-binding domain, anti-EGFRvIII ScFv antigen-binding domain, and anti-GD-2 Isolated nucleic acid molecules encoding CARs are provided, comprising an ScFv antigen-binding domain, an anti-NY-ESO-1 TCR ScFv antigen-binding domain, an anti-MAGE A3 TCR ScFv antigen-binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, or any combination thereof.

[0027] In one embodiment, the CAR provided herein further comprises a linker or spacer domain.

[0028] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, in which an extracellular CD19 / CD20 antigen-binding domain, an intracellular signaling domain, or both are connected to a transmembrane domain by a linker or spacer domain.

[0029] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded linker domain originates from the extracellular domain of CD8 or CD28 and is linked to a transmembrane domain.

[0030] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, further comprising a transmembrane domain containing a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, and CD154, or combinations thereof.

[0031] In yet another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded intracellular signaling domain further comprises a CD3 zeta intracellular domain.

[0032] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded intracellular signaling domain is located C-terminally relative to the CD3 zeta intracellular domain.

[0033] In another embodiment, at least one encoded intracellular signaling domain is co Isolated nucleic acid molecules encoding CARs are provided, including a stimulating domain, a primary signaling domain, or a combination thereof.

[0034] In further embodiments, isolated nucleic acid molecules encoding CARs are provided, wherein at least one co-stimulatory domain to be encoded comprises the functional signaling domains of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or a combination thereof.

[0035] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, further comprising a leader sequence or a signal peptide, wherein the nucleotide sequence of the leader or signal peptide comprises the nucleotide sequence of SEQ ID NO: 11.

[0036] In yet another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded leader sequence comprises the amino acid sequence of SEQ ID NO: 12.

[0037] In one embodiment, a chimeric antigen receptor (CAR) is provided herein, comprising at least one CD19 / CD20 antigen-binding domain, at least one transmembrane domain, and at least one intracellular signaling domain, from the N-terminus to the C-terminus.

[0038] In one embodiment, a CAR is provided in which the extracellular CD19 / CD20 antigen-binding domain comprises at least one single-chain variable fragment of an antibody that binds to the antigen, or at least one heavy-chain variable region of an antibody that binds to the antigen, or a combination thereof.

[0039] In another embodiment, a CAR is provided in which at least one transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, TNFRSF19, or a combination thereof.

[0040] In some embodiments, a CAR is provided that further encodes an extracellular antigen-binding domain comprising an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, or any combination thereof, of CD22, ROR1, mesoserine, CD33, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, TSLPR, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, TSLPR, NY-ESO-1 TCR, MAGE A3 TCR, or any combination thereof.

[0041] In one embodiment, the extracellular antigen-binding domains include: anti-CD22 ScFv antigen-binding domain, anti-ROR1 ScFv antigen-binding domain, anti-mesoserine ScFv antigen-binding domain, anti-CD33 ScFv antigen-binding domain, anti-CD38 ScFv antigen-binding domain, anti-CD123 (IL3RA) ScFv antigen-binding domain, anti-CD138 ScFv antigen-binding domain, anti-BCMA (CD269) ScFv antigen-binding domain, anti-GPC2 ScFv antigen-binding domain, anti-GPC3 ScFv antigen-binding domain, anti-FGFR4 ScFv antigen-binding domain, anti-TSLPR ScFv antigen-binding domain, anti-c-Met ScFv antigen-binding domain, anti-PMSA ScFv antigen-binding domain, anti-glycolipid F77 ScFv antigen-binding domain, anti-EGFRvIII ScFv antigen-binding domain, and anti-GD-2 ScFv antigen-binding domain, anti-NY-ESO-1 TCR ScFv antigen-binding domain, anti-MAGE A3 TCR ScFv antigen-binding domain, or 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the domain. A CAR is provided that contains amino acid sequences having % identity, or any combination thereof.

[0042] In another embodiment, a CAR is provided in which at least one intracellular signaling domain comprises a co-stimulatory domain and a primary signaling domain.

[0043] In yet another embodiment, a CAR is provided in which at least one intracellular signaling domain comprises a co-stimulatory domain containing a functional signaling domain of a protein selected from the group consisting of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or a combination thereof.

[0044] In one embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 1 (the nucleotide sequence of leader-CD19 VL-Whitlow linker CD19 VH(GGGGS)-5 CD20 VH(GGGGS)-3 CD20 VL CD8 hinge + TM-4-1BB-CD3z (construction CAR1920) (Figure 10A)). In one embodiment, the nucleic acid sequence encodes a CAR including the amino acid sequence of SEQ ID NO: 2 leader-CD19 VL-Whitlow linker CD19 VH(GGGGS)-5 CD20 VH(GGGGS)-3 CD20 VL CD8 hinge + TM-4-1BB-CD3z (construction CAR1920) ((Figure 10A)).

[0045] In another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 3 (leader-CD20 VH(GGGGS)3-CD20 VL-(GGGGS)5-CD19 VL-Whitlow linker-CD19 VH CD8 hinge+TM-4-1BB-CD3z (construct 2019) nucleotide sequence (Figure 10B)). In one embodiment, the nucleic acid sequence encodes a CAR including the amino acid sequence of SEQ ID NO: 4 (leader-CD20 VH(GGGGS)3-CD20 VL-(GGGGS)5-CD19 VL-Whitlow linker-CD19 VH CD8 hinge+TM-4-1BB-CD3z CAR amino acid sequence (Figure 10B)).

[0046] In one embodiment, the CAR disclosed herein is modified to express or contain a detectable marker for use in diagnosis, monitoring and / or prediction of treatment outcomes such as progression-free survival in cancer patients, or for monitoring the progression of such treatment.

[0047] In one embodiment, the nucleic acid molecule encoding the disclosed CAR may be contained in a vector such as a viral vector. The vector may be a DNA vector, RNA vector, plasmid vector, cosmid vector, herpesvirus vector, measles virus vector, lentivirus vector, adenovirus vector, or retrovirus vector, or a combination thereof.

[0048] In certain embodiments, the vector further includes a promoter which is an inducible promoter, a tissue-specific promoter, a constitutive promoter, a suicide promoter, or any combination thereof.

[0049] In yet another embodiment, the vector expressing CAR may be further modified to include one or more activatable elements to control the expression of CAR T cells or to eliminate CAR-T cells by suicide switches. Suicide switches may include, for example, apoptosis-inducible signaling cascades or drugs that induce cell death. In a preferred embodiment, the vector expressing CAR may include thymidine kinase (TK) Alternatively, it can be further modified to express enzymes such as cytosine deaminase (CD).

[0050] In another embodiment, a host cell containing a nucleic acid molecule encoding CAR is also provided. In some embodiments, the host cell is a T cell, for example, a primary T cell obtained from a subject. In one embodiment, the host cell is CD8 + These are T cells.

[0051] In yet another embodiment, a pharmaceutical composition is provided comprising an antitumor-effective population of human T cells, wherein the T cells comprise nucleic acid sequences encoding a chimeric antigen receptor (CAR) comprising the amino acid sequences of SEQ ID NOs: 2 and 4, the CAR comprising at least one extracellular antigen-binding domain comprising a CD19 / CD20 antigen-binding domain, at least one linker domain, at least one transmembrane domain, and at least one intracellular signaling domain, and the T cells are human T cells having cancer. Cancer includes, in particular, hematological cancers, e.g., leukemia (e.g., chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), or chronic myeloid leukemia (CML)), lymphoma (e.g., mantle cell lymphoma, non-Hodgkin lymphoma, or Hodgkin lymphoma), or multiple myeloma, or a combination thereof.

[0052] In one embodiment, a pharmaceutical composition is provided in which at least one transmembrane domain of CAR comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesoserine, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, and CD154, TNFRSF19, or a combination thereof.

[0053] In another embodiment, a pharmaceutical composition is provided that includes human cancers, including oral and pharyngeal cancers (tongue, mouth, pharynx, head and neck), gastrointestinal cancers (esophagus, stomach, small intestine, colon, rectum, anus, liver, intrahepatic bile duct, gallbladder, pancreas), respiratory cancers (larynx, lung, and bronchi), bone and joint cancers, soft tissue cancers, skin cancers (melanoma, basal cell carcinoma, and squamous cell carcinoma), pediatric tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma), central nervous system tumors (brain tumors, astrocytoma, glioblastoma, glioma), as well as adult cancers, including breast, reproductive system (cervix, uterine body, ovaries, vulva, vagina, prostate, testes, penis, endometrium), urinary system (bladder, kidneys and renal pelvis, ureters), eye and orbit, endocrine system (thyroid), and brain and other nervous system cancers, or any combination thereof.

[0054] In yet another embodiment, a pharmaceutical composition is provided comprising an antitumor effective amount of a population of human T cells of a person having cancer, wherein the cancer is a refractory cancer unresponsive to one or more chemotherapeutic agents. The cancer includes hematopoietic cancer, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumors, adult B-cell malignancies including acute lymphoblastic leukemia (ALL), minimal residual disease (MRD) in acute myeloid leukemia (AML), CLL (chronic lymphocytic leukemia), CML (chronic myeloid leukemia), non-Hodgkin lymphoma (NHL), pediatric B-cell malignancies (including B-cell lineage ALL (acute lymphocytic leukemia)), multiple myeloma, lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma, or other hematological cancers and solid tumors, or any combination thereof.

[0055] In another embodiment, a method for producing CAR-containing T cells (hereinafter referred to as "CAR T cells") is provided. This method comprises transduction of T cells with a disclosed CAR-encoding vector or nucleic acid molecule that specifically binds to CD19 / CD20, thereby producing CAR T cells.

[0056] In yet another embodiment, a method for generating a population of RNA-manipulated cells is disclosed. A method is provided for generating CAR cells by introducing in vitro transcribed RNA or synthetic RNA of a nucleic acid molecule encoding a CAR into target cells.

[0057] In one embodiment, the diseases, disorders, or conditions associated with CD19 expression include cancers such as hematopoietic cancers, myelodysplastic syndromes, pancreatic cancers, head and neck cancers, skin tumors, adult B-cell malignancies including acute lymphoblastic leukemia (ALL), minimal residual disease (MRD) in acute myeloid leukemia (AML), CLL (chronic lymphocytic leukemia), CML (chronic myeloid leukemia), and non-Hodgkin lymphoma (NHL), pediatric B-cell malignancies (including B-cell lineage ALL (acute lymphocytic leukemia)), multiple myeloma, lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma, or other hematological cancers and solid tumors, or any combination thereof.

[0058] In another embodiment, a method is provided for altering the tumor microenvironment to block T cell inhibition mediated by CD19 / CD20-expressing cells and inhibit tumor growth in mammals, the method comprising administering an effective amount of a composition comprising a CAR containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 4 to a mammal. In one embodiment, the cells are selected from the group consisting of tumor cells expressing CD19 and / or CD20, tumor-associated macrophages, and any combination thereof.

[0059] In another embodiment, a method is provided for inhibiting, suppressing or preventing immunosuppression of an antitumor or anticancer immune response in a mammal, comprising the step of administering to a mammal an effective amount of a composition comprising a CAR selected from the group consisting of SEQ ID NOs: 2 and 4. In one embodiment, the CAR inhibits the interaction between a first cell and a T cell, the first cell being selected from the group consisting of tumor cells expressing CD19 and / or CD20, tumor-associated macrophages, and any combination thereof.

[0060] In another embodiment, a method is provided for inducing antitumor immunity in a mammal, comprising the step of administering a therapeutically effective amount of T cells transduced with a vector or nucleic acid molecule encoding a disclosed CAR to the mammal.

[0061] In another embodiment, a method is provided for treating or preventing cancer in a mammal, comprising the step of administering one or more disclosed CARs to a mammal in an amount effective for treating or preventing cancer in the mammal. The method comprises the step of administering to a subject a therapeutically effective amount of host cells expressing a disclosed CAR that specifically binds to CD19 and / or CD20 and / or one or more of the aforementioned antigens, under conditions sufficient to form an immune complex between the antigen-binding domain of the CAR and the extracellular domains of CD19 and / or CD20 and / or one or more of the aforementioned antigens.

[0062] In yet another embodiment, a method is provided for treating a mammal having a disease, disorder or condition associated with elevated expression of a tumor antigen, comprising the step of administering a pharmaceutical composition comprising an antitumor-effective amount of a population of T cells to a subject, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprising at least one extracellular CD19 and / or CD20 antigen-binding domain or any combination thereof, at least one linker or spacer domain, at least one transmembrane domain, and at least one intracellular signaling domain, and the T cells are T cells of a subject having cancer.

[0063] In yet another embodiment, a method for treating cancer in a subject requiring it, comprising the step of administering a pharmaceutical composition comprising an antitumor-effective amount of T cell population to the subject, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), and the CAR comprises the amino acid sequences of SEQ ID NOs: 2 and 4, or any combination thereof, at least one linker or sperma A method is provided comprising a T cell domain, at least one transmembrane domain, and at least one intracellular signaling domain, wherein the T cell is a target T cell having cancer. In some embodiments of the method described above, the at least one transmembrane domain comprises the transmembrane alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD19, CD22, mesoserine, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, and CD154, TNFRSF16, TNFRSF19, or a combination thereof.

[0064] In yet another embodiment, a method is provided for generating a persistent population of genetically engineered T cells in a person diagnosed with cancer. In one embodiment, the method includes the step of administering T cells genetically engineered to express CAR to a human, wherein the CAR comprises the amino acid sequences of SEQ ID NOs. 2 and 4, or any combination thereof, at least one transmembrane domain, and at least one intracellular signaling domain, and the persistent population of genetically engineered T cells, or population of T cell offspring, persists in the human for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, or 3 years after administration.

[0065] In one embodiment, the descendant T cells in humans include memory T cells. In another embodiment, the T cells are autologous T cells.

[0066] In all aspects and embodiments of the methods described herein, any of the aforementioned cancers, diseases, disorders, or conditions associated with elevated tumor antigen expression may be treated, prevented, or remitted using one or more of the CARs disclosed herein.

[0067] In yet another embodiment, a kit is provided for producing the above-mentioned chimeric antigen receptor T cells, or for preventing, treating or relieving any of the cancers, diseases, disorders or conditions associated with elevated expression of a tumor antigen in the above-mentioned subject, the kit comprising a container containing one or any combination thereof of the nucleic acid molecules, vectors, host cells or compositions disclosed above, and instructions for using the kit.

[0068] It is understood that CARs, host cells, nucleic acids, and methods are useful beyond the specific embodiments and models described in detail herein. The aforementioned features and advantages of this disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying drawings. [Brief explanation of the drawing]

[0069] [Figure 1A] This document describes the construction of CARs targeting CD19 and CD20. Figure 1A: Anti-CD19 and anti-CD20 single-targeting CAR constructs were generated by linking the variable region of the single-chain fragment of the monoclonal antibody FMC-63 (CD19) of Leu-16 (CD20) in frame to the CD8 hinge and transmembrane domain, the 4-1BB (CD137) signaling domain, and the CD3 zeta signaling domain. Constructs 19A and 19B differ only in the linker sequence connecting the heavy and light chains of FMC63. Tandem targeting constructs 2019 and 1920 were generated in a similar manner to the single-targeting constructs, except that the variable regions of the single-chain fragments of CD20 and CD19 were continuously linked to each other by a flexible linker, and then linked to the CD8, 4-1BB, and CD3 zeta domains. [Figure 1B]This shows the construction of CARs that target CD19 and CD20. Figure 1B: Schematic diagram of tandem CAR T cells that target CD19 and CD20 tumor antigens. Tandem CARs 1920 (left) and 2019 (right) consist of a tandem extracellular targeting domain linked in-frame to a CD8-derived hinge and transmembrane domain, followed by a 4-1BB costimulatory domain and a CD3 zeta-activating domain. Each CAR T construct can be activated by binding to either or both of the CD19 or CD20 tumor antigens. [Figure 2A] Figure 2A: Surface expression of single and tandem CAR T constructs on human primary T cells. CAR T expression was determined by flow cytometry. As described in Materials and Methods, T cells were activated with Miltenyi Biotec TransAct® CD3 CD28 reagent in the presence of IL-2 and transduced with LV. On day 10 of culture, CAR surface expression of transduced live T cells (7-AAD negative) was assayed using one of three staining reagents: protein L (column 1); CD19 Fc, followed by anti-Fc-AF647 (column 2); or CD20-biotin, followed by streptavidin-PE staining (column 3). The LVs used for transduction are listed to the left of each row. The percentage of the CAR T-positive population related to the non-transduced T cell control is shown in the right corner of each histogram. GFP transduced cells served as an additional negative control. Representative data from three separate donors are shown. [Figure 2B] Figure 2B: The ratio of CD19 antigen binding to CD20 antigen binding by each tandem CAR is expressed as the ratio of the percentage of cells to which CD20 biotin binds to the percentage of cells to which CD19 Fc binds. The mean + SD of three separate experiments using three donors is shown (**p<0.01). [Figure 3A]This study demonstrates in vitro CAR T cell toxicity. Luciferase-based cytotoxicity assays were performed using K562, K562 CD19+, or K562 CD20+ cell lines (Figure 3A) or leukemia or lymphoma cell lines (Raji, NALM6, REH; Figure 3B) in which luciferase was stably transduced. CAR T cells and target tumor cells were co-incubated overnight at the enumerated effector-to-target (E:T) ratio (x-axis). Differences between groups were determined using one-way ANOVA and Dunnett's post-hoc test. Mean + SD, ****p<0.0001, **p<0.01 vs. untransduced control (NT) from the same donor. [Figure 3B] This study demonstrates in vitro CAR T cell toxicity. Luciferase-based cytotoxicity assays were performed using K562, K562 CD19+, or K562 CD20+ cell lines (Figure 3A) or leukemia or lymphoma cell lines (Raji, NALM6, REH; Figure 3B) in which luciferase was stably transduced. CAR T cells and target tumor cells were co-incubated overnight at the enumerated effector-to-target (E:T) ratio (x-axis). Differences between groups were determined using one-way ANOVA and Dunnett's post-hoc test. Mean + SD, ****p<0.0001, **p<0.01 vs. untransduced control (NT) from the same donor. [Figure 4] CAR T cytokine release in response to leukemia cell lines. Using a flow-based bead array, cytokine production by CAR-T cells (listed on the x-axis) was measured during overnight co-culture with the Raji leukemia cell line at an E:T ratio of 10:1. Bars represent the mean + SD of replicate samples. Data are representative of three independent experiments performed with CAR T cells from three separate donors. [Figure 5A] The in vivo activity of the CAR T construct is shown. Figure 5A shows the time course of tumor growth based on whole-body bioluminescence in mice. Ten mice were studied in the CAR T treatment group and five mice in the control group. [Figure 5B]The in vivo activity of the CAR T construct is shown. Figure 5B is a plot showing the mean ± SD of the signal per mouse. Statistical analysis at day 25 (the last time a subject in the untreated control group was still alive) is shown for the untreated group, using two-way ANOVA and Dunnett's multiple comparison test. Mean + SD, ***P<0.001. [Figure 6A] A single CAR19 construct that strongly selects the Raji tumor escape variant is shown. Figure 6A: Diagram of the experimental design for the tumor escape experiment. Raji and CAR T cells were co-cultured overnight or for 4 days in an E:T ratio of 1:1. After overnight incubation and on day 4, the cultures were harvested and live Raji cells were examined for CD19, CD20, and CD22 surface expression by flow cytometry. [Figure 6B] Figure 6B shows a single CAR19 construct that strongly selects Raji tumor escape variants. Gating methods for flow cytometry analysis used to analyze live Raji cells (7AAD- and CD3-) from co-cultures are shown for representative treatment groups, in column 1. Columns 2, 3, and 4 show the CD19, CD20, and CD22 expression levels, respectively, when Raji cells were co-cultured without T cells (column 1), with a 19A CAR (column 2), or with a 2019 CAR (column 3). [Figure 6C] This shows a single CAR19 construct that strongly selects the Raji tumor escape variant. Figure 6C: Graphs of CD19, CD20, and CD22 surface expression (black, white, and gray, respectively) in live Raji and NALM-6 cells after overnight or 4-day co-culture with CAR T cells, listed on the x-axis, as determined by flow cytometry. Bars represent group mean + SD. Statistical analysis was performed using one-way ANOVA and Dunnett's multiple comparison test against NT (non-transduced T cell) controls from the same donor (*p<0.05). TA-tumor-only control group. [Figure 7]Downregulation of CD19, CD20, and CD22 on the Raji surface requires direct contact with CART cells. A multiwell plate with a Transwell insert was used in this experiment. 5 x 10⁵ Raji and CAR T cells were combined in the lower part of each well, and 2.5 x 10⁵ Raji cells were cultured in the absence of T cells in the upper part of the Transwell. After overnight incubation, cells were harvested from the upper Transwell compartment and cells from the lower compartment, and live Raji cells were examined by flow cytometry for surface expression of CD19, CD20, and CD22 (black, light gray, and dark gray bars, respectively). Surface expression for each marker related to specific CAR T cells (x axis) contained in the lower compartment is shown. The bars represent the mean + SD of three independent experiments performed using CAR T cells from three different donors. One-way ANOVA, Dunnett's multiple comparison test *p<0.05. [Figure 8A] This study demonstrates the downregulation of full-length C19 protein and CD19 splice variants by a CAR19 construct. Raji cells were co-incubated with CAR T cells in an E:T ratio of 1:1. After overnight incubation, T cells were removed from the co-incubated cell population using magnetic beads. CD19 expression in the purified Raji population was studied by flow cytometry and Western blotting. Figure 8A: Raji cell samples were stained with anti-CD19 antibody and obtained by flow cytometry. Median fluorescence intensity for representative Raji cells from each treatment group is shown. Bars represent the mean + SD of three independent experiments performed using CAR T cells from three different donors. One-way ANOVA, Dunnett's multiple comparison test* p<0.05. [Figure 8B]This study demonstrates the downregulation of full-length C19 protein and CD19 splice variants by CAR19 constructs. Raji cells were co-incubated with CAR T cells in an E:T ratio of 1:1. After overnight incubation, T cells were removed from the co-incubated cell population using magnetic beads. CD19 expression on the purified Raji population was studied by flow cytometry and Western blotting. As described in Figure 8B: Materials and Methods, lysates of purified Raji cells from each co-incubation group (CAR-T identification is listed above each line) were degraded on 4%–12% SDS polyacrylamide gel and investigated with antibodies targeting the C-terminus of the CD19 molecule or β-actin (loading control). [Figure 8C] This study demonstrates the downregulation of full-length CD19 protein and CD19 splice variants by a CAR19 construct. Raji cells were co-incubated with CAR T cells in an E:T ratio of 1:1. After overnight incubation, T cells were removed from the co-incubated cell population using magnetic beads. CD19 expression in the purified Raji population was studied by flow cytometry and Western blotting. Figure 8C: The intensity of specific immunoreaction bands representing full-length CD19 protein (FL CD19) and exon 2 splice CD19 variant (Δ2CD19) was quantified using Image Studio software (LI-COR Biosciences). The relative band intensity of the CD19 bands was calculated as signal CD19 / signal β-actin. [Figure 9A] This figure shows the in vivo activity of CAR T cells in a high tumor burden model. On day 0, NSG mice (n=6) were intravenously injected with Raji-luciferase cells, and on day 12, they were treated with CAR T cells, as shown in the figure. Figure 9A: Disease burden is plotted as mean bioluminescence signal (mean radiance [p / s / cm2 / sr]) ± SEM. The group in which less than half of the mice survived by day 25 is plotted as a dotted line. The group in which more than half survived is plotted as a solid line. [Figure 9B]This shows the in vivo activity of CAR T cells in a high tumor burden model. On day 0, NSG mice (n=6) were intravenously injected with Raji-luciferase cells and treated with CAR T cells on day 12, as shown in the figure. Figure 9B: Bioluminescence images of tumor burden in mice treated with single and tandem CAR T constructs shown in the plot above, 25 days after tumor engraftment. Red X indicates mice that did not survive until day 25 of the study. [Figure 10A] Figure 10A shows the nucleic acid sequence and encoded amino acid sequence of the CAR T construct. It also shows the lentiviral vector expressing the CAR reader-CD19 VL-Whitlow linker CD19 VH(GGGGS)-5 CD20 VH(GGGGS)-3 CD20 VL CD8 hinge+TM-4-1BB-CD3z (CD1920 CAR construct) nucleic acid sequence and the encoded amino acid sequence. [Figure 10B] Figure 10B shows the nucleic acid sequence and encoded amino acid sequence of the CAR T construct. The lentiviral vector expressing a CAR containing the nucleic acid sequence leader-CD20 VH(GGGGS)3-CD20 VL-(GGGGS)5-CD19 VL-Whitlow linker CD19 VH CD8 hinge+TM-4-1BB-CD3z(CD2019 CAR construct) and the encoded amino acid sequence. [Figure 11A] Plasmid maps of the tandem CAR lentiviral vectors (LVs) encoding CD19_20 (Figure 11A) and CD20_19 (Figure 11B) tandem CARs are shown. The CD19_20 and CD20_19 tandem CARs were expressed using lentiviral backbone plasmids featuring a human EF-1 alpha internal promoter (EF1a), a leader sequence (leader), VH and VL sequences from FMC63 and Leu16 antibodies (CD19VL, CD19VH, CD20VL, and CD20VH, respectively), an interchain linker sequence, and an in-scFv sequence, ligated to the CD8, 41BB, and CD3 zeta signaling domains. [Figure 11B]Plasmid maps of the tandem CAR lentiviral vectors (LVs) encoding CD19_20 (Figure 11A) and CD20_19 (Figure 11B) tandem CARs are shown. The CD19_20 and CD20_19 tandem CARs were expressed using lentiviral backbone plasmids featuring a human EF-1 alpha internal promoter (EF1a), a leader sequence (leader), VH and VL sequences from FMC63 and Leu16 antibodies (CD19VL, CD19VH, CD20VL, and CD20VH, respectively), an interchain linker sequence, and an in-scFv sequence, ligated to the CD8, 41BB, and CD3 zeta signaling domains. [Modes for carrying out the invention]

[0070] Detailed explanation definition As used herein, the singular forms “a,” “an,” and “the” refer to both singular and plural forms unless the context clearly indicates otherwise. For example, the term “one antigen” may include one or more antigens and may be considered equivalent to the phrase “at least one antigen.” As used herein, the term “comprises” means “includes.” Therefore, “comprising one antigen” means “including one antigen” without excluding other elements. The phrase “and / or” means “and also "or" means "or". It should be further understood that any and all base sizes or amino acid sizes, as well as all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides, are approximate and provided for convenience unless otherwise noted. Many methods and materials similar or equivalent to those described herein may be used, but particularly suitable methods and materials are described below. In case of any inconsistency, this specification, including the explanation of terms, shall prevail. Furthermore, the materials, methods and examples are illustrative and not intended to be limiting. To facilitate a review of the various embodiments, the following explanations of terms are provided.

[0071] The term "approximately" means, when referring to measurable values ​​such as quantity or temporal duration, to include variations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% from the specified value, such variations being appropriate for carrying out the disclosed method.

[0072] Unless otherwise specified, technical terms in this specification shall be used in accordance with their conventional usage. Definitions of general terms in molecular biology are found in Benjamin Lewin, Genes VII, 1999, published by Oxford University Press; Kendrew et al., The Encyclopedia of Molecular Biology, 1994, published by Blackwell Science Ltd.; and Robert, published by VCH Publishers, Inc. A. Meyers (ed.), Molecular Biology and Biotechnology: A Comprehensive Desk Reference, 1995; and other similar references can be found.

[0073] This disclosure provides CD19 / CD20 antibodies or fragments thereof, and chimeric antigen receptors (CARs) having such CD19 / CD20 antigen-binding domains. Enhancement of the functional activity of CARs is directly related to the enhancement of the functional activity of T cells expressing CARs. As a result of one or more of these modifications, CARs are transduced into transduced CAR-expressing T cells. Along with T cell proliferation and sustained increases in vivo, the CART lentiviral product exhibits both high cytokine-induced cytolysis and cell surface expression in transduced T cells. The CARTs of this disclosure allow a single CART lentiviral product to be used to treat a large patient population (i.e., CD19+, CD20+, or dual CD19+CD20+ cancer patients), which is beneficial in that it allows for flexibility in resource-limited situations.

[0074] The unique ability to combine functional parts derived from various protein domains is a key and innovative feature of chimeric antigen receptors (CARs). The selection of each of these protein domains, as well as their specific combinations, are important design features. Each design domain is an essential component that can be used to manipulate lymphocyte function in various CAR platforms. For example, the selection of an extracellular binding domain can enable CARs that would otherwise be ineffective.

[0075] The immutable framework components of immunoglobulin-derived protein sequences used to create the extracellular antigen-binding domain of CARs can be either completely neutral or self-associate, leading to metabolic exhaustion of T cells, thus rendering therapeutic T cells expressing this CAR extremely ineffective. This occurs independently of the antigen-binding function of the CAR domain. Furthermore, the selection of intracellular signaling domains (multiple) can also determine the activity and durability of therapeutic lymphocyte populations used in immunotherapy. While the ability to bind to target antigens and transmit activation signals to T cells, respectively, are important aspects of CAR design, it has also become clear that the selection of the source of the extracellular antigen-binding fragment has a significant effect on the efficacy of CARs and may therefore play a decisive role in their function and clinical utility.

[0076] The CARs disclosed herein are expressed at high levels in cells. Cells expressing these CARs have high proliferation rates in vivo, produce large amounts of cytokines, and exhibit high cytotoxic activity against cells having the CD19 / CD20 antigen on their surface to which the CAR binds. The use of an extracellular CD19 / CD20 antigen-binding domain leads to the generation of CARs that function better in vivo, while avoiding the induction of anti-CAR immunity in the host immune response and the death of the CAR T cell population. CARs expressing an extracellular CD19 / CD20 ScFv antigen-binding domain exhibit superior activity / properties, including i) prevention of CAR T persistence and dysfunction seen in mouse-derived binding sequences; ii) absence of local (i.e., intrapleural) delivery of the CAR that should be effective; and iii) the ability to generate CAR T cell designs based on both high-affinity and low-affinity binders for CD19 / CD20. This last characteristic means that tumors express more CD19 / CD20 than normal tissues, allowing binders with lower affinity to have greater specificity to tumors than normal tissues. This can prevent toxicity to non-tumor cells and the death of bystander cells, thus allowing researchers to better control the toxicity and / or tissue specificity of CAR T products.

[0077] A detailed description of the CARs of the present invention, including a description of their extracellular CD19 / CD20 antigen-binding domains, transmembrane domains, and intracellular domains, is provided below, along with further descriptions of CARs, antibodies and their antigen-binding fragments, conjugates, nucleotides, expression, vectors and host cells, methods of treatment using the disclosed CARs, compositions and kits.

[0078] A. Chimeric antigen receptor (CAR) The CARs disclosed herein include at least one CD19 / CD20 antigen-binding domain capable of binding to CD19 / CD20, at least one transmembrane domain, and at least one intracellular domain.

[0079] Chimeric antigen receptors (CARs) are artificially constructed hybrid proteins or polypeptides containing an antigen-binding domain of an antibody (e.g., a single-chain variable fragment (ScFv)) linked to a T cell signaling domain via a transmembrane domain. Features of CARs include their ability to redirect the specificity and responsiveness of T cells toward a selected target by leveraging the antigen-binding properties of monoclonal antibodies in a non-MHC-restricted manner. Non-MHC-restricted antigen recognition gives T cells expressing CARs the ability to recognize antigens independently of antigen processing, thus bypassing the primary mechanism of tumor escape. Furthermore, when expressed in T cells, CARs advantageously do not dimerize with the alpha and beta chains of the endogenous T cell receptor (TCR).

[0080] As disclosed herein, the intracellular T cell signaling domain of a CAR may include, for example, a T cell receptor signaling domain, a T cell costimulatory signaling domain, or both. A T cell receptor signaling domain refers to a portion of a CAR that includes the intracellular domain of a T cell receptor, for example, the intracellular portion of the CD3 zeta protein, but not limited to that. A costimulatory signaling domain refers to a portion of a CAR that includes the intracellular domain of a costimulatory molecule, which is a cell surface molecule other than the antigen receptor or its ligand, required for an efficient lymphocyte response to an antigen.

[0081] 1. Extracellular domain In one embodiment, the CAR includes a target-specific binding element, otherwise called an antigen-binding domain or subdomain. The choice of domain depends on the type and number of ligands that define the surface of the target cell. For example, the antigen-binding domain may be selected to recognize ligands that act as cell surface markers on target cells associated with a particular disease state. Therefore, examples of cell surface markers that can act as ligands for antigen-binding domains in CARs include those associated with viral, bacterial and parasitic infections, autoimmune diseases, and cancer cells.

[0082] In one embodiment, a CAR may be engineered to target a desired tumor antigen by manipulating a desired antigen-binding domain that specifically binds to the antigen on tumor cells. The tumor antigen is a protein produced by tumor cells that elicits an immune response, particularly a T-cell-mediated immune response. The selection of the antigen-binding domain depends on the specific type of cancer being treated. Tumor antigens include, for example, glioma-associated antigens, carcinoembryonic antigens (CEAs), beta-human chorionic gonadotropins, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivorbin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin-like growth factor (IGF)-I, IGF-II, IGF-I receptor, and CD19 / CD20. The tumor antigens disclosed herein are for illustrative purposes only. This list is not intended to be exclusive, and further examples will be readily apparent to those skilled in the art.

[0083] In one embodiment, a tumor antigen comprises one or more antigenic cancer epitopes associated with a malignant tumor. Malignant tumors express several proteins that can function as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens, e.g., MART-1, tyrosinase, and GP 100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to a group of transformation-related molecules, such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens are carcinoembryonic antigens, such as carcinoembryonic antigens (CEAs). In B-cell lymphomas, tumor-specific idiotype immunoglobulins constitute truly tumor-specific immunoglobulin antigens unique to individual tumors. B-cell differentiation antigens, e.g., CD19, CD20, CD22, BCMA, ROR1, and CD37 are other candidate target antigens in B-cell lymphomas. Some of these antigens (CEA, HER-2, CD19, CD20, idiotypes) have been used as targets for passive immunotherapy with monoclonal antibodies, with limited success.

[0084] In one preferred embodiment, the tumor antigen is CD19 / CD20, and tumors associated with CD19 / CD20 expression include pulmonary mesothelioma, ovarian and pancreatic cancers, or any combination thereof, that express high levels of the extracellular proteins CD19 / CD20.

[0085] Tumor antigens can be either tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs). TSAs are unique to tumor cells and are not present on other cells throughout the body. TAAs are not unique to tumor cells and, instead, are expressed on normal cells under conditions that do not induce a state of immunological tolerance to the antigen. Antigen expression on tumors can occur under conditions that allow the immune system to respond to the antigen. TAAs may be antigens expressed on normal cells during fetal development when the immune system is immature and unresponsive, or they may be antigens that are normally present at very low levels on normal cells but expressed at considerably high levels on tumor cells.

[0086] Non-limiting examples of TSA or TAA include: differentiation antigens, e.g., MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multiseries antigens, e.g., MAGE-1, MAG E-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens, e.g., CEA; overexpressed oncogenes and mutated tumor suppressor genes, e.g., p53, Ras, HER-2 / neu; unique tumor antigens arising from chromosomal translocations, e.g., BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, e.g., Epstein-Barr virus antigen (EBVA) and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 / CA 27.29 / BCAA, CA 195, CA This includes 242, CA-50, CAM43, CD68 / P1, CO-029, FGF-5, G250, Ga733 / EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 / Mac-2 binding protein / cyclophyllin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0087] In one embodiment, the antigen-binding domain of the CAR targets antigens including, but not limited to, CD19, CD20, CD22, ROR1, CD33, CD38, CD123, CD138, BCMA, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, FGFR4, TSLPR, NY-ESO-1 TCR, and MAGE A3 TCR.

[0088] In a preferred embodiment, the antigen-binding domain of the CAR targets the extracellular CD19 / CD20 antigen.

[0089] In various embodiments of the CD19 / CD20-specific CARs disclosed herein, a general schematic is shown in Figures 1A and 1B, comprising, from N-terminus to C-terminus, a signal or leader peptide, anti-CD19 / CD20ScFv, extracellular linker, CD8 transmembrane, 4-1BB, and CD3 zeta.

[0090] In one embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 1 (leader-CD19 VL-Whitlow linker CD19 VH(GGGGS)-5 CD20 VH(GGGGS)-3 CD20 VL CD8 hinge+TM-4-1BB-CD3z(construction CAR1920)) and encodes a CAR that includes the amino acid sequence shown in SEQ ID NO: 2 (leader-CD19 VL-Whitlow linker CD19 VH(GGGGS)-5 CD20 VH(GGGGS)-3 CD20 VL CD8 hinge+TM-4-1BB-CD3z(construction CAR1920) (shown in Figure 10A).

[0091] In one embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 1, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the amino acid sequence shown in SEQ ID NO: 2, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto (leader-CD19 VL-Whitlow linker-CD19 VH(GGGGS)-5 CD20 VH(GGGGS)-3 CD20 VL CD8 hinge + TM-4-1BB-CD3z (construct CAR1920)) encodes a CAR containing the amino acid sequence (shown in Figure 10A).

[0092] In another embodiment, the nucleic acid sequence encoding CAR is sequence number 3 (leader-CD). It contains the nucleic acid sequence 20 VH(GGGGS)3-CD20 VL-(GGGGS)5-CD19 VL-Whitlow linker-CD19 VH CD8 hinge+TM-4-1BB-CD3z (construct 2019) (Figure 2B))) and encodes a CAR containing the amino acid sequence shown in SEQ ID NO: 4 [Leader-CD20 VH(GGGGS)3-CD20 VL-(GGGGS)5-CD19 VL-Whitlow linker-CD19 VH CD8 hinge+TM-4-1BB-CD3z CAR amino acid sequence (shown in Figure 10B)].

[0093] In another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 3, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR that includes the amino acid sequence shown in SEQ ID NO: 4, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto [leader-CD20 VH(GGGGS)3-CD20 VL-(GGGGS)5-CD19 VL-Whitlow linker-CD19 VH CD8 hinge+TM-4-1BB-CD3z CAR amino acid sequence (shown in Figure 10B)].

[0094] The surface expression of anti-CD19 / CD20 CARs incorporating immunoglobulin heavy chain variable domains (VH) and single-chain fragment variable (ScFv) sequences that react with the CD19 / CD20 antigen is shown in Example 1 below. The expression levels for each CAR containing ScFv were determined by flow cytometry analysis of healthy donor-derived LV transducer T cells using one of three detection methods: i) protein L-biotin followed by streptavidin PE; ii) CD19Fc recombinant protein followed by anti-Fc AF647 (APC); iii) recombinant CD20 biotin conjugate followed by streptavidin PE. The ScFv-based anti-CD19 / CD20 CAR constructs LTG1920 and LTG2019 were highly expressed in human primary T cells (shown by the gated population) compared to untransduced T cell controls (an ungated cell population).

[0095] As shown in Example 1 and Figures 3A and 3B, the high cytotoxic activity of the CD19 / CD20 CAR was demonstrated. Human primary T cells were transduced with LV encoding the CAR construct (see 19A, 19B, 20A, 1920, 2019, Methods) and then incubated for 18 hours with Raji, NALM-6, REH, K562, or 293T cell lines stably transduced with firefly luciferase, followed by in vitro cell death assays based on luminescence. All leukemia lines tested expressed CD19 on their surface, while negative controls, K562, and 293T did not. CD20 expression varied among tumor lines. The Raji line was CD20-positive, while REH was CD20-negative, as were the control lines K562 and 293T. The NALM-6 line had weak but detectable CD20 expression. As further controls, we created K562 strains expressing CD19 (K562-19+) or K562 strains expressing CD20 (K562-20+).

[0096] K562-19+ was lysed by CAR 19A and 18B constructs, and tandem CAR constructs 1920 and 2019, but not by the single 20A CAR (Figure 3A). K562-CD20+ was lysed by all CART constructs except the single CAR19 construct, demonstrating targeted antigen-limited death. Similar results were observed in other leukemia cell lines tested. CD19-targeting single and tandem CAR T constructs lysed Raji, NALM-6, and REH, but not 293T (Figure 3B) or K562 (Figure 3A). In particular, the 20A single-targeting CAR construct did not exhibit specific death activity against CD20-negative REH strains, but demonstrated death of NALM-6 with low but detectable levels of CD20 surface expression. Furthermore, tandem CAR 1920, which appeared by flow cytometry to show lower binding to the CD20 peptide than to CD19-Fc, exhibits lower cytotoxicity against K562-19+ and K562-20+, but lower cytotoxicity against CD19+CD20-REH. It does not exhibit cytotoxicity. This may suggest that 1920 tandem CARs are inferior to 2019 tandem CARs for some tumor targets.

[0097] Next, the cytokine secretion capacity of anti-CD19 / CD20 CAR T cells was evaluated. Tumor cells were co-incubated overnight with CAR T cells or control T cells at an effector-to-target ratio of 10:1, and the culture supernatant was analyzed for IFN gamma, TNF alpha, IL-2, and GM-CSF by ELISA (see Figure 4). All CAR T groups induced cytokines in response to tumor cells, but negative controls (non-transduced, NT) and GFP showed little cytokine induction. Of the five CAR T groups, single CAR T 20A produced the highest levels of cytokines, while 19A and 19B CAR T cells had the lowest levels of cytokine induction. In particular, tandem CAR T-expressing cells LTG1920 and / or LTG2019 showed moderate levels of IFN gamma, TNF alpha, IL-2, and GM-CSF, which may demonstrate usefulness in relation to clinical safety and avoidance of cytokine release syndrome.

[0098] While not intended to limit to any particular mechanism of action, possible reasons for the enhanced therapeutic function associated with the exemplary CARs of the present invention include, for example, but not as an limitation, a) improved lateral movement within the plasma membrane, enabling more efficient signaling; b) superior location within plasma membrane microdomains such as lipid rafts and a greater ability to interact with transmembrane signaling cascades associated with T cell activation; c) superior location within the plasma membrane, due to preferential movement away from inhibitory or downregulatory interactions, such as being less close to or less interacting with phosphatases such as CD45; and d) superior assembly to T cell receptor signaling complexes (i.e., immune synapses), or any combination thereof.

[0099] While this disclosure exemplifies the use of exemplary extracellular CD19 / CD20 variable heavy chains alone and ScFv antigen-binding domains, other nucleotides and / or amino acid variants within the CD19 / CD20 variable heavy chain alone and ScFv antigen-binding domains may also be used to obtain CD19 / CD20 antigen-binding domains for use in the CARs described herein.

[0100] Depending on the desired antigen to be targeted, the CAR may be further manipulated to include an appropriate antigen-binding domain specific to the desired antigen target. For example, if CD19 / CD20 is the desired antigen to be targeted, an antibody against CD19 / CD20 may be used to incorporate the antigen-binding domain into the CAR.

[0101] In one exemplary embodiment, the antigen-binding domain portion of the CAR further targets CD33. Preferably, the antigen-binding domain in the CAR is anti-CD33 ScFv, where the nucleic acid sequence of anti-CD33 ScFv includes the sequence shown in SEQ ID NO: 46. In one embodiment, anti-CD33 ScFv includes a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 46. In another embodiment, the anti-CD19 / CD20 ScFv portion of the CAR includes the amino acid sequence shown in SEQ ID NO: 47.

[0102] In one exemplary embodiment, the antigen-binding domain portion of the CAR further targets mesoserine. Preferably, the antigen-binding domain in the CAR is an anti-mesoserine ScFv, where the nucleic acid sequence of the anti-mesoserine ScFv includes the sequence shown in SEQ ID NO: 48. In one embodiment, the anti-mesoserine ScFv includes a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 48. In another embodiment, the anti-mesoserine ScFv portion of the CAR includes the amino acid sequence shown in SEQ ID NO: 49.

[0103] In one aspect of the present invention, a non-TSA or non-TAA CAR is provided that contains, for example, but is not limited to, a retroviridae (e.g., human immunodeficiency virus, e.g., HIV-1 and HIV-LP), picornaviridae (e.g., poliovirus, hepatitis A virus, enterovirus, human coxsackievirus, rhinovirus and echovirus), rubella virus, coronavirus, varicella stomatitis virus, rabies virus, Ebola virus, parainfluenza virus, mumps virus, measles virus, polynuclear respiratory virus, influenza virus, hepatitis B virus, parvovirus, adenoviridae, herpesviridae [e.g., herpes simplex virus type 1 and 2 (HSV), varicella-zoster virus, cytomegalovirus (CMV), and herpesviruses], poxviridae (e.g., smallpox virus, vaccinia virus and poxvirus), or hepatitis C virus antigens, or any combination thereof.

[0104] In another aspect of the present invention, CARs capable of binding to antigens derived from bacterial strains of Staphylococci, Streptococcus, Escherichia coli, Pseudomonas, or Salmonella are provided. In particular, CARs capable of binding to antigens derived from infectious bacteria, such as Helicobacter pyloris, Legionella pneumophilia, Mycobacteria sps. (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansaii, or M. gordonea), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitides, Listeria monocytogenes, Streptococcus pyogenes, Group A Streptococcus, Group B Streptococcus (Streptococcus agalactiae), Streptococcus pneumoniae, or Clostridium tetani, or combinations thereof, are provided.

[0105] 2. Transmembrane domain Regarding the transmembrane domain, the CAR contains one or more transmembrane domains fused to the extracellular CD19 / CD20 antigen-binding domain of the CAR.

[0106] The transmembrane domain may originate from either a natural or synthetic source. If the source is natural, the domain may originate from any membrane-bound or transmembrane protein.

[0107] The transmembrane domains particularly used in the CARs described herein may originate from the alpha, beta, or zeta chains of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesoserine, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF16, or TNFRSF19 (i.e., may include at least one of these transmembrane domains). Alternatively, the transmembrane domain may be synthetic, in which case it predominantly contains hydrophobic residues such as leucine and valine. Preferably, a triplicate of phenylalanine, tryptophan, and valine is found at each terminus of the synthetic transmembrane domain. Optionally, short oligopeptide linkers or polypeptide linkers, preferably between 2-amino acid and 10-amino acid lengths, may form a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. The glycine-serine duo provides a particularly suitable linker.

[0108] In one embodiment, a transmembrane domain naturally associated with one of the domains in the CAR is used in addition to the aforementioned transmembrane domain.

[0109] In some cases, transmembrane domains may be selected by amino acid substitutions to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, in order to minimize interaction with other members of the receptor complex.

[0110] In one embodiment, the transmembrane domain in the CAR of the present invention is a CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain contains the nucleic acid sequence of SEQ ID NO: 35. In one embodiment, the CD8 transmembrane domain contains a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 36. In another embodiment, the CD8 transmembrane domain contains the amino acid sequence of SEQ ID NO: 36.

[0111] In one embodiment, the encoded transmembrane domain includes an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) to the amino acid sequence of SEQ ID NO: 28, but with 20, 10, or 5 or fewer modifications (e.g., substitutions), or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO: 28.

[0112] In some cases, the transmembrane domain of the CAR includes a CD8 alpha-hinge domain. In one embodiment, the CD8 hinge domain includes the nucleic acid sequence of SEQ ID NO: 37. In another embodiment, the CD8 hinge domain includes a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 38. In yet another embodiment, the CD8 hinge domain includes the amino acid sequence of SEQ ID NO: 38, or a sequence having 95-99% identity thereto.

[0113] In one embodiment, an isolated nucleic acid molecule is provided in which the encoded linker domain originates from the extracellular domain of CD8 and is linked to a transmembrane CD8 domain, a transmembrane CD28 domain, or a combination thereof.

[0114] 3. Spacer Domain In CARs, the spacer domain may be located between the extracellular domain and the transmembrane domain, or between the intracellular domain and the transmembrane domain. The spacer domain refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular domain and / or to link the transmembrane domain to the intracellular domain. The spacer domain contains up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids.

[0115] In some embodiments, the linker may include a spacer element, if present, which increases the size of the linker, thereby increasing the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment. Exemplary spacers are known to those skilled in the art and include U.S. Patents 7,964,566, 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5,530,097, and 5,521 This includes U.S. Patent Nos. 284, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444 and 4,486,414, as well as those listed in U.S. Patent Application Publication Nos. 20110212088 and U.S. Patent Application Publication Nos. 20110070248, each of which is incorporated herein by reference in its entirety.

[0116] The spacer domain preferably promotes the binding of CAR to the antigen and the signal to enter the cell. It has sequences that enhance cellular signaling. Examples of amino acids predicted to promote binding include cysteine, charged amino acids, and serine and threonine in the potential glycosylation site, and these amino acids can be used as amino acids constituting the spacer domain.

[0117] As spacer domains, all or part of the hinge region of CD8 alpha (NCBI RefSeq: NP_001759.3), amino acids 137-206 (SEQ ID NO: 39), CD8 beta (GenBank: AAA35664.1), amino acids 135-195, CD4 (NCBI RefSeq: NP_000607.1), or CD28 (NCBI RefSeq: NP_006130.1), amino acids 137-152 may be used. Additionally, a portion of the constant region of the antibody H chain or L chain may be used as a spacer domain. Furthermore, the spacer domain may be an artificially synthesized sequence.

[0118] Furthermore, a signal peptide sequence can be ligated to the N-terminus of a CAR. Signal peptide sequences are present at the N-terminus of many secreted and membrane proteins and have a length of 15 to 30 amino acids. Since many of the protein molecules mentioned above as intracellular domains have signal peptide sequences, these signal peptides can be used as signal peptides for CARs. In one embodiment, the signal peptide includes the amino acid sequence shown in SEQ ID NO: 18.

[0119] 4. Intracellular domains The cytoplasmic domain or, otherwise, the intracellular signaling domain of a CAR is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is placed. The term “effector function” refers to a specialized function of a cell. For example, the effector function of a T cell may be cytolytic activity or helper activity, including cytokine secretion. Thus, the term “intracellular signaling domain” refers to the portion of a protein that transmits effector function signals and directs the cell to perform its specialized function. While the entire intracellular signaling domain can usually be used, it is often not necessary to use the entire chain. Insofar as a truncated portion of the intracellular signaling domain is used, such truncated portion can be used in place of the intact chain, as long as it transmits effector function signals. Thus, the term “intracellular signaling domain” means including any truncated portion of an intracellular signaling domain that is sufficient to transmit effector function signals.

[0120] Preferred examples of intracellular signaling domains for use in CARs include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that work together to initiate signaling after antigen receptor engagement, as well as any derivatives or variants of these sequences, and any synthetic sequences having the same functional capabilities.

[0121] It is known that the signals generated through the TCR alone are insufficient for complete T cell activation, and that secondary or co-stimulatory signals are also required. Therefore, it can be said that T cell activation is mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences) and those that act antigen-dependently to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences).

[0122] Primary cytoplasmic signaling sequences regulate the primary activation of the TCR complex either in a stimulative or inhibitory manner. Primary cytoplasmic signaling sequences that act in a stimulative manner are signaling motifs known as immunoreceptor-activated tyrosine motifs or ITAMs. It may contain.

[0123] Examples of ITAMs containing primary cytoplasmic signaling sequences particularly used in CARs disclosed herein include those derived from TCR zeta (CD3 zeta), FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Specific non-limiting examples of ITAM include amino acid numbers 51-164 of CD3 zeta (NCBI RefSeq: NP_932170.1), amino acid numbers 45-86 of Fc epsilon RI gamma (NCBI RefSeq: NP_004097.1), amino acid numbers 201-244 of Fc epsilon RI beta (NCBI RefSeq: NP_000130.1), amino acid numbers 139-182 of CD3 gamma (NCBI RefSeq: NP_000064.1), amino acid numbers 128-171 of CD3 delta (NCBI RefSeq: NP_000723.1), amino acid numbers 153-207 of CD3 epsilon (NCBI RefSeq: NP_000724.1), and CD5 (NCBI Amino acid numbers 402-495 of RefSeq:NP_055022.2, amino acid numbers 707-847 of 0022 (NCBI RefSeq:NP_001762.2), amino acid numbers 166-226 of CD79a (NCBI RefSeq:NP_001774.1), amino acid numbers 182-229 of CD79b (NCBI RefSeq:NP_000617.1), and CD66d (NCBI This includes peptides having sequences of amino acid numbers 177-252 (RefSeq:NP_001806.2), as well as variants of these peptides having the same function. The amino acid numbers based on NCBI RefSeq IDs or GenBank amino acid sequence information described herein are numbered based on the full length of each protein precursor (including signal peptide sequences, etc.). In one embodiment, the cytoplasmic signaling molecule in CAR includes a cytoplasmic signaling sequence derived from CD3 zeta.

[0124] In preferred embodiments, the intracellular domain of a CAR may be designed to include a CD3-zeta signaling domain, either by itself or in combination with any other desired cytoplasmic domain(s) useful in the context of the CAR. For example, the intracellular domain of a CAR may include a CD3 zeta chain portion and a costimulatory signaling region. The costimulatory signaling region refers to the portion of the CAR that includes the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is required for an efficient lymphocyte response to an antigen. Examples of such costimulatory molecules include ligands that specifically bind to CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83. Specific non-limiting examples of such co-stimulatory molecules include amino acid numbers 236-351 of CD2 (NCBI RefSeq: NP_001758.2), amino acid numbers 421-458 of CD4 (NCBI RefSeq: NP_000607.1), amino acid numbers 402-495 of CD5 (NCBI RefSeq: NP_055022.2), amino acid numbers 207-235 of CD8 alpha (NCBI RefSeq: NP_001759.3), amino acid numbers 196-210 of CD83 (GenBank: AAA35664.1), amino acid numbers 181-220 of CD28 (NCBI RefSeq: NP_006130.1), and CD137 (4-1BB, NCBI This includes peptides having the sequences of amino acid numbers 214-255 of RefSeq:NP_001552.2), amino acid numbers 241-277 of CD134 (OX40, NCBI RefSeq:NP_003318.1), and amino acid numbers 166-199 of ICOS (NCBI RefSeq:NP_036224.1), as well as variants of these peptides having the same functions. Therefore, while this disclosure primarily exemplifies 4-1BB as a co-stimulatory signaling element, other co-stimulatory elements are also within the scope of this disclosure.

[0125] The cytoplasmic signaling sequences within the cytoplasmic signaling region of a CAR can be linked to each other randomly or in a specified order. Optionally, short oligopeptide linkers or polypeptide linkers, preferably between 2-amino acid and 10-amino acid lengths, can form the linkage. Glycine-serine pairs provide particularly suitable linkers.

[0126] In one embodiment, the intracellular domain is designed to include a CD3-zeta signaling domain and a CD28 signaling domain. In another embodiment, the intracellular domain is designed to include a CD3-zeta signaling domain and a 4-1BB signaling domain. In yet another embodiment, the intracellular domain is designed to include a CD3-zeta signaling domain as well as CD28 and 4-1BB signaling domains.

[0127] In one embodiment, the intracellular domain in the CAR is designed to include a 4-1BB signaling domain and a CD3-zeta signaling domain, where the 4-1BB signaling domain includes the nucleic acid sequence shown in SEQ ID NO: 40, and the CD3-zeta signaling domain includes the nucleic acid sequence shown in SEQ ID NO: 42.

[0128] In one embodiment, the intracellular domain in the CAR is designed to include a 4-1BB signaling domain and a CD3-zeta signaling domain, where the 4-1BB signaling domain includes a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 41, and the CD3-zeta signaling domain includes a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 43.

[0129] In one embodiment, the intracellular domain in the CAR is designed to include a 4-1BB signaling domain and a CD3-zeta signaling domain, where the 4-1BB signaling domain includes the amino acid sequence shown in SEQ ID NO: 41, and the CD3-zeta signaling domain includes the amino acid sequence shown in SEQ ID NO: 43.

[0130] 5. Further description of CAR Functional parts of CARs disclosed herein are also expressly included within the scope of the invention. The term “functional part,” when used in relation to a CAR, means any part or fragment of one or more CARs disclosed herein, which retain the biological activity of the CAR from which it is a part (parent CAR). A functional part includes, for example, a part of a CRA that retains the ability to recognize target cells or to detect, treat, or prevent disease to a similar, equal, or greater degree than that of the parent CAR. With respect to a parent CAR, a functional part may constitute, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more of the parent CAR.

[0131] The functional moiety may contain additional amino acids at its amino-terminus, carboxy-terminus, or both, which are not found in the amino acid sequence of the parent CAR. Preferably, the additional amino acids do not interfere with the biological function of the functional moiety, such as recognizing target cells, detecting cancer, or treating or preventing cancer. More preferably, the additional amino acids enhance the biological activity of the functional moiety compared to that of the parent CAR.

[0132] Functional variants of CARs disclosed herein are included within the scope of this disclosure. The term “functional variant” as used herein means a CAR, polypeptide, or protein having substantial or significant sequence identity or similarity to the parent CAR, wherein the functional variant retains the biological activity of the CAR from which it is a variant. A functional variant may, for example, be similar to, the same as, or higher than the parent CAR. This includes variants of the CAR (parent CAR) described herein that retain the ability to recognize target cells. With respect to the parent CAR, functional variants may have an amino acid sequence identical to, for example, the parent CAR by at least about 30%, 50%, 75%, 80%, 90%, 98%, or more.

[0133] A functional variant may, for example, include the amino acid sequence of a parent CAR having at least one conserved amino acid substitution. Alternatively, the functional variant may further include the amino acid sequence of a parent CAR having at least one non-conserved amino acid substitution. In this case, it is preferable that the non-conserved amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. The non-conserved amino acid substitution may enhance the biological activity of the functional variant, resulting in an increased biological activity of the functional variant compared to the parent CAR.

[0134] The amino acid substitutions of CARs are preferably conservative amino acid substitutions. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having certain physical and / or chemical properties is replaced with another amino acid having the same or similar chemical or physical properties. For example, conservative amino acid substitutions may include an acidic / negatively charged polar amino acid substituting another acidic / negatively charged polar amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain substituting another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), a basic / positively charged polar amino acid substituting another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), an uncharged amino acid with a polar side chain substituting another uncharged amino acid with a polar side chain (e.g., Asn, Gin, Ser, Thr, Tyr, etc.), an amino acid with a beta-branched side chain substituting another amino acid with a beta-branched side chain (e.g., He, Thr, and Val), and an amino acid with an aromatic side chain substituting another amino acid with an aromatic side chain (e.g., His, Phe, Trp, and Tyr).

[0135] CARs may essentially consist of one or more of the specified amino acid sequences described herein, and as a result, other components, such as other amino acids, do not significantly alter the biological activity of the functional variant.

[0136] CARs (including functional parts and functional variants) can be of any length, i.e., they can contain any number of amino acids, provided that CARs (or their functional parts or functional variants) retain their biological activity, such as the ability to specifically bind to an antigen, the ability to detect diseased cells in mammals, or the ability to treat or prevent disease in mammals. For example, CARs can be approximately 50 to 5000 amino acid long, e.g., 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or longer.

[0137] CAR (including the functional parts and functional variants of the present invention) may include synthetic amino acids instead of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, -amino n-decanoic acid, homoserine, S-acetylaminomethylcysteine, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, -amino This includes nocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, γ-diaminobutyric acid, β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0138] CARs (including functional moieties and functional variants) can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via disulfide crosslinks, etc., or converted to acid addition salts and / or optionally dimerized, polymerized, or conjugated.

[0139] CARs (including their functional parts and functional variants) can be obtained by methods known in the art. CARs can be produced by any suitable method for producing polypeptides or proteins. Suitable methods for the de novo synthesis of polypeptides and proteins are described in references such as Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2000; Peptide and Protein Drug Analysis, edited by Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, edited by Westwood et al., Oxford University Press, Oxford, United Kingdom, 2001; and U.S. Patent No. 5,449,752. Polypeptides and proteins can also be recombinantly produced using standard recombination methods and nucleic acids described herein. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Furthermore, some CARs (including functional moieties and their functional variants) can be isolated and / or purified from sources such as plants, bacteria, insects, mammals, e.g., rats, humans. Methods for isolation and purification are well known in the art. Alternatively, CARs (including functional moieties and their functional variants) described herein can be commercially synthesized by companies. In this regard, CARs may be synthetic, recombinant, isolated, and / or purified.

[0140] B. Antibodies and antigen-binding fragments One embodiment further provides a CAR, a CAR-expressing T cell, and an antibody or its antigen-binding domain or portion that specifically binds to one or more of the antigens disclosed herein. As used herein, “CAR-expressing T cell” or “CAR T cell” means a T cell that expresses a CAR and has antigen specificity determined, for example, by the antibody-derived targeting domain of the CAR.

[0141] As used herein, “antigen-binding domain” may include an antibody and its antigen-binding fragments. The term “antibody” is used herein in its broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and their antigen-binding fragments, as long as they exhibit the desired antigen-binding activity. Non-limiting examples of antibodies include, for example, intact immunoglobulins known in the art, as well as their variants and fragments that retain binding affinity to an antigen.

[0142] A "monoclonal antibody" is an antibody obtained from a substantially homogeneous population of antibodies, that is, The individual antibodies constituting a population are identical except for possible naturally occurring mutations that may exist in trace amounts. Monoclonal antibodies are highly specific and target a single antigenic epitope. The modifier "monoclonal" indicates that the antibody is obtained from a substantially homogeneous population of antibodies, and should not be interpreted as requiring the production of the antibody by any particular method. In some cases, monoclonal antibodies are antibodies produced by a single clone of B lymphocytes, or antibodies produced by cells transfected with nucleic acids encoding the light and heavy chain variable regions of a single antibody (or its antigen-binding fragment), or by their offspring. In some cases, monoclonal antibodies are isolated from the subject. Monoclonal antibodies may have conserved amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions. Exemplary methods for the production of monoclonal antibodies are publicly known; see, for example, Harlow & Lane, Antibodies, A Laboratory Manual, 2nd edition, Cold Spring Harbor Publications, New York (2013).

[0143] Typically, immunoglobulins have heavy (H) and light (L) chains interconnected by disulfide bonds. Immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable domain genes. Two types of light chains exist: lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE.

[0144] Each heavy chain and light chain comprises a constant region (or constant domain) and a variable region (or variable domain; see, for example, Kindt et al., Kuby Immunology, 6th edition, WHFreeman and Co., p. 91 (2007)). In some embodiments, the variable regions of the heavy chain and light chain combine to specifically bind to the antigen. In further embodiments, only the variable region of the heavy chain is required. For example, naturally occurring camelid antibodies consisting only of heavy chains are functional and stable in the absence of the light chain. This is definitive (see, for example, Hamers-Casterman et al., Nature, Vol. 363: pp. 446-448, 1993; Sheriff et al., Nat. Struct. Biol., Vol. 3: pp. 733-736, 1996). A reference to "VH" or "VH" refers to the variable region of the antibody heavy chain, including antigen-binding fragments, e.g., those of Fv, ScFv, dsFv, or Fab. A reference to "VL" or "VL" refers to the variable domain of the antibody light chain, including those of Fv, ScFv, dsFv, or Fab.

[0145] The variable regions of the light and heavy chains contain a "framework" region interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs" (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, USD Department of Health and Human Services, 1991). The sequences of different light or heavy chain framework regions are relatively conserved within a species. The antibody framework region, which is the combined framework region of the constituent light and heavy chains, functions to position and align the CDRs in three-dimensional space.

[0146] CDRs are primarily responsible for binding antigens to epitopes. The amino acid sequence boundaries of a given CDR are described in Kabat et al. ("Sequences of Proteins of Immunological Interest," 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991; "Kabat" numbering scheme), Al-Lazikani et al. (JMB Vol. 273, pp. 927-948, 1997; "Chothia" numbering scheme), and Lefranc et al. ("IMGT unique numbering scheme"). g for immunoglobulin and T cell receptor The CDRs can be readily determined using one of several well-known schemes, including the one described in “Variable Domains and Ig Superfamily V-like Domains,” Dev. Comp. Immunol., Vol. 27: pp. 55–77, 2003 (the “IMGT” numbering scheme). The CDRs of each chain are typically called CDR1, CDR2, and CDR3 (from N-terminus to C-terminus) and are also typically identified by the chain on which a particular CDR is located. Thus, VH CDR3 is the CDR3 derived from the variable domain of the heavy chain of the antibody in which it is found, while VL CDR1 is the CDR1 derived from the variable domain of the light chain of the antibody in which it is found. Light chain CDRs are sometimes called LCDR1, LCDR2, and LCDR3. Heavy chain CDRs are sometimes called LCDR1, LCDR2, and LCDR3.

[0147] An "antigen-binding fragment" is a portion of a full-length antibody that retains the ability to specifically recognize a congener antigen, as well as various combinations of such portions. Non-limiting examples of antigen-binding fragments include Fv, Fab, Fab', Fab'-SH, F(ab')2; diabody; linear antibodies; single-chain antibody molecules (e.g., ScFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen-binding fragments produced by modification of the entire antibody, or antigen-binding fragments synthesized de novo using recombinant DNA methodologies (see, e.g., Kontermann and Dubel (eds.), Antibody Engineering, vols. 1-2, 2nd edition, Springer Press, 2010).

[0148] Single-chain antibodies (ScFv) are genetically engineered molecules containing the VH and VL domains of one or more antibodies linked by a suitable polypeptide linker as a single-chain molecule (see, e.g., Bird et al., Science, vol. 242: pp. 423-426, 1988; Huston et al., Proc. Natl. Acad. Sci., vol. 85: pp. 5879-5883, 1988; Ahmad et al., Clin. Dev. Immunol., 2012, doi:10.1155 / 2012 / 980250; Marbry, IDrugs, vol. 13: pp. 543-549, 2010). The intramolecular orientation of the VH and VL domains in ScFv is typically not definitive for ScFv. Therefore, ScFv can be used with both possible configurations (VH domain-linker domain-VL domain; VL domain-linker domain-VH domain).

[0149] In dsFv, the variable chains of the heavy and light chains are mutated to introduce disulfide bonds to stabilize chain association. Diabodies are also included, which are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but use a linker that is too short to allow pairing between the two domains on the same chain, thereby pairing that domain with a complementary domain on another chain and creating two antigen-binding sites (see, e.g., Holliger et al., Proc. Natl. Acad. Sci., vol. 90: pp. 6444-6448, 1993; Poljak et al., Structure, vol. 2: pp. 1121-1123, 1994).

[0150] Antibodies also include genetically modified forms such as chimeric antibodies (e.g., humanized mouse antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies). See also Pierce Catalog and Handbook, 1994–1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd edition, WH Freeman & Co., New York, 1997.

[0151] Antibodies not found in nature can be constructed using solid-phase peptide synthesis, recombinantly produced, or obtained by screening combinatorial libraries consisting of variable heavy and variable light chains, as described, for example, in Huse et al., Science Vol. 246: pp. 1275-1281 (1989), incorporated herein by reference. These and other methods for producing chimeric, humanized, CDR-grafted, single-chain, and bifunctional antibodies are well known to those skilled in the art (Winter and Harris, Immunol. Today Vol. 14: pp. 243-246 (1993); Ward et al., Nature). Volume 341: pp. 544-546 (1989); Harlow and Lane, above, 1988; Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrabeck, Antibody Engineering, 2nd edition (Oxford University Press 1995); each of these is incorporated herein by reference).

[0152] A reference antibody and an antibody that "binds to the same epitope" refer to an antibody that blocks 50% or more of the binding of the reference antibody to its antigen in a competitive assay, and conversely, a reference antibody blocks 50% or more of the binding of its antibody to its antigen in a competitive assay. Antibody competitive assays are well known, and exemplary competitive assays are provided herein.

[0153] A “humanized” antibody or antigen-binding fragment comprises a human framework region and one or more CDRs derived from a non-human (e.g., mouse, rat, or synthetic) antibody or antigen-binding fragment. The non-human antibody or antigen-binding fragment providing the CDRs is called the “donor,” and the human antibody or antigen-binding fragment providing the framework is called the “acceptor.” In one embodiment, all CDRs are derived from the donor immunoglobulin in the humanized immunoglobulin. A constant region is not required to be present, but if present, it may be substantially identical to the human immunoglobulin constant region, e.g., at least about 85–90%, e.g., about 95% or more identical. Thus, all parts of the humanized antibody or antigen-binding fragment are substantially identical to the corresponding parts of the natural human antibody sequence, except perhaps for the CDRs.

[0154] A "chimeric antibody" is an antibody that contains sequences derived from two different antibodies, typically from different species. In some cases, a chimeric antibody contains one or more CDRs and / or framework regions from one human antibody as well as CDRs and / or framework regions from another human antibody.

[0155] A “fully human antibody” or “human antibody” is an antibody that contains sequences derived from (or originating from) the human genome but does not contain sequences from another species. In some embodiments, a human antibody contains CDRs, framework regions, and (if present) Fc regions derived from (or originating from) the human genome. Human antibodies can be identified and isolated, for example, by phage display, using technologies for generating sequences based on sequences derived from the human genome, or by using transgenic animals (see, e.g., Barbas et al. Phage display: A Laboratory Manuel. 1st ed. New York: Cold Spring Harbor Laboratory Press, 2004 Print.; Lonberg, Nat. Biotech., vol. 23: pp. 1117-1125, 2005; Lonberg, Curr. Opin. Immunol., vol. 20: pp. 450-459, 2008).

[0156] Antibodies may have one or more binding sites. If there are more than one binding sites, these sites may be identical or different. For example, naturally occurring immunoglobulins have two identical binding sites, while single-chain antibodies or Fab fragments have one. While some antibodies have two binding sites, bispecific or bifunctional antibodies have two different binding sites.

[0157] Methods for testing antibodies for their ability to bind to any functional portion of a CAR are known in the art and include any antibody-antigen binding assays, such as radioimmunoassays (RIA), ELISA, Western blotting, immunoprecipitation, and competitive inhibition assays (see, for example, Janeway et al., U.S. Patent Application Publication No. 2002 / 0197266Al, and U.S. Patent No. 7,338,929).

[0158] Furthermore, CAR, CAR-expressing T cells, antibodies, or their antigen-binding moieties may be modified to include detectable labels, such as radioisotopes, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles).

[0159] C. Conjugate CARs, CAR-expressing T cells, or monoclonal antibodies or antigen-binding fragments thereof specific to one or more of the antigens disclosed herein may be conjugated to agents such as effector molecules or detectable markers by means of several means known to those skilled in the art. Both covalent and non-covalent means may be used. The conjugate includes, but is not limited to, molecules in which an effector molecule or detectable marker is covalently linked to an antibody or antigen-binding fragment that specifically binds to one or more of the antigens disclosed herein. Those skilled in the art will know that chemotherapeutic agents, anti-angiogenic agents, toxins, radioactive agents, etc., may be conjugated. 125 I, 32 P, 14 C, 3 H and 35 It is understood that various effector molecules and detectable markers, including (but not limited to) S, other labels, target moieties, and ligands, may be used.

[0160] The selection of a specific effector molecule or detectable marker depends on the specific target molecule or cell and the desired biological effect. For example, an effector molecule could be a cytotoxin used to induce the death of a specific target cell (e.g., tumor cells).

[0161] The procedure for conjugating an effector molecule or detectable marker to an antibody or antigen-binding fragment varies depending on the chemical structure of the effector. Polypeptides typically contain various functional groups; for example, carboxylic acid (COOH), free amine (-NH2), or sulfhydryl (-SH) groups, which are available for reaction with suitable functional groups on the antibody to result in the binding of the effector molecule or detectable marker. Alternatively, the antibody or antigen-binding fragment is derivatized to expose or bind further reactive functional groups. Derivatization may involve the binding of one of several known linker molecules, such as those available from Pierce Chemical Company, Rockford, IL. The linker can be any molecule used to conjugate the antibody or antigen-binding fragment to the effector molecule or detectable marker. The linker can form a covalent bond to both the antibody or antigen-binding fragment and the effector molecule or detectable marker. Suitable linkers are well known to those skilled in the art and include, but are not limited to, linear or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. When the antibody or antigen-binding fragment and the effector molecule or detectable marker are polypeptides, the linker may be attached to the constituent amino acids via their side groups (e.g., via disulfide linkage to cysteine) or to the amino and carboxyl groups of the alpha carbon of the terminal amino acids.

[0162] In some embodiments, the linker may include a spacer element, if present, which increases the size of the linker, thereby increasing the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment. Exemplary spacers are known to those skilled in the art and include U.S. Patents 7,964,566, 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5,530,097, and 5,521 This includes U.S. Patent Nos. 284, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444 and 4,486,414, as well as those listed in U.S. Patent Application Publication Nos. 20110212088 and U.S. Patent Application Publication Nos. 20110070248, each of which is incorporated herein by reference in its entirety.

[0163] In some embodiments, the linker is cleavable under intracellular conditions, and as a result, cleavage of the linker causes the release of an effector molecule or detectable marker from the antibody or antigen-binding fragment in the intracellular environment. In yet other embodiments, the linker is incleavable, and the effector molecule or detectable marker is released, for example, by antibody degradation. In some embodiments, the linker is cleavable by cleavage agents present in the intracellular environment (e.g., within lysosomes, endosomes, or caveolae). The linker may be a peptide linker cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, lysosomal or endosomal proteases. In some embodiments, the peptide linker is at least 2 amino acid long or at least 3 amino acid long. However, the linker may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid long, for example, 1-2, 1-3, 2-5, 3-10, 3-15, 1-5, 1-10, or 1-15 amino acid long. Proteases may include cathepsins B and D, as well as plasmin, all of which are known to hydrolyze dipeptide drug derivatives, resulting in the release of active drugs inside target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics, Vol. 83: pp. 67-123). For example, peptide linkers cleavable by cathepsin B, a thiol-dependent protease, may be used (e.g., phenylalanine-leucine or glycine-phenylalanine-leucine-glycine linkers). Other examples of such linkers are described, for example, in U.S. Patent No. 6,214,345, incorporated herein by reference. In specific embodiments, the peptide linker cleavable by intracellular proteases may be a valine-citrulline linker or a phenylalanine-lysine linker (see, for example, U.S. Patent No. 6,214,345, which describes the synthesis of doxorubicin using a valine-citrulline linker).

[0164] In other embodiments, the cleavable linker is pH-sensitive, i.e., susceptible to hydrolysis at a specific pH value. Typically, pH-sensitive linkers are hydrolyzable under acidic conditions. For example, acid-unstable linkers hydrolyzable in lysosomes (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, etc.) may be used (see, e.g., U.S. Patent No. 5,122,368; U.S. Patent No. 5,824,805; U.S. Patent No. 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics Vol. 83: pp. 67-123; Neville et al., 1989, Biol. Chem. Vol. 264: pp. 14653-14661). Such linkers are relatively stable under neutral pH conditions, such as in blood, but are not suitable for lysosomes. It is unstable at pH levels below its pH of 5.5 or 5.0. In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether bonded to the therapeutic agent via an acylhydrazone linkage) (see, for example, U.S. Patent No. 5,622,929).

[0165] In other embodiments, the linker is cleavable under reducing conditions (e.g., a disulfide linker). Various disulfide linkers are known in the field, including, for example, SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyldithio)toluene), which can be formed using SPDB and SMPT (e.g., Thorpe et al., 1987, Cancer Res. Vol. 47: pp. 5924-5931; Wawrzynczak et al., Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (CWVogel, ed., Oxford U. Press, 1987); Phillips et al., Cancer See Res. 68: pp. 9280-9290, 2008. Also see U.S. No. 4,880,935.

[0166] In yet other specific embodiments, the linker is a malonate linker (Johnson et al., 1995, Anticancer Res. Vol. 15: pp. 1387-1393), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. Vol. 3 (No. 10): pp. 1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. Vol. 3 (No. 10): pp. 1305-1312).

[0167] In yet another embodiment, the linker is incapable of cleavage, and the effector molecule or detectable marker is released by antibody degradation (see U.S. Patent Application Publication 2005 / 0238649, the full contents of which are incorporated herein by reference).

[0168] In some embodiments, the linker is resistant to cleavage in the extracellular environment. For example, when the conjugate is present in the extracellular environment (e.g., plasma), approximately 20%, 15%, 10%, 5%, 3%, or 1% of the linker in the conjugate sample is cleaved. Whether the linker is resistant to cleavage in the extracellular environment can be determined, for example, by incubating the conjugate containing the linker of interest with plasma for a predetermined period (e.g., 2, 4, 8, 16, or 24 hours) and then quantifying the amount of free effector molecules or detectable markers present in the plasma. Various exemplary linkers that may be used in a conjugate are described in WO2004-010957, U.S. Patent Application Publication 2006 / 0074008, U.S. Patent Application Publication 20050238649, and U.S. Patent Application Publication 2006 / 0024317, each of which is incorporated herein by reference in its entirety.

[0169] In some embodiments, CAR conjugates, CAR-expressing T cells, antibodies or their antigen-binding moieties, and one or more small molecule toxins, such as calitiamycin, maytansinoid, drastatin, auristatin, trichothecin, and CC1065, as well as derivatives of these toxins having toxic activity.

[0170] Maytansine, suitable for use as the toxin portion of a mytansinoid. The compounds are well known in the art and can be isolated from natural sources according to known methods, produced using genetic engineering techniques (see Yu et al. (2002) PNAS Vol. 99: pp. 7968-7973), or synthesized according to known methods using maytansinol and maytansinol analogs. Maytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansin was first isolated from the East African shrub Maytenus serrata (U.S. Patent No. 3,896,111). Subsequently, it was discovered that certain microorganisms also produce maytansinoids such as maytansinol and C-3 maytansinol ester (U.S. Patent No. 4,151,042). Synthetic mytansinol and its derivatives and analogs are, for example, U.S. Patent Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,31 U.S. Patent No. 3,946; U.S. Patent No. 4,315,929; U.S. Patent No. 4,317,821; U.S. Patent No. 4,322,348; U.S. Patent No. 4,331,598; U.S. Patent No. 4,361,650; U.S. Patent No. 4,364,866; U.S. Patent No. 4,424,219; U.S. Patent No. 4,450,254; U.S. Patent No. 4,362,663; and U.S. Patent No. 4,371,533, each of which is incorporated herein by reference. Conjugates containing mytansinoids, methods for preparing them, and their therapeutic uses are disclosed, for example, U.S. Patent No. 5,208,020; U.S. Patent No. 5,416,064; U.S. Patent No. 6,441,163 and European Patent EP0 425 235 B1, the disclosures of which are expressly incorporated herein by reference.

[0171] Further toxins may be used in conjunction with CARs, CAR-expressing T cells, antibodies, or their antigen-binding moieties. Exemplary toxins include Pseudomonas exotoxin (PE), hematopoxin, abrin, diphtheria toxin and its subunits, ribotoxin, ribonuclease, saporin and calitiamycin, and botulinum toxins A–F. These toxins are well known in the art and many are readily available from commercial sources (e.g., Sigma Chemical Company, St. Louis, MO). The toxins intended may also include variants of these toxins (see, for example, U.S. Patent Nos. 5,079,163 and 4,689,401).

[0172] Saporins are toxins derived from Saponaria officinalis that disrupt protein synthesis by inactivating the 60S portion of ribosome complexes (Stirpe et al., Bio / Technology, Vol. 10: pp. 405-412, 1992). However, these toxins lack a mechanism for specific entry into cells and therefore require conjugation to antibodies or antigen-binding fragments that recognize internalized cell surface proteins in order to be efficiently taken up by cells.

[0173] Diphtheria toxin is isolated from Corynebacterium diphtheriae. Typically, diphtheria toxin for use in immunotoxins is mutated to reduce or eliminate nonspecific toxicity. A variant known as CRM107, which has full enzymatic activity but significantly reduced nonspecific toxicity, has been known since the 1970s (Laird and Groman, J. Virol. 19:220, 1976) and has been used in human clinical trials. See U.S. Patents No. 5,792,458 and 5,208,021.

[0174] Castor bean toxin is derived from the lectin RCA60 from Ricinus communis (the red bean plant). For examples of hematopoiesis, see U.S. Patent Nos. 5,079,163 and 4,689,401. Ricinus communis aglutinin (RCA) is a compound of RCA, with molecular weights of approximately 65 kD and 120 kD, respectively. 60 and RCA 120 It exists in two forms, known as (Nicholson and Blaustein, J. Biochim. Biophys. Acta vol. 266: p. 543, 1972). Chain A is responsible for inactivating protein synthesis and cell death. Chain B binds hematin to galactose residues on the cell surface and promotes the transport of chain A into the cytosol (Olsnes et al., Nature vol. 249: pp. 627-631, 1974 and US Patent No. 3,060,165).

[0175] Ribonucleases have also been conjugated into targeted molecules for use as immunotoxins (see Suzuki et al., Nat. Biotech. Vol. 17: pp. 265-267, 1999). Exemplary ribotoxins, such as α-sarcin and restrictocin, are discussed, for example, in Rathore et al., Gene Vol. 190: pp. 31-35, 1997; and in Goyal and Batra, Biochem. Vol. 345 Part 2: pp. 247-244, 2000. Kalithiamycin, first isolated from Micromonospora echinospora, is a member of the enediin antitumor antibiotic family, inducing double-strand breaks in DNA that lead to apoptosis (see, for example, Lee et al., J. Antibiot. Vol. 42: pp. 1070-1087, 1989). This drug is the toxic portion of an immunotoxin in clinical trials (see, for example, Gillespie et al., Ann. Oncol. Vol. 11: pp. 735-741, 2000).

[0176] Abrin contains toxic lectins derived from Abrus precatorius. The toxic elements abrins a, b, c, and d have molecular weights of approximately 63 kD and 67 kD, and are composed of two disulfide-linked polypeptide chains A and B. Chain A inhibits protein synthesis; chain B (abrin-b) binds to D-galactose residues (see Funatsu et al., Agr. Biol. Chem. Vol. 52: p. 1095, 1988; and Olsnes, Methods Enzymol. Vol. 50: pp. 330-335, 1978).

[0177] CARs, CAR-expressing T cells, monoclonal antibodies specific to one or more of the antigens disclosed herein, and their antigen-binding fragments may also be conjugated with detectable markers; for example, detectable markers detectable by ELISA, spectrophotometry, flow cytometry, microscopy or imaging techniques (e.g., computed tomography (CT), computed axial tomography (CAT) scans, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance imaging (MTR), ultrasound, fiber optic testing and laparoscopic testing). Specific non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioactive isotopes and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds such as fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, and lanthanidrin photopolymers. Bioluminescent markers such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP) are also used. CARs, CAR-expressing T cells, antibodies, or their antigen-binding moieties can also be conjugated to enzymes useful for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, and glucose oxidase. When CARs, CAR-expressing T cells, antibodies, or their antigen-binding moieties are conjugated with a detectable enzyme, they produce a identifiable reaction product. It can be detected by adding additional reagents used by the enzyme. For example, when the agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine results in a visually detectable colored reaction product. The CAR, T cells expressing the CAR, an antibody or an antigen-binding portion thereof can also be conjugated to biotin and detected through an indirect measurement of the binding of avidin or streptavidin. It should be noted that avidin itself can be conjugated to an enzyme or a fluorescent label.

[0178] The CAR, T cells expressing the CAR, an antibody or an antigen-binding portion thereof can be conjugated to a paramagnetic agent such as gadolinium. Paramagnetic agents such as superparamagnetic iron oxide are also used as labels. The antibody can also be conjugated to lanthanides (e.g., europium and dysprosium) and manganese. The antibody or antigen-binding fragment can also be labeled with a predetermined polypeptide epitope (e.g., leucine zipper pairing sequence, binding site for a secondary antibody, metal-binding domain, epitope tag) recognized by a secondary reporter.

[0179] The CAR, T cells expressing the CAR, an antibody or an antigen-binding portion thereof can also be conjugated to radiolabeled amino acids. The radiolabel can be used for both diagnostic and therapeutic purposes. For example, the radiolabel can be used to detect one or more of the antigens and antigen-expressing cells disclosed herein by x-ray, luminescence spectrum or other diagnostic techniques. Further, the radiolabel can be used therapeutically as a toxin for the treatment of tumors in a subject, e.g., for the treatment of neuroblastoma. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionucleotides: 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I.

[0180] Means for detecting such detectable markers are well known to those skilled in the art. For example, radioactive labels can be detected using photographic film or a scintillation counter, and fluorescent markers can be detected using a photodetector to detect the emitted illumination. Enzymatic labels are typically detected by providing a substrate to an enzyme and detecting the reaction product produced by the enzyme's action on the substrate, and chromogenic labels are detected by simply visualizing the colored label.

[0181] D. Nucleotides, expression, vectors, and host cells A nucleic acid comprising a nucleotide sequence encoding any of the CARs, antibodies or their antigen-binding portions (including their functional portions and functional variants) described herein is further provided by one embodiment of the present invention. The nucleic acid of the present invention may comprise a nucleotide sequence encoding any of the leader sequences, antigen-binding domains, transmembrane domains and / or intracellular T cell signaling domains described herein.

[0182] In some embodiments, nucleotide sequences may be codon-modified. While not bound by any particular theory, codon optimization of nucleotide sequences is thought to increase the translation efficiency of mRNA transcripts. Codon optimization of nucleotide sequences may involve replacing native codons with other codons that encode the same amino acid but can be translated by tRNA that is more readily available in the cell, thereby increasing translation efficiency. Optimization of nucleotide sequences may also reduce secondary mRNA structures that interfere with translation, thereby increasing translation efficiency.

[0183] In embodiments of the present invention, the nucleic acid may comprise a codon-modified nucleotide sequence encoding the antigen-binding domain of the CAR of the present invention. In another embodiment of the present invention, the nucleic acid comprises any of the CARs described herein (including their functional portions and functional variants). It may contain a nucleotide sequence with modified codons that encode it.

[0184] As used herein, “nucleic acid” includes “polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” and generally refers to a polymer of DNA or RNA that may be single-stranded or double-stranded, synthetic or derived from natural sources (e.g., isolated and / or purified), may contain natural, unnatural or modified nucleotides, and may contain natural, unnatural or modified nucleotide linkages, such as phosphoramidate linkages or phosphorothioate linkages, instead of phosphodiesters found between nucleotides in unmodified oligonucleotides. In some embodiments, the nucleic acid does not contain any insertions, deletions, inversions, and / or substitutions. However, in some cases, as discussed herein, the nucleic acid may appropriately contain one or more insertions, deletions, inversions, and / or substitutions.

[0185] Recombinant nucleic acids may have sequences that do not exist in nature, or sequences that are created by artificial combinations of two or otherwise separated segments of a sequence. These artificial combinations are often achieved by chemical synthesis, or more generally, by artificial manipulation of isolated segments of nucleic acids by genetic engineering techniques, such as those described above by Sambrook et al. Nucleic acids can be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. See, for example, Sambrook et al., and Ausubel et al., above. For example, nucleic acids can be chemically synthesized using naturally occurring nucleotides, or variously modified nucleotides (e.g., phosphorothioate derivatives and acridine-substituted nucleotides) designed to increase the biological stability of the molecule or the physical stability of the double helix formed during hybridization.Examples of modified nucleotides that can be used to generate nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted adenine, 7-methylguanine This includes, but is not limited to, nin, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid(v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of the present invention can be purchased from companies such as Integrated DNA Technologies (Coralville, IA, USA).

[0186] Nucleic acids may include any isolated or purified nucleotide sequences encoding either a CAR or a functional portion or functional variant thereof. Alternatively, a nucleotide sequence may include a nucleotide sequence that is degenerate with any of the sequences, or a combination of degenerate sequences.

[0187] One embodiment is complementary to any of the nucleotide sequences of the nucleic acids described herein. We also provide isolated or purified nucleic acids, which include a nucleotide sequence, or a nucleotide sequence that hybridizes under stringent conditions with any of the nucleotide sequences of the nucleic acids described herein.

[0188] Nucleotide sequences that hybridize under stringent conditions may also hybridize under high stringency conditions. “High stringency conditions” means that the nucleotide sequence hybridizes specifically to a target sequence (a nucleotide sequence of any of the nucleic acids described herein) in a detectably strong amount compared to nonspecific hybridization. High stringency conditions include conditions that allow for the identification of polynucleotides with precisely complementary sequences, or polynucleotides containing only a few scattered mismatches, from random sequences that coincidentally have several subregions (e.g., 3–10 bases) that match the nucleotide sequence. These complementary subregions are more readily melted than full-length complements of 14–17 or more bases, and high stringency hybridization makes them readily distinguishable. Relatively high stringency conditions include low-salt and / or high-temperature conditions, such as those provided at a temperature of about 50–70°C with about 0.02–0.1 M NaCl or equivalent. Such highly stringent conditions tolerate little to no mismatch between the nucleotide sequence and the template or target strand, if any, and are particularly suitable for detecting the expression of any of the CARs of the present invention. Generally, it is understood that the conditions can be made more stringent by the addition of gradually increasing amounts of formamide.

[0189] Also provided are nucleic acids having nucleotide sequences that are identical to any of the nucleic acids described herein by at least about 70% or more, for example, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%.

[0190] In one embodiment, nucleic acids may be incorporated into a recombinant expression vector. In this regard, one embodiment provides a recombinant expression vector comprising any of the nucleic acids. For the purposes of this specification, the term “recombinant expression vector” means a genetically modified oligonucleotide or polynucleotide construct that enables the expression of mRNA, protein, polypeptide or peptide by a host cell, provided that the construct comprises a nucleotide sequence encoding mRNA, protein, polypeptide or peptide, and the vector is brought into contact with the cell under conditions sufficient for the mRNA, protein, polypeptide or peptide to be expressed in the cell. Vectors do not exist in nature as a whole.

[0191] However, some vector components may be naturally occurring. Recombinant expression vectors may be single-stranded or double-stranded, synthetic or partially derived from natural sources, and may contain any type of nucleotide, including but not limited to DNA and RNA, which may contain natural, non-natural, or modified nucleotides. Recombinant expression vectors may contain naturally occurring or non-natural nucleotide linkages, or both. Preferably, non-natural or modified nucleotides or nucleotide linkages do not interfere with the transcription or replication of the vector.

[0192] In one embodiment, the recombinant expression vector can be any suitable recombinant expression vector that can be used to transform or transfect any suitable host cell. Suitable vectors include plasmids and viruses, which are designed for reproduction and growth, or for expression, or both. Examples of vectors include the pUC series (Fermentas Life Sciences, Glen Burnie, MD), the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), and the pGEX series (Ph The group may be selected from armacia (Biotech, Uppsala, Sweden) and the pEX series (Clontech, Palo Alto, CA).

[0193] Bacteriophage vectors, such as λυTI O, λυTI 1, λZapII (Stratagene), EMBL4, and λNM149, may also be used. Examples of plant expression vectors include pBIOl, pBI101.2, pBHOl.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). Recombinant expression vectors may be viral vectors, such as retroviral vectors or lentiviral vectors. Lentiviral vectors are vectors derived from at least a portion of a lentiviral genome, including self-inactivating lentiviral vectors, such as those provided in Milone et al., Mol. Ther. Vol. 17 (No. 8): pp. 1453-1464 (2009). Other examples of lentiviral vectors that may be used in clinics include, but are not limited to, Oxford BioMedica plc's LENTIVECTOR® gene delivery technology and Lentigen's LENTIMAX® vector system. Non-clinical lentiviral vectors are also available and known to those skilled in the art.

[0194] Several transfection techniques are generally known in the field (see, for example, Graham et al., Virology, Vol. 52: pp. 456-467 (1973); Sambrook et al., above; Davis et al., Basic Methods in Molecular Biology, Elsevier (1986); and Chu et al., Gene, Vol. 13: p. 97 (1981)).

[0195] Transfection methods include calcium phosphate coprecipitation (e.g., Graham et al., see above), direct microinjection into cultured cells (e.g., Capecchi, Cell, Vol. 22: pp. 479-488 (1980)), electroporation (e.g., Shigekawa et al., BioTechniques, Vol. 6: pp. 742-751 (1988)), liposome-mediated gene transfer (e.g., Mannino et al., BioTechniques, Vol. 6: pp. 682-690 (1988)), lipid-mediated transduction (e.g., Feigner et al., Proc. Natl. Acad. Sci. USA, Vol. 84: pp. 7413-7417 (1987)), and high-velocity micropropellants. This includes nucleic acid delivery using microprojectiles (see, for example, Klein et al., Nature, Vol. 327: pp. 70-73 (1987)).

[0196] In one embodiment, recombinant expression vectors may be prepared using standard recombinant DNA techniques, such as those described above by Sambrook et al. and Ausubel et al. Circular or linear expression vector constructs may be prepared to contain a functional replication system in prokaryotic or eukaryotic host cells. The replication system may be derived from, for example, ColEl, 2μ plasmid, λ, SV40, bovine papillomavirus, etc.

[0197] Recombinant expression vectors, taking into account whether the vector is DNA-based or RNA-based, may, if necessary, include regulatory sequences specific to the type of host cell into which the vector is introduced (e.g., bacteria, fungi, plants, or animals), such as transcription and translation start and termination codons. Recombinant expression vectors may also include restriction sites to facilitate cloning.

[0198] Recombinant expression vectors may contain one or more marker genes that allow for the selection of transformed or transfected host cells. These marker genes may include those related to biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., and those related to nutritional requirements for providing protrophotometry. This primarily includes complementation. Suitable marker genes for the expression vector of the present invention include, for example, neomycin / G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0199] Recombinant expression vectors may include native or non-native promoters operably ligated to nucleotide sequences encoding CARs (including their functional portions and functional variants), or to nucleotide sequences complementary to or hybridizing to CAR-encoding nucleotide sequences. The choice of promoter, e.g., strong, weak, inducible, tissue-specific, and developmentally specific, is within the scope of the art. Similarly, combining nucleotide sequences with promoters is also within the scope of the art. Promoter may be a non-viral promoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, or a promoter found in the terminal repeat sequences of mouse stem cell viruses.

[0200] Recombinant expression vectors can be designed for transient expression, stable expression, or both. Furthermore, recombinant expression vectors can be constructed for constitutive or inducible expression.

[0201] Furthermore, recombinant expression vectors can be constructed to contain suicide genes. As used herein, the term “suicide gene” refers to a gene that causes cells expressing a suicide gene to die. A suicide gene may be a gene that confers sensitivity to drugs or other agents to cells expressing it, or a gene that causes cells to die when they come into contact with or are exposed to a drug. Suicide genes are publicly known in the art (see, for example, Suicide Gene Therapy: Methods and Reviews, Springer, Caroline J. (Cancer Research UK Centre for Cancer Therapeutics at the Institute of Cancer Research, Sutton, Surrey, UK), Humana Press, 2004), and include, for example, the herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase (daminase), purine nucleoside phosphorylase, and nitroreductase.

[0202] One embodiment further provides a host cell containing any of the recombinant expression vectors described herein. As used herein, the term “host cell” means any type of cell that may contain the recombinant expression vectors of the present invention. The host cell may be a eukaryotic cell, e.g., a plant, animal, fungus, or algae, or a prokaryotic cell, e.g., a bacterium or protist. The host cell may be a cultured cell or a primary cell, i.e., it may be isolated directly from an organism such as a human. The host cell may be an adherent cell or a suspension cell, i.e., a cell that grows in a suspension. Suitable host cells are known in the art and include, for example, DH5a E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, etc. If the purpose is to amplify or replicate the recombinant expression vector, the host cell may be a prokaryotic cell, e.g., a DH5a cell. If the purpose is to produce recombinant CAR, the host cell may be a mammalian cell. The host cell may be a human cell. The host cell can be of any cell type, originate from any type of tissue, and be at any developmental stage, but the host cell may be a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). The host cell may be a T cell.

[0203] For the purposes of this specification, T cells refer to any T cells, such as cultured T cells, such as primary T cells, or T cells derived from cultured T cell lines, such as Jurkat, SupTl, etc. Alternatively, they may be T cells obtained from mammals. When obtained from mammals, T cells can be obtained from a number of sources including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells can also be enriched or purified. T cells may be human T cells. T cells may be T cells isolated from humans. T cells are CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells, e.g., Th1 and Th2 cells, CD8 +T cells (e.g., cytotoxic T cells), tumor-infiltrating cells, memory T cells, memory stem cells, i.e., Tscm, naive T cells, etc., can be any type of T cell and can be at any developmental stage. T cells are CD8 + T cells or CD4 + It could be a T cell.

[0204] In one embodiment, the CAR described herein may be used in appropriate non-T cells. Such cells are cells with immune effector function, such as NK cells and T-like cells generated from pluripotent stem cells.

[0205] A population of cells comprising at least one host cell described herein is also provided by one embodiment. The population of cells may be a heterogeneous population comprising at least one other cell, e.g., a host cell (e.g., a T cell), or a cell other than a T cell, e.g., a B cell, macrophage, neutrophil, erythrocyte, hepatocyte, endothelial cell, epithelial cell, muscle cell, brain cell, etc., in addition to a host cell comprising one of the recombinant expression vectors described herein. Alternatively, the population of cells may be a substantially homogeneous population, where the population mainly comprises host cells comprising (e.g., essentially consisting of) recombinant expression vectors. The population may also be a clonal population of cells, where all cells in the population are clones of a single host cell comprising a recombinant expression vector, and as a result all cells in the population contain that recombinant expression vector. In one embodiment of the present invention, the population of cells is a clonal population comprising host cells comprising the recombinant expression vectors described herein.

[0206] CARs (including their functional parts and variants), nucleic acids, recombinant expression vectors, host cells (including their populations), and antibodies (including their antigen-binding parts) can be isolated and / or purified. For example, a purified (or isolated) host cell preparation is a preparation in which the host cells are purer than those in their natural environment within the body. Such host cells can be produced, for example, by standard purification techniques. In some embodiments, the host cell preparation is purified so that the host cells exhibit at least about 50% of the total cell content of the preparation, for example, at least about 70%. For example, purity can be at least about 50%, and can be above about 60%, about 70%, or about 80%, or it can be about 100%.

[0207] E. Treatment Method The CARs disclosed herein are intended to be used in methods for treating or preventing disease in mammals. In this regard, one embodiment provides a method for treating or preventing cancer in a mammal, comprising the step of administering to a mammal an effective amount for treating or preventing cancer in the mammal, a CAR, a nucleic acid, a recombinant expression vector, a host cell, a population of cells, an antibody and / or its antigen-binding moiety, and / or a pharmaceutical composition.

[0208] One embodiment further includes a step of lymphodepleting the mammal prior to the step of administering the CAR disclosed herein. Examples of lymphodepletion may include, but are not limited to, non-myeloablative lymphodepletion chemotherapy, myeloablative lymphodepletion chemotherapy, and total body irradiation.

[0209] For a method of administering host cells or a population of cells, the cells may be homogeneous or autologous to the mammal. Preferably, the cells are autologous to the mammal. Yes. As used herein, "homogeneous" means any material originating from different animals of the same species as the individual into which the material is introduced. Two or more individuals are said to be homogeneous if their genes are not identical at one or more loci. In some embodiments, homogeneous material from individuals of the same species may be genetically distinct enough to interact antigenically. As used herein, "autogeneous" means any material originating from the same individual that is later reintroduced into the individual.

[0210] Mammals referred to herein may be any mammal. As used herein, the term “mammal” means any mammal, including but not limited to rodents, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. Mammals may be of Carnivora origin, including felines (cats) and canines (dogs). Mammals may be of Artiodactyla origin, including bovines (cows) and swines (pigs), or Perissodactyla origin, including equines (horses). Mammals may be of Primates, New World Cebioids or Simoids (monkeys) or Anthropoids (humans and apes). Preferably, the mammal is human.

[0211] Regarding these methods, cancers include acute lymphoblastic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder cancer), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, cancer of the anus, anal canal or anorectum, eye cancer, intrahepatic bile duct cancer, joint cancer, cancer of the neck, gallbladder or pleura, cancer of the nose, nasal cavity or middle ear, oral cancer, vulvar cancer, chronic lymphocytic leukemia, and chronic myeloid cancer. It may be any cancer, including any of the following: cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia, humoral neoplasm, liver cancer, lung cancer (e.g., non-small cell lung cancer and lung adenocarcinoma), lymphoma, mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin lymphoma, B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia (ALL) and Burkitt lymphoma, ovarian cancer, pancreatic cancer, cancer of the peritoneum, retinoplasm and mesentery, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumor, synovial sarcoma, gastric cancer, testicular cancer, thyroid cancer and ureteral cancer.

[0212] The terms “treat” and “prevent,” and words derived therefrom, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that a person skilled in the art would recognize as having a potentially beneficial or therapeutic effect. In this regard, this method may provide treatment or prevention of cancer in mammals of any amount or level.

[0213] Furthermore, the treatment or prevention provided by this method may include the treatment or prevention of one or more conditions or symptoms of a disease, such as cancer, being treated or prevented. Also, for the purposes of this specification, “prevention” may include delaying the onset of the disease, or its symptoms or conditions.

[0214] Another embodiment provides a method for detecting the presence of cancer in a mammal, comprising the steps of (a) contacting a sample comprising one or more cells of mammalian origin with a CAR, nucleic acid, recombinant expression vector, host cell, population of cells, antibody and / or its antigen-binding moiety, or pharmaceutical composition to form a complex, and (b) detecting the complex, wherein the detection of the complex indicates the presence of cancer in the mammal.

[0215] Samples can be obtained by any suitable method, such as biopsy or autopsy. A biopsy is the removal of tissue and / or cells from an individual. Such removal may involve collecting tissue and / or cells from an individual in order to perform experiments on the removed tissue and / or cells. These experiments may include experiments to determine whether an individual has and / or is suffering from a particular condition or disease. The condition or disease may be, for example, cancer.

[0216] With regard to embodiments of a method for detecting the presence of proliferative disorders in mammals, such as cancer, a sample containing mammalian cells may include whole cells, their lysates, or fractions of whole cell lysates, such as nuclear or cytoplasmic fractions, whole protein fractions, or nucleic acid fractions. If the sample contains whole cells, these cells may be any cells of a mammal, such as blood cells or cells of any organ or tissue, including endothelial cells.

[0217] The contact step can occur in vitro or in vivo for mammals. Preferably, the contact step is in vitro.

[0218] Furthermore, the detection of the complex can be carried out by many methods known in the art. For example, the CARs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, cell populations, or antibodies or their antigen-binding moieties described herein and disclosed herein can be labeled with detectable labels, such as the radioisotopes disclosed herein, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles).

[0219] Methods for testing CARs for their ability to recognize target cells and antigen specificity are well known in the field. For example, Clay et al., J.Immunol, Vol. 163: pp. 507-513 (1999) teach a method for measuring the release of cytokines (e.g., interferon-γ, granulocyte / monocyte colony stimulating factor (GM-CSF), tumor necrosis factor α (TNF-α), or interleukin-2 (IL-2)). Furthermore, CAR function can be evaluated by measuring cytotoxicity, as described by Zhao et al., J.Immunol, Vol. 174: pp. 4415-4423 (2005).

[0220] Another embodiment provides the use of the CARs, nucleic acids, recombinant expression vectors, host cells, cell populations, antibodies or their antigen-binding moieties, and / or pharmaceutical compositions of the present invention to treat or prevent proliferative disorders such as cancer in mammals. Cancer may be any of the cancers described herein.

[0221] Any method of administration, including topical and systemic administration, may be used for the disclosed therapeutic agent. For example, topical, oral, intravascular, intramuscular, intraperitoneal, intranasal, intradermal, subarachnoid, and subcutaneous administration may be used. The specific mode of administration and drug regimen will be selected by the attending clinician, taking into account the characteristics of the case (e.g., the subject, the disease, the disease state involved, and whether the treatment is prophylactic). If more than one agent or composition is administered, one or more routes of administration may be used; for example, chemotherapeutic agents may be administered orally, and antibodies or antigen-binding fragments or conjugates or compositions may be administered intravenously. Methods of administration include injections in which CARs, CAR T cells, conjugates, antibodies, antigen-binding fragments or compositions are delivered in a non-toxic, pharmaceutically acceptable carrier, such as water, saline, Ringer's solution, dextrose solution, 5% human serum albumin, solid oil, ethyl oleate, or liposomes. In some embodiments, topical administration of the disclosed compound is, for example, For example, it may be used by applying an antibody or antigen-binding fragment to an area of ​​tissue from which a tumor has been removed, or to an area suspected of having a tendency toward tumor development. In some embodiments, sustained intratumoral (or near-tumor) release of a pharmaceutical preparation containing a therapeutically effective amount of antibody or antigen-binding fragment may be beneficial. In other examples, the conjugate is applied topically as eye drops to the cornea or intravitreally to the eye.

[0222] The disclosed therapeutic agents can be formulated in unit dosage forms appropriate for individual administrations of precise dosages. Furthermore, the disclosed therapeutic agents can be administered in single doses or in a multi-dose schedule. A multi-dose schedule is one in which the main course of treatment may consist of more than one separate dose, e.g., 1 to 10 doses, followed by other doses given at subsequent time intervals as needed to maintain or enhance the effect of the composition. Treatment may involve a daily dose or multiple daily doses of the compound over a period of several days to several months or even several years. Thus, the dosing regimen is also determined at least in part based on the specific requirements of the subject being treated and depends on the judgment of the practitioner administering it.

[0223] Typical dosages of antibodies or conjugates may range from approximately 0.01 to approximately 30 mg / kg, for example, from approximately 0.1 to approximately 10 mg / kg.

[0224] In certain cases, subjects are administered a therapeutic composition comprising one or more of the following: conjugates, antibodies, compositions, CARs, CAR T cells, or further drugs, in multiple daily dosing schedules, for example, over periods of several weeks, months, or years, such as at least two consecutive days or ten consecutive days. In one example, subjects are administered the conjugates, antibodies, compositions, or further drugs over periods of at least 30 days, for example, at least two months, at least four months, at least six months, at least twelve months, at least 24 months, or at least 36 months.

[0225] In some embodiments, the disclosed methods include providing surgical, radiotherapy, and / or chemotherapeutic agents to a target (e.g., sequentially, substantially simultaneously, or concurrently) in combination with disclosed antibodies, antigen-binding fragments, conjugates, CARs, or CAR-expressing T cells. Such agents and methods of treatment and therapeutic dosages are known to those skilled in the art and can be determined by a skilled clinician. Preparation and dosing schedules for further agents may be used according to the manufacturer's instructions or may be determined experimentally by those skilled in the art. Preparation and dosing schedules for such chemotherapeutic agents are also described in Chemotherapy Service, (1992), edited by MCPerry, Williams & Wilkins, Baltimore, Md.

[0226] In some embodiments, combination therapy may involve administering a therapeutically effective dose of an additional cancer inhibitor to the subject. Non-limiting examples of additional therapeutic agents that may be used in combination therapy include microtubule-binding agents, DNA intercalators or crosslinkers, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors. These agents (administered in therapeutically effective doses) and treatments may be used alone or in combination. For example, any suitable anticancer or anti-angiogenic agent may be administered in combination with CARs, CAR-T cells, antibodies, antigen-binding fragments, or conjugates disclosed herein. Methods and therapeutic doses of such agents are known to those skilled in the art and may be determined by a skilled clinician.

[0227] Further chemotherapeutic agents include alkylating agents, such as nitrogen mustards (e.g., chlorambucil, chlormethine, cyclophosphamide, ifosfamide, and melphalan), nitrosoureas (e.g., carmustine, fotemustine, lomustine, and streptozocin), and platinum compounds (e.g., carboplatin, cisplatin, oxaliplatin). (and BBR3464), busulfan, dacarbazine, mechloretamine, procarbazine, temozolomide, thiotepa and uramustine; antimetabolites, e.g., folic acid (e.g., methotrexate, pemetrexed and larcitrexed), purines (e.g., cladribine, clopharabine, fludarabine, mercaptopurine and thioguanine), pyrimidines (e.g., capecitabine), cytarabine, fluorouracil and gemcitabine; plant alka Lloyds, e.g., podofilms (e.g., etoposide and teniposide), taxanes (e.g., docetaxel and paclitaxel), vinca (e.g., vinblastine, vincristine, vindesine and vinorelbine); cytotoxic / antitemonic antibiotics, e.g., members of the anthracycline family (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone and barurubicin), bleomycin, rifampicin, hydroxyureas and Mitomycin; topoisomerase inhibitors, e.g., topotecan and irinotecan; monoclonal antibodies, e.g., alemtuzumab, bevacizumab, cetuximab, gemtuzumab, rituximab, panitumumab, pertuzumab and trastuzumab; tumor affinity photosensitive dyes, e.g., aminolevulinic acid, methyl aminolevulinic acid, sodium porfimer and verteporfin; and other drugs, e.g., alitretinoin, altretamine, amsacrine, anagrelide, triglycerides. This list includes, but is not limited to, arsenic, asparaginase, axitinib, bexarotene, bevacizumab, bortezomib, celecoxib, denileukin difutitox, erlotinib, estramustine, gefitinib, hydroxycarbamide, imatinib, lapatinib, pazopanib, pentostatin, masopropyl alcohol, mitotane, pegaspargase, tamoxifen, sorafenib, sunitinib, vemurafenib, vandetanib, and tretinoin. The selection and therapeutic dosage of such drugs are known to those skilled in the art and can be determined by a skilled clinician.

[0228] Combination therapy can provide and prove to be synergistic; that is, the effect achieved when active ingredients are used together is greater than the sum of the effects that could result from using those compounds separately. Synergistic effects can be achieved when active ingredients are (1) co-formulated and administered or delivered simultaneously with the unit dose formulations used in combination; (2) delivered alternately or in parallel as separate formulations; or (3) in part with other regimens. When delivered alternately, synergistic effects can be achieved when the compounds are administered or delivered sequentially, for example, by different injections in separate syringes. Generally, during alternation, effective doses of each active ingredient are administered sequentially, i.e., in order; however, in combination therapy, effective doses of two or more active ingredients are administered together.

[0229] In one embodiment, an effective amount of an antibody or antigen-binding fragment or conjugate that specifically binds to one or more of the antigens disclosed herein is administered to a subject with a tumor after anticancer treatment. After a sufficient amount of time has elapsed for the administered antibody or antigen-binding fragment or conjugate to form immune complexes with the antigens expressed on each cancer cell, the immune complexes are detected. The presence (or absence) of immune complexes indicates the effectiveness of the treatment. For example, an increase in immune complexes compared to a control taken before the treatment indicates that the treatment is ineffective, while a decrease in immune complexes compared to a control taken before the treatment indicates that the treatment is effective.

[0230] F. Biopharmaceutical Compositions Provided herein are biopharmaceutical compositions or biologic compositions (hereinafter referred to as "Compositions") for use in gene therapy, immunotherapy and / or cell therapy, comprising, in a carrier (e.g., a pharmaceutically acceptable carrier), one or more of the disclosed CARs, or T cells expressing CARs, antibodies, antigen-binding fragments, conjugates, or CARs that specifically bind to one or more antigens disclosed herein. These compositions may be prepared in unit dose form for administration to a subject. Desired outcome The dosage and timing of administration to achieve the desired effect are at the discretion of the clinician performing the treatment. These compositions may be formulated for systemic (e.g., intravenous) or topical (e.g., intratumoral) administration. In one example, the disclosed CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, and conjugates, are formulated for parenteral administration, such as intravenous administration. Compositions comprising CARs, or CAR-expressing T cells, conjugates, antibodies, or antigen-binding fragments disclosed herein are used for the treatment and detection of tumors, such as neuroblastoma, not limited to these. In some examples, these compositions are useful for the treatment or detection of cancer. Compositions comprising CARs, or CAR-expressing T cells, conjugates, antibodies, or antigen-binding fragments disclosed herein are also used, for example, for the detection of pathological angiogenesis.

[0231] The compositions for administration may include a solution of CAR, or CAR-expressing T cells, conjugates, antibodies, or antigen-binding fragments dissolved in a pharmaceutically acceptable carrier such as an aqueous carrier. Various aqueous carriers, such as buffered saline, may be used. These solutions are sterile and generally free of undesirable substances. The compositions may be sterilized by conventional, well-known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusters and buffers, toxicity modifiers, and adjuvants, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. The concentrations of CAR, or CAR-expressing T cells, antibodies, or antigen-binding fragments or conjugates in these formulations can vary widely and are selected primarily on the basis of volume, viscosity, body weight, etc., according to the requirements of the selected specific mode of administration and target. Practical methods for preparing such drug formulations for use in gene therapy, immunotherapy, and / or cell therapy are known or will become apparent to those skilled in the art.

[0232] Typical compositions for intravenous administration contain approximately 0.01 to 30 mg / kg of antibody or antigen-binding fragment or conjugate per subject per day (or a corresponding dose of CAR, or a conjugate containing CAR-expressing T cells, antibody or antigen-binding fragment). Practical methods for preparing administerable compositions are known or obvious to those skilled in the art and are described in more detail in publications such as Remington's Pharmaceutical Science, 19th edition, Mack Publishing Company, Easton, PA (1995).

[0233] CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, or conjugates, may be supplied in lyophilized form and rehydrated with sterile water before administration, or they may also be supplied in sterile solutions of known concentrations. The solution of CARs, or CAR-expressing T cells, antibodies, or antigen-binding fragments or conjugates, is then added to an infusion bag containing 0.9% sodium chloride, USP, and administered in some cases at doses of 0.5–15 mg / kg body weight. Considerable experience in the administration of antibody or antigen-binding fragments and conjugate drugs is available in this field; for example, antibody drugs have been commercially available in the United States since the approval of Rituxan® in 1997. CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, and their conjugates may be administered by slow infusion rather than by intravenous injection or intravenous bolus. In one example, a higher loading dose is administered along with a subsequent maintenance dose administered at a lower level. For example, an initial loading dose of 4 mg / kg of antibody or antigen-binding fragment (or a conjugate containing the antibody or antigen-binding fragment at a corresponding dose) may be infused over a period of approximately 90 minutes, followed by a weekly maintenance dose of 2 mg / kg over 30 minutes for 4 to 8 weeks, provided that the previous dose was well tolerated.

[0234] Controlled-release parenteral formulations can be prepared as implants, oily injections, or granular systems. For a broad overview of protein delivery systems, see Banga, AJ, Thera. See peutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA (1995). Granular systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain a therapeutic protein, such as a cytotoxin or drug, as a central core. In microspheres, the therapeutic agent is dispersed throughout the particle. Particles smaller than about 1 μm, microspheres and microcapsules, are generally called nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of about 5 μm so that only nanoparticles are administered intravenously. Microparticles are typically about 100 μm in diameter and are administered subcutaneously or intramuscularly. See, for example, Kreuter, J., Colloidal Drug Delivery Systems, edited by J. Kreuter, Marcel Dekker, Inc., New York, NY, pp. 219–342 (1994); and Tice and Tabibi, Treatise on Controlled Drug Delivery, edited by A. Kydonieus, Marcel Dekker, Inc., New York, NY, pp. 315–339 (1992).

[0235] The polymers may be used for the ion-controlled release of CARs disclosed herein, or T cells expressing CARs, antibodies, or antigen-binding fragments or conjugate compositions. Various degradable and non-degradable polymer matrices used for controlled drug delivery are known in the art (Langer, Accounts Chem. Res. Vol. 26: pp. 537-542, 1993). For example, the block copolymer polaxamer 407 exists as a viscous but mobile liquid at low temperatures, but forms a semi-fluid gel at body temperature. It has been shown to be an effective vehicle for the formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. Vol. 9: pp. 425-434, 1992; and Pec et al., J.Parent. Sci. Tech. Vol. 44 (No. 2): pp. 58-65, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for the controlled release of proteins (Ijntema et al., Int. J. Pharm. Vol. 112: pp. 215-224, 1994). In yet another embodiment, liposomes are used for the controlled release and drug targeting of lipid-encapsulated drugs (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Numerous further systems for the controlled delivery of therapeutic proteins are known (see U.S. Patent Nos. 5,055,303; 5,188,837; 4,235,871; 4,501,728; 4,837,028; 4,957,735; 5,019,369; 5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697; 4,902,505; 5,506,206; 5,271,961; 5,254,342 and 5,534,496).

[0236] G. Kitt In one embodiment, kits using the CARs disclosed herein are also provided. For example, a kit for treating a tumor in a subject or a kit for producing CAR T cells expressing one or more of the CARs disclosed herein. The kit typically includes, as disclosed herein, a disclosed antibody, antigen-binding fragment, conjugate, nucleic acid molecule, CAR, or CAR-expressing T cells. More than one of the disclosed antibodies, antigen-binding fragment, conjugate, nucleic acid molecule, CAR, or CAR-expressing T cells may be included in the kit.

[0237] The kit may include a container and labels or accompanying documents on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. Containers may be formed from a variety of materials, such as glass or plastic. Typically, a container holds a composition comprising one or more of the disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or CAR-expressing T cells. In some embodiments, the container may have a sterile access port (for example, the container may be an intravenous solution bag or vial with a stopper that can be pierced by a subcutaneous needle). Labels or accompanying documents indicate that the composition is used to treat a particular condition.

[0238] Labels or accompanying documents typically further include instructions for the use of disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or CAR-expressing T cells in, for example, methods for treating or preventing tumors or methods for producing CAR T cells. Accompanying documents typically include instructions customarily included in the market packaging of a therapeutic product, including information on indications, usage, dosage, administration, contraindications, and / or warnings relating to the use of the therapeutic product. Educational materials may be written in electronic form (e.g., computer diskettes or compact disks) or visual (e.g., video files). Kits may also include further components to facilitate the specific application for which the kit is designed. Thus, for example, a kit may further include means for detecting labels (e.g., enzyme substrates for enzyme labeling, filter sets for detecting fluorescent labels, appropriate secondary labels such as secondary antibodies, etc.). Kits may further include buffers and other reagents conventionally used for carrying out a particular method. Such kits and suitable contents are well known to those skilled in the art. [Examples]

[0239] The present invention is further illustrated by the following embodiments, which should not be construed as limiting the scope of the invention. On the contrary, it will be readily apparent that various other embodiments, modifications, and equivalents can be conceived after reading this specification without departing from the spirit of the invention and / or the appended claims, and these may come to the mind of those skilled in the art.

[0240] Example 1 Tandem CD19 / CD20 CAR lentiviral vectors drive on-target and off-target antigen modulation in leukemia cell lines. Adoptive immunotherapy for cancer using genetically engineered autologous human T cells is currently being evaluated at many sites. A common method for creating cell populations for adoptive immunotherapy is to isolate T cells from a patient by apheresis, as outlined in [1], and to transduce these cells ex vivo with a retroviral or lentiviral vector that is integrated into the host genome and expresses a chimeric antigen receptor (CAR). Chimeric antigen receptors are created by ligating functional sequence domains from various subunits of immunologically active proteins. For example, an scFv domain created from the VH and VL domains of an anti-CD19 or anti-CD20 antibody can be ligated to a transmembrane sequence derived from CD28 or CD8, and then to an intracellular signaling domain derived from the CD3-zeta chain and CD28 or CD137 [2, 3]. Thus, a CAR provides both a binding domain and a ligated signaling domain derived from scFv within a single transmembrane protein, which allows the vector to activate transduced T cells. Thus, this transduced T cell population (CAR-T) is indirectly organized by activated cell lysis and the production of cytokines such as interferon-gamma (IFNγ), interleukin-2 (IL-2), and tumor necrosis factor-alpha (TNFα). Regarding disruption by immune effector mechanisms, cells possessing congeneral antigens can be functionally targeted. Adoptive immunotherapy with chimeric antigen receptor-modified T cells that specifically target CD19 has demonstrated efficacy against pediatric pre-B ALL [4, 5]. The efficacy of CAR-modified T cells in adult hematological malignancies is more heterogeneous.

[0241] Experience with anti-CD19 CAR-T therapy in three CLL patients at the University of Pennsylvania appears to show a universally positive response, and the NCI's Surgery Branch reported a mixture of partial responses, stable disease, and one complete response in a diverse collection of eight patients with adult B-cell malignancies [6, 7]. Thus, anti-CD19 CARs are not unbiasedly effective and can benefit from further enhancement of their antitumor targeting potential. The Thomas-Tikhonenko lab brilliantly described the escape mechanisms used by B-ALL during anti-CD19 CAR-T therapy, which include alternative splicing, frameshift mutations, and missense mutations of CD19 [8]. One means of broadening the targeting range of CAR-T products and targeting malignancies with greater efficacy is to include two binding domains within a single CAR structure. Tandem CD19 and CD20-expressing malignancies include chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), mantle cell lymphoma (MCL), prolymphocytic leukemia (PLL), and splenic lymphoma with choriolymphocytes (SLVL) [9]. A single CAR vector targeting both antigens could target a broader variety of hematological malignancies and potentially target them more effectively.

[0242] In this example, the increased efficacy of the tandem CAR construct was modeled by testing the expression of both target antigens on the CD19+CD20+ Raji cell line during co-culture with a series of single-specific and tandem-specific CAR-T constructs. The rapid CD19 target antigen downregulation mechanism that exists was demonstrated in this leukemia cell line. Surprisingly, target antigen downregulation included non-targeted B cell receptor molecules, including CD22, which were not targeted by any of the vector constructs. The more rapid decrease in the number of target cells by our tandem targeting vector indicates the presence of stronger anti-leukemia immune pressure. In particular, the kinetics of escape from immune pressure by the leukemia cell line when a single-specific CAR was used, in contrast to the mechanism of target antigen regulation or loss that depends on selection for genetic escape variants, indicates that antigenic modulation is an existing property of leukemia cells.

[0243] Materials and Methods Cell Lines (PBMC and Targets) Unless otherwise noted, all cell lines and reagents were purchased from the American Tissue Culture Collection (ATCC, Manassas, VA). The Burkitt lymphoma cell line Raji, acute lymphoblastic leukemia cell lines REH and NALM-6 (ACC-128 DSMZ, Leibniz Institute DSMZ, Braunschweig, Germany), and the chronic myeloid leukemia line K562 were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT) and 2 mM L-Glutamax (Thermo Fisher Scientific, Grand Island, NY). The human embryonic kidney cell line 293T was grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated FBS.

[0244] A single cell clone of a luciferase-expressing cell line was generated by stably transducing a lentiviral vector encoding firefly luciferase (Lentigen Technology, Inc., Gaithersburg, MD) into a wild-type tumor strain, followed by cloning and selection of luciferase-positive clones. NALM6 cell line As previously reported

[10] for the Raji cell line , Raji clones stably expressing firefly luciferase were engrafted into NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ, The Jackson Laboratory Sacramento, CA), and Raji-luc tumor cells engrafted from mouse spleen were isolated by either positive selection (CD19 microbeads, human, Miltenyi Biotec, Bergisch Gladbach, Germany) or negative selection (mouse cell depletion kit, Miltenyi Biotec), grown in culture, and recloned to facilitate selection of clones with high firefly luciferase expression, generating a Raji-luc strain adapted to mice.

[0245] Whole blood was collected from healthy volunteers with donor consent forms at the Oklahoma Blood Institute (OBI). Treated meninges were purchased from OBI (Oklahoma City, OK). CD4-positive and CD8-positive human T cells were purified from meninges by positive selection using a 1:1 mixture of CD4 microbeads and CD8 microbeads (Miltenyi Biotec) according to the manufacturer's protocol.

[0246] Generation of chimeric antigen receptor (CAR) expression vectors The CAR antigen-binding domain, scFv, sequences were derived from mouse hybridoma FMC-63 (FMC-63: AA 1-267, GenBank ID: HM852952.1) for CD19 and Leu-16

[15] (full sequences of VL and VH) for CD20, as described in the Additional File section.

[0247] CAR19A, CAR19B, and CAR20A were generated by ligating the scFv of each antibody in frame to the CD8 hinge and transmembrane domain (AA 123-191, reference sequence ID NP_001759.3), the 4-1BB (CD137, AA 214-255, UniProt sequence ID Q07011) transactivation domain, and the CD3 zeta signaling domain (CD247, AA 52-163, reference sequence ID: NP_000725.1.). Constructs 19A and 19B were identical except for the flexible linker connecting the variable H and L chains of the scFv-binding domain; 19A used the Whitlow linker

[11] and 19B used the (GGGGS)3 (SEQ ID NO: 13) linker. Tandem-targeted constructs, CAR1920 and CAR2019, were generated in a similar manner. The scFv regions 19A and 20A were sequentially ligated with the flexible interchain linker (GGGGS) 5 (SEQ ID NO: 14), and then ligated to the CD8, 4-1BB, and CD3 zeta domains. As reported in

[12] , a leader sequence from the human granulocyte-macrophage colony-stimulating factor receptor alpha subunit was included in all constructs. The CAR construct sequences were codon-optimized (DNA2.0, Newark, CA) and cloned into a third-generation lentiviral plasmid skeleton (Lentigen Technology Inc., Gaithersburg, MD) under the control of the human EF-1α promoter. As previously reported

[13] , transient transfection of HEK293T cells generated supernatant containing the lentiviral vector (LV). The collected and pelleted supernatant containing the lentiviral vector was stored at -80°C.

[0248] Primary T cell transduction Selected CD4+ and CD8+ human primary T cells derived from normal donors are introduced at a density of 0.3-2x10⁻¹⁶. 6Cells were cultured at a concentration of 1 / ml in TexMACS medium (serum-free) supplemented with 40 IU / ml IL-2, activated with CD3 / CD28MACS® GMP TransAct® reagent (Miltenyi Biotec), transduced overnight on day 3 with a lentiviral vector encoding the CAR construct in the presence of 10 μg / ml protamine sulfate (Sigma-Aldrich, St. Louis, MO), and the medium was changed on day 4. On the fifth day, the cultures were transferred to TexMACS medium supplemented with 200 IU / ml IL-2 and allowed to grow until harvesting on the 10th to 13th day.

[0249] Immunoeffector assays (CTLs and cytokines) To determine cell-mediated cytotoxicity (CTL assay), 5,000 target cells stably transduced with firefly luciferase were combined with CAR T cells in various effector-to-target ratios and incubated overnight. SteadyGlo reagent (Promega, Madison WI) was added to each well, and the resulting luminescence was analyzed using an EnSpire plate reader (Perkin Elmer, Shelton, Connecticut) and recorded as counts per second (sample CPS). The assay range was determined using wells containing only targets (maximum CPS) and wells containing only targets with 1% Tween-20 added (minimum CPS). The percentage of specific lysis was calculated as (1 - (sample CPS - minimum CPS) / (maximum CPS - minimum CPS)). For cytokine release assays, effector and target cells were mixed in a 10:1 ratio and incubated overnight. The supernatant collected for secreted cytokines was analyzed using the MACSplex Human 12 Cytokine Bead Array Kit (Miltenyi Biotec) according to the manufacturer's instructions. Strong induction of IFNγ, TNFα, IL-2, and GM-CSF was detected in the CAR T-treated group. The following cytokines were not detected: IL-4, IL-5, IL-6, IL-12p70, IL-17A, IL-10, and IFNα. IL-9 was detected at low levels in some samples and was not reported. All samples were duplicated or tripled. Unless otherwise noted, all data presented are representative of three or more independent experiments.

[0250] Western blot Two million CAR T cells were washed twice with cold PBS (Lonza, Walkersville, MD), and then lysed in 100 μl of cold RIPA buffer (Sigma-Aldrich, St. Louis, MO) containing a mixture of protease and phosphatase inhibitors (Thermo-Fisher Scientific, Grand Island, NY). The lysates were incubated at 4°C for 20 minutes, pelletized at 13000 RPM for 10 minutes in a benchtop centrifuge at 4°C, the supernatant was collected, and frozen at -20°C. The sample was denatured in reducing loading buffer (Invitrogen) at 70°C for 10 minutes, and then denatured in MOPS buffer (Thermo-Fisher Scientific, Grand Under reducing conditions in Island, NY, the cells were developed on a 4%–12% gradient SDS-PAGE gel according to the manufacturer's protocol. Proteins were transferred to a 0.45 micron nitrocellulose transfer membrane (BioRad, Hercules, CA) and investigated with antibodies against all CD3 zeta (clone ab40804, Abcam, Cambridge, MA). Bands were developed using the Vectastain ABC-AMP reagent kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's protocol, and the bands were visualized and quantified using the Odyssey imaging system and Image Studio lite software (LI-COR, Lincoln, Nebraska). Western blotting for CD19 was also performed on Raji tumor cells. In short, after overnight incubation of Raji cells with CAR T cells, CD3-positive cells were depleted using an LD column with CD3 magnetic beads (Miltenyi Biotec) according to the manufacturer's protocol, and the recovered Raji cells were processed as described above. Specific bands were detected using antibodies against the CD19 C-terminus (sc-69735, Santa Cruz, CA) and beta-actin (8457, Cell Signaling Technology, Danvers, MA). Band intensity was quantified using Image Studio software (LI-COR, Lincoln, Nebraska). The relative band intensities of full-length CD19 and Δ-exon 2 CD19 isotype were calculated as signal CD19 / signal β-actin.

[0251] Flow cytometry analysis Unless otherwise noted, all cell staining reagents for flow cytometry were from Miltenyi Biotec. One million CAR T transduced cells were harvested from the culture, washed twice with cold staining buffer (AutoMACS solution containing 0.5% bovine serum albumin), and pelleted at 350xg at 4°C for 5 minutes.

[0252] CAR surface expression on transduced T cells was detected by staining with protein L-biotin conjugate (stock 1 mg / ml, 1:1000 dilution, GenScript, Piscataway, NJ) at 4°C for 30 minutes, followed by two washes, and then staining with streptavidin-PE conjugate (stock: 1.0 ml, 1:200 dilution, Jackson ImmunoResearch Laboratories, West Grove, PA) at 4°C for 30 minutes. Untransduced cells and transduced cells stained with streptavidin-PE alone were used as negative controls. Anti-CD4 antibody was used to determine the CD4 vs. CD8 ratio of the CAR T-positive population and was added during the second incubation step. Dead cells were excluded by 7AAD staining (BD Biosciences, San Jose, CA). Cells were washed twice, resuspended in 200 μl of staining buffer, and then quantitatively analyzed by flow cytometry.

[0253] Fc-tagged CD19 peptide (described below, 1 μg / ml) was incubated with cells at 4°C for 15 minutes, followed by incubation with the anti-Fc-AF647F(ab')2 fragment (Jackson). Specific CAR T staining was performed by incubation with Immuno Research (1:200) at 4°C for 15 minutes, and detection was performed in APC channels. Biotinylated CD20 peptide (Bachem, Torrance, CA) and streptavidin PE (both 1 μg / ml) were simultaneously added to cells and incubated in a dark room at room temperature for 10 minutes. Flow cytometry analysis was performed using a MACSQuant® 10 analyzer (Miltenyi Biotec). Target tumor lines and luciferase-positive subclones were characterized using CD19-FITC, CD20 VioBlue, and CD22-APC antibodies. Dead cells were excluded from the analysis by 7AAD staining (BD Biosciences, San Jose, CA).

[0254] Generation of Fc-tagged CD19 peptide To produce recombinant human CD19 peptide, the extracellular domain (amino acids 20-291, Uniprot P15391) was fused to human IgG1 Fc (CD19-Fc), and this was expressed in a CMV-driven mammalian expression vector by transduction in HEK293 cells. Transfected cells were cultured in DMEM and 5% FBS, and after incubation for 10 and 20 days in HYPERFlask® cell culture vessels (Corning), the cell culture supernatant containing CD19-Fc was collected. After centrifugation to remove cell debris and 0.22 μm sterile filtration, CD19-Fc was purified by protein A chromatography (HiTrap MabSelect, GE Healthcare) and stored in PBS at 4°C. Purity exceeded 97%, as determined by SDS-PAGE and Coomassie blue staining. The identity of CD19-Fc was confirmed by intact mass spectrometry and peptide mass fingerprinting after trypsin digestion (Miltenyi Biotec, Bergisch Gladbach, Germany).

[0255] In vivo analysis of CAR-T activity All animal research is conducted by Jackson Laboratory Animal Care. Approved by the and Use Committee (Sacramento, CA). NSG(NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ) 500,000 mouse-compatible Raji-luc cells were injected into the tail vein of mice. Six days after Raji-luc injection, tumor engraftment was measured by ip injection of 150 mg / kg luciferin and 40-second imaging using a Xenogen IVIS-200 instrument (Caliper Biosciences, now Perkin Elmer, Shelton, Connecticut) 10 minutes later. Images were analyzed using Living Image, version 4.1 software (Perkin Elmer), and the bioluminescence signal bundle for each mouse was expressed as radiance average (photons / sec / cm2 / steradians). On day 7, CAR T cells were administered to mice by tail vein injection. Imaging was performed on days 4, 6, 11, 14, 18, 25, 32, and 46 after injection to establish the dynamics of tumor growth and eradication by CAR T cells.

[0256] For high tumor burden in vivo studies, NSG mice were intravenously injected with Raji-luciferase cells on day 0. On day 11, mice were evenly distributed to the study group based on tumor burden. Then, on day 12 of the study, tandem CAR 2019 CAR T cell preparations, or two single CAR T combination preparations mixed with equal CART+ cell counts (19A+20A, 19B+20A), were intravenously administered. All CAR T preparations totaled 5 x 10⁶ cells. 6 CAR T cells / mice were tested. Non-transduced T cells (NTs) from the same donor and tumor-only groups served as controls. Tumor growth was assessed based on mean whole-body radiance in mice on days 18 and 25 of the study. N=6 / group.

[0257] In vitro analysis of leukemia immune evasion To analyze Raji tumor escape variants, CAR T and Raji cells were mixed in vitro at an effector-to-target ratio of 1:1. After overnight incubation of the co-cultures and on day 4, the surface expression of CD19, CD20, and CD22 was determined for viable Raji cells by flow cytometry. The cultures were harvested, washed, and stained with antibodies specific to CD3-PE, CD19-FITC, CD20-VioBlue, CD22-APC (Miltenyi Biotec), and 7AAD. To facilitate the analysis of viable Raji cells in each Raji:T cell co-culture, we gated the cell population to CD3-negative and 7AAD-negative (i.e., viable Raji) cells, and then analyzed this population for residual surface expression of CD19, CD20, and CD22.

[0258] In the Transwell co-culture assay, each was 5 x 10 5 CAR T and Raji cells were seeded in 1 ml of TexMACS medium in the lower compartment of a 24-transwell plate (Costar, REF 3470, 0.4 μM pore membrane). 2.5 x 10 5 Raji cells were seeded in the upper transwell compartment in TexMACS medium in the absence of T cells. After overnight incubation, cells from the upper and lower compartments were analyzed by flow cytometry as described above. The expression percentages of CD19, CD20, and CD22 on Raji cells were measured for each group. The data shown represent the mean + SD from three independent experiments from three different donors. Statistical analysis was performed against Raji alone using one-way ANOVA and Dunnett's multiple comparison test (*p<0.01).

[0259] statistical analysis Statistical analysis was performed using GraphPad Prism 7.01 software. In the in vitro cell death assay, group mean values ​​for repeated decisions were compared using two-way analysis of variance (ANOVA), followed by Dunnett's multiple comparison test, to identify differences between individual treatment groups and non-transduction controls. In the first in vivo study, IVIS radiance data on day 25 of the experiment, the last measurement day when all groups had surviving mice, were analyzed against the untreated group using two-way ANOVA, followed by Dunnett's multiple comparison test. Raji and NALM-6 cells after overnight and 4-day co-culture with CAR T cells were then analyzed. CD19, CD20, and CD22 expression were analyzed against NT (non-transduced T cells from the same donor) controls using one-way ANOVA followed by Dunnett's multiple comparison test. The CD20 / CD19 binding ratio in tandem CARs was analyzed using Student's t-test.

[0260] result To study the efficacy of tandem CD19 and CD20-targeted CARs, sequences encoding antigen-binding domains from mouse antibodies FMC63 and Leu16 were ligated with the (GGGGS)5 (SEQ ID NO: 14) sequence. For all constructs in this study, the ligation, transmembrane, and signaling domains were identical and, as previously reported

[12] (Figure 1A), encode human CD8-derived hinge and transmembrane domains, CD137 signaling domain, and CD3-zeta-derived signaling domain, respectively.

[11] As first published (GenBank ID HM852952.1, AA 130-148, known as the Whitlow linker),

[11] the heavy and light chains of FMC63 were both ligated to the scFv structure, while the heavy and light chains of Leu16 were ligated by the (GGGGS)3 (SEQ ID NO: 13) sequence. Furthermore, tandem CARs were created by linking heavy and light chain sequences derived from FMC63 and Leu16 with a polyGGGS (SEQ ID NO: 15) sequence in a single transcript. Unlike their single-specific counterparts, tandem CARs can induce activation of CAR-expressing T cells upon encountering target cells expressing either CD19 or CD20.

[0261] In 40 IU / ml IL-2, primary human T cells were activated by anti-CD3 / CD28 nanomatrix, and 3 days later, transduced with a lentiviral vector encoding CAR, and then grown in 200 IU / ml IL-2 maintained throughout the culture period. Flow cytometry using biotinylated protein L, followed by staining with streptavidin-PE, was used to measure CAR expression on the surface of transduced T cells (Figure 2A). CAR expression on the surface of transduced T cells ranged from 61% to 93%. To confirm that both scFv binding domains were intact, we also co-incubated CAR T cells with CD19-Fc fusion protein or biotinylated CD20 peptide (see methods). In the tandem specific construct, both domains retained the ability to bind to the target antigen. For CAR2019, protein L staining gave 89% expression, CD19-Fc expression was 80%, and CD20 peptide expression was 85%. For CAR 1920, protein L staining gave 85% CAR expression, CD19-Fc staining yielded 80%, and CD20 peptide was 68%. This observation was reproduced in CAR T cells generated from three separate donors.

[0262] To compare the differences in antigen binding, the CAR T CD20 / CD19 binding ratio was calculated. This ratio was defined as the percentage of tandem CAR T cells positively stained with CD20 soluble peptide divided by the percentage of CAR T cells positively stained with CD19-Fc peptide in the same sample. The mean values of the CD20 / CD19 binding ratio for T cell transduction from three separate donors were 0.74 ± 0.06 for CAR 1920 and 1.05 ± 0.02 for CAR 2019 (Figure 2B). Thus, in the CAR T construct 2019, the binding of soluble CD19 and CD20 peptides was similar and comparable to protein L binding, but CD20 binding in the CAR 1920 construct may be partially sterically hindered.

[0263] To confirm that the flow profile was due to a single, larger transcript, the molecular weight of the CAR protein was confirmed by Western blotting under reducing conditions (data not shown). Specific bands of the predicted size were detected for single (54 kDa) and tandem (81 kDa) CARs. To understand the relative expression levels, endogenous Band intensities were compared by calculating the ratio of CAR-associated zeta chain signals to CD3 zeta. When the CAR expression level in the CAR 19B single-chain vector was normalized to 1 (arbitrary unit), the expressions for 19A, 20A, 1920, and 2019 were 1.8, 1.5, 0.9, and 1.2, respectively. Therefore, the expression levels of the tandem CAR fall within the range of variability observed in single-CAR expression vectors. Since it is possible to create LV expressing the tandem CAR, and the expression of full-length CAR proteins on the surface of T cells expressing two functional scFvs has now been demonstrated, the anti-leukemic activity of the tandem CAR construct was then evaluated in an in vitro assay.

[0264] Human primary T cells were transduced with LV encoding CAR constructs (see 19A, 19B, 20A, 1920, 2019, Methods) and then incubated for 18 hours with Raji, NALM-6, REH, K562, or 293 T cell lines stably transduced with firefly luciferase, followed by in vitro cell death assays based on luminescence. All leukemia lines tested expressed CD19 on their surface, except for negative controls, K562, and 293T. CD20 expression varied among tumor lines. The Raji line was CD20-positive, while REH was CD20-negative, as were the control lines K562 and 293T. The NALM-6 line had weak but detectable CD20 expression. As further controls, we created K562 strains expressing CD19 (K562-19+) or K562 strains expressing CD20 (K562-20+).

[0265] K562-19+ was lysed by CAR 19A and 18B constructs, and tandem CAR constructs 1920 and 2019, but not by the single 20A CAR (Figure 3A). K562-CD20+ was lysed by all CART constructs except the single CAR19 construct, demonstrating targeted antigen-limited death. Similar results were observed in other leukemia cell lines tested. CD19-targeting single and tandem CAR T constructs lysed Raji, NALM-6, and REH, but not 293T (Figure 3B) or K562 (Figure 3A). In particular, the 20A single-targeting CAR construct did not exhibit specific death activity against CD20-negative REH strains, but demonstrated death of NALM-6 with low but detectable levels of CD20 surface expression. Furthermore, flow cytometry revealed that tandem CAR 1920 exhibited lower binding to the CD20 peptide than to CD19-Fc, and while it showed lower cytotoxicity against K562-19+ and K562-20+, it did not exhibit lower cytotoxicity against CD19+CD20-REH. This suggests that 1920 tandem CAR may be inferior to 2019 tandem CAR for some tumor targets.

[0266] Next, cytokine secretion by tumor-activated CAR T cells was investigated. After overnight incubation with CAR-T effectors and Raji cell lines in a 10:1 ratio, the supernatant of the co-culture medium was collected and analyzed using a MACSPlex cytokine-specific bead array, which allows for the simultaneous detection of 12 different cytokines (Figure 4). All CAR T constructs, when co-cultured with Raji cells, showed increased cytokine levels for IFN-gamma, TNF-alpha, IL-2, and GM-CSF compared to untransduced T cells, although cytokine levels for the negative control groups NT and GFP were undetectable. In particular, 20A CAR consistently produced the highest levels of IL-2, IFN-gamma, TNF-alpha, and GM-CSF. This was not due to preferential proliferation of the CD4+ T cell population, as the observed minor changes in the CD4 / CD8 ratio were inconsistent (see Supplementary Table 1 below).

[0267] [Table 1]

[0268] For Supplementary Table 1, the CD4 / CD8 ratio in CAR T cell preparations from three different donors was determined by flow cytometry. + and CD8 + T cells were co-purified from the pia mater, activated with TransAct CD3 CD28 reagent in the presence of IL-2, transduced with LV as described in Materials and Methods, and assayed for immune function. CD4 / CD8 composition was then assayed on day 10 of culture. The CD4 / CD8 cell ratio for each of the three donors is shown in the vertical columns, and the CAR T group is shown in the horizontal columns.

[0269] Both tandem constructs, CAR 1920 and CAR 2019, produced similar-sized and significantly stronger cytokine induction compared to non-transduced controls in the presence of tumor targets. The cytokines IFN-α, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, and IL-17A, which were also investigated in the MACSPlex cytokine array, were not detected at significant levels in our samples.

[0270] To evaluate the in vivo activity of CAR-modified human T cells, on day 0, 0.5 x 10⁶ cells were used. 6 Raji-Luc cells were intravenously injected into NSG mice. On day 6, the presence of engrafted leukemia was confirmed by imaging, and the mice were randomized to the experimental group. On day 7, 10x10 cells transduced with a CAR construct were introduced. 6Human T cells were intravenously injected, and disease was monitored by IVIS imaging. Representative images show disease progression or regression in each group (Figure 5A). In animals treated with 20A single CAR or tandem CAR 1920 or 2019, tumor burden peaked on day 11 and decreased to below pre-treatment levels by day 18. Tumor clearance was comparatively slower in the single CAR 19 group. Interestingly, CAR 19B, in which the ScFv heavy and light chains were linked by a Gly-Ser linker, functioned better than CAR 19A with a Whitlow linker (Figure 5B). Overall, both the CAR 1920 and CAR 2019 tandem CAR constructs demonstrated superior in vivo activity compared to the single CAR 19 in this model system. Furthermore, while this data highlights the importance of cytotoxicity, cytokine production, and in vivo activity in the NSG mouse line, the data must be interpreted collectively to understand the biology of transduced T cell populations.

[0271] Having established a series of single and double-specific CARs, we further utilized their activity. In particular, in light of recent explanations of leukemia escape mechanisms, to explore the relative efficacy of tandem CAR constructs in vitro, we used the Raji cell line (CD19, C). We explored the ability of leukemia cell lines to evade strong CAR-T effector activity using CAR-T (expressing D20 and CD22) and NALM-6 cell lines (expressing CD19, CD22, and low levels of CD20) (Figure 6A). To model leukemia cell escape under CAR-T immunosuppression, CAR-T cells were co-incubated with leukemia cells at a low effector-to-target (E:T) ratio of 1:1. At higher E:T ratios, all leukemia cells were eliminated. After short-term (overnight) or longer-term (4 days) co-culture, the expression of three B-cell markers on the surface of the leukemia cell lines was analyzed by flow cytometry (Figures 6B, 6C). CD22 was not specifically targeted by our CAR-T cells, and its loss was interesting because it may indicate the initiation of a more holistic immune escape program that generates multi-antigen loss variants.

[0272] When Raji cells were co-cultured overnight with CAR19-T cells, CD19 antigen was rapidly downregulated from the cell surface (Figures 6B, 6C). CD20 remained relatively constant, and for CD22, small populations of antigen-negative cells began to emerge. When the tandem construct 2019 was co-cultured with Raji cells, CD19 was downregulated, CD20 was moderately downregulated, and nearly half of CD22 expression was lost. When examined on day 4, all B cell antigens were significantly reduced (Figure 6C). Targeting CD19 alone (constructs 19A and 19B) had the greatest effect on CD19 expression. The tandem construct 2019 had a greater effect than the alternative 1920 construct, indicating that stronger immunosuppression was mediated by 2019. Regardless of whether it was short-term or long-term co-culture, CD20 was not lost much from cells that survived immunosuppression. Compared to the single CD20-targeted construct 20A, the tandem construct 2019 produced more CD19 epitope loss, but appeared to exert similar combined pressure against other targeted antigens CD20 and untargeted antigen CD22.

[0273] When the NALM-6 leukemia cell line was used as a target, there was little effect on CD20 expression, except when CD20-targeted CAR 20A was utilized (Figure 6C). This again may reflect a greater resistance to altering CD20 expression on the surface of the leukemia cell line. In overnight co-cultures, CD19 expression was again highly plastic, and antigen loss on the viable cell population was readily demonstrated. On day 4, there were very few NALM-6 cells available for study. This appears to be due to NALM-6's much greater sensitivity to both indirect cytokine and direct cell-mediated cell death effects. In particular, no cells positive only for CD22 were detected, indicating the overall sensitivity of this line to the immune effector mechanism, i.e., non-selective cell loss.

[0274] Regulation of tumor surface molecule expression by pro-inflammatory cytokines in chronic lymphocytic leukemia has been previously reported

[30] . As shown in Figure 4, CAR T cells produce high levels of pro-inflammatory cytokines when exposed to tumor cells, so it was necessary to determine whether the downregulation of CD19, CD22, or CD20 on Raji cells after co-incubation with CAR T cells was a direct effect of CAR T-tumor cell contact or due to soluble factors released into the culture medium by CAR T cells. Raji and CAR T cells were mixed in an effector-to-target (E:T) ratio of 1:1 in the lower part of a Transwell plate. In the upper compartment, we placed only Raji cells. After overnight incubation, cells were harvested from the Transwell compartment and from the lower part of the wells, and cell surface expression of CD19, CD20, and CD22 on live Raji cells was analyzed by flow cytometry (Figure 7). Consistent with our previous results, Raji cells co-incubated with CAR T cells demonstrated a dramatic reduction in CD19 surface expression by CAR 19A and 19B, and a more moderate but significant reduction in CD20 and CD22. In contrast, Raji cells recovered from the upper transwell compartment preserved full expression of CD19, CD20, and CD22, and negative control group: NT, GFP. It was indistinguishable from Raji alone. Therefore, the downregulation of CD19 Raji expression by CAR T cells 19A and 19B is a direct effect of tumor:CAR T contact.

[0275] It has recently been reported that CD19-possessing tumors can avoid CAR19 elimination through the preferential expression of a splice variant lacking exon 2, which removes a portion of the extracellular domain of CD19 containing an FMC63-binding epitope [8]. To explore CD19 plasticity on Raji leukemia cell lines, co-culture experiments were performed. Raji cells were co-cultured overnight with CAR T cells and then analyzed by flow cytometry to assay CD19 expression. Significant reductions in CD19 MFI were measured in both the 19A and 19B experimental groups (Figure 8A). Immunomagnetic beads were then used to deplete the co-culture, and purified Raji cells were analyzed by Western blotting (Figure 8B, C).

[0276] To assess whether exon 2 splicing contributed to the decrease in full-length CAR 19 protein expression, we performed Western blot analysis as previously reported [8]. Raji cells expressed both full-length and Δ2 splice CD19 isotypes in standard culture (Raji alone) (Figure 8B). To quantify the relationship between full-length CD19 expression and its Δ2 splice variant expression, we analyzed the intensity of Western blot bands (Figure 8C). The decrease in full-length CD19 isotype expression occurred in the groups treated with CAR19 constructs 19A and 19B, consistent with our flow cytometry results. Conversely, Δ2 splice CD19 expression remained relatively stable regardless of CAR T treatment (Figure 8C). When CAR T was removed from the co-culture and purified Raji was incubated alone, all treatment groups reexpressed the target antigens CD19, CD20, and CD22 at untreated levels (≥98%, not shown) by day 4. This demonstrates highly dynamic regulation of cell surface protein expression.

[0277] To further explore the differences in tumor clearance between single CAR and tandem CAR, we conducted further studies in which Raji tumors were allowed to grow until day 12 instead of day 7 before CAR T administration (Figures 9A and 9B). Tumor-bearing mice on day 12 were administered transduction T cell products, and 5 x 10⁶ cells were administered per mouse for single CAR or tandem 2019 CAR. 6 These were CAR T+ cells. Two T cell products containing separate transductions of CD19A+20A or CD19B+20A CARs were co-administered simultaneously. A total of 2.5 x 10⁶ cells were administered for each construct. 6 CAR T cells were obtained (5 x 10 in total) 6 Tandem CAR2019 achieved a strong reduction in tumor burden without mortality, but the combined treatments 19A+20A and 19B+20A were effective but highly toxic, with only 2 out of 6 mice surviving to day 25 in each group. In the single CAR 19A and 19B groups, tumor burden remained relatively high, with only 5 out of 6 mice surviving to day 25 in each group. In the single 20A group, tumors were efficiently removed, but only 2 out of 6 mice survived. Surprisingly, the study highlighted the toxicity of CAR administration, which was observed in the single 20A group, or when 20A was administered with 19A or 19B They were largest when mixed with CAR T cells.

[0278] Consideration The ligation of anti-CD19 and anti-Her2 domains to a single tandem CAR (referred to by the authors as TanCAR) has put into practice the theoretical proposal by many researchers that it is possible to generate a bispecific chimeric antigen receptor (CAR)

[14] . In an attempt to create improved CARs for adoptive immunotherapy of hematological malignancies, anti-CD19 and anti-CD20-based binding motifs were ligated to a single transmembrane glycoprotein to create a series of tandem CARs. As shown in Figures 1A and 1B, these CAR constructs can be activated via binding to either CD19 or CD20 tumor molecules and are effective both in vitro and in vivo against model leukemia cell lines. Animal models did not demonstrate a clear advantage or establish a preferred order of CD19 and CD20 scFv within the CAR structure itself. Nevertheless, the 2019 CAR construct showed better binding of CD20 peptide staining reagents by flow cytometry and improved killing of some tumor cell lines in vitro (Figures 2A, 2B, 3A, and 3B). Furthermore, immunostimulus analysis in overnight and 4-day co-culture experiments showed that the 2019 CAR may exert stronger immunostimulus against target leukemia cell lines (Figures 6A–6C and 8A–8C).

[0279] Regarding the polypeptide sequences of the CAR protein, the effectiveness of linking both the VH and VL domains with a polyglycine linker (GGGGS; SEQ ID NO: 15) and linking two independent scFv domains in a tandem CAR structure was demonstrated. This is important because the lengths of the VH and VL linking sequences, and their amino acid compositions, have been shown to govern diabody formation and proper folding of the scFv domains

[15] . Our data also support the findings of Zah et al.

[16] , who were able to link independent CD19 and CD20 scFv domains using the (GGGGS; SEQ ID NO: 15) sequence. As demonstrated by Western blot analysis, the intensity of the native CD3-zeta chain band indicates that the transcription or translation of TCR-related transcripts such as the zeta chain is not overwhelmed or substituted by CAR transcription using the EF-1-alpha driven CAR payload in the LV system (Figure 3 in Schneider et al., Journal for ImmunoTherapy of Cancer (2017), Vol. 5:42, is incorporated herein by reference in its entirety).

[0280] Using a set of unique tools generated by the expression of recombinant CD19 and CD20 fragments, the ability of scFv encoded by CAR and tandem CAR vectors to bind to target proteins was demonstrated (Figures 2A and 2B). Previous reports have used protein L, which binds to the copper light chain sequence, to stain cell surface CAR expression

[12] . For both CD19-specific FMC63-based scFv CARs (constructs 19A, 19B), protein L bound well, although the original Whitlow linker had a brighter MFI. This was also true for staining with recombinant CD19 fragments, although the difference was not as large. Protein L and recombinant CD20 peptide gave very similar results for the Leu16-based anti-CD20 CAR (construct 20A). For tandem CARs, protein L staining was equally strong. Anti-CD19 scFv binding of the target peptide was essentially equivalent in tandem and single CARs, but the signal of CD20-biotin-based staining was reduced in construct 1920 compared to 2019 (Figure 2B), which may indicate steric hindrance to binding to CD20 in this construct. In CD20-negative leukemia cell lines, we observed somewhat lower lysis levels with CAR 1920, in contrast to CAR 2019 tandem vector. One possible explanation for this functional difference is that in tandem CARs, the CD19 binder must be located closer to the T cell membrane to match the distance of the CD19 epitope from the tumor cell surface. This idea is supported by the example of ROR1 CAR

[17] , where the length of the extracellular spacer was decisive in determining CAR tumor recognition. Interestingly, the placement of the CD19 binder proximal to the T cell membrane was also required for the optimal function of another tandem CAR currently under development, the CD22_CD19 CAR (W. Haso, confidential observation).It is hypothesized that the CD20 binder needs to have an unbound carboxyl terminus for proper VH vs. VL folding to occur, or that in the 1920 construct, the CD20 binding site is obscured by the adjacent linker-CD19 domain, and that better antigen binding by the 2019 CAR may reflect this.

[0281] The in vitro cytokine release activity also demonstrated tandem CAR activity (Figure 4). However, the CD20-specific CAR 20A was superior to tandem and single CD19 CAR constructs in the production of Th-1-like cytokines: IFN-gamma, IL-2, and TNF-alpha, as well as GM-CSF. Both tandem CARs were similar in cytokine production, and were greater than that of the single 19A and 19B constructs. The cytokine levels produced by tandem CAR constructs 1920 and 2019 were primarily driven by CD20 recognition and may reflect other tandem CAR advantages; that is, the optimal features from each binder were conserved for better therapeutic efficacy potential.

[0282] In vivo, CAR 19B functioned better than CAR 19A (Figures 5A and 5B). This may be partly due to 19B's superior ability to produce IL-2 and thus maintain T cell activity in the NSG mouse model, which we did not complement with human cytokine supplementation. Together, these data suggest that cytokine production is important in predicting the in vivo activity of anti-leukemic CARs in the NSG mouse lineage.

[0283] Next, we tested the ability of CARs to regulate leukemia phenotypes through immunosuppression. To achieve this goal, we found that an effector-to-target ratio of ≥ 5:1 in the CTL assay effectively eliminated leukemia cells, but even with a lower effector-to-target ratio of 1:1, we were still able to collect viable leukemia cells and analyze their surface antigen expression. Flow cytometry allowed operators to gate viable cells, eliminate T cells, and quantify antigen expression (Figure 6A, 6B). After overnight incubation with 19A and 19B CARs, a strong downregulation of CD19 was observed. Tandem CARs induced less antigen loss, but far fewer cells remained. Regulation of CD20 expression was reduced. Interestingly, some CD22 downregulation was also detected. This may indicate that both antigen-specific and non-antigen-specific mechanisms of escape are possible in the analyzed Raji cell population. Next, both overnight and 4-day CAR co-incubations were performed in Raji and NALM-6 cell lines (Figure 6C). Analysis of NALM-6 was limited by the more general sensitivity of NALM-6 to activated lymphocytes. Even with a low E:T ratio of 1:1, NALM-6 leukemia cells from the co-culture were largely unusable for analysis on day 4. After overnight incubation, a strong downregulation of CD19 expression was observed, with CAR 19A and 19B LV transducible T cells having the greatest effect. Tandem CARs and, surprisingly, all 20A CD20 CARs had a measurable effect on CD19 (Figure 6C). It should also be noted that CD22 expression was reduced by 15–20%, indicating that untargeted B cell differentiation antigens were also affected. Observing the effect of CAR-mediated immunosuppression on CD20 was easier using the Raji cell line. In CAR-T co-culture experiments, CD20 showed less variability than the untargeted antigen CD22 in terms of loss of surface antigen expression. Similar to NALM-6, CD19 downregulation was rapid and occurred in the majority of cells. After 4 days of immunosuppression in Raji cells, all three B cell antigens were moderately downregulated, thus tandem CAR Cells 2019 and 1920 appeared to maintain immunopressure. In particular, with the CD19 single-specific vector, CD19 expression remained strongly downregulated. Cell 20A CAR immunopressure showed less ability to select escape variants on both overnight and day 4, and likely indicates a less ability of Raji cells to either omit or modify its expression.

[0284] Downregulation of tumor antigen surface expression by soluble cytokines produced by inflammatory cells has been documented in chronic lymphocytic leukemia

[30] . Next, in a Transwell co-culture assay, we investigated whether soluble factors present in the culture medium of co-cultured CAR T and tumor cells are responsible for the downregulation of CD19, CD20, and CD22 on Raji cells. We found that direct contact between Raji tumor cells and CAR T cells is responsible for C We found that it is necessary to downregulate the surface expression of D19, CD20, and CD22 in Raji.

[0285] To investigate the mechanism of CD19 loss related to antibody staining by flow cytometry, we studied the occurrence rate of CD19 isotypes in Raji cells exposed to CAR-T cells using a discovery by the Thomas-Tikhonenko lab [8]. To achieve this goal, Raji cells were co-cultured with CAR-T cells and then analyzed by flow cytometry at the end of an overnight incubation to demonstrate a reduction in CD19 expression. Subsequently, we depleted the T cell co-culture using immunomagnetic beads. The isolated Raji cells were analyzed by Western blotting. Surprisingly, we found that a reduction in full-length CD19 isotypes was the main cause of the decrease observed in CD19 staining, and that levels of Δ2 CD19 isotypes remained relatively stable regardless of the treatment.

[0286] The fluctuations in B cell surface antigen expression were so significant that we wanted to study their relative permanence. In other words, was this a true mutation effect or a phenotypic adaptation? To address this question, we returned Raji cells from which CAR T cells had been isolated to culture and then analyzed again by flow cytometry on day 4. All tumor populations demonstrated complete recovery, with CD19, CD20, and CD22 expression exceeding 98%.

[0287] Tumor antigen escape is one of the major challenges facing the field of adoptive cell therapy today. Despite the significant progress made in the treatment of relapsed or refractory ALL with CAR19, tumor escape by downregulating CD19 epitopes on tumor cells has been reported [5, 18, 19] and is responsible for a significant portion of disease relapse. In particular, combinatorial approaches have proven necessary to overcome tumor escape in hematological malignancies, especially in disease types that are more difficult to treat than pediatric pre-B-ALL, such as NHL, as outlined in

[20] . Furthermore, as demonstrated for CAR19 treatment of leukemia and also shown in in vivo models of PSCA and MUC1-positive tumors

[21] , heterogeneous target antigen expression can lead to tumor escape variants when a single CAR T therapy is used. Therefore, targeting multiple tumor antigens with a single CAR T therapy product may mitigate tumor antigen escape.

[0288] Sotillo and collaborators [8] demonstrated that CD19 loss occurs under CAR19 pressure in primary disease as well as leukemia and lymphoma cell lines through a variety of mechanisms, including both mutation and proliferation of CD19-negative variants, as well as alternative splicing that produces exon 2-deficient CD19 variants. Recent studies have shown that after prolonged CAR-T immunosuppression against the CD19 antigen, tumors can also evade detection by reverting to a CD19-negative myeloid phenotype [22, 23]. In the experimental model used, CD19 downregulation on Raji leukemia in the presence of CD19 CAR constructs 19A and 19B was rapid, with a marked loss of detectable CD19 expression occurring after only one night of co-culture with CAR T cells. The loss of CD19 expression was completely restored within 4 days after the CAR T cells were removed from the culture. Given the rapid nature of the changes and the fact that Raji cells replicate approximately once every 20 hours, CD19 downregulation due to actual CD19 loss mutations and preferential proliferation of CD19-negative lymphoma clones is unlikely to occur in our model. Furthermore, consistent with reduced CD19 staining investigated by flow cytometry, it was determined that while the amount of full-length CD19 protein in whole Raji cell lysates was reduced in the presence of single CAR19 constructs 19A and 19B, the amount of exon 2 spliced ​​CD19 remained relatively stable, regardless of the type of CAR T used. Therefore, alternative splicing of CD19 exon 2 is not related to full-length CD It is not modified by CAR-mediated downregulation.

[0289] CD19 is a B cell coreceptor that acts as a positive regulator of mutation, proliferation, and survival in early and late B cell development [24, 25]. CD19 is internalized along with the B cell receptor after its mating by a ligand. Similarly, CD19 may be internalized after binding by a specific antibody, a fact that is utilized in antibody conjugate (ADC) therapy. Internalization of CD20 from the B cell surface is also known to occur after the use of the therapeutic antibody rituximab

[26] . It could also be hypothesized that CAR19 pressure could cause CD19 internalization. However, in our system, mere CD19 internalization could not explain the fact that full-length CD19 protein levels decreased after 19A and 19B treatment, as shown by Western blotting. Therefore, CD19 protein downregulation must occur at the transcriptional or translational level, or by increased degradation of CD19 protein, during CAR-driven internalization. This effect is highly dynamic and immediately reverts after the removal of CAR19 pressure.

[0290] The significance of reduced CD19 expression due to exposure to CAR T therapy for tumor growth is not yet fully understood. Mechanistically, CD19 lowers the B cell activation threshold and initiates several downstream signaling pathways by promoting B cell receptor-antigen microcluster formation

[27] . CD19 is typically considered a promoter of lymphoma formation and has been shown to drive B cell proliferation via a positive feedback loop by the MYC oncoplastic protein [28, 29]. However, CD19 is downregulated on certain types of leukemia B cells, including CLL, B-PLL, SLVL, and MCL [9]. Therefore, in some cases, such as when selective pressure is applied by CD19-targeted CART cells, CD19 loss may provide a tumor growth benefit. The plasticity of CD19 demonstrated in leukemia cells may be a conserved feature from the biology of normal B cells, which here provides an additional survival benefit to malignancies.

[0291] Finally, the activity of tandem CARs versus single CARs was tested in more advanced disease environments with higher tumor burden. A simplified approach was also compared, where two CAR products expressing either a single CD19-CAR or a CD20-CAR were mixed to form a single effector population (Figures 9A and 9B). The CD2019 CAR was the only treatment group that was able to control the tumor, and all subjects in this group survived until day 25 of the study. The single CARs 19A and 19B themselves did not have a strong anti-disease effect, and one mouse was lost from each group. The 20A CAR, either alone or in combination with either CD19 CAR, clearly eliminated the disease from the mice, but four out of six mice did not survive until day 25. This may be due to the greater potential of the tandem 2019 CAR to be activated by tumor cells and its more moderate cytokine production profile compared to the 20A CAR (Figure 4). In this study, we were unable to distinguish between death due to progressive disease and death due to CAR-related toxicity. However, we suspect that 20A CAR-related toxicity may have played a role in the complete elimination of the disease from surviving mice. Since we use model tumor cell lines, alloantigen reactivity may play a role in all of our studies, but the lack of activity of GFP or NT controls in any of the assays used demonstrates that it is not the primary effect.

[0292] conclusion While the studies described herein do not prove that CAR T immunosuppression generates persistent escape variants, clinical experience to date indicates that CD19-negative relapse is not a rare event [8, 18, 19]. Furthermore, data suggest that CD19 antigen modulation is a very rapid event, and that targeting both antigens simultaneously addresses this issue. This demonstrates that it can be a rational method for integrating. High tumor burden studies use tandem CAR. This study highlights the intriguing new biology that may arise from T products and may demonstrate an excellent CAR design format for translational studies. Alternatively, in this study, a unique equilibrium is achieved between effective in vitro activity, particularly cytokine production and cell death activity, which needs to be confirmed for optimal in vivo activity by considering both the CAR expressed by effector cells and the overall disease burden.

[0293] Abbreviation AA - amino acids, 7AAD - 7-actinomycin D, AF - alexa fluor, ALL - acute lymphoblastic leukemia, ANOVA - analysis of variance, APC - allophycocyanin, CAR T - chimeric antigen receptor T cells, CD - surface antigen classification, CLL - chronic lymphocytic leukemia, CPS - counts per second, CTL - cytotoxic T lymphocytes, ELISA - enzyme-linked immunosorbent assay, FBS - fetal bovine serum, Fc - Fc region (fragment crystallizable) (region), FITC - Fluorescein isothiocyanate, GFP - Green fluorescent protein, GM - CSF - Granulocyte-macrophage colony-stimulating factor, GMP - Good Manufacturing Practice, HCL - Hair cell leukemia, HEK - Human embryonic kidney cell, Her2 - Human epidermal growth factor receptor 2, IFNγ - Interferon gamma, IL-2 - Interleukin 2, IU - International unit, LV - Lentiviral vector, Luc - Firefly luciferase, MCL - Mantle cell lymphoma, MOPS - 3-(N-morpholino)propanesulfonic acid, NSG - NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ, NT - Non-transduced T cell control from the same donor, OBI - Oklahoma Blood Institute, PE-phycoerythrin, PLL-prolymphocytic leukemia, ROR1-receptor tyrosine kinase-like orphan receptor 1, RPM-turns per minute, scFv-single chain variable fragment, SDS-PAGE-sodium dodecyl sulfate polyacrylamide gel electrophoresis, SLVL-splenic lymphoma with chorionic lymphocytes, TA-tumor-only control group, TNFα-tumor necrosis factor alpha, VH-variable heavy chain domain, VL-variable light chain domain.

[0294] References for Example 1 1. Lee, D.W., et al., The future is now: chimeric antigen receptors as new targeted therapies for childhood cancer. Clinical Cancer Research, 2012. 18(10): p. 2780-2790. 2. Kochenderfer, J.N., et al., Construction and pre-clinical evaluation of an anti-CD19 chimeric antigen receptor. Journal of immunotherapy (Hagerstown, Md.: 1997), 2009. 32(7): p. 689. 3. Jensen, M., et al., CD20 is a molecular target for scFvFc: zeta receptor redirected T cells: implications for cellular immunotherapy of CD20+ malignancy. Biology of Blood and Marrow Transplantation, 1998. 4(2): p. 75-83. 4. Lee, D.W., et al., T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial. The Lancet, 2015. 385(9967): p. 517-528. 5. Grupp, S.A., et al., Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. New England Journal of Medicine, 2013. 368(16): p. 1509-1518. 6. Porter, D.L., et al., Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. New England Journal of Medicine, 2011. 365(8): p. 725-733. 7. Kochenderfer, J.N., et al., B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells. Blood, 2012. 119(12): p. 2709-2720. 8. Sotillo, E., et al., Convergence of acquired mutations and alternative splicing of CD19 enables resistance to CART-19 immunotherapy. Cancer discovery, 201 5. 5(12): p. 1282-1295. 9. Ginaldi, L., et al., Levels of expression of CD19 and CD20 in chronic B cell leukaemias. Journal of clinical pathology, 1998. 51(5): p. 364-369. 10. Barrett, D.M., et al., Noninvasive bioluminescent imaging of primary patient acute lymphoblastic leukemia: a strategy for preclinical modeling. Blood, 2011. 118(15): p. e112-e117. 11. Whitlow, M., et al., An improved linker for single-chain Fv with reduced aggregation and enhanced proteolytic stability. Protein engineering, 1993. 6(8): p. 989-995. 12. Haso, W., et al., Anti-CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia. Blood, 2013. 121(7): p. 1165-1174. 13. Kuroda, H., et al., Simplified lentivirus vector production in protein-free media using polyethylenimine-mediated transfection. Journal of virological methods, 2009. 157(2): p. 113-121. 14. Grada, Z., et al., TanCAR: a novel bispecific chimeric antigen receptor for cancer immunotherapy. Molecular Therapy-Nucleic Acids, 2013. 2(7): p. e105. 15. Wu, A.M., et al., Multimerization of a chimeric anti-CD20 single-chain Fv-Fc fusion protein is mediated through variable domain exchange. Protein engineering, 2001. 14(12): p. 1025-1033. 16. Zah, E., et al., T cells expressing CD19 / CD20 bi-specific chimeric antigen receptors prevent antigen escape by malignant B cells. 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Maintenance of follicular and marginal zone B cells requires CD19-dependent survival signals. The Journal of Immunology, 2003. 170(1): p. 73-83. 26. Beers, S.A., et al., Antigenic modulation limits the efficacy of anti-CD20 antibodies: implications for antibody selection. Blood, 2010. 115(25): p. 5191-5201. 27. Depoil, D., et al., CD19 is essential for B cell activation by promoting B cell receptor-antigen microcluster formation in response to membrane-bound ligand. Nature immunology, 2008. 9(1): p. 63-72. 28. Chung, E.Y., et al., CD19 is a major B cell receptor-independent activator of MYC-driven B-lymphomagenesis. The Journal of clinical investigation, 2012. 122(6): p. 2257-2266. 29. Poe, J.C., et al., A c-Myc and surface CD19 signaling amplification loop promotes B cell lymphoma development and progression in mice. The Journal of Immunology, 2012. 189(5): p. 2318-2325. 30. Vilpo J, et al., Surface membrane antigen expression changes induced in vitro by exogenous growth factors in chronic lymphocytic leukemia cells. Leukemia, 2002 (16), 1691-1698. Equivalent Each application and patent cited herein, and each document or reference cited in each application and patent (including each granted patent in litigation, “Application Reference Documents”), and each PCT and foreign application or patent corresponding to and / or claiming priority to any of these applications and patents, and each document cited or referenced in each Application Reference Document, are incorporated herein by express reference and can be used in practicing the present invention. More generally, documents or references are cited in the text, in the list of references preceding the claims, or in the text itself, and each of these documents or references (“In-Specified References”), and each document or reference cited in each In-Specified Reference (including any manufacturer’s specifications, instructions, etc.), are incorporated herein by express reference.

[0295] The aforementioned descriptions of some specific embodiments provide sufficient information to enable others to easily modify or adapt the invention to various applications, such as specific embodiments, without departing from the general concept, by applying the knowledge of the invention. Therefore, such adaptations and modifications should and are intended to be understood as being within the meaning and scope of equivalents of the disclosed embodiments. It is understood that the terms or predicates used herein are for descriptive purposes only, not limiting purposes. Exemplary embodiments are disclosed in the drawings and description, and specific terminology may be used, but unless otherwise noted, they are used only in a general and descriptive sense, not limiting purposes, and therefore the claims are not so limited. Furthermore, those skilled in the art will understand that certain steps of the methods discussed herein can be arranged in a different order or combined. Therefore, the appended claims are not intended to be limited to the detailed embodiments disclosed herein. Those skilled in the art can understand and grasp many equivalents of the embodiments of the invention described herein using commonplace experiments. Such equivalents are encompassed by the following claims.

[0296] The array of the present disclosure The nucleic acid and amino acid sequences listed below are shown using standard abbreviations for nucleotide bases and three-letter codes for amino acids, as specified in 37C.FR1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is shown. It is understood to be included by reference. In the attached sequence listing: Sequence ID 1: Nucleotide sequence of Leader-CD19 VL-Whitlow linker CD19 VH(GGGGS)-5 CD20 VH(GGGGS)-3 CD20 VL CD8 hinge + TM-4-1BB-CD3z (construct CAR1920) ATGCTCCTTCTCGTGACCTCCCTGCTTCTCTGCGAACTGCCCCATCCCTGCCTTCCTGCTG ATTCCCGACATTCAGATGACTCAGACCACCTCCTCCCTGTCCGCCTCCCTGGGCGACCGC GTGACCATCTCATGCCGCGCCAGCCAGGACATCTCGAAGTACCTCAACTGGTACCAGCAG AAGCCCGACGGAACCGTGAAGCTCCTGATCTACCACACCTCCCGGCTGCACAGCGGAGTG CCGTCTAGATTCTCGGGTTCGGGGTCGGGAACTGACTACTCCCTTACTATTTCCAACCTG GAGCAGGAGGATATTGCCACCTACTTCTGCCAACAAGGAAACACCCTGCCGTACACTTTT GGCGGGGGAACCAAGCTGGAAATCACTGGCAGCACATCCGGTTCCGGGAAGCCCGGCTCC GGAGAGGGCAGCACCAAGGGGGAAGTCAAGCTGCAGGAATCAGGACCTGGCCTGGTGGCC CCGAGCCAGTCACTGTCCGTGACTTGTACTGTGTCCGGAGTGTCGCTCCCGGATTACGGA GTGTCCTGGATCAGGCAGCCACCTCGGAAAGGATTGGAATGGCTCGGAGTCATCTGGGGT TCCGAAACCACCTATTACAACTCGGCACTGAAATCCAGGCTCACCATTATCAAGGATAAC TCCAAGTCACAAGTGTTCCTGAAGATGAATAGCCTGCAGACTGACGACACGGCGATCTAC TATTGCGCCAAGCACTACTACTACGGCGGATCCTACGCTATGGACTACTGGGGCCAGGGG ACCAGCGTGACCGTGTCATCCGGAGGCGGCGGCAGCGGCGGGGGAGGGTCCGGAGGGGGT GGTTCTGGTGGAGGAGGATCGGGAGGCGGTGGCAGCGAGGTGCAGTTGCAACAGTCAGGA GCTGAACTGGTCAAGCCAGGAGCCAGCGTGAAGATGAGCTGCAAGGCCTCCGGTTACACC TTCACCTCCTACAACATGCACTGGGTGAAACAGACCCCGGGACAAGGGCTCGAATGGATT GGCGCCATCTACCCCGGGAATGGCGATACTTCGTACAACCAGAAGTTCAAGGGAAAGGCC ACCCTGACCGCCGACAAGAGCTCCTCCACCGCGTATATGCAGTTGAGCTCCCTGACCTCC GAGGACTCCGCCGACTACTACTGCGCACGGTCCAACTACTATGGAAGCTCGTACTGGTTC TTCGATGTCTGGGGGGCCGGCACCACTGTGACCGTCAGCTCCGGGGGCGGAGGATCCGGT GGAGGCGGAAGCGGGGGTGGAGGATCCGACATTGTGCTGACTCAGTCCCCGGCAATCCTG TCGGCCTCACCGGGCGAAAAGGTCACGATGACTTGTAGAGCGTCGTCCAGCGTGAACTAC ATGGATTGGTACCAAAAGAAGCCTGGATCGTCACCCAAGCCTTGGATCTACGCTACATCT AACCTGGCCTCCGGCGTGCCAGCGCGGTTCAGCGGGTCCGGCTCGGGCACCTCATACTCG CTGACCATCTCCCGCGTGGAGGCTGAGGACGCCGCGACCTACTACTGCCAGCAGTGGTCC TTCAACCCGCCGACTTTTGGAGGCGGTACTAAGCTGGAGATCAAAGCGGCCGCAACTACC ACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTG CGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTT GCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCG CTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAG CCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCT GAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCC GCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAG TACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGG AAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTAC TCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAG GGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCC CGG Amino acid sequence of Sequence ID No. 2 Leader-CD19 VL-Whitlow linker CD19 VH(GGGGS)-5 CD20 VH(GGGGS)-3 CD20 VL CD8 hinge + TM-4-1BB-CD3z (construction CAR1920) MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLV APSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSGGGGSGGGSEVQLQQSGAELVKPGASVKMSCKASG YTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGGGGSGGGGSGGGG SDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKAAAT TTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELR VKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Nucleotide sequence of Sequence ID 3: Leader-CD20 VH(GGGGS)3-CD20 VL-(GGGGS)5-CD19 VL-Whitlow linker-CD19 VH CD8 hinge + TM-4-1BB-CD3z (construct 2019) ATGCTCCTTCTCGTGACCTCCCTGCTTCTCTGCGAACTGCCCCATCCCTGCCTTCCTGCTG ATTCCCGAGGTGCAGTTGCAACAGTCAGGAGCTGAACTGGTCAAGCCAGGAGCCAGCGTG AAGATGAGCTGCAAGGCCTCCGGTTACACCTTCACCTCCTACAACATGCACTGGGTGAAA CAGACCCCGGGACAAGGGCTCGAATGGATTGGCGCCATCTACCCCGGGAATGGCGATACT TCGTACAACCAGAAGTTCAAGGGAAAGGCCACCCTGACCGCCGACAAGAGCTCCTCCACC GCGTATATGCAGTTGAGCTCCCTGACCTCCGAGGACTCCGCCGACTACTACTGCGCACGG TCCAACTACTATGGAAGCTCGTACTGGTTCTTCGATGTCTGGGGGGCCGGCACCACTGTG ACCGTCAGCTCCGGGGGCGGAGGATCCGGTGGAGGCGGAAGCGGGGGTGGAGGATCCGAC ATTGTGCTGACTCAGTCCCCGGCAATCCTGTCGGCCTCACCGGGCGAAAAGGTCACGATG ACTTGTAGAGCGTCGTCCAGCGTGAACTACATGGATTGGTACCAAAAGAAGCCTGGATCG TCACCCAAGCCTTGGATCTACGCTACATCTAACCTGGCCTCCGGCGTGCCAGCGCGGTTC AGCGGGTCCGGCTCGGGCACCTCATACTCGCTGACCATCTCCCGCGTGGAGGCTGAGGAC GCCGCGACCTACTACTGCCAGCAGTGGTCCTTCAACCCGCCGACTTTTGGAGGCGGTACT AAGCTGGAGATCAAAGGAGGCGGCGGCAGCGGCGGGGGAGGGTCCGGAGGGGGTGGTTCT GGTGGAGGAGGATCGGGAGGCGGTGGCAGCGACATTCAGATGACTCAGACCACCTCCTCC CTGTCCGCCTCCCTGGGCGACCGCGTGACCATCTCATGCCGCGCCAGCCAGGACATCTCG AAGTACCTCAACTGGTACCAGCAGAAGCCCGACGGAACCGTGAAGCTCCTGATCTACCAC ACCTCCCGGCTGCACAGCGGAGTGCCGTCTAGATTCTCGGGTTCGGGGTCGGGAACTGAC TACTCCCTTACTATTTCCAACCTGGAGCAGGAGGATATTGCCACCTACTTCTGCCAACAA GGAAACACCCTGCCGTACACTTTTGGCGGGGGAACCAAGCTGGAAATCACTGGCAGCACA TCCGGTTCCGGGAAGCCCGGCTCCGGAGAGGGCAGCACCAAGGGGGAAGTCAAGCTGCAG GAATCAGGACCTGGCCTGGTGGCCCCGAGCCAGTCACTGTCCGTGACTTGTACTGTGTCC GGAGTGTCGCTCCCGGATTACGGAGTGTCCTGGATCAGGCAGCCACCTCGGAAAGGATTG GAATGGCTCGGAGTCATCTGGGGTTCCGAAACCACCTATTACAACTCGGCACTGAAATCC AGGCTCACCATTATCAAGGATAACTCCAAGTCACAAGTGTTCCTGAAGATGAATAGCCTG CAGACTGACGACACGGCGATCTACTATTGCGCCAAGCACTACTACTACGGCGGATCCTAC GCTATGGACTACTGGGGCCAGGGGACCAGCGTGACCGTGTCATCCGCGGCCGCAACTACC ACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTG CGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTT GCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCG CTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAG CCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCT GAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCC GCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAG TACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGG AAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTAC TCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAG GGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCC CGG SEQ ID NO:4 Leader - CD20 VH (GGGGS)3 - CD20 VL - (GGGGS)5 - CD19 VL - Whitlow linker - CD19 VH CD8 hinge + TM - 4 - 1BB - CD3z amino acid sequence (Construct CAR2019) MLLLVTSLLLCELPHPAFLLIPEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPAILSASPGEKVTMTCRASSSVNY MDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSGGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQ GNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAG GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Nucleotide sequence of sequence number 5 CD19 LTG1494 (CAR 19A) ATGGTCATGCTTCTCCTGGTCACCTCCCTGCTCCTCTGCGAACTGCCTCACCCTGCCTTC CTTCTGATTCCTGACACTGACATTCAGATGACTCAGACCACCTCTTCCTTGTCCGCGTCA CTGGGAGACAGAGTGACCATCTCGTGTCGCGCAAGCCAGGATATCTCCAAGTACCTGAAC TGGTACCAACAGAAGCCCGACGGGACTGTGAAGCTGCTGATCTACCACACCTCACGCCTG CACAGCGGAGTGCCAAGCAGATTCTCCGGCTCCGGCTCGGGAACCGATTACTCGCTTACC ATTAGCAACCTCGAGCAGGAGGACATCGCTACCTACTTCTGCCAGCAAGGAAATACCCTG CCCTACACCTTCGGCGGAGGAACCAAATTGGAAATCACCGGCTCCACGAGCGGCTCCGGG AAGCCTGGTTCCGGGGAAGGCTCCACTAAGGGTGAAGTGAAGCTCCAGGAGTCCGGCCCC GGCCTGGTGGCGCCGTCGCAATCACTCTCTGTGACCTGTACCGTGTCGGGAGTGTCCCTG CCTGATTACGGCGTGAGCTGGATTCGGCAGCCGCCGCGGAAGGGCCTGGAATGGCTGGGT GTCATCTGGGGATCCGAGACTACCTACTACAACTCGGCCCTGAAGTCCCGCCTGACTATC ATCAAAGACAACTCGAAGTCCCAGGTCTTTCTGAAGATGAACTCCCTGCAAACTGACGAC ACCGCCATCTATTACTGTGCTAAGCACTACTACTACGGTGGAAGCTATGCTATGGACTAC TGGGGCCAGGGGACATCCGTGACAGTCAGCTCCGCGGCCGCAACTACCACCCCTGCCCCT CGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCT TGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATC TACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACC CTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGG CCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAG GGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAG GGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTG GACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAG GAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGG ATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACC GCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGG Amino acid sequence of SEQ ID NO: 6 CD19 LTG1494 (CAR 19A) protein MVMLLLVTSLLLCELPHPAFLLIPDTDIQMTQTTSSLSASLGDRVTISCRASQDISKYLN WYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTL PYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSL PDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDD TAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEA CRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMR PVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRGKGHDGLYQGLST ATKDTYDALHMQALPPR Sequence ID 7 CD19 LTG1538 (CAR 19B) DNA nucleotide sequence ATGCTTCTCCTGGTCACCTCCCTGCTCCTCTGCGAACTGCCTCACCCTGCCTTCCTTCTG ATTCCTGACATTCAGATGACTCAGACCACCTCTTCCTTGTCCGCGTCACTGGGAGACAGA GTGACCATCTCGTGTCGCGCAAGCCAGGATATCTCCAAGTACCTGAACTGGTACCAACAG AAGCCCGACGGGACTGTGAAGCTGCTGATCTACCACACCTCACGCCTGCACAGCGGAGTG CCAAGCAGATTCTCCGGCTCCGGCTCGGGAACCGATTACTCGCTTACCATTAGCAACCTC GAGCAGGAGGACATCGCTACCTACTTCTGCCAGCAAGGAAATACCCTGCCCTACACCTTC GGCGGAGGAACCAAATTGGAAATCACCGGCGGAGGAGGCTCCGGGGGAGGAGGTTCCGGG GGCGGGGGTTCCGAAGTGAAGCTCCAGGAGTCCGGCCCCGGCCTGGTGGCGCCGTCGCAA TCACTCTCTGTGACCTGTACCGTGTCGGGAGTGTCCCTGCCTGATTACGGCGTGAGCTGG ATTCGGCAGCCGCCGCGGAAGGGCCTGGAATGGCTGGGTGTCATCTGGGGATCCGAGACT ACCTACTACAACTCGGCCCTGAAGTCCCGCCTGACTATCATCAAAGACAACTCGAAGTCC CAGGTCTTTCTGAAGATGAACTCCCTGCAAACTGACGACACCGCCATCTATTACTGTGCT AAGCACTACTACTACGGTGGAAGCTATGCTATGGACTACTGGGGGCAAGGCACTTCGGTG ACTGTGTCAAGCGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCA ACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGA GCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCC GGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGG AAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAA GAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTC AAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAAC GAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGAC CCGGAGATGGGGGGGAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTC CAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGG GGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGAT GCCTTGCATATGCAAGCACTCCCACCCCGG Amino acid sequence of sequence number 8 CD19 LTG1538 (CAR 19B) MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQ KPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTF GGGTKLEITGGGGSGGGGSGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSW IRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCA KHYYYGGSYAMDYWGQGTSVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG AVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQE EDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRD PEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPP Nucleotide sequence of Array No. 9 CD20A LTG1495 (CAR 20A) ATGCTCCTTCTCGTGACCTCCCTGCTTCTCTGCGAACTGCCCCATCCTGCCTTCCTGCTG ATTCCCGAGGTGCAGTTGCAACAGTCAGGAGCTGAACTGGTCAAGCCAGGAGCCAGCGTG AAGATGAGCTGCAAGGCCTCCGGTTACACCTTCACCTCCTACAACATGCACTGGGTGAAA CAGACCCCGGGACAAGGGCTCGAATGGATTGGCGCCATCTACCCCGGGAATGGCGATACT TCGTACAACCAGAAGTTCAAGGGAAAGGCCACCCTGACCGCCGACAAGAGCTCCTCCACC GCGTATATGCAGTTGAGCTCCCTGACCTCCGAGGACTCCGCCGACTACTACTGCGCACGG TCCAACTACTATGGAAGCTCGTACTGGTTCTTCGATGTCTGGGGGGCCGGCACCACTGTG ACCGTCAGCTCCGGGGGCGGAGGATCCGGTGGAGGCGGAAGCGGGGGTGGAGGATCCGAC ATTGTGCTGACTCAGTCCCCGGCAATCCTGTCGGCCTCACCGGGCGAAAAGGTCACGATG ACTTGTAGAGCGTCGTCCAGCGTGAACTACATGGATTGGTACCAAAAGAAGCCTGGATCG TCACCCAAGCCTTGGATCTACGCTACATCTAACCTGGCCTCCGGCGTGCCAGCGCGGTTC AGCGGGTCCGGCTCGGGCACCTCATACTCGCTGACCATCTCCCGCGTGGAGGCTGAGGAC GCCGCGACCTACTACTGCCAGCAGTGGTCCTTCAACCCGCCGACTTTTGGAGGCGGTACT AAGCTGGAGATCAAAGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCC CCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGT GGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTG GCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGC CGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAG GAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGC GTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTAC AACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGC GACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAA CTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGG AGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTAC GATGCCTTGCATATGCAAGCACTCCCACCCCGG Amino acid sequence of SEQ ID NO: 10 CD20A 1495 (CAR 20A) MLLLVTSLLLCELPHPAFLLIPEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVK QTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCAR SNYYGSSYWFFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPAILSASPGEKVTM TCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGSGTSYSLTISRVEAED AATYYCQQWSFNPPTFGGGGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAG GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQ EEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR DPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY DALHMQALPPR Sequence ID 11: Nucleotide sequence of the reader / signal peptide (LP) atgctgctgctggtgaccagcctgctgctgtgcgaactgccgcatccggcgtttctgctgattccg Sequence ID 12: Amino acid sequence of the reader / signal peptide (LP) MLLLVTSLLLCELPHPAFLLIP Sequence ID 35: Nucleotide sequence of the DNA CD8 transmembrane domain atttgggccccgctggccggcacttgcggcgtgctcctgctgtcgctggtcatcaccctt tactgc SEQ ID NO: 36 Amino acid sequence of the CD8 transmembrane domain Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Sequence ID 37: Nucleotide sequence of the DNA CD8 hinge domain actaccacccctgcccctcggccgccgactccggccccaaccatcgcaagccaacccctc tccttgcgccccgaagcttgccgcccggccgcgggtggagccgtgcatacccgggggctg gactttgcctgcgatatctac Amino acid sequence of the CD8 hinge domain (SEQ ID NO: 38) Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Amino acid sequence of the hinge and transmembrane region (amino acid numbers 137-206) of sequence number 39 CD8.alpha (NCBI reference sequence: NP.sub.--001759.3) Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Nucleotide sequence of the DNA signaling domain of SEQ ID NO: 40 4-1BB aagaggggccggaagaagctgctttacatcttcaagcagccgttcatgcggcccgtgcag acgactcaggaagaggacggatgctcgtgcagattccctgaggaggaagaggggggatgc surfaceactg Amino acid sequence of the signaling domain of SEQ ID NO: 41 4-1BB Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Sequence ID 42: Nucleotide sequence of the DNA signaling domain of CD3-zeta cgcgtcaagttctcacggtccgccgacgcccccgcatatcaacagggccagaatcagctc tacaacgagctgaacctgggaaggagagaggagtacgacgtgctggacaagcgacgcgga cgcgaccgggatgggggggaaaccacggcggaaaaaccctcaggaaggactgtacaac gaactccagaaagacaagatggcggaagcctactcagaaatcgggatgaagggagagcgg aggaggggaaagggtcacgacgggctgtaccagggactgagcaccgccactaaggatacc tacgatgccttgcatatgcaagcactcccaccccgg Amino acid sequence of SEQ ID NO: 43 CD3 zeta Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gly Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Gl Lys Gly Pro Glu Leu Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg The 44 ScFv CD19(FMC63) gacattcagatgactcagaccacccttccttgtccgcgtcactgggagacagagtgaccat ctcgtgtcgcgcaagccaggatatctccaagtacctgaactggtaccaacagaagccga cgggactgtgaagctgctgatctaccacacctcacgcctgcacagcggagtgcaagcag attctccggctccggctcgggaaccgattactcgcttaccattagcaacctcgagcagga ggacatcgctacctacttctgccagcaaggaaataccctgccctacaccttcggcggagg aaccaaattggaaatcaccggcggaggaggctccgggggggttccggggggggggg ttccgaagtgaagctccaggagtccggccccggcctggtggcgccgtcgcaatcactctc tgtgacctgtaccgtgtcgggagtgtccctgcctgattacggcgtgagctggattcggca gccgccgcggaagggcctggaatggctgggtgtcatctggggatccgagactacctacta caactcggccctgaagtcccgcctgactatcatcaaagacaactcgaagtcccaggtctt tctgaagatgaactccctgcaaactgacgacaccgccatctattactgtgctaagcacta ctactacggtggaagctatgctatggactactgggggcaaggcacttcggtgactgtgtc aagc Amino acid sequence of SEQ ID NO: 45 ScFv CD19 (FMV63) Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly GlySer Gly Gly Gly Gly Ser Gl yGly Gly Gly Ser Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Nucleotide sequence of Array No. 46 anti-CD33 CAR (LTG1936) ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAACTGCCGCATCCGGCGTTTCTGCTGATTCCGCAGGTGCAGCTGGTGCAATCTGGGGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAGGATCTCCTGTAAGGGTTCTGGATTCAGTTTTCCCACCTACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACTAGTTGGAGATGGCTACAATACGGGGGCTTTTGATATCT Amino acid sequence of SEQ ID NO: 47 Anti-CD33 CAR (LTG1936) MLLLVTSLLLCELPHPAFLLIPQVQLVQSGAEVKPGESLRISCKGSGFSFPTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARLVGD GYNTGAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTHTPLSLSVTPGQPASISCKSSQSLLHSNGKTYLYWYLQKPGQPPQLLIYGASNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVG VYYCMQSIQLPITFGQGTRLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCS CRFPEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP Sequence ID 48: Nucleotide sequence of anti-mesoserine CAR (LTG1904) Amino acid sequence of SEQ ID NO: 49 Anti-mesoserine CAR (LTG1904) MLLLVTSLLLCELPHPAFLLIPEVQLVQSGGGLVQPGGSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDLS SVAGPFNYWGQGTLVTVSSGGGGSGGGGSGGGGSSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNS RDSSGNHLVFGGGTQLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Claims

1. An isolated nucleic acid molecule encoding a CD19 / CD20 tandem chimeric antigen receptor (CAR), comprising at least one extracellular antigen-binding domain including a CD19 / CD20 antigen-binding domain, at least one transmembrane domain, and at least one intracellular signaling domain, wherein the CD19 / CD20 tandem chimeric antigen receptor (CAR) is encoded by a nucleotide sequence comprising SEQ ID NO: 1 or 3.

2. The isolated nucleic acid molecule according to claim 1, wherein the encoded at least one CD19 / CD20 antigen-binding domain comprises at least one single-chain variable fragment of an antibody that binds to CD19 / CD20.

3. The isolated nucleic acid molecule according to claim 1, wherein the encoded at least one CD19 / CD20 antigen-binding domain comprises at least one heavy chain variable region of an antibody that binds to CD19 / CD20.

4. The isolated nucleic acid molecule according to claim 1, wherein the encoded at least one CD19 / CD20 antigen-binding domain, the at least one intracellular signaling domain, or both are connected to a transmembrane domain by a linker or spacer domain.

5. The isolated nucleic acid molecule according to claim 4, wherein the encoded linker or spacer domain is derived from the extracellular domain of CD8 or CD28 and is transmembrane-linked.

6. The isolated nucleic acid molecule according to claim 1, wherein the extracellular CD19 / CD20 antigen-binding domain that is encoded is preceded by a leader nucleotide sequence that encodes a leader peptide.

7. The isolated nucleic acid molecule according to claim 6, wherein the leader nucleotide sequence comprises a nucleotide sequence including sequence number 11 which encodes the leader amino acid sequence of sequence number 12.

8. The isolated nucleic acid molecule according to claim 1, wherein the transmembrane domain comprises a transmembrane domain of a protein containing the alpha, beta, or zeta chain of a T cell receptor, CD8, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD19 / CD20, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, and TNFRSF19, or any combination thereof.

9. The isolated nucleic acid molecule according to claim 1, wherein the nucleic acid sequence encoding the CD19 / CD20 tandem chimeric antigen receptor (CAR) is encoded by a nucleotide sequence including SEQ ID NO: 1 or 3, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

10. The isolated nucleic acid molecule according to claim 1, wherein the at least one encoded intracellular signaling domain further comprises a CD3 zeta intracellular domain.

11. The isolated nucleic acid molecule according to claim 10, wherein the at least one encoded intracellular signaling domain is located C-terminally relative to the CD3 zeta intracellular domain. 。

12. The isolated nucleic acid molecule according to claim 1, wherein the at least one encoded intracellular signaling domain comprises a co-stimulatory domain, a primary signaling domain, or any combination thereof.

13. The isolated nucleic acid molecule according to claim 12, wherein the at least one co-stimulatory domain to be encoded comprises the functional signaling domains of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or any combination thereof.

14. A chimeric antigen receptor (CAR) encoded by an isolated nucleic acid molecule as described in claim 1.

15. The CAR according to claim 14, comprising at least one extracellular antigen-binding domain including a CD19 / CD20 antigen-binding domain having the amino acid sequence of SEQ ID NO: 6, 8, or 10, at least one transmembrane domain, and at least one intracellular signaling domain.

16. The CAR according to claim 15, wherein the CD19 / CD20 antigen-binding domain comprises at least one single-chain variable fragment of an antibody that binds to CD19 / CD20.

17. The CAR according to claim 15, wherein the CD19 / CD20 antigen-binding domain includes at least one heavy chain variable region of an antibody that binds to CD19 / CD20.

18. The CAR according to claim 15, wherein the transmembrane domain comprises a transmembrane domain of a protein containing the alpha, beta, or zeta chain of a T cell receptor, CD8, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, and TNFRSF19, or any combination thereof.

19. The CAR according to claim 18, wherein the CD8 transmembrane domain includes an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO:

28.

20. The CAR according to claim 15, wherein the at least one extracellular antigen-binding domain comprising a CD19 / CD20 antigen-binding domain having the amino acid sequence of SEQ ID NO: 6, 8, or 10, and the at least one intracellular signaling domain, or both, are connected to the transmembrane domain by a linker or spacer domain.

21. The CAR according to claim 20, wherein the linker or spacer domain is derived from the extracellular domain of CD8 or CD28 and is linked to the transmembrane domain.

22. The CAR according to claim 17, wherein the at least one intracellular signaling domain comprises a co-stimulatory domain and a primary signaling domain.

23. The at least one intracellular signaling domain is OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or the same. The CAR according to claim 22, comprising a co-stimulatory domain containing a functional signaling domain of a protein selected from the group consisting of combinations of the above.

24. A vector comprising the nucleic acid molecule described in claim 1.

25. The vector according to claim 24, selected from the group consisting of a DNA vector, an RNA vector, a plasmid vector, a cosmid vector, a herpesvirus vector, a measles virus vector, a lentivirus vector, an adenovirus vector, or a retrovirus vector, or a combination thereof.

26. The vector according to claim 24, further comprising a promoter.

27. The vector according to claim 26, wherein the promoter is an inducible promoter, a constitutive promoter, a tissue-specific promoter, a suicide promoter, or any combination thereof.

28. A cell comprising the vector according to claim 24.

29. The cell according to claim 28, which is a T cell.

30. The T cell is CD8 ＋ The cell according to claim 28, which is a T cell.

31. The cell according to claim 28, which is a human cell.

32. A method for producing cells, comprising the step of transducing the vector described in claim 24 into T cells.

33. A method for generating a population of RNA-modified cells, comprising the step of introducing in vitro transcribed RNA or synthetic RNA into the cells, wherein the RNA comprises the nucleic acid molecule described in claim 1.

34. A method for inducing antitumor immunity in a mammal, comprising the step of administering an effective amount of the cells described in claim 28 to the mammal.

35. A method for treating or preventing cancer in a mammal, comprising the step of administering the CAR described in claim 15 to the mammal in an amount effective for treating or preventing cancer in the mammal.

36. A pharmaceutical composition comprising an antitumor-effective population of human T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), and the CAR comprises at least one extracellular antigen-binding domain including a CD19 / CD20 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2 or 4, at least one linker domain, at least one transmembrane domain, and at least one intracellular signaling domain, and the T cells are T cells of a human having cancer.

37. The pharmaceutical composition according to claim 36, wherein the at least one transmembrane domain comprises a transmembrane domain of a protein containing the alpha, beta, or zeta chain of a T cell receptor, CD8, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, and TNFRSF19 or any combination thereof.

38. The pharmaceutical composition according to claim 36, wherein the T cells are human T cells having hematological cancer.

39. The pharmaceutical composition according to claim 38, wherein the hematological cancer is leukemia or lymphoma.

40. The pharmaceutical composition according to claim 39, wherein the leukemia is chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), or chronic myeloid leukemia (CML).

41. The pharmaceutical composition according to claim 39, wherein the lymphoma is mantle cell lymphoma, non-Hodgkin lymphoma, or Hodgkin lymphoma.

42. The pharmaceutical composition according to claim 38, wherein the hematological cancer is multiple myeloma.

43. The pharmaceutical composition according to claim 36, wherein human cancers include oral and pharyngeal cancers (tongue, mouth, pharynx, head and neck), digestive system cancers (esophagus, stomach, small intestine, colon, rectum, anus, liver, intrahepatic bile duct, gallbladder, pancreas), respiratory system cancers (larynx, lung, and bronchi), bone and joint cancers, soft tissue cancers, skin cancers (melanoma, basal cell carcinoma, and squamous cell carcinoma), pediatric tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma), central nervous system tumors (brain tumors, astrocytoma, glioblastoma, glioma), as well as adult cancers including breast, reproductive system (cervix, uterine body, ovaries, vulva, vagina, prostate, testes, penis, endometrium), urinary system (bladder, kidneys and renal pelvis, ureters), eye and orbit, endocrine system (thyroid), and brain and other nervous system cancers, or any combination thereof.

44. A method for treating a mammal having a disease, disorder or condition associated with elevated expression of a tumor antigen, comprising the step of administering a pharmaceutical composition comprising an antitumor-effective amount of a population of T cells to a subject, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprises at least one extracellular antigen-binding domain comprising a CD19 / CD20 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2 or 4, at least one linker or spacer domain, at least one transmembrane domain, and at least one intracellular signaling domain, and the T cells are T cells of a subject having cancer.

45. A method for treating cancer in a subject requiring such treatment, comprising the step of administering to the subject a pharmaceutical composition comprising an antitumor-effective amount of a population of T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprising at least one extracellular antigen-binding domain comprising a CD19 / CD20 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 2 or 4, at least one linker or spacer domain, at least one transmembrane domain, and at least one intracellular signaling domain, and the T cells are T cells of a subject having cancer.

46. The method according to claim 44 or 45, wherein the at least one transmembrane domain comprises a transmembrane domain of a protein containing the alpha, beta, or zeta chain of a T cell receptor, CD8, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or any combination thereof.

47. A method for producing chimeric antigen receptor-expressing cells, comprising the step of introducing the isolated nucleic acid described in claim 1 into cells.

48. A method for producing chimeric antigen receptor-expressing cells according to claim 47, wherein the cells are T A method comprising cells or a population of cells including T cells.

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