B cell-targeted parallel CAR (pCAR) therapeutic agents

Abstract

Provided herein are immunoresponsive cells that express B cell-targeting pCARs, including second-generation chimeric antigen receptors (CARs) and chimeric costimulatory receptors (CCRs). Also provided herein are methods for preparing immunoresponsive cells and for directing T cell-mediated immune responses using immunoresponsive cells.

Description

[Technical Field]

[0001] 1. Background Chimeric antigen receptors (CARs), sometimes called artificial T cell receptors, chimeric T cell receptors (cTCRs), or chimeric immune receptors, are engineered receptors that are currently well known in the art. They are primarily used to transform immune effector cells, particularly T cells, to confer desired engineered specificity to these cells. Adoptive cell therapy using CAR-T cells is particularly considered in the field of cancer therapy. In these therapies, T cells are removed from a patient and engineered to express a CAR specific for an antigen found in a particular form of cancer. The resulting CAR-T cells, capable of recognizing and killing cancer cells, are then reintroduced into the patient. [Background technology]

[0002] First-generation CARs most commonly use the CD3 zeta (z) intracellular signaling domain to provide TCR-like signals, thereby resulting in tumoricidal function.However, the binding of CD3z chain fusion receptors may be insufficient to cause substantial IL-2 secretion and / or T cell proliferation in the absence of accompanying costimulatory signals.In physiological T cell responses, optimal lymphocyte activation requires the binding of one or more costimulatory receptors, such as CD28 or 4-1BB.

[0003] 2nd (2 ndSecond-generation CARs have been engineered to deliver functional antigen-dependent costimulatory signals in human primary T cells in addition to antigen-dependent TCR-like signals, enabling T cell proliferation in addition to tumoricidal activity. Second-generation CARs most commonly provide costimulation using costimulatory domains (synonymously, costimulatory signaling regions) derived from CD28 or 4-1BB. The combined delivery of costimulatory and CD3 zeta signals makes second-generation CARs clearly superior in terms of functionality compared to their first-generation counterparts (CD3z signal only). Examples of second-generation CARs can be found in U.S. Patent No. 7,446,190, incorporated herein by reference.

[0004] Recently, so-called third-generation CARs have been prepared. These combine multiple costimulatory domains (synonymously, costimulatory signaling regions) in cis with a TCR-like signaling domain (e.g., CD28+4-1BB+CD3z or CD28+OX40+CD3z) to further enhance efficacy. In third-generation CARs, the costimulatory domains are arranged in tandem within the CAR endodomain, generally located upstream of CD3z or its equivalent.

[0005] In general, however, the results achieved by these third-generation CARs have been disappointing, showing only minimal improvement over second-generation configurations, and some third-generation CARs being inferior to second-generation configurations.

[0006] We recently described a novel format in which immunocompetent cells, such as T cells, are engineered to express two juxtaposed constructs: a second-generation CAR and a chimeric costimulatory receptor (CCR). The second-generation CAR contains, from C-terminus to N-terminus (intracellular to extracellular), the following domains: (a) a signaling region; (b) a costimulatory signaling region; (c) a transmembrane domain; and (d) a first binding element that specifically interacts with a first epitope on a first target antigen. The CCR contains, from C-terminus to N-terminus (intracellular to extracellular), (a) a costimulatory signaling region that is different from the costimulatory signaling region of the CAR; (b) a transmembrane domain; and (c) a second binding element that specifically interacts with an epitope on the target antigen. The CAR and CCR can recognize the same epitope on the same antigen, different epitopes, or epitopes found on two separate antigens. Unlike CARs, CCRs lack TCR-like signaling regions such as CD3z. These parallel CAR (pCAR)-modified T cells exhibit superior activity and resistance to depletion compared to first-, second-, and third-generation CAR-T cells. See U.S. Pregrant Publication 2019 / 0002521, incorporated herein by reference in its entirety.

[0007] These properties of pCAR-T cells make them attractive candidates for the treatment of refractory malignancies where first-, second-, and third-generation CAR-T cells have limited efficacy due in part to T cell exhaustion. However, there is a need for additional antigen-targeting combinations that will enable broad therapeutic application of this technology. Summary of the Invention [Problem to be solved by the invention]

[0008] 2. Summary of the Invention The present applicants have found that effective T cell responses can be induced using a combination of constructs in which multiple costimulatory regions are arranged in separate constructs. In particular, provided herein are effective pCAR-T cells having parallel CAR (pCAR) constructs that bind to one or more antigens present on target cells derived from the B cell lineage. In some embodiments, the pCAR construct comprises a CAR (chimeric antigen receptor) containing a binding element that specifically binds to an epitope found on CD19 on target cells, and a CCR (chimeric costimulatory receptor) that binds to CD19 or another B cell lineage-specific marker. Examples of the latter include, but are not limited to, CD20, CD22, CD23, CD79a, and CD79b. [Means for solving the problem]

[0009] Thus, according to some embodiments: i. Second generation chimeric antigen receptors (CARs), including: a) signaling region; b) the first costimulatory signaling region; c) the first transmembrane domain; and d) a first binding element that specifically interacts with an epitope on the CD19 target antigen; and ii. A chimeric costimulatory receptor (CCR) comprising: e) a second costimulatory signaling region, wherein the second costimulatory signaling region is different from the first costimulatory signaling region; f) a second transmembrane domain; and g) a second binding element that specifically interacts with a second epitope on a second target antigen, where the second target antigen is CD19 or another B cell-associated target antigen. Provided herein are immunoresponsive cells that express the

[0010] When a T cell expressing a B cell-targeting pCAR construct binds to a cell expressing one or more antigens that have both epitope targets (for the CAR and CCR), both the CAR and CCR deliver stimulatory signals to enhance the T cell response.

[0011] Constructs of the present type may be referred to as "parallel chimeric activating receptors" or "pCARs." The inventors have found that the pCARs described herein are superior to second-generation CAR-T cells with similar elements in both in vitro and in vivo experiments.

[0012] Furthermore, T cell proliferation, ability to maintain cytotoxic efficacy and ability to release IL-2 are maintained over many repeated rounds of stimulation with antigen-expressing tumor cells.

[0013] In some embodiments, the first epitope recognized by the CAR component of pCAR is an epitope on the CD19 target antigen. In some embodiments, the first binding element comprises the complementarity determining region (CDR) of the FMC63 antibody and has the sequence of SEQ ID NOs: 10, 11, 12, 13, 14, and 15. In some embodiments, the first binding element comprises the variable heavy (V) domain of the FMC63 antibody. H ;GenBank accession number CAA74659.1) and variable light (V L ; GenBank Accession No. CAA74660.1) domain and has the sequence of SEQ ID NOs: 16 and 17. In some embodiments, the first binding element comprises the variable heavy (V H ) and variable light (V L ) domain and having the sequence of SEQ ID NO: 18 or 19. In some embodiments, the FMC63 scFv is expressed from the polynucleotide or set of polynucleotides of SEQ ID NO: 118.

[0014] In some preferred embodiments, the first binding element has a single G to A or Y to A mutation that results in a V H The modified V sequences of SEQ ID NOs: 20, 21, 22, 23, 24, 25 and 26 are introduced into the CDR3 of the domain. H In some preferred embodiments, the mutant FMC63 scFv is expressed from a polynucleotide or set of polynucleotides of SEQ ID NOs: 119, 120, 121, 122, 123, 124, or 125.

[0015] In some embodiments, the second epitope recognized by the chimeric costimulatory receptor (CCR) component of pCAR is also an epitope on the CD19 target antigen. In some embodiments, the second binding element comprises the complementarity determining region (CDR) of the FMC63 antibody and has the sequence of SEQ ID NOs: 10, 11, 12, 13, 14, and 15. In some embodiments, the second binding element comprises the variable heavy (V) domain of the FMC63 antibody. H ) and variable light (V L ) domain of the FMC63 antibody and has the sequence of SEQ ID NOs: 16 and 17. In some preferred embodiments, the second binding element comprises the variable heavy (V H ) and variable light (V L ) domain and has the sequence of SEQ ID NO: 18 or 19. In some preferred embodiments, the FMC63 scFv is expressed from the polynucleotide or set of polynucleotides of SEQ ID NO: 118.

[0016] In some embodiments, the CAR and CCR bind to the same epitope within the CD19 antigen. In some preferred embodiments, the pCARs are designated FBB / G01, FBB / G02, FBB / Y01, FBB / Y02, FBB / Y03, FBB / Y04, and FBB / Y05, respectively, and have the sequence of SEQ ID NO: 47, 48, 49, 50, 51, 52, or 53. The nomenclature is derived from the abbreviations for the following elements: CCR binder ( FMC63 scFv), CCR signaling domain (4-1 BB ) / CAR binder (respectively, G01 ~ Y05 In some embodiments, the CARs of FBB / G01, FBB / G02, FBB / Y01, FBB / Y02, FBB / Y03, FBB / Y04, and FBB / Y05 comprise the sequences of SEQ ID NOs: 56, 58, 59, 60, 61, 62, and 63, respectively, and the CCR comprises the sequence of SEQ ID NO: 57. In some preferred embodiments, these pCARs are expressed from a polynucleotide or set of polynucleotides of SEQ ID NOs: 109, 110, 111, 112, 113, 114, or 115, respectively. In some embodiments, the pCAR is a polypeptide having at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 47, 48, 49, 50, 51, 52, or 53. In some embodiments, pCAR is a polypeptide having at least about 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 47, 48, 49, 50, 51, 52 or 53.

[0017] In some embodiments, the CAR and CCR bind to distinct epitopes within the CD19 antigen.

[0018] In some embodiments, the CAR binds to CD19 and the CCR binds to a distinct B-cell lineage antigen, such as CD20, CD22, CD23, CD79a, or CD79b.

[0019] In some embodiments, the CAR binds to CD19 and the CCR binds to CD20. In some preferred embodiments, the second binding element directing CCR specificity comprises the complementarity determining regions (CDRs) of the 1F5 antibody and has the sequence of SEQ ID NOs: 27, 28, 29, 30, 31, and 32. In some preferred embodiments, the second binding element comprises the variable heavy (V) domain of the 1F5 antibody. H ;GenBank accession number AAL27650.1) and variable light (V L; GenBank Accession No. AAL27649.1) domain and has the sequence of SEQ ID NOs: 33 and 34. In some preferred embodiments, the second binding element comprises the variable heavy (V H ) and variable light (V L ) domain and has the sequence of SEQ ID NO: 35 or 36. In some preferred embodiments, the 1F5 scFv is expressed from the polynucleotide or set of polynucleotides of SEQ ID NO: 126. In some preferred embodiments, the pCAR is designated 1BB / F and has the sequence of SEQ ID NO: 54. The nomenclature is derived from the abbreviations of the following elements: CCR binder ( 1 F5 scFv), CCR signaling domain (4-1 BB ) / CAR Binder( F MC63 scFv). In some embodiments, the CAR of 1BB / F comprises the sequence of SEQ ID NO: 64, and the CCR of 1BB / F comprises the sequence of SEQ ID NO: 65. In some preferred embodiments, 1BB / F pCAR is expressed from the polynucleotide or set of polynucleotides of SEQ ID NO: 116. In some embodiments, pCAR is a polypeptide having at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 54. In some embodiments, pCAR is a polypeptide having at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 54.

[0020] In some embodiments, the CAR binds to CD19 and the CCR binds to CD22. In some preferred embodiments, the second binding element directing CCR specificity comprises the complementarity determining regions (CDRs) of the RFB4 antibody and has the sequence of SEQ ID NOs: 37, 38, 39, 40, 41, and 42. In some preferred embodiments, the second binding element comprises the variable heavy (V) region of the RFB4 antibody. H ;GenBank accession number CAJ09937.1) and variable light (V L; GenBank Accession No. CAJ09936.1) domain and has the sequence of SEQ ID NOs: 43 and 44. In some preferred embodiments, the second binding element comprises the variable heavy (V H ) and variable light (V L ) domain and has the sequence of SEQ ID NO: 45 or 46. In some preferred embodiments, the RFB4 scFv is expressed from the polynucleotide or set of polynucleotides of SEQ ID NO: 127. In some preferred embodiments, the pCAR is designated RBB / F and has the sequence of SEQ ID NO: 55. The nomenclature is derived from the abbreviations for the following elements: CCR binder ( R FB4 scFv), CCR signaling domain (4-1 BB ) / CAR Binder( F MC63 scFv). In some embodiments, the CAR of 1BB / F comprises the sequence of SEQ ID NO: 64, and the CCR of 1BB / F comprises the sequence of SEQ ID NO: 66. In some preferred embodiments, RBB / F pCAR is expressed from the polynucleotide or set of polynucleotides of SEQ ID NO: 117. In some embodiments, pCAR is a polypeptide having at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 55. In some embodiments, pCAR is a polypeptide having at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 55.

[0021] In some embodiments, the immunoresponsive cells are αβ T cells, γδ T cells, or natural killer (NK) cells. In some embodiments, the T cells are αβ T cells. In some embodiments, the T cells are γδ T cells.

[0022] In some embodiments, the polynucleotide or set of polynucleotides comprises: (a) a first nucleic acid encoding a CCR that binds to a B cell lineage antigen; and (b) a second nucleic acid encoding a CAR that binds to CD19. In some embodiments, the first nucleic acid and the second nucleic acid are in a single vector. In some embodiments, the first nucleic acid and the second nucleic acid are in two separate vectors.

[0023] In one aspect, the present invention provides a method of preparing an immunoresponsive cell, the method comprising transfecting or transducing an immunoresponsive cell with a polynucleotide or set of polynucleotides provided herein.

[0024] In another aspect, the present disclosure provides a method of directing a T cell-mediated immune response to a target cell in a patient in need thereof, the method comprising administering to the patient an immunoresponsive cell, wherein the target cell is a B cell.

[0025] In yet another aspect, the present disclosure provides a method for treating cancer, the method comprising administering an effective amount of immunoresponsive cells to a patient. In some embodiments, the patient's cancer expresses CD19. In some embodiments, the patient has a cancer arising from the B-cell lineage. In some embodiments, the patient has a cancer selected from the group consisting of acute or chronic B-cell leukemia or B-cell lymphoma.

[0026] In another aspect, the disclosure provides the use of immunoresponsive cells in therapy or for use as a medicament. The disclosure further provides the immunoresponsive cells in the manufacture of a medicament for the treatment of a pathological disorder. In some embodiments, the pathological disorder is cancer.

[0027] 3. Brief description of the drawings The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of various embodiments of the present invention. [Brief explanation of the drawings]

[0028] [Figure 1] Figure 1A and Figure 1B—Flow cytometry analysis of malignant B cell lines. Figure 1A shows the expression profiles of CD19 and CD20 on a panel of human lymphoma and leukemia cell lines (Daudi, Nalm-6, and Raji). In accordance with published data, analysis confirmed that CD19 was detectable at high levels on the cell surface of these tumor cells, and that CD20 was detectable in both the Raji and Daudi cell lines. The Nalm-6 cell line had no detectable expression of CD20. To enable bioluminescence and fluorescence imaging of the cells and the resulting xenografts, tumor cell lines were transduced with a LT retroviral vector encoding both the firefly luciferase enzyme and the red fluorescent protein (RFP), tandem dimer (td)Tomato. RFP expression was confirmed using flow cytometry, as shown in Figure 1B. [Figure 2]CAR and pCAR Design and Construction Figures 2A-2E provide schematic diagrams showing the salient features of certain second-generation CAR and pCAR constructs used in the experiments described herein. The cell membrane is represented as a parallel horizontal line, with the extracellular domain shown above the membrane and the intracellular domain shown below the membrane. F-2 is a second-generation CAR similar to that originally described in Kochenderfer et al., J. Immunother. 32:689-702 (2009) (incorporated herein by reference in its entirety). From C-terminus to N-terminus (intracellular to extracellular), it contains a CD3z signaling region, a CD28 costimulatory and transmembrane domain, a CD28 hinge / spacer domain containing an embedded myc epitope tag, and a human CD19-targeting FMC63 single-chain antibody (scFv) domain. Cells transduced with F-2 alone are standard second-generation CAR-T cells and are used for comparison purposes. The CDR3 region of the VH domain within FMC63 scFv was identified using www.abysis.org. To generate mutants with altered CD19 binding ability, an alanine (A) residue was substituted for the first or second glycine (G01, G02) or the first, second, third, fourth, or fifth tyrosine (Y01-Y05) within the CDR3 of the VH domain, as shown in Figures 2A and 2B. These modified CD19-specific second-generation CARs are designated G01, G02, Y01, Y02, Y03, Y04, and Y05, respectively. 1-2 is a second-generation CAR for which targeting was achieved using 1F5 scFv, as described in Budde et al., PLoS One 8(12):e82742 (2013), incorporated herein by reference in its entirety. It contains, from C-terminus to N-terminus (intracellular to extracellular), the CD3z signaling region, the CD28 costimulatory and transmembrane domains, the CD28 hinge / spacer domain containing an embedded myc epitope tag, and the human CD20-targeting 1F5 single-chain antibody (scFv) domain. Cells transduced with 1-2 alone are standard second-generation CAR-T cells and are used for comparison purposes.R-2) is a second-generation CAR in which targeting was achieved using RFB4 scFv, as described in James et al., J. Immunol. 180(10):7028-38 (2008) and incorporated herein by reference in its entirety. It contains, from C-terminus to N-terminus (intracellular to extracellular), a CD3z signaling region, a CD28 costimulatory and transmembrane domain, a CD28 hinge / spacer domain containing an embedded myc epitope tag, and a human CD22-targeting RFB4 single-chain antibody (scFv) domain. Cells transduced with R-2 alone are standard second-generation CAR-T cells and are used for comparison purposes. A series of B cell-targeting pCARs (Figures 2C, 2D, and 2E) were engineered using combinations of the aforementioned binding moieties. The nomenclature is derived from the abbreviations of the following elements: CCR binder, CCR signaling domain / CAR binder. For example, 1BB / F is a pCAR in which a 4-1BB endodomain-containing CCR targeted by 1F5 scFv is co-expressed with a CD28-containing second-generation CAR targeted by FMC63 scFv. FBB / Y01 is a pCAR in which a CCR targeted by FMC63 scFv is co-expressed with a CD28-containing second-generation CAR targeted by FMC63 (Y01) scFv. Similarly, FTr / Y05 is a control pCAR in which a CCR (with a truncated signaling domain) targeted by FMC63 scFv is co-expressed with a CD28-containing second-generation CAR targeted by FMC63 (Y05) scFv. The general structures of B cell-targeting pCARs and truncated controls are shown in Figures 2C, 2D, and 2E. Figure 2F provides an overview of the CD19-CD22 dual-targeting pCARs (RBB / F, RBB / Y05, and RBB / G02) and second-generation CARs (F-2 and R-2) targeting CD19 and CD22, respectively. RBB / F is a pCAR with an RBB CCR (CD22-specific RFB4 scFv fused to the 4-1BB signaling domain via a CD8α spacer and transmembrane domain) and a CD19-specific CD28-containing second-generation CAR (F).RBB / Y05 is a pCAR in which a CCR targeted by RFB4 scFv is co-expressed with a CD28-containing second-generation CAR targeted by FMC63(Y05) scFv. RBB / G02 is a pCAR in which a CCR targeted by RFB4 scFv is co-expressed with a CD28-containing second-generation CAR targeted by FMC63(G02) scFv. [Figure 3] Expression of CD19-specific CARs in human T cells. Figures 3A-3B show two representative examples of CD19-specific second-generation CARs containing mutant FMC63 scFvs expressed in human CAR T cells. Cell surface expression was detected using the 9e10 antibody, which binds to a myc epitope tag (EQKLISEEDL) inserted within the CD28 spacer domain of the CAR. The F-2 second-generation CAR was expressed as a control. [Figure 4] Expression of CD19-specific pCAR in human T cells. Figures 4A and 4B show representative examples of CD19-specific pCARs expressed in human T cells. In all CD19-specific pCARs, the FBB CCR (CD19-specific FMC63 scFv fused to the 4-1BB signaling domain via a CD8α spacer and transmembrane domain) is coexpressed with a CD19-specific CD28-containing second-generation CAR containing the indicated CDR3 mutations in the FMC63 VH chain. The F-2 second-generation CAR was expressed here as a control. Expression of the CAR or the CAR component of pCAR was detected using the 9e10 antibody binding to the myc epitope tag (EQKLISEEDL) as described above (Figure 4A). Expression of the CCR component of pCAR was detected by intracellular staining using a FLAG epitope tag (DYKDDDDK)-specific antibody (Figure 4B). [Figure 5]Expression of pCAR co-targeting CD19 and CD20 in human T cells. Figure 5 shows a representative example of 1BB / F pCAR expressed in human T cells. In 1BB / F, 1BB CCR (CD20-specific 1F5 scFv fused to the 4-1BB signaling domain via a CD8α spacer and transmembrane domain) is co-expressed with a CD19-specific CD28-containing second-generation CAR (F). In 1Tr / F, 1BB CCR with a truncated signaling domain is co-expressed with a CD19-specific CD28-containing second-generation CAR (F). 1-2 is a CD28-containing second-generation CAR control that binds to CD20. Both F-2 and 1-2 were expressed here as controls. Expression of the CAR or the CAR component of pCAR was detected using the 9e10 antibody as described above. [Figure 6] Expression of pCAR cotargeting CD19 and CD22 in human T cells. Figure 6 shows a representative example of RBB / F pCAR expressed in human T cells. In RBB / F, the RBB CCR (CD22-specific RFB4 scFv fused to the 4-1BB signaling domain via a CD8α spacer and transmembrane domain) is coexpressed with a CD19-specific CD28-containing second-generation CAR (F). R-2 is a CD28-containing second-generation CAR control that binds to CD22. Both F-2 and R-2 were expressed as controls. Expression of the CAR or the CAR component of pCAR was detected using a 9e10 MYC epitope tag-specific antibody as described above. Expression of the CCR component of pCAR was detected by intracellular staining using a FLAG epitope tag-specific antibody. [Figure 7]CD19 Binding to Parental and CDR3 VH-Mutated FMC63-Based Second-Generation CARs. Figures 7A-7D show the results of a representative experiment in which T cells expressing F-2, G01, G02, Y01, Y02, Y03, Y04, or Y05 second-generation CARs were incubated with two concentrations of soluble CD19-Fc fusion protein—0.5 μg (Figure 7A, Figure 7C (right)) and 1.0 μg (Figure 7B, Figure 7C (left)). Binding was measured by flow cytometry after incubation with Alexa-fluor 488-conjugated anti-human IgG. The percentage of transduced T cells present in each case is shown in Figure 3A. T cell binding to CD19-Fc, measured by flow cytometry, is plotted in histogram format in Figure 7C and quantified as % binding and mean fluorescence intensity (MFI) after incubation with CD19-Fc (Figure 7D, left and right, respectively). Note the range of binding efficiency of the engineered CARs compared to F-2: low (e.g., G01, G02) to medium (e.g., Y04) to high (e.g., Y05). [Figure 8] Titration of tumor cell killing by F-2 and mutant derivative second-generation CAR T cells Figures 8A-8D show three experiments comparing the cytotoxic activity of F-2 and mutant derivative second-generation CAR T cells against malignant CD19-expressing B-cell lymphoma cell lines, Nalm-6 (Figure 8A and Figure 8C) or Raji (Figure 8B and Figure 8D), compared to untransduced (UT) control T cells. [Figure 9] In vitro cytokine release (at 24 hours) Figures 9A and 9B show pooled data showing the release of IFNγ (Figure 9A) and IL-2 (Figure 9B) by CD19-specific CAR-T cells when cultured with Nalm-6 cells. Figures 9C and 9D show pooled data showing the release of IFNγ (Figure 9C) and IL-2 (Figure 9D) by CD19-specific CAR-T cells when cultured with Raji cells. Comparison is made to the CD19-specific second-generation CAR, F-2. [Figure 10]In vitro cytokine release (at 24 hours) Figures 10A and 10B show pooled data showing the release of IFNγ by CD19-specific CAR-T cells when cultured with Nalm-6 cells (Figure 10A) or Raji cells (Figure 10B). Figures 10C and 10D show pooled data showing the release of IL-2 by CD19-specific CAR-T cells when cultured with Nalm-6 cells (Figure 10C) or Raji cells (Figure 10D). [Figure 11] Figure 11A shows the tumor cell killing activity of the second-generation CD19-specific CAR of Figure 3A when repeatedly restimulated by the addition of Nalm-6 cells. Figures 11B and 11C show the IFN-γ (Figure 11B) and IL-2 (Figure 11C) produced by repeatedly stimulated CAR T cells. [Figure 12] Titration of tumor cell killing by CD19-targeted CAR and pCAR T cells Figure 12 shows pooled data in which the cytotoxic activity of second-generation CAR T cells based on CDR3 VH mutated FMC63 and pCAR T cells against Nalm-6 leukemia cells was titrated compared to F-2 as a control. [Figure 13] In vitro restimulation potential of CD19-specific pCAR T cells Figure 13A shows the tumor cell killing activity of CD19-specific pCAR T cells when repeatedly restimulated by co-culture with CD19-expressing LO68 tumor cells. Figures 13B and 13C show IFN-γ and IL-2 production by repeatedly stimulated CAR-T and pCAR-T cells, respectively. [Figure 14] In vivo antitumor activity of CD19-specific CAR and pCAR T cells (NSG mice) Figures 14A and 14B show the results of therapeutic evaluation of CD19-specific CAR or pCAR-T cells against validated luciferase-expressing Nalm-6 leukemic xenografts in NSG mice. Figure 14A shows the total flux emission from mice treated with CD19-specific CAR or pCAR-T cells, and Figure 14B shows the percentage weight change of the mice before and after treatment. [Figure 15] In vitro restimulation potential and cytotoxicity of pCAR T cells co-targeting CD19 and CD20. Figure 15A shows the number of restimulation cycles completed after co-culture of LO68 tumor cells co-expressing both CD19 and CD20 with transduced 1BB / F pCAR T cells co-targeting CD19 (CAR) and CD20 (CCR). Every 72 hours, T cells were transferred to a fresh monolayer of LO68 cells. Figure 15B shows pooled data of the tumor cell-killing activity of 1BB / F pCAR T cells co-cultured with LO68 tumor cells. Figure 15C shows the amount of IFN-γ (left panel) and IL-2 (right panel) released after each stimulation cycle. Figure 15D shows a pooled experiment in which the cytotoxic activity of CAR and pCAR T cells was titrated using LO68 tumor cells co-expressing both CD19 and CD20. Figures 15E-15F show the amount of IFN-γ (Figure 15E) and IL-2 (Figure 15F) released after 24 hours of co-culture at effector:target ratios of 1:1 and 1:4. [Figure 16] Figures 16A-16B show the cytotoxic activity of RBB / F, RBB / G02, and RBB / Y05 pCAR T cells co-targeting CD19 (CAR) and CD22 (CCR) against Nalm-6 leukemia cells. Comparisons are made to second-generation CD19- and CD22-targeted second-generation CARs (F-2 and R-2, respectively). [Figure 17]Figures 17A-17D show the binding affinity of CD19-specific CAR-T cells to CD19+ LO68 tumor cells in a z-Movi microfluidic chip. T cells were engineered to express the CD19-specific F-2 CAR or a derivative containing a mutation in the VHCDR3 region. After purification by flow sorting, CAR T cells were incubated on a monolayer of CD19+ LO68 tumor cells in a z-Movi microfluidic chip. Increasing fluidic forces were applied, and the percentage of bound T cells was determined (median, n=3) (Figure 17A). Figure 17B shows the total average percentage of bound T cells after minimal force application to separate an average of 90% of non-transduced T cells. Figure 17C is a bar plot showing the avidity score or the average rForce required to detach T cells from a monolayer of CD19+ LO68 tumor cells compared to untransduced T cells (the black dotted line indicates the avidity score of untransduced T cells; the red dotted line indicates the avidity score of F-2 CAR T cells). The dot plot in Figure 17D shows the rForce per cell required to detach from a monolayer of tumor cells, with each dot representing a single cell. [Figure 18] Expression of CD19-specific CAR or pCAR. Figures 18A-18B show the expression of CD19-specific CAR or pCAR in human T cells analyzed by flow cytometry. Human T cells were modified by retroviral transduction to express the indicated CAR or pCAR. T cells were incubated with antibodies directed against both the MYC (CAR) and FLAG (CCR) epitope tags and then analyzed by flow cytometry. Data are representative of three independent replicate experiments (Figure 18B) or more than seven independent replicates (Figure 18A). [Figure 19]Experimental design for in vivo antitumor activity testing (NSG mice). Figure 19 shows the experimental design for testing modified CD19-specific CAR-T or pCAR-T cells in vivo. RFP / ffLuc+Nalm6 cells (5x10 cells) were injected i.v. into NSG mice. Mice were sorted into groups of equal disease burden using BLI. On day 5, 5x10 indicated CAR or pCAR T cells were administered i.v. Disease burden was monitored by BLI from day 8 onwards. [Figures 20A-20B] In vivo anti-tumor activity of CD19-specific CAR-T or pCAR-T cells Figures 20A and 20B show the anti-tumor activity of CD19-specific CAR-T or pCAR-T cells in NSG mice bearing demonstrated luciferase-expressing Nalm6 leukemic xenografts. Figure 20A shows the total luminous intensity emission (e.g., leukemic burden) of mice treated with the indicated CD19-specific CAR-T or pCAR-T cells. Figure 20B shows the p-values for the "selected pCAR" versus the "2G CAR with a mutation in its corresponding VHCDR3 region," and for "2G CAR F-2" versus the "2G CAR with a mutation in the indicated VHCDR3 region." [Figures 21A-21D] Survival of NSG mice treated with CD19-specific CAR-T or pCAR-T cells. Figures 21A, 21B, 21C, and 21D show survival curves for the PBS-treated group, the CD19-specific CAR-T cell-treated group, and the CD19-specific pCAR-T cell-treated group. Figure 21A shows survival curves for the PBS-treated group, the F-2 CAR-T cell-treated group, the Y04 CAR-T cell-treated group, and the FBB / Y04 pCAR-T cell-treated group. Figure 21B shows survival curves for the PBS-treated group, the F-2 CAR-T cell-treated group, the Y05 CAR-T cell-treated group, and the FBB / Y05 pCAR-T cell-treated group. Figure 21C shows survival curves for the PBS-treated group, the F-2 CAR-T cell-treated group, the G02 CAR-T cell-treated group, and the FBB / G02 pCAR-T cell-treated group. FIG. 21D shows the median survival for each treatment group shown in FIGS. 21A-21C. DETAILED DESCRIPTION OF THE INVENTION

[0029] 4. Detailed Description Details of various embodiments of the invention are set forth in the following description. Other features, objects, and advantages of the invention will be apparent from the specification and drawings, and from the claims.

[0030] 4.1.Definition Unless otherwise defined herein, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the following terms have the meanings ascribed to them below.

[0031] As used herein, the term "variant" refers to a polypeptide sequence that is a naturally occurring polymorphic form of the basic sequence, as well as a synthetic variant in which one or more amino acids in the chain are inserted, removed, or replaced.However, the variant produces the same biological effect as that of the basic sequence.For example, the variant of the intracellular domain of the human CD3 zeta chain acts in a similar manner to that of the intracellular domain of the human CD3 zeta chain.If an amino acid is replaced by a different amino acid within the same class with approximately the same properties, the amino acid substitution can be considered "conservative."Non-conservative substitution is when an amino acid is replaced by an amino acid of a different type or class.

[0032] As is well known to those skilled in the art, conservative substitutions may alter the primary structure of a peptide without significantly altering its activity, since the amino acid side chain inserted into the sequence may be able to form similar bonds and contacts as the substituted amino acid side chain. This is true even when the substitution is in a region important for determining the peptide's three-dimensional structure. Non-conservative substitutions are also possible as long as they do not disrupt the function of the polypeptide, as described above. Generally speaking, non-conservative substitutions are less likely to alter the biological activity of a polypeptide. Generally, variants have an amino acid sequence that is at least 70%, e.g., at least 71%, 75%, 79%, 81%, 84%, 87%, 90%, 93%, 95%, 96%, or 98% identical to the base sequence, e.g., SEQ ID NO: 1 or SEQ ID NO: 2. Identity in this context can be determined using the BLASTP computer program with SEQ ID NO: 1, SEQ ID NO: 2, or fragments thereof, particularly those described below, as the base sequence. BLAST software is publicly available.

[0033] As used herein, the term "antigen" refers to any member of a specific binding pair that binds to a binding element. The term includes receptors on target cells.

[0034] As used herein, with respect to a binding element for a target molecule, the terms "binds with," "specifically binds with," "specifically binds to," "specifically interacts with," "specific for," "selectively binds with," "selectively interacts with," and "selective for" a particular antigen (e.g., a polypeptide target) or epitope on a particular antigen refer to binding that is measurably different from a nonspecific or nonselective interaction (e.g., with a non-target molecule). Specific binding can be measured, for example, by measuring binding to a target molecule and comparing it to binding to a non-target molecule. Specific binding can also be determined by competition with a control molecule that mimics the recognized epitope on the target molecule.

[0035] As used herein, the term "pCAR" refers to a juxtaposed chimeric antigen receptor (PCAR) that includes a combination of a second-generation chimeric antigen receptor (CAR) and a juxtaposed chimeric costimulatory receptor (CCR). PCARs are described in WO2017 / 021701, the entire contents of which are incorporated herein by reference.

[0036] 4.2. Other interpretive practices In the claims, articles such as "a," "an," and "the" may mean one or more than one, unless indicated otherwise or clear from the context. A claim or specification containing "or" between one or more members of a group is considered to be satisfied when one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process, unless indicated otherwise or clear from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0037] It should also be noted that the term "comprising" is intended to be open, permitting but not requiring the inclusion of additional elements or steps. When the term "comprising" is used herein, the term "consisting of" is also included and disclosed.

[0038] When ranges are given, the endpoints are included. Furthermore, unless otherwise indicated, or otherwise apparent from the context and the understanding of one of ordinary skill in the art, it should be understood that values expressed as ranges can, in different embodiments of the invention, be extrapolated to one-tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0039] All cited sources, e.g., references, publications, databases, database entries, and art cited herein, are incorporated by reference into this application, even if not expressly stated in the citation. In the event of a conflict between the statements in a cited source and this application, the statements in this application will control.

[0040] Section and table headings are not intended to be limiting.

[0041] 4.3. Immunoresponsive cells In a first aspect, an immunoresponsive cell is provided, which expresses a pCAR comprising a combination of a second-generation chimeric antigen receptor (CAR) and a juxtaposed chimeric costimulatory receptor (CCR).

[0042] The CAR comprises, from C-terminus to N-terminus (intracellular to extracellular so as to be expressed in immunoresponsive cells): (a) a signaling region; (b) a first costimulatory signaling region; (c) a first transmembrane domain; and (d) a first binding element that specifically interacts with a first epitope on the CD19 target antigen.

[0043] The CCR comprises, from the C-terminus to the N-terminus (from the intracellular to the extracellular so as to be expressed in immunoresponsive cells): (e) a second costimulatory signaling region different from the first costimulatory signaling region of the CAR; (f) a second transmembrane domain; and (g) a second binding element that specifically interacts with a second epitope on a second antigen. The second epitope may be the same as or different from the first epitope. The second antigen may be CD19 or an alternative B-cell lineage-specific antigen.

[0044] 4.3.1.Cells In an exemplary embodiment, the immunoresponsive cell is a T cell.

[0045] In certain embodiments, the immunoresponsive cells are αβ T cells. In certain embodiments, the immunoresponsive cells are cytotoxic αβ T cells. In certain embodiments, the immunoresponsive cells are αβ helper T cells. In certain embodiments, the immunoresponsive cells are regulatory αβ T cells (Tregs).

[0046] In certain embodiments, the immunoresponsive cells are γδ T cells. In certain embodiments, the immunoresponsive cells are Vδ2 T cells. + In a specific embodiment, the immunoresponsive cells are V52 T cells. - In a specific embodiment, V52 - T cells are Vδ1 + It is a cell.

[0047] In certain embodiments, the immunoresponsive cells are natural killer (NK) cells.

[0048] In some embodiments, the immunoresponsive cells do not express additional exogenous proteins, hi other embodiments, the immunoresponsive cells are modified to express additional exogenous proteins, such as cytokines, receptors, or derivatives thereof.

[0049] In some embodiments, the immunoresponsive cells are obtained from peripheral blood mononuclear cells (PBMCs). In some embodiments, the immunoresponsive cells are obtained from a tumor. In certain embodiments, the immunoresponsive cells obtained from a tumor are tumor-infiltrating lymphocytes (TILs). In specific embodiments, the TILs are αβ T cells. In other specific embodiments, the TILs are γδ T cells, particularly Vδ2 T cells. + or Vδ2 - γδ T cells.

[0050] 4.4. Structure of Chimeric Antigen Receptors 4.4.1. Signaling Area The CAR construct comprises a signaling region at its C-terminus. In some embodiments, the signaling region comprises an immunoreceptor tyrosine-based activation motif (ITAM), e.g., as reviewed by Love et al., Cold Spring Harbor Perspect. Biol. 2(6)1a002485 (2010). In some embodiments, the signaling region comprises the intracellular domain of the human CD3 zeta chain, or a variant thereof, e.g., as described in U.S. Pat. No. 7,446,190 (incorporated herein by reference). In certain embodiments, the signaling region comprises a domain spanning amino acid residues 52-163 of the full-length human CD3 zeta chain. The CD3 zeta chain has multiple known polymorphic forms (e.g., sequence IDs: gb|AAF34793.1 and gb|AAA60394.1), all of which are useful herein and are set forth as SEQ ID NOS: 1 and 2, respectively:

[0051] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:1);

[0052] RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 2).

[0053] Alternative signaling regions to the CD3 zeta domain include Fcεr1γ, CD3ε, DAP12 and other ITAM containing units such as multi-ITAM.Eshhar Z et al., "Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the gamma or zeta subunits of the immunoglobulin and T-cell receptors" Proc Natl Acad Sci USA 90:720-724 (1993); Nolan et al., "Bypassing immunization: optimized design of 'designer T cells' against carcinoembryonic antigen(CEA)-expressing tumors,and lack of suppression by soluble CEA"Clin Cancer Res 5:3928-3941(1999);Zhao et al.,"A herceptin-based chimeric antigen receptor with modified signaling domains leads to enhanced survival of transduced T lymphocytes and antitumor activity"J Immunol 183: 5563-5574(2009),Topfer et al. al.DAP12-based activating chimeric antigen receptor for NK cell tumor immunotherapy. J Immunol 194:3201-3212 (2015); and James JR, "Tuning ITAM multiplicity on T cell receptors can control potency and selectivity to ligand density," Sci Signal 11(531)eaan1088 (2018), the disclosures of which are incorporated herein by reference in their entireties.

[0054] 4.4.2. Costimulatory Signaling Regions In CARs, the costimulatory signaling region is appropriately positioned between the signaling region and the transmembrane domain, and separate from the binding element.

[0055] In CCRs, the costimulatory signaling region is appropriately positioned adjacent to the transmembrane domain and distant from the binding element.

[0056] Suitable costimulatory signaling regions are well known in the art and include costimulatory signaling regions of B7 / CD28 family members, e.g., B7-1, B7-2, B7-H1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA, CD28, CTLA-4, Gi24, ICOS, PD-1, PD-L2, or PDCD6; or ILT / CD85 family proteins, e.g., LILRA3, LILRA4, LILRB1, LILRB2, LILRB3, or LILRB4; or tumor necrosis factor (TNF) superfamily members, e.g., 4-1BB, BAFF, BAFF R, CD27, CD30, CD40, DR3, GITR, HVEM, LIGHT, lymphotoxin-α, OX40, RELT, TACI, TL1A, TNF-α, or TNF RII; or a member of the SLAM family, e.g., 2B4, BLAME, CD2, CD2F-10, CD48, CD8, CD84, CD229, CRACC, NTB-A or SLAM; or a member of the TIM family, e.g., TIM-1, TIM-3 or TIM-4; or other costimulatory molecules, e.g., CD7, CD96, CD160, CD200, CD300a, CRTAM, DAP12, Dectin-1, DPPIV, EphB6, integrin α4β1, integrin α4β7 / LPAM-1, LAG-3 or TSLPR. Mondino A et al., “Surface proteins involved in T cell costimulation” J Leukoc Biol.55:805-815(1994);Thompson CB, “Distinct roles for the costimulatory ligands B7-1 and B7-2 in T helper cell differentiation?” Cell.81:979-982(1995);Somoza C and Lanier LL, “T-cell costimulation via CD28-CD80 / CD86 and CD40-CD40 ligand interactions”Res Immunol.146:171-176(1995);Rhodes DA et al.,「Regulation of immunity by butyrophilins」Annu Rev Immunol.34:151-172(2016);Foell J et al.,「T cell costimulatory and inhibitory receptors as therapeutic targets for inducing anti-tumor immunity」Curr Cancer Drug Targets.7:55-70(2007);Greenwald RJ et al.,Annu Rev Immunol.,「The B7 family revisited」23:515-548(2005);Flem-Karlsen K et al.,「B7-H3 in cancer - beyond immune regulation」Trends Cancer.4:401-404(2018);Flies DB et al.,「The new B7s:playing a pivotal role in tumor immunity」J Immunother.30:251-260(2007);Gavrieli M et al.,「BTLA and HVEM cross talk regulates inhibition and costimulation」Adv Immunol.92:157-185(2006);Zhu Y et al.,「B7-H5 costimulates human T cells via CD28H」Nat Commun.4:2043(2013);Omar HA et al.,「Tacking molecular targets beyond PD-1 / PD-L1:Novel approaches to boost patients’response to cancer immunotherapy」Crit Rev Oncol Hematol.135:21-29(2019);Hashemi M et al.,「Association of PDCD6 polymorphisms with the risk of cancer:Evidence from a meta-analysis」Oncotarget.9:24857-24868 (2018);Kang Immunol.23:23-68(2005);Bryceson YT et al., "Activation, coactivation, and costimulation of resting human natural killer cells" Immunol Rev.214:73-91(2006);Sharpe AH, "Analysis of lymphocyte costimulation in vivo using transgenic and 'knockout' mice" Curr Opin Immunol.7:389-395(1995);Wingren AG et al., “T cell activation "Pathways: B7, LFA-3, and ICAM-1 shape unique T cell profiles," Crit Rev Immunol. 15:235-253 (1995), the disclosures of which are incorporated herein by reference in their entirety.

[0057] The costimulatory signaling region can be selected depending on the particular use intended for the immunoresponsive cell. In particular, the costimulatory signaling region can be selected to act additively or synergistically. In some embodiments, the costimulatory signaling region is selected from the costimulatory signaling regions of CD28, CD27, ICOS, 4-1BB, OX40, CD30, GITR, HVEM, DR3, and CD40.

[0058] In certain embodiments, one costimulatory signaling region of the pCAR is the costimulatory signaling region of CD28 and the other is the costimulatory signaling region of 41BB. In specific embodiments, the costimulatory signaling region of the CAR is the costimulatory signaling region of CD28 and the costimulatory signaling region of the CCR is the costimulatory signaling region of 41BB.

[0059] In certain embodiments, one costimulatory signaling region of the pCAR is the costimulatory signaling region of CD28 and the other is the costimulatory signaling region of CD27. In specific embodiments, the costimulatory signaling region of the CAR is the costimulatory signaling region of CD28 and the costimulatory signaling region of the CCR is the costimulatory signaling region of CD27.

[0060] 4.4.3. Transmembrane Domain The transmembrane domains of the CAR and CCR constructs may be the same or different. In a currently preferred embodiment, when the CAR and CCR constructs are expressed from a single vector, the transmembrane domains of the CAR and CCR are different to ensure the separation of the constructs on the cell surface. Because the inclusion of direct repeat nucleic acid sequences in viral vectors involves the deletion of sequences between the direct repeats, making them prone to rearrangement, selecting different transmembrane domains can also increase the stability of the expression vector. In embodiments where the transmembrane domains of the CAR and CCR of pCAR are selected to be identical, this risk can be reduced by modifying or "wobbling" the selected codons to encode the same protein sequence.

[0061] Suitable transmembrane domains are known in the art, and include, for example, CD8α, CD28, CD4, or CD3z transmembrane domains. Selecting CD3z as the transmembrane domain may result in the association of CAR or CCR with other elements of the TCR / CD3 complex. This association may recruit more ITAMs, but may also result in competition between CAR / CCR and endogenous TCR / CD3.

[0062] In certain embodiments, one transmembrane domain of the pCAR is the transmembrane domain of CD28 and the other is the transmembrane domain of CD8α. In certain pCAR embodiments, the transmembrane domain of the CAR is the transmembrane domain of CD28 and the transmembrane domain of the CCR is the transmembrane domain of CD8α.

[0063] 4.4.4. Costimulatory Signaling Domains and Transmembrane Domains In embodiments where the costimulatory signaling region of a CAR or CCR is or includes the costimulatory signaling region of CD28, the CD28 transmembrane domain represents a suitable, and often preferred, choice for the transmembrane domain. The full-length CD28 protein is a 220 amino acid protein of SEQ ID NO: 3, with the transmembrane domain shown in bold:

[0064] JPEG0007723420000001.jpg34153

[0065] In some embodiments, one of the costimulatory signaling regions is based on the hinge region, and suitably also the transmembrane domain and endodomain of CD28. In some embodiments, the costimulatory signaling region comprises amino acids 114-220 of SEQ ID NO:3, shown below as SEQ ID NO:4:

[0066] IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 4).

[0067] In certain embodiments, one of the costimulatory signaling regions is a modified version of SEQ ID NO:4, which includes a c-myc tag of SEQ ID NO:5:

[0068] EQKLISEEDL (SEQ ID NO: 5).

[0069] The c-myc tag may be added to the costimulatory signaling region by insertion within the ectodomain or by substitution of a region within the ectodomain, thus within the region of amino acids 1-152 of SEQ ID NO:3.

[0070] In a particularly preferred embodiment, the c-myc tag replaces the MYPPPY motif in the CD28 sequence. This motif represents a potentially dangerous sequence. It is responsible for the interaction between CD28 and its natural ligands (CD80 and CD86), and therefore provides the potential for off-target toxicity when CAR-T cells or pCAR-T cells encounter target cells expressing either of these ligands. By replacing this motif with a tag sequence as described above, the potential for unwanted side effects is reduced. Thus, in a specific embodiment, the costimulatory signaling region of the CAR construct comprises SEQ ID NO:6:

[0071] IEVEQKLISEEDLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 6).

[0072] Furthermore, the inclusion of the c-myc epitope facilitates detection of pCAR-T cells using monoclonal antibodies against the c-myc epitope, which is extremely useful since flow cytometry detection has proven unreliable with some available antibodies.

[0073] Furthermore, the provision of a c-myc epitope tag can facilitate antigen-independent expansion of targeted CAR-T cells, for example, by cross-linking the CAR with a suitable monoclonal antibody in solution or immobilized on a solid phase (e.g., a bag).

[0074] Furthermore, expression of an epitope for the anti-human c-myc antibody (9e10) within the variable region of the TCR has previously been shown to be sufficient to enable antibody-mediated and complement-mediated cytotoxicity both in vitro and in vivo. See Kieback et al. Proc. Natl. Acad. Sci. USA, 105(2)623-8(2008). Thus, provision of such an epitope tag can also be used as a "suicide system," whereby the antibody can be used to deplete pCAR-T cells in vivo in the event of toxicity.

[0075] In some embodiments, one of the costimulatory signaling regions is based on the endodomain of 4-1BB. In some embodiments, the costimulatory signaling region comprises amino acids 214-255 of 4-1BB, shown below as SEQ ID NO:7:

[0076] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 7).

[0077] In certain embodiments, one of the costimulatory signaling regions is a modified form of SEQ ID NO:7 that includes the FLAG epitope tag of SEQ ID NO:8:

[0078] DYKDDDDK (SEQ ID NO: 8).

[0079] In a particularly preferred embodiment, a FLAG epitope tag is added to the C-terminus of the 4-1BB endodomain. Thus, in a particular embodiment, the costimulatory signaling region of the CCR comprises SEQ ID NO:9:

[0080] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELDYKDDDDK (SEQ ID NO: 9).

[0081] 4.4.5. Chimeric Antigen Receptor Binding Elements The binding elements of the CAR and CCR constructs of pCAR bind to a first epitope and a second epitope, respectively, which may be the same or different.

[0082] In some embodiments, the binding elements of the CAR and CCR constructs are identical, however, more commonly, these binding elements are different from each other.

[0083] In various embodiments, the binding elements of the CAR and CCR specifically bind to a first epitope and a second epitope of the same antigen. In certain of these embodiments, the binding elements of the CAR and CCR specifically bind to the same, overlapping, or different epitopes of the same antigen. In embodiments where the first and second epitopes are the same or overlapping, the binding elements on the CAR and CCR can compete in their binding. In such embodiments, elements that bind with different affinities are used to achieve an optimal balance of signaling by the CAR and CCR components of the pCAR.

[0084] In various embodiments, the binding elements of the CAR and CCR components of the pCAR bind to different antigens, which in certain embodiments may be different but associated with the same disease (e.g., the same specific cancer derived from the B-cell lineage).

[0085] In a preferred embodiment, the CAR binds to CD19 and the CCR binds to CD19 or another B cell lineage-specific marker, examples of the latter including, but not limited to, CD20, CD22, CD23, CD79a, and CD79b.

[0086] Thus, a suitable binding element can be any element that provides the pCAR with the ability to recognize a target of interest. The target to which the pCAR of the present invention is directed can be any target of clinical interest to which it is desirable to direct a T cell response.

[0087] In various embodiments, the binding elements used in the CARs and CCRs of the pCARs described herein are antibody antigen binding sites (ABS). In typical embodiments, the ABS used as binding elements are formatted as scFvs or are single domain antibodies from camelids, humans, or other species.

[0088] Alternatively, the binding element of pCAR may comprise a ligand or ligands that bind to a surface protein of interest.

[0089] Alternatively, the binding element of pCAR may comprise a peptide or peptides that bind to a surface protein of interest.

[0090] In some embodiments, the binding element is associated with a leader sequence that promotes expression on the cell surface. Many leader sequences are known in the art, including the macrophage colony-stimulating factor receptor (FMS) leader sequence, the CD8α leader sequence, or the CD124 leader sequence.

[0091] 4.5. Parallel CARs targeted to B cell antigens 4.5.1. Binding Elements for Use in pCAR In certain embodiments, the binding element of CAR or the binding element of CCR specifically interacts with the epitope on the CD19 target antigen.CD19 is a B lymphocyte antigen encoded by the CD19 gene and is found on the surface of B cells.It is a known target for the treatment of B cell malignancies, such as leukemia or non-Hodgkin's lymphoma.It is also involved in autoimmune diseases, and therefore can be a target in the treatment of such conditions.

[0092] In some embodiments, the binding element of the CAR specifically interacts with an epitope on the CD19 antigen. In some embodiments, the binding element of the CCR specifically interacts with an epitope on the CD19 target antigen. In certain embodiments, the binding element of the CAR specifically interacts with an epitope on the CD19 antigen, and the binding element of the CCR specifically interacts with the same, overlapping, or different epitopes on the CD19 target antigen.

[0093] In currently preferred embodiments, the CAR and / or CCR binding element specifically interacts with a first epitope on the CD19 target antigen. In some embodiments, the CAR or CCR binding element comprises the antigen-binding site of the FMC63 antibody. In certain embodiments, the CAR or CCR binding element comprises the CDRs of the FMC63 antibody. The CDR sequences of the FMC63 antibody were determined using www.abysis.org and are set forth below as SEQ ID NOS: 10-15.

[0094] V H CDR1 GVSLPDY (SEQ ID NO: 10). V H CDR2 WGSET (SEQ ID NO: 11). V H CDR3 HYYYGGSYAMDY (SEQ ID NO: 12). V L CDR1 RASQDISKYLN (SEQ ID NO: 13). V L CDR2 HTSRLHS (SEQ ID NO: 14). V L CDR3 QQGNTLPYT (SEQ ID NO: 15).

[0095] In certain embodiments, the CAR or CCR binding element is V of the FMC63 antibody. H and V L The V domain of the FMC63 antibody H and V L The domain sequences are shown below as SEQ ID NOs: 16-17.

[0096] V H : EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS (SEQ ID NO: 16).

[0097] V L : DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT (SEQ ID NO: 17).

[0098] In particularly preferred embodiments, the CAR or CCR binding element is V H -Linker-V L or V L -Linker-V H These sequences are shown below as SEQ ID NOs: 18 and 19. In each case, V H and V L Linker sequences between domains are underlined and italicized.

[0099] JPEG0007723420000002.jpg38153

[0100] JPEG0007723420000003.jpg37153

[0101] In certain embodiments, the CAR or CCR binding element comprises an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to the sequence of the scFv of the FMC63 antibody (SEQ ID NO: 18 or 19).

[0102] In a particularly preferred embodiment, the CAR or CCR binding element comprises a CDR3 region of a variant of FMC63 scFv. In particular, the variant comprises a CDR3 region of FMC63 scFv to alter the affinity of the scFv for CD19. H A particularly preferred embodiment includes mutations within the V domain (SEQ ID NO: 12). H These variants comprise substitutions of alanine (A) for either tyrosine (Y) or glycine (G) within the CDR3 of the domain. These variants are shown below as SEQ ID NOs: 20-26.

[0103] JPEG0007723420000004.jpg50158

[0104] In some embodiments, the CAR or CCR binding element specifically interacts with an epitope on the CD20 antigen. In some embodiments, the CAR or CCR binding element comprises the 1F5 antibody that binds to CD20. See Ledbetter and Clark, Hum. Immunol. 15(1):30-43 (1986), incorporated herein by reference in its entirety. CD20 is an integral membrane protein expressed on the surface of all B cells beginning at the pro-B phase, with concentrations gradually increasing until maturation. In humans, CD20 is encoded by the MS4A1 gene. Antibodies targeting CD20 are used in the treatment of B-cell lymphomas and leukemias, as well as autoimmune diseases such as arthritis, particularly rheumatoid arthritis, multiple sclerosis (MS), and systemic lupus erythematosus. In certain embodiments, the CCR binding element comprises the CDRs of the 1F5 antibody. The CDR sequences of the 1F5 antibody were determined using www.abysis.org and are shown below as SEQ ID NOs: 27 to 32.

[0105] V H CDR1 GYTFTSY (SEQ ID NO: 27). V H CDR2 YPGNGD (SEQ ID NO: 28). V H CDR3 SHYGSNYVDYFDY (SEQ ID NO: 29). V L CDR1 RASSSLSFMH (SEQ ID NO: 30). V L CDR2 ATSNLAS (SEQ ID NO: 31). V L CDR3 HQWSSNPLT (SEQ ID NO: 32).

[0106] In certain embodiments, the CAR or CCR binding element is V of the 1F5 antibody. H and V L The V domain of the 1F5 antibody H and V L The domain sequences are shown below as SEQ ID NOs: 33-34.

[0107] V H : QVQLRQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSHYGSNYVDYFDYWGQGTLVTVSTG (SEQ ID NO: 33).

[0108] V L : QIVLSQSPAILSASPGEKVTMTCRASSSLSFMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYFCHQWSSNPLTFGAGTKVEIKRK (SEQ ID NO: 34).

[0109] In particularly preferred embodiments, the CAR or CCR binding element is V H -Linker-V L or V L -Linker-V H These sequences are shown below as SEQ ID NOs: 35 and 36. In each case, V H and V LLinker sequences between domains are underlined and italicized.

[0110] JPEG0007723420000005.jpg47153

[0111] JPEG0007723420000006.jpg44153

[0112] In certain embodiments, the CAR or CCR binding element comprises a variant of the scFv of the 1F5 antibody, which variant is 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to SEQ ID NO: 35 or 36 shown above.

[0113] In some embodiments, the CAR or CCR binding element specifically interacts with an epitope on the CD22 antigen. In some embodiments, the CAR or CCR binding element is the RFB4 antibody, which binds to CD22. See Campana et al., J. Immunol. 134(3):1524-30 (1985), incorporated herein by reference in its entirety. CD22 is a 135 kDa B-cell-specific adhesion molecule expressed on 60% to 90% of cells in B-cell malignancies. It is not expressed on hematopoietic stem cells or any other non-lymphoid hematopoietic or non-hematopoietic cells. The CDR sequences of the RFB4 antibody were determined using www.abysis.org and are set forth below as SEQ ID NOS: 37-42.

[0114] V H CDR1 GFAFSIY (SEQ ID NO: 37). V H CDR2 SSGGGT (SEQ ID NO: 38). V H CDR3 HSGYGSSYGVLFAY (SEQ ID NO: 39). V L CDR1 RASQDISNYLN (SEQ ID NO: 40). V L CDR2 YTSILHS (SEQ ID NO: 41). VL CDR3 QQGNTLPWT (SEQ ID NO: 42).

[0115] In certain embodiments, the CAR or CCR binding element is V of the RFB4 antibody. H and V L The V domain of the RFB4 antibody H and V L The domain sequences are shown below as SEQ ID NOs: 43-44:

[0116] V H : EVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQTPEKRLEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARHSGYGSSYGVLFAYWGQGTLVTVS (SEQ ID NO: 43).

[0117] V L : DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSILHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQQGNTLPWTFGGGTKLEIK (SEQ ID NO: 44).

[0118] In particularly preferred embodiments, the CAR or CCR binding element is V H -Linker-V L or V L -Linker-V H These sequences are shown below as SEQ ID NOS: 45 and 46. In each case, V H and V L Linker sequences between domains are underlined and italicized.

[0119] JPEG0007723420000007.jpg46153

[0120] JPEG0007723420000008.jpg47153

[0121] In certain embodiments, the CAR or CCR binding element comprises a variant of the scFv of the RFB4 antibody, which variant is 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to SEQ ID NO: 45 or 46 shown above.

[0122] 4.5.2. Representative Examples of B Cell-Specific pCAR Encoding Sequences Combinations of the aforementioned B cell antigen-specific binding elements have been used to engineer pCARs, with the CAR and CCR elements binding to the same epitope within CD19 or to different epitopes found on CD19 and a second lineage-specific B cell antigen. Many additional moieties that specifically bind to CD19 and other lineage-specific B cell antigens are known in the art, meaning that numerous B cell-specific pCARs can be engineered using similar methodologies. Consequently, the following pCAR examples are provided for illustrative purposes only and are not intended to limit the scope of the invention. The nomenclature of pCARs is derived from the following order: CCR binder, CCR signaling domain / CAR binder.

[0123] The protein sequence of FBB / G01 pCAR is shown below as SEQ ID NO: 47. FBB / G01 pCAR contains: (i) a CCR containing a linear fusion of the following elements: macrophage colony-stimulating factor receptor leader peptide, FMC63 scFv binding domain (V L -V H (in order), CD8α spacer and transmembrane domain, 4-1BB costimulatory endodomain, FLAG epitope tag ("FBB") and (ii) a second-generation CAR containing a linear fusion of the following elements: CD8α leader peptide, V of FMC63 scFv; HA variant of FMC63 scFv in which the first glycine in CDR3 is replaced by alanine ("G01"; V L -V H (in order), CD28 spacer containing an embedded myc epitope tag, CD28 transmembrane and endodomain, and CD3z endodomain.

[0124] The CCR and CAR are linked by a furin cleavage site, a Ser-Gly linker (SGSG), and a P2A ribosomal skip peptide. Codon wobbling was used to minimize direct repeats within the scFv module. The V of the scFv sequence H and V L Domains are underlined and in bold, epitope tags are in italics.

[0125] JPEG0007723420000009.jpg95153

[0126] The protein sequence of FBB / G02 pCAR is shown below as SEQ ID NO: 48. FBB / G02 pCAR contains: (i) a CCR containing a linear fusion of the following elements: macrophage colony-stimulating factor receptor leader peptide, FMC63 scFv binding domain (V L -V H (in order), CD8α spacer and transmembrane domain, 4-1BB costimulatory endodomain, FLAG epitope tag ("FBB") and (ii) a second-generation CAR containing a linear fusion of the following elements: CD8α leader peptide, V of FMC63 scFv; H A variant of FMC63 scFv in which the second glycine in CDR3 is replaced by alanine ("G02"; V L -V H (in order), CD28 spacer containing an embedded myc epitope tag, CD28 transmembrane and endodomain, and CD3z endodomain.

[0127] The CCR and CAR are linked by a furin cleavage site, a Ser-Gly linker (SGSG), and a P2A ribosomal skip peptide. Codon wobbling was used to minimize direct repeats within the scFv module. The V of the scFv sequence H and V L Domains are underlined and in bold, epitope tags are in italics.

[0128] JPEG0007723420000010.jpg95153

[0129] The protein sequence of FBB / Y01 pCAR is shown below as SEQ ID NO: 49. FBB / Y01 pCAR contains: (i) a CCR containing a linear fusion of the following elements: macrophage colony-stimulating factor receptor leader peptide, FMC63 scFv binding domain (V L -V H (in order), CD8α spacer and transmembrane domain, 4-1BB costimulatory endodomain, FLAG epitope tag ("FBB") and (ii) a second-generation CAR containing a linear fusion of the following elements: CD8α leader peptide, V of FMC63 scFv; H A variant of FMC63 scFv in which the first tyrosine in CDR3 is replaced by alanine ("Y01"; V L -V H (in order), CD28 spacer containing an embedded myc epitope tag, CD28 transmembrane and endodomain, and CD3z endodomain.

[0130] The CCR and CAR are linked by a furin cleavage site, a Ser-Gly linker (SGSG), and a P2A ribosomal skip peptide. Codon wobbling was used to minimize direct repeats within the scFv module. The V of the scFv sequence H and V L Domains are underlined and in bold, epitope tags are in italics.

[0131] JPEG0007723420000011.jpg96153

[0132] The protein sequence of FBB / Y02 pCAR is shown below as SEQ ID NO: 50. FBB / Y02 pCAR contains: (i) a CCR containing a linear fusion of the following elements: macrophage colony-stimulating factor receptor leader peptide, FMC63 scFv binding domain (V L -V H (in order), CD8α spacer and transmembrane domain, 4-1BB costimulatory endodomain, FLAG epitope tag ("FBB") and (ii) a second-generation CAR containing a linear fusion of the following elements: CD8α leader peptide, V of FMC63 scFv; H A variant of FMC63 scFv in which the second tyrosine in CDR3 is replaced by alanine ("Y02"; V L -V H (in order), CD28 spacer containing an embedded myc epitope tag, CD28 transmembrane and endodomain, and CD3z endodomain.

[0133] The CCR and CAR are linked by a furin cleavage site, a Ser-Gly linker (SGSG), and a P2A ribosomal skip peptide. Codon wobbling was used to minimize direct repeats within the scFv module. The V of the scFv sequence H and V L Domains are underlined and in bold, epitope tags are in italics.

[0134] JPEG0007723420000012.jpg98153

[0135] The protein sequence of FBB / Y03 pCAR is shown below as SEQ ID NO: 51. FBB / Y03 pCAR contains: (i) a CCR containing a linear fusion of the following elements: macrophage colony-stimulating factor receptor leader peptide, FMC63 scFv binding domain (VL -V H (in order), CD8α spacer and transmembrane domain, 4-1BB costimulatory endodomain, FLAG epitope tag ("FBB") and (ii) a second-generation CAR containing a linear fusion of the following elements: CD8α leader peptide, V of FMC63 scFv; H A variant of FMC63 scFv in which the third tyrosine in CDR3 is replaced by alanine ("Y03"; V L -V H (in order), CD28 spacer containing an embedded myc epitope tag, CD28 transmembrane and endodomain, and CD3z endodomain.

[0136] The CCR and CAR are linked by a furin cleavage site, a Ser-Gly linker (SGSG), and a P2A ribosomal skip peptide. Codon wobbling was used to minimize direct repeats within the scFv module. The V of the scFv sequence H and V L Domains are underlined and in bold, epitope tags are in italics.

[0137] JPEG0007723420000013.jpg98153

[0138] The protein sequence of FBB / Y04 pCAR is shown below as SEQ ID NO: 52. FBB / Y04 pCAR contains: (i) a CCR containing a linear fusion of the following elements: macrophage colony-stimulating factor receptor leader peptide, FMC63 scFv binding domain (V L -V H (in order), CD8α spacer and transmembrane domain, 4-1BB costimulatory endodomain, FLAG epitope tag ("FBB") and (ii) a second-generation CAR containing a linear fusion of the following elements: CD8α leader peptide, V of FMC63 scFv; H A variant of FMC63 scFv in which the fourth tyrosine in CDR3 is replaced by alanine ("Y04"; VL -V H (in order), CD28 spacer containing an embedded myc epitope tag, CD28 transmembrane and endodomain, and CD3z endodomain.

[0139] The CCR and CAR are linked by a furin cleavage site, a Ser-Gly linker (SGSG), and a P2A ribosomal skip peptide. Codon wobbling was used to minimize direct repeats within the scFv module. The V of the scFv sequence H and V L Domains are underlined and in bold, epitope tags are in italics.

[0140] JPEG0007723420000014.jpg96153

[0141] The protein sequence of FBB / Y05 pCAR is shown below as SEQ ID NO: 53. FBB / Y05 pCAR contains: (i) a CCR containing a linear fusion of the following elements: macrophage colony-stimulating factor receptor leader peptide, FMC63 scFv binding domain (V L -V H (in order), CD8α spacer and transmembrane domain, 4-1BB costimulatory endodomain, FLAG epitope tag ("FBB") and (ii) a second-generation CAR containing a linear fusion of the following elements: CD8α leader peptide, V of FMC63 scFv; H A mutant of FMC63 scFv in which the fifth tyrosine in CDR3 is replaced by alanine ("Y05"; V L -V H (in order), CD28 spacer containing an embedded myc epitope tag, CD28 transmembrane and endodomain, and CD3z endodomain.

[0142] The CCR and CAR are linked by a furin cleavage site, a Ser-Gly linker (SGSG), and a P2A ribosomal skip peptide. Codon wobbling was used to minimize direct repeats within the scFv module. The V of the scFv sequence H and V L Domains are underlined and in bold, epitope tags are in italics.

[0143] JPEG0007723420000015.jpg98153

[0144] The protein sequence of 1BB / F pCAR is shown below as SEQ ID NO: 54. 1BB / F pCAR contains: (i) a CCR containing a linear fusion of the following elements: macrophage colony-stimulating factor receptor leader peptide, 1F5 scFv binding domain (V L -V H (in order), CD8α spacer and transmembrane domain, 4-1BB costimulatory endodomain, FLAG epitope tag ("1BB") and (ii) a second-generation CAR containing a linear fusion of the following elements: CD8α leader peptide, FMC63 scFv (V L -V H (in order), CD28 spacer containing an embedded myc epitope tag, CD28 transmembrane and endodomain, and CD3z endodomain.

[0145] The CCR and CAR are linked by a furin cleavage site, a Ser-Gly linker (SGSG), and a T2A ribosomal skipping peptide. Codon wobbling was used to minimize direct repeats within the scFv module. Alternative spacers were used within both scFvs for the same purpose. The V of the scFv sequence H and V L Domains are underlined and in bold, epitope tags are in italics.

[0146] JPEG0007723420000016.jpg95153

[0147] The protein sequence of RBB / F pCAR is shown below as SEQ ID NO: 55. RBB / F pCAR contains: (i) a CCR containing a linear fusion of the following elements: macrophage colony-stimulating factor receptor leader peptide, RFB4 scFv binding domain (V L -V H (in order), CD8α spacer and transmembrane domain, 4-1BB costimulatory endodomain, FLAG epitope tag ("RBB") and (ii) a second-generation CAR containing a linear fusion of the following elements: CD8α leader peptide, FMC63 scFv (V L -V H (in order), CD28 spacer containing an embedded myc epitope tag, CD28 transmembrane and endodomain, and CD3z endodomain.

[0148] The CCR and CAR are linked by a furin cleavage site, a Ser-Gly linker (SGSG), and a T2A ribosomal skipping peptide. Codon wobbling was used to minimize direct repeats within the scFv module. Alternative spacers were used within both scFvs for the same purpose. The V of the scFv sequence H and V L Domains are underlined and in bold, epitope tags are in italics.

[0149] JPEG0007723420000017.jpg97153

[0150] 4.6. Nucleic Acids and Methods for Producing pCAR-T Cells Also provided herein is a combination of a first nucleic acid encoding the second-generation CAR described above and a second nucleic acid encoding the CCR described above. As mentioned above, for convenience, the combination of CAR and CCR will be referred to herein in the singular as pCAR, although the CAR and CCR are separate co-expressed proteins. Appropriate sequences for nucleic acids will be apparent to those skilled in the art based on the above description of CAR and CCR. The sequence may be optimized for use in the required immunoresponsive cells. However, in some cases, as mentioned above, codons may be suboptimal or "wobbled" to avoid repeat sequences. Specific examples of such nucleic acids encode the preferred embodiments described above. In some embodiments, a B cell-specific pCAR comprises a polypeptide of a sequence selected from SEQ ID NOs: 47-55. In some embodiments, the nucleic acid encoding pCAR is selected from the group consisting of SEQ ID NOs: 109-117.

[0151] In some embodiments, the nucleic acid encoding the CCR component of pCAR is selected from the group consisting of SEQ ID NOs: 128, 129 and 130.

[0152] In some embodiments, the nucleic acid encoding the CAR component of pCAR is selected from the group consisting of SEQ ID NOs: 101-108.

[0153] To achieve transduction, the nucleic acid encoding pCAR is suitably introduced into one or more vectors, such as a plasmid, a retroviral or lentiviral vector, or a non-viral vector. Such vectors, including plasmid vectors, or cell lines containing them, form further aspects of the present invention.

[0154] In an exemplary embodiment, immunoresponsive cells are subjected to genetic modification, e.g., by retroviral or lentiviral-mediated transduction, to introduce nucleic acids encoding the CAR and CCR into the host T cell genome, thereby allowing stable pCAR expression, which may then be reintroduced into the patient (possibly after expansion) to provide beneficial therapeutic effects, as described below.

[0155] The first and second nucleic acids encoding the CAR and CCR can be expressed from the same vector or different vectors. The vector or vectors comprising them can be combined in a kit provided to produce the immunoresponsive cells of the first aspect disclosed herein.

[0156] In some embodiments, T cells may be engineered to co-express a chimeric cytokine receptor, such as 4αβ, which comprises a fusion of the ectodomain of the IL-4 receptor-α with the transmembrane and endodomains of the shared IL-2 / 15 receptor-β chain. In this case, the expansion step may include ex vivo culture in a cytokine-containing medium (e.g., in the case of 4αβ, a medium containing IL-4 as the sole cytokine support). Alternatively, the chimeric cytokine receptor may comprise the ectodomain of the IL-4 receptor-α chain linked to a receptor endodomain used by a common gamma cytokine with distinct properties, such as IL-7. Growth of cells in IL-4 may result in less cell differentiation under these circumstances. In this way, selective expansion and enrichment of genetically engineered T cells with the desired differentiation state can be ensured.

[0157] 4.6. Treatment Methods Using pCAR T Cells Targeted Against B Cell Antigens As described above, immunoresponsive pCAR cells are useful in therapies for directing a T cell-mediated immune response to target cells. Accordingly, in another aspect, a method for directing a T cell-mediated immune response to target cells in a patient in need thereof is provided. The method comprises administering to the patient a population of immunoresponsive cells as described above, wherein the binding element is specific for the target cells. In some embodiments, the target cells express CD19 and / or other B cell antigens.

[0158] In another aspect, methods of treating cancer in a patient in need thereof are provided. The methods include administering to the patient a population of immunoresponsive cells described above, wherein the binding element is specific for target cells. In some embodiments, the target cells express CD19 and / or other B cell antigens. In some embodiments, the patient has acute or chronic B cell leukemia or B cell lymphoma.

[0159] In various embodiments, a therapeutically effective number of immunoresponsive cells are administered to a patient. In certain embodiments, the immunoresponsive cells are administered by intravenous infusion. In certain embodiments, the immunoresponsive cells are administered by intratumoral injection. In certain embodiments, the immunoresponsive cells are administered by peritumoral injection. In certain embodiments, the immunoresponsive cells are administered by multiple routes selected from intravenous infusion, intratumoral injection, and peritumoral injection. [Example]

[0160] 5. Working Example Below are examples of specific embodiments for carrying out the present invention. The examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0161] 5.1. Method 5.1.1. Cell line culture All tumor cells and 293T cells were grown in DMEM supplemented with L-glutamine and 10% FBS. Where indicated, tumor cells were transduced to express firefly luciferase and the tandem dimer Tomato red fluorescent protein (LT) SFG vector, followed by flow sorting for red fluorescent protein expression. LO68 CD19 + Cells and LO68 CD19 + / CD20 + The cells were produced by transduction of LO68-LT cells with SFG retroviral vectors encoding human CD19 and / or human CD20.

[0162] 5.1.2. Retrovirus Production 293T cells were triple transfected in Genejuice (MilliporeSigma, Merck KGaA, Darmstadt, Germany) with (i) the SFG retroviral vector encoding the indicated CAR / pCAR, (ii) the RDF plasmid encoding the RD114 envelope, and (iii) the Peq-Pam plasmid encoding gag-pol, as recommended by the manufacturer. 1.5 × 10 cells in 100 mm plates were transfected. 6 For transfection of 293T cells, 4.6875 μg of SFG retroviral vector, 4.6875 μg of Peq-Pam plasmid, and 3.125 μg of RDF plasmid were used. Media containing viral vectors were collected 48 and 72 hours posttransfection, flash-frozen, and stored at -80°C. In some cases, stable packaging cell lines were generated by transducing 293VEC GALV cells with retroviruses. Viruses prepared from either source were used interchangeably for transduction of target cells.

[0163] 5.1.3. T Cell Culture and Transduction Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor peripheral blood samples by density gradient centrifugation using Ficoll-Paque (ethical approval number 18 / WS / 0047). T cells were cultured in RPMI containing GlutaMax supplemented with 5% human AB serum. T cell activation was achieved by culturing for 24–48 hours in the presence of 5 μg / mL phytohemagglutinin leukoagglutinin (PHA-L). Cells were then expanded in IL-2 (100 U / mL) for an additional 24 hours before transduction. T cell transduction was achieved using RetroNectin (Takara Bio)-coated plates according to the manufacturer's protocol. Activated PBMCs (1 × 10) were cultured per well of a RetroNectin-coated 6-well plate. 6 Then, 3 mL of medium containing the retrovirus was added along with 100 U / mL of IL2 per well.

[0164] 5.1.4. Cytotoxicity Assay Tumor cells were plated at 2 x 10 in 24- or 96-well plates. 4 or 1×10 5 Tumor cells were seeded at 1000 μg / well and incubated with T cells at a defined target:effector ratio. In some cases, tumor cell destruction by T cells was quantified using an MTT assay. To achieve this, MTT (Sigma) was added at 500 μg / ml in D10 medium for 2 hours at 37°C and 5% CO2. After removing the supernatant, the formazan crystals were resuspended in 100 μL of DMSO. Absorbance was measured at 560 nm. Alternatively, tumor cell viability was monitored by luciferase assay. 150 mg / mL D-luciferin (PerkinElmer, Waltham MA) was added immediately before luminescence reading. In both cases, tumor cell viability was calculated as follows: (absorbance or luminescence of tumor cells cultured with T cells / absorbance or luminescence of untreated monolayers only) × 100%.

[0165] 5.1.5. Detection of CAR and CCR Expression by Flow Cytometry CAR expression was detected using the 9e10 antibody, which binds to the myc epitope tag (EQKLISEEDL), followed by a PE-conjugated goat anti-mouse antibody. Expression of the CCR component of pCAR was detected by intracellular staining using a PE-conjugated antibody that binds to the FLAG epitope tag (DYKDDDDK).

[0166] 5.1.6. Detection of IFN-γ and IL-2 by ELISA Supernatants were collected from CAR T cell and tumor cell cocultures at 24 hours, and cytokine levels were quantified using human IFN-γ (Bio-Techne) or human IL-2 ELISA kits (Invitrogen) according to the manufacturer's protocol.

[0167] 5.1.7. Repeated antigen stimulation assay Tumor cell suspensions were co-cultured with CAR-T / pCAR-T cells at an initial effector:target (E:T) ratio of 1:1 for 72–96 h. The viability of the remaining tumor cells was then assessed by luciferase assay. 150 mg / mL D-luciferin (PerkinElmer) was added immediately before luminescence reading. Fresh tumor cells (10 5 cells) was added and the procedure was repeated until the T cell culture could no longer be expanded.

[0168] Alternatively, the adherent LO68 tumor cell line was cultured at 1 × 10 in 24-well culture plates 24 hours before the addition of T cells. 5 Cells / well were plated in triplicate. CAR-T / pCAR-T cells were added at a 1:1 effector:target ratio. Tumor cell killing was measured 48-72 hours later using the MTT assay performed as described above. T cells were then harvested and restimulated by addition to a new tumor cell monolayer if >20% tumor cell death compared to untreated cells was observed. Tumor cell viability was calculated as described in section 5.1.4.

[0169] 5.1.8. In vivo testing PBMCs from healthy donors were modified to express the indicated CAR / pCAR or were untransduced. After 11 days of expansion in IL-2 (100 U / mL, added every 2-3 days), cells were analyzed by flow cytometry for CAR and CCR expression as described above. 5 × 10 5 Nalm-6 LT cells were injected i.v. Four days later, 5 × 10 5 Cars + (or non-transduced) T cells were injected intravenously in 200 μl of PBS to compare with PBS as a control. Tumor status was monitored by bioluminescence imaging (BLI) performed under isoflurane anesthesia 20 minutes after injection of StayBrite™ D-luciferin, potassium salt (150 mg / kg) in 200 μl of PBS. Images were acquired at the indicated time points using an IVIS® Lumina III (PerkinElmer) with Living Image software (PerkinElmer) set for automatically optimized exposure time, binning, and F / stop. Animals were humanely killed when the experimental endpoint was reached.

[0170] 5.1.9. CD19 CAR Binding Assay - z-Movi. CD19-modified L068 tumor cells were seeded into the z-Movi microfluidic chip and cultured for 16 hours. The following day, flow-sorted CAR-T cells were continuously flowed through the chip and incubated with target cells for 5 minutes prior to initializing a 3-minute linear force ramp. Cell separation was determined post-experimentally using image analysis techniques.

[0171] 5.2. Example 1: In vitro activity of CD19-specific CAR-T cells T cells were injected with a CD28-containing second generation CAR designated F-2 or a V designated Y01, Y02, Y03, Y04, Y05, G01, or G02. HThey were either modified by retroviral transduction to express CDR3 mutant derivatives or were not transduced (FIGS. 3A and 3B).

[0172] To test binding to CD19, transduced T cells were incubated with two different concentrations of CD19-Fc: 0.5 μg (FIG. 7A) and 1.0 μg (FIG. 7B). Binding of CD19-Fc to CAR was detected by flow cytometry after incubation with PE-conjugated anti-human IgG. Figures 7A-7D show that the F-2 variants with mutated CDR3 exhibit variable efficiency of binding to CD19.

[0173] To further test the relative CD19 binding ability of the 2G CAR, F-2, and its mutant derivatives, the engineered CAR-T cells were cultured in a z-Movi microfluidic chip to bind CD19 + The T cells were incubated on a monolayer of LO68 tumor cells. Increasing fluid forces were applied and the percentage of bound T cells was determined (median, n=3). H T cells expressing the CDR3-mutated CAR showed broad avidity for CD19. H The majority of CDR3-mutated derivatives showed reduced avidity for CD19 compared to 2G CAR F. Data from a representative experiment are shown in Figures 17A-17D. Data are presented as median rForce, the relative force required to separate cells (calibrated against 10 μM polystyrene beads). Data did not follow a normal distribution and are therefore presented as median rForce, the relative force required to separate cells, calibrated against 10 μM polystyrene beads. Figure 17B shows the overall mean percentage of bound T cells after applying the minimum rForce (210 pN) required to separate an average of 90% of non-transduced T cells. Data shown are the mean + standard error (n=11 runs, 3 independent donors). Figure 17C shows the avidity score (e.g., V compared to non-transduced T cells). HCD19 for each of the CDR3 mutated derivatives + Figure 17D shows the ratio of the mean rForce per cell required to detach T cells from a monolayer of LO68 tumor cells. The black dotted line indicates the avidity score of non-transduced T cells; the red dotted line indicates the avidity score of F-2 CAR T cells. Figure 17D shows the rForce per cell required to detach T cells from a monolayer of tumor cells, with each dot in the dot plot representing a single cell. Bars indicate the median + interquartile range. Statistical analysis was performed using a Kruskal-Wallis test with Dunnett's multiple comparison test. p=<0.0001( **** ), 0.0007( *** ), or 0.005( ** ). For clarity, a single representative run for one healthy donor is plotted, with each dot representing a single cell. Collectively, these dots generate the avidity curve shown in Figure 17A. The avidity curve therefore represents over 1000 single-cell observations per donor.

[0174] To evaluate antitumor activity, transduced T cells were cocultured in vitro with Nalm-6 LT or Raji LT cells, both of which naturally express CD19 (Figure 1A). The E:T ratio ranged from 10 to 0.31 (including 5, 2.5, 1.25, and 0.63). Data obtained using cells from three representative donors are shown in Figures 8A-8B. Viable cancer cells remaining after coculture were quantified by luciferase assay after 72 hours. The survival percentages of Nalm-6 and Raji tumor cells after coculture with CAR-T cells are shown in Figures 8A and 8B, respectively.

[0175] To further evaluate cytotoxic activity, 2 × 10 4Transduced T cells were co-cultured in duplicate with an equal number of Nalm-6 (Figure 8C) or Raji (Figure 8D) cells. After 72 hours, tumor cell viability was quantified by luciferase assay. The mean + standard error of the average levels of viable cancer cells (n = 3 biological replicates) are shown in Figures 8C-8D. Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison test. p < 0.0001 ( **** ), ≦0.001( *** ), or ≦0.01( ** ).

[0176] Transduced T cells were co-cultured in vitro with Nalm-6 LT (Figures 9A-9B) or Raji LT cells (Figures 9C-9D) at an effector:target ratio of 1.25 tumor cells:1 CAR-expressing T cell. The mean + standard error (n = 3 biological replicates) of the average levels of IFN-γ and IL-2 concentrations present in the supernatants collected after 24 hours are shown in Figures 9A-9D. In each case, statistical analysis was performed using a two-way ANOVA with Tukey's multiple comparison test. p = 0.033 ( * ), 0.002( ** ), 0.0002( *** ) or <0.0001( **** NS - not significant.

[0177] After the co-culture experiments described above (data shown in Figures 8C-8D), cell supernatants were collected 24 hours later and analyzed for IFN-γ (Figures 10A-10B) and IL-2 (Figures 10C-10D) by ELISA. Data shown are means + standard errors (n = 3 biological replicates). Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison test. p < 0.0001 ( **** ), ≦0.001( *** ), ≦0.01( ** ), or ≦0.05( * NS - not significant.

[0178] Transduced T cells were subjected to successive rounds of antigen (Ag) stimulation in the absence of exogenous cytokine IL-2.5 Triplicate cultures containing 10 engineered T cells were 5 The cells were restimulated twice weekly with the addition of Nalm-6 tumor cells. Figure 11A shows tumor cell viability measured by luciferase assay 72 hours after each tumor cell challenge. Figures 11B and 11C show IFN-γ and IL-2 levels, respectively, in supernatants collected 24 hours after each tumor cell challenge.

[0179] 5.3. Example 2: In vitro activity of CD19-specific pCAR-T cells Figures 4A-4B and Figures 18A-18B show the expression of a panel of CD19-specific pCARs in human T cells and compare them to a second-generation control CAR (F-2). The nomenclature of the pCARs is derived from the ordered abbreviations of the following elements: CCR binder ( F MC63 scFv), CCR signaling domain (4-1 BB ) / CAR binder (respectively G01 ~ Y05 ). Cell surface expression of the CD28-containing CAR in each pCAR was detected by flow cytometry after incubation of cells with the 9e10 antibody, which binds to the myc epitope tag within the CAR ectodomain. Expression of the CCR component in each pCAR was detected by intracellular staining of permeabilized cells with an anti-FLAG antibody, which binds to the distally located FLAG epitope tag within the CCR endodomain. The location of the epitope tag is shown schematically in Figures 2C-2F.

[0180] Figure 12 shows pooled data from five biological replicate experiments, each performed in duplicate, and shows the CDR3 V H Second-generation CAR and pCAR T cells based on the mutated FMC63 were co-cultured with Nalm-6 LT tumor cells in vitro and compared with parental second-generation CAR T cells (F-2). In each experiment, cultures were demonstrated, with T cells reaching 2 x 10 4The CAR-T cells were co-cultured with Nalm-6 tumor cells. The E:T ratio ranged from 1:1 to 1:128 (including 1:2, 1:4, 1:8, 1:16, 1:32, and 1:64). Remaining viable cancer cells were quantified by luciferase assay after 72 hours. The percentage of viable Nalm-6 tumor cells after co-culture with CAR-T cells is shown (mean + standard error of the mean, n = 10 data points). Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test. p = 0.033 ( * ), 0.002( ** ), 0.0002( *** ) or <0.0001( **** ) NS-Not significant. CDR 3V H Some second-generation CARs based on the mutated V FMC63 were superior to the parent second-generation CAR, F-2. Equal or even enhanced cytotoxicity was observed with the same mutated V H This was seen for the derived pCAR with CDR3.

[0181] 13A to 13C show the 5 Representative data are shown from an experiment in which pCAR-T cells were repeatedly restimulated on a monolayer of CD19-expressing LO68 tumors in the absence of exogenous cytokines. Figure 13A shows tumor cell viability measured by luciferase assay 24 hours after each round of stimulation. Figures 13B and 13C show the levels of IFN-γ and IL-2 present in supernatants collected 24 hours after each round of stimulation, respectively. Note the superior antitumor activity of some pCAR variants compared to the parental second-generation CAR, F-2.

[0182] 5.4. Example 3: In vivo activity of CD19-specific pCAR-T cells in NSG mice The antitumor activity of CD19-specific CAR-T and pCAR-T cells was evaluated in vivo in NSG mice bearing validated Nalm-6 leukemic xenografts.

[0183] RFP / ffLuc + Nalm6 cells (5 × 105 NSG mice were intravenously injected with 5 × 10 cells. On day 5, the animals were arranged into groups of 5–10 mice with comparable disease burden (by BLI). Mice were then injected intravenously with 5 × 10 cells. 5 Treatment with the indicated CAR or pCAR T-cells (iv administration) was compared using PBS. Pooled bioluminescence emission ("total intensity") from the leukemic xenografts was measured for each treatment starting on day 8 (Figure 19). Modest T-cell doses (5 x 10) were used. 5 In pCAR T cells (FBB / Y04, FBB / Y05, and FBB / G02), F-2 CAR T cells induced a transient delay in disease progression, while the mutant 2G derivatives (Y05 and G02) achieved superior anti-leukemic activity. All of the tested pCARs (FBB / Y04, FBB / Y05, and FBB / G02) achieved further enhanced disease control (Figure 14A, Figure 20A, and Figure 20B). The tested pCARs (FBB / Y04, FBB / Y05, and FBB / G02) also had excellent survival after treatment (Figures 21A-21D) and no weight loss (Figure 14B).

[0184] These results indicate that CD19-specific pCAR-T cells have superior antitumor activity compared to 2G CAR-T cells in vivo in NSG mice with a documented leukemic burden.

[0185] 5.6. Example 4: In vitro antitumor activity of pCAR-T cells co-targeting CD19 and CD20 T cells were engineered to express CD19- or CD20-specific second-generation CAR T cells (F-2 and 1-2, respectively) or 1BB / F pCAR. 5 transduced CAR or pCAR T cells in the same number of LO68-CD19 + CD20 + After 72 hours, T cells were co-cultured with tumor cells in triplicate. + CD20 +The cells were transferred to a fresh monolayer of cells. The data plotted in Figure 15A show the number of restimulation cycles completed for each co-culture condition. Cultures were terminated when tumor cell viability was 80% or greater.

[0186] Figure 15B shows the 10 5 Data are shown for 1BB / F pCAR-T cells combined with an equal number of LO68 tumor cells co-expressing CD19 and CD20. Culture was performed in the absence of exogenous cytokines. Comparisons are made with CD19- or CD20-specific second-generation CAR T cells (F-2 and 1-2, respectively) or non-transduced T cells. Tumor cell cytotoxicity was assessed by MTT assay after 72 hours. T cells were cultured at 10 5 Tissues were restimulated twice a week by co-culture with tumor cells, and tumor cell viability was assessed 72 hours later by MTT assay. This procedure was repeated until T cell cultures were exhausted. Data shown are mean ± SEM, n = 4. Supernatants were removed from the co-cultures one day after each cycle of stimulation and analyzed by ELISA for the release of IFN-γ and IL-2 (Figure 15C). Data shown are mean + standard error (n = 4).

[0187] In Figure 15D, T cells were cultured using 10 5 CAR- and pCAR-modified T cells were co-cultured with LO68 tumor cells in vitro for 72 hours. The effector:target (T cell:tumor cell) ratios ranged from 1:1 to 1:128 (including 1:2, 1:4, 1:8, 1:16, 1:32, and 1:64). Cultures were established in the absence of exogenous cytokines. Remaining viable cancer cells were quantified by MTT assay after 72 hours. Similar antitumor activity was observed in vitro for CAR- and pCAR-modified T cells. Data shown are mean ± SEM, n=4.

[0188] CAR or pCAR T cells with LO68-CD19 + CD20 +T cells were co-cultured with tumor cells at effector:target (T cell:tumor cell) ratios of 1:1 and 1:4. Supernatants were collected 24 hours after co-culture and analyzed for IFN-γ (Figure 15E) and IL-2 (Figure 15F).

[0189] 5.7. Example 5: In vitro antitumor activity of pCAR-T cells co-targeting CD19 and CD22 Figures 6A-6B show the expression of RBB / F pCAR, which cotargets CD19 and CD22, in human T cells, compared with CD19- or CD22-specific second-generation CAR T cells (F-2 and R-2, respectively) or untransduced T cells. Expression of the CAR or CAR component of pCAR was detected by flow cytometry after incubation of cells with the 9e10 antibody, which binds to the myc epitope tag within the CAR ectodomain. Expression of the CCR component of pCAR was detected by intracellular staining of permeabilized cells using an anti-FLAG antibody, which binds to the distally located FLAG epitope tag within the CCR endodomain. The location of the epitope tag is shown schematically in Figure 2F.

[0190] Figure 16A shows the cytotoxic activity of RBB / F pCAR T cells against Nalm-6 leukemia cells and compares them with CD19-specific (F-2) or CD22-specific (R-2) second-generation CAR T cells. T cells were co-cultured with tumor cells in vitro for 72 hours. The effector:target (T cell:tumor cell) ratio ranged from 1:1 to 1:128 (including 1:2, 1:4, 1:8, 1:16, 1:32, and 1:64). Remaining viable cancer cells were then quantified by luciferase assay. Note the enhanced antitumor activity of pCAR T cells at lower E:T ratios. Data shown are mean + SEM, n = 5. Statistical analysis was performed using two-way ANOVA followed by Tukey's post-hoc test. p = 0.0216 ( * ), 0.0021( ** ), 0.0002( *** ), <0.0001( **** )

[0191] Figure 16B shows the cytotoxic activity of RBB / G02 and RBB / Y05 pCAR T cells against Nalm-6 leukemia cells and compares them to RBB / F pCAR T cells and CD19-specific (F-2) or CD22-specific (R-2) second-generation CAR T cells. Experiments were performed as described above, and target cell viability was quantified by luciferase assay after 72 hours of co-culture. Data shown are the mean + standard error (n = 3-7 biological replicates).

[0192] 6. Arrays JPEG0007723420000018.jpg209158JPEG0007723420000019.jpg218158JPEG0007723420000020.jpg217158 JPEG0007723420000021.jpg216158JPEG0007723420000022.jpg216158JPEG0007723420000023.jpg215158 JPEG0007723420000024.jpg210158 JPEG0007723420000025.jpg216158JPEG0007723420000026.jpg216158JPEG0007723420000027.jpg214158 JPEG0007723420000028.jpg215158 JPEG0007723420000029.jpg215158JPEG0007723420000030.jpg213158 JPEG0007723420000031.jpg214158 JPEG0007723420000032.jpg214158JPEG0007723420000033.jpg214158 JPEG0007723420000034.jpg216158 JPEG0007723420000035.jpg212158JPEG0007723420000036.jpg217158 JPEG0007723420000037.jpg215158JPEG0007723420000038.jpg211158 JPEG0007723420000039.jpg217158 JPEG0007723420000040.jpg214158 JPEG0007723420000041.jpg214158 JPEG0007723420000042.jpg212158 JPEG0007723420000043.jpg214158 JPEG0007723420000044.jpg203158

[0193] 7. Equivalence and Scope Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments according to the invention described herein. The scope of the invention is not intended to be limited to the above description, but rather is as defined in the appended claims.

Claims

1. an immunoresponsive cell, i. A second generation chimeric antigen receptor (CAR) comprising: a) signaling region; b) a first costimulatory signaling region; c) a first transmembrane domain; and d) a first binding element that specifically interacts with a first epitope on the CD19 target antigen; and ii. A chimeric costimulatory receptor (CCR) comprising: e) a second costimulatory signaling domain; wherein said second costimulatory signaling domain is different from said first costimulatory signaling domain; f) a second transmembrane domain; and g) a second binding element that specifically interacts with a second epitope on a second target antigen; wherein said second target antigen is CD19 or another B cell lineage-specific target antigen. Expressing The first connecting element comprises: CDR1 V comprising the sequence of SEQ ID NO: 10 Ｈ , CDR2 V comprising the sequence of SEQ ID NO: 11 Ｈ , CDR3 V comprising one of the sequences selected from SEQ ID NOs: 20 to 26 Ｈ , CDR1 V comprising the sequence of SEQ ID NO: 13 Ｌ , CDR2 V comprising the sequence of SEQ ID NO: 14 Ｌ , and CDR3 V comprising the sequence of SEQ ID NO: 15 Ｌ Including, Optionally, The first connecting element comprises: V containing variants of the sequence of SEQ ID NO: 16 Ｈ , wherein said variant is the CDR3 V of SEQ ID NO: 16 Ｈ having a single amino acid mutation of G to A or Y to A in the region and comprising one of the sequences selected from SEQ ID NOs: 20-26; and V comprising the sequence of SEQ ID NO: 17 Ｌ and / or The first binding element may comprise a single chain variable fragment (scFv) comprising a variant of one of the sequences selected from SEQ ID NOs: 18-19; wherein said variant is CDR3 V Ｈ and may comprise one of the sequences selected from SEQ ID NOs: 20-26, with a single amino acid mutation of G to A or Y to A in the region. Immunoresponsive cells.

2. 10. The immunoresponsive cell of claim 1, the second target antigen comprising the second epitope is a B cell lineage-specific antigen selected from the group consisting of CD19, CD20, CD22, CD23, CD79a, and CD79b; Immunoresponsive cells.

3. 3. The immunoresponsive cell of claim 1 or 2, The second connecting element comprises: CDR1 V comprising the sequence of SEQ ID NO: 10 Ｈ , CDR2 V comprising the sequence of SEQ ID NO: 11 Ｈ , CDR3 V comprising the sequence of SEQ ID NO: 12 Ｈ , CDR1 V comprising the sequence of SEQ ID NO: 13 Ｌ , CDR2 V comprising the sequence of SEQ ID NO: 14 Ｌ , and CDR3 V comprising the sequence of SEQ ID NO: 15 Ｌ Including, Optionally, The second binding element has the sequence of SEQ ID NO:

16. Ｈ region and V having the sequence of SEQ ID NO: 17 Ｌ and / or The second binding element may comprise a single chain variable fragment (scFv) having the sequence of SEQ ID NO: 18 or 19; Immunoresponsive cells.

4. 3. The immunoresponsive cell of claim 1 or 2, The second connecting element comprises: CDR1 V comprising the sequence of SEQ ID NO: 27 Ｈ , CDR2 V comprising the sequence of SEQ ID NO: 28 Ｈ , CDR3 V comprising the sequence of SEQ ID NO: 29 Ｈ , CDR1 V comprising the sequence of SEQ ID NO: 30 Ｌ , CDR2 V comprising the sequence of SEQ ID NO: 31 Ｌ , and CDR3 V comprising the sequence of SEQ ID NO: 32 Ｌ Including, Optionally, The second binding element has the sequence of SEQ ID NO:

33. Ｈ region and V having the sequence of SEQ ID NO: 34 Ｌ and / or The second binding element may comprise a single chain variable fragment (scFv) having the sequence of SEQ ID NO: 35 or 36. Immunoresponsive cells.

5. 3. The immunoresponsive cell of claim 1 or 2, The second connecting element comprises: CDR1 V comprising the sequence of SEQ ID NO: 37 Ｈ , CDR2 V comprising the sequence of SEQ ID NO: 38 Ｈ , CDR3 V comprising the sequence of SEQ ID NO: 39 Ｈ , CDR1 V comprising the sequence of SEQ ID NO: 40 Ｌ , CDR2 V comprising the sequence of SEQ ID NO: 41 Ｌ , and CDR3 V comprising the sequence of SEQ ID NO: 42 Ｌ Including, Optionally, The second binding element has the sequence of SEQ ID NO:

43. Ｈ region and V having the sequence of SEQ ID NO: 44 Ｌ and / or The second binding element may comprise a single chain variable fragment (scFv) having the sequence of SEQ ID NO: 45 or 46; Immunoresponsive cells.

6. 10. The immunoresponsive cell of claim 1, the second generation CAR comprises the sequence of SEQ ID NO: 56, 58, 59, 60, 61, 62, or 63, and the CCR comprises the sequence of SEQ ID NO: 57; Immunoresponsive cells.

7. The immunoresponsive cell of any one of claims 1 to 6, the immunoresponsive cells are αβ T cells, γδ T cells, or natural killer (NK) cells; Immunoresponsive cells.

8. A polynucleotide or set of polynucleotides comprising: i. a first nucleic acid encoding a second generation chimeric antigen receptor (CAR) comprising: a) signaling region; b) a first costimulatory signaling region; c) a first transmembrane domain; and d) a first binding element that specifically interacts with a first epitope on the CD19 target antigen; and ii. A second nucleic acid encoding a chimeric costimulatory receptor (CCR) comprising: e) a second costimulatory signaling domain; wherein said second costimulatory signaling domain is different from said first costimulatory signaling domain; f) a second transmembrane domain; and g) a second binding element that specifically interacts with a second epitope on a second target antigen; wherein said second target antigen is CD19 or another B cell lineage-specific target antigen. Including, wherein the nucleic acid encoding a component of a second generation CAR is selected from the group consisting of SEQ ID NOs: 102 to 108. A polynucleotide or set of polynucleotides.

9. An immunoresponsive cell comprising the polynucleotide or set of polynucleotides of claim 8.

10. A method for preparing the immunoresponsive cells of any one of claims 1 to 7, comprising:

10. The method comprising transfecting or transducing the polynucleotide or set of polynucleotides of claim 8 into an immunoresponsive cell, wherein the method is not an in vivo method. method.

11. 8. The immunoresponsive cell of any one of claims 1 to 7 for use (i) in therapy or as a medicament, or (ii) in the treatment of cancer patients.

12. The immunoresponsive cell for use according to claim 11, wherein the patient has a cancer arising from the B-cell lineage.

13. 13. An immunoresponsive cell for use according to claim 12, comprising: The patient: a) acute or chronic B-cell leukemia; or b) B-cell lymphoma, having Immunoresponsive cells.

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