Compositions and methods for treating cancer with anti-CD19 / CD22 immunotherapy

Abstract

Chimeric antigen receptors containing CD19 / CD22 or CD22 / CD19 antigen-binding domains are disclosed. Nucleic acids, recombinant expression vectors, host cells, antigen-binding fragments, and pharmaceutical compositions related to the chimeric antigen receptors are also disclosed. Methods for treating or preventing cancer in a subject and methods for generating chimeric antigen receptor T cells are also disclosed.

Description

[Technical Field]

[0001] Cross-reference of related applications This application is a continuation of U.S. Patent Application No. 16 / 584,308 (currently U.S. Patent No. 10,822,412) filed on September 26, 2019, claiming priority to U.S. Provisional Patent Application No. 62 / 736,955 filed on September 26, 2018, under Section 119(e) of the U.S. Patent Act, and U.S. Provisional Patent Application No. 62 / 736,955 filed on September 26, 2018, under Section 119(e) of the U.S. Patent Act. This invention claims priority to U.S. Utility Patent Application No. 17 / 090,797, filed November 5, 2020, which is a continuation-in-part application of PCT Patent Application No. PCT / US19 / 53240 filed September 26, 2019, which is a continuation-in-part application of U.S. Patent Application No. 17 / 072,842 filed October 16, 2020, and which claims priority to U.S. Utility Patent Application No. 17 / 090,797 filed November 5, 2020, which is a continuation-in-part application of U.S. Patent Application No. 17 / 072,842 filed October 16, 2020, and which claims priority to the same invention claimed

[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 3 November 2021, is named Sequence_Listing.txt and is 220 kilobytes in size.

[0003] Areas of this disclosure This application relates to the field of cancer, particularly to CARs targeting (see TR12, fwigure1a) CD19 and CD22 B cell antigens, a simultaneous CD19 / CD22-mediated antigen targeting domain, and chimeric antigen receptors (CARs) containing such CD19 / CD22 antigen targeting 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 cancers, the five-year overall survival rate for all cancers has improved by only about 10% in the last 20 years. Cancer, or malignant tumors, metastasize and grow rapidly without control, making them extremely difficult to treat.

[0005] CD19 is an 85-95 kDa transmembrane cell surface glycoprotein receptor. CD19 is a member of the immunoglobulin (Ig) superfamily of proteins and contains two extracellular Ig-like domains, a transmembrane domain, and an intracellular signaling domain (Tedder TF, Isaacs, CM, 1989, J Immunol vol. 143: pp. 712-171). CD19 modifies B cell receptor signaling by lowering the antigen-induced threshold of the B cell receptor (Carter, RH and Fearon, DT, 1992, Science, vol. 256: pp. 105-107), and works in conjunction with CD81 and CD21 to regulate 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 individual development, CD19 can signal independently of antigen receptors in 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 in a strictly regulated form on normal B cells, limited to early progenitor B cells in the IgH gene rearrangement stage and mature B cells, but not on hematopoietic stem cells or mature plasma cells (Anderson, KC, Bates, MP, Slaughenhout BL, Pinkus GS, Schlossman SF, Nadler LM, 1984, Blood vol. 63: pp. 1424-1433).

[0006] CD22, also known as SIGLEC-2 (sialic acid-binding immunoglobulin-like lectin 2), is a 95 kDa transmembrane surface glycoprotein containing six Ig-like C2 domains and one Ig-like V domain (uniprot.org / uniprot / P20273#structure, accessed 07 / 12 / 2017). During B cell individual development, CD22 is expressed on the B cell surface from the pre-B cell stage, persists on mature B cells, and is lost on plasma cells (Nitschke L, 2009, Immunological Reviews, vol. 230: pp. 128-143). CD22 contains intracellular ITIM (immunoreceptor tyrosine-based inhibition motifs) domains that downregulate cell activation after the B cell receptor engages with an antigen. Antibody binding to CD22 induces co-localization with SHP-1 and other intracellular phosphatases that also downregulate signal transduction primarily through phosphorylation (Lumb S, Fleishcer SJ, Wiedemann A, Daridon C, Maloney A, Shock A, Dorner T, 2016, Journal of Cell Communication and Signaling, Vol. 10: pp. 143-151). An important point relevant to the treatment of B-cell malignancies is that CD22 is expressed in a tightly regulated form on normal B cells but not on hematopoietic stem cells or mature plasma cells, making it a suitable target antigen for B-cell leukemia.Because CD22 is expressed on (pre-B-ALL) B-cell malignancies in both adults and children, this target has been utilized in both antibody and chimeric antigen receptor (CAR) T-cell therapies (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, 2013, Blood, Vol. 121: pp. 1165-1174) (Wayne AS, Kreitman RJ, Findley HW, Lew G, Delbrook C, Steinberg SM, Stetler-Stevenson M, FitzGerald DJ, Pastan I, 2010, Clinical Cancer Research, Vol. 16: pp. 1894-1903).

[0007] Several novel approaches have been developed for the treatment of B-cell leukemia and lymphoma, including anti-CD22 antibodies linked to bacterial toxins or chemotherapeutic agents (Wayne AS, FitzGerald DJ, Kreitman RJ, Pastan I, 2014, Immunotoxins for leukemia, Blood, Vol. 123: pp. 2470-2477). Inotuzumab ozogamicin (CMC-544, a humanized version of the mouse monoclonal antibody G5 / 44) is an antibody-drug conjugate currently being evaluated in clinical trials as a monotherapy or in combination with chemotherapy (NCT01664910, sponsored by MD Anderson Cancer Center) (DiJoseph JF et al., 2004, Blood, Vol. 103: pp. 1807-1814). As a monotherapy, it showed greater efficacy than standard therapy, although significant hepatotoxicity was noted (Kantarjian H et al., 2016, Inotuzumb ozogamicin versus standard therapy for acute lymphoblastic leukemia, New England Journal of Medicine, vol. 375: pp. 740-753). Epratuzumab, an unmodified CD22 therapeutic antibody, is also being tested in combination with chemotherapy (NCT01219816, sponsored by Nantes University Hospital). Epratuzumab is a chimeric protein composed of mouse CDR grafted onto a human antibody framework. Moxetumomab pasudotox is effective in some leukemias, but has not entered widespread clinical development due to the immunogenicity of the bacterial toxin used to fuse the antibody, and issues with its modest or comparable level of activity to other drugs (see NCT01829711, sponsored by MedImmune, LLC).To date, many CD22 binding motifs used in CAR constructs utilize domains derived from these mouse antibodies and do not effectively activate T cells that target this CD22 domain (see, for example, HA22 anti-CD22 binder used as the base for moxetumomab pactotox, James SE, Greenberg PD, Jensen MC, Lin Y, Wang J, Till BG, Raubitschek AA, Forman SJ, Press OW, 2008, Journal of Immunology 180: pp. 7028-7038). One anti-CD22 binder effective as an anti-CD22CAR is currently in clinical trials at the National Institutes of Health (NIH), although the results have not yet been published (ClinicalTrials.gov Identifier: NCT02315612, Anti-CD22 Chimeric Receptor T Cells in Pediatric and Young Adults with Recurrent or Refractory CD22-expressing B Cell Malignancies, sponsor: NCI). This binder is primarily based on the m971 fully human antibody developed in the laboratory of Dr. Dimitrov, one of the inventors in this application (Xiao X, Ho M, Zhu Z, Pastan I, Dimitrov D, 2009, Identification and characterization of fully human anti-CD22 monoclonal antibodies, MABS, Vol. 1: pp. 297-303). The m971 domain was proven effective as a CAR in a study supervised by Dr. Rimas Orentas, another inventor in this application (Haso W et al., 2013, Anti-CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia, Blood, vol. 121: pp. 1165-1174).

[0008] Traditional treatment approaches for lineage B leukemia and lymphoma may include chemotherapy, radiotherapy, and stem cell transplantation (see mayclinic.org on the World Wide Web). The high toxicity associated with these treatments, as well as the risk of complications such as relapse, secondary malignancies, and GVHD, motivates the search for better treatment options. Because CD19 is expressed on both adult and pediatric (pre-B-ALL) B-cell malignancies, this target has been utilized in both antibody and chimeric antigen receptor (CAR) T-cell 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, the CD22 antigen is also present in lymphomas (DLBCL, FL) and leukemias (CLL), making it an attractive additional target for efficiently removing tumors and preventing tumor antigen evasion.

[0009] Current standard treatment for lineage B leukemia may consist of intensification following induction of remission with high-dose chemotherapy or radiation, and, if necessary, stem cell transplantation and additional courses of chemotherapy (see cancer.gov on the World Wide Web). The high toxicity associated with these treatments, as well as the risk of complications such as relapse, secondary malignancies, and GVHD, motivates the search for better treatment options. Because CD19 is expressed on both adult and pediatric (pre-B-ALL) B-cell malignancies, this target has been utilized in both antibody and chimeric antigen receptor (CAR) T-cell 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: 517-28).

[0010] Several novel approaches for treating B-cell leukemia and lymphoma have been developed, including bispecific antibodies that link anti-CD19 or anti-CD22 binding motifs to T-cell binding motifs (i.e., blinatumomab, Blincyto®, which has been shown to be necessary for the treatment of Philadelphia chromosome-negative relapsed or antitherapy-treated precursor B-cell acute lymphoblastic leukemia (ALL)). To date, many of the CD19 or CD22 binding portions used in CAR constructs have utilized domains derived from mouse antibodies. Several of these products, including those developed by Novartis and Kite Pharmaceuticals, are currently under consideration for approval. In April 2017, Novartis announced that CTL019 (tisagen recruitcel) had received FDA breakthrough designation for the treatment of adult patients with anti-treatment-resistant or relapsed (r / r) DLBCL (diffuse large B-cell lymphoma) who had failed two or more previous treatments. This designation has since been extended to include 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. In the JULIET trial, an overall response rate (ORR) of 45% was observed at 3 months, with 37% achieving complete remission (CR) and 8% achieving partial remission (PR). In the ELIANA study, 82% of patients who received the product achieved complete response (CR), including incomplete count recovery, and the relapse-free survival rate at 6 months was 60%. Kite Pharmaceuticals' CAR-T product (KTE-C19, axicapbutagen siloleucel) has been granted Breakthrough Therapy designation for diffuse large B-cell lymphoma (DLBLC), transformed follicular lymphoma (TFL), and primary mediastinal B-cell lymphoma (PMBCL). In the Kite ZUMA-3 Phase II trial of KTE-C19 in r / rALL, a 73% CR rate was reported (at 2 months or longer).Regardless of whether antibodies in CAR-T therapy are utilized or not, there still exists a significant number of patients who have not been helped by such therapies, and there is still room for improvement in the treatment methods.

[0011] Chimeric antigen receptor (CAR) is a hybrid molecule containing three essential units: (1) an extracellular antigen-binding motif, (2) a linker / 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;2(4):e23621). The antigen-binding motif of CAR is generally based on the single-chain variable fragment (scFv), which is the smallest binding domain of the immunoglobulin (Ig) molecule. As alternative antigen-binding motifs, for example, receptor ligands (i.e., IL-13 has been 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) have also been engineered. 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;18(8):2199-209; Lehner M et al. PLoS One. 2012;7(2):e31210). There are still quite a few things to be done regarding defining the most active T cell population for transducing CAR vectors, determining optimal culture and proliferation techniques, and defining the molecular details of the CAR protein structure itself.

[0012] The linking motif of a CAR can be designed to be a relatively stable structural domain, such as the constant domain of IgG, or an extended, flexible linker. Using structural motifs such as those derived from the constant domain of IgG, the scFv-binding domain can be extended away from the T 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 disialoganglioside 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 advancements in new technologies, the activation of T cells by beads linked to anti-CD3 and anti-CD28 antibodies, as well as the presence of the CD28-derived canonical "signal 2," no longer need 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 supported by the clinical success of CD19-specific CARs of 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 modes (Porter 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). The culture conditions under which the CAR T cell population was cultured, such as the inclusion of cytokines IL-2, IL-7, and / or IL-15, are equally important (Kaiser AD et al., Cancer Gene Ther. 2015; Vol. 22(2): pp. 72-78).

[0013] The current challenges in more extensive and effective applications of CAR therapy for cancer relate to the lack of convincing targets. Creating binding factors to cell surface antigens is currently easily achievable, but discovering cell surface antigens specific to tumors while avoiding normal tissues remains a troublesome issue. One potential way to confer high 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 a lower affinity and thus requires the alternative CAR to be bound 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 single scFv-based CARs as immunotherapeutic agents is the heterogeneity of tumor cells. At least one group is developing a CAR strategy for glioblastoma in which the effector cell population targets multiple antigens (HER2, IL-13Ra, EphA2) simultaneously, expecting to avoid the growth of target antigen-negative populations (Hegde M et al. Mol Ther. 2013;21(11):2087-101).

[0014] T cell-based immunotherapy is a new frontier in synthetic biology; multiple promoters and gene products are envisioned to guide these highly potent cells into the tumor microenvironment, where T cells can evade negative regulatory signals and mediate effective tumor death. Elimination of undesirable T cells via drug-induced dimerization of the inducible caspase-9 construct with chemical dimerizers such as AP1903 demonstrates one way in which a potent switch capable of controlling T cell populations can be pharmacologically activated (Di Stasi A et al., N Engl J Med. 2011; vol. 365(18): pp. 1673-1683). The creation of effector T cell populations immune to the negative regulatory effects of transforming growth factor-β through decoy receptor expression further demonstrates the extent to which effector T cells can be manipulated for optimal antitumor activity (Foster AE et al., J Immunother. 2008; vol. 31(5): pp. 500-555). Therefore, while CARs appear to be able to induce T cell activation in a manner similar to endogenous T cell receptors, the main obstacles 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 disappointing clinical activity. This may be partly due to the fact that some of the CAR sequences used are of mouse origin.

[0015] The use of blinatumomab (a bispecific anti-CD19 and anti-CD3 antibody) has shown remarkable results in severely ill patients who received this therapy. Nevertheless, the long-term remission rate is less than 40%, and at best only 50% of responders can be promoted to hematopoietic stem cell transplantation (HSCT) (see Gore et al., 2014, NCT01471782, and Von Stackelberg et al., 2014, NCT01471782, summarized in Therapeutic Advances in Hematology, Vol. 7: pp. 142-156, Benjamin, JE, Stein AS, 2016). The requirements for patients who have received either bispecific antibody therapy or CAR-T therapy to subsequently undergo HSCT in order to maintain a long-term response remain an area of ​​active debate. While high response rates, sometimes exceeding 90%, have been reported for CD19 CAR-T trials, the figures may approach 70% if the trials are recalculated as "intent-to-treat" trials (Davis KL, Mackall CL, 2016, Blood Advances Vol. 1: pp. 265-268). The best reported outcomes at 12 months post-CAR19 treatment were 55% RFS and 79% OS in patients who were able to accept T-cell products 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 Vol. 34, Supplement No. 15 (May 2016), pp. 3011-3011). [Overview of the project] [Problems that the invention aims to solve]

[0016] Therefore, there is an urgent and long-standing need in the field to discover novel compositions and methods for the treatment of B-ALL and other CD19 and / or CD22-expressing B-cell malignancies using techniques that can exhibit specific and effective antitumor effects without the aforementioned drawbacks. [Means for solving the problem]

[0017] 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 CD22 expression, wherein the CARs include tandem CD19 / CD22 antigen-binding domains that exhibit high surface expression in transduced T cells and show high cytolysis of CD19-expressing cells as well as in vivo proliferation and persistence of transduced T cells.

[0018] overview Novel tandem CD19 and CD22 targeted antibodies or their antigen-binding domains (hereinafter referred to as "CD19 / CD22") in which the CD19 targeting portion is positioned either before or after the CD22 targeting portion in the amino acid sequence, as well as chimeric antigen receptors (tandem CARs) containing such CD19 and / or CD22 antigen-binding domains, host cells expressing the receptors (e.g., T cells), and nucleic acid molecules encoding the receptors 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.

[0019] In one embodiment, an isolated nucleic acid molecule is provided encoding a tandem CD19 / CD22 chimeric antigen receptor (CAR) comprising, from N-terminus to C-terminus, at least one CD19 / CD22 antigen-binding domain, at least one transmembrane domain, and at least one intracellular signaling domain, wherein the tandem CD19 / CD22 CAR comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, and 86.

[0020] In one embodiment, an isolated nucleic acid molecule is provided encoding a tandem CD19 / CD22 chimeric antigen receptor (CAR) comprising at least one CD19 / CD22 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 / CD22CAR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, and 86 encodes a tandem CD19 / CD22CAR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, and 87.

[0021] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded extracellular CD19 / CD22 antigen-binding domain contains at least one single-chain variable fragment of an antibody that binds to CD19 / CD22.

[0022] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded extracellular CD19 / CD22 antigen-binding domain includes at least one heavy chain variable region of an antibody that binds to CD19 / CD22.

[0023] In yet another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded CAR extracellular CD19 / CD22 antigen-binding domain further comprises at least one lipocalin-based antigen-binding antigen (anticalin) that binds to CD19 / CD22.

[0024] In one embodiment, an isolated nucleic acid molecule is provided in which an encoded extracellular CD19 / CD22 antigen-binding domain is connected to a transmembrane domain by a linker domain.

[0025] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded CD19 / CD22 extracellular antigen-binding domain is preceded by a sequence encoding a leader or signal peptide.

[0026] In yet another embodiment, an isolated nucleic acid molecule is provided encoding a CAR comprising at least one CD19 / CD22 antigen-binding domain, each encoded by a nucleotide sequence including the CD19 / CD22 nucleotide sequence contained in SEQ ID NOs: 1, 3, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, and 86, 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.

[0027] In one embodiment, the CAR construct comprises two CAR chains co-expressed in the same cell via a 2A ribosome skipping element, one CAR chain containing a targeting domain directed to the CD19 antigen, and the other CAR chain containing a CAR targeting domain directed to the CD22 antigen. Each chain contains a hinge / linker / spacer domain, a transmembrane domain, and a CD3z activating domain fused in-frame to the targeting domain. Each CAR chain may not contain one or more co-stimulatory domains in-frame.

[0028] In one embodiment, the CAR chain contains two successively linked co-stimulatory domains (third-generation CAR).

[0029] In certain embodiments, further encoded extracellular antigen-binding domains include anti-CD22 scFV antigen-binding domains, anti-ROR1 scFV antigen-binding domains, anti-mesoserine scFV antigen-binding domains, anti-CD33 scFV antigen-binding domains, anti-CD38 scFV antigen-binding domains, anti-CD123(IL3RA) scFV antigen-binding domains, anti-CD138 scFV antigen-binding domains, anti-BCMA(CD269) scFV antigen-binding domains, anti-GPC2 scFV antigen-binding domains, anti-GPC3 scFV antigen-binding domains, anti-FGFR4 scFV antigen-binding domains, anti-TSLPR scFV antigen-binding domains, anti-c-Met scFV antigen-binding domains, anti-PMSA scFV antigen-binding domains, anti-glycolipid F77 scFV antigen-binding domains, anti-EGFRvIII scFV antigen-binding domains, anti-GD-2 scFV antigen-binding domains, and anti-NY-ESO-1 Isolated nucleic acid molecules encoding CARs are provided, comprising a 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, or any combination thereof.

[0030] In one embodiment, the CAR provided herein further comprises a linker or spacer domain.

[0031] In one embodiment, an isolated nucleic acid molecule encoding a CAR is provided, in which an extracellular CD19 / CD22 antigen-binding domain, an intracellular signaling domain, or both are connected to a transmembrane domain by a linker or spacer domain.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] In another embodiment, an isolated nucleic acid molecule encoding a CAR is provided, wherein the encoded intracellular signaling domain includes at least one co-stimulatory domain, a primary signaling domain, or a combination thereof.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] In one embodiment, a chimeric antigen receptor (CAR) is provided herein, comprising at least one extracellular CD19 / CD22 antigen-binding domain, at least one transmembrane domain, and at least one intracellular signaling domain, from the N-terminus to the C-terminus.

[0041] In one embodiment, a CAR is provided in which the extracellular CD19 / CD22 antigen-binding domain comprises at least one single-chain variable fragment of an antibody that binds to an antigen, or at least one heavy-chain variable region of an antibody that binds to an antigen, or a combination thereof.

[0042] 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.

[0043] In some embodiments, a CAR is provided which further encodes an extracellular antigen-binding domain comprising the amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, or any combination thereof.

[0044] 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, anti-GD-2 scFV antigen-binding domain, and anti-NY-ESO-1 TCR A CAR is provided comprising an 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, or any combination thereof.

[0045] 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.

[0046] 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.

[0047] In one embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of sequence number 1. In one embodiment, the nucleic acid sequence encodes a CAR containing the amino acid sequence of SEQ ID NO: 2.

[0048] In another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 3.

[0049] In one embodiment, the nucleic acid sequence encodes a CAR containing the amino acid sequence of SEQ ID NO: 4. In one embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of sequence number 60.

[0050] In one embodiment, the nucleic acid sequence encodes a CAR containing the amino acid sequence of SEQ ID NO: 61.

[0051] In one embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of sequence number 62. In one embodiment, the nucleic acid sequence encodes a CAR containing the amino acid sequence of SEQ ID NO: 63.

[0052] In one embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of sequence number 64. In one embodiment, the nucleic acid sequence encodes a CAR containing the amino acid sequence of SEQ ID NO: 65.

[0053] In one embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of sequence number 66. In one embodiment, the nucleic acid sequence encodes a CAR containing the amino acid sequence of SEQ ID NO: 67.

[0054] In one embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of sequence number 68. In one embodiment, the nucleic acid sequence encodes a CAR containing the amino acid sequence of SEQ ID NO: 69.

[0055] In one embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of sequence number 70. In one embodiment, the nucleic acid sequence encodes a CAR containing the amino acid sequence of SEQ ID NO: 71.

[0056] In one embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of sequence number 72. In one embodiment, the nucleic acid sequence encodes a CAR containing the amino acid sequence of SEQ ID NO: 73.

[0057] In one embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of sequence number 74. In one embodiment, the nucleic acid sequence encodes a CAR containing the amino acid sequence of SEQ ID NO: 75.

[0058] In one embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of sequence number 76. In one embodiment, the nucleic acid sequence encodes a CAR containing the amino acid sequence of SEQ ID NO: 77.

[0059] In one embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of sequence number 78. In one embodiment, the nucleic acid sequence encodes a CAR containing the amino acid sequence of SEQ ID NO: 79.

[0060] In one embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of sequence number 80. In one embodiment, the nucleic acid sequence encodes a CAR containing the amino acid sequence of SEQ ID NO: 81.

[0061] In one embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of sequence number 82. In one embodiment, the nucleic acid sequence encodes a CAR containing the amino acid sequence of SEQ ID NO: 83.

[0062] In one embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of sequence number 84. In one embodiment, the nucleic acid sequence encodes a CAR containing the amino acid sequence of SEQ ID NO: 85.

[0063] In one embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of sequence number 86. In one embodiment, the nucleic acid sequence encodes a CAR containing the amino acid sequence of SEQ ID NO: 87.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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 a suicide switch. The suicide switch may include, for example, an apoptosis-inducible signaling cascade or a drug that induces cell death. In a preferred embodiment, the vector expressing CAR may be further modified to express an enzyme such as thymidine kinase (TK) or cytosine deaminase (CD).

[0068] 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.

[0069] In yet another embodiment, a pharmaceutical composition is provided comprising an antitumor-effective population of human T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising the amino acid sequence of SEQ ID NOs: 2, 4, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, and 87, the CAR comprising at least one extracellular antigen-binding domain including a CD19 / CD22 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.

[0070] 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, CD154, TNFRSF19, or a combination thereof.

[0071] 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.

[0072] 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.

[0073] In another embodiment, a method for producing CAR-containing T cells (hereinafter referred to as "CAR T cells") is provided. This method comprises transducing T cells with a disclosed CAR-encoding vector or nucleic acid molecule that specifically binds to CD19 and / or CD22, thereby producing CAR T cells.

[0074] In yet another embodiment, a method is provided for generating a population of RNA-modified cells, comprising introducing in vitro transcribed RNA or synthetic RNA of a disclosed CAR-encoding nucleic acid molecule into target cells to generate CAR cells.

[0075] 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.

[0076] In another embodiment, a method is provided for altering the tumor microenvironment to block T cell inhibition mediated by cells expressing CD19 and / or CD22 and to inhibit tumor growth in a mammal, comprising the step of administering an effective amount of a composition containing a CAR to a mammal, wherein the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, and 87. In one embodiment, the cells are selected from the group consisting of tumor cells expressing CD19 and / or CD22, tumor-associated macrophages, and any combination thereof.

[0077] 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 an effective amount of a composition containing a CAR to a mammal, wherein the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, and 87. 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 CD22, tumor-associated macrophages, and any combination thereof.

[0078] 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.

[0079] 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 CD22 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 CD22 and / or one or more of the aforementioned antigens.

[0080] 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 CD22 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.

[0081] In yet another embodiment, a method is provided for treating cancer in a subject requiring it, 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 comprises the amino acid sequence of SEQ ID NOs: 2, 4, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, and 87, or any combination thereof, and the T cells are T cells of a subject having cancer. In some embodiments of the above-described method, at least one transmembrane domain includes 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, CD154, TNFRSF16, TNFRSF19, or a combination thereof.

[0082] 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 sequence of SEQ ID NOs: 2, 4, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, and 87, 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.

[0083] In one embodiment, the descendant T cells in humans include memory T cells. In another embodiment, the T cells are autologous T cells.

[0084] 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.

[0085] 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.

[0086] 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]

[0087] [Figure 1A] This figure shows a tandem CAR construct that targets CD22 and CD19. Figure 1A: The anti-CD22 and anti-CD19 dual-targeting CAR construct (CAR22-19) was constructed by linking a single-chain variable sequence membrane distal domain that targets the CD22 antigen to a single-chain variable sequence membrane proximal that targets the CD19 antigen via a mobile glycine-serine linker. The resulting CD22-CD19 dual-targeting domain was then in-frame ligated to the CD8 hinge and transmembrane domain, the 4-1BB (CD137) signaling domain, and the CD3 zeta signaling domain. [Figure 1B] This figure shows the tandem CAR constructs that target CD22 and CD19. Figure 1B: The tandem targeting construct CAR19-22 was constructed in the same manner, except that the variable region of the CD19 single-chain fragment (distal to the cell membrane) was linked to the variable region of the CD22 targeting sequence (proximal to the membrane) via a glycineserine mobile linker, and then linked to the CD8, 4-1BB, and CD3 zeta domains. Each CAR T construct can be activated by binding to either the CD19 or CD22 tumor antigen, or both. [Figure 2]This graph shows the surface expression of the tandem CAR T constructs LTG2681 (CAR22-19) and LTG2791 (CAR19-22) in 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 LVs. On day 8 of culture, viable transduced T cells (7-AAD negative) were tested for CAR surface expression using one of three staining methods: CD19 Fc followed by anti-Fc-AF647 (upper panel), or CD22-his reagent followed by anti-his-PE staining (lower panel). The LVs used for transduction are shown at the top of each column. The percentage of the CAR T-positive population relative to the untransduced T cell control (UTD) is shown above each histogram. This shows data representing three separate donors. [Figure 3] This graph shows the in vitro CAR T cell toxicity. Luciferase-based cytotoxicity assays were performed using Raji CD19+CD22+, REH CD19+CD22+, or CD19-CD22- cell line 293T, in which luciferase was stably transduced. Comparison of CAR 22-19 (LTG2681) and CAR19-22 (LTG2719), which differ only in the order of antigen targeting domains. Single-targeted CAR19 (pLTG1538) and CAR22 (pLTG2200) were included as comparison controls, as well as untransduced T cells as negative controls. CAR T cells and target tumor cells were co-incubated overnight at the effector-to-target (E:T) ratio (x axis) described. Error bars represent the mean from three technically replicated experiments. One experiment is shown that represents three standalone experiments using T cells from three donors. [Figure 4A]This graph shows in vitro CAR T cell toxicity against tumor lines A431 or 293T transduced to overexpress only one target antigen, in order to confirm the specificity of each binder domain in CD22 and CD19-targeted CAR T cells. Luciferase-based cytotoxicity assays were performed using 293T cells, 293T CD19+, 293T CD20+, or 293T CD22+ cell lines (Figure 4A), or A431, A431 CD19+, A431 CD22+, or A431 CD20+ cell lines (Figure 4B), in which luciferase was stably transduced. The data points represent the mean of three quantitative values ​​from one experiment that represents three independent experiments performed using CAR T cells from three separate donors. [Figure 4B] This graph shows in vitro CAR T cell toxicity against tumor lines A431 or 293T transduced to overexpress only one target antigen, in order to confirm the specificity of each binder domain in CD22 and CD19-targeted CAR T cells. Luciferase-based cytotoxicity assays were performed using 293T cells, 293T CD19+, 293T CD20+, or 293T CD22+ cell lines (Figure 4A), or A431, A431 CD19+, A431 CD22+, or A431 CD20+ cell lines (Figure 4B), in which luciferase was stably transduced. The data points represent the mean of three quantitative values ​​from one experiment that represents three independent experiments performed using CAR T cells from three separate donors. [Figure 5] This graph shows CAR T cytokine release in response to leukemia cell lines. Cytokine production by CAR-T cells (indicated on the x-axis) after overnight co-culture with Raji leukemia cell lines at a 10:1 E:T ratio was measured using ELISA. The bars represent the mean + SD of three replicated experimental samples. The data are representative of three independent experiments conducted using CAR T cells from three separate donors. [Figure 6A]This is a schematic diagram of various anti-CD22-19 CAR designs with different co-stimulatory domains. It shows second-generation CARs (Figure 6A), third-generation CARs (Figure 6B), or a two-cistronic CAR (Figure 6C) or two second-generation CAR chains (Figure 6D) that co-express in the same cell and possess one second-generation CAR chain targeting the CD19 antigen and another first-generation CAR chain targeting the CD22 antigen. Abbreviations: scFv CAR targeting domain that recognizes the CD19 antigen, a-CD22 - scFv CAR targeting domain that recognizes the CD22 antigen, H - hinge / linker domain, TM - transmembrane domain, Co-stim - co-stimulatory domain, CD3z - CD3 zeta-derived CAR activation domain, 2A - ribosome skip element for two-cistronic CAR expression. [Figure 6B] This is a schematic diagram of various anti-CD22-19 CAR designs with different co-stimulatory domains. It shows second-generation CARs (Figure 6A), third-generation CARs (Figure 6B), or a two-cistronic CAR (Figure 6C) or two second-generation CAR chains (Figure 6D) that co-express in the same cell and possess one second-generation CAR chain targeting the CD19 antigen and another first-generation CAR chain targeting the CD22 antigen. Abbreviations: scFv CAR targeting domain that recognizes the CD19 antigen, a-CD22 - scFv CAR targeting domain that recognizes the CD22 antigen, H - hinge / linker domain, TM - transmembrane domain, Co-stim - co-stimulatory domain, CD3z - CD3 zeta-derived CAR activation domain, 2A - ribosome skip element for two-cistronic CAR expression. [Figure 6C]This is a schematic diagram of various anti-CD22-19 CAR designs with different co-stimulatory domains. It shows second-generation CARs (Figure 6A), third-generation CARs (Figure 6B), or a two-cistronic CAR (Figure 6C) or two second-generation CAR chains (Figure 6D) that co-express in the same cell and possess one second-generation CAR chain targeting the CD19 antigen and another first-generation CAR chain targeting the CD22 antigen. Abbreviations: scFv CAR targeting domain that recognizes the CD19 antigen, a-CD22 - scFv CAR targeting domain that recognizes the CD22 antigen, H - hinge / linker domain, TM - transmembrane domain, Co-stim - co-stimulatory domain, CD3z - CD3 zeta-derived CAR activation domain, 2A - ribosome skip element for two-cistronic CAR expression. [Figure 6D] This is a schematic diagram of various anti-CD22-19 CAR designs with different co-stimulatory domains. It shows second-generation CARs (Figure 6A), third-generation CARs (Figure 6B), or a two-cistronic CAR (Figure 6C) or two second-generation CAR chains (Figure 6D) that co-express in the same cell and possess one second-generation CAR chain targeting the CD19 antigen and another first-generation CAR chain targeting the CD22 antigen. Abbreviations: scFv CAR targeting domain that recognizes the CD19 antigen, a-CD22 - scFv CAR targeting domain that recognizes the CD22 antigen, H - hinge / linker domain, TM - transmembrane domain, Co-stim - co-stimulatory domain, CD3z - CD3 zeta-derived CAR activation domain, 2A - ribosome skip element for two-cistronic CAR expression. [Figure 7] This graph shows the expression of anti-CD22-19 CARs with different transmembrane and costimulatory domains, as detected by flow cytometry. CAR T cells were simultaneously stained for CD19 scFv and CD22 scFv expression. Construct numbers and transmembrane and costimulatory domain arrangements are indicated above each flow chart. The data are representative of three transduction experiments using T cells from different healthy donors. [Figure 8]This graph shows the cell-lytic function of anti-CD22-19CARs with different costimulatory domains incorporated, after co-incubating Raji target cells with CAR T cells for 18 hours, at effector-to-target (ET) ratios of 10, 5, and 2.5. The y-axis shows the percentage of specific target lysis, and the x-axis shows the CAR construct designation. N=3 technically replicated experiments ± SEM. Data are from one experiment representative of three experiments using T cells from separate healthy donors. [Figure 9A] This graph shows the cytokine response to the Raji target for each anti-CD22-19 CAR with different co-stimulatory domain configurations. Effector and target cells were co-cultured at an E:T ratio of 10 for 18 hours, and the supernatant was analyzed for IL-2, TNFα, and IFNγ by ELISA. N=3 technically replicated experiments ± SEM. Data are from one experiment representative of three experiments using T cells from separate healthy donors. [Figure 9B] This graph shows the cytokine response to the Raji target for each anti-CD22-19 CAR with different co-stimulatory domain configurations. Effector and target cells were co-cultured at an E:T ratio of 10 for 18 hours, and the supernatant was analyzed for IL-2, TNFα, and IFNγ by ELISA. N=3 technically replicated experiments ± SEM. Data are from one experiment representative of three experiments using T cells from separate healthy donors. [Figure 9C] This graph shows the cytokine response to the Raji target for each anti-CD22-19 CAR with different co-stimulatory domain configurations. Effector and target cells were co-cultured at an E:T ratio of 10 for 18 hours, and the supernatant was analyzed for IL-2, TNFα, and IFNγ by ELISA. N=3 technically replicated experiments ± SEM. Data are from one experiment representative of three experiments using T cells from separate healthy donors. [Figure 9D]This graph shows the cytokine response to the Raji target for each anti-CD22-19 CAR with different co-stimulatory domain configurations. Effector and target cells were co-cultured at an E:T ratio of 10 for 18 hours, and the supernatant was analyzed for IL-2, TNFα, and IFNγ by ELISA. N=3 technically replicated experiments ± SEM. Data are from one experiment representative of three experiments using T cells from separate healthy donors. [Figure 10A] This figure shows the in vivo functionality of the CAR22_19 construct using a Raji animal model. Figure 10A: Raji-luc tumor cells were engrafted into NSG mice using 5 × 10⁵ Raji / mouse cells. On day 7, 5 × 10⁶ CAR T+ cells were injected into each mouse. The tumor burden measured by bioluminescence imaging at the indicated time points is shown. [Figure 10B] This figure shows the in vivo functionality of the CAR22_19 construct using the Raji animal model. Figure 10B: Graphical summary of tumor growth kinetics in mice treated with a CAR containing a TNFRSF costimulatory domain. N=6 mice / group, mean ± SEM. Bioluminescence data were logarithmically transformed before standard two-way ANOVA and Tukey's multiple comparisons. [Figure 10C] This figure shows the in vivo functionality of the CAR22_19 construct using the Raji animal model. Figure 10C: Graphical summary of tumor growth kinetics in mice treated with CARs containing Ig superfamily costimulatory domains, including both tandem and dual architectures. N=6 mice / group, mean ± SEM. Bioluminescence data were logarithmically transformed before standard two-way ANOVA and Tukey's multiple comparisons. [Figure 10D]This figure shows the in vivo functionality of the CAR22_19 construct using the Raji animal model. Figure 10D: Total Car+ T cells and Car+CD8 T cells in peripheral blood were measured on day 14 and are shown in a scatter dot plot. The lines represent the mean of each group. One-way ANOVA was performed with Dunnett's post-hoc test to compare with the UTD group at each time point. *: P<0.05; *** P<0.001; **** P<0.0001. [Figure 10E] This figure shows the in vivo functionality of the CAR22_19 construct using the Raji animal model. Figure 10E: Total Car+ T cells and Car+CD8 T cells in peripheral blood were measured on day 21 and are shown in a scatter dot plot. The lines represent the mean of each group. One-way ANOVA was performed with Dunnett's post-hoc test to compare with the UTD group at each time point. *: P<0.05; *** P<0.001; **** P<0.0001. [Figure 10F] This figure shows the in vivo functionality of the CAR22_19 construct using the Raji animal model. Figure 10F: Total Car+ T cells and Car+CD8 T cells in peripheral blood were measured on day 28 and are shown in a scatter dot plot. The lines represent the mean of each group. One-way ANOVA was performed with Dunnett's post-hoc test to compare with the UTD group at each time point. *: P<0.05; *** P<0.001; **** P<0.0001. [Figure 10G] This figure shows the in vivo functionality of the CAR22_19 construct using the Raji animal model. Figure 10G: Total Car+ T cells and Car+CD8 T cells in peripheral blood were measured on day 14 and are shown in a scatter dot plot. The lines represent the mean of each group. One-way ANOVA was performed with Dunnett's post-hoc test to compare with the UTD group at each time point. *: P<0.05; ***P<0.001; ****P<0.0001. [Figure 10H]This figure shows the in vivo functionality of the CAR22_19 construct using the Raji animal model. Figure 10H: Total Car+ T cells and Car+CD8 T cells in peripheral blood were measured on day 21 and are shown in a scatter dot plot. The lines represent the mean of each group. One-way ANOVA was performed with Dunnett's post-hoc test to compare with the UTD group at each time point. *: P<0.05; *** P<0.001; **** P<0.0001. [Figure 10I] This figure shows the in vivo functionality of the CAR22_19 construct using the Raji animal model. Figure 10I: Total Car+ T cells and Car+CD8 T cells in peripheral blood were measured on day 28 and are shown in a scatter dot plot. The lines represent the mean of each group. One-way ANOVA was performed with Dunnett's post-hoc test to compare with the UTD group at each time point. *: P<0.05; *** P<0.001; **** P<0.0001. [Figure 11A] This figure shows the characterization of selected CAR22_19 constructs in heterogeneous Raji models. Figure 11A: NSG mice were inoculated with a 1:1:1 mixture of 5 × 10⁵ cells each of Raji19KO, Raji22KO, and parental Raji clones expressing firefly luciferase. 5 million CAR T+ cells were injected per mouse 7 days after tumor inoculation. Tumor load was assessed by bioluminescence on the indicated days. In group D0140*, 6 mice were excluded by outlier testing (ROUT method, Q=0.1%). [Figure 11B] This figure shows the feature determination of selected CAR22_19 constructs in heterogeneous Raji models. Figure 11B: Graphological summary of tumor growth kinetics, N=6 mice / group, mean ± SEM. Bioluminescence data were logarithmically transformed before two-way ANOVA and subsequent Tukey multiple comparisons. [Figure 11C]This figure shows the feature determination of selected CAR22_19 constructs in heterogeneous Raji models. Figure 11C: Peripheral blood was collected on day 29. Genomic DNA was extracted from 1-2 million cells from bone marrow or spleen, or from 100 μl of whole blood. CAR T duration was analyzed using 50 ng of DNA per sample in real-time quantitative PCR. N=5-6 / group, mean ± SD. Statistical analysis was performed by one-way ANOVA followed by Tukey's multiple comparisons. *: P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 11D] This figure shows the feature determination of selected CAR22_19 constructs in heterogeneous Raji models. Figure 11D: Bone marrow and spleen were collected on day 29. Genomic DNA was extracted from 1-2 million cells in the bone marrow or spleen, or from 100 μl of whole blood. CAR T persistence was analyzed using 50 ng of DNA per sample in real-time quantitative PCR. N=5-6 / group, mean ± SD. Statistical analysis was performed by one-way ANOVA followed by Tukey's multiple comparisons. *: P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 11E] This figure shows the feature determination of selected CAR22_19 constructs in a heterogeneous Raji model. Figure 11E: Spleens were collected on day 29. Genomic DNA was extracted from 1-2 million cells of bone marrow or spleen, or from 100 μl of whole blood. CAR T duration was analyzed using 50 ng of DNA per sample in real-time quantitative PCR. N=5-6 / group, mean ± SD. Statistical analysis was performed by one-way ANOVA followed by Tukey's multiple comparisons. *: P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 12A]This figure shows CAR22-19CAR variants against Raji clones with high and low CD22 surface densities in an overnight cytotoxic assay. Figure 12A: Overlaid flow histograms of all Raji cell lines with different CD22 densities are shown. Surface antigen densities for high and low CD22 Raji cell lines are indicated at the top of the histograms. [Figure 12B] This figure shows CAR22-19 CAR variants against Raji clones with high and low CD22 surface density in an overnight cytotoxicity assay. Figure 12B: CAR22-19, CAR22, or UTD control T cells were co-cultured overnight with target cells at E:T ratios of 20:1, 10:1, and 5:1. The percentage of specific target lysis for each CAR construct is shown in panel B. Each data point represents the mean ± SEM of CAR T cells generated from different donors in separate experiments. The dataset represents four different donors: CD22 high cell lines (solid line) and CD22 low cell lines (dotted line). All other groups were compared to LTG2737 CAR by performing a two-way ANOVA test followed by Dunnett's multiple comparison. The asterisk next to the data point indicates the P value: *: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001. [Figure 12C]This figure shows CAR22-19 CAR variants against Raji clones with high and low CD22 surface density in an overnight cytotoxicity assay. Figure 12C: CAR22-19, CAR22, or UTD control T cells were co-cultured overnight with target cells at E:T ratios of 20:1, 10:1, and 5:1. The percentage of specific target lysis for each CAR construct is shown in panel C. Each data point represents the mean ± SEM of CAR T cells generated from different donors in separate experiments. The datasets represent four different donors: CD22 high cell lines (solid line) and CD22 low cell lines (dotted line). All other groups were compared to LTG2737 CAR by performing a two-way ANOVA test followed by Dunnett's multiple comparison. The asterisk next to the data point indicates the P value: *: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001. [Figure 12D] This figure shows CAR22-19 CAR variants against Raji clones with high and low CD22 surface density in an overnight cytotoxicity assay. Figure 12D: CAR22-19, CAR22, or UTD control T cells were co-cultured overnight with target cells at E:T ratios of 20:1, 10:1, and 5:1. The percentage of specific target lysis for each CAR construct is shown in panel D. Each data point represents the mean ± SEM of CAR T cells generated from different donors in separate experiments. Data from D0145 (Figure 12D) shows the mean of three different donor cells: CD22 high cell lines (solid line) and CD22 low cell lines (dotted line). All other groups were compared to LTG2737 CAR by performing a two-way ANOVA test followed by Dunnett's multiple comparisons. The asterisk next to the data point indicates the p-value. *:P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 12E]This figure shows CAR22-19 CAR variants against Raji clones with high and low CD22 surface density in an overnight cytotoxicity assay. Figure 12E: CAR22-19, CAR22, or UTD control T cells were co-cultured overnight with target cells at E:T ratios of 20:1, 10:1, and 5:1. The percentage of specific target lysis for each CAR construct is shown in panel E. Each data point represents the mean ± SEM of CAR T cells generated from different donors in separate experiments. Data from D0139 (Figure 12E) shows the mean from three different donors: CD22 high cell line (solid line) and CD22 low cell line (dotted line). All other groups were compared to LTG2737 CAR by two-way ANOVA and subsequent Dunnett's multiple comparisons. The asterisk next to the data point indicates the p-value. *:P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 12F] This figure shows CAR22-19 CAR variants against Raji clones with high and low CD22 surface density in an overnight cytotoxicity assay. Figure 12F: CAR22-19, CAR22, or UTD control T cells were co-cultured overnight with target cells at E:T ratios of 20:1, 10:1, and 5:1. The percentage of specific target lysis for each CAR construct is shown in panel F. Each data point represents the mean ± SEM of CAR T cells generated from different donors in separate experiments. The datasets represent four different donors: CD22 high cell lines (solid line) and CD22 low cell lines (dotted line). All other groups were compared to LTG2737 CAR by performing a two-way ANOVA test followed by Dunnett's multiple comparison. The asterisk next to the data point indicates the P value: *: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001. [Figure 12G]This figure shows CAR22-19 CAR variants against Raji clones with high and low CD22 surface density in an overnight cytotoxicity assay. Figure 12G: CAR22-19, CAR22, or UTD control T cells were co-cultured overnight with target cells at E:T ratios of 20:1, 10:1, and 5:1. The percentage of specific target lysis for each CAR construct is shown in panel G. Each data point represents the mean ± SEM of CAR T cells generated from different donors in separate experiments. The dataset represents four different donors: CD22 high cell lines (solid line) and CD22 low cell lines (dotted line). All other groups were compared to LTG2737 CAR by performing a two-way ANOVA test followed by Dunnett's multiple comparison. The asterisk next to the data point indicates the P value: *: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001. [Figure 12H] This figure shows CAR22-19 CAR variants against Raji clones with high and low CD22 surface density in an overnight cytotoxicity assay. Figure 12H: CAR22-19, CAR22, or UTD control T cells were co-cultured overnight with target cells at E:T ratios of 20:1, 10:1, and 5:1. The percentage of specific target lysis for each CAR construct is shown in panel H. Each data point represents the mean ± SEM of CAR T cells generated from different donors in separate experiments. The dataset represents four different donors: CD22 high cell lines (solid line) and CD22 low cell lines (dotted line). All other groups were compared to LTG2737 CAR by performing a two-way ANOVA test followed by Dunnett's multiple comparison. The asterisk next to the data point indicates the P value: *: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001. [Figure 12I]This figure shows CAR22-19 CAR variants against Raji clones with high and low CD22 surface density in an overnight cytotoxicity assay. Figure 12I: CAR22-19, CAR22, or UTD control T cells were co-cultured overnight with target cells at E:T ratios of 20:1, 10:1, and 5:1. The percentage of specific target lysis for each CAR construct is shown in Panel I. Each data point represents the mean ± SEM of CAR T cells generated from different donors in separate experiments. Data from D0138 (Figure 12I) shows the mean of three different donor cells: CD22 high cell lines (solid line) and CD22 low cell lines (dotted line). All other groups were compared to LTG2737 CAR by performing a two-way ANOVA test followed by Dunnett's multiple comparisons. The asterisk next to the data point indicates the p-value. *:P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 12J] This figure shows CAR22-19 CAR variants against Raji clones with high and low CD22 surface density in an overnight cytotoxicity assay. Figure 12J: CAR22-19, CAR22, or UTD control T cells were co-cultured overnight with target cells at E:T ratios of 20:1, 10:1, and 5:1. The percentage of specific target lysis for each CAR construct is shown in panel J. Each data point represents the mean ± SEM of CAR T cells generated from different donors in separate experiments. The datasets represent four different donors: CD22 high cell lines (solid line) and CD22 low cell lines (dotted line). All other groups were compared to LTG2737 CAR by performing a two-way ANOVA test followed by Dunnett's multiple comparison. The asterisk next to the data point indicates the P value: *: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001. [Figure 12K]This figure shows CAR22-19 CAR variants against Raji clones with high and low CD22 surface density in an overnight cytotoxicity assay. Figure 12K: CAR22-19, CAR22, or UTD control T cells were co-cultured overnight with target cells at E:T ratios of 20:1, 10:1, and 5:1. The percentage of specific target lysis for each CAR construct is shown in panel K. Each data point represents the mean ± SEM of CAR T cells generated from different donors in separate experiments. Data from D0147 (Figure 12K) shows the mean of two different donor cells: CD22 high cell line (solid line) and CD22 low cell line (dotted line). All other groups were compared to LTG2737 CAR by performing a two-way ANOVA test followed by Dunnett's multiple comparisons. The asterisk next to the data point indicates the P value. *:P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 12L] This figure shows CAR22-19 CAR variants against Raji clones with high and low CD22 surface density in an overnight cytotoxicity assay. Figure 12L: CAR22-19, CAR22, or UTD control T cells were co-cultured overnight with target cells at E:T ratios of 20:1, 10:1, and 5:1. The percentage of specific target lysis for each CAR construct is shown in panel L. Each data point represents the mean ± SEM of CAR T cells generated from different donors in separate experiments. Data from D0148 (Figure 12L) shows the mean of three different donor cells: CD22 high cell lines (solid line) and CD22 low cell lines (dotted line). All other groups were compared to LTG2737 CAR by performing a two-way ANOVA test followed by Dunnett's multiple comparisons. The asterisk next to the data point indicates the p-value. *:P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 12M]This figure shows CAR22-19 CAR variants against Raji clones with high and low CD22 surface density in an overnight cytotoxicity assay. Figure 12M: CAR22-19, CAR22, or UTD control T cells were co-cultured overnight with target cells at E:T ratios of 20:1, 10:1, and 5:1. The percentage of specific target lysis for each CAR construct is shown in panel M. Each data point represents the mean ± SEM of CAR T cells generated from different donors in separate experiments. Data from D0149 (Figure 12M) shows the mean of two different donor cells: CD22 high cell line (solid line) and CD22 low cell line (dotted line). All other groups were compared to LTG2737 CAR by performing a two-way ANOVA test followed by Dunnett's multiple comparisons. The asterisk next to the data point indicates the P value. *:P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 12N] This figure shows CAR22-19 CAR variants against Raji clones with high and low CD22 surface density in an overnight cytotoxic assay. Figure 12N: CAR22-19, CAR22, or UTD control T cells were co-cultured overnight with target cells at E:T ratios of 20:1, 10:1, and 5:1. The percentage of specific target lysis for each CAR construct is shown in panel N. Each data point represents the mean ± SEM of CAR T cells generated from different donors in separate experiments. Data from D0186 (Figure 12N) shows the mean of three different donor cells: CD22 high cell lines (solid line) and CD22 low cell lines (dotted line). All other groups were compared to LTG2737 CAR by performing a two-way ANOVA test followed by Dunnett's multiple comparisons. The asterisk next to the data point indicates the p-value. *:P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 12O]This figure shows CAR22-19 CAR variants against Raji clones with high and low CD22 surface density in an overnight cytotoxicity assay. Figure 12O: CAR22-19, CAR22, or UTD control T cells were co-cultured overnight with target cells at E:T ratios of 20:1, 10:1, and 5:1. The percentage of specific target lysis for each CAR construct is shown in panel O. Each data point represents the mean ± SEM of CAR T cells generated from different donors in separate experiments. The dataset represents four different donors: CD22 high cell lines (solid line) and CD22 low cell lines (dotted line). All other groups were compared to LTG2737 CAR by performing a two-way ANOVA test followed by Dunnett's multiple comparison. The asterisk next to the data point indicates the P value: *: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001. [Figure 12P] This figure shows CAR22-19 CAR variants against Raji clones with high and low CD22 surface density in an overnight cytotoxicity assay. Figure 12P: CAR22-19, CAR22, or UTD control T cells were co-cultured overnight with target cells at E:T ratios of 20:1, 10:1, and 5:1. The percentage of specific target lysis for each CAR construct is shown in panel P. Each data point represents the mean ± SEM of CAR T cells generated from different donors in separate experiments. The dataset represents four different donors: CD22 high cell lines (solid line) and CD22 low cell lines (dotted line). All other groups were compared to LTG2737 CAR by performing a two-way ANOVA test followed by Dunnett's multiple comparison. The asterisk next to the data point indicates the P value: *: P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 13A] This figure shows the sensitivity of CAR22-19 variants to Raji clone strains with high and low CD19 densities. Figure 13A. Flow overlay histograms show the surface CD19 expression levels of each cell line, with the calculated surface target molecule counts indicated on the panel. [Figure 13B]This figure shows the sensitivity of CAR22-19 variants to Raji clone strains with high and low CD19 densities. Figure 13B shows that CAR22-19, CAR19, or UTD control T cells were co-cultured overnight with target cells at E:T ratios of 20:1, 10:1, and 5:1. The percentage of specific target lysis for each construct is shown individually. Solid lines represent high CD19 cell lines, and dotted lines represent low CD19 cell lines. Datasets were generated from four different donors in separate experiments. Each data point shows the mean ± SEM. All other groups were compared to BB CAR by two-way ANOVA and subsequent Dunnett's multiple comparisons. The asterisks next to the data points indicate the P-values: *:P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 13C] This figure shows the sensitivity of CAR22-19 variants to Raji clone strains with high and low CD19 densities. Figure 13C shows that CAR22-19, CAR19, or UTD control T cells were co-cultured overnight with target cells at E:T ratios of 20:1, 10:1, and 5:1. The percentage of specific target lysis for each construct is shown individually. Solid lines represent high CD19 cell lines, and dotted lines represent low CD19 cell lines. Datasets were generated from four different donors in separate experiments. Each data point shows the mean ± SEM. All other groups were compared to BB CAR by two-way ANOVA and subsequent Dunnett's multiple comparisons. The asterisks next to the data points indicate the P-values: *:P<0.05;**:P<0.01;***:P<0.001;****:P<0.0001. [Figure 13D]This figure shows the sensitivity of CAR22-19 variants to Raji clone strains with high and low CD19 densities. Figure 13D shows that CAR22-19, CAR19, or UTD control T cells were co-cultured overnight with target cells at E:T ratios of 20:1, 10:1, and 5:1. The percentage of specific target lysis for each construct is shown individually. Solid lines represent high CD19 cell lines, and dotted lines represent low CD19 cell lines. Datasets were generated from four different donors in separate experiments. Each data point shows the mean ± SEM. All other groups were compared to BB CAR by performing a two-way ANOVA test followed by Dunnett's multiple comparisons. The asterisk next to the data point indicates the P-value: *:P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 13E] This figure shows the sensitivity of CAR22-19 variants to Raji clone strains with high and low CD19 densities. Figure 13E shows that CAR22-19, CAR19, or UTD control T cells were co-cultured overnight with target cells at E:T ratios of 20:1, 10:1, and 5:1. The percentage of specific target lysis for each construct is shown individually. Solid lines represent high CD19 cell lines, and dotted lines represent low CD19 cell lines. Data from D0139 (Figure 13E) show the mean from three different donors. Each data point shows mean ± SEM. All other groups were compared to BB CAR by performing a two-way ANOVA test followed by Dunnett's multiple comparisons. The asterisk next to the data point indicates the P value: *:P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 13F]This figure shows the sensitivity of CAR22-19 variants to Raji clone strains with high and low CD19 densities. Figure 13F shows that CAR22-19, CAR19, or UTD control T cells were co-cultured overnight with target cells at E:T ratios of 20:1, 10:1, and 5:1. The percentage of specific target lysis for each construct is shown individually. Solid lines represent high CD19 cell lines, and dotted lines represent low CD19 cell lines. Datasets were generated from four different donors in separate experiments. Each data point shows the mean ± SEM. All other groups were compared to BB CAR by two-way ANOVA and subsequent Dunnett's multiple comparisons. The asterisks next to the data points indicate the P-values: *:P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 13G] This figure shows the sensitivity of CAR22-19 variants to Raji clone strains with high and low CD19 densities. Figure 13G shows that CAR22-19, CAR19, or UTD control T cells were co-cultured overnight with target cells at E:T ratios of 20:1, 10:1, and 5:1. The percentage of specific target lysis for each construct is shown individually. Solid lines represent high CD19 cell lines, and dotted lines represent low CD19 cell lines. Data from D0145 (Figure 13G) show the mean from three different donors. Each data point shows mean ± SEM. All other groups were compared to BB CAR by performing a two-way ANOVA test followed by Dunnett's multiple comparisons. The asterisk next to the data point indicates the P value: *:P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 13H]This figure shows the sensitivity of CAR22-19 variants to Raji clone strains with high and low CD19 densities. Figure 13H, CAR22-19, CAR19, or UTD control T cells were co-cultured overnight with target cells at E:T ratios of 20:1, 10:1, and 5:1. The percentage of specific target lysis for each construct is shown individually. Solid lines represent high CD19 cell lines, and dotted lines represent low CD19 cell lines. Datasets were generated from four different donors in separate experiments. Each data point shows mean ± SEM. All other groups were compared to BB CAR by two-way ANOVA and subsequent Dunnett's multiple comparisons. The asterisk next to the data point indicates the P value: *:P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 13I] This figure shows the sensitivity of CAR22-19 variants to Raji clone strains with high and low CD19 densities. Figure 13I shows that CAR22-19, CAR19, or UTD control T cells were co-cultured overnight with target cells at E:T ratios of 20:1, 10:1, and 5:1. The percentage of specific target lysis for each construct is shown individually. Solid lines represent high CD19 cell lines, and dotted lines represent low CD19 cell lines. Data from D0138 (Figure 13I) show the mean from three different donors. Each data point shows mean ± SEM. All other groups were compared to BB CAR by performing a two-way ANOVA test followed by Dunnett's multiple comparisons. The asterisk next to the data point indicates the P value: *:P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 13J]This figure shows the sensitivity of CAR22-19 variants to Raji clone strains with high and low CD19 densities. Figure 13J shows that CAR22-19, CAR19, or UTD control T cells were co-cultured overnight with target cells at E:T ratios of 20:1, 10:1, and 5:1. The percentage of specific target lysis for each construct is shown individually. Solid lines represent high CD19 cell lines, and dotted lines represent low CD19 cell lines. Datasets were generated from four different donors in separate experiments. Each data point shows the mean ± SEM. All other groups were compared to BB CAR by performing a two-way ANOVA test followed by Dunnett's multiple comparisons. The asterisk next to the data point indicates the P value: *:P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 13K] This figure shows the sensitivity of CAR22-19 variants to Raji clone strains with high and low CD19 densities. Figure 13K shows that CAR22-19, CAR19, or UTD control T cells were co-cultured overnight with target cells at E:T ratios of 20:1, 10:1, and 5:1. The percentage of specific target lysis for each construct is shown individually. Solid lines represent high CD19 cell lines, and dotted lines represent low CD19 cell lines. Data from D0147 (Figure 13K) were derived from two different donors. Each data point shows mean ± SEM. All other groups were compared to BB CAR by performing a two-way ANOVA test followed by Dunnett's multiple comparisons. The asterisk next to the data point indicates the P value: *:P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 13L]This figure shows the sensitivity of CAR22-19 variants to Raji clone strains with high and low CD19 densities. Figure 13L shows that CAR22-19, CAR19, or UTD control T cells were co-cultured overnight with target cells at E:T ratios of 20:1, 10:1, and 5:1. The percentage of specific target lysis for each construct is shown individually. Solid lines represent high CD19 cell lines, and dotted lines represent low CD19 cell lines. Data from D0148 (Figure 13L) show the mean from three different donors. Each data point shows mean ± SEM. All other groups were compared to BB CAR by performing a two-way ANOVA test followed by Dunnett's multiple comparisons. The asterisk next to the data point indicates the P value. *:P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 13M] This figure shows the sensitivity of CAR22-19 variants to Raji clone strains with high and low CD19 densities. Figure 13M shows that CAR22-19, CAR19, or UTD control T cells were co-cultured overnight with target cells at E:T ratios of 20:1, 10:1, and 5:1. The percentage of specific target lysis for each construct is shown individually. Solid lines indicate high CD19 cell lines, and dotted lines indicate low CD19 cell lines. Data from D0149 (Figure 13M) were derived from two different donors. Each data point shows mean ± SEM. All other groups were compared to BB CAR by performing a two-way ANOVA test followed by Dunnett's multiple comparisons. The asterisk next to the data point indicates the P value: *:P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 13N]This figure shows the sensitivity of CAR22-19 variants to Raji clone strains with high and low CD19 densities. Figure 13N, CAR22-19, CAR19, or UTD control T cells were co-cultured overnight with target cells at E:T ratios of 20:1, 10:1, and 5:1. The percentage of specific target lysis for each construct is shown individually. Solid lines represent high CD19 cell lines, and dotted lines represent low CD19 cell lines. Data from D0186 (Figure 13N) show the mean from three different donors. Each data point shows mean ± SEM. All other groups were compared to BB CAR by performing a two-way ANOVA test followed by Dunnett's multiple comparisons. The asterisk next to the data point indicates the P value. *:P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 13O] This figure shows the sensitivity of CAR22-19 variants to Raji clone strains with high and low CD19 densities. Figure 130 shows that CAR22-19, CAR19, or UTD control T cells were co-cultured overnight with target cells at E:T ratios of 20:1, 10:1, and 5:1. The percentage of specific target lysis for each construct is shown individually. Solid lines represent high CD19 cell lines, and dotted lines represent low CD19 cell lines. Datasets were generated from four different donors in separate experiments. Each data point shows the mean ± SEM. All other groups were compared to BB CAR by performing a two-way ANOVA test followed by Dunnett's multiple comparisons. The asterisk next to the data point indicates the P value: *:P<0.05;**P<0.01;***P<0.001;****P<0.0001. [Figure 13P]This figure shows the sensitivity of CAR22-19 variants to Raji clone strains with high and low CD19 densities. Figure 13P shows that CAR22-19, CAR19, or UTD control T cells were co-cultured overnight with target cells at E:T ratios of 20:1, 10:1, and 5:1. The percentage of specific target lysis for each construct is shown individually. Solid lines represent high CD19 cell lines, and dotted lines represent low CD19 cell lines. Datasets were generated from four different donors in separate experiments. Each data point shows the mean ± SEM. All other groups were compared to BB CAR by two-way ANOVA and subsequent Dunnett's multiple comparisons. The asterisks next to the data points indicate the P-values: *:P<0.05;**:P<0.01;***:P<0.001;****:P<0.0001. [Modes for carrying out the invention]

[0088] Detailed explanation definition As used herein, the singular forms “a,” “an,” and “the” refer to both the 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” or “or.” 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, should be understood to be 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 conflict, 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 further consideration of the various embodiments, the following definitions of terms are provided.

[0089] 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.

[0090] Unless otherwise specified, technical terms herein are used in accordance with their conventional usage. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes VII, 1999, published by Oxford University Press; Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, 1994, published by Blackwell Science Ltd.; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, 1995, published by VCH Publishers, Inc.; and other similar references.

[0091] This disclosure provides CD19 / CD22 antibodies or fragments thereof, and chimeric antigen receptors (CARs) having such CD19 / CD22 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 exhibit both high levels of cytokine-induced cytolysis and cell surface expression in transduced T cells, along with increased T cell proliferation and sustained levels in vivo of transduced CAR-expressing T cells. The CARs of this disclosure can be used to treat multiple patient populations (i.e., CD19+, CD22+, or dual CD19+CD22+ cancer patients) using a single CART lentiviral product, which is advantageous in terms of flexibility in resource-limited situations.

[0092] 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.

[0093] 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.

[0094] 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 / CD22 antigen on their surface to which the CAR binds. The use of an extracellular CD19 / CD22 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 / CD22ScFv 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 / CD22. This last characteristic means that tumors express more CD19 / CD22 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.

[0095] A detailed description of the CARs of the present invention, including a description of their extracellular CD19 / CD22 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.

[0096] A. Chimeric antigen receptor (CAR) The CARs disclosed herein include at least one CD19 / CD22 antigen-binding domain capable of binding to CD19 / CD22, at least one transmembrane domain, and at least one intracellular domain.

[0097] 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).

[0098] 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.

[0099] 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. Thus, examples of cell surface markers that can act as ligands for the antigen-binding domain in the CAR include those associated with viruses, bacterial and parasitic infections, autoimmune diseases, and cancer cells.

[0100] 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, survivor 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 / CD22. 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.

[0101] 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, CD22, BCMA, ROR1, and CD37 are other candidate target antigens in B-cell lymphomas. Some of these antigens (CEA, HER-2, CD19, CD22, idiotypes) have been used as targets for passive immunotherapy with monoclonal antibodies, with limited success.

[0102] In one preferred embodiment, the tumor antigen is CD19 / CD22, and tumors associated with CD19 / CD22 expression include pulmonary mesothelioma, ovarian and pancreatic cancers, or any combination thereof, that express high levels of the extracellular protein CD19 / CD22.

[0103] 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.

[0104] 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, MAGE-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.

[0105] 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.

[0106] In a preferred embodiment, the antigen-binding domain of the CAR targets the extracellular CD19 / CD22 antigen.

[0107] In various embodiments of the CD19 / CD22-specific CARs disclosed herein, the general scheme is shown in Figures 1A and 1B and comprises, from N-terminus to C-terminus, a signal or leader peptide, an anti-CD19 / CD22 ScFv (where the CD19 binder is distal to the T cell membrane and the CD22 binder is proximal to the T cell membrane, or where the CD22 binder is distal to the T cell membrane and the CD19 binder is proximal to the T cell membrane), a CD8 extracellular linker, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta-activating domain.

[0108] In one embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 1 (leader-CD22VH-(GGGGS)-3 CD22VL(GGGGS)-5 CD19VH(GGGGS)-3 CD19VL CD8 hinge+TM-4-1BB-CD3z(construct 2219)) and encodes a CAR that includes the amino acid sequence shown in SEQ ID NO: 2 (leader-CD22VH-(GGGGS)-3 CD22VL(GGGGS)-5 CD19VH(GGGGS)-3 CD19VL CD8 hinge+TM-4-1BB-CD3z(construct 2219).

[0109] 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 thereof, and encodes a CAR including the amino acid sequence shown in SEQ ID NO: 2, or a sequence leader having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereof -CD22VH-(GGGGS)-3 CD22VL(GGGGS)-5 CD19VH(GGGGS)-3 CD19VL CD8 hinge+TM-4-1BB-CD3z (construct 2219).

[0110] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 3 (leader-CD19VH(GGGGS)3-CD19VL-(GGGGS)5-CD22VL-(GGGGS)3-CD22VH CD8 hinge+TM-4-1BB-CD3z(construct 1922) (Figure 2)) and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 4 [leader-CD19VH(GGGGS)3-CD19VL-(GGGGS)5-CD22VL-(GGGGS)3-CD22VH CD8 hinge+TM-4-1BB-CD3z(construct 1922)].

[0111] 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 thereof, and encodes a CAR including the amino acid sequence shown in SEQ ID NO: 4, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereof (leader-CD19VH(GGGGS)3-CD19VL-(GGGGS)5-CD22VL-(GGGGS)3-CD22VHCD8 hinge+TM-4-1BB-CD3z(construct 1922)).

[0112] The surface expression of anti-CD19 / CD22 CARs incorporating a single-chain fragment variable (ScFv) sequence that is reactive with the CD19 / CD22 antigen is shown below in Example 2. The expression levels for each ScFv-containing CAR were determined by flow cytometry analysis of LV transducible T cells from healthy donors using one of two detection methods: i) CD22 his followed by anti-his-FL, or ii) CD19 Fc recombinant protein followed by anti-Fc-FL. The ScFv-based anti-CD19 / CD22 CAR constructs, CAR22-19 and CAR19-22, were highly expressed in human primary T cells compared to non-transduced T cell controls.

[0113] As shown in Example 2 and Figure 3, the high cytolytic activity of CD19 / CD22CAR was demonstrated. Figure 3: Human primary T cells were transduced with LV encoding the CAR construct (CAR22-19 (LTG2681, D0023), CAR19-22 (D0024), CAR19 (LTG1538), or CAR22 (LTG2200); see Methods), and then incubated for 18 hours with Raji, REH, K562, or 293T cell lines stably transduced with firefly luciferase for a fluorescence-based in vitro cell death assay. Raji and Reh leukemia lines express CD19 and CD22 on their surface, while the negative control 293T does not. Raji and REH cells were effectively lysed by tandem CAR22-19 (LTG2681), tandem CAR19-22 (LTG2719), and single-targeted CAR19 (LTG1538) and CAR22 (LTG2200). These results demonstrate targeted antigen-limited cell death by single-CAR controls and tandem CD19-targeted and CD22-targeted CARs.

[0114] As additional specificity controls, 293T-luc and A431-luc strains expressing CD19, CD20, or CD22 were generated (Figures 4A and 4B). 293T-19+ cells were lysed by CAR19 (LTG1538) and tandem CAR22-19 (LTG2681), but not by CAR22 LTG2200 or non-transduced T cell controls (Figure 3). 293T-CD22+ cells were lysed by tandem CAR22-19 (LTG2681) and single CAR20 LTG2200, but not by single CAR19 (LTG1538) or non-transduced controls, demonstrating the antigen specificity of the tandem CAR. Finally, 293T luc CD20 cells were not lysed by the CAR construct because this antigen was not targeted (Figure 4A). Similarly, the A431-luc 19 strain was lysed by tandem CAR LTG2681 or single CAR19 LTG1538, but not by CAR22 LTG2200 or UTD control. Conversely, A431-luc CD22 cells were lysed by tandem CAR LTG2681 or single CAR22 LTG2200, but not by CD19 CAR LTG1538 or UTD control. In particular, the A431-luc CD20 strain, which expresses an unrelated antigen, was not lysed by the CAR construct because the CD20 antigen was not targeted (Figure 4B). This result highlights the independent functionality and specificity of each targeting domain of tandem CAR22-19 (Figure 4). Furthermore, this experiment demonstrated that tandem CAR22-19 is effective against tumor cells even when one of the two antigens (CD19 CD22) is downregulated and no longer expressed. Therefore, tandem CARs, in contrast to single CARs, may mitigate tumor antigen escape.

[0115] Next, the cytokine secretion capacity of anti-CD19 / CD22CAR T cells was evaluated (Figure 5). CD19+CD22+Raji tumor cells were co-incubated overnight with tandem 22-19CAR T cells (LTG2681), positive control CAR19 (LTG1538), positive control CAR22 (LTG2200), or negative control untransduced T cells (UTD) in an effector-to-target ratio of 10:1. The culture supernatant was analyzed by ELISA for IFN-gamma, TNF-alpha, and IL-2. Tandem CAR22-19 (LTG2681) strongly induced cytokines in response to tumor cells, while the negative control (untransduced, UTD) did not produce any detectable cytokine induction. In particular, LTG2681, a tandem CAR T-expressing cell, showed cytokine responses of IFN-gamma, TNF-alpha, and IL-2 at similar levels to the CAR22 control (LTG2200), and slightly higher than the single CAR19 (LTG1538) control, demonstrating the high efficacy of tandem CAR. Importantly, CAR22-19 did not produce cytokine secretion in the absence of tumor cells (CART monotherapy group), further confirming CAR specificity and suggesting the absence of sustained tonic signaling by tandem CAR.

[0116] In another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 60 [CD22-19 CD8 BBz (Construct LTG2737) (Figures 6, 7, 8, and 9, respectively)] and encodes a CAR including the amino acid sequence [CD22-19 CD8 BBz (Construct LTG2737)] shown in SEQ ID NO: 61.

[0117] In another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 60, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereof, and encodes a CAR including the amino acid sequence shown in SEQ ID NO: 61, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereof (CD22-19 CD8 BBz (Construct LTG2737)).

[0118] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 64 [CD22-19 CD8 ICOSz DNA (Construct D0136) (Figures 6, 7, 8, and 9, respectively)] and encodes a CAR comprising the amino acid sequence [CD22-19 CD8 ICOSz DNA (Construct D0136)] shown in SEQ ID NO: 65.

[0119] In another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 64, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereof, and encodes a CAR including the amino acid sequence shown in SEQ ID NO: 65, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereof (CD22-19 CD8 ICOSz DNA (Construct D0136)).

[0120] In another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 70 [CD22-19 CD28 CD28z (Construct D0139) (Figures 6, 7, 8, and 9, respectively)] and encodes a CAR including the amino acid sequence [CD22-19 CD28 CD28z (Construct D0139)] shown in SEQ ID NO: 71.

[0121] In another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 70, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereof, and encodes a CAR including the amino acid sequence shown in SEQ ID NO: 71, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereof (CD22-19 CD28 CD28z (Construct D0139)).

[0122] In another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 76 [CD19 CD8H&TM ICOS z_CD22 CD8H&TM 3z (Construct D0146) (Figures 6, 7, 8, and 9, respectively)] and encodes a CAR including the amino acid sequence shown in SEQ ID NO: 77 [CD19 CD8H&TM ICOS z_CD22 CD8H&TM 3z (Construct D0146)].

[0123] In another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NO: 76, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereof, and encodes a CAR including the amino acid sequence shown in SEQ ID NO: 77, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereof (CD19 CD8H&TM ICOS z_CD22 CD8H&TM 3z (Construct D0146)).

[0124] In another embodiment, the nucleic acid sequence encoding the CAR includes the nucleic acid sequence of SEQ ID NOs. 62, 66, 68, 72, 74, 78, 80, 82, 84, and 86, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity, and encodes a CAR including the amino acid sequence shown in SEQ ID NOs. 63, 67, 69, 73, 75, 79, 81, 83, 85, and 87, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.

[0125] The surface expression of anti-CD22 / CD19CARs incorporating a single-chain fragment variable (ScFv) sequence that is reactive with the CD22 / CD19 antigen is shown below in Example 3.

[0126] In one embodiment, a dual-targeting CAR construct composed of different co-stimulatory domains was designed. The CAR constructs are listed in Table 1 of Example 3 below. Schematic diagrams of the CAR design configurations are shown in Figures 6A-C. In some embodiments, a tandem CAR antigen-binding domain composed of anti-CD22 ScFv and anti-CD19 scFv sequences was linked in tandem in the order of anti-CD22 scFv-anti-CD19-scFv-hinge-transmembrane domain-internal domain (Figures 6A, 6B). This targeted tandem domain configuration is based on CAR22-19 (LTG2681) in all cases. The anti-CD22-19 CAR construct LTG2737 is identical to the anti-CD22-19 CAR construct LTG2681, but it contained a CAR sequence that did not use the woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE) during its construction.

[0127] In another embodiment, several CAR T constructs were prepared by in-frame ligation of the CAR antigen-binding domain to the CD8a hinge and transmembrane domain, as described in Example 3 below (constructs LTG2737, D0135, D0136, D0137, D0145, D0146, D0147, D0148, and D0149). In other constructs, transmembrane domain sequences matching the co-stimulatory domain were used, namely CD28 for D139 and D140, and OX40 for D0137, D0147, and D0148. The transmembrane domains were in-frame linked to co-stimulatory domains derived from 4-1BB (LTG2737), CD28 (D0135, D0139, D0140), ICOS (D0136, D0146, D0148, D0149), OX40 (D0137, D0145, D0147, D0148), or CD27 (D0138, D0149). All CAR molecules contained a CD3 zeta signaling domain (CD247, aa52-163, Ref sequence ID: NP_000725.1).

[0128] In further embodiments, the internal domain of the CAR consisted of two tandem-connected costimulatory domains, namely CD28 and 4-1BB (D0140, Figure 6B). In some embodiments, two distinct CAR molecules were co-expressed in the same T cell using a 2A ribosome skipping element for dicistronic expression (D146, D147, D148, D149, Figures 6C, 6D). In this configuration, each CAR chain contained only one scFv targeting either the CD19 or CD22 antigen, and both chains were co-expressed in each transduced T cell by transduction of a single lentiviral vector encoding a dicistronic sequence.

[0129] In yet another embodiment, no co-stimulatory domain was used for at least one of the CAR chains, resulting in the CD3ζ activation domain being directly ligated in-frame to one of the transmembrane domains of the CAR chains co-expressed in the same cell (D0146, D0147, Figure 6C). The CAR construct sequences were cloned into a third-generation lentiviral plasmid backbone under the control of the human EF-1α promoter (Lentigen Technology Inc., Gaithersburg, MD).

[0130] Figure 7 shows the surface expression of anti-CD22-19 CARs incorporating various co-stimulatory domains. Expression levels for each CAR, including ScFv, were determined by flow cytometry analysis of LV transducer T cells from healthy donors, using simultaneous staining for two scFv CAR targeting domains: i) CD22-his followed by anti-his-PE, and ii) CD19 Fc recombinant protein followed by anti-Fc-A647. All anti-CD22-19 CARs were highly expressed in human primary T cells compared to non-transducer T cell controls. CAR expression levels ranged from 71% to 90% (Figure 7).

[0131] As shown in Figures 9A–9D, the high cytolytic activity of anti-CD22-19CAR was demonstrated. Human primary T cells were transduced with LV encoding the CAR constructs LTG2737, D0135, D0136, D0137, D0138, D0139, D0140, D0145, D0146), and then incubated for 18 hours with Raji, 293T, 293TCD19, or 293TCD22 cell lines stably transduced with firefly luciferase for a fluorescence-based in vitro cell death assay. Effector-to-target (ET) ratios of 2.5:1, 5:1, or 10:1 were used, as shown in the legend to the right of each plot. Raji cells express CD19 and CD22 on their surface, while the negative control 293T does not. Target strains 293TCD19 and 293TCD22 were constructed to stably express either the CD19 or CD22 target antigen, respectively. Using these strains, the ability of dual-targeting CAR constructs with different costimulatory domains to achieve targeted lysis when each single target antigen, CD19 or CD22, is induced independently of the other antigen was evaluated.

[0132] Raji cells were effectively lysed by all dual-targeted CARs but not by negative control non-transduced T cells (UTD) (Figure 9A). For comparison, all dual-targeted CAR constructs lysed single-antigen strains 293TCD19 and 293TCD22, demonstrating the ability of these CAR constructs to induce their antitumor-lytic function when activated by either the CD19 antigen alone or the CD22 antigen alone (Figures 9B and 9D, respectively). On the other hand, none of the dual-targeted anti-CD22-19 CARs lysed the antigen-negative cell line 293T (Figure 9C). Therefore, all anti-CD22-19CARs functioned in tumor Raji strains co-expressing CD19 and CD22 antigens, as well as in 293T CD19 and 293T CD22 strains expressing either CD19 or CD22 monoantigens, but did not exhibit spontaneous cell death activity against the D19-CD22-cell line 293T, highlighting the target specificity of these constructs.

[0133] Next, the cytokine secretion capacity of anti-CD22-19CARs with various co-stimulatory domains was evaluated (Figure 8). CD19+CD22+Raji tumor cells were co-incubated overnight in an effector-to-target ratio of 10:1 with tandem anti-CD22-19CAR T cells expressing LTG2737, D0135, D0136, D0137, D0138, D0139, D0140, D0145, and D0146 constructs, or with negative control non-transduced T cells (UTDs). The culture supernatant was analyzed by ELISA for IFN-gamma, TNF-alpha, and IL-2 (Figure 8). All dual-targeted CARs potently induced IL-2 and TNF-α in response to tumor cells, while the negative control (non-transduced, UTD) did not produce any detectable cytokine induction (Figure 8). In particular, all CAR22-19s strongly induced subtle levels of IFN-gamma, but especially high for CAR constructs D0146, D0139, and D0136, suggesting that the strength of the cytokine response of anti-CD22-19 CARs can be regulated by the composition of the costimulatory domains used in CAR design. Overall, the secretion profiles of induced IFN-gamma, TNF-alpha, and IL-2 demonstrated the high potency of all CAR22-19 constructs. Importantly, in the absence of tumor cells (CAR T alone group), anti-CD22-19 CARs produced little to no cytokine secretion, further confirming CAR specificity and suggesting that there is no sustained signaling by tandem anti-CD22-19 CARs with various costimulatory domains.

[0134] While not intended to limit to any particular mechanism of action, possible reasons for the enhanced therapeutic function associated with the exemplary tandem CD22 and CD19 targeted CARs of the present invention include, for example, but not limited to, 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 higher 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 downmodulatory interactions, such as being less close to or less interacting with phosphatases such as CD45; d) superior assembly to T cell receptor signaling complexes (i.e., immune synapses); or e) superior association ability with tumor antigens due to two distinct targeting domains present in each CAR molecule; or any combination thereof.

[0135] While this disclosure exemplifies the use of an exemplary single extracellular CD19 / CD22 variable heavy chain and ScFv antigen-binding domain, other nucleotide and / or amino acid variants within the single CD19 / CD22 variable heavy chain and ScFv antigen-binding domain may be used to obtain the CD19 / CD22 antigen-binding domain used in the CAR described herein.

[0136] 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 / CD22 is the desired antigen to be targeted, an antibody against CD19 / CD22 may be used to incorporate the antigen-binding domain into the CAR.

[0137] In one exemplary embodiment, the antigen-binding domain portion of the CAR further targets CD33. Preferably, the antigen-binding domain in the CAR is an anti-CD33 scFV, where the nucleic acid sequence of the anti-CD33 scFV includes the sequence shown in SEQ ID NO: 46. In one embodiment, the anti-CD33 scFV includes a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 46. In another embodiment, the anti-CD19 / CD22 scFV portion of the CAR includes the amino acid sequence shown in SEQ ID NO: 47.

[0138] In one exemplary embodiment, the antigen-binding domain of the CAR further targets mesoserine. Preferably, the antigen-binding domain in the CAR is an anti-mesoserine ScFv, and the nucleic acid sequence of the anti-mesoserine ScFv includes the sequence specified 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.

[0139] 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.

[0140] 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.

[0141] 2. Transmembrane domain Regarding the transmembrane domain, the CAR contains one or more transmembrane domains fused to the extracellular CD19 / CD22 antigen-binding domain of the CAR.

[0142] 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.

[0143] The transmembrane domains particularly used in the CARs described herein may be derived 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 their transmembrane domains). Alternatively, the transmembrane domain may be synthetic, in which case it predominantly contains hydrophobic residues such as leucine and valine. Preferably, a triplet 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. Glycine-serine doublets provide a particularly suitable linker.

[0144] 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.

[0145] In some cases, the transmembrane domain may be selected or obtained by amino acid substitution to avoid binding of such domain to the transmembrane domain of the same or different surface membrane proteins, in order to minimize interaction with other members of the receptor complex.

[0146] 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.

[0147] 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: 36, 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: 36.

[0148] 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 one embodiment, the CD8 hinge domain includes a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 38. In another embodiment, the CD8 hinge domain includes the amino acid sequence of SEQ ID NO: 38, or a sequence having 95-99% identity.

[0149] 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.

[0150] 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.

[0151] 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.

[0152] The spacer domain preferably has a sequence that promotes CAR binding to the antigen and enhances signaling into the cell. Examples of amino acids that are expected 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.

[0153] 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.

[0154] Furthermore, in CARs, a signal peptide sequence may be ligated to the N-terminus. Signal peptide sequences are present at the N-terminus of many secretory 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 comprises the amino acid sequence shown in SEQ ID NO: 12.

[0155] 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.

[0156] 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.

[0157] 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).

[0158] 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 may contain signaling motifs known as immunoreceptor-activating tyrosine motifs or ITAMs.

[0159] 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 This includes peptides having the sequences of 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 amino acid numbers 177-252 of CD66d (NCBI RefSeq:NP_001806.2), as well as their variants having the same functions as these peptides. 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.

[0160] 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.

[0161] 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 doublets provide particularly suitable linkers.

[0162] 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.

[0163] 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.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] 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.

[0168] 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. Functional variants include, for example, variants of the CARs described herein (parent CARs) that retain the ability to recognize target cells to a similar, identical, or greater degree than the parent CAR. With respect to the parent CAR, a functional variant may, for example, have amino acid sequence identity with respect to the parent CAR by at least about 30%, 50%, 75%, 80%, 90%, 98%, or more.

[0169] 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.

[0170] 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).

[0171] 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.

[0172] 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.

[0173] 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, and 1,2,3,4-tetrahydroxy It contains diisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine,-aminocyclopentanecarboxylic 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.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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.

[0178] A "monoclonal antibody" is an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies constituting the 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 the characteristic of the antibody being obtained from a substantially homogeneous population of antibodies, and should not be interpreted as requiring antibody production 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 substantially have no effect on antigen binding or other immunoglobulin functions. Exemplary methods for producing monoclonal antibodies are publicly known; see, for example, Harlow & Lane, Antibodies, A Laboratory Manual, 2nd edition, Cold Spring Harbor Publications, New York (2013).

[0179] 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.

[0180] Each heavy and light chain comprises a constant region (or constant domain) and a variable region (or containing a variable domain; see, e.g., Kindt et al., Kuby Immunology, 6th edition, WHFreeman and Co., p. 91 (2007)). In some embodiments, the variable regions of the heavy and light chains combine to specifically bind to the antigen. In further embodiments, only the heavy chain variable region is required. For example, naturally occurring camelid antibodies consisting only of the heavy chain are functional and stable in the absence of the light chain (see, e.g., Hamers-Casterman et al., Nature, vol. 363: pp. 446-448, 1993; Sheriff et al., Nat. Struct. Biol., vol. 3: pp. 733-736, 1996). References to "VH" or "VH" refer to the variable region of the antibody heavy chain, including antigen-binding fragments, e.g., Fv, scFv, dsFv, or Fab. "VL" or reference to "VL" refers to the variable domain of the antibody light chain, including those of Fv, scFv, dsFv, or Fab.

[0181] 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.

[0182] CDRs are primarily responsible for binding antigens to epitopes. The amino acid sequence boundaries of a given CDR can be readily determined using one of several well-known schemes, including those described by 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 for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev.Comp.Immunol., Vol. 27: pp. 55-77, 2003; "IMGT" numbering scheme). The CDRs in each chain are typically referred to as CDR1, CDR2, and CDR3 (from N-terminus to C-terminus) and are also typically identified by the chain in 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 referred to as LCDR1, LCDR2, and LCDR3. Heavy chain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3.

[0183] 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).

[0184] 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 the scFv. Therefore, scFv can be used with both possible configurations (VH domain - linker domain - VL domain; VL domain - linker domain - VH domain).

[0185] 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).

[0186] 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.

[0187] Antibodies that do not exist in nature may be constructed using solid-phase peptide synthesis, recombinantly produced, or obtained by screening a combinatorial library consisting of variable heavy and variable light chains, as described, for example, in Huse et al., Science 246: pp. 1275-1281 (1989), incorporated herein by reference. For example, these and other methods for producing chimeric, humanized, CDR-transplanted, 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, Vol. 341: pp. 544-546 (1989); Harlow and Lane, ibid., 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).

[0188] 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.

[0189] 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.

[0190] 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.

[0191] 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).

[0192] 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, single-chain antibodies or Fab fragments have one binding site, while bispecific or bifunctional antibodies have two different binding sites.

[0193] 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).

[0194] 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).

[0195] 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.

[0196] 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).

[0197] 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.

[0198] 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.

[0199] 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 a cleaving agent present in the intracellular environment (e.g., within lysosomes, endosomes, or caveoleas). The linker may be a peptide linker that is 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 lengths, e.g., 1-2, 1-3, 2-5, 3-10, 3-15, 1-5, 1-10, or 1-15 amino acid lengths. 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, a peptide linker cleavable by cathepsin B, a thiol-dependent protease, may be used (e.g., a phenylalanine-leucine or glycine-phenylalanine-leucine-glycine linker). 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 is 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).

[0200] 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 unstable at pH levels below 5.5 or 5.0, which is the approximate pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether conjugated to the therapeutic agent via an acylhydrazone linkage) (see, for example, U.S. Patent No. 5,622,929).

[0201] 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.

[0202] 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).

[0203] 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).

[0204] 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 2005 / 0238649, and U.S. Patent Application Publication 2006 / 0024317, each of which is incorporated herein by reference in its entirety.

[0205] 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, maytansinoids, drastatin, auristatin, trichothecin, and CC1065, as well as derivatives of these toxins having toxic activity.

[0206] Maytansine compounds suitable for use as the toxin portion of a maytansinoid 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 using known methods to produce maytansinol and maytansinol analogs. Maytansinoids are mitototic inhibitors that act by inhibiting tubulin polymerization. Maytansine 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.

[0207] 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).

[0208] 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.

[0209] 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.

[0210] Castor bean toxin is lectin RCA60 derived from Ricinus communis (castor bean). For examples of castor bean toxin, see U.S. Patent Nos. 5,079,163 and 4,689,401. Ricinus communis aglutinin (RCA) has 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).

[0211] 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 currently undergoing clinical trials (see, for example, Gillespie et al., Ann. Oncol. Vol. 11: pp. 735-741, 2000).

[0212] 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).

[0213] 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 photomeridians. 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 can be detected by adding further reagents used by the enzyme to produce a identifiable reaction product. For example, in the presence of the drug horseradish peroxidase, the addition of hydrogen peroxide and diaminobenzidine results in a visually detectable colored reaction product. CARs, CAR-expressing T cells, antibodies, or their antigen-binding moieties can also be conjugated with biotin and detected via indirect measurement of avidin or streptavidin binding. It should be noted that avidin itself can be conjugated with enzymes or fluorescent labels.

[0214] A CAR, a T cell 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. Antibodies can also be conjugated to lanthanides (e.g., europium and dysprosium) and manganese. An antibody or antigen-binding fragment can also be labeled with a predetermined polypeptide epitope (e.g., leucine zipper pair sequence, binding site for a secondary antibody, metal-binding domain, epitope tag) recognized by a secondary reporter.

[0215] A CAR, a T cell 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 spectroscopy 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, and 131 I.

[0216] Means for detecting such detectable markers are well known to those of skill in the art. Thus, for example, a radiolabel can be detected using a photographic film or a scintillation counter, and a fluorescent marker can be detected using a photodetector to detect the emitted illumination. Enzyme labels are typically detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and chromogenic labels are detected by simply visualizing the colored label.

[0217] 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.

[0218] 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.

[0219] 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 may comprise a codon-modified nucleotide sequence encoding any of the CARs (including their functional portions and functional variants) described herein.

[0220] 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.

[0221] 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).

[0222] 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.

[0223] One embodiment also provides isolated or purified nucleic acids comprising a nucleotide sequence complementary to any of the nucleotide sequences of the nucleic acids described herein, or a nucleotide sequence that hybridizes with any of the nucleotide sequences of the nucleic acids described herein under stringent conditions.

[0224] 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.

[0225] 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%.

[0226] In one embodiment, the nucleic acid can 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 that when the construct contains a nucleotide sequence encoding mRNA, protein, polypeptide or peptide, and when the vector is contacted with a cell under conditions sufficient for the mRNA, protein, polypeptide or peptide to be expressed intracellularly, it is a genetically modified oligonucleotide or polynucleotide construct that enables the expression of mRNA, protein, polypeptide or peptide by the host cell. The vector as a whole is not naturally present.

[0227] However, a part of the vector can be naturally present. The recombinant expression vector can be single-stranded or double-stranded, obtained synthetically or from a partially natural source, and can contain DNA and RNA, including but not limited to any type of nucleotide that can contain natural, non-natural or modified nucleotides. The recombinant expression vector can contain natural or non-natural nucleotide linkages, or both types of linkages. Preferably, the non-natural or modified nucleotides or nucleotide linkages do not interfere with the transcription or replication of the vector.

[0228] In one embodiment, the recombinant expression vector can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host cell. Suitable vectors include those designed for propagation and growth, or for expression, or for both, such as plasmids and viruses. The vector can be selected from the group consisting of the pUC series (Fermentas Life Sciences, Glen Burnie, MD), the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, CA).

[0229] Bacteriophage vectors, such as, for example, λΤΙ Ο, λΤΙ 1, λZapII (Stratagene), EMBL4, and λΝΜΙ 149 can 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). The recombinant expression vector can be a viral vector, such as, for example, a retroviral vector or a lentiviral vector. Lentiviral vectors are vectors derived from at least a portion of the lentiviral genome, including, in particular, self-inactivating lentiviral vectors such as those provided by Milone et al., Mol. Ther. 17(8):1453 - 1464 (2009). Other examples of lentiviral vectors that can be used in the clinic include, for example, without limitation, the LENTIVECTOR® gene delivery technology of Oxford BioMedica plc, the LENTIMAX™ vector system of Lentigen, etc. Non-clinical lentiviral vectors are also available and are known to those skilled in the art.

[0230] 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)).

[0231] 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)).

[0232] 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. The 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.

[0233] 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.

[0234] Recombinant expression vectors may contain one or more marker genes that allow for the selection of transformed or transfected host cells. Marker genes may include those for biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., and nutritional complementation in the host to provide protrophotrophy. Suitable marker genes for the expression vectors of the present invention include, for example, neomycin / G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0235] 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.

[0236] 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.

[0237] 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.

[0238] 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 any cell type, may originate from any type of tissue, and may 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.

[0239] For the purposes of this specification, T cells may be any T cells, e.g., cultured T cells, e.g., primary T cells, or cultured T cell lines, e.g., T cells derived from Jurkat, SupTl, etc., or T cells obtained from mammals. When obtained from mammals, T cells may be obtained from a number of sources, including but not limited to blood, bone marrow, lymph nodes, thymus, or other tissues or body fluids. T cells may also be enriched or purified. T cells may be human T cells. T cells may be T cells isolated from humans. T cells may be any type of T cell, including but not limited to 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., and may be at any developmental stage. T cells may be CD8+ T cells or CD4+ T cells.

[0240] 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.

[0241] 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.

[0242] 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%.

[0243] 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.

[0244] 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.

[0245] For methods of administering host cells or populations of cells, the cells may be homogeneous or autologous to the mammal. Preferably, the cells are autologous to the mammal. 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, autologous means any material originating from the same individual that is subsequently reintroduced into the individual.

[0246] 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.

[0247] 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.

[0248] As used herein, the terms "treat" and "prevent" and words derived therefrom do not necessarily mean 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that one of ordinary skill in the art would recognize as having a potential beneficial or therapeutic effect. In this regard, the method can provide treatment or prevention of cancer in a mammal at any amount or level.

[0249] Furthermore, the treatment or prevention provided by this method can include 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" can include delaying the onset of a disease, or its symptoms or conditions.

[0250] Another embodiment is a method of detecting the presence of cancer in a mammal, comprising: (a) contacting a sample comprising one or more cells derived from the mammal with a CAR, nucleic acid, recombinant expression vector, host cell, population of cells, antibody and / or an antigen-binding portion thereof, or a pharmaceutical composition, thereby forming a complex; and (b) detecting the complex, wherein detection of the complex indicates the presence of cancer in the mammal.

[0251] The sample can be obtained by any suitable method, such as by biopsy or autopsy. A biopsy is the removal of tissue and / or cells from an individual. Such removal can be the collection of tissue and / or cells from an individual for the purpose of performing experiments on the removed tissue and / or cells. Such experiments can include experiments to determine whether an individual has a particular condition or disease state and / or is suffering from a particular condition or disease state. The condition or disease can be, for example, cancer.

[0252] 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.

[0253] The contact step can occur in vitro or in vivo for mammals. Preferably, the contact step is in vitro.

[0254] 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).

[0255] 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).

[0256] 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.

[0257] 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 drug 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 may be used, for example, 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 being prone to 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 may be applied topically as eye drops to the cornea, or intravitreally to the eye.

[0258] 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 multi-dose schedules. 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 drug 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.

[0259] 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.

[0260] 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, ten consecutive days, etc. 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.

[0261] 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.

[0262] 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.

[0263] Further chemotherapeutic agents include alkylating agents, e.g., nitrogen mustards (e.g., chlorambucil, chlormethine, cyclophosphamide, ifosfamide, and melphalan), nitrosoureas (e.g., carmustine, fotemustine, lomustine, and streptozocin), 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 alkaloids, e.g. podofilms (e.g., etoposide and teniposide), taxanes (e.g., docetaxel and paclitaxel), vinca (e.g., vinblastine, vincristine, vindesine and vinorelbine); cytotoxic / antitemocyte antibiotics, e.g., members of the anthracycline family (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone and barurubicin), bleomycin, rifampicin, hydroxyurea 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 aminolevulinate, porfimer sodium and verteporfin;This also includes, but is not limited to, other drugs such as alitretinoin, altretamine, amsacrin, anagrelide, arsenic trioxide, 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.

[0264] 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.

[0265] 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.

[0266] 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 CAR-expressing T cells, antibodies, antigen-binding fragments, conjugates, or CAR-expressing T cells that specifically bind to one or more antigens disclosed herein. These compositions may be prepared in unit dose forms for administration to a subject. The amount and timing of administration to achieve the desired outcome is 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, conjugates, etc., may be formulated for parenteral administration, such as intravenous administration. Compositions comprising the CARs, or CAR-expressing T cells, conjugates, antibodies, or antigen-binding fragments disclosed herein may be used for the treatment and detection of tumors, such as, but not limited to, neuroblastoma. In some cases, these compositions are useful for treating or detecting cancer. Compositions comprising CARs, or CAR-expressing T cells, conjugates, antibodies, or antigen-binding fragments disclosed herein, can also be used, for example, for the detection of pathological angiogenesis.

[0267] 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.

[0268] 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).

[0269] 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.

[0270] 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, Therapeutic 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).

[0271] 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).

[0272] 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.

[0273] 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.

[0274] 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]

[0275] 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.

[0276] Example 1A Isolation of CD19-specific antibodies from scFv libraries presenting fully human phages and yeast. material and method: a) Production of human scFv and CD19-specific antibodies A naive human ScFv (variable recombinant single-chain immunoglobulin fragment) phage display library constructed from peripheral blood B cells of 50 healthy donors (approximate diversity, 10 10Species-specific specificity (ZYZhu and DSDimitrov, unpublished data) was used for the selection of ScFv for recombinant human CD19 protein (Miltenyi Biotec, unpublished). Phage-displayed scFv10 12The amplified libraries of the species were incubated with 5, 3, and 1 μg of coated CD19 in 5 × 100 μl volumes, equally distributed into five wells of a 96-well plate during each of the first, second, and third rounds of biopanning, and left at room temperature for 2 hours. After each round of incubation, the wells were washed with phosphate-buffered saline (PBST) containing 0.05% Tween20 five times in the first round and ten times in subsequent rounds to remove nonspecifically bound phages. The bound phages were mixed with TG1-competent cells at 37°C for 1 hour, and the phages from the infected cells were amplified and used for biopanning in the next round. After the third round of biopanning, 380 clones were randomly selected from the infected TG1 cells and each was inoculated into 150 μl of 2YT medium containing 100 μg / ml carbenicillin and 0.2% glucose in a 96-well plate using an automated BioRobotics BioPick colony picking system (Genomic Solutions, Ann Arbor, MI). After the bacterial culture reached an optical density (OD600) of 0.5 at 600 nm, helper phage M13K07 with a MOI of 10 and kanamycin at a final concentration of 50 μg / ml were added to the medium, and the plates were further incubated overnight at 30°C at 250 rpm in a shaker. The phage supernatant was mixed with 3% skim milk solution in PBS in a volume ratio of 4:1 and used for enzyme-linked immunosorbent assay (ELISA) to identify phage clones presenting scFv or VH with high CD19 binding affinity. The supernatant was incubated in a 96-well plate with 50 ng of coated recombinant human CD19 per well at room temperature for 2 hours, washed five times with PBST (incubated overnight at 4°C, then blocked with 3% skim milk solution in PBS, and washed three times with PBS containing 0.05% Tween20). Phages bound to CD19 were detected using goat anti-M13 antibody conjugated with horseradish peroxidase.After incubation with the antibody, nonspecifically bound antibody was removed by washing the wells, and a 3,3',5,5'-tetramethylbenzidine (TMB) substrate was added. The solution absorbance (A450) at 450 nm was measured. CD19-bound clones with an A450 greater than 1.0 were selected for further characterization.

[0277] b) Expression and purification of selected soluble scFv The VH and VL sequences of the selected clones were DNA-sequenced, and the scFv encoded by the clones with unique sequences was expressed and purified as described below. Plasmids extracted from these clones were used to transform HB2151 cells. Single colonies were selected from plates containing newly transformed cells and inoculated into 200 ml of 2YT medium containing 100 μg / ml ampicillin and 0.2% glucose, and incubated at 37°C with shaking at 250 rpm. When the culture OD at 600 nm reached 0.90, isopropyl-β-d-thiogalactopyranoside was added at a final concentration of 0.5 mM, and the culture was incubated further overnight at 30°C. After centrifugation at 8,000 × g for 20 minutes, the bacterial pellet was collected and resuspended in PBS buffer containing 0.5 mU of polymixin B (Sigma-Aldrich, St. Louis, MO). After incubation at room temperature for 30 minutes while rotating at 50 rpm, the resuspended pellet was centrifuged at 25,000 × g for 25 minutes at 4°C, and the supernatant was used for ScFv purification using Ni-NTA resin according to the supplier's protocol (Qiagen).

[0278] c) ELISA binding assay ELISA binding assay: 50 μl of recombinant human CD19, diluted to 2 μg / ml in PBS, was coated into a 96-well plate overnight at 4°C. Purified ScFv with His and Flag tags was serially diluted and added to the wells coated with the target protein. After washing, anti-Flag antibody conjugated with HRP, diluted to 1:3000, was added at RT for 1 hour. After washing, 3,3,5,5'-tetramethylbenzidine (TMB) substrate was added and incubated at room temperature for 10 minutes. The reaction was then stopped by adding 1N H2SO4, and the relative ability of ScFv to bind to CD19 was quantified by reading the OD at 450 nm.

[0279] d) Yeast display in the scFv library The same ScFv starting material used for phage display was also incorporated into the yeast ScFv display system. To complement the phage-based scFv analysis, yeast libraries expressing human scFv libraries were also screened. Cell panning was performed on CD19-transfected CHOK1 cells to enrich yeast expressing scFv that binds to both recombinant CD19-Fc and CD19 expressed on the cell surface of CHOK1 cells. For the first round of panning on the cell surface, CHOK1-CD19 cells were seeded in 6-well plates and grown in F12K medium to a concentration of 50% two days prior to panning. Subsequently, 5 × 10⁶ cells were panned. 7Each yeast cell was washed twice with PBSA buffer, resuspended in 3 mL of F12K medium, and then gently added dropwise to the CHOK1-CD19 cells. The CHOK1-CD19 cells were gently agitated on ice for 2 hours, then washed three times with ice-cold PBSA to remove yeast cells that did not bind to CHOK1-CD19, and then 0.05% trypsin-EDTA (Gibco) was used to dissociate the CHOK1-CD19 cells and bound yeast cells from the plate. The cell mix containing yeast cells and CHOK1 cells was then inoculated into 10 mL of SDCAA medium and amplified overnight at 30°C, followed by induction in SGCAA medium at 30°C for 16 hours. For the second round of cell panning, the same protocol as above was followed, but with more stringent washing conditions. This panning method yielded the m19217 binder. Further characterization of this binder, as well as other binders from the phage display, revealed that the biological characteristics of the CARs generated from this hit were still not optimal, indicating that affinity maturation was necessary.

[0280] To enhance the affinity of m19217, a yeast-display m19217 mutant scFv library was created using error-prone PCR, inducing random point mutations in the scFv gene sequence. The resulting mutant library was electroporated, then grown overnight in SDCAA medium at 30°C for 16 hours, and then switched to SGCAA medium and incubated at 30°C for another 16 hours. The mutant library was then sorted by MACS (immunomagentic column, Miltenyi Biotec) using CD19-Fc as the capture antigen to reduce the library size and increase the population of mutants capable of binding to CD19-Fc. The pool was then double-stained with anti-c-Myc-Alexa488 and CD19-Fc / anti-Hu-Fc, and the most potent binder was selected by choosing the binder that showed the highest binding affinity and c-Myc expression level. Next, this process was repeated two more times by flow cytometry of yeast particles using fluorescently tagged antigens until the average binding affinity of the mutant pool was greater than that of the starting construct. Binding affinity was estimated by flow cytometry of the yeast pool using gradually decreasing amounts of labeled CD19. As a result of this process, the EC50 (effective concentration for binding 50% of labeled CD19 on yeast presenting ScFv) of 0.5 μg / ml for M19217 was increased to less than 0.01 μg / ml for binders with matured affinity (M19217-1, 19217-2, M19217-7, M19217-23, M19217-29, M19217-38, M19217-40).

[0281] result: Because the antigen loading of the CD19 structure is unique, no biologically functional CAR constructs have emerged from phage display candidates. Therefore, we achieved the identification of ScFvs that yield biologically active binders using yeast display. Based on flow cytometry analysis of ScFv using yeast display, eight ScFv clones specific to recombinant human CD19 were identified and named as follows: human anti-CD19 ScFv binder M19217 (LTG2050, primary clone, EC50 0.5 μg / ml), and subsequent binders with matured affinity (EC50 less than 0.01 μg / ml), namely M19217-1 (LTG2065), M19217-2 (LTG2066), M19217-7 (LTG2067), M19217-23 (LTG2068), M19217-29 (LTG2069), M19217-38 (LTG2070), and M19217-40 (LTG2071). The generation of tandem CARs incorporating the anti-CD19 scFv M19217-1 sequence will be outlined in Example 2 below.

[0282] Example 1B Isolation of CD22-specific antibodies from ScFv libraries using full human phage and yeast display. material and method: a) Production of human ScFv and CD22-specific antibodies A naive human ScFv (variable recombinant single-chain immunoglobulin fragment) phage display library constructed from peripheral blood B cells of 50 healthy donors (approximate diversity, 10 10 Species-specific specificity (ZYZhu and DSDimitrov, unpublished data) was used for the selection of ScFvs for recombinant human CD19 protein (Miltenyi Biotec, unpublished). Phage-displayed ScFv10 12The amplified libraries of the species were incubated with 5 × 100 μl volumes of coated CD22, 5, 3, and 1 μg, and equally distributed into five wells of a 96-well plate during each of the first, second, and third rounds of biopanning, and left at room temperature for 2 hours. After each round of incubation, the wells were washed with phosphate-buffered saline (PBST) containing 0.05% Tween20 five times in the first round and ten times in subsequent rounds to remove nonspecifically bound phages. The bound phages were mixed with TG1 competent cells at 37°C for 1 hour, and the phages from the infected cells were amplified and used for biopanning in the next round. After the third round of biopanning, 380 clones were randomly selected from the infected TG1 cells and each was inoculated into 150 μl of 2YT medium containing 100 μg / ml carbenicillin and 0.2% glucose in a 96-well plate using an automated BioRobotics BioPick colony picking system (Genomic Solutions, Ann Arbor, MI). After the bacterial culture reached an optical density (OD600) of 0.5 at 600 nm, helper phage M13K07 with a MOI of 10 and kanamycin at a final concentration of 50 μg / ml were added to the medium, and the plates were further incubated overnight at 30°C at 250 rpm in a shaker. The phage supernatant was mixed with 3% skim milk solution in PBS in a volume ratio of 4:1 and used for enzyme-linked immunosorbent assay (ELISA) to identify phage clones presenting scFv with high CD22 binding affinity. The supernatant was incubated in a 96-well plate with 50 ng of coated recombinant human CD22 per well at room temperature for 2 hours, and washed five times with PBST (incubated overnight at 4°C, then blocked with 3% skim milk solution in PBS, and washed three times with PBS containing 0.05% Tween20). Phages bound to CD22 were detected using goat anti-M13 antibody conjugated with horseradish peroxidase.After incubation with the antibody, nonspecifically bound antibody was removed by washing the wells, and a 3,3',5,5'-tetramethylbenzidine (TMB) substrate was added. The solution absorbance (A450) at 450 nm was measured. CD22-bound clones with an A450 greater than 1.0 were selected for further characterization.

[0283] b) Expression and purification of selected soluble ScFv The VH and VL sequences of the selected clones were DNA-sequenced, and ScFvs encoded by the clones with unique sequences were expressed and purified as described below. Plasmids extracted from these clones were used to transform HB2151 cells. Single colonies were selected from plates containing newly transformed cells, inoculated into 200 ml of 2YT medium containing 100 μg / ml ampicillin and 0.2% glucose, and incubated at 37°C with shaking at 250 rpm. When the culture OD at 600 nm reached 0.90, isopropyl-β-d-thiogalactopyranoside was added at a final concentration of 0.5 mM, and the culture was incubated further overnight at 30°C. After centrifugation at 8,000 × g for 20 minutes, the bacterial pellet was collected and resuspended in PBS buffer containing 0.5 mU polymyxin B (Sigma-Aldrich, St. Louis, MO). After incubation at room temperature for 30 minutes while rotating at 50 rpm, the resuspended pellet was centrifuged at 25,000 × g for 25 minutes at 4°C, and the supernatant was used for scFv purification using Ni-NTA resin according to the supplier's protocol (Qiagen).

[0284] c) ELISA binding assay For ELISA analysis, 50 μl of recombinant human CD22 diluted to 2 μg / ml in PBS was coated into a 96-well plate overnight at 4°C. Purified scFv with His and Flag tags were serially diluted and added to the wells coated with the target protein. After washing, anti-Flag antibody conjugated with HRP, diluted to 1:3000, was added at room temperature for 1 hour. After washing, 3,3,5,5'-tetramethylbenzidine (TMB) substrate was added and incubated at room temperature for 10 minutes. The reaction was then stopped by adding 1N H2SO4, and the relative ability of scFv to bind to CD22 was quantified by reading the OD at 450 nm.

[0285] d) Yeast display in the scFv library The same ScFv starting material used for phage display was also incorporated into the yeast ScFv display system. To complement the phage-based scFv analysis, yeast libraries expressing human scFv libraries were also screened. Cell panning was performed on CD22-transfected CHOK1 cells to enrich yeast expressing scFv that binds to both recombinant CD22-Fc and CD19 expressed on the cell surface of CHOK1 cells. For the first round of panning on the cell surface, CHOK1-CD22 cells were seeded in 6-well plates and grown in F12K medium to a concentration of 50% two days prior to panning. Subsequently, 5 × 10⁶ cells were panned. 7Each yeast cell was washed twice with PBSA buffer, resuspended in 3 mL of F12K medium, and then gently added dropwise to the CHOK1-CD22 cells. The CHOK1-CD22 cells were gently agitated on ice for 2 hours, then washed three times with ice-cold PBSA to remove yeast cells that did not bind to CHOK1-CD22, and then 0.05% trypsin-EDTA (Gibco) was used to dissociate the CHOK1-CD22 cells and bound yeast cells from the plate. The cell mix containing yeast cells and CHOK1 cells was then inoculated into 10 mL of SDCAA medium and amplified overnight at 30°C, and then induced in SGCAA medium at 30°C for 16 hours. For the second round of cell panning, the same protocol as above was followed, but with more stringent washing conditions. This panning method yielded 16P, 24P, 25P, 11S, and 12S binders. In a series of in vitro CART functional assays, the binder sequences were incorporated into the CART construct as described in Example 2 below. When these binders from phage display were characterized in CART format, only the 16P binder showed specific oncolytic activity in vitro, but its activity was lower than that of CAR-positive controls. Furthermore, when 16P-derived CART cells were tested in an in vivo xenograft model, their antitumor function was very weak (Example 2, shown below). In summary, these results indicate that the biological characteristics of CARs generated from this binder set were still not optimal, and that the affinity of the anti-CD22 ScFv binder needs to be matured.

[0286] To enhance 16P affinity, a yeast display mutant scFv library was created using error-prone PCR, inducing random point mutations in the scFv gene sequence. The resulting mutant library was electroporated, then grown overnight in SDCAA medium at 30°C for 16 hours, and then switched to SGCAA medium and incubated at 30°C for another 16 hours. The mutant library was then sorted by MACS (immunomagnetic column, Miltenyi Biotec) using CD22-Fc as the capture antigen to reduce library size and increase the population of mutants capable of binding to CD22-Fc. The pool was then double-stained with anti-c-Myc-Alexa488 and CD19-Fc / anti-Hu-Fc, and the most potent binder was selected by choosing the binder that showed the highest binding affinity and c-Myc expression level. Next, this process was repeated two more times by flow cytometry of yeast particles using fluorescently tagged antigens until the average binding affinity of the mutant pool was greater than that of the starting construct. Binding affinity was estimated by flow cytometry of the yeast pool using gradually decreasing amounts of labeled CD22. As a result of this process, the EC50 (effective concentration for binding 50% of labeled CD19 on yeast presenting ScFv) of 0.5 μg / ml for 16P was increased to less than 0.01 μg / ml for binders with matured affinity (16P1, 16P2, 16P3, 16P3v2, 16P6, 16P8, 16P10, 16P13, 16P15, 16P16, 16P17, 16P20, 16P20v2).

[0287] result: Due to the unique antigen loading of the CD22 structure, no fully functional CAR structures with high biological activity and specificity were obtained from phage display candidates. Therefore, ScFv for biologically active and highly specific binders was obtained by yeast display. Based on flow cytometry analysis of ScFv by yeast display, 13 ScFv clones specific to recombinant human CD22 were identified and named as the human anti-CD22 ScFv binder 16P (LTG2202, primary clone, EC50 0.5 μg / ml), and the following binders with matured affinity (EC50 < 0.01 μg / ml), namely 16P1, 16P2, 16P3, 16P3v2, 16P6, 16P8, 16P10, 16P13, 16P15, 16P17, 16P20, and 16P20v2. The generation of tandem CARs incorporating the anti-CD22 scFv 16P17 sequence will be outlined in Example 2 below.

[0288] Example 2 Fully human anti-CD22 and anti-CD19 scFv binding sequences expressing dual-targeting tandem CARs This example discusses the creation of a dual-targeting CAR that simultaneously targets tumor antigens CD19 and CD22. The requirement for this approach is to help mitigate tumor antigen escape, which is a significant cause of failure in single-CAR approaches, i.e., CAR therapies using anti-CD19 CAR or anti-CD22 CAR therapy (Sotillo, Elena et al., "Convergence of acquired mutations and alternative splicing of CD19 enables resistance to CAR-19 immunotherapy," Cancer Discovery (2015); Gardner, Rebecca et al., "Acquisition of a CD19 negative myeloid phenotype allows immune escape of MLL-rearranged B-ALL from CD19 CAR-T cell therapy," Blood (2016):blood-2015; Fry, Terry J. et al., "CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19-targeted CAR immunotherapy," Nature Medicine 24.1 (2018): No. 20).

[0289] The use of mouse scFv sequences as CAR components has been shown to cause immune rejection or allergic anaphylactic reactions in patients, thus limiting the continuation and use of CAR T therapy and increasing the risk of toxicity. Therefore, the use of fully human scFv binder sequences in CAR design should be a high priority for future CAR development.

[0290] CD19 is an 85-95 kDa transmembrane cell surface glycoprotein receptor. CD19 is a member of the immunoglobulin (Ig) superfamily of proteins and contains two extracellular Ig-like domains, a transmembrane domain, and an intracellular signaling domain (Tedder TF, Isaacs, CM, 1989, J Immunol vol. 143: pp. 712-171). CD19 modifies B cell receptor signaling by lowering the antigen-induced threshold of the B cell receptor (Carter, RH and Fearon, DT, 1992, Science, vol. 256: pp. 105-107), and works in conjunction with CD81 and CD21 to regulate 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 individual development, CD19 can signal independently of antigen receptors in 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 in a strictly regulated form on normal B cells, limited to early progenitor B cells in the IgH gene rearrangement stage and mature B cells, but not on hematopoietic stem cells or mature plasma cells (Anderson, KC, Bates, MP, Slaughenhout BL, Pinkus GS, Schlossman SF, Nadler LM, 1984, Blood vol. 63: pp. 1424-1433).

[0291] Homo sapiens CD22 (SIGLEC-2, Leu14) is a well-studied cell surface glycoprotein expressed in B-cell leukemia and lymphoma. At least two anti-CD22 antibody drugs (inotuzumab ozogamicin) or immunotoxin conjugates (moxetumomab pasudotox) have been included in clinical trials (NCT02981628, NCT00659425). These approaches have shown some success and are still being studied, for example, in combination with other chemotherapeutic agents (Muller F, Stookey S, Cunningham T, Pastan I, 2017, Paclitaxel synergizes with exposure tume adjusted CD22-targeted immunotoxins against B-cell malignancies, Oncotarget vol. 8: pp. 30644-30655). However, given the current advances in CD19 CAR-based T-cell therapy, cell-based immunotherapy is the best approach to target CD22-expressing malignancies. A therapy featuring an m971-based anti-CD22 CAR is currently in clinical trials at the National Cancer Institute (NCT02315612, PI: Terry Fry, MD). The tandem CD22 CD19-targeted CAR construct presented here is an innovative new approach that creates and implements a novel, fully human CD19 and CD22 binding moiety in a single construct to achieve complete and permanent remission and prevent tumor antigen evasion.

[0292] In this example, a single CAR control is used for comparison with a tandem CAR targeting CD19 and CD22. The CAR construct LTG1538, utilizing scFv derived from the mouse hybridoma FMC63, is used as the active control. This mouse-derived sequence is a currently used binder in commercial development (see KTE-C19, Kite Pharma, and CTL019, Novartis). LTG2200, an m971 CAR, is equivalent to a CAR evaluated by the NCI and is used as an anti-CD22 CAR control.

[0293] material and method: (a) Cell line The Burkitt lymphoma cell line Raji and the chronic myeloid leukemia cell line K562 were purchased from the American Tissue Culture Collection (ATCC, Manassass, VA). The REH leukemia cell line was purchased from DSMZ (Leibniz Institute DSMZ, Braunschwieg, Germany). Cells were cultured in RPMI-1640 medium supplemented with 10% thermoinactivated fetal bovine serum (FBS, Hyclone, Logan, UT) and 2 mM L-Glutamax (Thermo Fisher Scientific, Grand Island, NY). The human fetal kidney cell line 293T was purchased from ATCC (Gibco / Thermo Fisher Scientific, Grand Island, NY). Luciferase-expressing cell lines were generated by stably transducing wild-type tumor cells with a lentiviral vector encoding firefly luciferase (Lentigen Technology, Inc., Gaithersburg, MD), cloning them, and selecting luciferase-positive clones. Raji clones were generated by passaged luciferase-transduced Raji cells in mice and selected for their proliferative capacity. Whole blood was collected from healthy volunteers at the Oklahoma Blood Institute (OBI) with written donor consent. Processed buffy coat was purchased from OBI (Oklahoma City, OK). CD4-positive and CD8-positive human T cells were purified from the buffy coat by positive selection using a 1:1 mixture of CD4 and CD8 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's protocol.

[0294] (b) Creation of chimeric antigen receptors (CARs) - expression vectors The CAR antigen-binding domain, ScFv, and sequence were derived from human anti-CD22 ScFv or heavy chain variable fragments. The binder sequence was ligated in-frame to the CD8a ligation and transmembrane domain (aa123-191, Ref sequence ID NP_001759.3), and then to the 4-1BB (CD137, aa214-255, UniProt sequence ID Q07011) signaling domain and the CD3 zeta signaling domain (CD247, aa52-163, Ref sequence ID: NP_000725.1) to generate the CAR T construct. The CAR construct sequence was cloned into a third-generation lentiviral plasmid backbone (Lentigen Technology Inc., Gaithersburg, MD). Transient transfection of HEK 293T cells generated a supernatant containing a lentiviral vector (LV). The supernatant containing the lentiviral vector was then centrifuged to form a vector pellet, which was stored at -80°C.

[0295] (c) Purification and transduction of primary T cells CD4 according to the manufacturer's protocol (Miltenyi Biotec, Bergisch Gladbach, Germany) + and CD8 + Human primary T cells derived from healthy volunteers were purified from whole blood or buffy coat (purchased from private donors with written donor consent) using immunomagnetic bead selection of cells. The T cells were cultured in TexMACS medium supplemented with 200 IU / ml IL-2, resulting in 0.3–2 × 10⁶ cells. 6 Cells were cultured at a density of cells / ml, activated with CD3 / CD28 MACS® GMP T Cell TransAct reagent (Miltenyi Biotec), transduced overnight on day 2 with a lentiviral vector encoding the CAR construct in the presence of 10 ug / ml protamine sulfate (Sigma-Aldrich, St. Louis, MO), and the culture medium was changed on day 3. The cultures were grown in TexMACS medium supplemented with 200 IU / ml IL-2 until harvesting on days 8-13.

[0296] (d) 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 quantified 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). Specific lysis percentages were calculated as (1 - (sample CPS - minimum CPS) / (maximum CPS - minimum CPS)). Supernatants from co-cultures with a 10:1 E:T ratio were taken and analyzed for IFNγ, TNFα, and IL-2 concentrations by ELISA (eBioscience, San Diego, CA).

[0297] (e) Flow cytometry analysis For cell staining, 500,000 CAR T transduced cells were isolated from the culture and washed twice in cold AutoMACS buffer supplemented with 0.5% bovine serum albumin (Miltenyi Biotec). CAR surface expression was detected by staining with CD22-Fc peptide followed by anti-Fc-PE conjugate (Jackson ImmunoResearch, West Grove, PA). Where indicated in the supplier's protocol, anti-CD4 antibody conjugated to VioBlue fluorophores (Miltenyi Biotec) was used. Non-transduced cells were used as a negative control. In all studies, dead cells were excluded by 7AAD staining (BD Biosciences, San Jose, CA). Cells were washed twice and resuspended in 200 μl of staining buffer before quantitative analysis by flow cytometry. Flow cytometry analysis was performed using MACSQuant® 10 Analyzer (Miltenyi Biotec), and data plots were created using FlowJo software (Ashland, OR).

[0298] result To overcome tumor antigen evasion, we developed a tandem CAR construct for dual targeting of CD22 and CD19 tumor antigens. A schematic of the tandem CAR design is shown in Figures 1A and 1B. Fully human scFv binders targeting CD19 and CD22 were tandemly linked by a mobile linker in either a 22-19 or 19-22 orientation, shown distal to proximal to the membrane. The resulting CAR-binding segments were then in-frame ligated to the CD8 hinge and transmembrane domain, the 4-1BB costimulatory domain, and the CD3 zeta-activating domain to create CAR22-19 (LTG2681, Figure 1A) or CAR19-22 (LTG2719, Figure 1B). The CAR sequences were incorporated into third-generation lentiviral vectors and transduced into primary human T cells.

[0299] In this example, a single CAR control is used for comparison with a tandem CAR targeting CD19 and CD22. The CAR construct LTG1538, utilizing scFv derived from the mouse hybridoma FMC63, is used as the active control. This mouse-derived sequence is a currently used binder in commercial development (see KTE-C19, Kite Pharma, and CTL019, Novartis). LTG2200, an m971 CAR, is equivalent to a CAR evaluated by the NCI and is used as an anti-CD22 CAR control.

[0300] Figure 2 shows the surface expression of anti-CD19 / CD22 CARs incorporating a single-chain fragment variable (ScFv) sequence that is reactive with the CD19 / CD22 antigen. The expression levels for each CAR containing ScFv were determined by flow cytometry analysis of LV transducible T cells from healthy donors using one of two detection methods: i) CD22-his followed by anti-his-PE, or ii) CD19 Fc recombinant protein followed by anti-Fc-A647. The ScFv-based anti-CD19 / CD22 CAR constructs, CAR22-19 LTG2681 and CAR19-22 LTG2719, were highly expressed in human primary T cells compared to non-transduced T cell controls.

[0301] As shown in Figure 3, the high cytolytic activity of CD19 / CD22CAR was demonstrated. Human primary T cells were transduced with LV encoding the CAR construct (CAR22-19 (LTG2681, D0023), CAR19-22 (LTG2791, D0024), CAR19 (LTG1538), or CAR22 (LTG2200); see Methods), and then incubated for 18 hours with Raji, REH, or 293T cell lines stably transduced with firefly luciferase for a fluorescence-based in vitro cell death assay. Raji and Reh leukemia strains express CD19 and CD22 on their surface, while the negative control 293T does not. Raji and REH cells were effectively lysed by tandem CAR22-19 (LTG2681), tandem CAR19-22 (LTG27190), and single-target controls CAR19 (LTG1538) and CAR22 (LTG2200) (Figure 3). Thus, both tandem CAR22-19 and CAR19-22 functioned in tumor lines co-expressing CD19 and CD22 antigens and did not exhibit spontaneous cell death activity against the CD19-CD22-cell line 293T, highlighting the target specificity of these constructs.

[0302] Next, the functionality and specificity of each scFv in tandem CARs were isolated and tested using tumor lines A431 or 293T that were engineered to express only a single target antigen (CD19 or CD22, or the irrelevant antigen CD20). 293T-luc strains expressing CD19 (293T luc CD19+) or CD22 (293T luc-20+) were created, and CD20+ (293T luc CD20) was used as an irrelevant target control (Figure 4A). 293T-19+ was lysed by CAR19 (LTG1538) and tandem CAR22-19 (LTG2681), but not by CAR22 LTG2200 or a non-transduced T cell control (Figure 3). 293T-CD22+ cells were lysed by tandem CAR22-19 (LTG2681) and single CAR20 LTG2200, but not by single CAR19 (LTG1538) or non-transduced controls, demonstrating the antigen specificity of the tandem CARs. Finally, 293T luc CD20 cells were not lysed by the CAR construct because this antigen was not targeted (Figure 4A). Similarly, tandem CAR T cells were tested in an overnight death assay against A431 clones expressing only the CD19 antigen (A431 luc-CD29) or only the CD22 antigen (A431 luc-CD22), or against an unrelated target control strain, A431 luc-CD20, which expresses the CD20 antigen not intended to be recognized by tandem CAR19-22 and 22-19 (Figure 4B). Here again, the A431 luc CD19 cell was lysed only by CAR19 (LTG1538) and tandem CAR22-19 (LTG2681), but not by CAR22 LTG2200 or a non-transduced T cell control. In contrast, the A431 luc CD22 cell was lysed by tandem CAR22-19 (LTG2681) and single CAR20 LTG2200, but not by single CAR19 (LTG1538) or a non-transduced control, demonstrating the antigen specificity of the tandem CAR. Furthermore, A431 luc CD20 cells were not lysed by the CAR construct because this antigen was not targeted (Figure 4B).These results highlight the independent functionality and specificity of each targeting domain of tandem CAR22-19 (Figures 4A-4B), as well as the potential for tandem CARs targeting CD19 and CD22 antigens to mitigate tumor antigen escape.

[0303] Next, the cytokine secretion capacity of anti-CD19 / CD22CAR T cells was evaluated (Figure 5). CD19+CD22+Raji tumor cells were co-incubated overnight with tandem 22-19CAR T cells (LTG2681), positive control CAR19 (LTG1538), positive control CAR22 (LTG2200), or negative control untransduced T cells (UTD) in an effector-to-target ratio of 10:1. The culture supernatant was analyzed by ELISA for IFN-gamma, TNF-alpha, and IL-2. Tandem CAR22-19 (LTG2681) strongly induced cytokines in response to tumor cells, while the negative control (untransduced, UTD) did not produce any detectable cytokine induction. In particular, LTG2681, a tandem CAR T-expressing cell, showed similar levels of IFN-gamma, TNF-alpha, and IL-2 as the CAR22 control (LTG2200), and a slightly higher cytokine response than the single CAR19 (LTG1538) control, demonstrating the high efficacy of tandem CAR. Importantly, CAR22-19 did not secrete cytokines in the absence of tumor cells (CART monotherapy group), further confirming CAR specificity and suggesting that there is no sustained signaling by tandem CAR.

[0304] Example 3 Dual-targeted CAR22-19 constructs incorporating various co-stimulatory domains exhibit robust antitumor activity. This example describes a tandem CD22 and CD19 dual-targeting CAR construct incorporating various co-stimulatory domains. The co-stimulatory domains used in the CAR22-19 design in this example include ICOS, OX40, CD27, CD137 / 4-1BB, and CD28. These co-stimulatory domain sequences are derived from T cell surface molecules known to be involved in the positive regulation of T cell function, including T cell activation, proliferation, persistence, phenotypic differentiation, memory formation, and antitumor response (Zhao Z, Condomines M, van der Stegen SJ et al., Structural design of engineered costimulation determines tumor rejection kinetics and persistence of CAR T cells. Cancer Cell Vol. 28: pp. 415-428, 2015; Guedan S, Posey AD, Jr., Shaw C et al., Enhancing CAR T cell persistence through ICOS and 4-1BB costimulation. JCI Insight Vol. 3, 2018; Song DG, Ye Q, Poussin M et al., CD27 costimulation augments the survival and antitumor activity of redirected human T cells in vivo. Blood Vol. 119: pp. 696-706, 2012; Hombach AA, Heiders J, Foppe M et al., OX40 costimulation by a chimeric antigen receptor abrogates CD28 and IL-2 induced IL-10 secretion by redirected CD4+ T cells. Oncoimmunology Vol. 1: pp. 458-466, 2012; Yoshinaga SK, Whoriskey JS, Khare SD et al., T-cell co-stimulation through B7RP-1 and ICOS. Nature Vol. 402: pp. 827-832, 1999).In some cases, the CAR linker / hinge region was also derived from the co-stimulatory domain used.

[0305] Simultaneous dual targeting of CD19 and CD22 antigens is designed to mitigate tumor antigen escape, which hinders therapeutic benefit in a subpopulation of patients who have previously received mono-CAR therapy targeting either CD19 or CD22 (Sotillo, Elena et al., "Convergence of acquired mutations and alternative splicing of CD19 enables resistance to CART-19 immunotherapy," Cancer Discovery (2015); Gardner, Rebecca et al., "Acquisition of a CD19-negative myeloid phenotype allows immune escape of MLL-rearranged B-ALL from CD19 CAR-T cell therapy," Blood (2016):blood-2015; Fry, Terry J. et al., "CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19-targeted CAR immunotherapy," Nature Medicine 24.1 (2018): No. 20).

[0306] In CAR design, avoiding non-human sequences that could trigger an anti-"foreign" immune response in the host is considered desirable as it contributes to improving the persistence of CAR T cells. All CAR components used in this example were of human origin. A CAR binder configuration based on the anti-CD22-19CAR (LTG2737), consisting of a T cell membrane-proximal human scFv-targeted CD19 and a T cell membrane-distal human scFv-targeted CD22 linked in-frame, was used for all CAR constructs in Example 3.

[0307] material and method: (a) Cell line The Burkitt lymphoma cell line, Raji, was purchased from the American Tissue Culture Collection (ATCC, Manassass, VA). Cells were cultured in RPMI-1640 medium supplemented with 10% thermoinactivated fetal bovine serum (FBS, Hyclone, Logan, UT) and 2 mM L-Glutamax (Thermo Fisher Scientific, Grand Island, NY). The human fetal kidney cell line 293T was purchased from ATCC (Gibco / Thermo Fisher Scientific, Grand Island, NY). After stably transducing wild-type tumor cells with a lentiviral vector encoding firefly luciferase (Lentigen Technology, Inc., Gaithersburg, MD), cloning was performed, and luciferase-positive clones were selected to generate single-cell clones of luciferase-expressing cell lines. CD19 and CD22-expressing 293T cell line clones, designated 293TCD19 and 293TCD22 respectively, were generated by transducing human CD19 and human CD22 proteins into parental 293T-luciferase-expressing clones using lentiviruses, followed by single-cell cloning, selection and proliferation of CD19 or CD22-positive target cell clones as needed. Stable luciferase-expressing Raji clones were generated by passage of luciferase-transduced Raji cells in mice, and their proliferative capacity was selected. Whole blood was collected from healthy volunteers at the Oklahoma Blood Institute (OBI) with written donor consent. Processed buffy coat was purchased from OBI (Oklahoma City, OK). CD4-positive and CD8-positive human T cells were purified from buffy coat by positive selection using a 1:1 mixture of CD4 and CD8 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's protocol.

[0308] (b) Creation of chimeric antigen receptors (CARs) - expression vectors Tandem CAR antigen-binding domains were obtained from human anti-CD22 ScFv and human anti-CD19 scFv sequences and tandem-linked in the configuration of anti-CD22 scFv-anti-CD19 scFv-hinge-transmembrane domain-internal domain. Some CAR T constructs were created by in-frame linking the CAR antigen-binding domain to the CD8a hinge and transmembrane domain (aa123-191, Ref sequence ID: NP_001759.3), and then to the 4-1BB (CD137, aa214-255, UniProt sequence ID: Q07011) signaling domain and CD3 zeta signaling domain (CD247, aa52-163, Ref sequence ID: NP_000725.1). In other constructs, the 4-1BB co-stimulatory domain was replaced with human ICOS, CD27, CD28, and OX-40 co-stimulatory domains, along with the complete signaling domain sequences of each molecule. In some embodiments, the CD8 transmembrane domain was replaced with a transmembrane sequence derived from the same protein as the co-stimulatory domain. In some embodiments, the internal domain of the CAR contained two tandemly linked co-stimulatory domains. In some embodiments, no co-stimulatory domains were used, and the CD3ζ activation domain was directly in-frame ligated to the transmembrane domain. In some embodiments, the two CAR chains were encoded within the same 2-cistronic expression cassette, separated by a 2A ribosome skipping element, thus enabling the simultaneous expression of the two CAR chains in each T cell transduced with a lentiviral construct encoding a 2-cistronic CAR. The CAR construct sequences were cloned into a third-generation lentiviral plasmid backbone (Lentigen Technology Inc., Gaithersburg, MD) under the control of the human EF-1α promoter. Transient transfection of HEK293T cells generated a supernatant containing a lentiviral vector (LV). The supernatant containing the lentiviral vector was then centrifuged to form a vector pellet, which was stored at -80°C.

[0309] (c) Purification and transduction of primary T cells CD4 according to the manufacturer's protocol (Miltenyi Biotec, Bergisch Gladbach, Germany) + and CD8 + Human primary T cells derived from healthy volunteers were purified from whole blood or buffy coat (purchased from private donors with written donor consent) using immunomagnetic bead selection of cells. The T cells were raised to 0.3–2 × 10⁶ in TexMACS medium supplemented with 200 IU / ml IL-2. 6 Cells were cultured at a density of cells / ml, activated with CD3 / CD28 MACS® GMP T Cell TransAct reagent (Miltenyi Biotec), transduced overnight on day 2 with a lentiviral vector encoding the CAR construct in the presence of 10 ug / ml protamine sulfate (Sigma-Aldrich, St. Louis, MO), and the culture medium was changed on day 3. The cultures were grown in TexMACS medium supplemented with 200 IU / ml IL-2 until harvesting on days 8-13.

[0310] (d) 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 quantified 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)). Supernatants from co-cultures with a 10:1 E:T ratio were taken and analyzed for IFNγ, TNFα, and IL-2 concentrations by ELISA (eBioscience, San Diego, CA).

[0311] (e) Flow cytometry analysis For cell staining, 500,000 CAR T transduced cells were isolated from the culture and washed twice in cold AutoMACS buffer supplemented with 0.5% bovine serum albumin (Miltenyi Biotec). CAR surface expression was detected by staining with CD22-His peptide followed by anti-His secondary detection reagent, and simultaneously with CD19Fc peptide followed by anti-Fc conjugate (secondary detection reagents were purchased from Jackson ImmunoResearch, West Grove, PA). Where indicated in the supplier's protocol, anti-CD4 antibody conjugated to VioBlue fluorophores (Miltenyi Biotec) was used. Non-transduced cells were used as a negative control. In all studies, dead cells were excluded by 7AAD staining (BD Biosciences, San Jose, CA). Cells were washed twice and resuspended in 200 μl of staining buffer before quantitative analysis by flow cytometry. Flow cytometry analysis was performed using MACSQuant® 10 Analyzer (Miltenyi Biotec), and data plots were generated using FlowJo software (Ashland, OR).

[0312] result: We designed dual-targeting CAR constructs composed of different co-stimulatory domains. The CAR constructs are listed in Table 1. Schematic diagrams of the CAR design configurations are shown in Figures 6A-D. In some embodiments, a tandem CAR antigen-binding domain composed of anti-CD22 ScFv and anti-CD19 scFv sequences was tandem-linked in the order of anti-CD22 scFv-anti-CD19-scFv-hinge-transmembrane domain-internal domain (Figures 6A and 6B). This targeted tandem domain configuration is based on CAR22-19 (LTG2681) in all cases. The anti-CD22-19 CAR construct LTG2737 is identical to the anti-CD22-19 CAR construct LTG2681, but it contained a CAR sequence that did not use the woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE) during its construction.

[0313] Some CAR T constructs were created by in-frame ligation of the CAR antigen-binding domain to the CD8a hinge and transmembrane domain (constructs LTG2737, D0135, D0136, D0137, D0145, D0146, D0147, D0148, and D0149). For other constructs, transmembrane domain sequences matching the co-stimulatory domain were used, namely CD28 for D139 and D140, and OX40 for D0137, D0147, and D0148. The transmembrane domain was in-frame linked to a co-stimulatory domain derived from 4-1BB (LTG2737), CD28 (D0135, D0139, D0140), ICOS (D136, D146, D148, D149), OX40 (D0137, D0145, D0147, D0148), or CD27 (D0138, D0149). All CAR molecules contained a CD3 zeta signaling domain (CD247, aa52-163, Ref sequence ID: NP_000725.1). In one embodiment, the internal domain of the CAR consisted of two tandemly linked co-stimulatory domains, namely CD28 and 4-1BB (D0140, Figure 6B). In some embodiments, a 2A ribosome skipping element for dicistronic expression was used to co-express two distinct CAR molecules in the same T cell (D146, D147, D148, D149, Figures 6C, 6D). In this configuration, each CAR chain contained only one scFv targeting either the CD19 or CD22 antigen, and both chains were co-expressed in each transduced T cell by transduction of a single lentiviral vector encoding a dicistronic sequence. In some embodiments, a co-stimulatory domain was not used for at least one of the CAR chains, resulting in the CD3ζ activation domain being directly ligated in-frame to one of the transmembrane domains of the CAR chains co-expressed in the same cell (D0146, D0147, Figure 6C). The CAR construct sequences were cloned into a third-generation lentiviral plasmid skeleton under the control of a human EF-1α promoter (Lentigen Technology Inc., Gaithersburg, MD).

[0314] [Table 1]

[0315] Figure 7 shows the surface expression of anti-CD22-19 CARs incorporating various co-stimulatory domains. Expression levels for each CAR, including ScFv, were determined by flow cytometry analysis of LV transducer T cells from healthy donors, using simultaneous staining for two scFv CAR targeting domains: i) CD22-his followed by anti-his-PE, and ii) CD19 Fc recombinant protein followed by anti-Fc-A647. All anti-CD22-19 CARs were highly expressed in human primary T cells compared to non-transducer T cell controls. CAR expression levels ranged from 71% to 90% (Figure 7).

[0316] As shown in Figures 9A–D, the high cytolytic activity of anti-CD22-19CAR was demonstrated. Human primary T cells were transduced with LV encoding the CAR constructs LTG2737, D0135, D0136, D0137, D0138, D0139, D0140, D0145, D0146), and then incubated for 18 hours with Raji, 293T, 293TCD19, or 293TCD22 cell lines stably transduced with firefly luciferase for a fluorescence-based in vitro cell death assay. Effector-to-target (ET) ratios of 2.5:1, 5:1, or 10:1 were used, as shown in the legend to the right of each plot. Raji cells express CD19 and CD22 on their surface, while the negative control 293T does not. Target strains 293TCD19 and 293TCD22 were constructed to stably express either the CD19 or CD22 target antigen, respectively. Using these strains, the ability of dual-targeting CAR constructs with different costimulatory domains to achieve targeted lysis when each single target antigen, CD19 or CD22, is induced independently of the other antigen was evaluated.

[0317] Raji cells were effectively lysed by all dual-targeted CARs but not by negative control non-transduced T cells (UTD) (Figure 9A). For comparison, all dual-targeted CAR constructs lysed single-antigen strains 293TCD19 and 293TCD22, demonstrating the ability of these CAR constructs to induce their antitumor-lytic function when activated by either the CD19 antigen alone or the CD22 antigen alone (Figures 9B and 9D, respectively). On the other hand, none of the dual-targeted anti-CD22-19 CARs lysed the antigen-negative cell line 293T (Figure 9C). Therefore, all anti-CD22-19CARs functioned in tumor lines co-expressing CD19 and CD22 antigens, as well as in 293T CD19 and 293T CD22 cell lines expressing either CD19 or CD22 monoantigens, but did not exhibit spontaneous cell death activity against the D19-CD22-cell line 293T, highlighting the target specificity of these constructs.

[0318] Next, the cytokine secretion capacity of anti-CD22-19CARs with various co-stimulatory domains was evaluated (Figure 8). CD19+CD22+Raji tumor cells were co-incubated overnight in an effector-to-target ratio of 10:1 with tandem anti-CD22-19CAR T cells expressing LTG2737, D0135, D0136, D0137, D0138, D0139, D0140, D0145, and D0146 constructs, or with negative control non-transduced T cells (UTDs). The culture supernatant was analyzed by ELISA for IFN-gamma, TNF-alpha, and IL-2 (Figure 8). All dual-targeted CARs potently induced IL-2 and TNF-α in response to tumor cells, while the negative control (non-transduced, UTD) did not produce any detectable cytokine induction (Figure 8). In particular, all CAR22-19s strongly induced subtle levels of IFN-gamma, but especially high for CAR constructs D0146, D0139, and D0136, suggesting that the strength of the cytokine response of anti-CD22-19 CARs can be regulated by the composition of the costimulatory domains used in CAR design. Overall, the secretion profiles of induced IFN-gamma, TNF-alpha, and IL-2 demonstrated the high potency of all CAR22-19 constructs. Importantly, in the absence of tumor cells (CAR T alone group), anti-CD22-19 CARs produced little to no cytokine secretion, further confirming CAR specificity and suggesting that there is no sustained signaling by tandem anti-CD22-19 CARs with various costimulatory domains.

[0319] Example 4 In vivo trials of dual CD22-CD19 targeted tandem CARs and dual cistronic CARs material and method: Cell line: The Burkitt lymphoma cell line Raji was cultured in Corning Glutgro RPMI-1640 (Corning, NY) and 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT). Raji cells were transduced with the firefly luciferase gene. Raji clones used herein were generated by transplanting luciferase-expressing Raji cells into mice and selected based on their proliferative capacity.

[0320] Derivation of Raji CD19 or CD22 knockout strains and strains with different target densities CD19 knockout (KO), CD22 KO, and CD19 / CD22 double KO strains were generated using CRISPR Cas9 gene editing techniques based on luciferase-expressing Raji cells. For the Raji 19 KO strain, a LentiCRISPR v2 plasmid containing human single guide RNA (sgRNA, AAGCGGGGACTCCCGAGACC, Genscript, Piscataway, NJ) was electroporated into Raji cells using a Lonza 4D-Nuclefector X unit. For the Raji CD22 KO strain, an Alt-R CRISPR-Cas9 crRNA RNP complex (guide RNA: CCCGAGTGCTGGACCTTCAC, PAM CGG, IDT, Coralville, Iowa) was constructed and introduced into the Raji cell line by electroporation as instructed by the supplier. Single-cell cloning was achieved by limiting dilution (0.5 cells per 96-well) before CD19 or CD22 microbead depletion (Miltenyi, Bergisch Gladbach, Germany). All knockout cell lines were validated using surface protein staining and DNA sequencing by flow cytometry. CD19 and CD22 dual knockout lines were generated based on CD22 knockout lines using the same procedure as the CD19 knockout strategy described above.

[0321] To generate Raji cell lines with different CD19 or CD22 densities, CD19 / CD22 dual knockout Raji cell lines were stably transduced with lentiviral vectors encoding CD19 or CD22 fused to puromycin via F2A peptide. Before limiting dilution (0.5 cells / well), each available clone was screened for target molecule expression by flow cytometry staining.

[0322] Immunoeffector assay (cytotoxic) The functionality of different CAR constructs was evaluated by performing a cytotoxic overnight cell death assay as described elsewhere (13). Briefly, CAR T cells were co-cultured with 5,000 target cells possessing firefly luciferase activity for 16–18 hours at various effector-to-target ratios. SteadyGlo reagent (Promega, Madison WI) was added to each well, and the resulting luminescence was quantified using a GloMax Luminometer, which was then used to calculate the specific percentage of lysis.

[0323] Flow cytometry analysis Flow cytometry analysis was performed to detect cell surface molecule expression. Cells were washed and stained with cold AutoMACS buffer supplemented with 0.5% bovine serum albumin (Miltenyi Biotec), resuspended in 200 μl of running buffer, and then acquired using a MACSQuant® 10 Analyzer (Miltenyi Biotec). For CAR surface expression, CD22-his peptide (Abcam, Cambridge, MA) and CD19Fc peptide (R&D System, Minneapolis, MN) were used, followed by anti-His-PE or anti-Fc-AF647 (Jackson ImmunoResearch, West Grove, PA). T cell subtypes were further identified using anti-CD4 antibody conjugated to a VioBlue fluorophore. In all studies, dead cells were excluded by 7AAD staining (BD Biosciences, San Jose, CA) or Viobility 405 / 520 Fixable Dye. For CD19 or CD22 surface expression on cell lines, we used the anti-human CD19 antibody LT19 conjugated to a PE fluorophore and the anti-human CD22 antibody REA340 conjugated to an APC or APC-Vio770 fluorophore. To quantify the CD19 or CD22 surface molecular density, antibody-binding capacity (ABC) assays were performed according to the manufacturer's protocol using the PE Fluorescence Quantitation Kit from BD Bioscience (San Jose, CA) and BD's anti-human CD19-PE (SJ25C1) and anti-human CD22-PE (S-HCL-1). All antibodies and staining reagents listed were from Miltenyi Biotec unless otherwise noted. Flow data were analyzed using FlowJo software (Ashland, OR).

[0324] in vivo NSG xenograft model All animal research was conducted by the MI Bioresearch Animal Care and Use Committee (Ann Arbor, MI). NSG (NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ) Mouse, 5x10 5 One mouse-adapted WT Raji-luc cells, or a Raji mixture containing equal proportions of Raji, Raji19KO, and Raji22KO for heterogeneous models, were injected. Tumor burden was measured using bioluminescence imaging with a Xenogen IVIS-200 instrument (Perkin Elmer, Shelton, Connecticut). Mice with comparable tumor burden were randomly assigned to each group for CAR T administration on day 7. Tumor development kinetics were measured by live animal imaging at indicated time points. Tissue samples, including blood, bone marrow, and spleen, were collected upon request.

[0325] Analysis of viral copy number in animal tissues Mouse blood, bone marrow, and spleen were collected at the end of the animal study. Tissue samples were extracted using the DNeasy Blood & Tissue Kit (Qiagen, Germantown, MD) according to the instructed manual, and genomic DNA was quantified using Nanodrop (ThermoFisher, Carlsbad, CA). 50 ng of DNA per sample per reaction was used for real-time PCR with TaqMan Fast Advanced Master (ThermoFisher, Carlsbad, CA) on a BioRad CFX qPCR instrument. Primers and probes were synthesized by IDT Technology. The temperature cycling conditions consisted of a three-step amplification cycle: 2 minutes at 50°C, followed by 40 repeats at 95°C for 20 seconds, 95°C for 5 seconds, 56°C for 20 seconds, and 65°C for 20 seconds. The viral copy number (VCN) for each sample was calculated based on a standard curve.

[0326] TaqMan primer and probe set: Regarding tandem cars, Tan For:5'-GAGCGACATAGGCAACAAGA-3', Tan Rev:5'-GGCGATCGTAGTCATCATACAC-3', Tan probe: 5'-ACAGAAACCAGGTCAAGCACCTGT-3'.

[0327] Regarding 2-cistronic dual-targeting CARs, Dual targeting For:5'-CACTTACTACCGGTCCAAAT-3' Dual targeting Rev:5'-GAGTGAGAACTGGTTCTTCGAG-3' Dual-targeting probe: 5'-ACTACGCCGTGTCCGTGAAGAATC-3'.

[0328] statistical analysis Statistical analysis was performed using GraphPad Prism 8 software. Both biological replicates showing different donors and technical replicates showing repeated measures were included in the statistical analysis. Bioluminescence data, as log-normal parametric data, were log-transformed before analysis. Differences between groups were determined by two-way or one-way ANOVA followed by Dunnett or Tukey post-hoc tests. Paired t-tests were used to compare data between two groups. P-values ​​were reported as follows: ns - no significance, p>0.05. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Outlier tests were used to exclude mice with results inconsistent with the group.

[0329] result We constructed a series of CAR constructs capable of simultaneously targeting CD19 and CD22 B cell antigens. All constructs were fully human, utilizing human scFv sequences for CD19 (m19217-1) and CD22 (16P17). We constructed second-generation CARs, third-generation CARs, and bi-cistronic dual-targeting CARs for CD19 and CD22 (Table 2).

[0330] [Table 2]

[0331] The CAR co-stimulatory domain modulates the antitumor efficacy of tandem CAR22-19 in Raji NSG xenografts. The in vivo tumor rejection functionality of CARs with different TNFR superfamily-derived or Ig superfamily-derived costimulatory domains was further investigated using wild-type NSG Raji xenograft and CD19 or CD22-antigen heterogeneous Raji xenograft mouse models (Figures 10A-I). In wild-type Raji xenograft, the highest tumor regression was achieved with the dual-targeted CAR construct D0146, followed by the third-generation CAR D0140 and second-generation D0135, and then a third group with D0136, D0137, D0138, and D0139, while the LTG2737 CAR produced only weak antitumor activity (Figures 10A, 10B, 10C). Unexpectedly, among the two OX40-costimulating CARs that differed only in hinge and transmembrane domain composition, CAR D0145 showed higher expression and functionality than D0137 in vitro (Figures 9A-9D), yet was the least effective CAR in vivo (Figures 10A and 10B). When comparing CD28 domain 22-19 tandem CARs, CAR D0135 performed better in vivo than D0139 despite similar in vitro function (Figures 10A, 10C, 9A-9D).

[0332] In vivo proliferation and persistence of 22-19CAR variants in total human T cells (Figure 10D-10F) and CD8 + (Figures 10G-10I) Flow cytometry counts of T cell populations were measured in peripheral blood on days 14, 21, and 28 of the study. On day 14, the dual-targeted CAR D0146, which had the fastest tumor clearance, also showed the most significant total T cell proliferation in peripheral blood. Several CAR22-19 costimulatory domain variants also showed evidence of proliferation in the blood, including increases in total T cells for D0135, D0136, D0137, and D0138 (Figure 10D). Although not statistically significant, D0140 showed a tendency toward T cell proliferation. CD8 + A corresponding increase in T cell count was detected for these CAR treatments (Figure 10G). However, the total T cell levels and CD8 of 22-19CAR LTG2737, D0139, and D0145 were not detected. + T levels remained low on day 14 (Figure 10D, 10G). On day 21, two CARs containing BB-costimulatory sequences, LTG2737 and D0140, were found to be low in total T cells and CD8 + Both CAR variants elevated T cell levels, while other CAR variants had decayed in peripheral blood by this point (Figure 10E, 10H). By day 28 of the study, third-generation CAR D0140 elevated total T cells and CD8 + While both levels remained elevated at the T cell level, CAR LTG2737 maintained an elevated total T cell count, but CD8 + This is not maintained in the T subset, this is CD4 + This suggests that T cells were responsible for the persistence of the LTG2737 CAR at this point (Figures 10F, 10I). Clearly, third-generation CAR22-19 with 28-BB costimulation was responsible for total T cells and CD8 +The longest persistence of the T portion was maintained, which is thought to contribute significantly to the overall antitumor function of CARs through differentiation into CTLs (cytotoxic T lymphocytes, an effector subset capable of killing tumor cells). In particular, early CAR proliferation on day 14 was necessary for effective tumor clearance, even in the absence of prolonged blood persistence, as observed for CARs D0146, D0135, D0136, D0137, and D0138. In comparison, prolonged persistence in the absence of early CAR T proliferation was insufficient for tumor clearance, as observed for second-generation 22-19BB tandem CARs LTG2737 and CAR D0145.

[0333] To investigate the ability of bispecific CAR22-19 variants to eradicate antigen-negative, antigen-low, or antigen-heterogeneous tumors, CAR constructs that mediated strong tumor regression in wild-type Raji xenografts were further investigated in a Raji antigen-heterogeneous model. Mice were transplanted with an equal-proportion mixture of Raji 19KO, Raji22KO, and wild-type Raji tumor cells, all stably expressing firefly luciferase, to facilitate detection of tumor progression. This included alternative costimulatory domain variant CARs D0140, D0135, D0136, D0137, bitargeted CARs D0146, D0148, and D0186, the control CAR LTG2737, as well as experimental groups treated with single-targeted CAR19 and CAR22, UTDs, and tumor-negative controls (Figure 11). Tumor progression was monitored by bioluminescence imaging. Mouse number 6 in group D0140 developed a high tumor burden, possibly due to a drug delivery error, and was excluded from the data plot by outlier testing (Figures 11A, 11B). All bispecific CD22 and CD19-targeted CARs, D0140, D0135, D0136, D0137, D0146, D0148, and D0186, as well as the control CAR LTG2737, strongly rejected antigen-heterogeneous Raji tumors, while single-targeted CARs 22 and CAR19 allowed tumor progression (Figures 11A, 11B). Therefore, bispecific 22-19 CARs prevented tumor escape from removal in this xenograft model, while single-CAR controls did not.

[0334] As seen from bioluminescence data on day 9 of the study, the most rapid tumor rejection was mediated by CARs D0135, D0137, D0148, and D0186, followed shortly thereafter by CARs D0136, D0140, and D0146. Meanwhile, CAR LTG2737 lagged behind other CAR22-19 variants on both day 9 and also day 14, although the differences between tandem CARs were not statistically significant (Figure 11B).

[0335] The persistence of CD19 and CD22-targeted CARs was investigated in this model by real-time PCR analysis of the presence of CAR DNA in mouse peripheral blood, bone marrow, and spleen at the conclusion of the study on day 29 (Figures 11C-11E). Complete tumor rejection was achieved in all tandem 22-19CAR groups by day 14, and measurable CAR copy numbers were detected in blood, bone marrow, and spleen on day 29, clearly indicating the persistence of the 22-19CAR construct (Figures 11C-11E). The highest CAR copy numbers were found for CARs D0140 and D0137, followed by the dual-targeted CAR D0148, then D0136, D0186, and LTG2737 CAR, while D0135 and D0146 were barely detectable (Figures 11C-11E). Therefore, results from in vivo models of both wild-type and heterogeneous Raji support the efficacy and long-term persistence of D0140 and D0145. Furthermore, the combination of the CD28 and BB costimulatory domains in the third-generation tandem 22-19CAR construct had a dramatic synergistic effect on its persistence in peripheral blood, bone marrow, and spleen.

[0336] These results demonstrate that the antitumor function of tandem CAR22-19BBζ (LTG2737) in vivo can be modulated and dramatically improved by utilizing alternative costimulatory domains. Specifically, optimization of the CAR22-19 costimulatory sequence, hinge, and transmembrane domain, as well as the implementation of a third-generation CAR architecture, yielded tandem 22-19 CAR candidates with superior antitumor functionality. Therefore, it is crucial to determine whether these improved CAR constructs are better suited to detecting low-antigen-density tumor cell variants, which remain a troublesome obstacle to the clinical efficacy of CARs.

[0337] Alternative co-stimulatory domains improved the response of 22-19BBz CAR T cells to tumor targets with low antigen density. Reduced CD22 surface expression has been sufficient to cause tumor recurrence due to tumor antigen evasion in recent clinical trials of CD22 CARs using single-targeted CAR constructs with BB costimulatory domains and CD3ζ activating domains (6). To determine whether tandem CAR22-19 constructs with alternative costimulatory domains, which have been highly effective in tumor killing in vitro and in vivo, may have greater sensitivity to reduced levels of CD22, single-antigen Raji clones with high or low surface density CD22 were subjected to CRISPR-Cas9 dual knockout of CD19 and CD22, as well as CD19 neg CD22 neg CD22 was prepared by lentiviral transduction of CD22 into a Raji background (Figure 12A). Tandem CAR constructs and single-targeted CAR22 controls were subjected to overnight Raji exposure at E:T ratios of 5:1, 10:1, or 20:1. hi Or Raji lowCo-incubation with the target was performed. The reduction in lysis efficacy due to reduced CD22 surface density was most pronounced in the LTG2737 tandem 22-19 CAR construct with a BB co-stimulatory domain, and a particularly dramatic loss of cytotoxicity was observed against strains with low CD22 target density (Figure 12B). The tandem 22-19 third-generation CAR D0140, the tandem CARs D0135 and D0139 with 28 co-stimulatory domains, and the dual-targeted CARs D0146, D0147, D0148, D0149 and D0186 showed high killing efficacy at most E:T ratios (Figures 12C-12E, 12J-12N). In terms of lysis efficiency, the next most effective were the tandem CARs D0136 and D0145 with ICOS co-stimulation (Figure 12F, 12G), followed by CAR D0137 and CAR D0138 (Figure 12H, 12I). The lysis capacity of single CAR22 controls was lower than that of most 22-19 CAR variants, with the exception of LTG2737 CAR (Figure 12O). Thus, compared to single CAR22, the reduction in lysis capacity and sensitivity of CARs to low target density clones observed in tandem CAR22-19BBζ LTG2737 was modified and enhanced beyond the original CAR22 by the incorporation of the CAR co-stimulatory domain. In particular, the order of tandem CAR22-19 variants against CD22 antigen density closely followed their potency in in vivo xenograft models: the Ig-superfamily CD28 and 28-BB CARs were the strongest, followed by ICOS. The TNFR superfamily CAR variants ranked lower than the Ig superfamily group, and OX co-stimulation was comparable to or somewhat more efficient than ICOS, followed by CD27 and BB (Figure 12). Interestingly, all dual CAR constructs were lower than single CAR22. low They demonstrated superior cytotoxicity and sensitivity to the target. Consistently, in xenograft models, the tested dual-targeted CAR constructs performed comparably to or better than the best tandem constructs D0135 and D0140, demonstrating rapid tumor eradication (Figure 11).

[0338] In particular, when tested against Raji clones with high and reduced CD19 density, the same efficacy sequence of tandem 22-19CARs emerged (Figure 13A-P). This observation suggests that while the binding properties of individual binders in the tandem CAR form contribute to their lysis ability, the sensitivity of each binder to low antigen density targets can be significantly improved by substitution of the co-stimulatory domain.

[0339] Equal parts 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.

[0340] 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.

[0341] Sequence List 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 understood to be included by referring to the shown strand. In the attached sequence listing: Sequence ID 1 is the nucleotide sequence LTG2681 D0023 leader-CD22VH-(GGGGS)-3 CD22VL(GGGGS)-5 CD19VH(GGGGS)-3 CD19VL CD8 hinge+TM-4-1BB-CD3z (construct CAR2219). Sequence ID 2 is the amino acid sequence LTG2681 D0023 leader-CD22VH-(GGGGS)-3 CD22VL(GGGGS)-5 CD19VH(GGGGS)-3 CD19VL CD8 hinge+TM-4-1BB-CD3z (construct CAR2219). MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKHSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQEVEPHDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVYASVGDKVTITCRASQDVSGW LAWYQQKPGLAPQLLISGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKYFPYTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGLINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDT AVYYCARSDRGITATDAFDIWGQGTMVTVSSGGGGSGGGGSGGGSQSVLTQPPSVSVAPGRMAKITCGGSDIGNKNVHWYQQKPGQAPVLVVYDDYDRPSGIPERFSGSNSGDAATLTISTVEVGDEADYFCQVWDGSGDPYWMFGGGTQLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAG GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 3 is the nucleotide sequence LTG2791 D0024 leader-CD19VH(GGGGS)3-CD19VL-(GGGGS)5-CD22VH(GGGGS)3-CD22VH CD8 hinge+TM-4-1BB-CD3z(construct CAR1922). Sequence ID 4 is the amino acid sequence LTG2719 D0024 leader-CD19VH(GGGGS)3-CD19VL-(GGGGS)5-CD22VH(GGGGS)3-CD22VH CD8 hinge+TM-4-1BB-CD3z(construct CAR1922). MLLLVTSLLLCELPHPAFLLIPEVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGLINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSDRGITATDAFDIWGQGTMVTVSSGGGGSGGGGSGGGSQSVLTQPPSVSVAPGRMAKITCGGSDIGNKNV HWYQQKPGQAPVLVVYDDYDRPSGIPERFSGSNSGDAATLTISTVEVGDEADYFCQVWDGSGDPYWMFGGGTQLTVLGGGGGSGGGGSGGGGSGGGGSGGGSQVQLQQSGPGLVKHSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNS VTPEDTAVYYCAQEVEPHDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVYASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLISGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKYFPYTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAG GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 5 is the nucleotide sequence of the complete human CAR19 LTG2065 (M19217-1-CD8TM-4-1BB zeta). Sequence ID 6 is the amino acid sequence of the complete human CAR19 LTG2065 (M19217-1-CD8TM-4-1BB zeta). MLLLVTSLLLCELPHPAFLLIPEVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGLINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSDR GITATDAFDIWGQGTMVTVSSGGGGSGGGGSGGGSQSVLTQPPSVSVAPGRMAKITCGGSDIGNKNVHWYQQKPGQAPVLVVYDDYDRPSGIPERFSGSNSGDAATLTISTVEVGDEADYFCQ VWDGSGDPYWMFGGGTQLTVLGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 7 is the nucleotide sequence of mouse scFv CAR19 LTG1538. Sequence ID 8 is the amino acid sequence of mouse scFv CAR19 LTG1538. MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGG GTKLEITGGGGSGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYY YGGSYAMDYWGQGTSVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP Sequence ID 9 is the nucleotide sequence of CAR22 LTG2209. Sequence ID 10 is the amino acid sequence of CD22A 1495 (CAR20A). MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKHSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQ EVEPHDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVYASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLISGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQAKYFPYTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID 11 is the nucleotide sequence of the leader / signal peptide (LP). atgctgctgctggtgaccagcctgctgctgtgcgaactgccgcatccggcgtttctgctgattccg Sequence ID 12 is the amino acid sequence of the leader / signal peptide (LP). MLLLVTSLLLCELPHPAFLLIP Sequence ID 35 is the nucleotide sequence of the DNA CD8 transmembrane domain. atttgggccccgctggccggcacttgcggcgtgctcctgctgtcgctggtcatcaccctt tactgc Sequence ID 36 is the 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 is the nucleotide sequence of the DNA CD8 hinge domain. actaccacccctgcccctcggccgccgactccggccccaaccatcgcaagccaacccctc tccttgcgccccgaagcttgccgcccggccgcgggtggagccgtgcatacccgggggctg gactttgcctgcgatatctac Sequence ID 38 is the amino acid sequence of the CD8 hinge domain. 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 Sequence ID 39 contains the amino acid sequence of the hinge (amino acids 137-206) and the transmembrane region of CD8α (NCBI RefSeq: NP). - 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 Sequence ID 40 is the nucleotide sequence of the DNA signaling domain at 4-1BB. aagaggggccggaagaagctgctttacatcttcaagcagccgttcatgcggcccgtgcag acgactcaggaagaggacggatgctcgtgcagattccctgaggaggaagaggggggatgc surfaceactg Sequence ID 41 is the amino acid sequence of the signaling domain at 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 is the nucleotide sequence of the DNA signaling domain of CD3-zeta. cgcgtcaagttctcacggtccgccgacgcccccgcatatcaacagggccagaatcagctc tacaacgagctgaacctgggaaggagagaggagtacgacgtgctggacaagcgacgcgga cgcgaccgggatgggggggaaaccacggcggaaaaaccctcaggaaggactgtacaac gaactccagaaagacaagatggcggaagcctactcagaaatcgggatgaagggagagcgg aggaggggaaagggtcacgacgggctgtaccagggactgagcaccgccactaaggatacc tacgatgccttgcatatgcaagcactcccaccccgg Sequence ID 43 is the amino acid sequence of CD3 zeta. Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr 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 Lys Asp Thr Tyr Asp With Leu His Met Gln Ala Leu Pro Pro Arg The 44 scvf CD19 (FMC63) is located on the screen. gatatcagatgactcagaccacctctctctgtccgcgtcactgggagacagtgaccat ctcgtgtcgcgcaagccaggatatctccaagtacctgactgtaccacagaagcccga cgggactgtgaagctgctgatctaccaccacctcacgcctgcacagcggagtgccaagcag attctccggctccggctcgggaaccgattactcgcttaccattagcacctcgagcagga ggacatcgctacctactctctgccagcaaggaaataccctgcctacaccttcggcggagg aaccaaattggaaatcaccggcggaggaggctccggggggagggttccggggcgggg ttccgaagtgaagctccaggagtccggccccggcctggtggcgccgtcgcaatcactctc tgtgacctgtaccgtgtcgggagtgtccctgcctgattacggcgtgagctggattcggca gccgccgcggaagggcctggaatggctgggtgtcatctggggatccgagactacctacta caactcggccctgaagtcccgcctgactatcatcaaagacaactcgaagtcccaggtctt tctgaagatgaactccctgcaaactgacgacaccgccatctattactgtgctaagcacta ctactacggtggaagctatgctatggactactggggcaaggcacttcggtgactgtgtc aagc Amino acid sequence of Scvf CD19 (FMC63) is sequence number 45. Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val ...

Claims

1. An isolated nucleic acid molecule encoding a CD19 / CD22 tandem chimeric antigen receptor (CAR) comprising at least one extracellular antigen-binding domain including a CD19 / CD22 antigen-binding domain, at least one transmembrane domain, and at least one intracellular signaling domain, wherein the CD19 / CD22 tandem chimeric antigen receptor (CAR) is encoded by a nucleotide sequence comprising SEQ ID NO: 84 or 86.

2. A vector comprising the nucleic acid molecule described in Claim 1.

3. The vector according to claim 2, selected from the group consisting of 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.

4. A cell comprising the vector according to claim 2.

5. A method for producing cells, comprising the step of transducing the vector described in Claim 2 into T cells.

6. A pharmaceutical composition comprising an antitumor-effective amount of human T cell population, 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 including a CD19 / CD22 antigen-binding domain, at least one transmembrane domain, and at least one intracellular signaling domain, the CD19 / CD22 tandem CAR comprises an amino acid sequence including SEQ ID NO: 85 or 87, and the T cells are T cells of a human having cancer.

7. Use of a population of T cells for the manufacture of a pharmacopoeia for treating cancer, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising at least one extracellular antigen-binding domain including a CD19 / CD22 antigen-binding domain, at least one transmembrane domain, and at least one intracellular signaling domain, wherein the CD19 / CD22 tandem CAR comprises an amino acid sequence comprising SEQ ID NO: 85 or 87, thereby treating a cancer of interest.

8. The use according to claim 7, wherein the at least one transmembrane domain comprises a transmembrane domain of a protein comprising the alpha, beta, or zeta chain of a T cell receptor, CD8, CD28, CD3 epsilon, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, and TNFRSF19, or any combination thereof.

9. The use according to claim 7, wherein the at least one CD19 / CD22 antigen-binding domain, the at least one intracellular signaling domain, or both are connected to the transmembrane domain by a linker or spacer domain.

10. The use according to claim 9, wherein the linker or spacer domain is derived from the extracellular domain of CD8 or CD28 and is transmembrane-linked.

11. The use according to claim 7, wherein the at least one extracellular CD19 / CD22 antigen-binding domain is preceded by a leader nucleotide sequence encoding a leader peptide.

12. The isolated use according to claim 7, wherein the at least one intracellular signaling domain comprises a co-stimulatory domain comprising a functional signaling domain 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 any combination thereof.

13. The use according to claim 7, wherein the nucleic acid sequence encoding the CD19 / CD22 tandem chimeric antigen receptor (CAR) is encoded by a nucleotide sequence comprising SEQ ID NO: 84 or 86, or by a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.

14. The use according to claim 7, wherein the at least one intracellular signaling domain comprises a co-stimulatory domain, a primary signaling domain, or any combination thereof.

15. The use according to claim 7, wherein the at least one intracellular signaling domain further comprises a CD3 zeta intracellular domain.

16. The use according to claim 7, wherein the cancer is a hematological cancer.

17. The use according to claim 16, wherein the hematological cancer is leukemia or lymphoma.

18. The use according to claim 17, wherein the leukemia is chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), or chronic myeloid leukemia (CML).

19. The use according to claim 17, wherein the lymphoma is mantle cell lymphoma, non-Hodgkin lymphoma, or Hodgkin lymphoma.

20. The use according to claim 7, wherein the cancer is selected from the group consisting of oral and pharyngeal cancers, digestive system cancers, respiratory system cancers, bone and joint cancers, soft tissue cancers, skin cancers, pediatric tumors, central nervous system tumors, and cancers of the breast, reproductive system, urinary system, eye and orbit, endocrine system, and brain.

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