B cell targeted parallel car (pCAR) therapeutic agents

Abstract

Provided herein are immuno-responsive cells expressing a B cell targeting pCAR comprising a 2nd generation chimeric antigen receptor (CAR) and a chimeric co-stimulatory receptor (CCR). Also provided herein are methods of preparing the immuno-responsive cells and methods of directing T cell mediated immune response using the immuno-responsive cells.

Claims

1. An immuno-responsive cell expressing: (i) a 2.sup.nd generation chimeric antigen receptor (CAR) comprising a) a signaling region; b) a first co-stimulatory signaling region; c) a first transmembrane domain; and d) a first binding element that specifically interacts with a first epitope on a CD19 target antigen, wherein said first binding element comprises: CDR1 V.sub.H comprising the sequence of SEQ ID NO: 10, CDR2 V.sub.H comprising the sequence of SEQ ID NO: 11, CDR3 V.sub.H comprising one of the sequences selected from SEQ ID NOs: 20-26, CDR1 V.sub.L comprising the sequence of SEQ ID NO: 13, CDR2 V.sub.L comprising the sequence of SEQ ID NO: 14, and CDR3 V.sub.L comprising the sequence of SEQ ID NO: 15; and (ii) a chimeric co-stimulatory receptor (CCR) comprising e) a second co-stimulatory signaling region, wherein the second co-stimulatory signaling region is different from the first co-stimulatory 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, wherein the second target antigen is CD19 or another B cell lineage-specific target antigen.

2. The immuno-responsive cell of claim 1, wherein said first binding element comprises: (i) V.sub.H comprising a variant of the sequence of SEQ ID NO: 16, wherein the variant has a G to A or Y to A single-amino acid mutation in CDR3 V.sub.H region of SEQ ID NO: 16 to comprise one of the sequences selected from SEQ ID NOs: 20-26, and V.sub.L comprising the sequence of SEQ ID NO: 17; or (ii) a single-chain variable fragment (scFv) comprising a variant of one of the sequences selected from SEQ ID NOs: 18-19, wherein the variant has a G to A or Y to A single amino-acid mutation in CDR3 V.sub.H region to comprise one of the sequences selected from SEQ ID NOs: 20-26.

3. The immuno-responsive cell of claim 1, wherein said second target antigen comprising said second epitope is a B cell lineage-specific antigen selected from the group consisting of CD19, CD20, CD22, CD23, CD79a and CD79b.

4. The immuno-responsive cell of claim 1, wherein said second binding element comprises: CDR1 V.sub.H comprising the sequence of SEQ ID NO: 10, CDR2 V.sub.H comprising the sequence of SEQ ID NO: 11, CDR3 V.sub.H comprising the sequence of SEQ ID NO: 12, CDR1 V.sub.L comprising the sequence of SEQ ID NO: 13, CDR2 V.sub.L comprising the sequence of SEQ ID NO: 14, and CDR3 V.sub.L comprising the sequence of SEQ ID NO: 15.

5. The immuno-responsive cell of claim 4, wherein said second binding element comprises (i) the V.sub.H region having the sequence of SEQ ID NO: 16 and V.sub.L region having the sequence of SEQ ID NO: 17 or (ii) the single-chain variable fragment (scFv) having the sequence of SEQ ID NO: 18 or 19.

6. The immuno-responsive cell of claim 1, wherein said second binding element comprises: CDR1 V.sub.H comprising the sequence of SEQ ID NO: 27, CDR2 V.sub.H comprising the sequence of SEQ ID NO: 28, CDR3 V.sub.H comprising the sequence of SEQ ID NO: 29, CDR1 V.sub.L comprising the sequence of SEQ ID NO: 30, CDR2 V.sub.L comprising the sequence of SEQ ID NO: 31, and CDR3 V.sub.L comprising the sequence of SEQ ID NO: 32.

7. The immuno-responsive cell of claim 6, wherein said second binding element comprises (i) the V.sub.H region with the sequence of SEQ ID NO: 33 and V.sub.L region with the sequence of SEQ ID NO: 34 or (ii) the single-chain variable fragment (scFv) having the sequence of SEQ ID NO: 35 or 36.

8. The immuno-responsive cell of claim 1, wherein said second binding element comprises: CDR1 V.sub.H comprising the sequence of SEQ ID NO: 37, CDR2 V.sub.H comprising the sequence of SEQ ID NO: 38, CDR3 V.sub.H comprising the sequence of SEQ ID NO: 39, CDR1 V.sub.L comprising the sequence of SEQ ID NO: 40, CDR2 V.sub.L comprising the sequence of SEQ ID NO: 41, and CDR3 V.sub.L comprising the sequence of SEQ ID NO: 42.

9. The immuno-responsive cell of claim 8, wherein said second binding element comprises (i) the V.sub.H region with the sequence of SEQ ID NO: 43 and Vi region with the sequence of SEQ ID NO: 44 or (ii) the single-chain variable fragment (scFv) having the sequence of SEQ ID NO: 45 or 46.

10. The immuno-responsive cell of claim 1, wherein the 2.sup.nd 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.

11. The immuno-responsive cell of claim 1, wherein the 2.sup.nd generation CAR comprises the sequence of SEQ ID NO: 63 and the CCR comprises the sequence of SEQ ID NO: 65 or 66.

12. The immuno-responsive cell of claim 1, wherein said immuno-responsive cell is an T cell, T cell, or a Natural Killer (NK) cell.

13. A polynucleotide or set of polynucleotides comprising: (i) a first nucleic acid encoding a 2.sup.nd generation chimeric antigen receptor (CAR) comprising a) a signaling region; b) a first co-stimulatory signaling region; c) a first transmembrane domain; and d) a first binding element that specifically interacts with a first epitope on a CD19 target antigen, wherein said first binding element comprises: CDR1 V.sub.H comprising the sequence of SEO ID NO: 10, CDR2 V.sub.H comprising the sequence of SEQ ID NO: 11, CDR3 V.sub.H comprising one of the sequences selected from SEO ID NOs: 20-26, CDR1 V.sub.L comprising the sequence of SEQ ID NO: 13, CDR2 V.sub.L comprising the sequence of SEQ ID NO: 14, and CDR3 V.sub.L comprising the sequence of SEQ ID NO: 15; and (i) a second nucleic acid encoding a chimeric co-stimulatory receptor (CCR) comprising e) a second co-stimulatory signaling region, wherein the second co-stimulatory signaling region is different from the first co-stimulatory 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, wherein the second target antigen is CD19 or another B cell lineage specific target antigen.

14. A method of preparing a modified immuno-responsive cell, said method comprising transfecting or transducing said polynucleotide or set of polynucleotides of claim 13 into an immuno-responsive cell.

15. A method for directing a T cell-mediated immune response to a target cell in a patient in need thereof, said method comprising administering to the patient the immuno-responsive cell of claim 1, wherein the target cell expresses CD19.

16. A method of treating cancer, said method comprising administering to the patient an effective amount of the immuno-responsive cell of claim 1, wherein the patient's cancer expresses CD19.

Description

3. BRIEF DESCRIPTION OF THE DRAWINGS

(1) The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of various embodiments of the invention.

(2) FIGS. 1A and 1BFlow Cytometric Analysis of Malignant B Cell Lines

(3) FIG. 1A demonstrates the expression profile of CD19 and CD20 on a panel of human lymphoma and leukemia cell lines (Daudi, Nalm-6 and Raji). In accordance with published data, the analysis confirmed that CD19 was detectable on the cell surface of these tumor cells at a high level and CD20 was detectable on both Raji and Daudi cell lines. The Nalm-6 cell line had no detectable expression of CD20.

(4) To enable bioluminescence and fluorescence imaging of cells and derived xenografts, tumor cell lines were transduced with the LT retroviral vector that encodes for both firefly luciferase enzyme and the red fluorescent protein (RFP), tandem dimer (td) Tomato. Expression of RFP was confirmed using flow cytometry, as indicated in FIG. 1B.

(5) FIG. 2Design and Construction of CARs and pCARs

(6) FIGS. 2A-2E provide schematic diagrams showing salient features of certain 2.sup.nd generation CAR and pCAR constructs used in the experiments described herein. The cell membrane is shown as parallel horizontal lines, with the extracellular domains depicted above the membrane and intracellular domains shown below the membrane. F-2 is a 2.sup.nd generation CAR similar to that originally described in Kochenderfer et al., J. Immunother. 32:689-702 (2009), incorporated herein by reference in its entirety. It comprises, from C-terminus to N-terminus (intracellular to extracellular), a CD3z signaling region, CD28 co-stimulatory and transmembrane domains, a CD28 hinge/spacer domain that contains an embedded myc epitope tag and a human CD19-targeting FMC63 single chain antibody (scFv) domain. Cells transduced with F-2 alone are standard 2.sup.nd generation CAR-T cells and are used for comparative purposes.

(7) The CDR3 region of the V.sub.H domain within the FMC63 scFv was identified usingabysis.org. To generate variants with an altered ability to bind CD19, an alanine (A) residue was substituted for the first or second glycine (G01, G02) or alternatively for the first, second, third, fourth or fifth tyrosine (Y01-Y05) within CDR3 of the said V.sub.H domain, as illustrated in FIGS. 2A and 2B. These modified CD19-specific 2.sup.nd generation CARs are designated G01, G02, Y01, Y02, Y03, Y04 and Y05 respectively.

(8) 1-2 is a 2.sup.nd generation CAR in which targeting is achieved using the 1F5 scFv, as described in Budde et al., PLOS One 8 (12): e82742 (2013) and incorporated herein by reference in its entirety. It comprises, from C-terminus to N-terminus (intracellular to extracellular), a CD3z signaling region, CD28 co-stimulatory and transmembrane domains, a CD28 hinge/spacer domain that contains an embedded myc epitope tag and a human CD20-targeting 1F5 single chain antibody (scFv) domain. Cells transduced with 1-2 alone are standard 2.sup.nd generation CAR-T cells and are used for comparative purposes.

(9) R-2) is a 2nd generation CAR in which targeting is achieved using the RFB4 scFv, as described in James et al., J. Immunol. 180 (10): 7028-38 (2008) and incorporated herein by reference in its entirety. It comprises, from C-terminus to N-terminus (intracellular to extracellular), a CD3z signaling region, CD28 co-stimulatory and transmembrane domains, a CD28 hinge/spacer domain that contains an embedded myc epitope tag and a human CD22-targeting RFB4 single chain antibody (scFv) domain. Cells transduced with R-2 alone are standard 2.sup.nd generation CAR-T cells and are used for comparative purposes.

(10) A series of B cell targeted pCARs (FIGS. 2C, 2D and 2E) have been engineered using combinations of the aforementioned binding moieties. Nomenclature derives from an ordered abbreviation of the following elements: CCR binder, CCR signaling domain/CAR binder. For example, 1BB/F is a pCAR in which a CCR targeted by a 1F5 scFv and containing a 4-1BB endodomain is co-expressed with an FMC63 scFv-targeted CD28-containing 2.sup.nd generation CAR. FBB/Y01 is a pCAR in which an FMC63 scFv-targeted CCR is co-expressed with a CD28-containing 2.sup.nd generation CAR that is targeted by a FMC63 (Y01) scFv. Similarly, FTr/Y05 is a control pCAR in which an FMC63 scFv-targeted CCR that has a Truncated signaling domain is co-expressed with a CD28-containing 2.sup.nd generation CAR that is targeted by an FMC63(Y05) scFv. Generic structure of B cell targeted pCARs and truncated controls is indicated in FIGS. 2C, 2D and 2E. FIG. 2F provides a schematic of the CD19-CD22 dual targeting pCARs RBB/F, RBB/Y05, and RBB/G02, as well as the 2.sup.nd generation CARs F-2 and R-2 that target CD19 and CD22, respectively. RBB/F is a pCAR with the RBB CCR (CD22-specific RFB4 scFv fused via a CD8a spacer and transmembrane domain to a 4-1BB signaling domain) and a CD19-specific CD28-containing 2.sup.nd generation CAR (F). RBB/Y05 is a pCAR in which an RFB4 scFv-targeted CCR is co-expressed with a CD28-containing 2.sup.nd generation CAR that is targeted by an FMC63(Y05) scFv. RBB/G02 is a pCAR in which an RFB4 scFv-targeted CCR is co-expressed with a CD28-containing 2.sup.nd generation CAR that is targeted by an FMC63(G02) scFv.

(11) FIG. 3Expression of CD19-Specific CARs in Human T Cells

(12) FIGS. 3A-3B show two representative examples in which CD19-specific 2.sup.nd generation CARs containing a mutated FMC63 scFv were expressed in human CAR T cells. Cell surface expression was detected using 9e10 antibody, which binds to a myc epitope tag (EQKLISEEDL) that had been inserted into the CD28 spacer domain of the CAR. The F-2 2.sup.nd generation CAR was expressed as a control.

(13) FIG. 4Expression of CD19-Specific pCARs in Human T Cells

(14) FIGS. 4A and 4B show representative examples in which CD19-specific pCARs were expressed in human T cells. In all CD19-specific pCARs, the FBB CCR (CD19-specific FMC63 scFv fused via a CD8 spacer and transmembrane domain to a 4-1BB signaling domain) is co-expressed with a CD19-specific CD28-containing 2.sup.nd generation CAR in which the FMC63 V.sub.H chain contains the indicated CDR3 mutation. The F-2 2.sup.nd generation CAR was expressed as a control here. Expression of CARs, or the CAR component of pCARs, was detected using 9e10 antibody which binds to a myc epitope tag (EQKLISEEDL), as explained above (FIG. 4A). Expression of the CCR component of pCARs was detected by intracellular staining using a FLAG epitope tag (DYKDDDDK)-specific antibody (FIG. 4B).

(15) FIG. 5Expression of pCARs that Co-Target CD19 and CD20 in Human T Cells

(16) FIG. 5 shows a representative example in which the 1BB/F pCAR was expressed in human T cells. In 1BB/F, the 1BB CCR (CD20-specific 1F5 scFv fused via a CD8a spacer and transmembrane domain to a 4-1BB signaling domain) is co-expressed with a CD19-specific CD28-containing 2.sup.nd generation CAR (F). In 1Tr/F, the 1BB CCR having a Truncated signaling domain is co-expressed with a CD19-specific CD28-containing 2.sup.nd generation CAR (F). 1-2 is a CD28-containing 2.sup.nd generation CAR control that binds to CD20. Both F-2 and 1-2 were expressed as controls here. Expression of CARs, or the CAR component of pCARs, was detected using 9e10 antibody, as explained above.

(17) FIG. 6Expression of pCAR that Co-Targets CD19 and CD22 in Human T Cells

(18) FIG. 6 shows a representative example in which the RBB/F pCAR was expressed in human T cells. In RBB/F, the RBB CCR (CD22-specific RFB4 scFv fused via a CD8a spacer and transmembrane domain to a 4-1BB signaling domain) is co-expressed with a CD19-specific CD28-containing 2.sup.nd generation CAR (F). R-2 is a CD28-containing 2.sup.nd generation CAR control that binds to CD22. Both F-2 and R-2 were expressed as controls. Expression of CARs, or the CAR component of pCARs, was detected using the 9e10 MYC epitope tag-specific antibody as explained above. Expression of the CCR component of pCARs was detected by intracellular staining using a FLAG epitope tag-specific antibody.

(19) FIG. 7Binding of CD19 to Parental and CDR3 V.sub.H mutated FMC63-Based 2.sup.nd Generation CARs

(20) FIGS. 7A-7D show the results of a representative experiment in which T cells that express F-2, G01, G02, Y01, Y02, Y03, Y04 or Y05 second generation CARs were incubated with two concentrations of a soluble CD19-Fc fusion protein0.5 g (FIG. 7A, FIG. 7C (right)) and 1.0 g (FIG. 7B, FIG. 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 FIG. 3A. Binding of the T cells to CD19-Fc as measured by flow cytometry is plotted in histogram form in FIG. 7C and numeralized as percent binding and mean fluorescence intensity (MFI) after incubation with CD19-Fc (FIG. 7D left and right respectively). Note the spectrum of binding efficiencies of the mutated CARs from low (e.g. G01, G02) to intermediate (e.g. Y04) to increased (e.g. Y05), when compared to F-2.

(21) FIG. 8Titration of Tumor Cell Killing by F-2 and Mutant Derivative 2.sup.nd Generation CAR T Cells

(22) FIGS. 8A-8D show three experiments that compare the cytotoxic activity of F-2 and mutant derivative 2.sup.nd generation CAR T cells against the malignant CD19-expressing B cell lymphoma cell lines, Nalm-6 (FIG. 8A and FIG. 8C) or Raji (FIG. 8B and FIG. 8D), making comparison with untransduced (UT) control T cells.

(23) FIG. 9In Vitro Cytokine Release (at 24 Hours)

(24) FIGS. 9A and 9B show pooled data indicating the release of IFN- (FIG. 9A) and IL-2 (FIG. 9B) by CD19-specific CAR-T cells when cultured with Nalm-6 cells. FIGS. 9C and 9D show pooled data indicating the release of IFN- (FIG. 9C) and IL-2 (FIG. 9D) by CD19-specific CAR-T cells when cultured with Raji cells. Comparison is made to the CD19-specific 2.sup.nd generation CAR, F-2.

(25) FIG. 10In Vitro Cytokine Release (at 24 Hours)

(26) FIGS. 10A and 10B show pooled data indicating the release of IFN- by CD19-specific CAR-T cells when cultured with Nalm-6 cells (FIG. 10A) or Raji cells (FIG. 10B). FIGS. 10C and 10D show pooled data indicating the release of IL-2 by CD19-specific CAR-T cells when cultured with Nalm-6 cells (FIG. 10C) or Raji cells (FIG. 10D).

(27) FIG. 11In Vitro Re-Stimulation Potential

(28) FIG. 11A show tumor cell killing activity of the 2.sup.nd generation CD19-specific CARs of FIG. 3A when iteratively re-stimulated by addition of Nalm-6 cells. FIGS. 11B and 11C shows IFN- (FIG. 11B) and IL-2 (FIG. 11C) production by the iteratively stimulated CAR T cells.

(29) FIG. 12Titration of Tumor Cell Killing by CAR and pCAR T Cells Targeted Against CD19

(30) FIG. 12 shows pooled data in which cytotoxic activity of CDR3 V.sub.H-mutated FMC63-based 2.sup.nd generation CAR T cells and pCAR T cells was titrated against Nalm-6 leukemic cells, making comparison with F-2 as control.

(31) FIG. 13In Vitro Re-Stimulation Potential of CD19-Specific pCAR T Cells

(32) FIG. 13A shows tumor cell killing activity of CD19-specific pCAR T cells when iteratively re-stimulated by co-culture with CD19-expressing LO68 tumor cells. FIGS. 13B and 13C respectively show IFN- and IL-2 production by the iteratively stimulated CAR-T and pCAR-T cells.

(33) FIG. 14In Vivo Anti-Tumor Activity of CD19-Specific CAR and pCAR T-Cells (NSG Mice)

(34) FIGS. 14A and 14B show the results of therapeutic evaluation of CD19-specific CAR or pCAR-T cells against an established luciferase-expressing Nalm-6 leukemic xenograft in NSG mice. FIG. 14A shows total flux emission from mice treated with CD19-specific CAR or pCAR-T cells and FIG. 14B shows percentage weight change of mice before and after the treatment.

(35) FIG. 15In Vitro Re-Stimulation Potential and Cytotoxicity of pCAR T Cells that Co-Target CD19 and CD20

(36) FIG. 15A shows number of restimulation cycles completed following co-cultivation of transduced 1BB/F pCAR T cells which co-target CD19 (CAR) and CD20 (CCR) with LO68 tumor cells that co-express both CD19 and CD20. Every 72 hours, T cells were transferred to a fresh monolayer of LO68 cells. FIG. 15B shows the pooled data of tumor cell killing activity of 1BB/F pCAR T cells co-cultivated with LO68 tumor cells. FIG. 15C shows the amount of IFN- (left panel) and IL-2 (right panel) released following each stimulation cycle. FIG. 15D shows pooled experiments in which cytotoxic activity of CAR and pCAR T cells was titrated using LO68 tumor cells that co-express both CD19 and CD20. FIGS. 15E-15F show the amount of IFN- (FIG. 15E) and IL-2 (FIG. 15F) released following 24 hours of co-cultivation at effector to target ratios of 1:1 and 1:4.

(37) FIG. 16Titration of Tumor Cell Killing by pCAR T Cells Co-Targeted Against CD19 and CD22

(38) FIG. 16A-16B show the cytotoxic activity of RBB/F, RBB/G02 and RBB/Y05 pCAR T cells that co-target CD19 (CAR) and CD22 (CCR) against Nalm-6 leukemic cells. Comparison is made to the 2.sup.nd generation CD19 and CD22 targeting second generation CARs, F-2 and R-2 respectively.

(39) FIG. 17Binding Affinity of CD19-Specific CAR-T Cells

(40) FIGS. 17A-17D show the binding affinity of CD19-specific CAR-T cells to CD19.sup.+ LO68 tumor cells in a z-Movi microfluidic chip. T-cells were engineered to express the CD19-specific F-2 CAR or derivates that contain a mutation in V.sub.H CDR3 region. After flow sorting to purity, CAR T-cells were incubated on a monolayer of CD19.sup.+ LO68 tumor cells in a z-Movi microfluidic chip. Increasing fluidic force was applied and the percentage of bound T-cells was determined (median, n=3) (FIG. 17A). FIG. 17B shows the overall mean percentage of bound T cells after applying minimal force to detach a mean of 90% untransduced T cells. FIG. 17C is a bar plot representing the avidity score or the mean rForce required to detach T cells from the monolayer of CD19.sup.+ LO68 tumor cells compared to untransduced T cells (the black dotted line represents the avidity score of untransduced T cells; the red dotted line represents the avidity score of F-2 CAR T cells. The dot plot in FIG. 17D shows the rForce per cell required to detach from the tumor cell monolayer where each dot represents a single cell.

(41) FIG. 18Expression of CD19-Specific CAR or pCARs

(42) FIGS. 18A-18B show the expression of CD19-specific CAR or pCARs in human T cells analyzed by flow cytometry. Human T-cells were engineered by retroviral transduction to express the indicated CAR or pCAR. T-cells were incubated with antibodies directed against both MYC (CAR) and FLAG (CCR) epitope tags and then analyzed by flow cytometry. Data are representative of three independent replicate experiments (FIG. 18B) or greater than seven independent replicate experiments (FIG. 18A).

(43) FIG. 19Experimental Design of In Vivo Anti-Tumor Activity Testing (NSG Mice)

(44) FIG. 19 shows the experimental design for testing the engineered CD19-specific CAR-T or pCAR-T cells in vivo. RFP/ffLuc.sup.+ Nalm6 cells (510.sup.5 cells) were injected i.v. in NSG mice. Mice were sorted into groups of equal disease burden using BLI. On day 5, 510.sup.5 of the indicated CAR or pCAR T-cells were administered i.v. Disease burden was monitored by BLI from day 8.

(45) FIGS. 20A and 20BIn Vivo Anti-Tumor Activity of CD19-Specific CAR-T or pCAR-T Cells

(46) FIGS. 20A and 20B show the anti-tumor activity of CD19-specific CAR-T or pCAR-T cells in NSG mice bearing established luciferase-expressing Nalm6 leukemic xenografts. FIG. 20A shows the total flux emission (e.g. leukemic burden) of mice treated with indicated CD19-specific CAR-T or pCAR-T cells. FIG. 20B shows the p values of selected pCAR vs its corresponding 2G CAR with a mutation in V.sub.H CDR3 region and the p values of 2G CAR F-2 vs the indicated 2G CAR with a mutation in V.sub.H CDR3 region.

(47) FIGS. 21A, 21B, 21C, and 21DSurvival of NSG Mice Treated with CD19-Specific CAR-T or pCAR-T Cells

(48) FIGS. 21A, 21B, 21C, and 21D show the survival curves of PBS, CD19 specific CAR-T cell- and CD19 specific pCAR-T cell-treated groups. FIG. 21A shows the survival curves of PBS, F-2 CAR-T cells, Y04 CAR-T cells, and FBB/Y04 pCAR-T cells-treated groups. FIG. 21B shows the survival curves of PBS, F-2 CAR-T cells, Y05 CAR-T cells, and FBB/Y05 pCAR-T cells treated-groups. FIG. 21C shows the survival curves of PBS, F-2 CAR-T cells, G02 CAR-T cells, and FBB/G02 pCAR-T cells-treated groups. FIG. 21D shows the median survival of each treatment group shown in FIGS. 21A-21C.

4. DETAILED DESCRIPTION

(49) The details of various embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and the drawings, and from the claims.

4.1. Definitions

(50) Unless otherwise defined herein, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. As used herein, the following terms have the meanings ascribed to them below.

(51) As used herein, the term variant refers to a polypeptide sequence which is a naturally occurring polymorphic form of the basic sequence as well as synthetic variants, in which one or more amino acids within the chain are inserted, removed or replaced. However, the variant produces a biological effect which is similar to that of the basic sequence. For example, a variant of the intracellular domain of human CD3 zeta chain will act in a manner similar to that of the intracellular domain of human CD3 zeta chain. Amino acid substitutions may be regarded as conservative where an amino acid is replaced with a different amino acid in the same class with broadly similar properties. Non-conservative substitutions are where amino acids are replaced with amino acids of a different type or class.

(52) As is well known to those skilled in the art, altering the primary structure of a peptide by a conservative substitution may not significantly alter the activity of that peptide because the side-chain of the amino acid which is inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted out. This is so even when the substitution is in a region which is critical in determining the peptide's conformation. Non-conservative substitutions may also be possible provided that these do not interrupt the function of the polypeptide as described above. Broadly speaking, fewer non-conservative substitutions will be possible without altering the biological activity of the polypeptides. In general, variants will have amino acid sequences that will be at least 70%, for instance at least 71%, 75%, 79%, 81%, 84%, 87%, 90%, 93%, 95%, 96% or 98% identical to the basic sequence, for example SEQ ID NO: 1 or SEQ ID NO: 2. Identity in this context may be determined using the BLASTP computer program with SEQ ID NO: 1, SEQ ID NO: 2, or a fragment thereof, in particular a fragment as described below, as the base sequence. The BLAST software is publicly available.

(53) As used herein, the term antigen refers to any member of a specific binding pair that will bind to the binding elements. The term includes receptors on target cells.

(54) As used herein and with regard to the binding element to a target molecule, the terms bind, specific binding, specifically binds to, specifically interacts with, specific for, selectively binds, selectively interacts with, and selective for a particular antigen (e.g., a polypeptide target) or an epitope on a particular antigen mean binding that is measurably different from a non-specific or non-selective 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 epitope recognized on the target molecule.

(55) The term pCAR as used herein refers to a parallel chimeric antigen receptor which comprises the combination of a 2.sup.nd generation chimeric antigen receptor (CAR) and, in parallel, a chimeric co-stimulatory receptor (CCR). pCAR has been described in WO2017/021701, which is incorporated by reference in its entirety herein.

4.2. Other Interpretational Conventions

(56) In the claims, articles such as a, an, and the may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include or between one or more members of a group are considered satisfied if 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 to the contrary or otherwise evident 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.

(57) It is also noted that the term comprising is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term comprising is used herein, the term consisting of is thus also encompassed and disclosed.

(58) Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

(59) All cited sources, for example, references, publications, databases, database entries, and art cited herein, are incorporated into this application by reference, even if not expressly stated in the citation. In case of conflicting statements of a cited source and the instant application, the statement in the instant application shall control.

(60) Section and table headings are not intended to be limiting.

4.3. Immuno-Responsive Cells

(61) In a first aspect, immuno-responsive cells are provided. The immuno-responsive cells express a pCAR which comprises the combination of a 2.sup.nd generation chimeric antigen receptor (CAR) and, in parallel, a chimeric co-stimulatory receptor (CCR).

(62) The CAR comprises, from C-terminus to N-terminus (from intracellular to extracellular as expressed within the immuno-responsive cell), (a) a signaling region; (b) a first co-stimulatory signaling region; (c) a first transmembrane domain; and (d) a first binding element that specifically interacts with a first epitope on a CD19 target antigen.

(63) The CCR comprises, from C-terminus to N-terminus (from intracellular to extracellular as expressed within the immuno-responsive cell), (e) a second co-stimulatory signaling region which is different from that of the first co-stimulatory 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 can be identical to or distinct from the first epitope. The second antigen can be CD19 or an alternative B cell lineage-specific antigen.

(64) 4.3.1. Cells

(65) In typical embodiments, the immuno-responsive cells are T cells.

(66) In certain embodiments, the immuno-responsive cells are T cells. In particular embodiments, the immuno-responsive cells are cytotoxic T cells. In particular embodiments, the immuno-responsive cells are helper T cells. In particular embodiments, the immuno-responsive cells are regulatory T cells (Tregs).

(67) In certain embodiments, the immuno-responsive cells are T cells. In particular embodiments, the immuno-responsive cells are V2.sup.+ T cells. In particular embodiments, the immuno-responsive cells are V2.sup. T cells. In specific embodiments, the V2.sup. T cells are V1.sup.+ cells.

(68) In certain embodiments, the immuno-responsive cells are Natural Killer (NK) cells.

(69) In some embodiments, the immuno-responsive cell expresses no additional exogenous proteins. In other embodiments, the immuno-responsive cell is engineered to express additional exogenous proteins, such as a cytokine, receptor or derivative thereof.

(70) In some embodiments, the immuno-responsive cells are obtained from peripheral blood mononuclear cells (PBMCs). In some embodiments, the immuno-responsive cells are obtained from tumors. In particular embodiments, the immuno-responsive cells obtained from tumors are tumor infiltrating lymphocytes (TILs). In specific embodiments, the TILs are T cells. In other specific embodiments, the TILs are T cells, and in particular, V2.sup.+ or V2.sup. T cells.

4.4. Chimeric Antigen Receptor Structure

(71) 4.4.1. Signaling Region

(72) The CAR construct comprises a signaling region at its C-terminus. In some embodiments, the signaling region comprises an Immune-receptor-Tyrosine-based-Activation-Motif (ITAM), as reviewed for example by Love et al., Cold Spring Harbor Perspect. Biol. 2(6)1 a002485 (2010). In some embodiments, the signaling region comprises the intracellular domain of human CD3 zeta chain, as described for example in U.S. Pat. No. 7,446,190, incorporated by reference herein, or a variant thereof. In particular embodiments, the signaling region comprises the domain which spans amino acid residues 52-163 of the full-length human CD3 zeta chain. The CD3 zeta chain has a number of known polymorphic forms, (e.g. Sequence ID: gb|AAF34793.1 and gb|AAA60394.1), all of which are useful herein, and shown respectively as SEQ ID NO: 1 and 2:

(73) TABLE-US-00001 (SEQIDNO:1) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR; (SEQIDNO:2) RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR.

(74) Alternative signaling regions to the CD3 zeta domain include other ITAM containing units such as Fcr1, CD3, DAP12 and multi-ITAM. See 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), Tpfer et al. DAP12-based activating chimeric antigen receptor for NK cell tumor immunotherapy. J Immunol 194:3201-3212 (2015); and James J R, 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.

(75) 4.4.2. Co-Stimulatory Signaling Region

(76) In the CAR, the co-stimulatory signaling region is suitably located between the signaling region and transmembrane domain, and remote from the binding element.

(77) In the CCR, the co-stimulatory signaling region is suitably located adjacent the transmembrane domain and remote from the binding element.

(78) Suitable co-stimulatory signaling regions are well known in the art, and include the co-stimulatory signaling regions of members of the B7/CD28 family such as 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 such as LILRA3, LILRA4, LILRB1, LILRB2, LILRB3 or LILRB4; or tumor necrosis factor (TNF) superfamily members such as 4-1BB, BAFF, BAFF R, CD27, CD30, CD40, DR3, GITR, HVEM, LIGHT, Lymphotoxin-alpha, OX40, RELT, TACI, TLIA, TNF-alpha, or TNF RII; or members of the SLAM family such as 2B4, BLAME, CD2, CD2F-10, CD48, CD8, CD84, CD229, CRACC, NTB-A or SLAM; or members of the TIM family such as TIM-1, TIM-3 or TIM-4; or other co-stimulatory molecules such as CD7, CD96, CD160, CD200, CD300a, CRTAM, DAP12, Dectin-1, DPPIV, EphB6, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7/LPAM-1, LAG-3 or TSLP R. See Mondino A et al., Surface proteins involved in T cell costimulation, J Leukoc Biol. 55:805-815 (1994); Thompson C B, 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 L L, T-cell costimulation via CD28-CD80/CD86 and CD40-CD40 ligand interactions, Res Immunol. 146:171-176 (1995); Rhodes D A 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 R J et al., Annu Rev Immunol., The B7 family revisited, 23:515-548 (2005); Flem-Karlsen K et al., B7-H3 in cancerbeyond immune regulation, Trends Cancer. 4:401-404 (2018); Flies D B 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 H A 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 X et al., Inhibitory leukocyte immunoglobulin-like receptors: Immune checkpoint proteins and tumor sustaining factors, Cell Cycle. 15:25-40 (2016); Watts T H, TNF/TNFR family members in costimulation of T cell responses, Annu Rev Immunol. 23:23-68 (2005); Bryceson Y T et al., Activation, coactivation, and costimulation of resting human natural killer cells, Immunol Rev. 214:73-91 (2006); Sharpe A H, Analysis of lymphocyte costimulation in vivo using transgenic and knockout mice, Curr Opin Immunol. 7:389-395 (1995); Wingren A G 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 entireties.

(79) The co-stimulatory signaling regions may be selected depending upon the particular use intended for the immuno-responsive cell. In particular, the co-stimulatory signaling regions can be selected to work additively or synergistically together. In some embodiments, the co-stimulatory signaling regions are selected from the co-stimulatory signaling regions of CD28, CD27, ICOS, 4-1BB, OX40, CD30, GITR, HVEM, DR3 and CD40.

(80) In a particular embodiment, one co-stimulatory signaling region of the pCAR is the co-stimulatory signaling region of CD28 and the other is the co-stimulatory signaling region of 4-1BB. In a specific embodiment, the co-stimulatory signaling region of the CAR is the co-stimulatory signaling region of CD28 and the co-stimulatory signaling region of the CCR is the co-stimulatory signaling region of 4-1BB.

(81) In a particular embodiment, one co-stimulatory signaling region of the pCAR is the co-stimulatory signaling region of CD28 and the other is the co-stimulatory signaling region of CD27. In a specific embodiment, the co-stimulatory signaling region of the CAR is the co-stimulatory signaling region of CD28 and the co-stimulatory signaling region of the CCR is the co-stimulatory signaling region of CD27.

(82) 4.4.3. Transmembrane Domains

(83) The transmembrane domains for the CAR and CCR constructs may be the same or different. In currently preferred embodiments, when the CAR and CCR constructs are expressed from a single vector, the transmembrane domains of the CAR and CCR are different, to ensure separation of the constructs on the surface of the cell. Selection of different transmembrane domains may also enhance stability of the expression vector since inclusion of a direct repeat nucleic acid sequence in the viral vector renders it prone to rearrangement, with deletion of sequences between the direct repeats. In embodiments in which the transmembrane domains of the CAR and CCR of the pCAR are chosen to be the same, this risk can be reduced by modifying or wobbling the codons selected to encode the same protein sequence.

(84) Suitable transmembrane domains are known in the art and include for example, the transmembrane domains of CD8, CD28, CD4 or CD3z. Selection of CD3z as transmembrane domain may lead to the association of the CAR or CCR with other elements of TCR/CD3 complex. This association may recruit more ITAMs but may also lead to the competition between the CAR/CCR and the endogenous TCR/CD3.

(85) In certain embodiments, one transmembrane domain of the pCAR is the transmembrane domain of CD28 and the other is the transmembrane domain of CD8a. In a particular pCAR embodiment, the transmembrane domain of the CAR is the transmembrane domain of CD28 and the transmembrane domain of the CCR is the transmembrane domain of CD8a.

(86) 4.4.4. Co-Stimulatory Signal Domain and Transmembrane Domain

(87) In embodiments in which the co-stimulatory signaling region of the CAR or CCR is, or comprises, the co-stimulatory signaling region of CD28, the CD28 transmembrane domain represents a suitable, often preferred, option for the transmembrane domain. The full length CD28 protein is a 220 amino acid protein of SEQ ID NO: 3, where the transmembrane domain is shown in bold type:

(88) TABLE-US-00002 (SEQIDNO:3) MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSR EFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFY LQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFP GPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTP RRPGPTRKHYQPYAPPRDFAAYRS.

(89) In some embodiments, one of the co-stimulatory signaling regions is based upon the hinge region and suitably also the transmembrane domain and endodomain of CD28. In some embodiments, the co-stimulatory signaling region comprises amino acids 114-220 of SEQ ID NO: 3, shown below as SEQ ID NO: 4:

(90) TABLE-US-00003 (SEQIDNO:4) IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGV LACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAP PRDFAAYRS.

(91) In a particular embodiment, one of the co-stimulatory signaling regions is a modified form of SEQ ID NO: 4 which includes a c-myc tag of SEQ ID NO: 5:

(92) TABLE-US-00004 (SEQIDNO:5) EQKLISEEDL.

(93) The c-myc tag may be added to the co-stimulatory signaling region by insertion into the ectodomain or by replacement of a region in the ectodomain, which is therefore within the region of amino acids 1-152 of SEQ ID NO: 3.

(94) In a particularly preferred embodiment, the c-myc tag replaces MYPPPY motif in the CD28 sequence. This motif represents a potentially hazardous sequence. It is responsible for interactions between CD28 and its natural ligands, CD80 and CD86, so that it provides potential for off-target toxicity when CAR-T cells or pCAR-T cells encounter a target cell that expresses either of these ligands. By replacement of this motif with a tag sequence as described above, the potential for unwanted side-effects is reduced. Thus, in a particular embodiment, the co-stimulatory signaling region of the CAR construct comprises SEQ ID NO: 6:

(95) TABLE-US-00005 (SEQIDNO:6) IEVEQKLISEEDLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVV VGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQ PYAPPRDFAAYRS.

(96) Furthermore, the inclusion of a c-myc epitope facilitates detection of the pCAR-T cells using a monoclonal antibody to the c-myc epitope. This is very useful since flow cytometric detection had proven unreliable when using some available antibodies.

(97) In addition, the provision of a c-myc epitope tag could facilitate the antigen-independent expansion of targeted CAR-T cells, for example by cross-linking of the CAR using the appropriate monoclonal antibody, either in solution or immobilized onto a solid phase (e.g., a bag).

(98) Moreover, expression of the epitope for the anti-human c-myc antibody, 9e10, within the variable region of a 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, the provision of such epitope tags could also be used as a suicide system, whereby an antibody could be used to deplete pCAR-T cells in vivo in the event of toxicity.

(99) In some embodiments, one of the co-stimulatory signaling regions is based upon the endodomain of 4-1BB. In some embodiments, the co-stimulatory signaling region comprises amino acids 214-255 of 4-1BB shown below as SEQ ID NO: 7:

(100) TABLE-US-00006 (SEQIDNO:7) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

(101) In a particular embodiment, one of the co-stimulatory signaling regions is a modified form of SEQ ID NO: 7 which includes a FLAG epitope tag of SEQ ID NO: 8:

(102) TABLE-US-00007 (SEQIDNO:8) DYKDDDDK.

(103) In a particularly preferred embodiment, the FLAG epitope tag is appended to the C-terminus of the 4-1BB endodomain. Thus, in a particular embodiment, the co-stimulatory signaling region of the CCR comprises SEQ ID NO: 9:

(104) TABLE-US-00008 (SEQIDNO:9) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELDYKDDDDK.
4.4.5. Chimeric Antigen Receptor Binding Elements

(105) The binding elements of the CAR and CCR constructs of the pCAR respectively bind a first epitope and a second epitope which may be identical or distinct.

(106) In some embodiments, the binding elements of the CAR and CCR constructs are identical. More commonly however, these binding elements are different from one another.

(107) In various embodiments, the binding elements of the CAR and CCR specifically bind to a first epitope and 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 in which 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 affinity may be employed in order to achieve an optimal balance of signaling by the CAR and CCR components of the pCAR.

(108) In various embodiments, the binding elements of the CAR and CCR components of the pCAR bind to different antigens. In certain embodiments, the antigens are different but may be associated with the same disease, such as the same specific cancer derived from the B cell lineage.

(109) In a preferred embodiment, the CAR binds to CD19 while the CCR binds either to CD19 or to another B cell lineage-specific marker. Examples of the latter include, but are not restricted to CD20, CD22, CD23, CD79a and CD79b.

(110) Thus, suitable binding elements may be any element which provides the pCAR with the ability to recognize a target of interest. The target to which the pCARs of the invention are directed can be any target of clinical interest to which it would be desirable to direct a T cell response.

(111) In various embodiments, the binding elements used in the CARs and CCRs of the pCARs described herein are antigen binding sites (ABS) of antibodies. In typical embodiments, the ABS used as the binding element is formatted into an scFv or is a single domain antibody from a camelid, human or other species.

(112) Alternatively, a binding element of a pCAR may comprise ligand(s) that bind to a surface protein of interest.

(113) Alternatively, a binding element of a pCAR may comprise peptide(s) that bind to a surface protein of interest.

(114) In some embodiments, the binding element is associated with a leader sequence which facilitates expression on the cell surface. Many leader sequences are known in the art, and these include the macrophage colony stimulating factor receptor (FMS) leader sequence, the CD8a leader sequence or the CD124 leader sequence.

4.5. Parallel CARs Targeted Against B Cell Antigens

(115) 4.5.1. Binding Elements for Use in pCARs

(116) In particular embodiments, the binding element of CAR or the binding element of CCR specifically interacts with an 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 has also been implicated in autoimmune diseases and so may be a target in the treatment of such conditions.

(117) 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 epitope on the CD19 target antigen.

(118) In currently preferred embodiments, the CAR and/or the CCR binding element specifically interacts with a first epitope on the CD19 target antigen. In some embodiments, the CAR or the CCR binding element comprises the antigen binding site of the FMC63 antibody. In certain embodiments, the CAR or the CCR binding element comprises the CDRs of the FMC63 antibody. The CDR sequences of the FMC63 antibody were determined using abysis.org and are shown as SEQ ID NO: 10-15 below.

(119) TABLE-US-00009 V.sub.HCDR1 (SEQIDNO:10) GVSLPDY. V.sub.HCDR2 (SEQIDNO:11) WGSET. V.sub.HCDR3 (SEQIDNO:12) HYYYGGSYAMDY. V.sub.LCDR1 (SEQIDNO:13) RASQDISKYLN. V.sub.LCDR2 (SEQIDNO:14) HTSRLHS. V.sub.LCDR3 (SEQIDNO:15) QQGNTLPYT.

(120) In certain embodiments, the CAR or the CCR binding element comprises the V.sub.H and V.sub.L domains of the FMC63 antibody. The V.sub.H and V.sub.L domain sequences of the FMC63 antibody are shown below as SEQ ID NO: 16-17.

(121) TABLE-US-00010 V.sub.H: (SEQIDNO:16) EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVI WGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYG GSYAMDYWGQGTSVTVSS. V.sub.L: (SEQIDNO:17) DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHT SRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGT KLEIT.

(122) In particularly preferred embodiments, the CAR or the CCR binding element comprises the antigen binding site of the FMC63 antibody formatted as an scFv, either arranged as V.sub.H-linker-V.sub.L or as V.sub.L-linker-V.sub.H. These sequences are presented as SEQ ID NO: 18 and 19 below. In each case, the linker sequence between the V.sub.H and V.sub.L domains has been underlined and italicized.

(123) TABLE-US-00011 V.sub.H-V.sub.L: (SEQIDNO:18) EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVI WGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYG GSYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDR VTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGT DYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT. V.sub.L-V.sub.H: (SEQIDNO:19) DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHT SRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGT KLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD YGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKM NSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS.

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

(125) In particularly preferred embodiments, the CAR or the CCR binding element comprises the CDR3 region of a variant of an FMC63 scFv. Specifically, the variant includes a mutation within the FMC V.sub.H domain (SEQ ID NO: 12) in order to modify affinity of the scFv for CD19. Particularly preferred embodiments contain a substitution of alanine (A) for either tyrosine (Y) or glycine (G) within CDR3 of the V.sub.H domain. These variants are shown below as SEQ ID NO: 20-26.

(126) TABLE-US-00012 Y01 (SEQIDNO:20) HAYYGGSYAMDY. Y02 (SEQIDNO:21) HYAYGGSYAMDY. Y03 (SEQIDNO:22) HYYAGGSYAMDY. G01 (SEQIDNO:23) HYYYAGSYAMDY. G02 (SEQIDNO:24) HYYYGASYAMDY. Y04 (SEQIDNO:25) HYYYGGSAAMDY. Y05 (SEQIDNO:26) HYYAGGSYAMDA.

(127) In some embodiments, the CAR or the CCR binding element specifically interacts with an epitope on the CD20 antigen. In some embodiments, the CAR or the CCR binding element comprises the 1F5 antibody, which 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 and progressively increasing in concentration until maturity. In humans, CD20 is encoded by the MS4A1 gene. Antibodies which target CD20 are used in treatment of B cell lymphomas and leukemias, as well as in the treatment of autoimmune diseases such as arthritis, in particular 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 abysis.org and are shown below as SEQ ID NO: 27-32.

(128) TABLE-US-00013 V.sub.HCDR1 (SEQIDNO:27) GYTFTSY. V.sub.HCDR2 (SEQIDNO:28) YPGNGD. V.sub.HCDR3 (SEQIDNO:29) SHYGSNYVDYFDY. V.sub.LCDR1 (SEQIDNO:30) RASSSLSFMH. V.sub.LCDR2 (SEQIDNO:31) ATSNLAS. V.sub.LCDR3 (SEQIDNO:32) HQWSSNPLT.

(129) In certain embodiments, the CAR or the CCR binding element comprises the V.sub.H and V.sub.L domains of the 1F5 antibody. The V.sub.H and V.sub.L domain sequences of the 1F5 antibody are shown below as SEQ ID NO: 33-34.

(130) TABLE-US-00014 V.sub.H: (SEQIDNO:33) QVQLRQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAI YPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLISEDSAVYYCARSHYG SNYVDYFDYWGQGTLVTVSTG. V.sub.L: (SEQIDNO:34) QIVLSQSPAILSASPGEKVTMTCRASSSLSFMHWYQQKPGSSPKPWIYATS NLASGVPARFSGSGSGTSYSLTISRVEAEDAATYFCHQWSSNPLTFGAGTK VEIKRK.

(131) In particularly preferred embodiments, the CAR or the CCR binding element comprises the antigen binding site of the 1F5 antibody formatted as a scFv, either arranged as V.sub.H-linker-V.sub.L or V.sub.L-linker-V.sub.H. These sequences are presented as SEQ ID NO: 35 and 36 below. In each case, the linker sequence between the V.sub.H and V.sub.L domains has been underlined and italicized.

(132) TABLE-US-00015 V.sub.H-V.sub.L: (SEQIDNO:35) QVQLRQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAI YPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSHYG SNYVDYFDYWGQGTLVTVSTGGSTSGSGKPGSGEGSTKGQIVLSQSPAILS ASPGEKVTMTCRASSSLSFMHWYQQKPGSSPKPWIYATSNLASGVPARFSG SGSGTSYSLTISRVEAEDAATYFCHQWSSNPLTFGAGTKVEIKRK. V.sub.L-V.sub.H: (SEQIDNO:36) QIVLSQSPAILSASPGEKVTMTCRASSSLSFMHWYQQKPGSSPKPWIYATS NLASGVPARFSGSGSGTSYSLTISRVEAEDAATYFCHQWSSNPLTFGAGTK VEIKRKGSTSGSGKPGSGEGSTKGQVQLRQPGAELVKPGASVKMSCKASGY TFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSST AYMQLSSLTSEDSAVYYCARSHYGSNYVDYFDYWGQGTLVTVSTG.

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

(134) In some embodiments, the CAR or the CCR binding element specifically interacts with an epitope on the CD22 antigen. In some embodiments, the CAR or the CCR binding element is 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 that is expressed on the cells of 60% to 90% of B cell malignancies. It is not expressed on hematopoietic stem cells or on any other non-lymphoid hematopoietic or nonhematopoietic cells. The CDR sequences of the RFB4 antibody were determined using abysis.org and are shown below as SEQ ID NO: 37-42.

(135) TABLE-US-00016 V.sub.HCDR1 (SEQIDNO:37) GFAFSIY. V.sub.HCDR2 (SEQIDNO:38) SSGGGT. V.sub.HCDR3 (SEQIDNO:39) HSGYGSSYGVLFAY. V.sub.LCDR1 (SEQIDNO:40) RASQDISNYLN. V.sub.LCDR2 (SEQIDNO:41) YTSILHS. V.sub.LCDR3 (SEQIDNO:42) QQGNTLPWT.

(136) In certain embodiments, the CAR or the CCR binding element comprises the V.sub.H and V.sub.L domains of the RFB4 antibody. The V.sub.H and V.sub.L domain sequences of the RFB4 antibody are shown below as SEQ ID NO: 43-44:

(137) TABLE-US-00017 V.sub.H: (SEQIDNO:43) EVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQTPEKRLEWVAYI SSGGGTTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARHSGY GSSYGVLFAYWGQGTLVTVS. VL: (SEQIDNO:44) DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYT SILHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQQGNTLPWTFGGGT KLEIK.

(138) In particularly preferred embodiments, the CAR or the CCR binding element comprises the antigen binding site of the RFB4 antibody formatted as a scFv, either arranged as V.sub.H-linker-V.sub.L or V.sub.L-linker-V.sub.H. These sequences are presented as SEQ ID NO: 45 and 46 below. In each case, the linker sequence between the V.sub.H and V.sub.L domains has been underlined and italicized.

(139) TABLE-US-00018 V.sub.H-V.sub.L: (SEQIDNO:45) EVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQTPEKRLEWVAYI SSGGGTTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARHSGY GSSYGVLFAYWGQGTLVTVSGSTSGSGKPGSGEGSTKGDIQMTQTTSSLSA SLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSILHSGVPSRFSG SGSGTDYSLTISNLEQEDFATYFCQQGNTLPWTFGGGTKLEIK. V.sub.L-V.sub.H: (SEQIDNO:46) DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYT SILHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQQGNTLPWTFGGGT KLEIKGSTSGSGKPGSGEGSTKGEVQLVESGGGLVKPGGSLKLSCAASGFA FSIYDMSWVRQTPEKRLEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNTL YLQMSSLKSEDTAMYYCARHSGYGSSYGVLFAYWGQGTLVTVS.

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

(141) 4.5.2. Representative Examples of Sequences Encoding B Cell Specific pCARs

(142) Combinations of the aforementioned B cell antigen-specific binding elements have been used to engineer pCARs in which the CAR and CCR elements bind to an identical epitope within CD19, or to different epitopes found on CD19 and a second lineage-specific B cell antigen. Many additional moieties that bind specifically to CD19 and to other lineage-specific B cell antigens are known in the art, meaning that a large number of B cell specific pCARs could be engineered using similar methodologies. Consequently, the following pCAR examples are offered for illustrative purposes only and are not intended to limit the scope of the present invention. Nomenclature of pCARs derives from the following order: CCR binder, CCR signaling domain/CAR binder.

(143) The protein sequence of FBB/G01 pCAR is shown below as SEQ ID NO: 47. The FBB/G01 pCAR comprises: (i) a CCR comprising a linear fusion of the following elements: a macrophage colony-stimulating factor receptor leader peptide, FMC63 scFv binding domain (V.sub.L-V.sub.H order), CD8a spacer and transmembrane domain, a 4-1BB co-stimulatory endodomain, FLAG epitope tag (FBB) and (ii) a 2.sup.nd generation CAR comprising a linear fusion of the following elements: a CD8a leader peptide, a variant of FMC63 scFv in which the first glycine in V.sub.H CDR3 of FMC63 scFv has been substituted by alanine (G01; V.sub.L-V.sub.H order), CD28 spacer containing an embedded myc epitope tag, CD28 transmembrane and endodomain, CD3z endodomain.
The CCR and the CAR are linked by a furin cleavage site, Ser-Gly linker (SGSG), and P2A ribosomal skip peptide. Codon wobbling has been used to minimize direct repeats within the scFv modules. The V.sub.H and the V.sub.L domains of scFv sequences are underlined and in bold. Epitope tags are italicized.

(144) TABLE-US-00019 (SEQIDNO:47) MGPGVLLLLLVATAWHGQGGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNELQEDIATYFCQQGNTL PYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSL PDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQ TDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAPTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHKRGRK KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELDYKDDDDKRRKRSGSGATNF SLLKQAGDVEENPGPNMALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVT ISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISN LEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLV APSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTI IKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYAGSYAMDYWGQGTSVTVSSAAAIEV EQKLISEEDLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLL VTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSR SADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

(145) The protein sequence of FBB/G02 pCAR is shown below as SEQ ID NO: 48. The FBB/G02 pCAR comprises: (i) a CCR comprising a linear fusion of the following elements: a macrophage colony-stimulating factor receptor leader peptide, FMC63 scFv binding domain (V.sub.L-V.sub.H order), CD8a spacer and transmembrane domain, a 4-1BB co-stimulatory endodomain, FLAG epitope tag (FBB) and (ii) a 2.sup.nd generation CAR comprising a linear fusion of the following elements: a CD8a leader peptide, a variant of FMC63 scFv in which the second glycine in V.sub.H CDR3 of FMC63 scFv has been substituted by alanine (G02; V.sub.L-V.sub.H order), CD28 spacer containing an embedded myc epitope tag, CD28 transmembrane and endodomain, CD3z endodomain.
The CCR and the CAR are linked by a furin cleavage site, Ser-Gly linker (SGSG), and P2A ribosomal skip peptide. Codon wobbling has been used to minimize direct repeats within the scFv modules. The V.sub.H and the V.sub.L domains of scFv sequences are underlined and in bold. Epitope tags are italicized.

(146) TABLE-US-00020 (SEQIDNO:48) MGPGVLLLLLVATAWHGQGGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNELQEDIATYFCQQGNTL PYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSL PDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQ TDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAPTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHKRGRK KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELDYKDDDDKRRKRSGSGATNF SLLKQAGDVEENPGPNMALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVT ISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISN LEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLV APSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTI IKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYAGSYAMDYWGQGTSVTVSSAAAIEV EQKLISEEDLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLL VTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSR SADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

(147) The protein sequence of FBB/Y01 pCAR is shown below as SEQ ID NO: 49. The FBB/Y01 pCAR comprises: (i) a CCR comprising a linear fusion of the following elements: a macrophage colony-stimulating factor receptor leader peptide, FMC63 scFv binding domain (V.sub.L-V.sub.H order), CD8a spacer and transmembrane domain, a 4-1BB co-stimulatory endodomain, FLAG epitope tag (FBB) and (ii) a 2.sup.nd generation CAR comprising a linear fusion of the following elements: a CD8a leader peptide, a variant of FMC63 scFv in which the first tyrosine in V.sub.H CDR3 of FMC63 scFv has been substituted by alanine (Y01; V.sub.L-V.sub.H order), CD28 spacer containing an embedded myc epitope tag, CD28 transmembrane and endodomain, CD3z endodomain.
The CCR and the CAR are linked by a furin cleavage site, Ser-Gly linker (SGSG), and P2A ribosomal skip peptide. Codon wobbling has been used to minimize direct repeats within the scFv modules. The V.sub.H and the V.sub.L domains of scFv sequences are underlined and in bold. Epitope tags are italicized.

(148) TABLE-US-00021 (SEQIDNO:49) MGPGVLLLLLVATAWHGQGGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTL PYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSL PDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQ TDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAPTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHKRGRK KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELDYKDDDDKRRKRSGSGATNF SLLKQAGDVEENPGPNMALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVT ISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISN LEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLV APSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTI IKDNSKSQVFLKMNSLQTDDTAIYYCAKHAYYGGSYAMDYWGQGTSVTVSSAAAIEV EQKLISEEDLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLL VTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSR SADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

(149) The protein sequence of FBB/Y02 pCAR is shown below as SEQ ID NO: 50. The FBB/Y02 pCAR comprises: (i) a CCR comprising a linear fusion of the following elements: a macrophage colony-stimulating factor receptor leader peptide, FMC63 scFv binding domain (V.sub.L-V.sub.H order), CD8a spacer and transmembrane domain, a 4-1BB co-stimulatory endodomain, FLAG epitope tag (FBB) and (ii) a 2.sup.nd generation CAR comprising a linear fusion of the following elements: a CD8a leader peptide, a variant of FMC63 scFv in which the second tyrosine in V.sub.H CDR3 of FMC63 scFv has been substituted by alanine (Y02; V.sub.L-V.sub.H order), CD28 spacer containing an embedded myc epitope tag, CD28 transmembrane and endodomain, CD3z endodomain.
The CCR and the CAR are linked by a furin cleavage site, Ser-Gly linker (SGSG), and P2A ribosomal skip peptide. Codon wobbling has been used to minimize direct repeats within the scFv modules. The V.sub.H and the V.sub.L domains of scFv sequences are underlined and in bold. Epitope tags are italicized.

(150) TABLE-US-00022 (SEQIDNO:50) MGPGVLLLLLVATAWHGQGGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTL PYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSL PDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQ TDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAPTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHKRGRK KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELDYKDDDDKRRKRSGSGATNF SLLKQAGDVEENPGPNMALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVT ISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISN LEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLV APSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTI IKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAIEV EQKLISEEDLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLL VTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSR SADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

(151) The protein sequence of FBB/Y03 pCAR is shown below as SEQ ID NO: 51. The FBB/Y03 pCAR comprises: (i) a CCR comprising a linear fusion of the following elements: a macrophage colony-stimulating factor receptor leader peptide, FMC63 scFv binding domain (V.sub.L-V.sub.H order), CD8a spacer and transmembrane domain, a 4-1BB co-stimulatory endodomain, FLAG epitope tag (FBB) and (ii) a 2.sup.nd generation CAR comprising a linear fusion of the following elements: a CD8a leader peptide, a variant of FMC63 scFv in which the third tyrosine in V.sub.H CDR3 of FMC63 scFv has been substituted by alanine (Y03; V.sub.L-V.sub.H order), CD28 spacer containing an embedded myc epitope tag, CD28 transmembrane and endodomain, CD3z endodomain.
The CCR and the CAR are linked by a furin cleavage site, Ser-Gly linker (SGSG), and P2A ribosomal skip peptide. Codon wobbling has been used to minimize direct repeats within the scFv modules. The V.sub.H and the V.sub.L domains of scFv sequences are underlined and in bold. Epitope tags are italicized.

(152) TABLE-US-00023 (SEQIDNO:51) MGPGVLLLLLVATAWHGQGGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTL PYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSL PDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQ TDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAPTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHKRGRK KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELDYKDDDDKRRKRSGSGATNF SLLKQAGDVEENPGPNMALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVT APSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTI LEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLV APSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTI IKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAIEV EQKLISEEDLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLL VTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSR SADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

(153) The protein sequence of FBB/Y04 pCAR is shown below as SEQ ID NO: 52. The FBB/Y04 pCAR comprises: (i) a CCR comprising a linear fusion of the following elements: a macrophage colony-stimulating factor receptor leader peptide, FMC63 scFv binding domain (V.sub.L-V.sub.H order), CD8a spacer and transmembrane domain, a 4-1BB co-stimulatory endodomain, FLAG epitope tag (FBB) and (ii) a 2.sup.nd generation CAR comprising a linear fusion of the following elements: a CD8a leader peptide, a variant of FMC63 scFv in which the fourth tyrosine in V.sub.H CDR3 of FMC63 scFv has been substituted by alanine (Y04; V.sub.L-V.sub.H order), CD28 spacer containing an embedded myc epitope tag, CD28 transmembrane and endodomain, CD3z endodomain.
The CCR and the CAR are linked by a furin cleavage site, Ser-Gly linker (SGSG), and P2A ribosomal skip peptide. Codon wobbling has been used to minimize direct repeats within the scFv modules. The V.sub.H and the V.sub.L domains of scFv sequences are underlined and in bold. Epitope tags are italicized.

(154) TABLE-US-00024 (SEQIDNO:52) MGPGVLLLLLVATAWHGQGGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTL PYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSL PDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQ TDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAPTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHKRGRK KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELDYKDDDDKRRKRSGSGATNF SLLKQAGDVEENPGPNMALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVT APSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTI LEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLV APSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTI IKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAIEV EQKLISEEDLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLL VTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSR SADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

(155) The protein sequence of FBB/Y05 pCAR is shown below as SEQ ID NO: 53. The FBB/Y05 pCAR comprises: (i) a CCR comprising a linear fusion of the following elements: a macrophage colony-stimulating factor receptor leader peptide, FMC63 scFv binding domain (V.sub.L-V.sub.H order), CD8a spacer and transmembrane domain, a 4-1BB co-stimulatory endodomain, FLAG epitope tag (FBB) and (ii) a 2.sup.nd generation CAR comprising a linear fusion of the following elements: a CD8a leader peptide, a variant of FMC63 scFv in which the fifth tyrosine in V.sub.H CDR3 of FMC63 scFv has been substituted by alanine (Y05; V.sub.L-V.sub.H order), CD28 spacer containing an embedded myc epitope tag, CD28 transmembrane and endodomain, CD3z endodomain.
The CCR and the CAR are linked by a furin cleavage site, Ser-Gly linker (SGSG), and P2A ribosomal skip peptide. Codon wobbling has been used to minimize direct repeats within the scFv modules. The V.sub.H and the V.sub.L domains of scFv sequences are underlined and in bold. Epitope tags are italicized.

(156) TABLE-US-00025 (SEQIDNO:53) MGPGVLLLLLVATAWHGQGGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTL PYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSL PDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQ TDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAPTTTPAPRPPTPAPTIASQP LSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHKRGRK KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELDYKDDDDKRRKRSGSGATNF SLLKQAGDVEENPGPNMALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVT APSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTI LEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLV APSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTI IKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAIEV EQKLISEEDLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLL VTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSR SADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

(157) The protein sequence of 1BB/F pCAR is shown below as SEQ ID NO: 54. The 1BB/F pCAR comprises: (i) a CCR comprising a linear fusion of the following elements: a macrophage colony-stimulating factor receptor leader peptide, 1F5 scFv binding domain (V.sub.L-V.sub.H order), CD8a spacer and transmembrane domain, a 4-1BB co-stimulatory endodomain, FLAG epitope tag (1BB) and (ii) a 2.sup.nd generation CAR comprising a linear fusion of the following elements: a CD8a leader peptide, FMC63 scFv (V.sub.L-V.sub.H order), CD28 spacer containing an embedded myc epitope tag, CD28 transmembrane and endodomain, CD3z endodomain.
The CCR and the CAR are linked by a furin cleavage site, Ser-Gly linker (SGSG), and T2A ribosomal skip peptide. Codon wobbling has been used to minimize direct repeats within the scFv modules. Alternative spacers have been used within both scFvs for the same motive. The V.sub.H and the V.sub.L domains of scFv sequences are underlined and in bold. Epitope tags are italicized.

(158) TABLE-US-00026 (SEQIDNO:54) MGPGVLLLLLVATAWHGQGGQIVLSQSPAILSASPGEKVTMTCRASSSLSFMHWYQQKPGSS PKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYFCHQWSSNPLTFGAGTKVE IKRKGSTSGSGKPGSGEGSTKGQVQLRQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQT PGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSHYG SNYVDYFDYWGQGTLVTVSTGAAAPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHT RGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHKRGRKKLLYIFKQPFMRPVQTTQEEDGC SCRFPEEEEGGCELDYKDDDDKRRKRSGSGATNFSLLKQAGDVEENPGPNMALPVTALLLPL ALLLHAARPDIQMTQTTSSLSASLGDRVYISCRASQDISKYLNWYQQKPDGTVKLLIYHTSR LHSGVPSRFSGSGSGTDYLSTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGG GSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSE TTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVT VSSAAAIEVEQKLISEEDLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLAC YSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRS ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

(159) The protein sequence of RBB/F pCAR is shown below as SEQ ID NO: 55. The RBB/F pCAR comprises: (i) a CCR comprising a linear fusion of the following elements: a macrophage colony-stimulating factor receptor leader peptide, RFB4 scFv binding domain (V.sub.L-V.sub.H order), CD8a spacer and transmembrane domain, a 4-1BB co-stimulatory endodomain, FLAG epitope tag (RBB) and (ii) a 2.sup.nd generation CAR comprising a linear fusion of the following elements: a CD8a leader peptide, FMC63 scFv (V.sub.L-V.sub.H order), CD28 spacer containing an embedded myc epitope tag, CD28 transmembrane and endodomain, CD3z endodomain.
The CCR and the CAR are linked by a furin cleavage site, Ser-Gly linker (SGSG), and T2A ribosomal skip peptide. Codon wobbling has been used to minimize direct repeats within the scFv modules. Alternative spacers have been used within both scFvs for the same motive. The V.sub.H and the V.sub.L domains of scFv sequences are underlined and in bold. Epitope tags are italicized.

(160) TABLE-US-00027 (SEQIDNO:55) MGPGVLLLLLVATAWHGQGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGT VKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLE IKGSTSGSGKPGSGEGSTKGEVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQTPE KRLEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARHSGYGS SYGVLFAYWGQGTLVTVSAAAAPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG LDFACDIYIWAPLAGTCGVLLLSLVITLYCNHKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELDYKDDDDKRRKRSGSGATNFSLLKQAGDVEENPGPNMALPVTALLLPLAL LLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLH SGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGS GGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETT YYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVS SAAAIEVEQKLISEEDLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYS LLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSAD APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAY SEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

4.6. Nucleic Acids and Methods of Making pCAR-T Cells

(161) Also provided herein is a combination of a first nucleic acid encoding a 2.sup.nd generation CAR as described above and a second nucleic acid encoding a CCR as described above. As indicated above, for convenience herein, the CAR and CCR combination is referred to in the singular as a pCAR, although the CAR and CCR are separate, co-expressed, proteins. Suitable sequences for the nucleic acids will be apparent to a skilled person based on the description of the CAR and CCR above. The sequences may be optimized for use in the required immuno-responsive cell. However, in some cases, as discussed above, codons may be varied from the optimum or wobbled in order to avoid repeat sequences. Particular examples of such nucleic acids will encode the preferred embodiments described above. In some embodiments, the B cell specific pCAR comprises the polypeptide of a sequence selected from SEQ ID NOs: 47-55. In some embodiments, the nucleic acid which encodes for the pCAR is selected from the group consisting of SEQ ID NOs: 109-117.

(162) In some embodiments, the nucleic acid which encodes the CCR component of the pCAR is selected from the group consisting of SEQ ID NOs: 128, 129 and 130.

(163) In some embodiments, the nucleic acid which encodes the CAR component of the pCAR is selected from the group consisting of SEQ ID NOs: 101-108.

(164) In order to achieve transduction, the nucleic acids encoding the pCAR are 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 a further aspect of the invention.

(165) In typical embodiments, the immuno-responsive cells are subjected to genetic modification, for example by retroviral or lentiviral mediated transduction, to introduce CAR and CCR coding nucleic acids into the host T cell genome, thereby permitting stable pCAR expression. They may then be reintroduced into the patient, optionally after expansion, to provide a beneficial therapeutic effect, as described below.

(166) The first and second nucleic acids encoding the CAR and CCR can be expressed from the same vector or from different vectors. The vector or vectors containing them can be combined in a kit, which is supplied with a view to generating immuno-responsive cells of the first aspect disclosed herein.

(167) In some embodiments, the T cells may also be engineered to co-express a chimeric cytokine receptor such as 4, which comprises a fusion of the ectodomain of IL-4 receptor- and the transmembrane and endodomain of the shared IL-2/15 receptor- chain. In this case, the expansion step may include an ex vivo culture step in a medium which comprises the cytokine, such as a medium comprising IL-4 as the sole cytokine support in the case of 4. Alternatively, the chimeric cytokine receptor may comprise the ectodomain of the IL-4 receptor- chain joined to the receptor endodomain used by a common cytokine with distinct properties, such as IL-7. Expansion of the 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 state of differentiation can be ensured.

4.6. Methods of Treatment Using pCAR T Cells Targeted Against B Cell Antigens

(168) As discussed above, the immuno-responsive pCAR cells are useful in therapy to direct a T cell-mediated immune response to a target cell. Thus, in another aspect, methods for directing a T cell-mediated immune response to a target cell in a patient in need thereof are provided. The method comprises the administration to the patient a population of immuno-responsive cells as described above, wherein the binding elements are specific for the target cell. In some embodiments, the target cell expresses CD19 and/or other B cell antigens.

(169) In another aspect, methods for treating cancer in a patient in need thereof are provided. The method comprises administering to the patient a population of immuno-responsive cells as described above, wherein the binding elements are specific for the target cell. In some embodiments, the target cell expresses CD19 and/or other B cell antigens. In some embodiments, the patient has acute or chronic B cell leukemia or B cell lymphoma.

(170) In various embodiments, a therapeutically effective number of the immuno-responsive cells is administered to the patient. In certain embodiments, the immuno-responsive cells are administered by intravenous infusion. In certain embodiments, the immuno-responsive cells are administered by intratumoral injection. In certain embodiments, the immuno-responsive cells are administered by peritumoral injection. In certain embodiments, the immuno-responsive cells are administered by a plurality of routes selected from intravenous infusion, intratumoral injection, and peritumoral injection.

5. EXAMPLES

(171) Below are examples of specific embodiments for carrying out the present invention. The examples are offered 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.

(172) 5.1. Methods

(173) 5.1.1. Culture of Cell Lines

(174) 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 a firefly luciferase and tandem dimer Tomato red fluorescent protein (LT) SFG vector, followed by flow sorting for red fluorescent protein expression. LO68 CD19.sup.+ cells and LO68 CD19.sup.+/CD20.sup.+ cells were generated by transduction of LO68-LT cells with an SFG retroviral vector that encodes for human CD19 and/or human CD20.

(175) 5.1.2. Retrovirus Production

(176) 293T cells were triple transfected in Genejuice (MilliporeSigma, Merck KGaA, Darmstadt, Germany) with (i) SFG retroviral vectors encoding the indicated CAR/pCAR, (ii) RDF plasmid encoding the RD114 envelope and (iii) Peq-Pam plasmid encoding gag-pol, as recommended by the manufacturers. For transfection of 1.510.sup.6 293T cells in a 100 mm plate, 4.6875 g SFG retroviral vector, 4.6875 g Peq-Pam plasmid, and 3.125 g RDF plasmid were used. Viral vector containing medium was collected 48 and 72 h post-transfection, snap-frozen and stored at 80 C. In some cases, stable packaging cell lines were then created by transduction of 293 VEC GALV cells with retrovirus. Virus prepared from either source was used interchangeably for transduction of target cells.

(177) 5.1.3. T Cell Culture and Transduction

(178) Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor peripheral blood samples by density gradient centrifugation using Ficoll-Paque (Ethical approval no. 18/WS/0047). T cells were cultured in RPMI with GlutaMax supplemented with 5% human AB serum. Activation of T cells was achieved by culture in the presence of 5 g/mL phytohemagglutinin leucoagglutinin (PHA-L) for 24-48 h after which the cells were grown in IL-2 (100 U/mL) for a further 24 h prior to gene transfer. T cell transduction was achieved using RetroNectin (Takara Bio) coated-plates according to the Manufacturer's protocol. Activated PBMCs (110.sup.6 cells) were added per well of a RetroNectin coated 6-well plate. Retrovirus-containing medium (3 mL) was then added per well with 100 U/mL IL2.

(179) 5.1.4. Cytotoxicity Assays

(180) Tumor cells were seeded at 210.sup.4 or 110.sup.5 cells/well in a 24 or 96 well plate and were incubated with T cells at specified target to effector ratios. In some cases, destruction of tumor cells by T cells was quantified using an MTT assay. To achieve this, MTT (Sigma) was added at 500 g/ml in D10 medium for 2 hours at 37 C. and 5% CO2. After removal of the supernatant, formazan crystals were re-suspended in 100 L DMSO. Absorbance was measured at 560 nm. Alternatively, tumor cell viability was monitored by luciferase assays. D-luciferin (PerkinElmer, Waltham MA) was added at 150 mg/mL immediately prior to luminescence reading. In either case, tumor cell viability was calculated as follows: (absorbance or luminescence of tumor cells cultured with T cells/absorbance or luminescence of untreated monolayer alone)100%.

(181) 5.1.5. Detection of CAR and CCR Expression by Flow Cytometry

(182) Expression of CARs was detected using 9e10 antibody, which binds to a myc epitope tag (EQKLISEEDL), followed by PE-conjugated goat anti-mouse antibody. Expression of the CCR component of pCARs was detected by intracellular staining using a PE-conjugated antibody, which binds to a FLAG epitope tag (DYKDDDDK).

(183) 5.1.6. Detection of IFN- and IL-2 by ELISA

(184) Supernatant was collected at 24 hours from co-cultures of tumor cells with CAR T cells. Cytokine levels were quantified using a human IFN- (Bio-Techne) or human IL-2 ELISA kit (Invitrogen) according to the Manufacturer's protocol.

(185) 5.1.7. Repeated Antigen Stimulation Assays

(186) Suspension tumor cells were co-cultured with CAR-T/pCAR-T cells at an initial effector:target (E:T) ratio of 1:1 for 72-96 h. Residual tumor cell viability was then assessed by luciferase assay. D-luciferin (PerkinElmer) was added at 150 mg/mL immediately prior to luminescence reading. Fresh tumor cells (10.sup.5 cells) were then added and this procedure was repeated until T cell cultures failed to expand.

(187) Alternatively, adherent LO68 tumor cell lines were plated in triplicate at 110.sup.5 cells per well in a 24-well culture plate 24 h prior to addition of T cells. CAR-T/pCAR-T cells were added at a 1:1 effector:target ratio. Tumor cell killing was measured after 48-72 h using an MTT assay, performed as described above. T cells were then collected and restimulated by addition to a new tumor cell monolayer provided that >20% tumor cells were killed compared to untreated cells. Tumor cell viability was calculated as described in section 5.1.4.

(188) 5.1.8. In Vivo Study

(189) PBMCs from healthy donors were engineered to express the indicated CARs/pCARs 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 expression of the CAR and CCR as described above. Female NSG mice were injected i.v. with 510.sup.5 cells Nalm-6 LT cells. After 4 days, 510.sup.5 CAR.sup.+ (or untransduced) T cells were injected i.v. in 200 l of PBS, making comparison with PBS as control. Tumor status was monitored by bioluminescence imaging (BLI), performed under isoflurane anaesthesia 20 minutes after injection of StayBrite D-Luciferin, Potassium Salt in 200 l PBS (150 mg/kg). Image acquisition was performed 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 experimental endpoints had been reached.

(190) 5.1.9. CD19 CAR Binding Studiesz-Movi.

(191) CD19-engineered L068 tumor cells were seeded in a z-Movi microfluidic chip and cultured for 16 hours. The next day, flow sorted CAR-T cells were serially flowed in the chips and incubated with the target cells for 5 minutes prior to initializing a 3-minute linear force ramp. Cell detachment was determined using post-experiment using image analysis techniques.

5.2. Example 1: In Vitro Activity of CD19-Specific CAR-T Cells

(192) T cells were engineered by retroviral transduction to express a CD28-containing 2.sup.nd generation CAR designated F-2, or V.sub.H CDR3 mutated derivatives designated Y01, Y02, Y03, Y04, Y05, G01 or G02, or were untransduced (FIGS. 3A and 3B).

(193) To test binding to CD19, transduced T cells were incubated with CD19-Fc at two different concentrations, 0.5 g (FIG. 7A) and 1.0 g (FIG. 7B). Binding of CD19-Fc to CARs was detected by flow cytometry, following incubation with PE-conjugated anti-human IgG. FIGS. 7A-7D show that CDR3 mutated variants of F-2 exhibit variable efficiency of binding to CD19.

(194) To further test the relative CD19 binding capacity of the 2G CAR, F-2, and its mutated derivatives, the engineered CAR-T cells were incubated on a monolayer of CD19+LO68 tumor cells in a z-Movi microfluidic chip. Increasing fluidic force was applied and the percentage of bound T-cells was determined (median, n=3). T cells expressing the V.sub.H CDR3 mutated CARs presented a spectrum of binding activities for CD19. Most of the V.sub.H CDR3 mutated derivatives exhibited reduced binding activity for CD19 as compared with the 2G CAR F. Data from representative experiments are shown in FIGS. 17A-17D. Data are presented as median rForce which is the relative force required to elicit cell detachment, calibrated to 10 M polystyrene beads. Since data were not normally distributed, they are presented as median rForce which is the relative force required to elicit cell detachment and calibrated to 10 M polystyrene beads. FIG. 17B shows the overall mean percentage of bound T cells after applying the minimal rForce (210 pN) required to detach a mean of 90% untransduced T cells. Data shown are meanstandard error (n=11 runs, 3 independent donors). FIG. 17C shows the avidity score (e.g., the ratio of the mean rForce per cell required to detach T cells from the monolayer of CD19.sup.+ LO68 tumor cells for each of the V.sub.H CDR3 mutated derivatives compared to untransduced T cells. The black dotted line represents the avidity score of untransduced T cells; the red dotted line represents the avidity score of F-2 CAR T cells. FIG. 17D shows the rForce per cell required to detach from the tumor cell monolayer where each dot in the dot plot represents a single cell. Bars indicate the median+interquartile range. Statistical analysis was performed using Kruskal-Wallis test with Dunnett's multiple comparisons test where p=<0.0001 (****), 0.0007 (***), or 0.005 (**). For clarity, a single representative run for one healthy donor is plotted in which each dot represents a single cell. Collectively, these dots generate the avidity curve shown in FIG. 17A. The avidity curves therefore represent more than 1000 single cell observations per donor.

(195) To evaluate anti-tumor activity, transduced T cells were co-cultivated in vitro with Nalm-6 LT or Raji LT cells, both of which naturally express CD19 (FIG. 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 FIGS. 8A-8B. Residual viable cancer cells after the co-culture were quantified by luciferase assay after 72 hours. The percentage survival of Nalm-6 and Raji tumor cells after co-culture with the CAR-T cells is presented in FIGS. 8A and 8B respectively.

(196) To further evaluate cytotoxic activity, 210.sup.4 transduced T cells were co-cultivated in duplicate with an equal number of Nalm-6 (FIG. 8C) or Raji (FIG. 8D) cells. After 72 hours, viability of tumor cells was quantified by luciferase assay. Meanstandard error (n=3 biological replicates) of the mean levels of viable cancer cells are shown in FIGS. 8C-8D. Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison test where p<0.0001 (****), 0.001 (***), or 0.01 (**).

(197) Transduced T cells were co-cultivated in vitro with Nalm-6 LT (FIG. 9A-9B) or Raji LT cells (FIG. 9C-9D), at an effector:target ratio of 1.25 tumor cells:1 CAR-expressing T cell. Meanstandard error (n=3 biological replicates) of the mean levels of IFN- and IL-2 concentration present in supernatant collected after 24 hours are presented in FIGS. 9A-9D. In each case, statistical analysis was performed using two-way ANOVA with Tukey's multiple comparisons test where p=0.033 (*), 0.002 (**), 0.0002 (***) or <0.0001 (****). NSnot significant.

(198) Following the co-cultivation experiments described above (data shown in FIGS. 8C-8D), cell supernatants were collected after 24 hours and analyzed for IFN- (FIGS. 10A-10B) and IL-2 (FIGS. 10C-10D) by ELISA. Data shown are meanstandard error (n=3 biological replicates). Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison test where p<0.0001 (****), 0.001 (***), 0.01 (**), or 0.05 (*). NSnot significant.

(199) Transduced T cells were subjected to successive rounds of antigen (Ag) stimulation in the absence of exogenous cytokine IL-2. Triplicate cultures containing 10.sup.5 engineered T cells were re-stimulated twice weekly by addition of 10.sup.5 Nalm-6 tumor cells. FIG. 11A shows tumor cell viability which was measured by luciferase assay 72 hours after each tumor cell challenge. FIGS. 11B and 11C respectively show IFN- and IL-2 levels in supernatant, which was collected 24 hours after each tumor cell challenge.

5.3. Example 2: In Vitro Activity of CD19-Specific pCAR-T Cells

(200) FIGS. 4A-4B and FIGS. 18A-18B show expression of a panel of CD19-specific pCARs in human T cells, making comparison with the 2.sup.nd generation control CAR, (F-2). Nomenclature of pCARs derives from an ordered abbreviation of the following elements: CCR binder (FMC63 scFv), CCR signaling domain (4-1BB)/CAR binder (G01-Y05 respectively). Cell surface expression of the CD28-containing CAR within each pCAR was detected by flow cytometry after incubation of cells with 9e10 antibody, which binds to a myc epitope tag within the CAR ectodomain. Expression of the CCR component within each pCAR was detected by intracellular staining of permeabilized cells using anti-FLAG antibody, which binds to a FLAG epitope tag located at a distal position in the CCR endodomain. Locations of epitope tags are illustrated schematically in FIGS. 2C-2F.

(201) FIG. 12 shows pooled data from five biological replicate experiments, each conducted in duplicate in which CDR3 V.sub.H-mutated FMC63-based 2.sup.nd generation CAR and pCAR T cells were co-cultivated in vitro with Nalm-6 LT tumor cells, making comparison with parental 2.sup.nd generation CAR T cells (F-2). In each experiment, cultures were established in which T cells were co-cultivated with 210.sup.4 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. Residual viable cancer cells were quantified by luciferase assay after 72 hours. The percentage survival of Nalm-6 tumor cells after co-culture with the CAR-T cells is presented (meanstandard error of the mean, n=10 data points). Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparisons test where p=0.033 (*), 0.002 (**), 0.0002 (***) or <0.0001 (****). NSnot significant. Some of the CDR3 V.sub.H-mutated FMC63-based 2.sup.nd generation CARs outperformed the parental 2.sup.nd generation CAR, F-2. Equivalent or further enhancement of cytotoxicity was noted for derived pCARs with the same mutated V.sub.H CDR3.

(202) FIGS. 13A-13C show representative data from experiments in which 10.sup.5 pCAR-T cells were iteratively restimulated on LO68 tumor monolayers that express CD19 in the absence of exogenous cytokine. FIG. 13A shows tumor cell viability which was measured by luciferase assay 24 hours after each round of stimulation. Levels of IFN- and IL-2 present in supernatants collected 24 hours after each round of stimulation are shown in FIG. 13B and FIG. 13C respectively. Note the superior anti-tumor activity of some pCAR variants when compared to the parental 2.sup.nd generation CAR, F-2.

5.4. Example 3: In Vivo Activity of CD19-Specific pCAR-T Cells in NSG Mice

(203) The anti-tumor activity of the CD19-specific CAR-T and pCAR-T cells was assessed in vivo in NSG mice bearing established Nalm-6 leukemic xenografts. RFP/ffLuc.sup.+ Nalm6 cells (510.sup.5 cells) were injected i.v. in NSG mice. On day 5, animals were arranged into groups of 5-10 mice with equal disease burden (according to BLI). Mice were then treated with 510.sup.5 of the indicated CAR or pCAR T-cells, administered i.v. Comparison was made with PBS. Pooled bioluminescence emission (total flux) from leukemic xenografts was measured for each treatment from day 8 (FIG. 19). At the modest T-cell dose employed (510.sup.5 cells), F-2 CAR T-cells elicited transient delay in disease progression while the mutated 2G derivates, Y05 and G02, achieved superior anti-leukemic activity. All of the tested pCARs, FBB/Y04, FBB/Y05, and FBB/G02, achieved further enhancement of disease control (FIG. 14A, FIGS. 20A and 20B). The tested pCARs FBB/Y04, FBB/Y05, and FBB/G02 also achieved superior after treatment survival (FIGS. 21A-21D) without weight loss (FIG. 14B).

(204) These results indicate that the CD19-specific pCAR-T cells have superior anti-tumor activity in vivo compared to the 2G CAR-T cells in NSG mice with established leukemic burden.

5.6. Example 4: In Vitro Anti-Tumor Activity of pCAR-T Cells that Co-Target CD19 and CD20

(205) T cells were engineered to express the CD19- or CD20-specific 2.sup.nd generation CAR T cells (F-2 and 1-2, respectively) or the 1BB/F pCAR. 110.sup.5 transduced CAR or pCAR T cells were co-cultivated in triplicate with an equal number of LO68-CD19.sup.+CD20.sup.+ tumor cells. After 72 hours, T cells were transferred to a fresh monolayer of LO68-CD19.sup.+CD20.sup.+ cells. Data plotted in FIG. 15A show the number of re-stimulation cycles completed for each co-cultivation condition. Cultures were terminated when tumor cell viability was 80% or greater.

(206) FIG. 15B shows data in which 10.sup.5 of 1BB/F pCAR-T cells were combined with an equal number of LO68 tumor cells that co-express CD19 and CD20. Cultures were established in the absence of exogenous cytokine. Comparison is made with CD19- or CD20-specific 2.sup.nd generation CAR T cells (F-2 and 1-2, respectively) or untransduced T cells. Tumor cell cytotoxicity was assessed after 72 hours by MTT assay. T cells were re-stimulated twice weekly by co-culture with 10.sup.5 tumor cells and MTT assay performed after 72 hours to assess tumor cell viability. This procedure was repeated until T cell cultures failed. Data shown are meanSEM, n=4. Supernatant was removed from the co-cultures one day after each cycle of stimulation and was analyzed by ELISA for release of IFN- and IL-2 (FIG. 15C). Data shown are meanstandard error (n=4).

(207) In FIG. 15D, T cells were co-cultivated in vitro for 72 hours with 10.sup.5 of LO68 tumor cells that co-express CD19 and CD20. 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. Cultures were established in the absence of exogenous cytokine. Residual viable cancer cells were quantified by MTT assay after 72 hours. Similar anti-tumor activity was noted in vitro for CAR- and pCAR-engineered T cells. Data shown are meanSEM, n=4.

(208) CAR or pCAR T cells were co-cultivated with LO68-CD19.sup.+CD20.sup.+ tumor cells at an effector:target (T cell:tumor cell) ratio of 1:1 and 1:4. Supernatants were collected after 24 hours of co-cultivations and were analyzed for IFN- (FIG. 15E) and IL-2 (FIG. 15F).

5.7. Example 5: In Vitro Anti-Tumor Activity of pCAR-T Cells that Co-Target CD19 and CD22

(209) FIGS. 6A-6B show expression in human T cells of the RBB/F pCAR that co-targets CD19 and CD22, making comparison with CD19- or CD22-specific 2.sup.nd generation CAR T cells (F-2 and R-2, respectively) or untransduced T cells. Expression of CARs, or the CAR component of pCARs was detected by flow cytometry after incubation of cells with 9e10 antibody, which binds to a myc epitope tag within the CAR ectodomain. Expression of the CCR component within the pCAR was detected by intracellular staining of permeabilized cells using anti-FLAG antibody, which binds to a FLAG epitope tag located at a distal position in the CCR endodomain. Locations of epitope tags are illustrated schematically in FIG. 2F.

(210) FIG. 16A shows the cytotoxic activity of RBB/F pCAR T cells against Nalm-6 leukemic cells, making comparisons with CD19-specific (F-2) or CD22-specific (R-2) 2.sup.nd generation CAR T cells. T cells were co-cultivated in vitro for 72 hours with tumor cells. 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. Residual viable cancer cells were then quantified by luciferase assay. Note the enhanced anti-tumor activity of pCAR T cells at lower E:T ratios. Data shown are meanSEM, n=5. Statistical analysis was performed with two-way ANOVA followed by Tukey post hoc test. p=0.0216 (*), 0.0021 (**), 0.0002 (***), <0.0001 (****)

(211) FIG. 16B shows the cytotoxic activity of RBB/G02 and RBB/Y05 pCAR T cells against Nalm-6 leukemic cells, making comparisons with RBB/F pCAR T cells and CD19-specific (F-2) or CD22-specific (R-2) 2.sup.nd generation CAR T cells. Experiments were performed as described above, with viability of the target cells quantified by luciferase assay following 72 hours of co-cultivation. Data shown are meanstandard error (n=3-7 biological replicates).

(212) TABLE-US-00028 6.SEQUENCES ID SEQ SEQIDNO:1 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPE CD3zetachain(a MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD polymorphicform) GLYQGLSTATKDTYDALHMQALPPR SEQIDNO:2 RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEM CD3zetachain(a GGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL polymorphicform) YQGLSTATKDTYDALHMQALPPR SEQIDNO:3 MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNL CD28protein FSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLG NESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKG KHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRS RLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS SEQIDNO:4 IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVG Hinge, GVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHY transmembraneand QPYAPPRDFAAYRS co-stimulatoryregion inCD28protein SEQIDNO:5 EQKLISEEDL c-myctag SEQIDNO:6 IEVEQKLISEEDLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLV Hinge(containingmyc VVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTR tag),transmembrane KHYQPYAPPRDFAAYRS andco-stimulatory regioninCD28 protein SEQIDNO:7 KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 4-1BBendodomain SEQIDNO:8 DYKDDDDK FLAGepitopetag SEQIDNO:9 KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELDYKD 4-1BBendodomain DDDK fusedtoFLAGepitope tag SEQIDNO:10 GVSLPDY V.sub.HCDR1FMC63 SEQIDNO:11 WGSET V.sub.HCDR2FMC63 SEQIDNO:12 HYYYGGSYAMDY V.sub.HCDR3FMC63 SEQIDNO:13 RASQDISKYLN V.sub.LCDR1FMC63 SEQIDNO:14 HTSRLHS V.sub.LCDR2FMC63 SEQIDNO:15 QQGNTLPYT V.sub.LCDR3FMC63 SEQIDNO:16 EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLE V.sub.HoftheFMC63 WLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAI antibody YYCAKHYYYGGSYAMDYWGQGTSVTVSS SEQIDNO:17 DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKL V.sub.LoftheFMC63 LIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTL antibody PYTFGGGTKLEIT SEQIDNO:18 EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLE scFvoftheFMC63 WLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAI antibody(V.sub.H-V.sub.L YYCAKHYYYGGSYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGS format) DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKL LIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTL PYTFGGGTKLEIT SEQIDNO:19 DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKL scFvoftheFMC63 LIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTL antibody(V.sub.L-V.sub.H LPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSL format) SVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSAL KSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMD YWGQGTSVTVSS SEQIDNO:20 HAYYGGSYAMDY V.sub.HCDR3FMC63Y01 (Y->Amutation) SEQIDNO:21 HYAYGGSYAMDY V.sub.HCDR3FMC63Y02 (Y->Amutation) SEQIDNO:22 HYYAGGSYAMDY V.sub.HCDR3FMC63Y03 (Y->Amutation) SEQIDNO:23 HYYYAGSYAMDY V.sub.HCDR3FMC63G01 (G->Amutation) SEQIDNO:24 HYYYGASYAMDY V.sub.HCDR3FMC63G02 (G->Amutation) SEQIDNO:25 HYYYGGSAAMDY V.sub.HCDR3FMC63Y04 (Y->Amutation) SEQIDNO:26 HYYAGGSYAMDA V.sub.HCDR3FMC63Y05 (Y->Amutation) SEQIDNO:27 GYTFTSY V.sub.HCDR11F5 SEQIDNO:28 YPGNGD V.sub.HCDR21F5 SEQIDNO:29 SHYGSNYVDYFDY V.sub.HCDR31F5 SEQIDNO:30 RASSSLSFMH V.sub.LCDR11F5 SEQIDNO:31 ATSNLAS V.sub.LCDR21F5 SEQIDNO:32 HQWSSNPLT V.sub.LCDR31F5 SEQIDNO:33 QVQLRQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQG V.sub.H1F5 LEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSED SAVYYCARSHYGSNYVDYFDYWGQGTLVTVSTG SEQIDNO:34 QIVLSQSPAILSASPGEKVTMTCRASSSLSFMHWYQQKPGSSPKPWI V.sub.L1F5 YATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYFCHQWSSN PLTFGAGTKVEIKRK SEQIDNO:35 QVQLRQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQG scFvofthe1F5 LEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSED antibody(V.sub.H-V.sub.L SAVYYCARSHYGSNYVDYFDYWGQGTLVTVSTGGSTSGSGKPGS format) GEGSTKGQIVLSQSPAILSASPGEKVTMTCRASSSLSFMHWYQQKP GSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYF CHQWSSNPLTFGAGTKVEIKRK SEQIDNO:36 QIVLSQSPAILSASPGEKVTMTCRASSSLSFMHWYQQKPGSSPKPWI scFvofthe1F5 YATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYFCHQWSSN antibody(V.sub.L-V.sub.H PLTFGAGTKVEIKRKGSTSGSGKPGSGEGSTKGQVQLRQPGAELVK format) PGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGD TSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSHYG SNYVDYFDYWGQGTLVTVSTG SEQIDNO:37 GFAFSIY V.sub.HCDR1RFB4 SEQIDNO:38 SSGGGT V.sub.HCDR2RFB4 SEQIDNO:39 HSGYGSSYGVLFAY V.sub.HCDR3RFB4 SEQIDNO:40 RASQDISNYLN V.sub.LCDR1RFB4 SEQIDNO:41 YTSILHS V.sub.LCDR2RFB4 SEQIDNO:42 QQGNTLPWT V.sub.LCDR3RFB4 SEQIDNO:43 EVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQTPEKRLEV V.sub.HRFB4 VAYISSGGGTTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMY CARHSGYGSSYGVLFAYWGQGTLVTVSA SEQIDNO:44 DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLI V.sub.LRFB4 YTSILHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQQGNTLPWTI GGTKLEIK SEQIDNO:45 EVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQTPEKRLE scFvoftheRFB4 WVAYISSGGGTTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTA antibody(V.sub.H-V.sub.L MYYCARHSGYGSSYGVLFAYWGQGTLVTVSAGSTSGSGKPGSGE format) GSTKGDIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPD GTVKLLIYYTSILHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQ QGNTLPWTFGGGTKLEIK SEQIDNO:46 DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKL scFvoftheRFB4 LIYYTSILHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQQGNTL antibody(V.sub.L-V.sub.H PWTFGGGTKLEIKGSTSGSGKPGSGEGSTKGEVQLVESGGGLVKPG format) GSLKLSCAASGFAFSIYDMSWVRQTPEKRLEWVAYISSGGGTTYYP DTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARHSGYGSSY GVLFAYWGQGTLVTVSA SEQIDNO:47 MGPGVLLLLLVATAWHGQGGDIQMTQTTSSLSASLGDRVTISCRA FBB/G01pCAR SQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDY SLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSG GGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPR KGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD DTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAPTTTPAPRPP TPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTC GVLLLSLVITLYCNHKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELDYKDDDDKRRKRSGSGATNFSLLKQAGDVEENPG PNMALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCR ASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTD YSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGS GGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPP RKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQT DDTAIYYCAKHYYYAGSYAMDYWGQGTSVTVSSAAAIEVEQKLIS EEDLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLA CYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYA PPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQIDNO:48 MGPGVLLLLLVATAWHGQGGDIQMTQTTSSLSASLGDRVTISCRA FBB/G02pCAR SQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDY SLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSG GGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPR KGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD DTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAPTTTPAPRPP TPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTC GVLLLSLVITLYCNHKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELDYKDDDDKRRKRSGSGATNFSLLKQAGDVEENPG PNMALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCR ASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTD YSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGS GGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPP RKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQT DDTAIYYCAKHYYYGASYAMDYWGQGTSVTVSSAAAIEVEQKLIS EEDLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLA CYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYA PPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQIDNO:49 MGPGVLLLLLVATAWHGQGGDIQMTQTTSSLSASLGDRVTISCRA FBB/Y01pCAR SQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDY SLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSG GGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPR KGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD DTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAPTTTPAPRPP TPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTC GVLLLSLVITLYCNHKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELDYKDDDDKRRKRSGSGATNFSLLKQAGDVEENPG PNMALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCR ASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTD YSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGS GGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPP RKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQT DDTAIYYCAKHAYYGGSYAMDYWGQGTSVTVSSAAAIEVEQKLIS EEDLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLA CYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYA PPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQIDNO:50 MGPGVLLLLLVATAWHGQGGDIQMTQTTSSLSASLGDRVTISCRA FBB/Y02pCAR SQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDY SLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSG GGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPR KGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD DTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAPTTTPAPRPP TPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTC GVLLLSLVITLYCNHKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELDYKDDDDKRRKRSGSGATNFSLLKQAGDVEENPG PNMALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCR ASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTD YSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGS GGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPP RKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQT DDTAIYYCAKHYAYGGSYAMDYWGQGTSVTVSSAAAIEVEQKLIS EEDLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLA CYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYA PPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQIDNO:51 MGPGVLLLLLVATAWHGQGGDIQMTQTTSSLSASLGDRVTISCRA FBB/Y03pCAR SQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDY SLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSG GGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPR KGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD DTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAPTTTPAPRPP TPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTC GVLLLSLVITLYCNHKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELDYKDDDDKRRKRSGSGATNFSLLKQAGDVEENPG PNMALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCR ASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTD YSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGS GGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPP RKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQT DDTAIYYCAKHYYAGGSYAMDYWGQGTSVTVSSAAAIEVEQKLIS EEDLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLA CYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYA PPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQIDNO:52 MGPGVLLLLLVATAWHGQGGDIQMTQTTSSLSASLGDRVTISCRA FBB/Y04pCAR SQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDY SLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSG GGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPR KGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD DTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAPTTTPAPRPP TPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTC GVLLLSLVITLYCNHKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELDYKDDDDKRRKRSGSGATNFSLLKQAGDVEENPG PNMALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCR ASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTD YSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGS GGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPP RKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQT DDTAIYYCAKHYYYGGSAAMDYWGQGTSVTVSSAAAIEVEQKLIS EEDLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLA CYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYA PPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQIDNO:53 MGPGVLLLLLVATAWHGQGGDIQMTQTTSSLSASLGDRVTISCRA FBB/Y05pCAR SQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDY SLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSG GGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPR KGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD DTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAPTTTPAPRPP TPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTC GVLLLSLVITLYCNHKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELDYKDDDDKRRKRSGSGATNFSLLKQAGDVEENPG PNMALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCR ASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTD YSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGS GGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPP RKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQT DDTAIYYCAKHYYYGGSYAMDAWGQGTSVTVSSAAAIEVEQKLIS EEDLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLA CYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYA PPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQIDNO:54 MGPGVLLLLLVATAWHGQGGQIVLSQSPAILSASPGEKVTMTCRA 1BB/FpCAR SSSLSFMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYS LTISRVEAEDAATYFCHQWSSNPLTFGAGTKVEIKRKGSTSGSGKP GSGEGSTKGQVQLRQPGAELVKPGASVKMSCKASGYTFTSYNMH WVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAY MQLSSLTSEDSAVYYCARSHYGSNYVDYFDYWGQGTLVTVSTGA AAPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC DIYIWAPLAGTCGVLLLSLVITLYCNHKRGRKKLLYIFKQPFMRPV QTTQEEDGCSCRFPEEEEGGCELDYKDDDDKRRKRSGSGATNFSLL KQAGDVEENPGPNMALPVTALLLPLALLLHAARPDIQMTQTTSSLS ASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGV PSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEI TGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLP DYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKS QVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS AAAIEVEQKLISEEDLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFW VLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRP GPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMA EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQIDNO:55 MGPGVLLLLLVATAWHGQGDIQMTQTTSSLSASLGDRVTISCRAS RBB/FpCAR QDISNYLNWYQQKPDGTVKLLIYYTSILHSGVPSRFSGSGSGTDYSL TISNLEQEDFATYFCQQGNTLPWTFGGGTKLEIKGSTSGSGKPGSGE GSTKGEVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQT PEKRLEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNTLYLQMSSL KSEDTAMYYCARHSGYGSSYGVLFAYWGQGTLVTVSAAAAPTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWA PLAGTCGVLLLSLVITLYCNHKRGRKKLLYIFKQPFMRPVQTTQEE DGCSCRFPEEEEGGCELDYKDDDDKRRKRSGSGATNFSLLKQAGD VEENPGPNMALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGD RVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSG SGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGG SGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVS WIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLK MNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAIE VEQKLISEEDLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVV VGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRK HYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRR EEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQIDNO:56 MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRAS CARofFBB/G01 QDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYS LTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGG GGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRK GLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDD TAIYYCAKHYYYAGSYAMDYWGQGTSVTVSSAAAIEVEQKLISEE DLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACY SLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPP RDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGER RRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQIDNO:57 MGPGVLLLLLVATAWHGQGGDIQMTQTTSSLSASLGDRVTISCRA CCRofFBB/G01, SQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDY FBB/G02,FBB/Y01, SLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSG FBB/Y02,FBB/Y03, GGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPR FBB/Y04,and KGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD FBB/Y05 DTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAPTTTPAPRPP TPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTC GVLLLSLVITLYCNHKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELDYKDDDDK SEQIDNO:58 MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRAS CARofFBB/G02 QDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYS LTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGG GGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRK GLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDD TAIYYCAKHYYYGASYAMDYWGQGTSVTVSSAAAIEVEQKLISEE DLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACY SLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPP RDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGER RRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQIDNO:59 MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRAS CARofFBB/Y01 QDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYS LTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGG GGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRK GLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDD TAIYYCAKHAYYGGSYAMDYWGQGTSVTVSSAAAIEVEQKLISEE DLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACY SLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPP RDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGER RRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQIDNO:60 MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRAS CARofFBB/Y02 QDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYS LTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGG GGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRK GLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDD TAIYYCAKHYAYGGSYAMDYWGQGTSVTVSSAAAIEVEQKLISEE DLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACY SLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPP RDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGER RRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQIDNO:61 MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRAS CARofFBB/Y03 QDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYS LTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGG GGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRK GLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDD TAIYYCAKHYYAGGSYAMDYWGQGTSVTVSSAAAIEVEQKLISEE DLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACY SLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPP RDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGER RRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQIDNO:62 MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRAS CARofFBB/Y04 QDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYS LTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGG GGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRK GLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDD TAIYYCAKHYYYGGSAAMDYWGQGTSVTVSSAAAIEVEQKLISEE DLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACY SLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPP RDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGER RRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQIDNO:63 MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRAS CARofFBB/Y05 QDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYS LTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGG GGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRK GLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDD TAIYYCAKHYYYGGSYAMDAWGQGTSVTVSSAAAIEVEQKLISEE DLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACY SLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPP RDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGER RRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQIDNO:64 MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRAS CARof1BB/Fand QDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYS RBB/F LTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGG GGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRK GLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDD TAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAIEVEQKLISEE DLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACY SLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPP RDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGER RRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQIDNO:65 MGPGVLLLLLVATAWHGQGGQIVLSQSPAILSASPGEKVTMTCRA CCRof1BB/F SSSLSFMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYS LTISRVEAEDAATYFCHQWSSNPLTFGAGTKVEIKRKGSTSGSGKP GSGEGSTKGQVQLRQPGAELVKPGASVKMSCKASGYTFTSYNMH WVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAY MQLSSLTSEDSAVYYCARSHYGSNYVDYFDYWGQGTLVTVSTGA AAPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC DIYIWAPLAGTCGVLLLSLVITLYCNHKRGRKKLLYIFKQPFMRPV QTTQEEDGCSCRFPEEEEGGCELDYKDDDDK SEQIDNO:66 MGPGVLLLLLVATAWHGQGDIQMTQTTSSLSASLGDRVTISCRAS CCRofRBB/F QDISNYLNWYQQKPDGTVKLLIYYTSILHSGVPSRFSGSGSGTDYSL TISNLEQEDFATYFCQQGNTLPWTFGGGTKLEIKGSTSGSGKPGSGE GSTKGEVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQT PEKRLEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNTLYLQMSSL KSEDTAMYYCARHSGYGSSYGVLFAYWGQGTLVTVSAAAAPTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWA PLAGTCGVLLLSLVITLYCNHKRGRKKLLYIFKQPFMRPVQTTQEE DGCSCRFPEEEEGGCELDYKDDDDK SEQIDNO:101 ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTG Nucleotidesequence CTGCACGCCGCTAGACCCGACATCCAGATGACCCAGACCACCAG encodingforF-2 CAGCCTGAGCGCCAGCCTGGGCGACAGAGTGACCATCAGCTGC AGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGC AGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAG CAGACTGCACAGCGGCGTGCCCAGCAGATTCAGCGGCAGCGGC AGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCAGG AGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCC TACACCTTCGGCGGAGGCACCAAGCTGGAGATCACCGGCGGAG GAGGAAGCGGAGGAGGAGGCAGCGGAGGAGGAGGCAGCGAGG TGAAGCTGCAGGAGAGCGGCCCCGGCCTGGTGGCCCCCAGCCA GAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCG ACTACGGCGTGAGCTGGATCAGACAGCCTCCCAGAAAGGGCCT GGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTAC AACAGCGCCCTGAAGAGCAGACTGACCATCATTAAGGACAACA GCAAGTCCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGAC GACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGG AAGCTACGCTATGGACTACTGGGGCCAGGGAACCAGCGTGACC GTGAGCAGCGCGGCCGCTATCGAGGTGGAGCAGAAGCTGATCA GCGAGGAGGACCTGCTAGACAATGAGAAGAGCAATGGAACCAT TATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTC CCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGA GTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATT TTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACT ACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCAT TACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTC CAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAG CAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAA GAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCC TGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGG CCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTAC AGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGC ACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACC TACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA SEQIDNO:102 ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTG Nucleotidesequence CTGCACGCCGCTAGACCCGACATCCAGATGACCCAGACCACCAG encodingforG01 CAGCCTGAGCGCCAGCCTGGGCGACAGAGTGACCATCAGCTGC AGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGC AGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAG CAGACTGCACAGCGGCGTGCCCAGCAGATTCAGCGGCAGCGGC AGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCAGG AGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCC TACACCTTCGGCGGAGGCACCAAGCTGGAGATCACCGGCGGAG GAGGAAGCGGAGGAGGAGGCAGCGGAGGAGGAGGCAGCGAGG TGAAGCTGCAGGAGAGCGGCCCCGGCCTGGTGGCCCCCAGCCA GAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCG ACTACGGCGTGAGCTGGATCAGACAGCCTCCCAGAAAGGGCCT GGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTAC AACAGCGCCCTGAAGAGCAGACTGACCATCATTAAGGACAACA GCAAGTCCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGAC GACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGCCGG AAGCTACGCTATGGACTACTGGGGCCAGGGAACCAGCGTGACC GTGAGCAGCGCGGCCGCTATCGAGGTGGAGCAGAAGCTGATCA GCGAGGAGGACCTGCTAGACAATGAGAAGAGCAATGGAACCAT TATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTC CCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGA GTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATT TTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACT ACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCAT TACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTC CAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAG CAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAA GAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCC TGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGG CCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTAC AGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGC ACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACC TACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA SEQIDNO:103 ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTG Nucleotidesequence CTGCACGCCGCTAGACCCGACATCCAGATGACCCAGACCACCAG encodingforG02 CAGCCTGAGCGCCAGCCTGGGCGACAGAGTGACCATCAGCTGC AGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGC AGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAG CAGACTGCACAGCGGCGTGCCCAGCAGATTCAGCGGCAGCGGC AGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCAGG AGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCC TACACCTTCGGCGGAGGCACCAAGCTGGAGATCACCGGCGGAG GAGGAAGCGGAGGAGGAGGCAGCGGAGGAGGAGGCAGCGAGG TGAAGCTGCAGGAGAGCGGCCCCGGCCTGGTGGCCCCCAGCCA GAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCG ACTACGGCGTGAGCTGGATCAGACAGCCTCCCAGAAAGGGCCT GGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTAC AACAGCGCCCTGAAGAGCAGACTGACCATCATTAAGGACAACA GCAAGTCCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGAC GACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGC CAGCTACGCTATGGACTACTGGGGCCAGGGAACCAGCGTGACC GTGAGCAGCGCGGCCGCTATCGAGGTGGAGCAGAAGCTGATCA GCGAGGAGGACCTGCTAGACAATGAGAAGAGCAATGGAACCAT TATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTC CCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGA GTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATT TTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACT ACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCAT TACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTC CAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAG CAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAA GAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCC TGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGG CCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTAC AGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGC ACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACC TACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA SEQIDNO:104 ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTG Nucleotidesequence CTGCACGCCGCTAGACCCGACATCCAGATGACCCAGACCACCAG encodingforY01 CAGCCTGAGCGCCAGCCTGGGCGACAGAGTGACCATCAGCTGC AGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGC AGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAG CAGACTGCACAGCGGCGTGCCCAGCAGATTCAGCGGCAGCGGC AGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCAGG AGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCC TACACCTTCGGCGGAGGCACCAAGCTGGAGATCACCGGCGGAG GAGGAAGCGGAGGAGGAGGCAGCGGAGGAGGAGGCAGCGAGG TGAAGCTGCAGGAGAGCGGCCCCGGCCTGGTGGCCCCCAGCCA GAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCG ACTACGGCGTGAGCTGGATCAGACAGCCTCCCAGAAAGGGCCT GGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTAC AACAGCGCCCTGAAGAGCAGACTGACCATCATTAAGGACAACA GCAAGTCCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGAC GACACCGCCATCTACTACTGCGCCAAGCACGCCTACTACGGCGG AAGCTACGCTATGGACTACTGGGGCCAGGGAACCAGCGTGACC GTGAGCAGCGCGGCCGCTATCGAGGTGGAGCAGAAGCTGATCA GCGAGGAGGACCTGCTAGACAATGAGAAGAGCAATGGAACCAT TATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTC CCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGA GTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATT TTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACT ACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCAT TACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTC CAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAG CAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAA GAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCC TGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGG CCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTAC AGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGC ACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACC TACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA SEQIDNO:105 ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTG Nucleotidesequence CTGCACGCCGCTAGACCCGACATCCAGATGACCCAGACCACCAG encodingforY02 CAGCCTGAGCGCCAGCCTGGGCGACAGAGTGACCATCAGCTGC AGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGC AGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAG CAGACTGCACAGCGGCGTGCCCAGCAGATTCAGCGGCAGCGGC AGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCAGG AGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCC TACACCTTCGGCGGAGGCACCAAGCTGGAGATCACCGGCGGAG GAGGAAGCGGAGGAGGAGGCAGCGGAGGAGGAGGCAGCGAGG TGAAGCTGCAGGAGAGCGGCCCCGGCCTGGTGGCCCCCAGCCA GAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCG ACTACGGCGTGAGCTGGATCAGACAGCCTCCCAGAAAGGGCCT GGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTAC AACAGCGCCCTGAAGAGCAGACTGACCATCATTAAGGACAACA GCAAGTCCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGAC GACACCGCCATCTACTACTGCGCCAAGCACTACGCCTACGGCGG AAGCTACGCTATGGACTACTGGGGCCAGGGAACCAGCGTGACC GTGAGCAGCGCGGCCGCTATCGAGGTGGAGCAGAAGCTGATCA GCGAGGAGGACCTGCTAGACAATGAGAAGAGCAATGGAACCAT TATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTC CCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGA GTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATT TTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACT ACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCAT TACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTC CAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAG CAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAA GAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCC TGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGG CCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTAC AGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGC ACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACC TACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA SEQIDNO:106 ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTG Nucleotidesequence CTGCACGCCGCTAGACCCGACATCCAGATGACCCAGACCACCAG encodingforY03 CAGCCTGAGCGCCAGCCTGGGCGACAGAGTGACCATCAGCTGC AGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGC AGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAG CAGACTGCACAGCGGCGTGCCCAGCAGATTCAGCGGCAGCGGC AGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCAGG AGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCC TACACCTTCGGCGGAGGCACCAAGCTGGAGATCACCGGCGGAG GAGGAAGCGGAGGAGGAGGCAGCGGAGGAGGAGGCAGCGAGG TGAAGCTGCAGGAGAGCGGCCCCGGCCTGGTGGCCCCCAGCCA GAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCG ACTACGGCGTGAGCTGGATCAGACAGCCTCCCAGAAAGGGCCT GGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTAC AACAGCGCCCTGAAGAGCAGACTGACCATCATTAAGGACAACA GCAAGTCCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGAC GACACCGCCATCTACTACTGCGCCAAGCACTACTACGCCGGCGG AAGCTACGCTATGGACTACTGGGGCCAGGGAACCAGCGTGACC GTGAGCAGCGCGGCCGCTATCGAGGTGGAGCAGAAGCTGATCA GCGAGGAGGACCTGCTAGACAATGAGAAGAGCAATGGAACCAT TATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTC CCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGA GTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATT TTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACT ACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCAT TACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTC CAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAG CAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAA GAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCC TGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGG CCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTAC AGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGC ACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACC TACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA SEQIDNO:107 ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTG Nucleotidesequence CTGCACGCCGCTAGACCCGACATCCAGATGACCCAGACCACCAG encodingforY04 CAGCCTGAGCGCCAGCCTGGGCGACAGAGTGACCATCAGCTGC AGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGC AGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAG CAGACTGCACAGCGGCGTGCCCAGCAGATTCAGCGGCAGCGGC AGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCAGG AGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCC TACACCTTCGGCGGAGGCACCAAGCTGGAGATCACCGGCGGAG GAGGAAGCGGAGGAGGAGGCAGCGGAGGAGGAGGCAGCGAGG TGAAGCTGCAGGAGAGCGGCCCCGGCCTGGTGGCCCCCAGCCA GAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCG ACTACGGCGTGAGCTGGATCAGACAGCCTCCCAGAAAGGGCCT GGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTAC AACAGCGCCCTGAAGAGCAGACTGACCATCATTAAGGACAACA GCAAGTCCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGAC GACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGG AAGCGCCGCTATGGACTACTGGGGCCAGGGAACCAGCGTGACC GTGAGCAGCGCGGCCGCTATCGAGGTGGAGCAGAAGCTGATCA GCGAGGAGGACCTGCTAGACAATGAGAAGAGCAATGGAACCAT TATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTC CCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGA GTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATT TTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACT ACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCAT TACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTC CAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAG CAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAA GAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCC TGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGG CCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTAC AGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGC ACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACC TACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA SEQIDNO:108 ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTG Nucleotidesequence CTGCACGCCGCTAGACCCGACATCCAGATGACCCAGACCACCAG encodingforY05 CAGCCTGAGCGCCAGCCTGGGCGACAGAGTGACCATCAGCTGC AGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGC AGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAG CAGACTGCACAGCGGCGTGCCCAGCAGATTCAGCGGCAGCGGC AGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCAGG AGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCC TACACCTTCGGCGGAGGCACCAAGCTGGAGATCACCGGCGGAG GAGGAAGCGGAGGAGGAGGCAGCGGAGGAGGAGGCAGCGAGG TGAAGCTGCAGGAGAGCGGCCCCGGCCTGGTGGCCCCCAGCCA GAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCG ACTACGGCGTGAGCTGGATCAGACAGCCTCCCAGAAAGGGCCT GGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTAC AACAGCGCCCTGAAGAGCAGACTGACCATCATTAAGGACAACA GCAAGTCCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGAC GACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGG AAGCTACGCTATGGACGCCTGGGGCCAGGGAACCAGCGTGACC GTGAGCAGCGCGGCCGCTATCGAGGTGGAGCAGAAGCTGATCA GCGAGGAGGACCTGCTAGACAATGAGAAGAGCAATGGAACCAT TATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTC CCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGA GTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATT TTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACT ACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCAT TACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTC CAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAG CAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAA GAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCC TGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGG CCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTAC AGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGC ACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACC TACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA SEQIDNO:109 ATGGGCCCAGGAGTTCTGCTGCTCCTGCTGGTGGCCACAGCTTG Nucleotidesequence GCATGGTCAGGGAGGTGACATCCAGATGACACAGACAACATCC encodingforFBB/G01 TCCCTCTCCGCTTCCCTCGGAGACAGGGTCACAATTTCCTGCAG GGCTTCCCAGGACATTTCCAAGTACCTCAACTGGTACCAGCAGA AGCCTGACGGAACAGTCAAGCTCCTCATTTACCACACATCCAGG CTCCACTCCGGAGTCCCTTCCAGGTTCTCCGGATCCGGATCCGG AACAGACTACTCCCTCACAATTTCCAACCTCGAGCAGGAGGACA TTGCTACATACTTCTGCCAGCAGGGAAACACACTCCCTTACACA TTCGGAGGAGGAACAAAGCTCGAGATTACAGGAGGTGGCGGTT CCGGTGGTGGAGGCTCCGGTGGTGGTGGATCTGAGGTCAAGCTC CAGGAGTCCGGACCTGGACTTGTCGCTCCTTCTCAGTCTCTCTCC GTCACATGCACAGTCTCCGGAGTCTCCCTCCCTGATTATGGAGT CTCCTGGATTAGGCAACCTCCTAGGAAAGGACTCGAATGGCTCG GAGTCATTTGGGGATCCGAAACAACATATTATAATTCCGCTCTC AAATCCAGGCTCACAATTATTAAAGATAATTCCAAATCCCAAGT CTTTCTCAAGATGAACTCCCTCCAGACAGACGACACAGCTATTT ACTACTGCGCTAAGCACTACTACTACGGGGGATCTTACGCTATG GACTACTGGGGACAGGGAACATCCGTCACAGTCTCTTCCGCTGC TGCTCCCACCACCACACCCGCTCCCAGACCCCCTACCCCTGCCC CCACCATCGCCAGCCAGCCCCTGAGCCTGAGACCCGAGGCCTGC AGACCTGCCGCCGGCGGAGCCGTGCACACCAGAGGCCTGGACT TCGCCTGCGACATCTACATCTGGGCTCCCCTGGCCGGCACCTGC GGCGTGCTGCTGCTGAGCCTGGTGATCACCCTGTACTGCAACCA CAAGAGAGGCAGAAAGAAGCTGCTGTACATCTTCAAGCAGCCC TTCATGAGACCCGTGCAGACCACCCAGGAGGAGGACGGCTGCA GCTGCAGATTCCCCGAGGAGGAGGAGGGCGGCTGCGAGCTGGA CTACAAAGACGATGACGACAAGCGGAGAAAGCGCAGCGGCTCC GGCGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGA AGAAAACCCCGGTCCCAACATGGCTCTGCCTGTGACAGCTCTGC TGCTGCCTCTGGCTCTGCTGCTGCACGCCGCTAGACCCGACATC CAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCG ACAGAGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAA GTACCTGAACTGGTACCAGCAGAAGCCCGACGGCACCGTGAAG CTGCTGATCTACCACACCAGCAGACTGCACAGCGGCGTGCCCAG CAGATTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACC ATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCA GCAGGGCAACACCCTGCCCTACACCTTCGGCGGAGGCACCAAG CTGGAGATCACCGGCGGAGGAGGAAGCGGAGGAGGAGGCAGC GGAGGAGGAGGCAGCGAGGTGAAGCTGCAGGAGAGCGGCCCC GGCCTGGTGGCCCCCAGCCAGAGCCTGAGCGTGACCTGCACCGT GAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCAGA CAGCCTCCCAGAAAGGGCCTGGAGTGGCTGGGCGTGATCTGGG GCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCAGACT GACCATCATTAAGGACAACAGCAAGTCCCAGGTGTTCCTGAAGA TGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCC AAGCACTACTACTACGCCGGAAGCTACGCTATGGACTACTGGGG CCAGGGAACCAGCGTGACCGTGAGCAGCGCGGCCGCTATCGAG GTGGAGCAGAAGCTGATCAGCGAGGAGGACCTGCTGGACAACG AGAAGAGCAACGGCACCATCATCCACGTGAAGGGCAAGCACCT GTGCCCCAGCCCCCTGTTCCCCGGCCCCAGCAAGCCCTTCTGGG TGCTGGTGGTGGTGGGCGGCGTGCTGGCCTGCTACAGCCTGCTG GTGACCGTGGCCTTCATCATCTTCTGGGTGCGGAGCAAGCGGAG CCGGCTGCTGCACAGCGACTACATGAACATGACCCCCCGGCGGC CTGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGC GACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGC AGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAAC GAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACA AGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAG GAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGAT AAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGC GCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAG TACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCC TGCCCCCTCGCTAA SEQIDNO:110 ATGGGCCCAGGAGTTCTGCTGCTCCTGCTGGTGGCCACAGCTTG Nucleotidesequence GCATGGTCAGGGAGGTGACATCCAGATGACACAGACAACATCC encodingforFBB/G02 TCCCTCTCCGCTTCCCTCGGAGACAGGGTCACAATTTCCTGCAG GGCTTCCCAGGACATTTCCAAGTACCTCAACTGGTACCAGCAGA AGCCTGACGGAACAGTCAAGCTCCTCATTTACCACACATCCAGG CTCCACTCCGGAGTCCCTTCCAGGTTCTCCGGATCCGGATCCGG AACAGACTACTCCCTCACAATTTCCAACCTCGAGCAGGAGGACA TTGCTACATACTTCTGCCAGCAGGGAAACACACTCCCTTACACA TTCGGAGGAGGAACAAAGCTCGAGATTACAGGAGGTGGCGGTT CCGGTGGTGGAGGCTCCGGTGGTGGTGGATCTGAGGTCAAGCTC CAGGAGTCCGGACCTGGACTTGTCGCTCCTTCTCAGTCTCTCTCC GTCACATGCACAGTCTCCGGAGTCTCCCTCCCTGATTATGGAGT CTCCTGGATTAGGCAACCTCCTAGGAAAGGACTCGAATGGCTCG GAGTCATTTGGGGATCCGAAACAACATATTATAATTCCGCTCTC AAATCCAGGCTCACAATTATTAAAGATAATTCCAAATCCCAAGT CTTTCTCAAGATGAACTCCCTCCAGACAGACGACACAGCTATTT ACTACTGCGCTAAGCACTACTACTACGGGGGATCTTACGCTATG GACTACTGGGGACAGGGAACATCCGTCACAGTCTCTTCCGCTGC TGCTCCCACCACCACACCCGCTCCCAGACCCCCTACCCCTGCCC CCACCATCGCCAGCCAGCCCCTGAGCCTGAGACCCGAGGCCTGC AGACCTGCCGCCGGCGGAGCCGTGCACACCAGAGGCCTGGACT TCGCCTGCGACATCTACATCTGGGCTCCCCTGGCCGGCACCTGC GGCGTGCTGCTGCTGAGCCTGGTGATCACCCTGTACTGCAACCA CAAGAGAGGCAGAAAGAAGCTGCTGTACATCTTCAAGCAGCCC TTCATGAGACCCGTGCAGACCACCCAGGAGGAGGACGGCTGCA GCTGCAGATTCCCCGAGGAGGAGGAGGGCGGCTGCGAGCTGGA CTACAAAGACGATGACGACAAGCGGAGAAAGCGCAGCGGCTCC GGCGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGA AGAAAACCCCGGTCCCAACATGGCTCTGCCTGTGACAGCTCTGC TGCTGCCTCTGGCTCTGCTGCTGCACGCCGCTAGACCCGACATC CAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCG ACAGAGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAA GTACCTGAACTGGTACCAGCAGAAGCCCGACGGCACCGTGAAG CTGCTGATCTACCACACCAGCAGACTGCACAGCGGCGTGCCCAG CAGATTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACC ATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCA GCAGGGCAACACCCTGCCCTACACCTTCGGCGGAGGCACCAAG CTGGAGATCACCGGCGGAGGAGGAAGCGGAGGAGGAGGCAGC GGAGGAGGAGGCAGCGAGGTGAAGCTGCAGGAGAGCGGCCCC GGCCTGGTGGCCCCCAGCCAGAGCCTGAGCGTGACCTGCACCGT GAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCAGA CAGCCTCCCAGAAAGGGCCTGGAGTGGCTGGGCGTGATCTGGG GCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCAGACT GACCATCATTAAGGACAACAGCAAGTCCCAGGTGTTCCTGAAGA TGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCC AAGCACTACTACTACGGCGCCAGCTACGCTATGGACTACTGGGG CCAGGGAACCAGCGTGACCGTGAGCAGCGCGGCCGCTATCGAG GTGGAGCAGAAGCTGATCAGCGAGGAGGACCTGCTGGACAACG AGAAGAGCAACGGCACCATCATCCACGTGAAGGGCAAGCACCT GTGCCCCAGCCCCCTGTTCCCCGGCCCCAGCAAGCCCTTCTGGG TGCTGGTGGTGGTGGGCGGCGTGCTGGCCTGCTACAGCCTGCTG GTGACCGTGGCCTTCATCATCTTCTGGGTGCGGAGCAAGCGGAG CCGGCTGCTGCACAGCGACTACATGAACATGACCCCCCGGCGGC CTGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGC GACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGC AGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAAC GAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACA AGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAG GAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGAT AAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGC GCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAG TACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCC TGCCCCCTCGCTAA SEQIDNO:111 ATGGGCCCAGGAGTTCTGCTGCTCCTGCTGGTGGCCACAGCTTG Nucleotidesequence GCATGGTCAGGGAGGTGACATCCAGATGACACAGACAACATCC encodingforFBB/Y01 TCCCTCTCCGCTTCCCTCGGAGACAGGGTCACAATTTCCTGCAG GGCTTCCCAGGACATTTCCAAGTACCTCAACTGGTACCAGCAGA AGCCTGACGGAACAGTCAAGCTCCTCATTTACCACACATCCAGG CTCCACTCCGGAGTCCCTTCCAGGTTCTCCGGATCCGGATCCGG AACAGACTACTCCCTCACAATTTCCAACCTCGAGCAGGAGGACA TTGCTACATACTTCTGCCAGCAGGGAAACACACTCCCTTACACA TTCGGAGGAGGAACAAAGCTCGAGATTACAGGAGGTGGCGGTT CCGGTGGTGGAGGCTCCGGTGGTGGTGGATCTGAGGTCAAGCTC CAGGAGTCCGGACCTGGACTTGTCGCTCCTTCTCAGTCTCTCTCC GTCACATGCACAGTCTCCGGAGTCTCCCTCCCTGATTATGGAGT CTCCTGGATTAGGCAACCTCCTAGGAAAGGACTCGAATGGCTCG GAGTCATTTGGGGATCCGAAACAACATATTATAATTCCGCTCTC AAATCCAGGCTCACAATTATTAAAGATAATTCCAAATCCCAAGT CTTTCTCAAGATGAACTCCCTCCAGACAGACGACACAGCTATTT ACTACTGCGCTAAGCACTACTACTACGGGGGATCTTACGCTATG GACTACTGGGGACAGGGAACATCCGTCACAGTCTCTTCCGCTGC TGCTCCCACCACCACACCCGCTCCCAGACCCCCTACCCCTGCCC CCACCATCGCCAGCCAGCCCCTGAGCCTGAGACCCGAGGCCTGC AGACCTGCCGCCGGCGGAGCCGTGCACACCAGAGGCCTGGACT TCGCCTGCGACATCTACATCTGGGCTCCCCTGGCCGGCACCTGC GGCGTGCTGCTGCTGAGCCTGGTGATCACCCTGTACTGCAACCA CAAGAGAGGCAGAAAGAAGCTGCTGTACATCTTCAAGCAGCCC TTCATGAGACCCGTGCAGACCACCCAGGAGGAGGACGGCTGCA GCTGCAGATTCCCCGAGGAGGAGGAGGGCGGCTGCGAGCTGGA CTACAAAGACGATGACGACAAGCGGAGAAAGCGCAGCGGCTCC GGCGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGA AGAAAACCCCGGTCCCAACATGGCTCTGCCTGTGACAGCTCTGC TGCTGCCTCTGGCTCTGCTGCTGCACGCCGCTAGACCCGACATC CAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCG ACAGAGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAA GTACCTGAACTGGTACCAGCAGAAGCCCGACGGCACCGTGAAG CTGCTGATCTACCACACCAGCAGACTGCACAGCGGCGTGCCCAG CAGATTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACC ATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCA GCAGGGCAACACCCTGCCCTACACCTTCGGCGGAGGCACCAAG CTGGAGATCACCGGCGGAGGAGGAAGCGGAGGAGGAGGCAGC GGAGGAGGAGGCAGCGAGGTGAAGCTGCAGGAGAGCGGCCCC GGCCTGGTGGCCCCCAGCCAGAGCCTGAGCGTGACCTGCACCGT GAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCAGA CAGCCTCCCAGAAAGGGCCTGGAGTGGCTGGGCGTGATCTGGG GCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCAGACT GACCATCATTAAGGACAACAGCAAGTCCCAGGTGTTCCTGAAGA TGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCC AAGCACGCCTACTACGGCGGAAGCTACGCTATGGACTACTGGG GCCAGGGAACCAGCGTGACCGTGAGCAGCGCGGCCGCTATCGA GGTGGAGCAGAAGCTGATCAGCGAGGAGGACCTGCTAGACAAT GAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACC TTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGG TGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTA GTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAG CAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCC CCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGC GACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGC AGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAAC GAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACA AGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAG GAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGAT AAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGC GCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAG TACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCC TGCCCCCTCGCTAA SEQIDNO:112 ATGGGCCCAGGAGTTCTGCTGCTCCTGCTGGTGGCCACAGCTTG Nucleotidesequence GCATGGTCAGGGAGGTGACATCCAGATGACACAGACAACATCC encodingforFBB/Y02 TCCCTCTCCGCTTCCCTCGGAGACAGGGTCACAATTTCCTGCAG GGCTTCCCAGGACATTTCCAAGTACCTCAACTGGTACCAGCAGA AGCCTGACGGAACAGTCAAGCTCCTCATTTACCACACATCCAGG CTCCACTCCGGAGTCCCTTCCAGGTTCTCCGGATCCGGATCCGG AACAGACTACTCCCTCACAATTTCCAACCTCGAGCAGGAGGACA TTGCTACATACTTCTGCCAGCAGGGAAACACACTCCCTTACACA TTCGGAGGAGGAACAAAGCTCGAGATTACAGGAGGTGGCGGTT CCGGTGGTGGAGGCTCCGGTGGTGGTGGATCTGAGGTCAAGCTC CAGGAGTCCGGACCTGGACTTGTCGCTCCTTCTCAGTCTCTCTCC GTCACATGCACAGTCTCCGGAGTCTCCCTCCCTGATTATGGAGT CTCCTGGATTAGGCAACCTCCTAGGAAAGGACTCGAATGGCTCG GAGTCATTTGGGGATCCGAAACAACATATTATAATTCCGCTCTC AAATCCAGGCTCACAATTATTAAAGATAATTCCAAATCCCAAGT CTTTCTCAAGATGAACTCCCTCCAGACAGACGACACAGCTATTT ACTACTGCGCTAAGCACTACTACTACGGGGGATCTTACGCTATG GACTACTGGGGACAGGGAACATCCGTCACAGTCTCTTCCGCTGC TGCTCCCACCACCACACCCGCTCCCAGACCCCCTACCCCTGCCC CCACCATCGCCAGCCAGCCCCTGAGCCTGAGACCCGAGGCCTGC AGACCTGCCGCCGGCGGAGCCGTGCACACCAGAGGCCTGGACT TCGCCTGCGACATCTACATCTGGGCTCCCCTGGCCGGCACCTGC GGCGTGCTGCTGCTGAGCCTGGTGATCACCCTGTACTGCAACCA CAAGAGAGGCAGAAAGAAGCTGCTGTACATCTTCAAGCAGCCC TTCATGAGACCCGTGCAGACCACCCAGGAGGAGGACGGCTGCA GCTGCAGATTCCCCGAGGAGGAGGAGGGCGGCTGCGAGCTGGA CTACAAAGACGATGACGACAAGCGGAGAAAGCGCAGCGGCTCC GGCGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGA AGAAAACCCCGGTCCCAACATGGCTCTGCCTGTGACAGCTCTGC TGCTGCCTCTGGCTCTGCTGCTGCACGCCGCTAGACCCGACATC CAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCG ACAGAGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAA GTACCTGAACTGGTACCAGCAGAAGCCCGACGGCACCGTGAAG CTGCTGATCTACCACACCAGCAGACTGCACAGCGGCGTGCCCAG CAGATTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACC ATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCA GCAGGGCAACACCCTGCCCTACACCTTCGGCGGAGGCACCAAG CTGGAGATCACCGGCGGAGGAGGAAGCGGAGGAGGAGGCAGC GGAGGAGGAGGCAGCGAGGTGAAGCTGCAGGAGAGCGGCCCC GGCCTGGTGGCCCCCAGCCAGAGCCTGAGCGTGACCTGCACCGT GAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCAGA CAGCCTCCCAGAAAGGGCCTGGAGTGGCTGGGCGTGATCTGGG GCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCAGACT GACCATCATTAAGGACAACAGCAAGTCCCAGGTGTTCCTGAAGA TGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCC AAGCACTACGCCTACGGCGGAAGCTACGCTATGGACTACTGGG GCCAGGGAACCAGCGTGACCGTGAGCAGCGCGGCCGCTATCGA GGTGGAGCAGAAGCTGATCAGCGAGGAGGACCTGCTGGACAAC GAGAAGAGCAACGGCACCATCATCCACGTGAAGGGCAAGCACC TGTGCCCCAGCCCCCTGTTCCCCGGCCCCAGCAAGCCCTTCTGG GTGCTGGTGGTGGTGGGCGGCGTGCTGGCCTGCTACAGCCTGCT GGTGACCGTGGCCTTCATCATCTTCTGGGTGCGGAGCAAGCGGA GCCGGCTGCTGCACAGCGACTACATGAACATGACCCCCCGGCGG CCTGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACG CGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCG CAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAA CGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGAC AAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAA GGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGA TAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAG CGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCA GTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCC CTGCCCCCTCGCTAA SEQIDNO:113 ATGGGCCCAGGAGTTCTGCTGCTCCTGCTGGTGGCCACAGCTTG Nucleotidesequence GCATGGTCAGGGAGGTGACATCCAGATGACACAGACAACATCC encodingforFBB/Y03 TCCCTCTCCGCTTCCCTCGGAGACAGGGTCACAATTTCCTGCAG GGCTTCCCAGGACATTTCCAAGTACCTCAACTGGTACCAGCAGA AGCCTGACGGAACAGTCAAGCTCCTCATTTACCACACATCCAGG CTCCACTCCGGAGTCCCTTCCAGGTTCTCCGGATCCGGATCCGG AACAGACTACTCCCTCACAATTTCCAACCTCGAGCAGGAGGACA TTGCTACATACTTCTGCCAGCAGGGAAACACACTCCCTTACACA TTCGGAGGAGGAACAAAGCTCGAGATTACAGGAGGTGGCGGTT CCGGTGGTGGAGGCTCCGGTGGTGGTGGATCTGAGGTCAAGCTC CAGGAGTCCGGACCTGGACTTGTCGCTCCTTCTCAGTCTCTCTCC GTCACATGCACAGTCTCCGGAGTCTCCCTCCCTGATTATGGAGT CTCCTGGATTAGGCAACCTCCTAGGAAAGGACTCGAATGGCTCG GAGTCATTTGGGGATCCGAAACAACATATTATAATTCCGCTCTC AAATCCAGGCTCACAATTATTAAAGATAATTCCAAATCCCAAGT CTTTCTCAAGATGAACTCCCTCCAGACAGACGACACAGCTATTT ACTACTGCGCTAAGCACTACTACTACGGGGGATCTTACGCTATG GACTACTGGGGACAGGGAACATCCGTCACAGTCTCTTCCGCTGC TGCTCCCACCACCACACCCGCTCCCAGACCCCCTACCCCTGCCC CCACCATCGCCAGCCAGCCCCTGAGCCTGAGACCCGAGGCCTGC AGACCTGCCGCCGGCGGAGCCGTGCACACCAGAGGCCTGGACT TCGCCTGCGACATCTACATCTGGGCTCCCCTGGCCGGCACCTGC GGCGTGCTGCTGCTGAGCCTGGTGATCACCCTGTACTGCAACCA CAAGAGAGGCAGAAAGAAGCTGCTGTACATCTTCAAGCAGCCC TTCATGAGACCCGTGCAGACCACCCAGGAGGAGGACGGCTGCA GCTGCAGATTCCCCGAGGAGGAGGAGGGCGGCTGCGAGCTGGA CTACAAAGACGATGACGACAAGCGGAGAAAGCGCAGCGGCTCC GGCGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGA AGAAAACCCCGGTCCCAACATGGCTCTGCCTGTGACAGCTCTGC TGCTGCCTCTGGCTCTGCTGCTGCACGCCGCTAGACCCGACATC CAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCG ACAGAGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAA GTACCTGAACTGGTACCAGCAGAAGCCCGACGGCACCGTGAAG CTGCTGATCTACCACACCAGCAGACTGCACAGCGGCGTGCCCAG CAGATTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACC ATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCA GCAGGGCAACACCCTGCCCTACACCTTCGGCGGAGGCACCAAG CTGGAGATCACCGGCGGAGGAGGAAGCGGAGGAGGAGGCAGC GGAGGAGGAGGCAGCGAGGTGAAGCTGCAGGAGAGCGGCCCC GGCCTGGTGGCCCCCAGCCAGAGCCTGAGCGTGACCTGCACCGT GAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCAGA CAGCCTCCCAGAAAGGGCCTGGAGTGGCTGGGCGTGATCTGGG GCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCAGACT GACCATCATTAAGGACAACAGCAAGTCCCAGGTGTTCCTGAAGA TGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCC AAGCACTACTACGCCGGCGGAAGCTACGCTATGGACTACTGGG GCCAGGGAACCAGCGTGACCGTGAGCAGCGCGGCCGCTATCGA GGTGGAGCAGAAGCTGATCAGCGAGGAGGACCTGCTGGACAAC GAGAAGAGCAACGGCACCATCATCCACGTGAAGGGCAAGCACC TGTGCCCCAGCCCCCTGTTCCCCGGCCCCAGCAAGCCCTTCTGG GTGCTGGTGGTGGTGGGCGGCGTGCTGGCCTGCTACAGCCTGCT GGTGACCGTGGCCTTCATCATCTTCTGGGTGCGGAGCAAGCGGA GCCGGCTGCTGCACAGCGACTACATGAACATGACCCCCCGGCGG CCTGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACG CGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCG CAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAA CGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGAC AAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAA GGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGA TAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAG CGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCA GTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCC CTGCCCCCTCGCTAA SEQIDNO:114 ATGGGCCCAGGAGTTCTGCTGCTCCTGCTGGTGGCCACAGCTTG Nucleotidesequence GCATGGTCAGGGAGGTGACATCCAGATGACACAGACAACATCC encodingforFBB/Y04 TCCCTCTCCGCTTCCCTCGGAGACAGGGTCACAATTTCCTGCAG GGCTTCCCAGGACATTTCCAAGTACCTCAACTGGTACCAGCAGA AGCCTGACGGAACAGTCAAGCTCCTCATTTACCACACATCCAGG CTCCACTCCGGAGTCCCTTCCAGGTTCTCCGGATCCGGATCCGG AACAGACTACTCCCTCACAATTTCCAACCTCGAGCAGGAGGACA TTGCTACATACTTCTGCCAGCAGGGAAACACACTCCCTTACACA TTCGGAGGAGGAACAAAGCTCGAGATTACAGGAGGTGGCGGTT CCGGTGGTGGAGGCTCCGGTGGTGGTGGATCTGAGGTCAAGCTC CAGGAGTCCGGACCTGGACTTGTCGCTCCTTCTCAGTCTCTCTCC GTCACATGCACAGTCTCCGGAGTCTCCCTCCCTGATTATGGAGT CTCCTGGATTAGGCAACCTCCTAGGAAAGGACTCGAATGGCTCG GAGTCATTTGGGGATCCGAAACAACATATTATAATTCCGCTCTC AAATCCAGGCTCACAATTATTAAAGATAATTCCAAATCCCAAGT CTTTCTCAAGATGAACTCCCTCCAGACAGACGACACAGCTATTT ACTACTGCGCTAAGCACTACTACTACGGGGGATCTTACGCTATG GACTACTGGGGACAGGGAACATCCGTCACAGTCTCTTCCGCTGC TGCTCCCACCACCACACCCGCTCCCAGACCCCCTACCCCTGCCC CCACCATCGCCAGCCAGCCCCTGAGCCTGAGACCCGAGGCCTGC AGACCTGCCGCCGGCGGAGCCGTGCACACCAGAGGCCTGGACT TCGCCTGCGACATCTACATCTGGGCTCCCCTGGCCGGCACCTGC GGCGTGCTGCTGCTGAGCCTGGTGATCACCCTGTACTGCAACCA CAAGAGAGGCAGAAAGAAGCTGCTGTACATCTTCAAGCAGCCC TTCATGAGACCCGTGCAGACCACCCAGGAGGAGGACGGCTGCA GCTGCAGATTCCCCGAGGAGGAGGAGGGCGGCTGCGAGCTGGA CTACAAAGACGATGACGACAAGCGGAGAAAGCGCAGCGGCTCC GGCGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGA AGAAAACCCCGGTCCCAACATGGCTCTGCCTGTGACAGCTCTGC TGCTGCCTCTGGCTCTGCTGCTGCACGCCGCTAGACCCGACATC CAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCG ACAGAGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAA GTACCTGAACTGGTACCAGCAGAAGCCCGACGGCACCGTGAAG CTGCTGATCTACCACACCAGCAGACTGCACAGCGGCGTGCCCAG CAGATTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACC ATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCA GCAGGGCAACACCCTGCCCTACACCTTCGGCGGAGGCACCAAG CTGGAGATCACCGGCGGAGGAGGAAGCGGAGGAGGAGGCAGC GGAGGAGGAGGCAGCGAGGTGAAGCTGCAGGAGAGCGGCCCC GGCCTGGTGGCCCCCAGCCAGAGCCTGAGCGTGACCTGCACCGT GAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCAGA CAGCCTCCCAGAAAGGGCCTGGAGTGGCTGGGCGTGATCTGGG GCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCAGACT GACCATCATTAAGGACAACAGCAAGTCCCAGGTGTTCCTGAAGA TGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCC AAGCACTACTACTACGGCGGAAGCGCCGCTATGGACTACTGGG GCCAGGGAACCAGCGTGACCGTGAGCAGCGCGGCCGCTATCGA GGTGGAGCAGAAGCTGATCAGCGAGGAGGACCTGCTGGACAAC GAGAAGAGCAACGGCACCATCATCCACGTGAAGGGCAAGCACC TGTGCCCCAGCCCCCTGTTCCCCGGCCCCAGCAAGCCCTTCTGG GTGCTGGTGGTGGTGGGCGGCGTGCTGGCCTGCTACAGCCTGCT GGTGACCGTGGCCTTCATCATCTTCTGGGTGCGGAGCAAGCGGA GCCGGCTGCTGCACAGCGACTACATGAACATGACCCCCCGGCGG CCTGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACG CGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCG CAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAA CGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGAC AAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAA GGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGA TAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAG CGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCA GTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCC CTGCCCCCTCGCTAA SEQIDNO:115 ATGGGCCCAGGAGTTCTGCTGCTCCTGCTGGTGGCCACAGCTTG Nucleotidesequence GCATGGTCAGGGAGGTGACATCCAGATGACACAGACAACATCC encodingforFBB/Y05 TCCCTCTCCGCTTCCCTCGGAGACAGGGTCACAATTTCCTGCAG GGCTTCCCAGGACATTTCCAAGTACCTCAACTGGTACCAGCAGA AGCCTGACGGAACAGTCAAGCTCCTCATTTACCACACATCCAGG CTCCACTCCGGAGTCCCTTCCAGGTTCTCCGGATCCGGATCCGG AACAGACTACTCCCTCACAATTTCCAACCTCGAGCAGGAGGACA TTGCTACATACTTCTGCCAGCAGGGAAACACACTCCCTTACACA TTCGGAGGAGGAACAAAGCTCGAGATTACAGGAGGTGGCGGTT CCGGTGGTGGAGGCTCCGGTGGTGGTGGATCTGAGGTCAAGCTC CAGGAGTCCGGACCTGGACTTGTCGCTCCTTCTCAGTCTCTCTCC GTCACATGCACAGTCTCCGGAGTCTCCCTCCCTGATTATGGAGT CTCCTGGATTAGGCAACCTCCTAGGAAAGGACTCGAATGGCTCG GAGTCATTTGGGGATCCGAAACAACATATTATAATTCCGCTCTC AAATCCAGGCTCACAATTATTAAAGATAATTCCAAATCCCAAGT CTTTCTCAAGATGAACTCCCTCCAGACAGACGACACAGCTATTT ACTACTGCGCTAAGCACTACTACTACGGGGGATCTTACGCTATG GACTACTGGGGACAGGGAACATCCGTCACAGTCTCTTCCGCTGC TGCTCCCACCACCACACCCGCTCCCAGACCCCCTACCCCTGCCC CCACCATCGCCAGCCAGCCCCTGAGCCTGAGACCCGAGGCCTGC AGACCTGCCGCCGGCGGAGCCGTGCACACCAGAGGCCTGGACT TCGCCTGCGACATCTACATCTGGGCTCCCCTGGCCGGCACCTGC GGCGTGCTGCTGCTGAGCCTGGTGATCACCCTGTACTGCAACCA CAAGAGAGGCAGAAAGAAGCTGCTGTACATCTTCAAGCAGCCC TTCATGAGACCCGTGCAGACCACCCAGGAGGAGGACGGCTGCA GCTGCAGATTCCCCGAGGAGGAGGAGGGCGGCTGCGAGCTGGA CTACAAAGACGATGACGACAAGCGGAGAAAGCGCAGCGGCTCC GGCGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGA AGAAAACCCCGGTCCCAACATGGCTCTGCCTGTGACAGCTCTGC TGCTGCCTCTGGCTCTGCTGCTGCACGCCGCTAGACCCGACATC CAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCG ACAGAGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAA GTACCTGAACTGGTACCAGCAGAAGCCCGACGGCACCGTGAAG CTGCTGATCTACCACACCAGCAGACTGCACAGCGGCGTGCCCAG CAGATTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACC ATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCA GCAGGGCAACACCCTGCCCTACACCTTCGGCGGAGGCACCAAG CTGGAGATCACCGGCGGAGGAGGAAGCGGAGGAGGAGGCAGC GGAGGAGGAGGCAGCGAGGTGAAGCTGCAGGAGAGCGGCCCC GGCCTGGTGGCCCCCAGCCAGAGCCTGAGCGTGACCTGCACCGT GAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCAGA CAGCCTCCCAGAAAGGGCCTGGAGTGGCTGGGCGTGATCTGGG GCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCAGACT GACCATCATTAAGGACAACAGCAAGTCCCAGGTGTTCCTGAAGA TGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCC AAGCACTACTACTACGGCGGAAGCTACGCTATGGACGCCTGGG GCCAGGGAACCAGCGTGACCGTGAGCAGCGCGGCCGCTATCGA GGTGGAGCAGAAGCTGATCAGCGAGGAGGACCTGCTGGACAAC GAGAAGAGCAACGGCACCATCATCCACGTGAAGGGCAAGCACC TGTGCCCCAGCCCCCTGTTCCCCGGCCCCAGCAAGCCCTTCTGG GTGCTGGTGGTGGTGGGCGGCGTGCTGGCCTGCTACAGCCTGCT GGTGACCGTGGCCTTCATCATCTTCTGGGTGCGGAGCAAGCGGA GCCGGCTGCTGCACAGCGACTACATGAACATGACCCCCCGGCGG CCTGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACG CGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCG CAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAA CGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGAC AAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAA GGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGA TAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAG CGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCA GTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCC CTGCCCCCTCGCTAA SEQIDNO:116 ATGGGACCTGGCGTGCTGCTGCTGCTGCTGGTGGCCACCGCTTG Nucleotidesequence GCATGGACAGGGAGGACAGATCGTGCTGAGCCAGAGCCCTGCC encodingfor1BB/F ATCCTGAGCGCCAGCCCTGGCGAGAAGGTGACCATGACCTGCA GAGCCAGCTCCAGCCTGAGCTTCATGCACTGGTACCAGCAGAAG CCCGGCAGCAGCCCTAAGCCCTGGATCTACGCCACCAGCAACCT GGCCAGCGGCGTGCCCGCCAGATTCAGCGGCAGCGGCAGCGGC ACCAGCTACAGCCTGACCATCAGCAGAGTGGAGGCCGAGGACG CCGCCACCTACTTCTGCCACCAGTGGAGCAGCAACCCCCTGACC TTCGGCGCCGGCACCAAGGTGGAGATCAAGAGAAAGGGCAGCA CCAGCGGAAGCGGCAAGCCTGGAAGCGGAGAGGGCAGCACCAA GGGACAGGTGCAGCTGAGACAGCCTGGCGCCGAGCTGGTGAAG CCCGGCGCCAGCGTGAAGATGAGCTGCAAGGCCAGCGGCTACA CCTTCACCAGCTACAACATGCACTGGGTGAAGCAGACCCCTGGC CAGGGCCTGGAGTGGATCGGCGCCATCTATCCCGGCAACGGCG ACACCAGCTACAACCAGAAGTTCAAGGGCAAGGCCACCCTGAC CGCCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGC CTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGAAGCC ACTACGGCAGCAACTACGTGGACTACTTCGACTACTGGGGCCAG GGCACCCTGGTGACCGTGAGCACCGGCGCGGCCGCCCCCACCAC CACACCCGCTCCCAGACCCCCTACCCCTGCCCCCACCATCGCCA GCCAGCCCCTGAGCCTGAGACCCGAGGCCTGCAGACCTGCCGCC GGCGGAGCCGTGCACACCAGAGGCCTGGACTTCGCCTGCGACAT CTACATCTGGGCTCCCCTGGCCGGCACCTGCGGCGTGCTGCTGC TGAGCCTGGTGATCACCCTGTACTGCAACCACAAGAGAGGCAG AAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCCG TGCAGACCACCCAGGAGGAGGACGGCTGCAGCTGCAGATTCCC CGAGGAGGAGGAGGGCGGCTGCGAGCTGGACTACAAAGACGAT GACGACAAGAGAAGAAAGAGAAGCGGCAGCGGCGCCACGAAC TTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCG GTCCCAACATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGG CTCTGCTGCTGCACGCCGCTAGACCCGACATCCAGATGACCCAG ACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACAGAGTGACCA TCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTG GTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACC ACACCAGCAGACTGCACAGCGGCGTGCCCAGCAGATTCAGCGG CAGCGGCAGCGGCACCGACTACAGCCTGACCATCAGCAACCTG GAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACA CCCTGCCCTACACCTTCGGCGGAGGCACCAAGCTGGAGATCACC GGCGGAGGAGGAAGCGGAGGAGGAGGCAGCGGAGGAGGAGGC AGCGAGGTGAAGCTGCAGGAGAGCGGCCCCGGCCTGGTGGCCC CCAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAG CCTGCCCGACTACGGCGTGAGCTGGATCAGACAGCCTCCCAGAA AGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCAC CTACTACAACAGCGCCCTGAAGAGCAGACTGACCATCATTAAGG ACAACAGCAAGTCCCAGGTGTTCCTGAAGATGAACAGCCTGCA GACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACT ACGGCGGAAGCTACGCTATGGACTACTGGGGCCAGGGAACCAG CGTGACCGTGAGCAGCGCGGCCGCTATCGAGGTGGAGCAGAAG CTGATCAGCGAGGAGGACCTGCTAGACAATGAGAAGAGCAATG GAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCC CTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTT GGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTT TATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACA GTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGC AAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTA TCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGT ACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGG ACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGG GACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGG AAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGC CTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAG GGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGA CACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA SEQIDNO:117 ATGGGCCCTGGAGTGCTGCTGCTCCTGCTGGTGGCCACAGCTTG Nucleotidesequence GCACGGCCAGGGAGATATCCAGATGACCCAGACTACATCCTCCC encodingforRBB/F TGTCTGCCTCTCTGGGAGACAGAGTCACCATTAGTTGCAGGGCA AGTCAGGACATTAGCAATTATTTAAACTGGTATCAGCAGAAACC AGATGGAACTGTTAAACTCCTGATCTACTACACATCAATATTAC ACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACA GATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATTTTGC CACTTACTTTTGCCAACAGGGTAATACGCTTCCGTGGACGTTCG GTGGAGGCACCAAGCTGGAAATCAAAGGCAGCACCAGCGGAAG CGGCAAGCCTGGAAGCGGAGAGGGCAGCACCAAGGGAGAAGT GCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGG TCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCGCTTTCAGTATC TATGACATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGA GTGGGTCGCATACATTAGTAGTGGTGGTGGTACCACCTACTATC CAGACACTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCC AAGAACACCCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAGG ACACAGCCATGTATTACTGTGCAAGACATAGTGGCTACGGTAGT AGCTACGGGGTTTTGTTTGCTTACTGGGGCCAAGGGACTCTGGT CACTGTCTCTGCCGCGGCCGCCCCCACCACCACACCCGCTCCCA GACCCCCTACCCCTGCCCCCACCATCGCCAGCCAGCCCCTGAGC CTGAGACCCGAGGCCTGCAGACCTGCCGCCGGCGGAGCCGTGC ACACCAGAGGCCTGGACTTCGCCTGCGACATCTACATCTGGGCT CCCCTGGCCGGCACCTGCGGCGTGCTGCTGCTGAGCCTGGTGAT CACCCTGTACTGCAACCACAAGAGAGGCAGAAAGAAGCTGCTG TACATCTTCAAGCAGCCCTTCATGAGACCCGTGCAGACCACCCA GGAGGAGGACGGCTGCAGCTGCAGATTCCCCGAGGAGGAGGAG GGCGGCTGCGAGCTGGACTACAAAGACGATGACGACAAGCGGA GAAAGCGCAGCGGCTCCGGCGCCACGAACTTCTCTCTGTTAAAG CAAGCAGGAGACGTGGAAGAAAACCCCGGTCCCAACATGGCTC TGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTGCTGCACG CCGCTAGACCCGACATCCAGATGACCCAGACCACCAGCAGCCTG AGCGCCAGCCTGGGCGACAGAGTGACCATCAGCTGCAGAGCCA GCCAGGACATCAGCAAGTACCTGAACTGGTACCAGCAGAAGCC CGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACTGC ACAGCGGCGTGCCCAGCAGATTCAGCGGCAGCGGCAGCGGCAC CGACTACAGCCTGACCATCAGCAACCTGGAGCAGGAGGACATC GCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCCTACACCTT CGGCGGAGGCACCAAGCTGGAGATCACCGGCGGAGGAGGAAGC GGAGGAGGAGGCAGCGGAGGAGGAGGCAGCGAGGTGAAGCTG CAGGAGAGCGGCCCCGGCCTGGTGGCCCCCAGCCAGAGCCTGA GCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGC GTGAGCTGGATCAGACAGCCTCCCAGAAAGGGCCTGGAGTGGC TGGGCGTGATCTGGGGCAGCGAGACCACCTACTACAACAGCGC CCTGAAGAGCAGACTGACCATCATTAAGGACAACAGCAAGTCC CAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCG CCATCTACTACTGCGCCAAGCACTACTACTACGGCGGAAGCTAC GCTATGGACTACTGGGGCCAGGGAACCAGCGTGACCGTGAGCA GCGCGGCCGCTATCGAGGTGGAGCAGAAGCTGATCAGCGAGGA GGACCTGCTAGACAATGAGAAGAGCAATGGAACCATTATCCAT GTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACC TTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGC TTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGT GAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAAC ATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCC CTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGA AGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCA GAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAG TACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGG GGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAA TGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATT GGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGC CTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGC CCTTCACATGCAGGCCCTGCCCCCTCGCTAA SEQIDNO:118 ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTG Nucleotidesequence CTGCACGCCGCTAGACCCGACATCCAGATGACCCAGACCACCAG encodingforFMC63 CAGCCTGAGCGCCAGCCTGGGCGACAGAGTGACCATCAGCTGC scFvusedinCARs AGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGC andpCARs AGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAG CAGACTGCACAGCGGCGTGCCCAGCAGATTCAGCGGCAGCGGC AGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCAGG AGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCC TACACCTTCGGCGGAGGCACCAAGCTGGAGATCACCGGCGGAG GAGGAAGCGGAGGAGGAGGCAGCGGAGGAGGAGGCAGCGAGG TGAAGCTGCAGGAGAGCGGCCCCGGCCTGGTGGCCCCCAGCCA GAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCG ACTACGGCGTGAGCTGGATCAGACAGCCTCCCAGAAAGGGCCT GGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTAC AACAGCGCCCTGAAGAGCAGACTGACCATCATTAAGGACAACA GCAAGTCCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGAC GACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGG AAGCTACGCTATGGACTACTGGGGCCAGGGAACCAGCGTGACC GTGAGCAGCG SEQIDNO:119 ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTG Nucleotidesequence CTGCACGCCGCTAGACCCGACATCCAGATGACCCAGACCACCAG encodingforFMC63 CAGCCTGAGCGCCAGCCTGGGCGACAGAGTGACCATCAGCTGC scFvcontainingV.sub.H AGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGC CDR3G01mutation AGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAG usedinCARsand CAGACTGCACAGCGGCGTGCCCAGCAGATTCAGCGGCAGCGGC pCARs AGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCAGG AGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCC TACACCTTCGGCGGAGGCACCAAGCTGGAGATCACCGGCGGAG GAGGAAGCGGAGGAGGAGGCAGCGGAGGAGGAGGCAGCGAGG TGAAGCTGCAGGAGAGCGGCCCCGGCCTGGTGGCCCCCAGCCA GAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCG ACTACGGCGTGAGCTGGATCAGACAGCCTCCCAGAAAGGGCCT GGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTAC AACAGCGCCCTGAAGAGCAGACTGACCATCATTAAGGACAACA GCAAGTCCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGAC GACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGCCGG AAGCTACGCTATGGACTACTGGGGCCAGGGAACCAGCGTGACC GTGAGCAGC SEQIDNO:120 ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTG Nucleotidesequence CTGCACGCCGCTAGACCCGACATCCAGATGACCCAGACCACCAG encodingforFMC63 CAGCCTGAGCGCCAGCCTGGGCGACAGAGTGACCATCAGCTGC scFvcontainingV.sub.H AGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGC CDR3G02mutation AGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAG usedinCARsand CAGACTGCACAGCGGCGTGCCCAGCAGATTCAGCGGCAGCGGC pCARs AGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCAGG AGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCC TACACCTTCGGCGGAGGCACCAAGCTGGAGATCACCGGCGGAG GAGGAAGCGGAGGAGGAGGCAGCGGAGGAGGAGGCAGCGAGG TGAAGCTGCAGGAGAGCGGCCCCGGCCTGGTGGCCCCCAGCCA GAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCG ACTACGGCGTGAGCTGGATCAGACAGCCTCCCAGAAAGGGCCT GGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTAC AACAGCGCCCTGAAGAGCAGACTGACCATCATTAAGGACAACA GCAAGTCCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGAC GACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGC CAGCTACGCTATGGACTACTGGGGCCAGGGAACCAGCGTGACC GTGAGCAGC SEQIDNO:121 ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTG Nucleotidesequence CTGCACGCCGCTAGACCCGACATCCAGATGACCCAGACCACCAG encodingforFMC63 CAGCCTGAGCGCCAGCCTGGGCGACAGAGTGACCATCAGCTGC scFvcontainingV.sub.H AGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGC CDR3Y01mutation AGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAG usedinCARsand CAGACTGCACAGCGGCGTGCCCAGCAGATTCAGCGGCAGCGGC pCARs AGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCAGG AGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCC TACACCTTCGGCGGAGGCACCAAGCTGGAGATCACCGGCGGAG GAGGAAGCGGAGGAGGAGGCAGCGGAGGAGGAGGCAGCGAGG TGAAGCTGCAGGAGAGCGGCCCCGGCCTGGTGGCCCCCAGCCA GAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCG ACTACGGCGTGAGCTGGATCAGACAGCCTCCCAGAAAGGGCCT GGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTAC AACAGCGCCCTGAAGAGCAGACTGACCATCATTAAGGACAACA GCAAGTCCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGAC GACACCGCCATCTACTACTGCGCCAAGCACGCCTACTACGGCGG AAGCTACGCTATGGACTACTGGGGCCAGGGAACCAGCGTGACC GTGAGCAGC SEQIDNO:122 ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTG Nucleotidesequence CTGCACGCCGCTAGACCCGACATCCAGATGACCCAGACCACCAG encodingforFMC63 CAGCCTGAGCGCCAGCCTGGGCGACAGAGTGACCATCAGCTGC scFvcontainingV.sub.H AGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGC CDR3Y02mutation AGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAG usedinCARsand CAGACTGCACAGCGGCGTGCCCAGCAGATTCAGCGGCAGCGGC pCARs AGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCAGG AGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCC TACACCTTCGGCGGAGGCACCAAGCTGGAGATCACCGGCGGAG GAGGAAGCGGAGGAGGAGGCAGCGGAGGAGGAGGCAGCGAGG TGAAGCTGCAGGAGAGCGGCCCCGGCCTGGTGGCCCCCAGCCA GAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCG ACTACGGCGTGAGCTGGATCAGACAGCCTCCCAGAAAGGGCCT GGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTAC AACAGCGCCCTGAAGAGCAGACTGACCATCATTAAGGACAACA GCAAGTCCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGAC GACACCGCCATCTACTACTGCGCCAAGCACTACGCCTACGGCGG AAGCTACGCTATGGACTACTGGGGCCAGGGAACCAGCGTGACC GTGAGCAGC SEQIDNO:123 ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTG Nucleotidesequence CTGCACGCCGCTAGACCCGACATCCAGATGACCCAGACCACCAG encodingforFMC63 CAGCCTGAGCGCCAGCCTGGGCGACAGAGTGACCATCAGCTGC scFvcontainingV.sub.H AGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGC CDR3Y03mutation AGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAG usedinCARsand CAGACTGCACAGCGGCGTGCCCAGCAGATTCAGCGGCAGCGGC pCARs AGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCAGG AGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCC TACACCTTCGGCGGAGGCACCAAGCTGGAGATCACCGGCGGAG GAGGAAGCGGAGGAGGAGGCAGCGGAGGAGGAGGCAGCGAGG TGAAGCTGCAGGAGAGCGGCCCCGGCCTGGTGGCCCCCAGCCA GAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCG ACTACGGCGTGAGCTGGATCAGACAGCCTCCCAGAAAGGGCCT GGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTAC AACAGCGCCCTGAAGAGCAGACTGACCATCATTAAGGACAACA GCAAGTCCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGAC GACACCGCCATCTACTACTGCGCCAAGCACTACTACGCCGGCGG AAGCTACGCTATGGACTACTGGGGCCAGGGAACCAGCGTGACC GTGAGCAGC SEQIDNO:124 ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTG Nucleotidesequence CTGCACGCCGCTAGACCCGACATCCAGATGACCCAGACCACCAG encodingforFMC63 CAGCCTGAGCGCCAGCCTGGGCGACAGAGTGACCATCAGCTGC scFvcontainingV.sub.H AGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGC CDR3Y04mutation AGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAG usedinCARsand CAGACTGCACAGCGGCGTGCCCAGCAGATTCAGCGGCAGCGGC pCARs AGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCAGG AGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCC TACACCTTCGGCGGAGGCACCAAGCTGGAGATCACCGGCGGAG GAGGAAGCGGAGGAGGAGGCAGCGGAGGAGGAGGCAGCGAGG TGAAGCTGCAGGAGAGCGGCCCCGGCCTGGTGGCCCCCAGCCA GAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCG ACTACGGCGTGAGCTGGATCAGACAGCCTCCCAGAAAGGGCCT GGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTAC AACAGCGCCCTGAAGAGCAGACTGACCATCATTAAGGACAACA GCAAGTCCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGAC GACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGG AAGCGCCGCTATGGACTACTGGGGCCAGGGAACCAGCGTGACC GTGAGCAGC SEQIDNO:125 ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTG Nucleotidesequence CTGCACGCCGCTAGACCCGACATCCAGATGACCCAGACCACCAG encodingforFMC63 CAGCCTGAGCGCCAGCCTGGGCGACAGAGTGACCATCAGCTGC scFvcontainingV.sub.H AGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGC CDR3Y05mutation AGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAG usedinCARsand CAGACTGCACAGCGGCGTGCCCAGCAGATTCAGCGGCAGCGGC pCARs AGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCAGG AGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCC TACACCTTCGGCGGAGGCACCAAGCTGGAGATCACCGGCGGAG GAGGAAGCGGAGGAGGAGGCAGCGGAGGAGGAGGCAGCGAGG TGAAGCTGCAGGAGAGCGGCCCCGGCCTGGTGGCCCCCAGCCA GAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCG ACTACGGCGTGAGCTGGATCAGACAGCCTCCCAGAAAGGGCCT GGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTAC AACAGCGCCCTGAAGAGCAGACTGACCATCATTAAGGACAACA GCAAGTCCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGAC GACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGG AAGCTACGCTATGGACGCCTGGGGCCAGGGAACCAGCGTGACC GTGAGCAGC SEQIDNO:126 ATGGGACCTGGCGTGCTGCTGCTGCTGCTGGTGGCCACCGCTTG Nucleotidesequence GCATGGACAGGGAGGACAGATCGTGCTGAGCCAGAGCCCTGCC encodingfor1F5scFv ATCCTGAGCGCCAGCCCTGGCGAGAAGGTGACCATGACCTGCA GAGCCAGCTCCAGCCTGAGCTTCATGCACTGGTACCAGCAGAAG CCCGGCAGCAGCCCTAAGCCCTGGATCTACGCCACCAGCAACCT GGCCAGCGGCGTGCCCGCCAGATTCAGCGGCAGCGGCAGCGGC ACCAGCTACAGCCTGACCATCAGCAGAGTGGAGGCCGAGGACG CCGCCACCTACTTCTGCCACCAGTGGAGCAGCAACCCCCTGACC TTCGGCGCCGGCACCAAGGTGGAGATCAAGAGAAAGGGCAGCA CCAGCGGAAGCGGCAAGCCTGGAAGCGGAGAGGGCAGCACCAA GGGACAGGTGCAGCTGAGACAGCCTGGCGCCGAGCTGGTGAAG CCCGGCGCCAGCGTGAAGATGAGCTGCAAGGCCAGCGGCTACA CCTTCACCAGCTACAACATGCACTGGGTGAAGCAGACCCCTGGC CAGGGCCTGGAGTGGATCGGCGCCATCTATCCCGGCAACGGCG ACACCAGCTACAACCAGAAGTTCAAGGGCAAGGCCACCCTGAC CGCCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGC CTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGAAGCC ACTACGGCAGCAACTACGTGGACTACTTCGACTACTGGGGCCAG GGCACCCTGGTGACCGTGAGCACCGGC SEQIDNO:127 ATGGGACCTGGCGTGCTGCTGCTGCTGCTGGTGGCCACCGCTTG Nucleotidesequence GCATGGACAGGGAGGAGATATCCAGATGACCCAGACTACATCC encodingforRFB4 TCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATTAGTTGCAG scFv GGCAAGTCAGGACATTAGCAATTATTTAAACTGGTATCAGCAGA AACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAATA TTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGG AACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATT TTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTGGACGT TCGGTGGAGGCACCAAGCTGGAAATCAAAGGCAGCACCAGCGG AAGCGGCAAGCCTGGAAGCGGAGAGGGCAGCACCAAGGGAGA AGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGA GGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCGCTTTCAGT ATCTATGACATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCT GGAGTGGGTCGCATACATTAGTAGTGGTGGTGGTACCACCTACT ATCCAGACACTGTGAAGGGCCGATTCACCATCTCCAGAGACAAT GCCAAGAACACCCTGTACCTGCAAATGAGCAGTCTGAAGTCTGA GGACACAGCCATGTATTACTGTGCAAGACATAGTGGCTACGGTA GTAGCTACGGGGTTTTGTTTGCTTACTGGGGCCAAGGGACTCTG GTCACTGTCTCTGCA SEQIDNO:128 ATGGGCCCAGGAGTTCTGCTGCTCCTGCTGGTGGCCACAGCTTG Nucleotidesequence GCATGGTCAGGGAGGTGACATCCAGATGACACAGACAACATCC encodingforFBB TCCCTCTCCGCTTCCCTCGGAGACAGGGTCACAATTTCCTGCAG (FMC63scFv-4-1BB GGCTTCCCAGGACATTTCCAAGTACCTCAACTGGTACCAGCAGA CCR) AGCCTGACGGAACAGTCAAGCTCCTCATTTACCACACATCCAGG CTCCACTCCGGAGTCCCTTCCAGGTTCTCCGGATCCGGATCCGG AACAGACTACTCCCTCACAATTTCCAACCTCGAGCAGGAGGACA TTGCTACATACTTCTGCCAGCAGGGAAACACACTCCCTTACACA TTCGGAGGAGGAACAAAGCTCGAGATTACAGGAGGTGGCGGTT CCGGTGGTGGAGGCTCCGGTGGTGGTGGATCTGAGGTCAAGCTC CAGGAGTCCGGACCTGGACTTGTCGCTCCTTCTCAGTCTCTCTCC GTCACATGCACAGTCTCCGGAGTCTCCCTCCCTGATTATGGAGT CTCCTGGATTAGGCAACCTCCTAGGAAAGGACTCGAATGGCTCG GAGTCATTTGGGGATCCGAAACAACATATTATAATTCCGCTCTC AAATCCAGGCTCACAATTATTAAAGATAATTCCAAATCCCAAGT CTTTCTCAAGATGAACTCCCTCCAGACAGACGACACAGCTATTT ACTACTGCGCTAAGCACTACTACTACGGGGGATCTTACGCTATG GACTACTGGGGACAGGGAACATCCGTCACAGTCTCTTCCGCTGC TGCTCCCACCACCACACCCGCTCCCAGACCCCCTACCCCTGCCC CCACCATCGCCAGCCAGCCCCTGAGCCTGAGACCCGAGGCCTGC AGACCTGCCGCCGGCGGAGCCGTGCACACCAGAGGCCTGGACT TCGCCTGCGACATCTACATCTGGGCTCCCCTGGCCGGCACCTGC GGCGTGCTGCTGCTGAGCCTGGTGATCACCCTGTACTGCAACCA CAAGAGAGGCAGAAAGAAGCTGCTGTACATCTTCAAGCAGCCC TTCATGAGACCCGTGCAGACCACCCAGGAGGAGGACGGCTGCA GCTGCAGATTCCCCGAGGAGGAGGAGGGCGGCTGCGAGCTG SEQIDNO:129 ATGGGACCTGGCGTGCTGCTGCTGCTGCTGGTGGCCACCGCTTG Nucleotidesequence GCATGGACAGGGAGGACAGATCGTGCTGAGCCAGAGCCCTGCC encodingfor1BB(1F5 ATCCTGAGCGCCAGCCCTGGCGAGAAGGTGACCATGACCTGCA scFv-4-1BBCCR) GAGCCAGCTCCAGCCTGAGCTTCATGCACTGGTACCAGCAGAAG CCCGGCAGCAGCCCTAAGCCCTGGATCTACGCCACCAGCAACCT GGCCAGCGGCGTGCCCGCCAGATTCAGCGGCAGCGGCAGCGGC ACCAGCTACAGCCTGACCATCAGCAGAGTGGAGGCCGAGGACG CCGCCACCTACTTCTGCCACCAGTGGAGCAGCAACCCCCTGACC TTCGGCGCCGGCACCAAGGTGGAGATCAAGAGAAAGGGCAGCA CCAGCGGAAGCGGCAAGCCTGGAAGCGGAGAGGGCAGCACCAA GGGACAGGTGCAGCTGAGACAGCCTGGCGCCGAGCTGGTGAAG CCCGGCGCCAGCGTGAAGATGAGCTGCAAGGCCAGCGGCTACA CCTTCACCAGCTACAACATGCACTGGGTGAAGCAGACCCCTGGC CAGGGCCTGGAGTGGATCGGCGCCATCTATCCCGGCAACGGCG ACACCAGCTACAACCAGAAGTTCAAGGGCAAGGCCACCCTGAC CGCCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGC CTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGAAGCC ACTACGGCAGCAACTACGTGGACTACTTCGACTACTGGGGCCAG GGCACCCTGGTGACCGTGAGCACCGGCGCGGCCGCCCCCACCAC CACACCCGCTCCCAGACCCCCTACCCCTGCCCCCACCATCGCCA GCCAGCCCCTGAGCCTGAGACCCGAGGCCTGCAGACCTGCCGCC GGCGGAGCCGTGCACACCAGAGGCCTGGACTTCGCCTGCGACAT CTACATCTGGGCTCCCCTGGCCGGCACCTGCGGCGTGCTGCTGC TGAGCCTGGTGATCACCCTGTACTGCAACCACAAGAGAGGCAG AAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCCG TGCAGACCACCCAGGAGGAGGACGGCTGCAGCTGCAGATTCCC CGAGGAGGAGGAGGGCGGCTGCGAGCTG SEQIDNO:130 ATGGGCCCTGGAGTGCTGCTGCTCCTGCTGGTGGCCACAGCTTG Nucleotidesequence GCACGGCCAGGGAGATATCCAGATGACCCAGACTACATCCTCCC encodingforRBB TGTCTGCCTCTCTGGGAGACAGAGTCACCATTAGTTGCAGGGCA (RFB4scFv-4-1BB AGTCAGGACATTAGCAATTATTTAAACTGGTATCAGCAGAAACC CCR) AGATGGAACTGTTAAACTCCTGATCTACTACACATCAATATTAC ACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACA GATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATTTTGC CACTTACTTTTGCCAACAGGGTAATACGCTTCCGTGGACGTTCG GTGGAGGCACCAAGCTGGAAATCAAAGGCAGCACCAGCGGAAG CGGCAAGCCTGGAAGCGGAGAGGGCAGCACCAAGGGAGAAGT GCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGG TCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCGCTTTCAGTATC TATGACATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGA GTGGGTCGCATACATTAGTAGTGGTGGTGGTACCACCTACTATC CAGACACTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCC AAGAACACCCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAGG ACACAGCCATGTATTACTGTGCAAGACATAGTGGCTACGGTAGT AGCTACGGGGTTTTGTTTGCTTACTGGGGCCAAGGGACTCTGGT CACTGTCTCTGCCGCGGCCGCCCCCACCACCACACCCGCTCCCA GACCCCCTACCCCTGCCCCCACCATCGCCAGCCAGCCCCTGAGC CTGAGACCCGAGGCCTGCAGACCTGCCGCCGGCGGAGCCGTGC ACACCAGAGGCCTGGACTTCGCCTGCGACATCTACATCTGGGCT CCCCTGGCCGGCACCTGCGGCGTGCTGCTGCTGAGCCTGGTGAT CACCCTGTACTGCAACCACAAGAGAGGCAGAAAGAAGCTGCTG TACATCTTCAAGCAGCCCTTCATGAGACCCGTGCAGACCACCCA GGAGGAGGACGGCTGCAGCTGCAGATTCCCCGAGGAGGAGGAG GGCGGCTGCGAGCTG

7. EQUIVALENTS AND SCOPE

(213) Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the appended claims.