Chimeric antigen receptor and method of use thereof

Abstract

To provide chimeric antibodies, nucleic acids encoding the same, and cells that express the chimeric antigen receptors.SOLUTION: The invention provides a chimeric antigen receptor comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain, where the intracellular signaling domain comprises a CD3 zeta chain, the transmembrane domain comprises CD28, the chimeric antigen receptor further comprises one or more additional co-stimulatory molecule of CD28, 4-1BB, ICOS or OX40 positioned between the transmembrane domain and the intracellular signaling domain, and the extracellular domain comprises an antibody such as a scFV that is specific to BCMA, CD138, CCR4, PD-1, PDL-1, D-L2, CXCR4, GITR, SARS virus, flavivirus, MERS virus, influenza virus or CAIX. The invention also provides a nucleic acid that encodes the chimeric antigen receptor and a cell that expresses the chimeric antigen receptor.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related applications This application claims priority and benefits from U.S. Provisional Application No. 62 / 094,625, filed on 19 December 2014, and U.S. Provisional Application No. 62 / 252,083, filed on 6 November 2015, incorporating by reference the entirety of the contents of each respective application.

[0002] Inclusion by referencing sequence listings The contents of the text file named "Omnibus Sequence Listing for filing with DFCI-102_001WO_ST25," created on December 21, 2015, are incorporated herein by reference in their entirety.

[0003] Field of Invention The present invention relates, in general, to chimeric antigen receptor cells for the treatment of cancer and other disorders, and to methods for using them for the treatment of cancer and other disorders.

[0004] Government interests This invention was made with government support under [] granted by []. The government has certain rights with respect to this invention. [Background technology]

[0005] Background of the Invention T lymphocytes recognize individual antigens through the interaction of short peptides presented by major histocompatibility complex (MHC) class I or II molecules with the T cell receptor (TCR). For initial activation and clonal expansion, naive T cells are dependent on professional antigen-presenting cells (APCs) that provide additional costimulatory signals. Activation of the TCR in the absence of costimulation can lead to unresponsiveness and clonal anergy. To evade immunity, various approaches have been developed to induce cytotoxic effector cells with transplanted recognition specificities. Chimeric antigen receptors (CARs) have been constructed that consist of natural ligands specific for cell surface components of the TCR-binding CD3 complex or antibody-derived binding domains. Upon antigen binding, such chimeric antigen receptors link to the effector cell's endogenous signaling pathways and generate activation signals similar to those initiated by the TCR complex. Since the first report of chimeric antigen receptors, this concept has been steadily improved and the molecular design of chimeric receptors has been optimized. Facilitated by advances in recombinant antibody technology, chimeric antigen receptors have been generated that target a wide variety of antigens on the surface of cancer cells and cells infected with the human immunodeficiency virus (HIV). Summary of the Invention

[0006] Abstract of the Invention In various aspects, the invention provides a chimeric antigen receptor (CAR) having an intracellular signaling domain, a transmembrane domain, and an extracellular domain.

[0007] In some instances, the transmembrane domain further includes a stalk region located between the extracellular domain and the transmembrane domain. The transmembrane domain includes CD28. In another aspect, the CAR further includes one or more additional co-stimulatory molecules located between the transmembrane domain and the intracellular signaling domain. The co-stimulatory molecule is CD28, 4-1BB, ICOS, or OX40. The intracellular signaling domain is, for example, the CD3 zeta chain. The extracellular domain is an antibody, such as a Fab or scFV. Preferably, the antibody is specific for BCMA, CA-9, CD138, CCR4, or influenza virus.

[0008] Also included in the present invention is a nucleic acid encoding a CAR of the present invention, which further includes a nucleic acid encoding a polypeptide located behind the intracellular signaling domain. The polypeptide is an antibody, such as scFV. Preferably, the antibody is specific for CCR4, PD-1, PDL-1, PD-L2, CXCR4, or GITR. Also included in the present invention are vectors containing the nucleic acids according to the present invention and cells containing this vector.

[0009] In yet a further aspect, the present invention provides a genetically engineered cell that expresses and retains the chimeric antigen receptor of the present invention on the cell surface membrane. The cell is a T cell or an NK cell. The T cell is CD4 + ,

[0010] , , or CD8 + The cell is a CD4 + cell and a mixed population of CD8 cells + The cell is further engineered to express and secrete a polypeptide, such as an antibody.

[0010] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by a person of ordinary skill in the art to which the present invention pertains. In carrying out the present invention, methods and materials similar to or equivalent to those described herein may be used, but suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety. In case of any conflict, this specification shall prevail, including definitions. Furthermore, the materials, methods, and examples described herein are illustrative and not intended to be limiting.

[0011] [Invention 1001] A chimeric antigen receptor (CAR) containing an intracellular signaling domain, a transmembrane domain, and an extracellular domain. [Invention 1002] The CAR of the present invention 1001, wherein the transmembrane domain further comprises a stalk region located between the extracellular domain and the transmembrane domain. [Invention 1003] A CAR according to the present invention 1001, wherein the transmembrane domain contains CD28. [Invention 1004] The CAR of the present invention 1001 further comprises one or more additional costimulatory molecules located between the transmembrane domain and the intracellular signaling domain. [Invention 1005] CAR of the present invention 1004, wherein the co-stimulatory molecule is CD28, 4-1BB, ICOS, or OX40. [Invention 1006] The CAR of the present invention 1001, wherein the intracellular signaling domain includes a CD3 zeta chain. [Invention 1007] CAR of the present invention 1001, wherein the extracellular domain is an antibody. [Invention 1008] CAR of Invention 1007, wherein the antibody is Fab or scFV. [Invention 1009] CAR of the present invention 1007, wherein the antibody is specific to BCMA, CD138, CCR4, PD-1, PDL-1, PD-L2, CXCR4, GITR, SARS virus, flavivirus, MERS virus, influenza virus, or CAIX. [Invention 1010] A nucleic acid encoding any of the CARs of the present invention, further comprising a nucleic acid encoding a polypeptide located after an intracellular signaling domain. [Invention 1011] The nucleic acid of the present invention 1010, wherein the polypeptide is an antibody. [Invention 1012] The nucleic acid of the present invention 1011, wherein the antibody is scFV. [Invention 1013] The nucleic acid of the present invention 1012, wherein the antibody is specific to BCMA, CD138, CCR4, PD-1, PDL-1, PD-L2, CXCR4, GITR, SARS virus, flavivirus, MERS virus, influenza virus, or CAIX. [Invention 1014] A vector comprising any nucleic acid according to invention 1011 to 1013. [Invention 1015] A cell containing the vector of the present invention 1014. [Invention 1016] A genetically modified cell that expresses one of the chimeric antigen receptors 1001 to 1009 of the present invention and retains it on the cell surface membrane. [Invention 1017] Genetically modified cells according to the present invention 1016, which are T cells or NK cells. [Invention 1018] T cells CD4 + or CD8 + The genetically modified cells of the present invention 1017. [Invention 1019] CD4 + Cells and CD8 cells + Genetically modified cells according to the present invention 1018, including a mixed population of the following. [Invention 1020] Cells of the present invention 1016, further manipulated to express and secrete polypeptides. [Invention 1021] Cells of the present invention 1020, wherein the polypeptide expressed and secreted is anti-PDL-1. Other features and advantages of the present invention are evident from and included in the following detailed description and appended claims. [Brief explanation of the drawing]

[0012] [Figure 1] ADCC with CAIX-specific Ab. 1 μg / ml of CAIX-specific scFv-Fc minibodies were added to target tumor cells in the presence of human PBMCs (E:T 25:1). Similar results were obtained in two experiments. Unrelated anti-SARS scFv-Fc(11A) and anti-CCR4 scFv-Fc(48) minibodies were used as negative controls. A, CAIX+ sk-rc-09 cells; B, CAIX+ sk-rc-52 cells; C, CAIX- sk-rc59 cells. [Figure 2A]Construction and expression of CAIX-specific CARs. A. Construction: The first-generation CAR, scFv-CD8-TCRζ (CD8 CAR), consists of a specific anti-CAIX scFv fused to the truncated human CD8α extracellular domain, hinge (H), transmembrane (TM), and intracellular domain, and then to the signaling domain of human TCRζ. The second-generation CAR, scFv-CD28-TCRζ (CD28 CAR), contains an anti-CAIX scFv fused to the human CD28 extracellular, TM, and intracellular signaling domains, and then to TCRζ. Both anti-CAIX CARs were cloned into a bisistronic self-inactivating (SIN) lentiviral vector whose expression is induced by an internal eF1-α promoter. The CAR control construct contains an unrelated anti-HIV CCR5-specific A8 scFv substitution. B. FACS analysis: The efficiency of primary T cell transduction by the lentiviral CAR constructs was quantified using the reporter gene ZsGreen. In addition, anti-CAIX scFv CARs were stained with CAIX-Fc fusion protein, and C9 tags (TETSQV APA) were stained with 1D4 antibody. Only LAK cells, which are non-transduced activated T cells, were used as an unstained cell control (i) or cells stained with secondary antibodies (ii. PE-anti-human IgG and iii. APC-anti-mouse IgG) were used as a staining control. C. Western blot: Shows the molecular sizes of monomeric / dimeric anti-CAIX (clone G36) CD28 and anti-CCR5 (clone A8) CD28 CARs, as well as the endogenous TCRζ chain of non-transduced T cells. [Figure 2B] See the explanation in Figure 2A. [Figure 2C] See the explanation in Figure 2A. [Figure 3A]Effector function of CAIX-specific CART. A. Cytokine secretion. Anti-CAIX CART, unrelated CART, or activated control T cells (LAK) were co-cultured overnight with kidney cancer cell lines sk-rc-52 (CAIX+) and sk-rc-59 (CAIX-) for cytokine production. One representative result from 2-3 experiments is shown. B. ELISPOT. G36 CART or control A8 CART cells were added to tumor cells overnight. IFN-γ or granzyme B secreting T cells were detected by ELISPOT. Similar results were obtained in 2-3 experiments. C. Specific antitumor cytotoxicity of CAIX-specific CART cells. Control A8 CART cells or LAK cells were incubated under different amounts of target tumor cells in the indicated ratios in a 4-hour cytotoxicity assay. One representative result from 2 experiments is shown. Clone 4-1 is an in vivo passaged subclone of sk-rc-52. [Figure 3B] See the explanation in Figure 3A. [Figure 3C] See the explanation in Figure 3A. [Figure 4A] Clonal proliferation of CART cells after tumor contact. A. Proliferation. CAR-transduced T cells or non-transduced T cells (LAKs) were plated weekly with tumor cells (CAIX+ sk-rc-52 & CAIX- sk-rc-59) irradiated at three different tumor-to-T cell ratios shown. T cell counts were counted three times in two separate wells every 3-4 days. Similar results were obtained in two experiments. B. Clonal enrichment. In tumor stimulation experiments, cultures derived from CART- and LAK cells were assayed by flow cytometry for 1 and 2 weeks for the expression of CART and T cell subsets. One representative result is shown. [Figure 4B] See the explanation in Figure 4A. [Figure 4C] See the explanation in Figure 4A. [Figure 5]Regression of human RCC xenografts formed by CART cells. 7.5 x 10⁶ sk-rc-52 and 5 x 10⁶ sk-rc-59 RCC tumor cells were subcutaneously inoculated into the left and right flanks of athymic null mice. Six days after tumor transplantation, mice were intravenously injected with 50 x 10⁶ G36 CD28 CART cells, A8 CD28 CART cells (≥20% CAR+), LAK, or PBS. High doses of IL-2 (1 x 10⁵ U / ml) were injected every 2-3 days. Tumor size was measured by calipas every 2-3 days. Experiment 1, n=7 and Experiment 2, n=8. Tumor sizes from these two experiments are shown separately. In these two trials, the G36 tandem-treated mice versus the control non-T cell-treated mice groups showed +, p<0.05;*, p<0.01;**, p<0.001. Other statistical calculations are reported in the text. [Figure 6A]In vivo antitumor activity of CAR+ T cells. A. ZsGreen expression by CART cells is shown in the upper panel. CART cells were pre-stained with Far Red dye, cytospinned, and examined by fluorescence microscopy (lower panel). B. In-situ staining of G36 CD28 CART cells in degenerative tumors. CART cells were IV injected into RCC-forming mice, and tumor tissue was collected on days 1-3. Apoptosis of tumor cells was measured using a TUNNEL assay with PE-Cy5 dye (shown in red) using confocal microscopy. Transduced T cells are shown with ZsGreen. Nuclei were counterstained with DPAI. Two representative slides show apoptosis of tumor cells at the tumor margin (upper panel) and within the tumor bed (middle panel), respectively. Magnified images (lower panel) show that CART cells interacted with multiple tumors, killing some surrounding tumor cells. C. Granzyme B+ T cells and tumor necrosis. Following treatment with CART cells, degenerative CAIX+ sk-rc-52 tumors were stained with granzyme B antibody (brown) and H&E. Higher magnification images (center and lower panels of compartments a and b in the upper panel) show the location of granzyme B+ T cells (indicated by arrows), and the corresponding H&E slides show tumor necrosis (indicated by n). Granzyme B+ T cells are distributed at the tumor margin (center panel) and within the tumor (lower panel). [Figure 6B] See the explanation in Figure 6A. [Figure 6C] See the explanation in Figure 6A. [Figure 7] CAIX-sk-rc-52 tumors treated with control LAK cells showed negative granzyme B staining (left) (bottom panel), with corresponding histology shown in H&E (right). [Figure 8] Low-background staining of granzyme B in CAIX-sk-rc-59 tumors treated with G36 CD28z CART cells. [Figure 9] Low-background staining of granzyme B in CAIX-sk-rc-59 tumors treated with LAK cells. [Figure 10] A positive control for granzyme B staining was performed in sk-rc-52 tumors locally injected with G36 CD28z CART cells (left), and the tumor morphology is shown by H&E (right). [Figure 11] Expression of 293T cells transiently transfected with αCAIX CAR and αPD-L1 scFv-Fc. 106 293T cells were transfected with or without the lentiviral pHAGE-EF1αG36-C9 tag-CD28-CD3 zeta-IRES-anti-PDL1 scFv-Fc(IgG4)-WPRE plasmid or the control pHAGE-EF1α-A716-C9 tag-CD28-CD3 zeta-IRES-ZsGreen-WPRE plasmid. Cells and supernatants were collected 24 and 48 hours after transfection. Left: Purified CAIX(ECD)-Fc-biotin and streptavidin-APC were used for cell staining and flow cytometry analysis. Right: Total human IgG in the supernatant was quantified using a human IgG quantification kit. [Figure 12A] (A) Total PBMCs from four donors were stimulated with 50 ng / ml SEB and treated with 10 ug / ml anti-PDL1(#42)sIgG4 or anti-influenza sIgG4. Recovery of IFNγ production (top) or TNFα production (bottom) was measured. [Figure 12B] (B) The parental anti-CCR4 antibody, c1567IgG, inhibited the chemotaxis of Tregs to CCL22. [Figure 12C] (C)CD4+CD25- T cells were labeled with CFSE, incubated with Tregs in a 10:1 ratio, and then stimulated with anti-CCR4 mAbs and control mAbs in the presence of anti-CD3 / 28 co-stimulation. Cells were collected at 3 and 7 days and analyzed by flow cytometry. Percentage of cells was calculated in fluorescently positive CD4+CD25- T cells and count beads. Proliferation rates were standardized against CD4+CD25- T effector cells on day 0. The data shown were calculated from two independent experiments. Bars represent mean ± SD. [Figure 12D](D) IFNγ ELISPOT analysis of CD4+CD25- Teff cells treated with mAb2-3 and control antibodies. Data represent quantification from three separate experiments using blood from three different donors. [Figure 12E] (E) Left: CFSE-labeled Teff (5x10⁴) and unlabeled Treg (5x10³) were co-incubated in a 96-well plate with 20 μg / ml PHA for 5 days. CFSE-labeled Teff was collected and CFSE intensity was analyzed by flow cytometry. Teff growth was observed only when the co-culture was incubated with an anti-GITR mAb. Right: IFNγ production in the same culture was further measured by MSD. CFSE-labeled Teff (5x10⁴) and unlabeled Treg (5x10³) were co-incubated in a 96-well plate with 20 μg / ml PHA for 5 days. CFSE-labeled Teff was collected and CFSE intensity was analyzed by flow cytometry. Teff grew after 5 days of incubation with PHA, but not in the Teff / Treg co-culture. Left: IFNγ production in the same culture was measured by MSD. [Figure 13] This figure shows various CART configurations according to the present invention. [Figure 14] This figure shows various armed CART configurations according to the present invention. [Figure 15] Further diagrams illustrating various armed CART configurations according to the present invention. [Figure 16-1]A series of figures and graphs showing a chimeric antigen receptor (CAR) construct for transduction into CD8+ T cells. (A) Schematic diagram of a second-generation pHAGE lentiviral vector containing CD28 as a costimulatory domain. Anti-carbonic anhydrase IX (CAIX) or anti-B cell maturation antigen (BCMA) scFv (as a negative control) was inserted after the eIFα promoter to express the CAR-binding domain. The second cassette after the internal ribosome entry site (IRES) sequence is responsible for encoding soluble anti-PD-L1 IgG1 or IgG4 isotype or anti-severe acute respiratory syndrome (SARS) coronavirus IgG1. To produce this lentivirus, the pHAGE vector, along with packaging plasmids (Gag, Rev, Tat, and VSVG), was transfected into 293T LentiX cells using polyethyleneimine. Two days after transfection, the virus was collected, purified, and concentrated using LentiX Concentrator (Clontech®) according to the manufacturer's instructions. LTR: Long terminal repeat, eIFα: Eukaryotic translation initiation factor alpha, scFv: Single-chain variable fragment, C9 tag: C9 peptide TETSQVAPA, IRES: Internal ribosome entry site, WPRE: Woodchuck hepatitis virus post-transcriptional regulatory element. (B) This lentivirus was used to transduce T cells and generate anti-CAIX CART cells that can recognize CAIX-positive RCCs and release anti-PD-L1 IgG1 or IgG4 in the tumor microenvironment to prevent PD-1 / PD-L1-induced T cell exhaustion. (C) Percentage of CART cells 14 days after transduction, showing stable long-term expression of CAR by the incorporated lentivirus in CD8+ T cells. CD8+ T cells were selected using Dynabeads® CD8 Positive Isolation Kit (Life Technologies) and activated using Dynabeads® Human T Cell Activator CD3 / CD28 (Life Technologies) in the presence of IL-21 50 U / mL. IL-21 was added to the culture medium every two days.After 14 days, CART cells were incubated with human CAIX-Fc or BCMA-Fc, then incubated with APC-conjugated anti-human Fc IgG, and analyzed by FACS. (D) Concentration of IgG secreted into the culture medium of transduced T cells, as assessed by Human IgG ELISA Quantitation Set (Bethyl Laboratories). (E) Concentration of anti-PD-L1 antibody in the supernatant of 293 T cells transduced with lentiviruses containing anti-CAIX or anti-BCMA CAR and anti-PD-L1 IgG1, anti-PD-L1 IgG4, or nonspecific anti-SARS IgG1 sequences. Antibodies in the supernatant were purified using Protein A Sepharose beads (GE Healthcare) and biotinylated using EZ-Link Sulfo-NHS-LC-Biotin (Thermo Scientific®). These antibodies were incubated with 5 μg / mL human PD-L1 pre-immobilized in 96-well MaxiSorp plates (Nunc®). Biotinylated antibodies were detected by incubation with streptavidin-HRP for 1 hour and development with TMB. Absorbance was read at λ=450 nm. *P<0.001 compared to anti-BCMA / anti-SARS IgG1 and anti-CAIX / anti-SARS IgG1. **P<0.05 compared to anti-CAIX / anti-PD-L1 IgG1. (F and G) Clonal proliferation of CD8+ CART cells. (F) Concentration of CART cells as a percentage of days in the presence of skrc-52 CAIX+ / PD-L1- cells. *P<0.05 compared to all CARS compared to anti-BCMA / anti-SARS IgG1. (G) Percentage of transduced T cells as a percentage of days in the presence of skrc-52 CAIX-positive cells. *P<0.05 compared to all CARS compared to anti-BCMA / anti-SARS IgG1.CD8+ CART cells were transduced using an anti-CAIX CAR capable of expressing anti-PD-L1 IgG1 (anti-CAIX / anti-PD-L1 IgG1), IgG4 (anti-CAIX / anti-PD-L1 IgG4), or nonspecific anti-SARS Ab (anti-CAIX / anti-SARS IgG1), or an anti-BCMA CAR (negative control) capable of expressing nonspecific anti-SARS Ab (anti-BCMA / anti-SARS IgG1), and pre-cultured for 5 days. They were then activated using Dynabeads™ Human T Activator CD3 / CD28 (Life Technologies) in the presence of IL-21 50 U / mL. After removing the beads, the CART cells were cultured with skrc52 CAIX+ PD-L1- and IL-21 (50 U / mL) added to the culture medium every 2 days for 21 days. Results represent the mean ± SD of three donors in a two-cycle study. [Figure 16-2] See the explanation in Figure 16-1. [Figure 16-3] See the explanation in Figure 16-1. [Figure 17-1]This is a series of graphs showing the effector function of CART cells. (A) Skrc59 CAIX+ / PD-L1+ cells or (B) Skrc52 CAIX- / PD-L1- cells incubated overnight (ON) with anti-CAIX / anti-PD-L1 IgG1, anti-CAIX / anti-PD-L1 IgG4, anti-CAIX / anti-SARS IgG1, or anti-BCMA / anti-SARS IgG1. These CART cells were used 4 days after lentiviral transduction. Cell viability was assessed by MTT (Molecular Probes®). *P<0.05 for all CART cells compared to anti-BCMA / anti-SARS IgG1. (C) IL2 released by CART cells after overnight contact with Skrc59 CAIX+ / PD-L1+ cells or (D) Skrc52 CAIX- / PD-L1- cells. IL-2 secretion was evaluated using the Human IL-2 ELISA Ready-SET-Go Kit (eBioscience®). *P<0.001 for all CART cells compared to anti-BCMA / anti-SARS IgG1. (E)IFNγ released by CART cells after overnight contact with Skrc59 CAIX+ / PD-L1+ or (F)Skrc52 CAIX- / PD-L1- cells. IFNγ secretion was evaluated using the Human IFNγ ELISA Ready-SET-Go Kit (eBiosciences®). *P<0.05 for all CART cells compared to anti-BCMA / anti-SARS IgG1. Antibody-dependent cell-mediated cytotoxicity of (G)skrc59 CAIX+ / PD-L1+ or (H)skrc52 CAIX- / PD-L1- after incubation with the supernatant (SN) of CART cells containing 500 ng / mL anti-PD-L1 IgG1, anti-PD-L1 IgG4 or anti-SARS IgG1. To obtain antibodies, CART cells were incubated for 6 days with Dynabeads™ Human T Activator CD3 / CD28 (Life Technologies) in the presence of IL-21 50 U / mL.After 7 days, the medium containing the mAbs was collected, and the NK cells were purified using the EasySep® Human NK Cell Enrichment Kit (StemCell® Technologies). RCC cell lines Skrc59 CAIX+ PD-L1+ and Skrc52 CAIX- PD-L1- were used as target cells, and 1.5 x 10³ cells / well were plated into 96-well plates. The RCC cells were incubated at 37°C for 1 hour with 50 μL of CART cell supernatant adjusted to contain 500 ng / mL of each Ab anti-PD-L1 IgG1, anti-PD-L1 IgG4, or anti-SARS IgG1. After incubation, the cells were washed with medium and incubated with NK cells in 12.5:1, 25:1, or 50:1 ratios at 37°C for 4 hours. The culture supernatant was collected by centrifugation, and LDH in the supernatant was measured at 490 nm using the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega®). These results represent the mean ± SD of three donors in a two-cycle study. [Figure 17-2] See the explanation in Figure 17-1. [Figure 18-1]This is a series of graphs showing the expression of fatigue markers in CART cells. (A) Lag3, (B) Tim3, and (C) PD-1 expression. *P<0.05 compared to anti-CAIX / anti-SARS IgG1 and anti-BCMA / anti-SARS IgG1. CD8+ CART cells were selected using the Dynabeads® CD8 Positive Isolation Kit (Life Technologies), activated with Dynabeads® Human T Activator CD3 / CD28 (Life Technologies), and transduced with the following CARs: anti-CAIX / anti-PD-L1 IgG1, anti-CAIX / anti-PD-L1 IgG4, anti-CAIX / anti-SARS IgG1, or anti-BCMA / anti-SARS IgG1. These cells were cultured for 5 days in the presence of IL-21 50 U / mL and Dynabeads® Human T Activator CD3 / CD28. After this period, CART cells were co-cultured with Skrc-59 CAIX+ PD-L1+ for 2 days to stimulate exhaustion. CART cells were stained with FITC-conjugated anti-human PD-1, PE-conjugated anti-human Tim3, and PerCP / Cy5.5 anti-human Lag3, and analyzed by FACS. (D) Viability of Skrc59 CAIX-positive / PD-L1-positive cells after incubation with exhausted CART cells. Cell viability was assessed by MTT (Molecular Probes). *P<0.05 compared to both anti-CAIX / anti-SARS IgG1 and anti-BCMA / anti-SARS IgG1. **P<0.05 compared to anti-CAIX / anti-PD-L1 IgG1. These results represent the mean ± SD of the three donors. [Figure 18-2] See the explanation in Figure 18-1. [Figure 18-3] See the explanation in Figure 18-1. [Figure 19A]This is a series of images and graphs demonstrating the effects of CART cells in an orthotopic model of human RCC. (A) NSG mice (N=35) were injected with 5.0 x 10⁴ skrc-59 CAIX-positive, PD-L1-positive, and luciferase-positive RCC cells. One week later, the mice were intravenously injected with 1.0 x 10⁷ CART or non-transduced T cells (day 0). CART cells were pre-transduced using the following lentiviral sequences: anti-BCMA CAR / anti-SARS IgG1, anti-CAIX CAR / anti-SARS IgG1, anti-CAIX CAR / anti-PD-L1 IgG1, and anti-CAIX CAR / anti-PD-L1 IgG4 (N=6 mice per group). Tumor bioluminescence was quantified using IVIS after a 5-minute luciferin IP injection. Images of the tumor before CART injection (day 0) and 7, 14, 23, and 30 days after the first CART cell injection. A second injection of 2.5 x 10⁶ cells was administered on day 17. (B) Image of the tumor after resection on day 30. Scale bar = 1 cm. (C) Tumor growth curve. *P<0.05 when comparing the anti-PD-L1 IgG1 and IgG4 groups to anti-BCMA / anti-SARS IgG1 and **P<0.05 when comparing the anti-PD-L1 IgG1 and IgG4 groups to anti-CAIX / anti-SARS IgG1. (D) Tumor weight 30 days after treatment. *P<0.05 compared to anti-BCMA / anti-SARS IgG1 CAR. **P<0.05 compared to anti-CAIX / anti-SARS IgG1. [Figure 19B] See the explanation in Figure 19A. [Figure 19C] See the explanation in Figure 19A. [Figure 19D] See the explanation in Figure 19A. [Figure 20A]A series of graphs and histological images showing the antitumor activity from CART cells. (A) Expression of exhaustion markers in tumor-infiltrating lymphocytes (TILs). Kidney tumors from all mice were divided into two parts, one of which was fragmented into small pieces, and TILs were extracted by digestion with collagenase and DNAse. CART cells were analyzed for exhaustion markers PD-1, Tim-3, and Lag3. *P<0.05 compared to non-transduced, anti-BCMA / anti-SARS IgG1 CAR and anti-CAIX / anti-SARS IgG1. (B) Detection of Ki67 and granzyme B as tumor cell proliferation markers for analysis of CART cell activity in tissue. 4-micrometer pieces of formalin-fixed, paraffin-embedded tissue were dewaxed and rehydrated with a decreasing ethanol series. Endogenous peroxidase activity was eliminated using 3% hydrogen peroxide. Antigen recovery was performed in citrate buffer (pH = 6.0) at 123°C, 15 PSI for 45 seconds using a pressure cooker. These tissue sections were incubated for 45 minutes with rabbit anti-human Ki67 polyclonal Ab 1:2000 (Vector, VP-K451), mouse anti-human PD-L1 mAb 10.4 μg / mL (clone 405.9A11) developed by Dr. Gordon Freeman (Boston, MA), biotinylated CAIX-Fc protein 17 μg / mL (prepared in our laboratory), rabbit anti-human granzyme B polyclonal Ab (Abcam, ab4059) 1:100, or rabbit anti-human NCAM (CD56) mAb 1:100 (Abcam, ab133345), followed by secondary HRP-conjugated anti-rabbit Ab or HRP-avidin. The slides were developed with 3,3'-diaminobenzidine (DAB) and counterstained with hematoxylin. Images were obtained using an Olympus BX51 microscope with a DP71 digital camera (Olympus) and analyzed with DP Controller Software (Olympus®). The scale bars represent the magnification of the image for each column [500 μm (40X), 100 μm (200X), or 50 μm (400X)]. (C) The series of images shows the quantification of TILs positively stained for granzyme B, PD-L1-IHC, and Ki67 by percentage.(C) also shows the number of Ki67-DAB pixels in TIL. [Figure 20B] See the explanation in Figure 20A. [Figure 20C-1] See the explanation in Figure 20A. [Figure 20C-2] See the explanation in Figure 20A. [Figure 20C-3] See the explanation in Figure 20A. [Figure 20C-4] See the explanation in Figure 20A. [Figure 21]A series of graphs showing the evaluation of IL-2 versus IL-21 for the proliferation of CD8+ CART cells. A and B. Proliferation of CART transduced cells in the presence of IL-2 or IL-21, evaluated 48, 72, and 120 hours after lentiviral transduction. (A) Anti-CAIX CART cells or (B) Nonspecific anti-BCMA CART cells (both with ZsGreen in the second cassette). CD8+ T cells were selected using the Dynabeads® CD8 Positive Isolation Kit (Life Technologies) and activated with Dynabeads® Human T Activator CD3 / CD28 (Life Technologies) in the presence of IL-2 or IL-21 50 U / mL (Peprotech®). Transduction of CART cells was evaluated by ZsGreen expression using FACS. Data represent mean ± SD from two donors. *IL-21 compared to untreated control (Ctr) p<0.05. **IL21 compared to IL-2 (p<0.05). C and D. Viability of RCC cells treated with CD8+ CART cells cultured in the presence of IL-2 or IL-21. Viability was assessed by MTT after incubation overnight with CART cells, anti-BCMA, anti-CAIX, or untransduced T cells, along with (C)Skrc-59 CAIX+ / PD-L1+ and (F)Skrc-52 CAIX- / PD-L1- RCC cells. CART cells were cultured for 120 hours prior to the study in the presence of IL2 or IL-21 50 U / mL. These results represent the mean ± SD of two donors performed in triplicates. *P<0.05 compared to anti-CAIX CARs compared to anti-BCMA CARs or untransduced T cells. [Figure 22]These are a series of flow cytometry graphs showing PD-1 and CAIX expression in renal cell carcinoma (RCC) strains. (A) Negative control, (B) Skrc52 CAIX- PD-L1-, (C) Skrc52 CAIX+ PD-L1-, (D) Skrc59 CAIX+ PD-L1+. Cells were stained with anti-human CAIX antibody developed with APC anti-human Fc IgG and biotinylated anti-human PD-L1 antibody developed with PE-avidin. Analysis was performed by FACS. [Figure 23] This is a series of graphs illustrating the characterization of CART cells. (A) Proliferation of total CD8+ T cells 2 or 4 days after transduction of anti-CAIX CAR / anti-PD-L1 IgG1), anti-CAIX CAR / anti-PD-L1 IgG4, anti-CAIX CAR / anti-SARS IgG1), or anti-BCMA CAR / anti-SARS IgG1). CD8+ T cells were selected using the Dynabeads® CD8 Positive Isolation Kit (Life Technologies) and activated with the Dynabeads® Human T Activator CD3 / CD28 (Life Technologies) in the presence of IL-21 50 U / mL. IL-21 was added to the culture medium every 2 days. Proliferation was assessed by FACS using Counting Beads (Molecular Probes). (B) Concentration of CAR transduced T cells 2 and 4 days after transduction. CART cells were incubated with human CAIX-Fc or BCMA-Fc, then incubated with APC-bound anti-human Fc IgG, and analyzed by FACS. (C) Percentage of CART cells 2 and 4 days after transduction. Results represent the mean ± SD of three donors in a two-sequence study. [Figure 24A]This is a series of graphs showing the effects of CART cells in an orthotopic model of human RCC. (A) Comparison of tumor size detected by bioluminescence as a function of days in the CART cell group. 5.0 x 10⁴ skrc-59 CAIX+, PD-L1+, and luciferase+ RCC cells were injected into the renal capsule of NSG mice (N=35). After one week, 1.0 x 10⁷ CART or non-transduced T cells were intravenously injected into the mice. CART cells were pre-transduced using the following lentiviral sequences: anti-BCMA CAR / anti-SARS IgG1, anti-CAIX CAR / anti-SARS IgG1, anti-CAIX CAR / anti-PD-L1 IgG1, and anti-CAIX CAR / anti-PD-L1 IgG4 (N=6 mice per group). Tumor bioluminescence was quantified using IVIS 5 minutes after IP injection of luciferin. On day 17, an additional 2.5 x 10⁶ CART cells were injected. *P<0.05 compared to anti-BCMA CART cells. **P<0.05 compared to untransduced T cells, ***P<0.05 compared to anti-CAIX / anti-SARS IgG1. (B) Percentage of T cells in mouse blood 8 days after treatment. *P<0.05 compared to untransduced T cells. **P<0.05 compared to all anti-CAIX CARs. Erythrocytes were lysed with ACK Lysing Buffer (Lonza™), and the remaining cells were stained with Pacific Blue-conjugated anti-human CD45 and analyzed by FACS. (C) Total tumor-infiltrating lymphocytes (TILs) 30 days after CART cell treatment. Tumors and kidneys from all mice were divided into two parts, one of which was fragmented into small pieces, digested with collagenase and DNAse to extract TILs. Cells were stained with Pacific Blue-conjugated anti-human CD45 and analyzed by FACS. [Figure 24B] See the explanation in Figure 24A. [Figure 24C] See the explanation in Figure 24A. [Figure 25A]A series of graphs and histological images showing human natural killer (NK) cells in tumors treated with anti-CAIX CART cells releasing anti-PD-L1 IgG1 Ab. (A) Percentage of CD56+ cells (NK marker) in tumors. Two mice from each group were injected with 4.5 x 10⁶ NK cells one day before euthanasia. Renal tumors from all mice were divided into two sections, one of which was fragmented into small pieces and digested with collagenase and DNAse to extract NK. NK cells present in the tumors were stained with APC-anti-CD56 Ab and analyzed by FACS. *P<0.05. (B) CD56+ cells in excised tumors detected by IHC and quantified using the IHC Profiler Plugin in ImageJ Software. 4-micrometer pieces of formalin-fixed, paraffin-embedded tissue were dewaxed and rehydrated with a decreasing ethanol series. Endogenous peroxidase activity was eliminated using 3% hydrogen peroxide. Antigen recovery was performed in citrate buffer (pH = 6.0) at 123°C and 15 PSI for 45 seconds using a pressure cooker. These tissue sections were incubated for 45 minutes with rabbit anti-human CD56 mAb 1:100 (Abcam, ab133345), followed by secondary HRP-conjugated anti-rabbit or anti-mouse Ab. Slides were developed with 3,3'-diaminobenzidine (DAB) and counterstained with hematoxylin. Images were obtained using an Olympus BX51 microscope with a DP71 digital camera (Olympus) and analyzed with DP Controller Software (Olympus®). Quantification was performed using the IHC Profiler Plugin in ImageJ Software (23). The scale bar represents the magnification of the image (400X). *P<0.05 compared to non-transduced, **P<0.05 compared to non-transduced and anti-BCMA / anti-SARS IgG1. [Figure 25B] See the explanation in Figure 25A. [Modes for carrying out the invention]

[0013] Detailed description of the invention This invention relates particularly to chimeric antigen receptors (CARs) adapted to immune cells used in immunotherapy.

[0014] In one embodiment, a dual immunotherapy strategy is described that is based on inhibiting T cell exhaustion using an anti-PD-L1 antibody secreted by targeted anti-CAIX CAR T cells combined in a single lentiviral construct to improve the treatment of cancer.

[0015] A new mechanism associated with the progression of RCC and other tumors is an immune checkpoint pathway present in cell interactions that prevents excessive T cell activation under normal conditions, thereby limiting T cell function. As an evasion mechanism, many tumors can stimulate the expression of immune checkpoint molecules, inducing anerogenic phenotype T cells that cannot suppress tumor progression. New clinical data highlight the importance of a single inhibitory ligand and receptor pair as immune checkpoints in preventing cancer cell death by cytotoxic T lymphocytes: programmed death ligand 1 (PD-L1; B7-H1 and CD274) and programmed death receptor 1 (PD-1; CD279). The PD1 receptor is expressed by many cell types and host tissues, including T cells, B cells, and natural killer cells (NK). Tumor and antigen-presenting cells (APCs) expressing PD-L1 can block T cell receptor (TCR) signaling of cytotoxic T lymphocytes through binding to the receptor PD-1, thereby reducing cytokine production and T cell proliferation. Overexpression of PD-L1 can be found in many tumor types, and it also mediates immunosuppressive functions through its interaction with other proteins, including CD80(B7.1), which inhibits its ability to activate T cells through binding to CD28.

[0016] By genetically engineering human lymphocytes to express tumor-specific chimeric antigen receptors (CARs), it is possible to generate anti-tumor effector cells that evade tumor immune evasion mechanisms resulting from abnormalities in protein-antigen processing and presentation. Furthermore, these transgenic receptors can target tumor-associated antigens that are not protein-derived. In certain embodiments of the present invention, lymphocytes (CARTS) are modified to contain at least one CAR, and in certain embodiments of the present invention, one CAR targets two or more antigens. In a preferred embodiment, the CARTS are further modified to express and secrete one or more polypeptides, such as antibodies or cytokines. Such CARTS are referred to herein as armed CARTS. Armed CARTS achieve localized simultaneous secretion of polypeptides at the targeting site (i.e., the tumor site).

[0017] Modified TCRs called chimeric antigen receptors (CARs), which contain pre-selected single-chain variable antibody fragments (scFv) with high affinity for specific tumor-associated antigens, represent a powerful new approach to cancer. The scFv present in the CAR is linked to an intracellular signaling block containing CD3ζ to induce T cell activation and subsequent antigen binding. This structure is characteristic of first-generation CARs, which evolved into second-generation CARs linking signaling costimulatory internal domains of CD28, 4-1BB, or OX40 to CD3, or third-generation CARs linking two elements in series to CD3ζ. These internal domains are required for complete T cell activation upon TCR recognition by antigen-presenting cells (APCs), improving cytokine production and proliferation of CAR-T cells. Due to the difficulty in discovering specific tumor-associated antigens, inefficient T cell homing to tumor sites, low persistence of T cells in the body, and the immunosuppressive microenvironment of solid tumors, the effectiveness of CAR cells in treating solid tumors has been limited until now.

[0018] In certain cases, these lymphocytes are chimeric, non-natural, and contain receptors that are at least partially manipulated by humans. In certain cases, the manipulated chimeric antigen receptor (CAR) has one, two, three, four or more elements, and in some embodiments, one or more elements facilitate the targeting or binding of the lymphocyte to one or more tumor antigen-containing cancer cells.

[0019] A CAR according to the present invention generally comprises at least one transmembrane polypeptide containing at least one extracellular ligand-binding domain, and one transmembrane polypeptide containing at least one intracellular signaling domain, wherein these polypeptides are combined to form a chimeric antigen receptor.

[0020] As used herein, the term “extracellular ligand-binding domain” is defined as an oligopeptide or polypeptide capable of binding to a ligand. Preferably, this domain would be capable of interacting with cell surface molecules. For example, an extracellular ligand-binding domain may be selected to recognize a ligand that functions as a cell surface marker on target cells associated with a particular disease condition.

[0021] In particular, the extracellular ligand-binding domain may include an antigen-binding domain derived from an antibody against the target antigen.

[0022] As a non-limiting example, target antigens include tumor-associated surface antigens, such as ErbB2 (HER2 / neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40, disialoganglioside GD2, ductal epithelial mucin, gp36, TAG-72, glycosphingolipids, glioma-associated antigens, beta-human chorionic gonadotropins, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, and mut. hsp70-2, M-CSF, prostase, prostase-specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, prostain, PSMA, survival and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF1)-I, IGF-II, IGFI receptor, mesothelium, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigen, extra domain A (EDA) and extra domain B (EDB) of fibronectin, and A1 domain (TnC) of tenascin C A1) and fibroblast-associated proteins (fap); lineage-specific or tissue-specific antigens, e.g., CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, CTLA-4, B7-1 (CD80), B7-2 (CD86), endoglin, major histocompatibility complex (MHC) molecules, BCMA (CD269, TNFRSF 17), or virus-specific surface antigens, e.g., HIV-specific antigen (e.g., HIV gp120); EBV-specific antigen, CMV-specific antigen, HPV-specific antigen, Lassa virus-specific antigen, influenza virus-specific antigen, and any derivatives or variants of these surface markers.

[0023] Preferably, the CAR is specific for BMCA, CAIX, CCR4, PD-L1, PD-L2, PD1, glucocorticoid-induced tumor necrosis factor receptor (GITR), severe acute respiratory syndrome (SARS), influenza, flavivirus or Middle East respiratory syndrome (MERS).

[0024] In a preferred embodiment, the extracellular ligand-binding domain is a single-chain antibody fragment (scFv) comprising the light chain variable fragment (V L ) and heavy chain variable fragment (V H ) of a target antigen-specific monoclonal antibody connected by a flexible linker.

[0025] In a more preferred embodiment, the scFv antibody is specific for BMCA, CAIX, CCR4, PD-L1, PD-L2, PD1, GITR, SARS, influenza, flavivirus or MERS.

[0026] Exemplary antibodies useful in constructing a CAR according to the present invention include, for example, the antibodies disclosed in WO / 2005 / 060520, WO / 2006 / 089141, WO / 2007 / 065027, WO / 2009 / 086514, WO / 2009 / 079259, WO / 2011 / 153380, WO / 2014 / 055897, WO 2015 / 143194, WO 2015 / 164865, WO 2013 / 166500 and WO 2014 / 144061, PCT / US2015 / 054202, PCT / US2015 / 054010 and 62 / 144,729, the entire contents of which are incorporated herein by reference.

[0027] PDL1(68) Exemplary anti-PDL1 antibodies include a VH nucleotide sequence having SEQ ID NO:1485 and a VL nucleotide sequence having SEQ ID NO:1487, a VH nucleotide sequence having SEQ ID NO:1485 and a VL nucleotide sequence having SEQ ID NO:1487, a VH nucleotide sequence having SEQ ID NO:1489 and a VL nucleotide sequence having SEQ ID NO:1491, a VH nucleotide sequence having SEQ ID NO:1493 and a VL nucleotide sequence having SEQ ID NO:1495, a VH nucleotide sequence having SEQ ID NO:1497 and a VL nucleotide sequence having SEQ ID NO:1499, a VH nucleotide sequence having SEQ ID NO:1501 and a VL nucleotide sequence having SEQ ID NO:1503, a VH nucleotide sequence having SEQ ID NO:1505 and a VL nucleotide sequence having SEQ ID NO:1507, a VH nucleotide sequence having SEQ ID NO:1509 and SEQ ID The antibody contains a VL nucleotide sequence having NO:1511, a VH nucleotide sequence having SEQ ID NO:1513 and a VL nucleotide sequence having SEQ ID NO:1515, a VH nucleotide sequence having SEQ ID NO:1517 and a VL nucleotide sequence having SEQ ID NO:1519, a VH nucleotide sequence having SEQ ID NO:1521 and a VL nucleotide sequence having SEQ ID NO:1523, a VH nucleotide sequence having SEQ ID NO:1525 and a VL nucleotide sequence having SEQ ID NO:1527, a VH nucleotide sequence having SEQ ID NO:1529 and a VL nucleotide sequence having SEQ ID NO:1531, a VH nucleotide sequence having SEQ ID NO:1533 and a VL nucleotide sequence having SEQ ID NO:1535, a VH nucleotide sequence having SEQ ID NO:1537 and a VL nucleotide sequence having SEQ ID NO:1539.

[0028] An example of an anti-PDL1 antibody is a VH amino acid sequence having SEQ ID NO:970 and a VL amino acid sequence having SEQ ID NO:971, a VH amino acid sequence having SEQ ID NO:1486 and a VL polypeptide sequence having SEQ ID NO:1488, a VH amino acid sequence having SEQ ID NO:1490 and a VL polypeptide sequence having SEQ ID NO:1492, a VH amino acid sequence having SEQ ID NO:1494 and a VL polypeptide sequence having SEQ ID NO:1496, a VH amino acid sequence having SEQ ID NO:1498 and a VL polypeptide sequence having SEQ ID NO:1500, a VH amino acid sequence having SEQ ID NO:1502 and a VL polypeptide sequence having SEQ ID NO:1504, a VH amino acid sequence having SEQ ID NO:1506 and a VL polypeptide sequence having SEQ ID NO:1508, a VH amino acid sequence having SEQ ID NO:1510 and a VL polypeptide sequence having SEQ ID NO:1512, SEQ ID The antibody contains a VH amino acid sequence having NO:1514 and a VL polypeptide sequence having SEQ ID NO:1516, a VH amino acid sequence having SEQ ID NO:1518 and a VL polypeptide sequence having SEQ ID NO:1520, a VH amino acid sequence having SEQ ID NO:1522 and a VL polypeptide sequence having SEQ ID NO:1524, a VH amino acid sequence having SEQ ID NO:1526 and a VL polypeptide sequence having SEQ ID NO:1528, a VH amino acid sequence having SEQ ID NO:1530 and a VL polypeptide sequence having SEQ ID NO:1532, a VH amino acid sequence having SEQ ID NO:1534 and a VL polypeptide sequence having SEQ ID NO:1536, and a VH amino acid sequence having SEQ ID NO:1538 and a VL polypeptide sequence having SEQ ID NO:1540.

[0029] In other embodiments, the anti-PDL1 antibody comprises a heavy chain having three CDRs containing amino acid sequences SEQ ID NO: 1541, 1554, and 1569, respectively, and a light chain having three CDRs containing amino acid sequences 1584, 1599, and 1610, respectively; or a heavy chain having three CDRs containing amino acid sequences 1543, 1556, and 1571, and a light chain having three CDRs containing amino acid sequences 1586, 1600, and 1612; or a heavy chain having three CDRs containing amino acid sequences 1544, 1557, and 1572, and a light chain having three CDRs containing amino acid sequences 1587, 1601, and 1613, or amino A heavy chain having three CDRs including amino acid sequences 1545, 1558, 1573 and a light chain having three CDRs including amino acid sequences 1588, 1602, 1614, or a heavy chain having three CDRs including amino acid sequences 1546, 1559, 1574 and a light chain having three CDRs including amino acid sequences 1589, 1603, 1615, or a heavy chain having three CDRs including amino acid sequences 1547, 1560, 1575 and a light chain having three CDRs including amino acid sequences 1590, 1604, 1616, or amino acid sequence 1 A heavy chain having three CDRs including 548, 1561, and 1576, and a light chain having three CDRs including amino acid sequences 1591, 1605, and 1617, or a heavy chain having three CDRs including amino acid sequences 1541, 1562, and 1577, and a light chain having three CDRs including amino acid sequences 1592, 1599, and 1618, or a heavy chain having three CDRs including amino acid sequences 1549, 1563, and 1578, and a light chain having three CDRs including amino acid sequences 1593, 1606, and 1619, or amino acid sequence 1550, A heavy chain having three CDRs including 1564 and 1579 and a light chain having three CDRs including amino acid sequences 1594, 1607, and 1620, or a heavy chain having three CDRs including amino acid sequences 1551, 1565, and 1580 and a light chain having three CDRs including amino acid sequences 1595, 1599, and 1621, or a heavy chain having three CDRs including amino acid sequences 1542, 1566, and 1581 and a light chain having three CDRs including amino acid sequences 1596, 1599, and 1622, or amino acid sequences 1552 and 1567,It has a heavy chain with three CDRs including 1582 and a light chain with three CDRs including amino acid sequences 1597, 1608, and 1623, or a heavy chain with three CDRs including amino acid sequences 1553, 1568, and 1583 and a light chain with three CDRs including amino acid sequences 1598, 1609, and 1624.

[0030] SARS (26) Exemplary SARS neutralizing antibodies include the VH nucleotide sequence with SEQ ID NO:1626 and the VL nucleotide sequence with SEQ ID NO:1628, the VH nucleotide sequence with SEQ ID NO:1630 and the VL nucleotide sequence with SEQ ID NO:1639, the VH nucleotide sequence with SEQ ID NO:1634 and the VL nucleotide sequence with SEQ ID NO:1640, the VH nucleotide sequence with SEQ ID NO:1632 and the VL nucleotide sequence with SEQ ID NO:1641, the VH nucleotide sequence with SEQ ID NO:1633 and the VL nucleotide sequence with SEQ ID NO:1642, the VH nucleotide sequence with SEQ ID NO:1634 and the VL nucleotide sequence with SEQ ID NO:1643, the VH nucleotide sequence with SEQ ID NO:1635 and the VL nucleotide sequence with SEQ ID NO:1644, the VH nucleotide sequence with SEQ ID NO:1636 and SEQ ID This antibody contains a VL nucleotide sequence having NO:1645, a VH nucleotide sequence having SEQ ID NO:1637, and a VL nucleotide sequence having SEQ ID NO:1646.

[0031] CXCR4(33) Exemplary anti-CXCR4 antibodies include antibodies having a VH amino acid sequence with SEQ ID NO:771 and a VL amino acid sequence with SEQ ID NO:779, a VH amino acid sequence with SEQ ID NO:772 and a VL amino acid sequence with SEQ ID NO:780, a VH amino acid sequence with SEQ ID NO:773 and a VL amino acid sequence with SEQ ID NO:781, a VH amino acid sequence with SEQ ID NO:774 and a VL amino acid sequence with SEQ ID NO:782, a VH amino acid sequence with SEQ ID NO:775 and a VL amino acid sequence with SEQ ID NO:783, a VH amino acid sequence with SEQ ID NO:776 and a VL amino acid sequence with SEQ ID NO:784, a VH amino acid sequence with SEQ ID NO:777 and a VL amino acid sequence with SEQ ID NO:785, or a VH amino acid sequence with SEQ ID NO:778 and a VL amino acid sequence with SEQ ID NO:786.

[0032] In other embodiments, the anti-CXCR4 antibody comprises a heavy chain having three CDRs containing amino acid sequences SEQ ID NO: 803, 804, and 805, respectively, and a light chain having three CDRs containing amino acid sequences 806, 807, and 808, respectively; or a heavy chain having three CDRs containing amino acid sequences 809, 810, and 811, respectively, and a light chain having three CDRs containing amino acid sequences 812, 813, and 814, respectively; or a heavy chain having three CDRs containing amino acid sequences 815, 816, and 817, respectively, and a light chain having three CDRs containing amino acid sequences 818, 819, and 820, respectively; or each It has a heavy chain having three CDRs containing amino acid sequences 827, 828, and 829 and a light chain having three CDRs containing amino acid sequences 830, 831, and 832, respectively; or a heavy chain having three CDRs containing amino acid sequences 833, 834, and 835, respectively and a light chain having three CDRs containing amino acid sequences 836, 837, and 838, respectively; or a heavy chain having three CDRs containing amino acid sequences 839, 840, and 841, respectively and a light chain having three CDRs containing amino acid sequences 842, 843, and 844, respectively.

[0033] Carbonic anhydrase IX (40) Exemplary anti-CA IX antibodies include VH amino acid sequences having SEQ ID NO:845 and VL amino acid sequences having SEQ ID NO:846, VH amino acid sequences having SEQ ID NO:847 and VL amino acid sequences having SEQ ID NO:868, VH amino acid sequences having SEQ ID NO:848 and VL amino acid sequences having SEQ ID NO:869, VH amino acid sequences having SEQ ID NO:849 and VL amino acid sequences having SEQ ID NO:870, VH amino acid sequences having SEQ ID NO:850 and VL amino acid sequences having SEQ ID NO:871, VH amino acid sequences having SEQ ID NO:851 and VL amino acid sequences having SEQ ID NO:872, VH amino acid sequences having SEQ ID NO:852 and VL amino acid sequences having SEQ ID NO:873, VH amino acid sequences having SEQ ID NO:853 and VL amino acid sequences having SEQ ID NO:874, SEQ ID VH amino acid sequence having NO:854 and VL amino acid sequence having SEQ ID NO:875, VH amino acid sequence having SEQ ID NO:855 and VL amino acid sequence having SEQ ID NO:876, VH amino acid sequence having SEQ ID NO:856 and VL amino acid sequence having SEQ ID NO:877, VH amino acid sequence having SEQ ID NO:857 and VL amino acid sequence having SEQ ID NO:878, VH amino acid sequence having SEQ ID NO:858 and VL amino acid sequence having SEQ ID NO:879, VH amino acid sequence having SEQ ID NO:859 and VL amino acid sequence having SEQ ID NO:880, VH amino acid sequence having SEQ ID NO:860 and VL amino acid sequence having SEQ ID NO:881, VH amino acid sequence having SEQ ID NO:861 and VL amino acid sequence having SEQ ID NO:882, VH amino acid sequence having SEQ ID NO:862 and SEQ ID VL amino acid sequence having NO:883, SEQ ID VH amino acid sequence having NO:863 and VL amino acid sequence having SEQ ID NO:884, SEQ IDThe antibody contains a VH amino acid sequence having NO:864 and a VL amino acid sequence having SEQ ID NO:885, a VH amino acid sequence having SEQ ID NO:865 and a VL amino acid sequence having SEQ ID NO:886, a VH amino acid sequence having SEQ ID NO:866 and a VL amino acid sequence having SEQ ID NO:887, and a VH amino acid sequence having SEQ ID NO:867 and a VL amino acid sequence having SEQ ID NO:888.

[0034] In another embodiment, the anti-CA IX antibody comprises a heavy chain having three CDRs containing amino acid sequences SEQ ID NO: 803, 804, and 805, and a light chain having three CDRs containing amino acid sequences 806, 807, and 808, respectively; or a heavy chain having three CDRs containing amino acid sequences 899, 915, and 909 and a light chain having three CDRs containing amino acid sequences 905, 906, and 952; or a heavy chain having three CDRs containing amino acid sequences 899, 915, and 909 and a light chain having three CDRs containing amino acid sequences 935, 943, and 953; or containing amino acid sequences 899, 915, and 909 A heavy chain with three CDRs and a light chain with three CDRs containing amino acid sequences 935, 906, 954, or a heavy chain with three CDRs containing amino acid sequences 910, 916, 923 and a light chain with three CDRs containing amino acid sequences 936, 944, 955, or a heavy chain with three CDRs containing amino acid sequences 899, 915, 909 and a light chain with three CDRs containing amino acid sequences 936, 944, 956, or a heavy chain with three CDRs containing amino acid sequences 911, 917, 924 and amino acid sequence 9 A light chain having three CDRs including 37, 945, and 957, or a heavy chain having three CDRs including amino acid sequences 899, 915, and 909 and a light chain having three CDRs including amino acid sequences 935, 946, and 958, or a heavy chain having three CDRs including amino acid sequences 899, 915, and 909 and a light chain having three CDRs including amino acid sequences 938, 946, and 959, or a heavy chain having three CDRs including amino acid sequences 899, 915, and 909 and three CDRs including amino acid sequences 905, 946, and 960 A light chain having three CDRs including amino acid sequences 899, 918, 925 and a light chain having three CDRs including amino acid sequences 937, 947, 955, or a heavy chain having three CDRs including amino acid sequences 899, 918, 926 and a light chain having three CDRs including amino acid sequences 937, 945, 957, or a heavy chain having three CDRs including amino acid sequences 912, 919, 927 and a light chain having three CDRs including amino acid sequences 937, 943, 961, or amino acid sequences 899, 918,A heavy chain having three CDRs including 928 and a light chain having three CDRs including amino acid sequences 937, 906, 960, or a heavy chain having three CDRs including amino acid sequences 899, 918, 928 and a light chain having three CDRs including amino acid sequences 937, 906, 960, or a heavy chain having three CDRs including amino acid sequences 913, 920, 929 and a light chain having three CDRs including amino acid sequences 939, 948, 962, or a heavy chain having three CDRs including amino acid sequences 899, 918, 930 and a light chain having three CDRs including amino acid sequences 935, 944, 955, or a heavy chain having three CDRs including amino acid sequences 899, 921, 931 and amino acid sequence 93 It has a light chain with three CDRs including amino acid sequences 5, 944, and 955, or a heavy chain with three CDRs including amino acid sequences 912, 919, and 932 and a light chain with three CDRs including amino acid sequences 940, 949, and 963, or a heavy chain with three CDRs including amino acid sequences 899, 915, and 909 and a light chain with three CDRs including amino acid sequences 935, 943, and 960, or a heavy chain with three CDRs including amino acid sequences 914, 922, and 933 and a light chain with three CDRs including amino acid sequences 941, 950, and 964, or a heavy chain with three CDRs including amino acid sequences 912, 918, and 934 and a light chain with three CDRs including amino acid sequences 942, 951, and 965.

[0035] CC chemokine receptor 4 (CCR4) (048) Exemplary CC chemokine receptor 4 (CCR4) antibodies include antibodies having a VH nucleotide sequence with SEQ ID NO: 969 and a VL nucleotide sequence with SEQ ID NO: 971; and antibodies having a VH nucleotide sequence with SEQ ID NO: 969 and a VL nucleotide sequence with SEQ ID NO: 972.

[0036] Exemplary CCR4 antibodies include antibodies having a VH amino acid sequence with SEQ ID NO:970 and a VL amino acid sequence with SEQ ID NO:971.

[0037] In another embodiment, the CCR4 antibody has a heavy chain having three CDRs containing amino acid sequences SEQ ID NO: 973, 974, and 975, and a light chain having three CDRs containing amino acid sequences 976, 977, and 978, respectively.

[0038] Middle East Respiratory Syndrome Coronavirus (MERS-CoV) (85) Exemplary anti-Middle East Respiratory Syndrome Coronavirus (MERS-CoV) antibodies include antibodies having a VH nucleotide sequence with SEQ ID NO:677 and a VL nucleotide sequence with SEQ ID NO:679, a VH nucleotide sequence with SEQ ID NO:681 and a VL nucleotide sequence with SEQ ID NO:683, a VH nucleotide sequence with SEQ ID NO:685 and a VL nucleotide sequence with SEQ ID NO:687, a VH nucleotide sequence with SEQ ID NO:689 and a VL nucleotide sequence with SEQ ID NO:692, a VH nucleotide sequence with SEQ ID NO:693 and a VL nucleotide sequence with SEQ ID NO:695, a VH nucleotide sequence with SEQ ID NO:697 and a VL nucleotide sequence with SEQ ID NO:699, and a VH nucleotide sequence with SEQ ID NO:701 and a VL nucleotide sequence with SEQ ID NO:703.

[0039] Exemplary anti-Middle East Respiratory Syndrome Coronavirus (MERS-CoV) antibodies include antibodies having VL amino acid sequences with VH amino acid sequences SEQ ID NO:678 and SEQ ID NO:680, VL amino acid sequences with VH amino acid sequences SEQ ID NO:682 and SEQ ID NO:684, VL amino acid sequences with VH amino acid sequences SEQ ID NO:686 and SEQ ID NO:688, VL amino acid sequences with VH amino acid sequences SEQ ID NO:690 and SEQ ID NO:692, VL amino acid sequences with VH amino acid sequences SEQ ID NO:694 and SEQ ID NO:696, VL amino acid sequences with VH amino acid sequences SEQ ID NO:698 and SEQ ID NO:700, and VL amino acid sequences with VH amino acid sequences SEQ ID NO:702 and SEQ ID NO:704.

[0040] In another embodiment, the anti-Middle East Respiratory Syndrome Coronavirus (MERS-CoV) antibody comprises a heavy chain having three CDRs including amino acid sequences 705, 706, and 707; a light chain having three CDRs including amino acid sequences 722, 723, and 724; a heavy chain having three CDRs including amino acid sequences 708, 709, and 710; a light chain having three CDRs including amino acid sequences 725, 726, and 727; a heavy chain having three CDRs including amino acid sequences 711, 712, and 713; a light chain having three CDRs including amino acid sequences 728, 729, and 730; and amino acid sequences 711, 735, and 715. It has a heavy chain containing three CDRs and a light chain containing three CDRs containing amino acid sequences 731, 732, and 733; a heavy chain containing three CDRs containing amino acid sequences 711, 735, and 716 and a light chain containing three CDRs containing amino acid sequences 737, 738, and 739; a heavy chain containing three CDRs containing amino acid sequences 717, 718, and 719 and a light chain containing three CDRs containing amino acid sequences 736, 742, and 743; and a heavy chain containing three CDRs containing amino acid sequences 714, 720, and 721 and a light chain containing three CDRs containing amino acid sequences 740, 729, and 741.

[0041] GITR(93) Exemplary anti-human GITR antibodies include VH nucleotide sequences having SEQ ID NO: 1361 and VL nucleotide sequences having SEQ ID NO: 1363, VH nucleotide sequences having SEQ ID NO: 1365 and VL nucleotide sequences having SEQ ID NO: 1367, VH nucleotide sequences having SEQ ID NO: 1369 and VL nucleotide sequences having SEQ ID NO: 1371, VH nucleotide sequences having SEQ ID NO: 1381 and VL nucleotide sequences having SEQ ID NO: 1375, VH nucleotide sequences having SEQ ID NO: 1377 and VL nucleotide sequences having SEQ ID NO: 1379, VH nucleotide sequences having SEQ ID NO: 1381 and VL nucleotide sequences having SEQ ID NO: 1383, VH nucleotide sequences having SEQ ID NO: 1385 and VL nucleotide sequences having SEQ ID NO: 1387, VH nucleotide sequences having SEQ ID NO: 1389 and SEQ ID The antibody comprises a VL nucleotide sequence having NO:1391, a VH nucleotide sequence having SEQ ID NO:1393 and a VL nucleotide sequence having SEQ ID NO:1395, a VH nucleotide sequence having SEQ ID NO:1397 and a VL nucleotide sequence having SEQ ID NO:1398, or a VH nucleotide sequence having SEQ ID NO:1401 and a VL nucleotide sequence having SEQ ID NO:1403.

[0042] Exemplary anti-human GITR antibodies include VH amino acid sequences with SEQ ID NO:1362 and VL amino acid sequences with SEQ ID NO:1364, VH amino acids with SEQ ID NO:1366 and VL polypeptide sequences with SEQ ID NO:1368, VH amino acid sequences with SEQ ID NO:1371 and VL amino acid sequences with SEQ ID NO:1372, VH amino acid sequences with SEQ ID NO:1382 and VL amino acid sequences with SEQ ID NO:1376, VH nucleotide sequences with SEQ ID NO:1378 and VL nucleotide sequences with SEQ ID NO:1380, VH amino acids with SEQ ID NO:1382 and VL polypeptide sequences with SEQ ID NO:1384, VH amino acid sequences with SEQ ID NO:1386 and VL amino acid sequences with SEQ ID NO:1388, VH amino acid sequences with SEQ ID NO:1390 and SEQ ID The antibody contains a VL amino acid sequence having NO:1392, a VH amino acid sequence having SEQ ID NO:1394 and a VL polypeptide sequence having SEQ ID NO:1396, a VH amino acid sequence having SEQ ID NO:1399 and a VL amino acid sequence having SEQ ID NO:1400, or a VH amino acid sequence having SEQ ID NO:1402 and a VL amino acid sequence having SEQ ID NO:1404.

[0043] In another embodiment, the anti-human GITR antibody comprises a heavy chain having three CDRs containing amino acid sequences 1405, 1406, and 1407, a light chain having three CDRs containing amino acid sequences 1408, 1409, and 1410, a heavy chain having three CDRs containing amino acid sequences 1411, 1412, and 1413, a light chain having three CDRs containing amino acid sequences 1414, 1415, and 1416, a heavy chain having three CDRs containing amino acid sequences 1417, 1418, and 1419, and amino acids A light chain having three CDRs containing sequences 1420, 1421, and 1422; a heavy chain having three CDRs containing amino acid sequences 1423, 1424, and 1425, respectively; a light chain having three CDRs containing amino acid sequences 1426, 1427, and 1428; a heavy chain having three CDRs containing amino acid sequences 1429, 1430, and 1431, respectively; a light chain having three CDRs containing amino acid sequences 1432, 1433, and 1434; and three CDRs containing amino acid sequences 1435, 1436, and 1437, respectively. A heavy chain having a heavy chain and a light chain having three CDRs containing amino acid sequences 1438, 1439 and 1440, a heavy chain having three CDRs containing amino acid sequences 1441, 1442 and 1443 respectively, a light chain having three CDRs containing amino acid sequences 1444, 1445 and 1446, a heavy chain having three CDRs containing amino acid sequences 1447, 1448 and 1449 respectively, a light chain having three CDRs containing amino acid sequences 1450, 1451 and 1452 respectively, and amino acid sequences 1453, 1454 and The structure has a heavy chain having three CDRs including 1455 and a light chain having three CDRs including amino acid sequences 1456, 1457, and 1458, a heavy chain having three CDRs including amino acid sequences 1459, 1460, and 1461 and a light chain having three CDRs including amino acid sequences 1462, 1463, and 1464, or a heavy chain having three CDRs including amino acid sequences 1465, 1466, and 1467 and a light chain having three CDRs including amino acid sequences 1468, 1469, and 1470.

[0044] Flavivirus (73) Exemplary anti-West Nile virus envelope protein E (WNE) antibodies include antibodies having a VH nucleotide sequence with a VH amino acid sequence having SEQ ID NO: 1224 and a VL amino acid sequence having SEQ ID NO: 1226.

[0045] Exemplary anti-West Nile virus envelope protein E (WNE) antibodies include antibodies having a VH nucleotide sequence with SEQ ID NO: 1225 and a VL nucleotide sequence with SEQ ID NO: 1227.

[0046] In another embodiment, the anti-West Nile virus envelope protein E (WNE) antibody comprises a heavy chain having three CDRs containing amino acid sequences 1244, 1245, and 1246, and a light chain having three CDRs containing amino acid sequences 1247, 1248, and 1249.

[0047] CCR4(65) Exemplary anti-CC chemokine receptor 4 (CCR4) antibodies include antibodies having a VH nucleotide sequence with SEQ ID NO: 1329 and a VL nucleotide sequence with SEQ ID NO: 1331, a VH nucleotide sequence with SEQ ID NO: 1333 and a VL nucleotide sequence with SEQ ID NO: 1335, a VH nucleotide sequence with SEQ ID NO: 1337 and a VL nucleotide sequence with SEQ ID NO: 1192, a VH nucleotide sequence with SEQ ID NO: 1341 and a VL nucleotide sequence with SEQ ID NO: 1343, or a VH nucleotide sequence with SEQ ID NO: 1357 and a VL nucleotide sequence with SEQ ID NO: 1359.

[0048] An exemplary anti-CC chemokine receptor 4 (CCR4) antibody has SEQ ID NO: 1330. H V with amino acid sequence and SEQ ID NO:1332 L V with amino acid sequence, SEQ ID NO:1334 HV with amino acid sequence and SEQ ID NO:1336 L V with amino acid sequence, SEQ ID NO:1338 H V with amino acid sequence and SEQ ID NO:1340 L V with amino acid sequence, SEQ ID NO:1342 H V with amino acid sequence and SEQ ID NO:1344 L V having an amino acid sequence or SEQ ID NO:1358 H V with amino acid sequence and SEQ ID NO:1360 L Contains antibodies having an amino acid sequence.

[0049] In other embodiments, the anti-CC chemokine receptor 4 (CCR4) antibody comprises a heavy chain having three CDRs containing amino acid sequences 1203, 1208, and 1211, and a light chain having three CDRs containing amino acid sequences 1207, 1209, and 1216, respectively; or a heavy chain having three CDRs containing amino acid sequences 1204, 1208, and 1212, respectively, and a light chain having three CDRs containing amino acid sequences 1207, 1209, and 1217, respectively; or a heavy chain having three CDRs containing amino acid sequences 1204, 1208, and 1213, respectively, and three CDRs containing amino acid sequences 1207, 1209, and 1217. The device has a light chain with one CDR, or a heavy chain with three CDRs containing amino acid sequences 1205, 1208, and 1214, and a light chain with three CDRs containing amino acid sequences 1207, 1209, and 1218, or a heavy chain with three CDRs containing amino acid sequences 1206, 1208, and 1210, and a light chain with three CDRs containing amino acid sequences 1207, 1209, and 1220, or a heavy chain with three CDRs containing amino acid sequences 1202, 1208, and 1210, and a light chain with three CDRs containing amino acid sequences 1207, 1209, and 1219.

[0050] Human immunoglobulin heavy chain variable region germline gene VH1-69(57)

[0051] Exemplary anti-human immunoglobulin heavy chain variable region germline gene VH1-69 antibodies include antibodies having a VH nucleotide sequence with SEQ ID NO:1153 and a VL nucleotide sequence with SEQ ID NO:1155, or a VH nucleotide sequence with SEQ ID NO:1163 and a VL nucleotide sequence with SEQ ID NO:1155.

[0052] An example of an anti-human immunoglobulin heavy chain variable region germline gene VH1-69 antibody is V with SEQ ID NO:1154. H V with amino acid sequence and SEQ ID NO:1156 L V having an amino acid sequence or SEQ ID NO:1164 H V with amino acid sequence and SEQ ID NO:1156 L Contains antibodies having an amino acid sequence.

[0053] In another embodiment, the anti-human immunoglobulin heavy chain variable region germline gene VH1-69 antibody comprises a heavy chain having three CDRs containing amino acid sequences 1157, 1158, and 1159, and a light chain having three CDRs containing amino acid sequences 1160, 1161, and 1162.

[0054] Influenza (49) Exemplary anti-influenza antibodies include: VH nucleotide sequence with SEQ ID NO:981 and VL nucleotide sequence with SEQ ID NO:983, VH nucleotide sequence with SEQ ID NO:985 and VL nucleotide sequence with SEQ ID NO:989, VH nucleotide sequence with SEQ ID NO:987 and VL nucleotide sequence with SEQ ID NO:991, VH nucleotide sequence with SEQ ID NO:993 and VL nucleotide sequence with SEQ ID NO:997, VH nucleotide sequence with SEQ ID NO:995 and VK nucleotide sequence with SEQ ID NO:999, VH nucleotide sequence with SEQ ID NO:1001 and VL nucleotide sequence with SEQ ID NO:1005, VH nucleotide sequence with SEQ ID NO:1003 and VL nucleotide sequence with SEQ ID NO:1007, VH nucleotide sequence with SEQ ID NO:1009 and VL nucleotide sequence with SEQ ID NO:1011, SEQ ID The antibody comprises a VH nucleotide sequence having NO:1013 and a VL nucleotide sequence having SEQ ID NO:1015, as well as a VH nucleotide sequence having SEQ ID NO:1017 and a VK nucleotide sequence having SEQ ID NO:1019, a VH nucleotide sequence having SEQ ID NO:1020 and a VL nucleotide sequence having SEQ ID NO:1022.

[0055] Exemplary anti-influenza antibodies include VH amino acid sequences with SEQ ID NO:982 and VL amino acid sequences with SEQ ID NO:984, VH amino acid sequences with SEQ ID NO:986 and VL amino acid sequences with SEQ ID NO:988, VH amino acid sequences with SEQ ID NO:986 and VL amino acid sequences with SEQ ID NO:990, VH amino acid sequences with SEQ ID NO:992 and VL amino acid sequences with SEQ ID NO:994, VH amino acid sequences with SEQ ID NO:992 and VK amino acid sequences with SEQ ID NO:996, VH amino acid sequences with SEQ ID NO:998 and VL amino acid sequences with SEQ ID NO:1000, VH amino acid sequences with SEQ ID NO:998 and VL amino acid sequences with SEQ ID NO:1002, VH amino acid sequences with SEQ ID NO:1004 and VL amino acid sequences with SEQ ID NO:1006, SEQ ID The antibody comprises a VH amino acid sequence having NO:1008 and a VL amino acid sequence having SEQ ID NO:1010, a VH amino acid sequence having SEQ ID NO:1012 and a VK amino acid sequence having SEQ ID NO:1014, and an antibody having a VH amino acid sequence having SEQ ID NO:1016 and a VL amino acid sequence having SEQ ID NO:1018.

[0056] In another embodiment, the anti-influenza antibody comprises a heavy chain having three CDRs containing amino acid sequences 1023, 1031 and 1039, a light chain having three CDRs containing amino acid sequences 1047, 1059 and 1071, a heavy chain having three CDRs containing amino acid sequences 1023, 1032 and 1040, a light chain having three CDRs containing amino acid sequences 1048, 1060 and 1072, a heavy chain having three CDRs containing amino acid sequences 1025, 1032 and 1040, and amino acids A light chain having three CDRs containing sequences 1057, 1069, and 1081; a heavy chain having three CDRs containing amino acid sequences 1026, 1033, and 1041; a light chain having three CDRs containing amino acid sequences 1049, 1061, and 1073; a heavy chain having three CDRs containing amino acid sequences 1026, 1033, and 1041; a light chain having three CDRs containing amino acid sequences 1054, 1066, and 1078; and three CDRs containing amino acid sequences 1027, 1034, and 1042. A heavy chain having a light chain with three CDRs containing amino acid sequences 1050, 1062 and 1074, a heavy chain having three CDRs containing amino acid sequences 1027, 1034 and 1042, a light chain having three CDRs containing amino acid sequences 1056, 1068 and 1080, a heavy chain having three CDRs containing amino acid sequences 1028, 1035 and 1043, and a light chain having three CDRs containing amino acid sequences 1051, 1063 and 1065, and amino acids 1028, 1036 and 1044. It has a heavy chain with three CDRs containing an acid sequence and a light chain with three CDRs containing amino acid sequences 1052, 1064, and 1076; a heavy chain with three CDRs containing amino acid sequences 1029, 1037, and 1045 and a light chain with three CDRs containing amino acid sequences 1053, 1065, and 1077; or a heavy chain with three CDRs containing amino acid sequences 1030, 1038, and 1046 and a light chain with three CDRs containing amino acid sequences 1058, 1070, and 1082.

[0057] Influenza (78) An exemplary anti-influenza antibody is a VH nucleotide sequence with SEQ ID NO:397 and a VL nucleotide sequence with SEQ ID NO:398, a VH nucleotide sequence with SEQ ID NO:399 and a VL nucleotide sequence with SEQ ID NO:400. L VH nucleotide sequence with nucleotide sequence, SEQ ID NO:401 and V with SEQ ID NO:402 LNucleotide sequence, VH nucleotide sequence having SEQ ID NO:403 and VL nucleotide sequence having SEQ ID NO:404, or VH nucleotide sequence having SEQ ID NO:405 and VL nucleotide sequence having SEQ ID NO:406, or VH nucleotide sequence having SEQ ID NO:407 and VL nucleotide sequence having SEQ ID NO:408, or VH nucleotide sequence having SEQ ID NO:409 and VL nucleotide sequence having SEQ ID NO:410, or VH nucleotide sequence having SEQ ID NO:411 and VL nucleotide sequence having SEQ ID NO:412, or VH nucleotide sequence having SEQ ID NO:413 and VL nucleotide sequence having SEQ ID NO:414, or VH nucleotide sequence having SEQ ID NO:415 and VL nucleotide sequence having SEQ ID NO:416, or VH nucleotide sequence having SEQ ID NO:417 and VL nucleotide sequence having SEQ ID NO:418, or SEQ ID VH nucleotide sequence having NO:419 and VL nucleotide sequence having SEQ ID NO:420, or VH nucleotide sequence having SEQ ID NO:421 and VL nucleotide sequence having SEQ ID NO:422, or VH nucleotide sequence having SEQ ID NO:423 and VL nucleotide sequence having SEQ ID NO:424, or VH nucleotide sequence having SEQ ID NO:425 and VL nucleotide sequence having SEQ ID NO:426, or VH nucleotide sequence having SEQ ID NO:427 and VL nucleotide sequence having SEQ ID NO:428, or VH nucleotide sequence having SEQ ID NO:429 and VL nucleotide sequence having SEQ ID NO:430, or VH nucleotide sequence having SEQ ID NO:431 and VL nucleotide sequence having SEQ ID NO:432, or VH nucleotide sequence having SEQ ID NO:433 and VL nucleotide sequence having SEQ ID NO:434, or SEQ IDVH nucleotide sequence having NO:435 and VL nucleotide sequence having SEQ ID NO:436, or VH nucleotide sequence having SEQ ID NO:437 and VL nucleotide sequence having SEQ ID NO:438, or VH nucleotide sequence having SEQ ID NO:439 and VL nucleotide sequence having SEQ ID NO:440, or VH nucleotide sequence having SEQ ID NO:441 and VL nucleotide sequence having SEQ ID NO:442, or VH nucleotide sequence having SEQ ID NO:541 and VL nucleotide sequence having SEQ ID NO:542, or VH nucleotide sequence having SEQ ID NO:543 and VL nucleotide sequence having SEQ ID NO:544, or VH nucleotide sequence having SEQ ID NO:545 and VL nucleotide sequence having SEQ ID NO:546, or VH nucleotide sequence having SEQ ID NO:547 and VL nucleotide sequence having SEQ ID NO:548, or VH nucleotide sequence having SEQ ID NO:549 and SEQ ID VL nucleotide sequence having NO:550, or VH nucleotide sequence having SEQ ID NO:551 and VL nucleotide sequence having SEQ ID NO:552, or VH nucleotide sequence having SEQ ID NO:553 and VL nucleotide sequence having SEQ ID NO:554, or VH nucleotide sequence having SEQ ID NO:555 and VL nucleotide sequence having SEQ ID NO:556, or VH nucleotide sequence having SEQ ID NO:557 and VL nucleotide sequence having SEQ ID NO:558, or VH nucleotide sequence having SEQ ID NO:559 and VL nucleotide sequence having SEQ ID NO:560, or VH nucleotide sequence having SEQ ID NO:561 and VL nucleotide sequence having SEQ ID NO:562, or VH nucleotide sequence having SEQ ID NO:563 and VL nucleotide sequence having SEQ ID NO:564, or SEQ IDVH nucleotide sequence having NO:565 and VL nucleotide sequence having SEQ ID NO:566, or VH nucleotide sequence having SEQ ID NO:567 and VL nucleotide sequence having SEQ ID NO:568, or VH nucleotide sequence having SEQ ID NO:569 and VL nucleotide sequence having SEQ ID NO:570, or VH nucleotide sequence having SEQ ID NO:571 and VL nucleotide sequence having SEQ ID NO:572, or VH nucleotide sequence having SEQ ID NO:573 and VL nucleotide sequence having SEQ ID NO:574, or VH nucleotide sequence having SEQ ID NO:575 and VL nucleotide sequence having SEQ ID NO:576, or VH nucleotide sequence having SEQ ID NO:577 and VL nucleotide sequence having SEQ ID NO:578, or VH nucleotide sequence having SEQ ID NO:579 and VL nucleotide sequence having SEQ ID NO:580, or VH nucleotide sequence having SEQ ID NO:581 and SEQ ID VL nucleotide sequence having NO:582, or VH nucleotide sequence having SEQ ID NO:583 and VL nucleotide sequence having SEQ ID NO:584, or VH nucleotide sequence having SEQ ID NO:585 and VL nucleotide sequence having SEQ ID NO:586, or VH nucleotide sequence having SEQ ID NO:587 and VL nucleotide sequence having SEQ ID NO:588, or VH nucleotide sequence having SEQ ID NO:589 and VL nucleotide sequence having SEQ ID NO:590, or VH nucleotide sequence having SEQ ID NO:591 and VL nucleotide sequence having SEQ ID NO:592, or VH nucleotide sequence having SEQ ID NO:593 and VL nucleotide sequence having SEQ ID NO:594, or VH nucleotide sequence having SEQ ID NO:595 and VL nucleotide sequence having SEQ ID NO:596, or SEQ IDThe antibody comprises a VH nucleotide sequence having NO:597 and a VL nucleotide sequence having SEQ ID NO:598, or a VH nucleotide sequence having SEQ ID NO:599 and a VL nucleotide sequence having SEQ ID NO:600.

[0058] Exemplary anti-influenza antibodies include a VH amino acid sequence with SEQ ID NO: 469 and a VL amino acid sequence with SEQ ID NO: 470, a VH amino acid sequence with SEQ ID NO: 471 and a VL amino acid sequence with SEQ ID NO: 472. L The polypeptide sequence, the VH amino acid sequence having SEQ ID NO:473, and the V amino acid sequence having SEQ ID NO:474. LAmino acid sequence, VH amino acid sequence having SEQ ID NO:475 and VL amino acid sequence having SEQ ID NO:476, or VH nucleotide sequence having SEQ ID NO:477 and VL nucleotide sequence having SEQ ID NO:478, VH amino acid sequence having SEQ ID NO:479 and VL amino acid sequence having SEQ ID NO:480, VH amino acid sequence having SEQ ID NO:481 and VL amino acid sequence having SEQ ID NO:482, VH amino acid sequence having SEQ ID NO:483 and VL amino acid sequence having SEQ ID NO:484, VH amino acid sequence having SEQ ID NO:485 and VL amino acid sequence having SEQ ID NO:486, VH amino acid sequence having SEQ ID NO:487 and VL amino acid sequence having SEQ ID NO:488, VH amino acid sequence having SEQ ID NO:489 and VL amino acid sequence having SEQ ID NO:490, VH amino acid sequence having SEQ ID NO:491 and SEQ ID VL amino acid sequence having NO:492, VH amino acid sequence having SEQ ID NO:493 and VL amino acid sequence having SEQ ID NO:494, VH amino acid sequence having SEQ ID NO:495 and VL amino acid sequence having SEQ ID NO:496, VH amino acid sequence having SEQ ID NO:497 and VL amino acid sequence having SEQ ID NO:498, VH amino acid sequence having SEQ ID NO:499 and VL amino acid sequence having SEQ ID NO:500, VH amino acid sequence having SEQ ID NO:501 and VL amino acid sequence having SEQ ID NO:502, VH amino acid sequence having SEQ ID NO:503 and VL amino acid sequence having SEQ ID NO:504, VH amino acid sequence having SEQ ID NO:505 and VL amino acid sequence having SEQ ID NO:506, VH amino acid sequence having SEQ ID NO:507 and VL amino acid sequence having SEQ ID NO:508, SEQ ID VH amino acid sequence with NO:509 and VL amino acid sequence with SEQ ID NO:510, SEQ IDVH amino acid sequence having NO:511 and VL amino acid sequence having SEQ ID NO:512, VH amino acid sequence having SEQ ID NO:513 and VL amino acid sequence having SEQ ID NO:514, VH amino acid sequence having SEQ ID NO:515 and VL amino acid sequence having SEQ ID NO:516, VH amino acid sequence having SEQ ID NO:517 and VL amino acid sequence having SEQ ID NO:518, VH amino acid sequence having SEQ ID NO:519 and VL amino acid sequence having SEQ ID NO:520, VH amino acid sequence having SEQ ID NO:521 and VL amino acid sequence having SEQ ID NO:522, VH amino acid sequence having SEQ ID NO:523 and VL amino acid sequence having SEQ ID NO:524, VH amino acid sequence having SEQ ID NO:525 and VL amino acid sequence having SEQ ID NO:526, VH amino acid sequence having SEQ ID NO:527 and SEQ ID VL amino acid sequence having NO:528, VH amino acid sequence having SEQ ID NO:529 and VL amino acid sequence having SEQ ID NO:530, VH amino acid sequence having SEQ ID NO:531 and VL amino acid sequence having SEQ ID NO:532, VH amino acid sequence having SEQ ID NO:533 and VL amino acid sequence having SEQ ID NO:534, VH amino acid sequence having SEQ ID NO:535 and VL amino acid sequence having SEQ ID NO:536, VH amino acid sequence having SEQ ID NO:537 and VL amino acid sequence having SEQ ID NO:538, VH amino acid sequence having SEQ ID NO:539 and VL amino acid sequence having SEQ ID NO:540, VH amino acid sequence having SEQ ID NO:601 and VL amino acid sequence having SEQ ID NO:602, VH amino acid sequence having SEQ ID NO:603 and VL amino acid sequence having SEQ ID NO:604, SEQ ID VH amino acid sequence with SEQ ID NO:605 and VL amino acid sequence with SEQ ID NO:606, VH amino acid sequence with SEQ ID NO:607 and SEQ IDVL amino acid sequence having NO:608, VH amino acid sequence having SEQ ID NO:609 and VL amino acid sequence having SEQ ID NO:610, VH amino acid sequence having SEQ ID NO:611 and VL amino acid sequence having SEQ ID NO:612, VH amino acid sequence having SEQ ID NO:613 and VL amino acid sequence having SEQ ID NO:614, VH amino acid sequence having SEQ ID NO:615 and VL amino acid sequence having SEQ ID NO:616, VH amino acid sequence having SEQ ID NO:617 and VL amino acid sequence having SEQ ID NO:618, VH amino acid sequence having SEQ ID NO:619 and VL amino acid sequence having SEQ ID NO:620, VH amino acid sequence having SEQ ID NO:621 and VL amino acid sequence having SEQ ID NO:622, VH amino acid sequence having SEQ ID NO:623 and VL amino acid sequence having SEQ ID NO:624, SEQ ID VH amino acid sequence having NO:625 and VL amino acid sequence having SEQ ID NO:626, VH amino acid sequence having SEQ ID NO:627 and VL amino acid sequence having SEQ ID NO:628, VH amino acid sequence having SEQ ID NO:629 and VL amino acid sequence having SEQ ID NO:630, VH amino acid sequence having SEQ ID NO:631 and VL amino acid sequence having SEQ ID NO:632, VH amino acid sequence having SEQ ID NO:633 and VL amino acid sequence having SEQ ID NO:634, VH amino acid sequence having SEQ ID NO:635 and VL amino acid sequence having SEQ ID NO:636, VH amino acid sequence having SEQ ID NO:637 and VL amino acid sequence having SEQ ID NO:638, VH amino acid sequence having SEQ ID NO:639 and VL amino acid sequence having SEQ ID NO:640, VH amino acid sequence having SEQ ID NO:641 and SEQ ID VL amino acid sequence having NO:642, SEQ ID VH amino acid sequence having NO:643 and VL amino acid sequence having SEQ ID NO:644, SEQ IDThe antibody contains a VH amino acid sequence having NO:645 and a VL amino acid sequence having SEQ ID NO:646, a VH amino acid sequence having SEQ ID NO:647 and a VL amino acid sequence having SEQ ID NO:648, a VH amino acid sequence having SEQ ID NO:649 and a VL amino acid sequence having SEQ ID NO:650, a VH amino acid sequence having SEQ ID NO:651 and a VL amino acid sequence having SEQ ID NO:652, a VH amino acid sequence having SEQ ID NO:653 and a VL amino acid sequence having SEQ ID NO:654, a VH amino acid sequence having SEQ ID NO:655 and a VL amino acid sequence having SEQ ID NO:656, a VH amino acid sequence having SEQ ID NO:657 and a VL amino acid sequence having SEQ ID NO:658, a VH amino acid sequence having SEQ ID NO:659 and a VL amino acid sequence having SEQ ID NO:660.

[0059] In another embodiment, the anti-influenza antibody comprises a heavy chain having three CDRs containing amino acid sequences SEQ ID NO: 1, 37, and 73, and a light chain having three CDRs containing amino acid sequences 109, 145, and 181, respectively; or a heavy chain having three CDRs containing amino acid sequences 2, 38, and 74, respectively, and a light chain having three CDRs containing amino acid sequences 110, 146, and 182, respectively; or a heavy chain having three CDRs containing amino acid sequences 3, 39, and 75, respectively, and a light chain having three CDRs containing amino acid sequences 111, 147, and 183, respectively; or containing amino acid sequences 4, 40, and 76, respectively. A heavy chain having three CDRs and a light chain having three CDRs containing amino acid sequences 112, 148, and 184 respectively, or a heavy chain having three CDRs containing amino acid sequences 5, 41, and 77 respectively and a light chain having three CDRs containing amino acid sequences 113, 149, and 185 respectively, or a heavy chain having three CDRs containing amino acid sequences 6, 42, and 78 respectively and a light chain having three CDRs containing amino acid sequences 114, 150, and 186 respectively, or a heavy chain having three CDRs containing amino acid sequences 7, 43, and 79 respectively. and a light chain having three CDRs containing amino acid sequences 115, 151, and 187 respectively, or a heavy chain having three CDRs containing amino acid sequences 8, 44, and 80 respectively and a light chain having three CDRs containing amino acid sequences 116, 152, and 188 respectively, or a heavy chain having three CDRs containing amino acid sequences 9, 45, and 81 respectively and a light chain having three CDRs containing amino acid sequences 117, 153, and 189 respectively, or a heavy chain having three CDRs containing amino acid sequences 10, 46, and 82 respectively and each amino acid A light chain having three CDRs containing sequences 118, 154, and 190, or a heavy chain having three CDRs containing amino acid sequences 11, 47, and 83 respectively and a light chain having three CDRs containing amino acid sequences 119, 155, and 191 respectively, or a heavy chain having three CDRs containing amino acid sequences 12, 48, and 84 respectively and a light chain having three CDRs containing amino acid sequences 120, 156, and 192 respectively, or a heavy chain having three CDRs containing amino acid sequences 13, 49, and 85 respectively and amino acid sequences 121, 157,A light chain having three CDRs including 193, or a heavy chain having three CDRs each containing amino acid sequences 14, 50, and 86 and a light chain having three CDRs each containing amino acid sequences 122, 158, and 194, or a heavy chain having three CDRs each containing amino acid sequences 15, 51, and 87 and a light chain having three CDRs each containing amino acid sequences 123, 159, and 195, or a heavy chain having three CDRs each containing amino acid sequences 16, 52, and 88 and a light chain having three CDRs each containing amino acid sequences 124, 160, and 196, Or a heavy chain having three CDRs containing amino acid sequences 17, 53, and 89 respectively, and a light chain having three CDRs containing amino acid sequences 125, 161, and 197 respectively, or a heavy chain having three CDRs containing amino acid sequences 18, 54, and 90 respectively, and a light chain having three CDRs containing amino acid sequences 126, 162, and 198 respectively, or a heavy chain having three CDRs containing amino acid sequences 19, 55, and 91 respectively, and a light chain having three CDRs containing amino acid sequences 127, 163, and 199 respectively, or amino acid sequences 20, 56, A heavy chain having three CDRs including 92 and a light chain having three CDRs including amino acid sequences 128, 164, and 200 respectively, or a heavy chain having three CDRs including amino acid sequences 21, 57, and 93 respectively and a light chain having three CDRs including amino acid sequences 129, 165, and 201 respectively, or a heavy chain having three CDRs including amino acid sequences 22, 58, and 94 respectively and a light chain having three CDRs including amino acid sequences 130, 166, and 202 respectively, or a heavy chain having three CDRs including amino acid sequences 23, 59, and 95 respectively A light chain having three CDRs containing amino acid sequences 131, 167, and 203, respectively; or a heavy chain having three CDRs containing amino acid sequences 24, 60, and 96, respectively, and a light chain having three CDRs containing amino acid sequences 132, 168, and 204, respectively; or a heavy chain having three CDRs containing amino acid sequences 25, 61, and 95, respectively, and a light chain having three CDRs containing amino acid sequences 133, 169, and 205, respectively; or a heavy chain having three CDRs containing amino acid sequences 26, 62, and 96, respectively, and amino acid sequences 134, 170, respectively.A light chain having three CDRs including 206, or a heavy chain having three CDRs including amino acid sequences 27, 63, and 97 respectively and a light chain having three CDRs including amino acid sequences 135, 171, and 207 respectively, or a heavy chain having three CDRs including amino acid sequences 28, 64, and 98 respectively and a light chain having three CDRs including amino acid sequences 136, 172, and 208 respectively, or a heavy chain having three CDRs including amino acid sequences 29, 65, and 99 respectively and a light chain having three CDRs including amino acid sequences 137, 173, and 209 respectively, or A heavy chain having three CDRs containing amino acid sequences 30, 66, and 100, and a light chain having three CDRs containing amino acid sequences 138, 174, and 210, respectively; or a heavy chain having three CDRs containing amino acid sequences 31, 67, and 101, respectively, and a light chain having three CDRs containing amino acid sequences 139, 175, and 211, respectively; or a heavy chain having three CDRs containing amino acid sequences 32, 68, and 102, respectively, and a light chain having three CDRs containing amino acid sequences 140, 176, and 212, respectively; or amino acid sequences 33, 69, and 103 A heavy chain having three CDRs including and a light chain having three CDRs containing amino acid sequences 141, 177, and 213 respectively, or a heavy chain having three CDRs containing amino acid sequences 34, 70, and 104 respectively and a light chain having three CDRs containing amino acid sequences 142, 178, and 214 respectively, or a heavy chain having three CDRs containing amino acid sequences 35, 71, and 105 respectively and a light chain having three CDRs containing amino acid sequences 143, 179, and 215 respectively, or a heavy chain having three CDRs containing amino acid sequences 36, 72, and 106 respectively and A light chain having three CDRs containing amino acid sequences 144, 180, and 216, respectively; or a heavy chain having three CDRs containing amino acid sequences 217, 247, and 277, respectively, and a light chain having three CDRs containing amino acid sequences 307, 337, and 367, respectively; or a heavy chain having three CDRs containing amino acid sequences 218, 248, and 278, respectively, and a light chain having three CDRs containing amino acid sequences 308, 338, and 368, respectively; or a heavy chain having three CDRs containing amino acid sequences 219, 249, and 279, respectively, and an amino acid sequence 309, respectively.A light chain having three CDRs including 339 and 369, or a heavy chain having three CDRs including amino acid sequences 220, 250, and 280 respectively and a light chain having three CDRs including amino acid sequences 310, 340, and 370 respectively, or a heavy chain having three CDRs including amino acid sequences 221, 251, and 281 respectively and a light chain having three CDRs including amino acid sequences 311, 341, and 371 respectively, or a heavy chain having three CDRs including amino acid sequences 222, 252, and 282 respectively and 3 CDRs including amino acid sequences 312, 342, and 372 respectively A light chain having one CDR, or a heavy chain having three CDRs containing amino acid sequences 223, 253, and 283 respectively, and a light chain having three CDRs containing amino acid sequences 313, 343, and 373 respectively, or a heavy chain having three CDRs containing amino acid sequences 224, 254, and 284 respectively, and a light chain having three CDRs containing amino acid sequences 314, 344, and 374 respectively, or a heavy chain having three CDRs containing amino acid sequences 225, 255, and 285 respectively, and a light chain having three CDRs containing amino acid sequences 315, 345, and 375 respectively. , or a heavy chain having three CDRs containing amino acid sequences 226, 256, and 286 respectively and a light chain having three CDRs containing amino acid sequences 316, 346, and 376 respectively, or a heavy chain having three CDRs containing amino acid sequences 227, 257, and 287 respectively and a light chain having three CDRs containing amino acid sequences 317, 347, and 377 respectively, or a heavy chain having three CDRs containing amino acid sequences 228, 258, and 288 respectively and a light chain having three CDRs containing amino acid sequences 318, 348, and 378 respectively, or each amino A heavy chain having three CDRs containing amino acid sequences 229, 259, and 289 and a light chain having three CDRs containing amino acid sequences 319, 349, and 379 respectively, or a heavy chain having three CDRs containing amino acid sequences 230, 260, and 290 respectively and a light chain having three CDRs containing amino acid sequences 320, 350, and 380 respectively, or a heavy chain having three CDRs containing amino acid sequences 231, 261, and 291 respectively and a light chain having three CDRs containing amino acid sequences 321, 351, and 381 respectively, or amino acid sequences 232, 262,A heavy chain having three CDRs including 292 and a light chain having three CDRs each containing amino acid sequences 322, 352, and 382, ​​or a heavy chain having three CDRs each containing amino acid sequences 233, 263, and 293 and a light chain having three CDRs each containing amino acid sequences 323, 353, and 383, or a heavy chain having three CDRs each containing amino acid sequences 234, 273, and 294 and a light chain having three CDRs each containing amino acid sequences 324, 354, and 384, or three CDRs each containing amino acid sequences 235, 274, and 295 A heavy chain having a CDR and a light chain having three CDRs containing amino acid sequences 325, 355, and 385 respectively, or a heavy chain having three CDRs containing amino acid sequences 236, 275, and 296 respectively and a light chain having three CDRs containing amino acid sequences 326, 356, and 386 respectively, or a heavy chain having three CDRs containing amino acid sequences 237, 276, and 297 respectively and a light chain having three CDRs containing amino acid sequences 327, 357, and 387 respectively, or a heavy chain having three CDRs containing amino acid sequences 237, 277, and 298 respectively. A light chain having three CDRs containing amino acid sequences 328, 358, and 388 respectively, or a heavy chain having three CDRs containing amino acid sequences 238, 278, and 299 respectively and a light chain having three CDRs containing amino acid sequences 329, 359, and 389 respectively, or a heavy chain having three CDRs containing amino acid sequences 239, 279, and 300 respectively and a light chain having three CDRs containing amino acid sequences 330, 360, and 390 respectively, or a heavy chain having three CDRs containing amino acid sequences 240, 280, and 301 respectively and each amino acid A light chain having three CDRs containing sequences 331, 361, and 391, or a heavy chain having three CDRs containing amino acid sequences 241, 281, and 302 respectively, and a light chain having three CDRs containing amino acid sequences 332, 362, and 392, or a heavy chain having three CDRs containing amino acid sequences 242, 282, and 303 respectively, and a light chain having three CDRs containing amino acid sequences 333, 363, and 393, or a heavy chain having three CDRs containing amino acid sequences 243, 283, and 304 respectively, and amino acid sequences 334, 364,It has a light chain with three CDRs including 394, or a heavy chain with three CDRs containing amino acid sequences 244, 284, and 305, and a light chain with three CDRs containing amino acid sequences 335, 365, and 395, respectively, or a heavy chain with three CDRs containing amino acid sequences 245, 285, and 306, and a light chain with three CDRs containing amino acid sequences 336, 366, and 396, respectively.

[0060] Other anti-influenza antibodies include those having the amino acid or nucleic acid sequences shown in Table 1 below.

[0061] (Table 1A) Nucleic acid sequence of the variable region of antibody 3I14 TIFF0007833003000001.tif60152

[0062] (Table 1B) Amino acid sequence of the variable region of antibody 3I14 TIFF0007833003000002.tif37152

[0063] (Table 1C) Antibody 3I14V L Nucleic acid sequence of the variable region of D94N TIFF0007833003000003.tif25152

[0064] (Table 1C) Antibody 3I14V L Amino acid sequence of the variable region of D94N TIFF0007833003000004.tif15152

[0065] 3I14 and 3I14V L The amino acid sequences of the heavy and light chain complementarity-determining regions of the D94N neutralizing influenza antibody are shown in Table 2 below.

[0066] (Table 2) TIFF0007833003000005.tif32148

[0067] Other binding domains besides scFv, such as, in non-limiting examples, camel single-domain antibody fragments or receptor ligands, antibody-binding domains, antibody hypervariable loops, or CDRs, may also be used for predetermined targeting of lymphocytes.

[0068] In a preferred embodiment, the transmembrane domain further includes a stalk region between the extracellular ligand-binding domain and the transmembrane domain. As used herein, the term “stalk region” generally refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular ligand-binding domain. In particular, the stalk region is used to provide high flexibility and accessibility to the extracellular ligand-binding domain. The stalk region may contain up to 300 amino acids, preferably 10 to 100 amino acids and more preferably 25 to 50 amino acids. The stalk region may be derived from all or part of a native molecule, for example, all or part of the extracellular region of CD8, CD4, or CD28, or all or part of the antibody constant region. Alternatively, the stalk region may be a synthetic sequence corresponding to a naturally occurring stalk sequence, or a completely synthetic stalk sequence. In a preferred embodiment, the stalk region is a portion of a human CD8 alpha chain.

[0069] The signaling domain or intracellular signaling domain of the CAR of the present invention is responsible for intracellular signaling that activates immune cells and immune responses after the binding of the extracellular ligand-binding domain to a target. In other words, the signaling domain is responsible for the activation of at least one of the normal effector functions of immune cells expressing the CAR. For example, the effector function of a T cell may be helper activity including cytolytic activity or cytokine secretion. Therefore, the term “signaling domain” refers to a part of a protein that transmits effector signaling function and causes that cell to exert a specific function.

[0070] The signaling domain comprises two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation and those that act in an antigen-independent manner to provide secondary or co-stimulatory signals. The primary cytoplasmic signaling sequence may include signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs. ITAMs are well-defined signaling motifs found in the cytoplasmic tails of various receptors that function as binding sites for syk / zap70 class tyrosine kinases. Examples of ITAMs used in the present invention, in non-limiting examples, may include those derived from TCR zeta, FcR gamma, FcR beta, FcR epsilon, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In a preferred embodiment, the signaling domain of the CAR may include a CD3 zeta signaling domain or an intraplasmic domain of an Fc epsilon RI beta or gamma chain. In another preferred embodiment, the signaling domain is provided by CD3 zeta, and the co-stimulation is provided by CD28 and tumor necrosis factor receptor (TNFr), e.g., 4-1BB or OX40.

[0071] In certain embodiments, the intracellular signaling domain of the CAR of the present invention includes a co-stimulatory signaling molecule. In some embodiments, the intracellular signaling domain includes two, three, four or more co-stimulatory molecules in series. The co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands required for an effective immune response.

[0072] A "costimulatory ligand" refers to a molecule on an antigen-presenting cell that specifically binds to an allogeneic costimulatory molecule on a T cell, thereby providing a signal that mediates a T cell response, including but not limited to activation of proliferation and differentiation, in addition to the primary signal provided by, for example, the binding of a peptide-loaded MHC molecule to the TCR / CD3 complex. Costimulatory ligands include CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intracellular adhesion molecules (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, Toll ligand receptor, and agonists or antibodies that bind to these receptors. The costimulatory ligands may include, but are not limited to, ligands that specifically bind to B7-H3. The costimulatory ligands also include antibodies that specifically bind to costimulatory molecules present on T cells, such as, non-limitingly, ligands that specifically bind to CD27, CD28, 4-IBB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, and CD83.

[0073] A “costimulatory molecule” represents an allobinding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by that cell, such as, not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class 1 molecules, BTLA, and Toll ligand receptors. Examples of costimulatory molecules include ligands that specifically bind to CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83. In another specific embodiment, the signaling domain is a TNFR-related factor 2 (TRAF2) binding motif, which is the cytoplasmic tail of the costimulatory TNFR member family. The cytoplasmic tails of co-stimulatory TNFR family members contain a TRAF2-binding motif consisting of a major conserved motif (P / S / A)X(Q / E)E) or a minor motif (PXQXXD), where X is any amino acid. TRAF proteins are induced in the intracellular tails of many TNFRs in response to receptor trimerization.

[0074] Notable features of a suitable transmembrane polypeptide include its ability to be expressed on the surface of immune cells, particularly lymphocytes or natural killer (NK) cells, and its interaction to direct the immune cell's cellular response to a predetermined target cell. Different transmembrane polypeptides of the CAR of the present invention, including an extracellular ligand-binding domain and / or a signaling domain, are involved in signaling after binding to a target ligand and interact to induce an immune response. The transmembrane domain may be derived from either a natural or synthetic source. The transmembrane domain may be derived from any membrane-bound or transmembrane protein.

[0075] As used herein, the term “part” refers to any subset of the molecule that is a shorter polypeptide. Alternatively, an amino acid sequence functional variant of a polypeptide may be prepared by mutations in the DNA encoding that polypeptide. Such variants or functional variants include, for example, deletions or insertions or substitutions of residues in the amino acid sequence. Any combination of deletions, insertions, and substitutions may also be made to exhibit particularly specific anti-target cell immune activity to reach the final construct, insofar as the final construct has the desired activity. The function of the CAR of the present invention in the host is detectable in assays suitable for demonstrating the signaling ability of this CAR when bound to a specific target. Such assays are available to those skilled in the art. For example, an assay including, for example, the measurement of calcium ion release, intracellular tyrosine phosphorylation, inositol phosphate turnover or the resulting increase in interleukin (IL) 2, interferon gamma, GM-CSF, IL-3, and IL-4 production allows for the detection of signaling pathways induced by target binding.

[0076] cell Aspects of the present invention include cells expressing a CAR (i.e., CARTS). The cells may be immune cells capable of expressing a CAR for cancer treatment or cells holding an expression vector encoding a CAR, including, for example, bacterial cells, of any species. As used herein, the terms “cell,” “cell line,” and “cell culture” may be used interchangeably. Furthermore, all these terms encompass their offspring, which are any and all subsequent generations. It is understood that all offspring may not be identical due to intentional or unintentional mutations. In relation to the expression of heterologous nucleic acid sequences, “host cell” refers to a eukaryotic cell capable of replicating a vector and / or expressing the heterologous gene encoded by the vector. A host cell is one that can and has been used as a recipient of a vector. A host cell may be “transfected” or “transformed,” these referring to the process by which a foreign nucleic acid is introduced into or introduced into its host cell. Transformed cells include primary relevant cells and their offspring. As used herein, the terms “engineered” and “recombinant” cell or host cell are intended to refer to cells into which a foreign nucleic acid sequence, such as a vector, has been introduced. Therefore, recombinant cells can be distinguished from naturally occurring cells that do not contain recombinant nucleic acids. In aspects of the present invention, the host cells are T cells, including cytotoxic T cells (also known as TCs, cytotoxic T lymphocytes, CTLs, T killer cells, cytolytic T cells, CD8+ T cells, or killer T cells), and NK cells and NKT cells are also included in the present invention.

[0077] Some vectors may utilize regulatory sequences that allow them to be replicated and / or expressed in both prokaryotic and eukaryotic cells. Those skilled in the art will further understand the conditions for maintaining them and incubating all of the aforementioned host cells to replicate the vectors. Techniques and conditions for achieving large-scale production of vectors and the production of nucleic acids encoded by the vectors and their homonucleotides, proteins, or peptides are also understood and known.

[0078] Cells can be self-cells, syngeneic cells, allogeneic cells, and in some cases even heterogeneic cells.

[0079] In many situations, a person skilled in the art may want to be able to kill modified CTLs when it is desired to terminate a procedure, when the cells become neoplastic, in research where the absence of the cells after their presence is of interest, or in other cases. For this purpose, a person skilled in the art may provide the expression of a specific gene product, such as an inducible suicide gene, that can kill modified cells under controlled conditions.

[0080] Armed Carts The present invention further comprises CARTS modified to secrete one or more polypeptides. The polypeptides may be, for example, antibodies or cytokines. Preferably, the antibodies are specific to CAIX, GITR, PD-L1, PD-L2, PD-1, or CCR4.

[0081] Armed CARTS have the advantage of simultaneously secreting polypeptides at the target site, such as a tumor site.

[0082] Armed CARTs can be constructed by including a nucleic acid encoding a polypeptide of interest after an intracellular signaling domain. Preferably, an internal ribosome entry site (IRES) is positioned between the intracellular signaling domain and the polypeptide of interest. Those skilled in the art will understand that two or more polypeptides can be expressed by using multiple IRES sequences in series.

[0083] In one embodiment, the methods and compositions described herein provide second-generation target-specific anti-CAIX CAR T cells armed with the ability to secrete anti-PD-L1 IgG in an RCC environment to address T cell exhaustion. Human anti-CAIX CARs containing a CD28 costimulatory domain were selected based on their killing activity and low immunogenicity in human cRCC xenografts in mice. Anti-CAIX CAR T cells secreting anti-PD-L1 IgG1 or IgG4 were extensively compared with unrelated anti-BCMA CARs or anti-CAIX CARs secreting unrelated anti-SARS IgG1, respectively. We demonstrated stable expression of CARs and high proliferative capacity of all CAR cells. Anti-CAIX CAR T cells were activated to release high levels of IFN□ and IL-2, which may enhance tumor suppression, and possess the ability to induce clonal proliferation upon contact with CAIX+ RCC cells. These anti-CAIX CART cells can also secrete high levels of anti-PD-L1 IgG1 or IgG4, and these antibodies can specifically interact with PD-L1 in vitro and in vivo to induce downregulation of exhaustion markers PD-1, Tim-3, and Lag-3. These results indicate that anti-PD-L1 antibodies secreted into the tumor microenvironment can reverse T cell exhaustion and enhance the antitumor activity of anti-CAIX CART cells. These anti-CAIX CART cells, primarily those secreting anti-PD-L1, can also suppress the proliferation of CAIX+ RCC cells in an NSG orthotopic mouse model of RCC, slowing tumor growth and reducing tumor size and weight. In addition, anti-CAIX CART cells secreting the anti-PD-L1 IgG1 isotype were also able to induce an increase in the number of human NK cells infiltrating ADCC in vitro and in vivo at tumor sites. To determine the ability of NK cells to recognize anti-PD-L1 IgG1 isotypes in vivo, human NK cells were injected into only two mice in each group two days before their euthanasia, and this was demonstrated. Since our previous experiments with this mouse model showed that these cells are maintained in their blood for only a few days, the injection of human NK cells was not performed at the start of the procedure.

[0084] In one embodiment, CART cells were injected into mice without the addition of interleukins to avoid affecting the therapeutic effect of CART cells alone.

[0085] In another embodiment, CART cells may be maintained by the use of cytokines, such as IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21.

[0086] Cytokines that share the γc receptor, such as IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, are important for the development and maintenance of memory T cells. Among these, IL-21 promotes a poorly differentiated phenotype, thereby increasing tumor-specific CD8 T cells and enhancing the antitumor effect in a mouse melanoma model compared to IL-2 or IL-15.

[0087] In certain embodiments, CART cells are maintained using IL-21.

[0088] Although CAIX is physiologically expressed in some tissues, its overexpression in many tumors, particularly cRCCs, makes it a consistent marker in the development of cancer-targeted systemic therapies.

[0089] In one embodiment, the anti-CAIX scFv within the CAR recognizes the catalytic domain of CAIX located in the central part of the protein, which may enhance its specificity to the high-expression site of CAIX.

[0090] The selection of CD28 as the co-stimulatory domain for CARs as described herein is based on the fact that CD28 CARs elicit a vigorous proliferation response and enhance effector function, while 4-1BB-based CARs induce a more progressive accumulation of T cells that can respond to less immediate effects. In one embodiment, CD28 is replaced by 41BB in the CAR construct.

[0091] T cell exhaustion is common in cancer, and these T cells exhibit low proliferative and cytokine-producing capacity, associated with high apoptosis rates and the expression of inhibitory receptors such as PD-1, Tim-3, and Lag3. Novel strategies to prevent T cell exhaustion include those related to the PD-1 / PD-L1 axis.

[0092] The methods and compositions described herein provide anti-CAIX CART cells that secrete anti-PD-L1 IgG1 and IgG4, which restore T cell exhaustion and improve the efficacy of CART cells in in vitro and in vivo cRCC treatment.

[0093] Introducing constructs to CTLs An expression vector encoding a CAR may be introduced as one or more DNA molecules or constructs, which may include at least one marker that allows for the selection of host cells containing those constructs.

[0094] The construct can be prepared by conventional or other convenient means, in which the gene and regulatory regions are isolated, ligated where appropriate, cloned in a suitable cloning host, and analyzed by restriction or sequencing. In particular, individual fragments containing all or part of the functional unit can be isolated using PCR, where one or more mutations can be introduced using primer repair, ligating, in vitro mutagenesis, etc., where appropriate. The completed construct, demonstrated to have a suitable sequence, can then be introduced into CTLs by any convenient means. The construct can be incorporated and packaged into a non-replicating incomplete viral genome, such as an adenovirus, adeno-associated virus (AAV), or herpes simplex virus (HSV), or other retroviral or lentiviral vectors, for infection or transduction into cells. The construct may contain a viral sequence for transfection, if desired. Alternatively, the construct can be introduced by fusion, electroporation, microparticle gun, transfection, lipofection, etc. Host cells may be grown and proliferated in culture before construct introduction, after which appropriate treatment is performed for construct introduction and construct integration. The cells are then proliferated and screened for markers present in the construct. Various vectors that may be used appropriately include HPRT, neomycin resistance, thymidine kinase, and hygromycin resistance.

[0095] In some cases, a construct may have a target site for homologous recombination if it is desired to be incorporated into a specific locus. For example, using materials and methods for homologous recombination known in the art, an endogenous gene can be knocked out and replaced with a gene encoded by the construct (at the same locus or at a different location). For homologous recombination, those skilled in the art may use either an OMEGA or an O-vector. See, for example, Thomas and Capecchi, Cell (1987) 51, 503-512; Mansour, et al., Nature (1988) 336, 348-352; and Joyner, et al., Nature (1989) 338, 153-156.

[0096] The constructs can be introduced as a single DNA molecule encoding at least one CAR and optionally another gene, or as different DNA molecules having one or more genes. Other genes may include, for example, genes encoding therapeutic molecules or suicide genes. The constructs can be introduced simultaneously or sequentially, each using the same or different markers.

[0097] Vectors containing useful elements that can be used to prepare stocks of construct DNA and to carry out transfection, such as bacterial or yeast replication origins, selectable and / or amplified markers, and promoter / enhancer elements for expression in prokaryotes or eukaryotes, are well known in the art and many are commercially available.

[0098] How to use Cells according to the present invention may be used to treat cancer, viral infections, or autoimmune disorders in patients who require them. In another embodiment, isolated cells according to the present invention may be used in the manufacture of pharmaceuticals for treating cancer, viral infections, or autoimmune disorders in patients who require them.

[0099] The present invention is based on a method for treating a patient in need, comprising at least one of the steps of (a) providing chimeric antigen receptor cells in accordance with the present invention and (b) administering the cells to the patient.

[0100] The treatment may be improvement, cure, or prevention. It may be part of either autoimmunotherapy or alloimmunotherapy. Auto means that the cells, cell lines, or cell population used in the patient's treatment are derived from that patient or from a human leukocyte antigen (HLA)-matched donor. Allogeneic means that the cells or cell population used in the patient's treatment are derived from a donor and not from that patient.

[0101] The present invention is particularly suitable for allogeneic immunotherapy, insofar as T cells typically obtained from a donor can be transformed into non-allogeneic reactive cells. This can be done under a standard protocol and replicated as many times as needed. The resulting modified T cells can be pooled and administered to one or more patients, thus becoming available as an "off-the-shelf" therapeutic product.

[0102] The cells that may be used in the disclosed method are described in the preceding section. The treatment may be used to treat patients diagnosed with cancer, viral infection, autoimmune disorder, or graft-versus-host disease (GvHD). Cancers that can be treated include tumors that do not exhibit or have not yet substantially exhibited angiogenesis, and tumors that exhibit angiogenesis. Cancers may include non-solid tumors (e.g., hematological malignancies, e.g., leukemia and lymphoma) or solid tumors. The types of cancer treated with the CAR of the present invention include, but are not limited to, carcinomas, blastomas and sarcomas, as well as certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant tumors, e.g., sarcomas, carcinomas and melanomas. Adult tumors / cancers and pediatric tumors / cancers are also included.

[0103] This may be a treatment in combination with one or more treatments for cancer selected from the group consisting of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser phototherapy, and radiotherapy.

[0104] In a preferred embodiment of the present invention, the treatment may be performed on a patient undergoing immunosuppressive treatment. In fact, the present invention is preferably based on cells or cell populations conferred resistance to at least one immunosuppressant by inactivation of genes encoding receptors for such immunosuppressants. In this aspect, the immunosuppressive treatment should assist in the selection and proliferation of T cells in the patient's body according to the present invention.

[0105] In a further embodiment, the cell composition of the present invention is administered to a patient in combination with (e.g., before, simultaneously with, or after) bone marrow transplantation, T-cell depletion therapy using any of the following chemotherapeutic agents, e.g., fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies, e.g., OKT3 or CAM PATH. In another embodiment, the cell composition of the present invention is administered after B-cell depletion therapy, e.g., a drug that reacts with CD20, e.g., rituximab. For example, in one embodiment, the subject may undergo standard treatment using high-dose chemotherapy followed by peripheral blood stem cell transplantation. In a specific embodiment, after transplantation, the subject receives an infusion of the proliferated immune cells of the present invention. In a further embodiment, the proliferated cells are administered before or after surgery. Modified cells obtained by any one of the methods described herein may be used in a particular aspect of the present invention to treat patients in need of it for host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD). Therefore, a method for treating patients in need of it for host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD), comprising the step of treating a patient by administering to the patient an effective amount of modified cells containing inactivated TCR alpha and / or TCR beta genes, is within the scope of the present invention.

[0106] Cell administration The present invention is particularly well suited to allogeneic immunotherapy, insofar as T cells typically obtained from a donor can be transformed into non-allogeneic reactive cells. This can be done under a standard protocol and replicated as many times as needed. The resulting modified T cells can be pooled and administered to one or more patients, thus becoming available as an "in-stock" therapeutic product.

[0107] Depending on the properties of the cells, they can be introduced into host organisms, such as mammals, in a wide variety of ways. In certain embodiments, cells can be introduced into tumor sites, while in alternative embodiments, cells are targeted at cancer or modified to target cancer. The number of cells used depends on many factors, including the purpose of introduction, cell survival time, the protocol used, such as the number of doses, cell proliferation capacity, and the stability of the recombinant construct. Cells can be applied as a dispersion and are usually injected into or near the site of interest. Cells can be contained in physiologically acceptable media.

[0108] In some ways, cells are encapsulated to inhibit immune recognition and then delivered to the tumor site.

[0109] Cells can be administered as desired. Various protocols may be used depending on the desired response, mode of administration, cell viability, and the number of cells present. The number of doses depends, at least in part, on the factors mentioned above.

[0110] The administration of cells or cell populations according to the present invention may be carried out in any convenient manner, including by aerosol inhalation, injection, digestion, infusion, implantation, or transplantation. The compositions described herein may be administered to a patient by subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intravenous or intralymphatic injection, or intraperitoneal injection. In one embodiment, the cell composition of the present invention is preferably administered by intravenous injection.

[0111] The administration of cells or cell populations includes all integer values ​​of the cell number within that range, 10 per kg of body weight.4 ~10 9 individual cells, preferably 10 per kg of body weight 5 ~10 6 The administration may consist of individual cells. Cells or cell populations may be administered in one or more doses. In another embodiment, an effective amount of cells is administered as a single dose. In another embodiment, an effective amount of cells is administered over a period of time in two or more doses. The timing of administration is within the discretion of the attending physician and depends on the patient's clinical condition. Cells or cell populations may be from any source, e.g., a blood bank or donor. While individual needs vary, determining the optimal range of effective doses of a particular cell type for a specific disease or condition is within the scope of the art of this art. An effective dose means the amount that provides a therapeutic or preventive benefit. The dose administered depends on the recipient's age, health condition and weight, the type of treatment being performed concurrently, if any, the frequency of the treatment, and the nature of the desired effect.

[0112] It should be understood that the system is subject to many variable factors, such as cellular responses to ligands, expression efficiency and, where appropriate, secretion levels, activity of expression products, individual patient requirements which may vary with time and environment, and the rate of loss of cellular activity as a result of cell loss or the expression activity of individual cells. Therefore, for each individual patient, even if there are common cells that can be administered to the entire population, it is expected that each patient will be monitored for the appropriate dose for their individual, and the practice of such patient monitoring is customary in the art.

[0113] Nucleic acid-based expression systems The CAR of the present invention can be expressed from an expression vector. Recombination techniques for producing such expression vectors are well known in the art.

[0114] vector The term “vector” is used to describe a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell capable of replicating it. A nucleic acid sequence can be “exogenous,” meaning it is foreign to the cell into which the vector is introduced, or its sequence is homologous to a sequence within that cell but located in a host nucleic acid where the sequence is not normally found. Vectors include plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). Those skilled in the art are well familiar with the techniques for constructing vectors through standard recombination techniques (see, for example, Maniatis et al., 1988 and Ausubel et al., 1994, both incorporated herein by reference).

[0115] The term “expression vector” refers to any type of gene construct containing nucleic acids that encode transcriptionally transcribed RNA. In some examples, the RNA molecule is subsequently translated into a protein, polypeptide, or peptide. In other examples, these sequences are not translated, for example, in the production of antisense molecules or ribozymes. An expression vector may contain various “regulatory sequences” that represent nucleic acid sequences required for the transcription and, if possible, translation of a functionally linked coding sequence in a particular host cell. In addition to regulatory sequences that govern transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that perform other functions, which will be discussed later.

[0116] Promoter and enhancer A "promoter" is a regulatory sequence, a region of a nucleic acid sequence that controls the initiation and rate of transcription. It may contain a genetic element to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind to initiate the individual transcriptions of the nucleic acid sequence. The phrases "functionally positioned," "functionally linked," "regulated," and "transcriptionally regulated" mean that the promoter is in a precise functional position and / or orientation in relation to the nucleic acid sequence to control the transcription initiation and / or expression of that sequence.

[0117] A promoter typically contains a sequence that functions to locate the start site of RNA synthesis. The most well-known example is the TATA box, but in some promoters lacking a TATA box, such as the promoter of the mammalian terminal deoxynucleotidyltransferase gene and the promoter of the SV40 late gene, another element overlapping the start site itself helps to fix the start position. Further promoter elements regulate the frequency of transcription initiation. Typically, these are located in a region 30 to 110 bp upstream of the start site, but many promoters have been shown to also contain functional elements downstream of the start site. To place the coding sequence under the "control" of the promoter, those skilled in the art position the 5' end of the transcription start site in the transcription reading frame "downstream" (i.e., 3' side) of a selected promoter. This "upstream" promoter stimulates the transcription of DNA and promotes the expression of the encoded RNA.

[0118] The spacing between promoter elements is often elastic to preserve promoter function even if elements are inverted or moved relative to each other. In tk promoters, the spacing between promoter elements can be increased to 50 bp before activity begins to decline. Depending on the promoter, individual elements appear to function either cooperatively or independently to activate transcription. Promoters may or may not be used in combination with "enhancers," which represent cis-acting regulatory elements involved in the transcriptional activation of nucleic acid sequences.

[0119] Promoters may be naturally associated with nucleic acid sequences and can be obtained by isolating the 5' prime non-coding sequence located upstream of its coding segment and / or exon. Such promoters may be referred to as “endogenous.” Similarly, enhancers may also be naturally associated with nucleic acid sequences and located downstream or upstream of the sequence. Alternatively, certain benefits may be obtained by placing the coding nucleic acid segment under the control of a recombinant or heterologous promoter that represents a promoter not normally associated with the nucleic acid sequence in its natural environment. Recombinant or heterologous enhancers also represent enhancers not normally associated with the nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, as well as promoters or enhancers isolated from any other virus or prokaryotic or eukaryotic cell, and promoters or enhancers that are not “naturally present,” i.e., those containing mutations that alter different elements and / or expression of different transcriptional regulatory regions. For example, the most widely used promoters in recombinant DNA construction include the lactamase (penicillinase), lactose, and tryptophan (trp) promoter systems. With respect to the compositions disclosed herein, in addition to the synthetic production of promoter and enhancer nucleic acid sequences, the sequences may be produced using nucleic acid amplification techniques, including recombinant cloning and / or PCR® (see U.S. Patents No. 4,683,202 and No. 5,928,906, incorporated herein by reference). Furthermore, it is envisioned that regulatory sequences that induce transcription and / or expression of the sequences in non-nuclear organelles, such as mitochondria and chloroplasts, may also be used.

[0120] It is generally important to use promoters and / or enhancers that effectively induce the expression of a DNA segment in a selected organ, cell type, tissue, organ, or organism for expression. Those skilled in the field of molecular biology are generally familiar with the use of promoter, enhancer, and cell type combinations for protein expression (see, for example, Sambrook et al. 1989, incorporated herein by reference). The promoters used may be constitutive, tissue-specific, inducible, and / or useful under conditions favorable for inducing high-level expression of the introduced DNA segment, such as in the large-scale production of recombinant proteins and / or peptides. Promoter may be heterogeneous or endogenous.

[0121] Furthermore, any promoter / enhancer combination may also be used to induce expression. The use of T3, T7, or SP6 cytoplasmic expression systems is another possible embodiment. Eukaryotic cells may support cytoplasmic transcription from specific bacterial promoters when a suitable bacterial polymerase is provided, either as part of a delivery complex or as an additional gene expression construct.

[0122] Assays for identifying tissue-specific promoters or elements and characterizing their activity are well known to those skilled in the art.

[0123] Specific start signals may also be required for the effective translation of the code sequence. These signals may include an ATG start codon or an adjacent sequence. Exogenous translation control signals, including an ATG start codon, may need to be provided. Those skilled in the art can immediately determine this and provide the necessary signals.

[0124] In certain embodiments of the present invention, internal ribosome entry site (IRES) elements are used to generate polygenetic or polycistronic messages, and these can be used in the present invention.

[0125] A vector may contain a multi-cloning site (MCS), which is a nucleic acid region containing multiple restriction enzyme sites, any of which may be used to digest the vector in combination with standard recombination techniques. “Restriction enzyme digestion” refers to the catalytic cleavage of a nucleic acid molecule by an enzyme that functions only at specific locations within the nucleic acid molecule. Many of these restriction enzymes are commercially available. The use of such enzymes is well understood by those skilled in the art. Often, a vector may be linearized or fragmented using restriction enzymes that cleave within the MCS so that an exogenous sequence can be ligated into the vector. “Ligate” refers to the process of forming a phosphodiester bond between two nucleic acid fragments that may or may not be continuous with each other. Techniques related to restriction enzymes and ligating reactions are well known to those skilled in the art of recombination.

[0126] Splice sites, termination signals, replication origins, and selection markers may also be used.

[0127] Plasmid vectors In certain embodiments, plasmid vectors are intended to be used to transform host cells. Generally, plasmid vectors containing species-derived regulatory sequences compatible with the replicon and host cell are used in relation to these hosts. Vectors typically have replication sites and marking sequences that can provide phenotypic selection in transformed cells. In a non-limiting example, Escherichia coli (E. coli) is often transformed using derivatives of pBR322, a plasmid derived from the E. coli species. pBR322 contains genes for ampicillin and tetracycline resistance and thus provides a simple means of identifying transformed cells. pBR plasmids or other microbial plasmids or phages must also contain, or be modified to contain, promoters that can be used by the microorganism for the expression of its own proteins, for example.

[0128] In addition, phage vectors containing replicons and regulatory sequences adapted to host microorganisms can be used as transformation vectors in relation to these hosts. For example, phage-lambda GEM® 11 can be used in the preparation of recombinant phage vectors that can be used to transform host cells, such as E. coli LE392.

[0129] Further useful plasmid vectors include the pIN vector (Inouye et al., 1985) and the pGEX vector, which is used to construct glutathione S-transferase (GST) soluble fusion proteins for subsequent purification and isolation or cleavage. Other suitable fusion proteins include those containing galactosidase and ubiquitin.

[0130] Bacterial host cells containing the expression vector, such as E. coli, are grown in any many suitable culture media, e.g., LB. Expression of recombinant proteins within a particular vector can be induced, as is understood by those skilled in the art, by contacting the host cells with a promoter-specific active agent, e.g., by adding IPTG to the culture medium or by switching to incubation at a higher temperature. After further bacterial culture, typically 2–24 hours, the cells are harvested by centrifugation and washed to remove any remaining culture medium.

[0131] Viral vector The ability of certain viruses to infect cells or enter them through receptor-mediated endocytosis, integrate into the host cell's genome, and stably and efficiently express viral genes makes them attractive candidates for the delivery of foreign nucleic acids into cells (e.g., mammalian cells). An element of the present invention may be a viral vector encoding one or more CARs of the present invention. Non-limiting examples of viral vectors that may be used to deliver the nucleic acids of the present invention are described below.

[0132] Adenovirus vector Certain nucleic acid delivery methods utilize adenovirus expression vectors. While adenovirus vectors are known to have low loading capacity for integration into genomic DNA, this characteristic is offset by the high gene transfer efficiency they provide. “Adenovirus expression vector” means that it contains a construct that (a) supports the packaging of the construct and (b) ultimately expresses a tissue or cell-specific construct cloned within it, containing sufficient adenovirus sequences. Knowledge of genetic organization and adenoviruses shows that a 36 kb, linear double-stranded DNA virus can replace large portions of adenovirus DNA with foreign sequences of up to 7 kb (Grunhaus and Horwitz, 1992).

[0133] AAV Vector Nucleic acids can be introduced into cells using adenovirus-mediated transfection. High transfection efficiencies have been reported in cell systems using adenovirus-based systems (Kelleher and Vos, 1994; Cotten et al., 1992; Curiel, 1994). Adeno-associated viruses (AAVs) are attractive vector systems for use in the cells of the present invention because they have a high degree of integration, can infect non-dividing cells, and are therefore useful for gene delivery to mammalian cells, for example, under tissue culture (Muzyczka, 1992) or in vivo. AAVs have a broad host range in terms of infectivity (Tratschin et al., 1984; Laughlin et al., 1986; Lebkowski et al., 1988; McLaughlin et al., 1988). Details regarding the preparation and use of rAAV vectors are described in U.S. Patents No. 5,139,941 and No. 4,797,368, which are incorporated herein by reference, respectively.

[0134] Retrovirus vectors Retroviruses are useful as delivery vectors because they integrate their genes into the host genome, introduce large amounts of foreign genetic material, infect a wide range of species and cell types, and can be packaged in specific cell lines (Miller, 1992).

[0135] To construct a retroviral vector, nucleic acid (e.g., one encoding the desired sequence) is inserted into the viral genome, replacing a specific viral sequence to produce a replication-defective virus. To generate virions, a packaging cell line is constructed containing the gag, pol, and env genes but lacking the LTR and packaging elements (Mann et al., 1983). A recombinant plasmid containing cDNA along with the retroviral LTR and packaging sequence is introduced into a special cell line (e.g., by calcium phosphate precipitation). The packaging sequence packages the RNA transcript of the recombinant plasmid into viral particles, which are then secreted into the culture medium (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The medium containing the recombinant retrovirus is then collected, optionally enriched, and used for gene transfer. Retroviral vectors can infect a wide variety of cell types. However, host cell division is required for integration and stable expression (Paskind et al., 1975).

[0136] Lentiviruses are complex retroviruses that, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural functions. Lentiviral vectors are well known in the art (see, e.g., Naldini et al., 1996; Zufferey et al., 1997; Blomer et al., 1997; U.S. Patents 6,013,516 and 5,994,136). Some examples of lentiviruses include human immunodeficiency virus: HIV-1, HIV-2, and simian immunodeficiency virus: SIV. Lentiviral vectors are made biologically safe by multiple attenuations of HIV-toxic genes, for example, by removing genes env, vif, vpr, vpu, and nef.

[0137] Recombinant lentiviral vectors can infect non-dividing cells and can be used for gene transfer and nucleic acid sequence expression both in vivo and ex vivo. For example, a recombinant lentivirus that can infect non-dividing cells, in which a suitable host cell is transfected with two or more vectors having packaging functions, i.e., gag, pol, env and rev and tat, is described in U.S. Patent No. 5,994,136, incorporated herein by reference. Those skilled in the art may target recombinant viruses by linking the envelope protein with an antibody or specific ligand that targets a receptor of a particular cell type. For example, by inserting the sequence of interest (including regulatory regions) into a viral vector together with another gene encoding a ligand for a receptor on a particular target cell, the vector becomes target-specific.

[0138] Other viral vectors Other viral vectors can also be used as vaccine constructs in this invention. Virus-derived vectors such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988), Sindbis virus, cytomegalovirus, and herpes simplex virus can be used. They offer a variety of attractive properties for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990).

[0139] Delivery using modified viruses The delivered nucleic acid can be contained within an infectious virus engineered to express a specific binding ligand. The viral particle will then specifically bind to an allogeneic receptor on the target cell and deliver its contents to the cell. A novel approach designed to enable the specific targeting of retroviral vectors was developed based on the chemical modification of retroviruses by chemically adding lactose residues to the viral envelope. This modification can enable specific infection of hepatocytes via the sialycoprotein receptor.

[0140] Another approach to targeting recombinant retroviruses using biotinylated antibodies against retroviral envelope proteins and specific cell receptors was also designed. The antibodies were ligated via the biotin component by using streptavidin (Roux et al., 1989). Using antibodies against major histocompatibility complex class I and class II antigens, they demonstrated in vitro infection of various human cells possessing these surface antigens with ecotropic viruses (Roux et al., 1989).

[0141] Vector delivery and cell transformation Suitable methods for delivering nucleic acids for cell transfection or transformation are known to those skilled in the art. Such methods include, but are not limited to, direct delivery of DNA, such as by injection, or by ex vivotransfection. Cells can be transformed stably or transiently through the application of techniques known in the art.

[0142] Ex vivo transformation Methods for transfecting eukaryotic cells and tissues isolated from living organisms in an ex vivo setting are known to those skilled in the art. Therefore, it is assumed that cells or tissues can be isolated and then ex vivo transfected with the nucleic acids of the present invention. In certain aspects, the transplanted cells or tissues can be introduced into a living organism. In preferred aspects, the nucleic acids are expressed in the transplanted cells.

[0143] The present invention kit Any composition described herein may be included in the kit. In non-limiting examples, reagents for generating one or more cells for use in cell therapy and / or one or more cells for use in cell therapy having recombinant expression vectors may be included in the kit. The components of the kit may be provided by appropriate housing means.

[0144] Some components of the kit may be packaged in either an aqueous medium or in a lyophilized form. The kit's containment means typically include at least one vial, test tube, flask, bottle, syringe, or other containment means in which the components may be arranged, preferably appropriately subdivided. If the kit contains two or more components, the kit also typically includes a second, third, or further container in which the additional components may be arranged individually. However, various combinations of components may be contained in vials. The kits of the present invention also typically include means for tightly sealing and containing the components for commercial sale. Such containers may include injection-molded or blow-molded plastic containers in which the desired vials are held.

[0145] If the components of the kit are provided in one and / or more liquid solutions, the liquid solutions are aqueous solutions, and sterile aqueous solutions are particularly useful. In some examples, the means of containment may themselves be syringes, pipettes and / or other such similar devices from which the formulation may be applied to the infected area of ​​the body, injected into an animal, and / or applied together and / or even mixed with other components of the kit.

[0146] However, the components of the kit may be supplied in the form of dry powder. If the reagents and / or components are supplied as dry powder, the powder may be reconstituted by the addition of a suitable solvent. It is assumed that the solvent may also be supplied in a separate containment container. The kit may also include a second containment container for sterile, pharmaceutically acceptable buffers and / or other diluents.

[0147] In certain embodiments of the present invention, cells used in cell therapy are provided as a kit, and in some examples, the cells are essentially the sole component of the kit. The kit may include reagents and materials for producing the desired cells. In certain embodiments, the reagents and materials include primers, nucleotides, appropriate buffers or buffering reagents, salts, etc., for amplifying the desired sequence, and in some examples, the reagents include vectors and / or DNA encoding CARs and / or modulators therefor as described herein.

[0148] In certain embodiments, the kit includes one or more devices suitable for extracting one or more samples from an organism. These devices may include syringes, surgical scalpels, and the like.

[0149] In some examples of the present invention, the kit includes, in addition to the cell therapy, a second cancer therapy, such as chemotherapy, hormone therapy, and / or immunotherapy. The kit may be specialized for a particular cancer of an individual and may include each of the second cancer therapies for that individual.

[0150] Combination therapy In certain embodiments of the present invention, the method of the present invention for clinical applications is used in combination with other agents effective in treating hyperproliferative diseases, such as anticancer agents. “Anticancer” agents can negatively affect cancer in a subject by, for example, killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing blood supply to tumors or cancer cells, promoting an immune response against cancer cells or tumors, preventing or inhibiting cancer progression, or extending the lifespan of the subject with cancer. More generally, these other compositions may be provided in combination doses effective in killing cells or inhibiting cell proliferation. This process may involve contacting cancer cells with an expression construct and / or multiple elements simultaneously. This can be achieved by contacting cells with a single composition or pharmaceutical formulation containing both agents, or by contacting cells with two separate compositions or formulations simultaneously, one containing an expression construct and the other containing a second agent.

[0151] Tumor cells resistant to chemotherapy and radiotherapy agents represent a major challenge in clinical oncology. One goal of current cancer research is to find ways to improve the effectiveness of chemotherapy and radiotherapy by combining them with other therapies. In relation to this invention, it is envisioned that cell therapy may be used in conjunction with chemotherapy, radiotherapy, or immunotherapy interventions, as well as with apoptosis-promoting or cell cycle-modulating agents.

[0152] Alternatively, the treatment of the present invention may be administered before or after treatment with other agents, with intervals ranging from a few minutes to several weeks. In embodiments in which other agents and the present invention are applied separately to an individual, those skilled in the art would typically ensure that no significant time elapses between each delivery so that the agents and the treatment of the present invention can continue to exert a favorable synergistic effect on the cells. In such examples, those skilled in the art would assume that both forms can be brought into contact with the cells within about 12 to 24 hours from each other, more preferably within about 6 to 12 hours from each other. However, in some situations, it may be desirable to significantly extend the treatment period, in which case several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) may be provided between each administration.

[0153] It is anticipated that the treatment cycle may be repeated as needed. It is also anticipated that various standard treatments and surgical interventions may be applied in combination with the cell therapy of this invention.

[0154] chemotherapy Cancer treatment also includes a variety of combination therapies with both compound-based and radiation-based treatments. Combination chemotherapy includes, for example, Abraxane, Altretamine, Docetaxel, Herceptin, Methotrexate, Novantrone, Zoladex, Cisplatin (CDDP), Carboplatin, Procarbazine, Mechloretamine, Cyclophosphamide, Camptothecin, Ifosfamide, Melphalan, Chlorambucil, Busulfan, Nitrosourea, Dactinomycin, Daunorubicin, Doxorubicin, Bleomycin, Plicamycin, Mitomycin, Etoposide (VP16), Tamoxifen, Raloxifene, Estrogen receptor conjugates, Taxol, Gemcitabine, Navelbine, Farnesyl protein transferase inhibitors, Trans-platinum, 5-Fluorouracil, Vincristine, Vinblastine, and Methotrexate or any analogs or derivative variants of the above, as well as combinations thereof.

[0155] In certain embodiments, chemotherapy to an individual is performed in combination with the present invention, for example, before, during, and / or after the implementation of the present invention.

[0156] Radiation therapy Other widely used elements that cause DNA damage include gamma rays, X-rays, and / or direct delivery of radioisotopes to tumor cells, which are well known as such. Other forms of DNA damage elements, such as microwave and UV irradiation, have also been considered. All of these elements are thought to cause widespread damage to DNA, to DNA precursors, to DNA replication and repair, and to chromosome construction and maintenance. The dose range for X-rays ranges from a daily dose of 50–200 roentgens over a long period (3–4 weeks) to a single dose of 2000–6000 roentgens. The dose range for radioisotopes varies and depends on the half-life of the isotope, the intensity and type of radiation produced, and uptake by neoplastic cells.

[0157] The terms “contact” and “exposure” are used herein to describe the process of delivering a therapeutic construct and a chemotherapeutic or radiotherapeutic agent to or positioning them directly adjacent to target cells, when applied to cells. To achieve cell death or cessation, both agents are delivered to the cells in a combined dose effective in killing the cells or preventing their division.

[0158] immunotherapy Immunotherapy typically relies on the use of immune effector cells and molecules that target and destroy cancer cells. Immune effectors can be, for example, antibodies specific to certain markers on the surface of tumor cells. Antibodies can function alone as effectors of therapeutic agents, or they can induce other cells to actually undergo cell death. Antibodies can also be linked to drugs or toxins (chemotherapeutic agents, radionuclides, lysine A chain, cholera toxin, pertussis toxin, etc.) and used solely as targeting agents. Alternatively, effectors can be lymphocytes with surface molecules that interact directly or indirectly with tumor cell targets. Various effector cells include cytotoxic T cells and NK cells.

[0159] The immunotherapies of the treatments of the present invention described herein can therefore be used as part of a combination therapy in combination with the cell therapies of the present invention. A general approach to combination therapy is discussed below. Tumor cells typically have to possess several markers that can be targeted, i.e., markers that are not present in most other cells. Many tumor markers exist, and any of these may be suitable for targeting in the present invention. Common tumor markers include PD-1, PD-L1, CTLA4, carcinoembryonic antigen, prostate-specific antigen, urinary tract tumor-associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erbB, and p155.

[0160] gene In yet another embodiment, the secondary treatment is gene therapy involving the administration of therapeutic polynucleotides before, after, or concurrently with the clinical manifestations of the present invention. The present invention includes a variety of expression products, including cell proliferation inducers, cell proliferation inhibitors, or programmed cell death regulators.

[0161] surgery Approximately 60% of cancer patients will undergo several types of surgery, including preventive, diagnostic or staging, curative, and palliative surgery. Curative surgery is a cancer treatment that can be used in combination with other treatments, such as the procedures of the present invention, chemotherapy, radiotherapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies.

[0162] Curative surgery includes excision, which involves the physical removal, excision, and / or destruction of all or part of cancerous tissue. Tumor excision represents the physical removal of at least a portion of the tumor. In addition to tumor excision, surgical procedures include laser surgery, cryosurgery, electrosurgery, and microsurgery (Mohs procedure). It is further assumed that the present invention may be used in combination with the removal of superficial cancer, precancerous tissue, or an associated amount of normal tissue.

[0163] When all or part of cancer cells, tissue, or tumors are removed, cavities may form within the body. Treatment can be achieved by perfusion, direct injection, or local application to the area where further anticancer therapy is to be administered. Such treatments may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may also be performed with varying doses.

[0164] Other drugs Other agents may also be used in combination with the present invention to improve the therapeutic effect of the treatment. These further agents include immunomodulators, agents that upmodulate cell surface receptors and affect GAP binding, cell proliferation inhibitors and differentiation agents, cell adhesion inhibitors, or agents that increase the sensitivity of hyperproliferating cells to apoptosis inducers. Immunomodulators include tumor necrosis factor, interferon alpha, beta and gamma, IL-2 and other cytokines, F42K and other cytokine analogs, or MIP-1, MIP-1 beta, MCP-1, RANTES, and other chemokines. Upmodulation of cell surface receptors or their ligands, e.g., Fas / Fas ligand, DR4 or DR5 / TRAIL, may also be used to enhance the apoptosis-inducing capacity of the present invention by establishing autocrine or paracrine effects on hyperproliferating cells. Increased intracellular signaling by increasing the number of GAP bindings will enhance the anti-hyperproliferative effect on adjacent hyperproliferating cell populations. In other embodiments, cell proliferation inhibitors or differentiation agents may be used in combination with the present invention to improve the anti-hyperproliferative effect of the treatment. Inhibitors of cell adhesion are thought to improve the effects of the present invention. Examples of cell adhesion inhibitors are adhesion plaque kinase (FAK) inhibitors and lovastatin. It is also conceivable that other agents that enhance the sensitivity of overproliferating cells to apoptosis, such as the antibody c225, may be used in combination with the present invention to improve the effects of the treatment. [Examples]

[0165] Example 1: Materials and Method Cells, culture medium, and reagents. Human CAIX+ renal cell carcinoma cell lines sk-rc-52, sk-rc-09, and CAIX-sk-rc-59 were obtained from Dr. Gerd Ritter of Memorial Sloan-Kettering Cancer Center, New York. They were cultured at 37°C under 5% CO2 in R-10 complete medium containing RPMI 1640 medium (Life Technologies) supplemented with 10% FCS, 2 mmol / L L-glutamine, 100 U / ml penicillin, and 100 μg / ml streptomycin (Sigma). Primary human T cells were maintained in R-10 containing 10% human serum and 100 IU / ml recombinant human interleukin-2 (IL-2) (Chiron). Human embryonic kidney cell line 293T (ATCC) and mouse fibroblast cells NIH3T3 (ATCC) were grown in D-10 complete medium (Life Technologies) containing DMEM medium with 10% FCS, 100 U / ml penicillin, and 100 μg / ml streptomycin (Sigma). Leukopacks obtained from the Children's Hospital Boston blood bank were collected from healthy volunteers who provided written informed consent.

[0166] In one embodiment, human ccRCC cell lines, Skrc52, which is CAIX+ / PD-L1- in its initial state, and Skrc59, which is CAIX- / PD-L1+ in its initial state, were obtained from Dr. Gerd Ritter (Memorial Sloan-Kettering Cancer Center, New York). These cells were cultured in RPMI 1640 medium (Life Technologies) supplemented with 10% (v / v) heat-inactivated fetal bovine serum (FBS, Gibco), 100 IU / ml penicillin, and 100 μg / ml streptomycin. 293T (CRL-11268, ATCC) and Lenti-X 293T (Clontech) cells were grown in DMEM medium (Life Technologies) supplemented with 10% FBS, 100 IU / ml penicillin, and 100 μg / ml streptomycin. All cell lines used in this project were transduced with luciferase via lentiviral transduction and maintained at 37°C and 5% CO2. Skrc52 cells were selected for CAIX- / PD-L1- and CAIX+ / PD-L1- cell populations by fluorescence-activated cell sorting (FACS). Skrc59 cells were manipulated to express high levels of human CAIX, and CAIX+ / PD-L1+ cells were selected by FACS sorting.

[0167] Cloning of anti-PD-L1 scFv-Fc IgG1 and IgG4 into a bicistronic lentiviral vector encoding anti-CAIX second-generation CAR.

[0168] The anti-PD-L1 antibody (Ab) used to construct CART cells was previously selected using a 27 billion-member human scFv phage display library against full-length PD-L1 in the form of paramagnetic proteoliposomes (manuscript in preparation). The DNA sequence encoding anti-PD-L1 scFv(clone 42)-Fc IgG1 or IgG4 was codon-optimized and synthesized to include the 5' NdeI and 3' ClaI restriction sites, allowing for further scFv-Fc clonings that replace ZsGreen in the lentiviral vector pHAGE-eIFα signal-scFvG36(anti-CAIX)-C9 tag-linker-CD28-CD3ζ-IRES-ZsGreen (e.g., CD28z) (Genewiz). This original anti-CAIX vector had been previously constructed and published (21). For cloning a negative control, we first replaced the anti-PD-L1 scFv with anti-severe acute respiratory syndrome (SARS) scFv (clone 11A) in an IgG1-Fc lentiviral vector containing the anti-CAIX G36 scFv CAR. The DNA sequence encoding anti-SARS scFv was applied using MluI forward primers. TIFF0007833003000006.tif4128 and XbaI reverse primer Using TIFF0007833003000007.tif4128, plasmid pHAGE CMV-anti-SARS(11A)scFv-Fc-CD28-gp41-IRES-ZsGreen was amplified by PCR to insert the 5'MluI and 3'XbaI restriction sites for further cloning. The final pHAGE-eIFα-scFv G36(anti-CAIX)-C9 tag-linker-CD28-CD3ζ-IRES-anti-SARS(11A)IgG1 was used as one of the negative controls. We also replaced the anti-CAIX scFv within the CAR structure containing anti-SARS IgG1 with anti-B cell maturation antigen (BCMA) scFv by double digestion of pHAGE-eIFα-A716scFv(anti-BCMA)-C9 tag-linker-CD28-CD3ζ-IRES-ZsGreen and pHAGE-eIFα-G36scFv(anti-CAIX)-C9 tag-linker-CD28-CD3ζ-IRES-anti-SARS(11A)IgG1 with anti-SARS IgG1 by double digestion of pHAGE-eIFα-A716scFv(anti-BCMA)-C9 tag-linker-CD28-CD3ζ-IRES-anti-SARS(11A)IgG1 using NcoI and NotI. Following the cloning process, we obtained four main plasmids to initiate lentivirus production: an anti-CAIX CAR (anti-CAIX / anti-PD-L1 IgG1) capable of expressing anti-PD-L1 IgG1, an anti-CAIX CAR (anti-CAIX / anti-PD-L1 IgG4) capable of expressing anti-PD-L1 IgG4, an anti-CAIX CAR (anti-CAIX / anti-SARS IgG1) capable of expressing unrelated anti-SARS Ab, and an anti-BCMA CAR (anti-BCMA / anti-SARS IgG1) capable of expressing unrelated anti-SARS Ab.

[0169] Isolation of scFv and conversion of scFv to scFv-Fc. CAIX-specific scFv antibodies were isolated from a non-immune human scFv phage library, as previously reported and registered in GenBank with accession numbers GQ903548-GQ903561. 23The scFv coding DNA fragment was excised from the pFarber phagemide at the SfiI / NotI site and subcloned into a mammalian expression vector, pcDNA3.1-F105L-hinge-stuffer, which contains a human IgG1 F105 leader sequence and a human IgG1 hinge-CH2-CH3 Fc moiety expressing scFv-Fc antibodies. The scFv-Fc plasmid was transiently transfected into 293T cells with lipofectamine 2000 (Invitrogen), and the expressed antibodies were purified using Sepharose protein A beads (Amersham Bioscience). Specific binding to CAIX was tested by staining with scFv converted to scFv-Fc antibodies by incubation with phage scFv antibodies or CAIX-expressing 293T and sk-rc-52 cell lines, as well as CAIX-negative 293T and sk-rc-59 cell lines. In these experiments, unrelated anti-HIV CCR5 antibodies (clone A8) were used. 25 or anti-SARS antibody (11A) 24 Only fluorescently conjugated secondary antibodies were used as negative targets.

[0170] Construction of the scFv-CD8-TCRζ and scFv-CD28-TCRζ constructs. The DNA constructs Pz1,scFv-CD8-TCRζ and P28z,scFv-CD28-TCRζ, within the phagemide vector pSL1180, were obtained from Dr. Michel Sadelain of Memorial Sloan Kettering Cancer Center, New York. In Pz1, the scFv and TCRζ intracellular domains are attached to the N and C-terminuses of the human CD8α chain, respectively. Similarly, in P28z, the scFv and TCRζ sequences are attached to the N and C-terminuses of human CD28, respectively. The amino acid sequence of human CD8α is 71 residues long, consisting of 47 (aa 137-183), 23 (aa 184-206), and 2 (aa 207-208) residues in the extracellular, hinge, transmembrane, and cytoplasmic domains, respectively. The CD28 sequence within P28z is 107 residues long, consisting of 40 (aa 114-153), 23 (aa 154-176), and 44 (aa 177-220) residues in the extracellular, transmembrane, and cytoplasmic domains, respectively. The intracellular domain of human CD3ζ, common to both CARs, consists of 112 amino acids (aa 52-163).

[0171] The nucleic acid sequence encoding an internal C9 tag (a 9-amino acid peptide of human rhodopsin, TETSQVAPA) with a GGGGS linker was amplified by PCR and fused upstream of the CD8-TCRζ and CD28-TCRζ sequences using the 5' NotI and 3' PacI sites. The primers used for cloning the chimeric TCRζ construct were: TIFF0007833003000008.tif18142 (This is a forward primer for the CD8 construct; italics indicate NotI sites, capital letters indicate C9 tag sequences, and underlines indicate GGGGS linkers), TIFF0007833003000009.tif12128 (forward primer for CD28 construct) and reverse primers for both constructs TIFF0007833003000010.tif5128, the italicized part is the PacI site. These DNA fragments encode a functional feature arranged according to the following sequence: NotI-C9 tag (TETSQVQPQ)-GGGGS-CD8 or CD28-TCRζ-PacI. The chimeric TCR construct tagged with the internal C9 peptide was cloned into a pcDNA3.1-F105L-hinge-stuffer vector containing anti-CXCR4 scFv-Fc, clone 48, using the NotI and PacI restriction sites. With this design, we inserted the chimeric TCR receptor construct to replace the Fc partial fragment. Next, anti-CAIX scFv (clone G36) and anti-CCR5 scFv (clone A8 as an unrelated scFv control) antibody fragments were cloned to replace anti-CXCR4 scFv at the SfiI / NotI site, thereby creating a CAIX-specific chimeric TCR construct.

[0172] The lentiviral vector pHAGE-CMV-DsRed-IRES-ZsGreen and four HIV helper plasmids, pHDM-Hgpm2 (HIV gag-pol), pMD-tat, pRC / CMV-rev, and Env VSV-G pseudotypes, were obtained from Dr. Richard Mulligan of the Virus Production Core at The Harvard Gene Therapy Initiative in Boston. The CMV promoter in pHAGE-CMV-IRES-ZsGreen was replaced at the SpeI / NotI site with the EF1α promoter derived from the pSIN lentiviral vector. G36, one of five scFv-Fc antibodies exhibiting high affinity for CAIX+ cells and high ADCC specifically against CAIX+ tumor cells, was cloned into the pHAGE-EF1α lentiviral vector via AscI / BamHI to replace the first cassette of the DsRed protein.

[0173] Lentivirus production and transduction of primary human T cells. Lentiviruses were produced by transient transfection of 293 T cells with five plasmids using lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). Cells were prepared to 80% confluence in a 15 cm petri dish (Nalge Nunc) and transfected with 30 μg of total plasmid DNA. The vector-plasmid ratio (pHDM-Hgpm2 (HIV gag-pol): pMD-tat: pRC / CMV-rev: Env VSV-G pseudotype) was 20:1:1:1:2. After changing to D-10 medium, the viral supernatant was collected on day 3, filtered through a 0.45 μm filter, and concentrated by ultracentrifugation at 16,500 rpm (48,960 xg, Beckman SW28 rotor) and 4°C for 90 minutes (Beckman Coulter, Fullerton, CA). The virus pellet was resuspended in R-10 medium and kept frozen at -80°C.

[0174] In one embodiment, lentiviruses were produced by transient transfection of 293T cells with five plasmids using polyethyleneimine (PEI). Briefly, 293T cells at 80% confluence in 15 cm plates (Nalge Nunc) were transfected with 30 μg of five plasmids: 5 μg each of the structural plasmids pHDH-Hgpm2 (HIV gag-pol), pMD-tat, pRC / CMV-rev, and Env VSV-G, and 10 μg of the main plasmid containing CAR (anti-CAIX / anti-PD-L1 IgG1, anti-CAIX / anti-PD-L1 IgG4, anti-CAIX / anti-SARS IgG1, or anti-BCMA / anti-SARS IgG1). The viral supernatant was concentrated using Lenti-X Concentrator (Clontech) according to the manufacturer's instructions and kept frozen at -80°C.

[0175] Human PBMCs were isolated by Ficol density gradient separation and activated with 2 μg / ml PHA(Sigma) and 100 IU / ml human IL-2 for 4 days. The cells were infected with lentiviral transduction in 2 or 3 rounds at infection multiplicity (MOI) of 10-20 in the presence of 10 μg / ml DEAE. Three days after transduction, transduced T cells were harvested for in vitro phenotypic and functional analysis or grown for in vivo experiments.

[0176] Selection, activation, and lentivector transduction of CD8+ T cells. Blood collars were collected from healthy volunteers who provided written informed consent and obtained from the blood bank at Brigham and Woman's Hospital (Boston, MA). Human peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque PLUS (GE Healthcare, NJ). CD8-positive cells were isolated from PBMCs using Dynabeads from CD8 Positive Isolation (Life Technologies) and cultured in RPMI 1640 medium (Life Technologies) containing 10% thermoactivated fetal bovine serum, 20 mM HEPES, 100 IU / ml penicillin, and 100 μg / ml streptomycin, with 50 IU / mL of IL-21 (Peprotech) added to the medium every two days. Assays were performed using 50 IU / mL of each cytokine to determine whether IL-2 or IL-21 is the best cytokine for inducing the proliferation of anti-CAIX CART cells. CD8+ T cells were activated with Dynabeads Human T-Activator CD3 / CD28 (Life Technologies) in a 1:1 ratio. These cells were transduced using lentivirus under 20 infection multiplicities and 10 μg / mL diethylaminoethyl. All assays were performed in triplicate sets using T cells from three different healthy donors.

[0177] Flow cytometry analysis was performed. Transduction efficiency of human primary T cells was evaluated by the expression of a reporter gene (ZsGreen). CAIX-Fc protein was expressed from a pcDNA3.1 plasmid encoding CAIX amino acids 38-397, followed by the human IgG1 hinge, CH2 and CH3 domains, with the CAIX signal peptide (aa1-37) replaced by an Ig reader sequence. Expression of scFv(G250) in transduced T cells was tested by staining cells with 1 μg of CAIX-Fc protein, followed by APC-conjugated mouse anti-human IgG antibody (Jackson ImmunoResearch). Furthermore, expression of the internal rhodopsin nanopeptide (TETSQVAPA) C9 tag of the scFv domain of the TCR construct in transduced T cells was detected by staining with 5 μg of mouse 1D4 antibody, followed by APC-conjugated goat anti-mouse IgG antibody (Jackson ImmunoResearch). For analysis, a subset of human cells in cultures during clonal proliferation experiments was stained with a fluorescently conjugated mouse anti-human antibody (Invitrogen) against CD3 (clone S4.1), CD4 (clone S3.5), or CD8 (clone 3B5). For all cell staining, 500,000 cells were stained with the recommended antibody concentration as instructed by the company. Isotype-matched control antibodies were used for each sample, and cells were analyzed using a FACSCalibur cytometer (Beckton-Dickinson).

[0178] In one embodiment, transduction of 293 T cells or CD8+ T cells was confirmed by FACS analysis for anti-CAIX or anti-BCMA expression. Cells were stained with 10 μg / mL human CAIX-Fc or human BCMA-mouse-Fc (AB Bioscience) prepared in our laboratory, and then developed with 1:250 APC-conjugated mouse anti-human IgG Ab (Southern Biotech) or goat anti-mouse IgG Ab (Biolegend), respectively. CountBright® Absolute Counting Beads (Molecular Probes) were used for proliferation and clonal proliferation assays. All samples were analyzed using LSR Fortessa or FACSCalibur (BD Bioscience), and the data were analyzed using FlowJo software. To analyze the T cell exhaustion status of CART cells, they were cultured for 5 days in the presence of IL-21 50 U / mL (Peprotech) and Dynabeads Human T Activator CD3 / CD28. Following this period, CART cells were co-cultured with Skrc-59 CAIX+ PD-L1+ cells for 2 days to stimulate exhaustion. 1 x 10⁶ CART cells from this assay and tumor-infiltrating lymphocytes (TILs) recovered from the in vivo assay were stained with FITC-conjugated anti-human PD-1, PE-conjugated anti-human Tim3, PerCP / Cy5.5-conjugated anti-human Lag3 antibody (Biolegend), and Pacific Blue-conjugated anti-human CD45, and analyzed by FACS. To validate the expression levels of CAIX and PD-L1 in the different RCC cell lines used in this project, we used 10 μg / mL anti-human CAIX mAb (clone G36) and 10 μg / mL biotinylated mouse anti-human PD-L1 (Biolegend), both produced in our laboratory. Primary antibodies were detected using 1:250 APC-conjugated anti-human Ab and PE-conjugated avidin, respectively, and analyzed by FACS.

[0179] ADCC and cytotoxicity assays of lentiviral transducer T cells. The cytotoxicity assay was performed using the DELFIA EuTDA Cytotoxicity Kit (Perkin Elmer, Boston, MA) according to the manufacturer's instructions. Briefly, target tumor cells were labeled with a fluorescent ligand (BATDA) at 37°C for 30 minutes, and 1 x 10⁶ cells were measured. 4 Nine labeled cells were placed in each well of a 96-well U-bottom plate. For antibody-dependent cytotoxicity (ADCC) assays, a panel of anti-CAIX scFv-Fc antibodies at concentrations of 1 μg / ml or 5 μg / ml, or unrelated scFv-Fc antibodies, were added separately. Assays were prepared with effector cell (human PBMC) to target cell ratios (E:T) of 50:1, 25:1, and 12.5:1. For T-cell cytotoxicity assays, different effector cell (non-transduced or transduced T cells) to target cell ratios (E:T) were prepared (100:1, 50:1, and 25:1). Cultures were incubated at 37°C for 4 hours under humidified 5% CO2. After rotating the plates at 500 xg for 5 minutes, 20 μl of supernatant was transferred to a flat-bottom plate. 200 μl of europium solution was added, and the fluorescence emitted from the cells was read using a fluorometer (Victor®, PerkinElmer). A spontaneous emission control was prepared by culturing labeled cells alone, while a maximum emission control was prepared by adding lysis buffer (provided in the kit) to labeled cells.

[0180] ELISA, ELISPOT assay, and Western blot. For cytokine secretion, RCC cell lines sk-rc-52 (CAIX+) or sk-rc-59 (CAIX-) were used, 1 x 10⁶ cells per well in a 24-well plate. 6 Sow overnight to form individual plants, then 1 x 10 6Nine non-transduced or transduced T cells were seeded. Before co-culture with tumor cells, the T cells were washed twice with PBS to remove human IL-2. After overnight incubation, the supernatant was collected and analyzed for IL-2 and IFN-γ by ELISA (e-Bioscience). To detect T cells in the IFN-γ ELISPOT assay (e-Bioscience), the membrane was developed with AEC substrate solution and the number of spots was counted using an ELISPOT plate reader (CTL Cellular Technology).

[0181] For Western blotting, the preparation of non-transduced and transduced T cells was carried out as described. 50 One million cells were prepared in non-reducing and reducing buffer (0.1 M dithiothreitol) and electrophoresed on a 10-20% polyacrylamide gradient gel (Invitrogen). Proteins were transferred to polyvinylidene fluoride transfer membranes (NEN Life Science Products, Boston, MA) overnight at 100 V, 4°C. These membranes were incubated with a 1:2000 primary antibody, anti-human ζ-chain monoclonal antibody 8D3 (BD ​​Pharmingen, San Diego, CA), followed by a 1:3000 secondary antibody, horseradish peroxidase (Caltag). Immunodetection was performed using the ECL Plus Western blot detection system (GE Healthcare, Piscataway, NJ) and X-ray film irradiation.

[0182] Detection of IgG secreted by CART cells using ELISA. Total IgG levels secreted into the culture medium of transduced cells were detected using the Human IgG ELISA Quantitation Set (Bethyl Laboratories). Anti-PD-L1 antibody secreted by transduced CD8+ CART cells was purified using Protein A Sepharose beads (GE Healthcare) and biotinylated using EZ-Link Sulfo-NHS-LC-Biotin (Thermo Scientific). These antibodies were incubated with 5 μg / mL human PD-L1, prepared in our laboratory, pre-fixed on MaxiSorp plates (Nunc) at room temperature for 2 hours. The biotinylated antibodies were detected by incubation with streptavidin-HRP for 1 hour and developed using SureBlue® TMB Peroxidase Substrate and TMB Stop Solution (KPL). Absorbance was read at λ=450 nm.

[0183] Proliferation, clonal proliferation, and cytokine secretion after contact with tumor cells. Tumor cells were irradiated (3,000 rads) and 2.5 x 10⁶ wells were observed. 5 Individual seeds were sown. For one week of cultivation, 1 x 10 6 T cells were added to culture medium containing R-10 and 100 IU / ml of human IL-2. The T cells were divided to maintain appropriate density and re-stimulated with tumor cells weekly. The number of T cells was counted every 3 or 4 days for two weeks. ZsGreen expression rates in transduced T cells and T cell subsets were determined weekly by fluorescence-activated cell sorting (FACS). For the study of cytokine secretion after contact with tumor cells, T cells that had been in contact with irradiated tumor cells for 1 or 2 weeks were washed, incubated overnight with fresh tumor cells, and the culture supernatant was collected for analysis after 24 hours.

[0184] Clonal proliferation of CAIX+ CART cells In one embodiment, Skrc52 CAIX+ / PD-L1- and Skrc52 CAIX- / PD-L1- cells are irradiated with 3,000 rads, resulting in 2.5 x 10⁶ cells per well. 5 I sowed 1 x 10 6 T cells were added every two days to a culture medium containing 50 IU / ml of human IL-21. The T cells were divided to maintain an appropriate density and restimulated with tumor cells weekly. The number of T cells was counted weekly by FACS for three weeks.

[0185] The effect of anti-CAIX CART cells secreting anti-PD-L1 antibodies on the viability of RCC cells and antibody-dependent cytotoxicity (ADCC). 2.5 x 10³ Skrc59 CAIX+ / PD-L1+ and Skrc52 CAIX- / PD-L1- cells were plated overnight in 96-well plates. Four days after transduction of CART cells, they were added to RCC cells at effector cell:tumor cell (E:T) ratios of 25:1, 50:1, and 100:1 and incubated overnight. CART cells were removed, and tumor cell viability was assayed using MTT (Life Technologies). In the ADCC assay, RCC cells were incubated at 37°C for 1 hour with 50 μL of CART cell supernatant adjusted to contain 500 ng / mL of each anti-PD-L1 IgG1, anti-PD-L1 IgG4, or anti-SARS IgG1. The cells were then incubated with NK cells in a 12.5:1, 25:1, or 50:1 ratio at 37°C for 4 hours. Lactate dehydrogenase (LDH) in the supernatant was measured using CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega).

[0186] An ELISA assay to detect IL-2 and IFNγ released by functional CART cells. For cytokine secretion analysis, 2.5 x 10³ RCC cells, either Skrc59 CAIX+ / PD-L1+ or Skrc52 CAIX- / PD-L1-, were seeded overnight in 96-well plates, followed by the addition of CAR transduction T cells in 5:1, 25:1, and 50:1 ratios, and incubated overnight. The supernatant was collected and analyzed for IL-2 and IFNγ secretion using the Human IFNγ or Human IL-2 ELISA Ready-SET-Go-Kit (eBioscience).

[0187] Construction of an orthotopic renal cell carcinoma model and CART cell therapy. 5 x 10 4 10⁴ passaged Skrc59 CAIX+ / PD-L1+ cells were suspended in 10 μL of culture medium, diluted 1:1 with Matrigel® (Life Technologies), and injected subcapsularly into the left kidney capsule of 6-8 week old male NSG mice (N=35). One week later, tumor transplantation was confirmed by bioluminescence (BLI) imaging using the Xenogen IVIS imaging system (Life Technologies), and 1.0 x 10⁷ cells of each CAR T cell type (anti-BCMA CAR / anti-SARS IgG1, anti-CAIX CAR / anti-SARS IgG1, anti-CAIX CAR / anti-PD-L1 IgG1, and anti-CAIX CAR / anti-PD-L1 IgG4) or untransduced T cells were intravenously injected into the tail vein (day 0); N=6 mice per group. Tumor BLI was quantified 7, 14, 23, and 30 days after CAR cell injection. 6A second injection of CAR or non-transduced T cells was administered on day 17. Mice were sacrificed 30 days post-tumor transplantation by standard CO2 inhalation, and the tumors were collected and weighed. All mouse-derived kidney tumors were bisected; one half was fragmented into small pieces and digested with collagenase 0.5 U / mL and DNAse 1.0 mg / mL for TIL extraction, which was then analyzed by FACS for the expression of exhaustion markers and the percentage of CAR cells. The other half was fixed with 10% buffered formaldehyde and subjected to immunohistochemistry for different markers. Two days before euthanasia, 4.5 x 10⁶ NK cells were injected into two mice from each group. NK cells present in the tumors were stained with APC-anti-CD56 Ab and analyzed by FACS. Animal experiments were conducted in accordance with DFCI Animal Care Committee guidelines.

[0188] Tumor formation and T-cell therapy. In one embodiment, considering the immune rejection and rapid in vivo growth characteristics of sk-rc-52 in 6-8 week old female BALB / c nude mice, 5 million cells were subcutaneously inoculated into the mice, collected, and grown in vitro. This cell line was then passaged two more times in nude mice, and the passaged cells were grown for further experiments (subclone 4-1). In therapeutic experiments, 5 million sk-rc-59 and 7.5 million passaged sk-rc-52 cells were subcutaneously inoculated into the contralateral side of nude mice to achieve equivalent tumor growth rates. After 7 days, the tumors had grown to approximately 6 mm in size and were intravenously injected with 50 million non-transduced or transduced T cells. These mice were also treated with 20,000 IU of human IL-2 by intraperitoneal injection every two days. Tumor size was measured two-dimensionally using a caliper, and the average diameter of the two tumors is reported here. Animal studies were conducted in accordance with the guidelines of the Dana Farber Cancer Institute Animal Care Committee. Tumors were 15 mm in diameter or 2,000 mm in size. 3 The mice were sacrificed when they reached a certain stage, and the tumors were collected.

[0189] Immunohistochemistry and immunofluorescence staining. For in vitro testing of transduced T cells, cultured T cells were washed twice with PBS and resuspended in 2 μM Far Red DDAO-SE CellTrase dye (Molecular Probe) in PBS at 37°C for 15 minutes. The cells were then washed twice with culture medium and cytospinned on glass slides. CART cells pre-stained with Far red co-expressing ZsGreen were observed using a confocal microscope (Zeiss) at the Optical Imaging Core facility of the Harvard NeuroDiscovery Center.

[0190] To test the transduction T cell death effect in the tumor bed in situ, tumors were prepared as frozen sections for the ApopTag Peroxidase In Situ Apoptosis Detection Kit (Millipore). The frozen sections were incubated with TdT enzyme (Millipore) for 1 hour. Rabbit anti-DIG (Dako) was added and incubated for 30 minutes, followed by Cy3-conjugated anti-rabbit antibody (Invitrogen) and incubated for another 30 minutes. The sections were mounted using DAPI anti-fade mounting medium and fluorescence imaging was examined using a confocal microscope.

[0191] Xenograft tumors and mouse spleens were collected, fixed in 10% formalin / PBS solution, and provided to the Rodent Histopathology Core Facility, Harvard Medical School. Before staining, paraffin-embedded sections were dewaxed with xylene and rehydrated with graded alcohols. Immunohistochemical staining was performed by incubation with anti-human granzyme B antibody (Dako, clone GrB-7 (1:200)) as the primary antibody for 1 hour, followed by incubation with secondary anti-rabbit antibody (Pierce) or anti-mouse antibody (Dako) for 30 minutes. Sections were developed with DAB substrate and counterstained with hematoxylin.

[0192] In one embodiment, fixed tumors were paraffin-embedded, sectioned into 4-micrometer sections, placed on slides, and prepared for IHQ. This tissue was stained with anti-human Ki67 (Vector, VP-K451), PD-L1 (Clone 405.9A11, prepared in Dr. Gordon Freeman's laboratory), granzyme B (Abcam, ab4059), or NCAM (CD56) (Abcam, ab133345) antibodies, followed by secondary HRP-conjugated anti-rabbit Ab or HRP-avidin. Slides were developed using DAB and counterstained with hematoxylin. Images were acquired using a DP71 digital camera (Olympus) under an Olympus BX51 microscope and analyzed with DP Controller Software (Olympus). Image quantification was performed using the IHC Profiler Plugin for ImageJ Software, as described in Varghese F, Bukhari AB, Malhotra R, De A. IHC Profiler: an open source plugin for the quantitative evaluation and automated scoring of immunohistochemistry images of human tissue samples. PloS one. 2014;9;e96801.

[0193] statistical analysis Statistical significance was determined using a two-tailed Student's t-test.

[0194] The statistical analyses in Figures 16–23 represent at least three experiments unless otherwise noted. Statistical significance of the data was assessed using ANOVA and Tukey post-hoc tests. P<0.05 was considered significant. Statistical analyses were performed using IBM SPSS Statistics software version 20.

[0195] Example 2: Removal of anti-CAIX antibody via ADCC and selection of CAR targeting moiety We previously reported a panel of high-affinity human anti-CAIX antibodies that differed in their epitope mapping, expression levels, and ability to internalize CAIX. 23 Our initial objective was to investigate the antitumor activity of five of these anti-CAIX single-chain antibodies as candidates for CAR construction. To test ADCC through anti-CAIX mAbs, we converted scFv to scFv-Fc(hIgG1) minibodies. 23 We found that all scFv-Fc cells exhibited antigen-specific oncolysis. Under a background of <5% lysis against the CAIX-tumor cell line sk-rc-59, specific lysis ranged from 40-57% against the high-CAIX+-expressing tumor cell line sk-rc-09, and from 46-60% against the moderately-expressing sk-rc-52. Negative control scFv-Fc cells, e.g., anti-CXCR4 48-Fc cells, were also observed. 23 and anti-SARS 11A-Fc 24 Only background-level cell lysis was observed (Figure 1). Based on ADCC death and other reported analyses, scFvG36 was selected as the CAR-targeted portion for further evaluation.

[0196] Construction and expression of CAIX-specific chimeric receptors. Two generations of anti-CAIX CARs were constructed: a first-generation G36 CD8 CAR having scFv G36 (G36-CD8z) fused to the truncated extracellular, hinge, and transmembrane domains of CD8 and TCRζ, as well as the signaling domain. A second-generation CD28 CAR was created (G36-CD28z) (Figure 2A) consisting of scFvG36 fused to the truncated extracellular, transmembrane, and intracellular domains of CD28, as well as the signaling domain of TCRζ, to deliver co-stimulatory signals. An unrelated second-generation CD28 CAR was constructed by using anti-HIV CCR5 (clone A8) scFv instead. 25To detect the expression of these constructs, a human rhodopsin C9 tag was inserted between the scFv and either the CD8 or CD28 domain, respectively, and ZsGreen was expressed after the IRES sequence. Equivalent levels of high viral stocks were obtained between the different constructs, which were tested by co-transfection of the vector plasmid into 293T cells (data not shown).

[0197] Peripheral blood lymphocytes were stimulated for 3 days using PHA mitogen for transduction. Human primary T cells were infected with concentrated lentiviral supernatant in the presence of the cationic reagent DEAE, which improved the transduction rate by 1.5 to 2 times compared to polyblen (data not shown). The transduction rate of primary T cells ranged from 17% to 45% based on ZsGreen expression in FACS analysis. A representative experiment showing approximately 25% ZsGreen expression in primary CART cells after lentiviral transduction is shown in the left column of Figure 2B. The CAIX-Fc fusion protein can bind to G36-CD8z and -CD28z CART cells, but not to control A8-CD28z CART cells (Figure 2B, center column). C9 tag expression was detected at only about one-third the level of CAIX-Fc protein (Figure 2B, right column), which is thought to be related to the finding (data not shown) that mAb 1D4 preferentially recognizes rhodopsin nanopeptide C9 when presented as a carboxy-terminal polypeptide sequence relative to its internal polypeptide sequence. Transduced cells cultured in vitro for 6 weeks maintained their ZsGreen expression.

[0198] In Western blotting under reducing conditions, the G36 and A8 CD28z CARs migrated to a molecular weight of approximately 53 kDa, with an endogenous TCRζ of 16 kDa. The G36-CD8z CAR migrated to a molecular weight of approximately 48 kDa. Under non-reducing conditions, these two CD28z CARs formed homodimers (Figure 2C; data for the CD8z CAR are not shown).

[0199] Example 3: Enhanced cytokine secretion by transduced T cells upon contact with CAIX+ tumors. We conducted a study to compare the reported superior effects of second-generation G36-CD28z CART cells, which incorporate the signaling element of the co-stimulatory molecule CD28 to avoid MHC presentation and enhance T cell effector function, with first-generation G36-CD8z CART cells. As shown in Figure 3A, after overnight incubation with CAIX+ sk-rc-52 cells, only low levels of type I cytokine secretion (IL-2, IFNγ, and IL-17) were observed in control A8 CD28z CART cells or LAK cells alone. In contrast, both first-generation and second-generation G36-expressing CART cells showed high levels of cytokine secretion, with second-generation G36-CD28z CART cells secreting large amounts of type I cytokines, reflecting their higher activation state compared to first-generation G36-CD8z CART cells. Specifically, G36-CD28z CART cells secreted 6.5 times, 2.3 times, and 4 times more IL-2, IFNγ, and IL-17, respectively, than G36-CD8z CART cells. The specificity of cytokine secretion induction by these two G36 CART cells is observed even with minimal stimulation using CAIX-sk-rc-59 cells.

[0200] In the Elispot study, G36-CD28z CART cells became highly IFN-γ producing cells after interaction with CAIX+ sk-rc-52 tumors (Figure 3B). G36-CD28z CART cells generated six times more spots than G36-CD8z CART cells and twelve times more spots than those seen after interaction with CAIX+ sk-rc-59 tumor cells after interaction with CAIX+ sk-rc-52 tumor cells. Similarly, G36-CD28z CART cells had more granzyme B secreting spots after contact with CAIX+ tumors compared to G36-CD8z CART cells and control T cells. T cells stimulated with PMA and ionomycin produced the most IFN-γ and granzyme B secreting T cells. These studies demonstrate both the specificity and high productivity of G36-CD28z CART cells activated by contact with CAIX+ tumor cells.

[0201] Example 4: Specific cytotoxicity in transduced T cells via CAR signaling To further evaluate the tumor-killing activity of different G36 CART cells, an in vitro cytotoxicity assay was constructed. Using different effector-to-target ratios, G36-CD28z CART cells and their two-passage subclone 4-1 showed the highest cytotoxicity of CAIX+ tumor sk-rc-52 (Figure 3C). When using high ratios greater than 25:1, G36-CD28z CART cells showed 2-3 times higher cytotoxicity than G36-CD8z CART cells, and when using a low ratio of 5:1, G36-CD28z CART cells showed 8-9 times higher lysis than G36-CD8z CART cells. However, even when using an E:T ratio of 100:1, G36-CD28z CART cells still showed good cytotoxicity, exhibiting over 60% tumor lysis. Unrelated A8-CD28z CART cells and control T cells (LAK) showed background nonspecific tumor lysis with approximately 20% lysis when using an E:T ratio of up to 100:1. In all cases using CAIX-tumorsk-rc-59, transduced and non-transduced T cells showed background lysis.

[0202] Example 5: Improved in vitro proliferation of CART cells in the presence of CAIX+ tumor over a long period of time. Apart from the improved cytokine secretion and cytotoxicity observed in short-term contact with CAIX+ tumor cells, the incorporation of the CD28 co-stimulatory molecule into the CAR construct showed improved proliferation in long-term contact with antigen-specific tumor cells. Non-transduced and transduced (approximately 20%) T cells were mixed with tumor cells freshly irradiated weekly in the presence of 100 units / ml of human IL-2. To test different levels of antigen stimulation to a given amount of T cells, we used tumor cell-to-T cell ratios of 1:8, 1:4, and 1:2. T cell counts were determined by trypan removal, and the CART cell fraction was examined by flow cytometry. Transduced and non-transduced T cell counts were maintained under culture with CAIX-sk-rc-59 tumor cells (Figure 4A, bottom). The lack of baseline-level proliferation of control T cells is thought to be due to the large amount of inhibitory cytokines secreted by the tumor cell line. In contrast, after two weeks of culture with CAIX+ sk-rc-52 tumor cells, at a ratio of 1:8, the population of G36-CD28z CART cells increased 30-fold and G36-CD8z CART cells proliferated 17-fold. At a ratio of 1:4, the number of G36-CD28z CART cells increased 19-fold and G36-CD8z CART cells increased 4-fold. Neither G36-CD28z nor G36-CD8z CART cells were able to proliferate under a large number of tumor cells. Unrelated A8-CD28z CART cells and control T cells (LAK) did not show proliferation under tumor cells (Figure 4A, top).

[0203] Proliferative T cells were also collected to test their enrichment upon contact with CAIX+ tumor cells. Contact with CAIX− tumors did not change the percentage of any CART cells within this population. However, contact with CAIX+ sk-rc-52 tumor cells resulted in enrichment in both populations of G36 CART cells. In G36-CD28z CART cells, the positive population was enriched from 18% on day 0 to 52% on day 8 and 88% on day 16. Expression of G36-CD8z CART cells was enriched from 19% on day 0 (same level as T cells only) to 32% on day 8 and 72% on day 16. No proliferation of A8-CD28z CART cells was seen during this two-week study (Figure 4B). The percentage of CD8 cells was maintained constant throughout the 16-day study under all conditions (Figure 4C).

[0204] Example 6: Persistent effector function of CART cells after re-contact with tumors Transduced T cells that had been in contact with irradiated tumor cells for 1 or 2 weeks were also tested for cytokine secretion after 24 hours of contact with fresh non-irradiated tumor cells. Contact with CAIX+ tumors (sk-rc-52) for 1 or 2 weeks showed that co-stimulatory signals through the G36-CD28z CAR resulted in 2- to 2.5-fold more IFN-γ secretion than that seen with the G36-CD8z CAR, although G36-CD28z and G36-CD8z CART cells showed similar IFN-γ secretion levels (Table 1). With regard to IL-2 secretion, 2 weeks of tumor contact for G36-CD28z and G36-CD8z CART cells showed more IL-2 secretion than 1 week of contact. G36-CD28z CART cells produced 5-fold more IL2 after 1 week of contact and 2.5-fold more after 2 weeks of contact than G36-CD8z CART cells. In addition, G36-CD28z CART cells that had been in contact with tumor cells for 2 weeks secreted 3.3-fold more IL-2 than after a single tumor contact, and G36-CD8z CART showed 6.8-fold more IL-2 secretion 2 weeks after compared to 1 week of tumor contact. These results indicate that transduced CART cells did not become exhausted after the second tumor stimulation and maintained their functional activity. Treatment with A8-CD28z, LAK, and G36 CART cells in contact with CAIX- sk-rc59 cells showed only background levels of IFN-γ and IL-2 secretion.

[0205] Example 7: Suppression of tumors formed by CART cells We next tested whether CART cells inhibit the growth of formed tumor cells in nude mice inoculated with sk-rc-52 tumor cells formed to produce a similar tumor curve on the left flank and sk-rc-59 tumor cells on the right flank. On day 7 after tumor transplantation when typical tumor size had reached approximately 6x6 mm, 50 million G36-CD28z CART cells, A8-CD28z CART cells, or non-transduced T cells (LAK) were injected intravenously. Adoptive T cell therapy was performed in two separate experiments with group sizes of n = 7 in the first test and n = 8 in the second test, with high-dose IL-2 (2x10 5The procedure was performed in the presence of IU. T-cell treatment was not included to compare tumor growth and the effects of cell therapy.

[0206] In the first study, treated and untreated CAIX-sk-rc-59 tumors (within the four study groups) had mean sizes of 6.09±0.02 mm on day 4 and 9.29±0.12 mm on day 25. They showed the same tumor growth rate in the control and T-cell treated groups. Untreated CAIX+ tumors that did not receive T cells showed similar tumor sizes to CAIX- tumors, with mean sizes of 6.09±0.13 mm on day 4 and 9.15±0.11 mm on day 25. However, tumor size in G36-CD28z CART cell-treated mice showed a statistically significant size reduction compared to untreated mice at every time point tested during the 25-day study (Figure 5). G36-CD28z CART treatment also resulted in a greater reduction in tumor size than observed in A8-CD28z CART cell and LAK-treated mice, as calculated by two-sided t-tests, at day 7 (p<0.05) and day 25 (p<0.001). In the second study, tumor size in G36-CD28z CART cell-treated mice was significantly smaller than that of untreated mice throughout the 29-day experiment. G36-CD28z CART cell-treated mice also had smaller tumors than observed in A8-CD28z CART cell and LAK-treated mice at p<0.01 from day 8 to day 26 and at day 29 (p<0.001) (Figure 5).

[0207] Partial regression of the CAIX+ tumor was considered to occur when the tumor size was smaller than 30% of the volume of the control CAIX- tumor in the same mouse given the same T cells. Partial tumor regression was observed in a high proportion of cases using G36-CD28z CART cells (10 out of 15 (67%)), but only slightly in unrelated target A8-CD28z CART cells (1 out of 15 (7%)) and activated T cell LAK (2 out of 15 (13%)) (Table 2). The frequency of the partial regression response was found to be statistically significant, with p<0.001 and p<0.005, respectively, by Fisher's test in mice treated with G36-CD28z CART cells compared to control A8-CD28z CART cells and LAK.

[0208] Example 8: In-situ cytotoxicity by CART cells Samples of the total population of transduced T cells used in the in vivo study were pre-stained with Far red dye, and CART cells expressing ZsGreen protein within that population were analyzed by confocal microscopy. These results showed a transduction efficiency of approximately 30%, similar to our FACS analysis (Figure 6A).

[0209] Tumor sections were stained by the Tunnel assay to provide evidence that treatment of CAIX+ sk-rc-52 tumor cells with G36-CD28z CART cells in vivo resulted in apoptotic death. Three days after adoptive T cell treatment, Tunnel staining showed apoptotic tumor cells (red) in the tumor periphery (Figure 6B, bottom row) and within the tumor bed (Figure 6B, middle row). Apoptotic tumor cells lost DAPI nuclear staining. ZsGreen-expressing CART cells interacting with two apoptotic tumor cells are shown in the enlarged graph (Figure 6B, bottom row).

[0210] Due to the limitations of fluorescence signaling, ZsGreen-expressing CART cells could not be observed from total tissue sections. Therefore, tumors were collected on day 3 after G36-CD28z CART cell or LAK treatment, and sections were also stained with granzyme B antibody to identify activated T cells. In Figure 6C, the dark brown stained areas indicate granzyme B+ T cells, which are observed to infiltrate the CAIX+ sk-rc-52 tumor section (Figure 6C upper left). These granzyme B+ T cells were observed around the tumor (Figure 6C upper left (a) and center) and within the tumor (Figure 6C upper left (b) and bottom). Tumors with necrotic areas are shown in H&E stained slides (indicated as n in Figure 6C center right and bottom) and are located near the granzyme B+ T cells. In contrast, CAIX+ sk-rc-52 tumors treated with control activated T cells (LAK) (Figure 7) did not show granzyme B+ T cells. Similarly, CAIX- sk-rc-59 treated with G36-CD28z CART cells (Figure 8) or LAK (Figure 9) showed low background staining, and the tumors were proliferative. As a positive control for granzyme B staining, CART cells were locally injected into formed sk-rc-52 tumors in mice. After 1 day, the mice were sacrificed, and tumor tissue was excised for this staining (Figure 10).

[0211] (Table 1) Cytokine secretion after 1 or 2 weeks of contact with tumor cells * TIFF0007833003000011.tif67152 * Transduced T cells were incubated with irradiated tumor cells for 1 or 2 weeks, then collected, washed, and incubated overnight with fresh, unirradiated tumor cells. The supernatant was collected after 24 hours for cytokine analysis. In T cell cultures that did not interact with tumor cells, only background levels of cytokines were detected, with levels of <50 pg / ml IFN-γ and <10 pg / ml IL-2.

[0212] (Table 2) Frequency of partial regression of CAIX+ tumors by G36-CD28z CART cells Mice from the experiments reported in Figure 5 (Experiment 1, n = 7 and Experiment 2, n = 8) were scored for their response on day 10. Partial response was defined as tumor regression to less than 30% of the volume of the control tumor (same T-cell treatment in the same mice having sk-rc-52 in the left flank and control tumor sk-rc-59 in the right flank). Results of Fisher's test - * G36-CD28z vs. LAK; ** G36-CD28z vs. A8-CD28z;NS - There is no statistically significant relationship between the number of tumors and the partial response between LAK-transduced T cells and A8-CD28z-transduced T cells.

[0213] Example 9: Anti-PD-L1 antibody-releasing anti-carbonic anhydrase IX chimeric antigen receptor T cells restore cellular exhaustion and regress renal cell carcinoma in a humanized mouse model. Characterization of anti-CAIX CAR T cells that secrete anti-PDL1 IgG1 or IgG4

[0214] To develop a novel CAR therapy for CAIX+ RCC, we engineered a bicistronic lentiviral vector to express an anti-CAIX(G36)scFv linked to the CD28 and CD3-ζ signaling domains (G36-CD28z CAR in the first cassette and anti-PD-L1 IgG1 or IgG4 in the second expression cassette following the IRES site) (Figure 16A). The anti-PD-L1 IgG sequence was inserted into the lentiviral vector to prevent T cell exhaustion, which we plan may improve the efficacy of G36-CD28z CART cells (Figure 16B). As a control, we used an anti-CAIX CAR or anti-BCMA CAR containing an unrelated anti-SARS IgG1 mAb in the second cassette. Lentiviruses generated from these constructs were transduced into CD8 T cells and cultured in the presence of IL-21, thereby obtaining CART cells that showed slightly improved proliferation compared to those observed with IL-2 (Figures 21A and 21B) while maintaining the same specific killing activity against CAIX+ RCCs (Figures 21C and 21D). The percentages of CAIX and PD-L1 expression in all RCC lines used in our experiment are shown in Figure 22.

[0215] The function of CART cells is shown in Figure 23. Transduced CD8 T cells in all CARs were able to proliferate in the presence of IL-21 and anti-CD8 / CD28 beads (Figures 23A and 23B), achieving transduction levels of 65–90% after 4 days (Figure 23C). Fourteen days after transduction, we assessed the stable long-term expression of CARs by the incorporated lentivirus (Figure 16C), which maintained approximately 25–50% in all CARs. Total IgG levels secreted by CD8 T cells were also determined, ranging from approximately 300–650 ng / mL after 4 days (Figure 16D). Binding specificity of anti-PD-L1 IgG1 and IgG4 antibodies against human PD-L1 was also confirmed (Figure 16E). The levels of biotinylated anti-PD-L1 IgG1 and IgG4 were significantly lower than total IgG, which could be explained by the fact that a portion of IgG was lost during the purification process. The ability of anti-CAIX CART cells to undergo clonal proliferation in the presence of CAIX+ RCC cells was confirmed (Figures 16F and 16G). Anti-CAIX CART cells were significantly unable to proliferate in the presence of CAIX- RCC cells.

[0216] Effector activity of anti-CAIX CART cells. All anti-CAIX CART cells were able to induce a 50-70% reduction in the survival of Skrc59 CAIX+ / PD-L1+ cells, indicating that anti-PD-L1 IgG1 and IgG4 secreted by some CART cells did not increase cell death under these assay conditions (Figure 17A). Anti-CAIX CART cells induced IL-2 and IFNγ release in the presence of CAIX+ / PD-L1+ cells, demonstrating specific activation of anti-CAIX CART cells (Figures 17C and 17E, respectively). A unique differential effect was observed with anti-CAIX CART cells secreting anti-PD-L1 IgG, specifically with the IgG1 isoform, which was able to induce approximately 60% ADCC in CAIX+ / PD-L1+ RCC cells when incubated with natural killer cells (NK) (Figure 17G). In the presence of CAIX- / PD-L1- cells, no effect on cell survival, cytokine secretion, or ADCC was observed in any of the CART cells (Figures 17B, 17D, 17F, and 17H).

[0217] Anti-CAIX CART cells secreting anti-PD-L1 antibodies can reduce T cell exhaustion in vitro. In the anti-PD-L1 IgG1 and IgG4 anti-CAIX CART groups, approximately 50% reductions in exhaustion markers Lag-3, Tim3, and PD-1 were observed after exhaustion induction compared to parental anti-CAIX or unrelated anti-BCMA CART cells (Figures 18A, 18B, and 18C, respectively). At this point, the death-causing activity of anti-CAIX CART cells without anti-PD-L1 was re-examined in Skrc59 CAIX+ / PD-L1+ cells. As can be seen in Figure 18D, anti-CAIX CART cells lost their death-causing activity against CAIX+ / PD-L1+ RCCs in vitro to a level similar to that of the unrelated CAR group, confirming that anti-CAIX CART cells become anergenic. In contrast, RCC survival was reduced by 25–50% in the anti-CAIX CAR, anti-PD-L1 IgG1, and IgG4 CAR groups, providing evidence that checkpoint inhibition induced by the presence of anti-PD-L1 IgG secreted therefrom may reduce T cell exhaustion.

[0218] Anti-CAIX CART cells that secrete anti-PD-L1 antibodies can further reduce tumor growth in an orthotopic mouse model of human RCC. An orthotopic RCC model was constructed using NSG mice by injecting Skrc-59 CAIX+PD-L1+luciferase+positive RCC cells subcapsularly into the kidney capsule, followed by IV injection of 1.0 x 10⁷ CART or non-transduced T cells (day 0) and a low dose (2.5 x 10⁷) on day 17. 6Repeated treatments were performed using (1) of the same cells. To avoid any potential bias that this molecule may exert on tumor growth, we did not treat the mice with systemic IL-2 to maintain CART cell proliferation. The data in Figures 19A–19C demonstrate that all three CAIX CART cell groups showed reduced RCC growth compared to unrelated BCMA CART cells or non-transduced cells over the experimental period, and the significant antitumor effect of anti-CAEX CART cells secreting anti-PD-L1 IgG1 or IgG4 became evident on days 23 and 30 (Figures 19A and 19B). However, even one week after IV treatment with CART cells, we observed that tumors were 2–3 times smaller in anti-PD-L1 secreting CART cells compared to parental anti-CAIX CART cells and the two control groups (Figure 24A). We also analyzed the survival of CD45+ T cells in mouse blood to assess their survival in this passive transfer model. On day 8, we observed that the amount of human T cells in the PBMCs was only 10–15% (Figure 24B). For this reason, we decided to grow CART cells in vitro for a second injection into the tail blood on day 17. One week after the second injection (day 23), the anti-PD-L1 IgG1 and IgG4 groups had tumors at 1 / 15th the size of the control group and 1 / 5th the size of the anti-CAIX CAR T cells that did not secrete anti-PD-L1 (Figures 19C and 24A). On day 30, the group of mice treated with CART cells secreting anti-PD-L1 antibodies had tumors at 1 / 5th the size of the control group (Figures 19C and 24A). Tumor weights excised from mice treated with CART cells secreting anti-PD-L1 antibodies were also lower, which was particularly pronounced in the anti-PD-L1 IgG4 antibody group (Figures 19B and 19D).

[0219] Analysis of tumor infiltration by CART cells and evidence that anti-CAIX CART cells secreting anti-PD-L1 antibodies can improve T cell exhaustion. Analysis of resected tumors showed approximately 10% TILs in all groups (Figure 24C).

[0220] One of the most significant effects observed using anti-CAIX CAR cells secreting anti-PD-L1 IgG1 or IgG4 antibodies in vivo was their ability to reduce the expression of exhaustion markers PD-1, Tim-3, and Lag-3. As shown in Figure 20A, we observed a reduction of approximately 40–50% in the expression of these markers in TILs compared to treatment with non-transduced control cells at day 30. A reduction of approximately 30–70% was also observed compared to anti-CAIX CAR cells that did not secrete anti-PD-L1 antibodies, providing evidence that locally secreted antibodies have an effect on reducing T cell exhaustion.

[0221] The effector activity of CART cells in vivo and their effects on RCC proliferation were evaluated by immunohistochemical detection of granzyme B, the tumor growth marker Ki67, and the tumor immunosuppressive protein PD-L1 in TILs (Figures 20B and 20C). Granzyme B staining indicated effector activity of CD8+ cells, particularly in RCC tumors treated with anti-CAIX CART cells secreting anti-PD-L1 IgG4, as shown by an increased percentage of strongly stained cells (Figures 20B and 20C). PD-L1 expression was dramatically reduced in tumors treated with anti-CAIX-CART cells, and this was more pronounced in the anti-CAIX / anti-PD-L1 IgG secreting group (Figures 20B and 20C). Ki67 expression was significantly reduced in the anti-CAIX / anti-PD-L1 IgG secretion group, which we can visualize in the total DAB pixel count graph. However, when evaluating the intensity in positive nuclear staining (Ki67 IHC quantification graph), we can point out that anti-CAIX / anti-PD-L1 IgG4 showed the lowest intensity of Ki67 expression (Figures 20B and 20C). When quantifying nuclear proteins such as Ki67, the DAB staining pattern should be limited to the nucleus, and the positive staining region for quantification should be selected using ImageJ's thresholding function. For this reason, the Ki67 IHC quantification graph does not contain negative cells (Figure 20C), and the DAB pixel count is also shown to evaluate total Ki67 staining including negative cells (Figure 20C).

[0222] Anti-CAIX CART cells that secrete anti-PD-L1 IgG1 antibodies can induce NK cells to the tumor. Tumors in mice treated with anti-CAIX / anti-PD-L1 IgG1, in which NK cells were injected 2 days before euthanasia, showed the presence of over 40% more NK cells in the tumor detected by anti-CD56 staining by FACS compared to anti-BCMA / anti-SARS IgG1 (Figure 25A). The increase in NK cells in the anti-CAIX / antibody PD-L1 IgG1 group was also detected and quantified by IHC (Figure 25B). Very few NK cells were detected in the group treated with CART cells secreting non-specific IgG1 Ab.

[0223] Other aspects Although the present invention has been described in connection with its detailed description, the above description is intended to be illustrative and not to limit the scope of the present invention, which is defined by the appended claims. Other aspects, advantages and modifications are also within the scope of the appended claims.

[0224] References TIFF0007833003000013.tif114148TIFF0007833003000014.tif210148TIFF0007833003000015.tif217149TIFF0007833003000016.tif210148TIFF0007833003000017.tif203149TIFF0007833003000018.tif203149TIFF0007833003000019.tif210149TIFF0007833003000020.tif217148TIFF0007833003000021.tif210149TIFF0007833003000022.tif232148

[0225] Sequence information SEQUENCE LISTING <110> Dana-Farber Cancer Institute, Inc. <120> CHIMERIC ANTIGEN RECEPTORS AND METHODS OF USE THEREOF <150> US 62 / 094,625 <151> 2014-12-19 <150> US 62 / 252,083 <151> 2015-11-06 <160> 1664 <170> PatentIn version 3.5 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 1 Gly Gly Pro Phe Ser Ser Tyr Ala 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 2 Gly Gly Ile Phe Ser Ser Tyr Ala 1 5 <210> 3 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 3 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 4 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 4 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 5 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 5 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 6 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 6 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 7 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 7 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 8 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 8 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 9 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 9 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 10 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 10 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 11 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 11 Gly Val Thr Phe Arg Ser Tyr Ala 1 5 <210> 12 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 12 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 13 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 13 Gly Gly Pro Phe Ser Ser Tyr Ala 1 5 <210> 14 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 14 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 15 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 15 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 16 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 16 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 17 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 17 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 18 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 18 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 19 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 19 Gly Gly Pro Phe Ser Ser Tyr Ala 1 5 <210> 20 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 20 Gly Gly Pro Phe Ser Ser Tyr Ala 1 5 <210> 21 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 21 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 22 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 22 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 23 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 23 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 24 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 24 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 25 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 25 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 26 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 26 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 27 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 27 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 28 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 28 Gly Gly Ile Phe Ser Ser Tyr Ala 1 5 <210> 29 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 29 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 30 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 30 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 31 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 31 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 32 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 32 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 33 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 33 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 34 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 34 Gly Val Thr Phe Arg Ser Tyr Ala 1 5 <210> 35 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 35 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 36 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 36 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 37 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 37 Ile Ser Pro Met Phe Gly Thr Ala 1 5 <210> 38 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 38 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 39 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 39 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 40 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 40 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 41 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 41 Ile Met Pro Met Phe Gly Thr Ala 1 5 <210> 42 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 42 Ile Ser Pro Leu Phe Gly Thr Ala 1 5 <210> 43 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 43 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 44 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 44 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 45 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 45 Ile Ser Pro Met Phe Gly Thr Ala 1 5 <210> 46 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 46 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 47 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 47 Ile Ser Gly Ile Phe Gly Thr Ala 1 5 <210> 48 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 48 Ile Ser Pro Met Phe Gly Thr Ala 1 5 <210> 49 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 49 Ile Ser Gly Ile Phe Gly Thr Ala 1 5 <210> 50 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 50 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 51 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 51 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 52 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 52 Ile Ser Pro Met Phe Gly Thr Ala 1 5 <210> 53 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 53 Ile Thr Pro Met Phe Gly Thr Ala 1 5 <210> 54 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 54 Ile Ser Pro Leu Phe Gly Thr Ala 1 5 <210> 55 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 55 Ile Ser Pro Met Phe Gly Thr Ala 1 5 <210> 56 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 56 Ile Ser Pro Leu Phe Gly Thr Ala 1 5 <210> 57 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 57 Ile Ser Pro Met Phe Gly Thr Ala 1 5 <210> 58 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 58 Ile Ser Pro Leu Phe Gly Thr Ala 1 5 <210> 59 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 59 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 60 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 60 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 61 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 61 Ile Ser Pro Leu Phe Gly Thr Ala 1 5 <210> 62 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 62 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 63 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 63 Ile Ser Pro Met Phe Gly Thr Ala 1 5 <210> 64 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 64 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 65 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 65 Ile Thr Pro Ile Phe Gly Thr Ala 1 5 <210> 66 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 66 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 67 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 67 Ile Ile Pro Ile Phe Gly Thr Ala 1 5 <210> 68 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 68 Ile Thr Pro Met Phe Gly Thr Ala 1 5 <210> 69 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 69 Ile Ser Ala Met Phe Gly Thr Ala 1 5 <210> 70 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 70 Ile Ile Pro Ile Phe Gly Thr Ala 1 5 <210> 71 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 71 Ile Thr Pro Met Phe Gly Thr Ala 1 5 <210> 72 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 72 Ile Ser Pro Met Phe Gly Thr Ala 1 5 <210> 73 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 73 Ala Arg Asp Asp Gly Tyr Ala Pro Ser Gly Gly Leu Arg Glu Phe Asp 1 5 10 15 Val <210> 74 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 74 Ala Arg Gly Arg Gly Ala Tyr Met Gly Pro Ser Met Asp Val 1 5 10 <210> 75 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 75 Ala Arg Gly Ala Arg Tyr Tyr Ala Gly Gly Tyr Phe Asp Val 1 5 10 <210> 76 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 76 Ala Arg Asp Ser Gly Asn Tyr Asp Gly Tyr Gly Pro Gly Ser Arg Phe 1 5 10 15 Asp Val <210> 77 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 77 Ala Arg Glu Arg Gly Ser Trp Ser Phe Gly Tyr Phe Asp Val 1 5 10 <210> 78 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 78 Ala Arg Ser Arg Thr Tyr Ala Asp Gly Arg Thr Phe Asp Val 1 5 10 <210> 79 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 79 Ala Arg Glu Leu Gly Tyr Leu Ala Gly Ser Pro Ser Pro Gly Phe Asp 1 5 10 15 Tyr <210> 80 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 80 Ala Arg Ser Arg Arg Tyr Trp Ala Asp Gly Gly Phe Asp Tyr 1 5 10 <210> 81 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 81 Ala Arg Glu Gly Gly Tyr Ser Pro Gly Gly Val Asp Phe Asp Tyr 1 5 10 15 <210> 82 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 82 Ala Arg Gly Thr Thr Tyr Ser Thr Ala Arg Tyr Phe Asp Val 1 5 10 <210> 83 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 83 Ala Arg Ser Pro Ala Tyr Tyr Phe Gly Pro Asn Met Asp Val 1 5 10 <210> 84 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 84 Ala Arg Ser Ser Arg Tyr Ala Pro Ser Asp Ser Thr Asn Phe Asp Gln 1 5 10 15 <210> 85 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 85 Ala Arg Gly Asp Arg Phe Tyr Val Gly Glu Arg Phe Asp Val 1 5 10 <210> 86 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 86 Ala Arg Gly Gly Gly Val Gly Arg Ile Trp Ile Ala Gly Tyr Gly Phe 1 5 10 15 Asp Gln <210> 87 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 87 Ala Arg Gly Pro Gly Tyr His Pro Ala Gly Ala Ser Gly Gln Phe Phe 1 5 10 15 Asp Leu <210> 88 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 88 Ala Arg Gly Arg Gly Tyr Ala Pro Asp Ala Leu Thr Asn Phe Asp Val 1 5 10 15 <210> 89 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 89 Ala Arg Gly Arg Gly Tyr Ile Ala Val Ala Gly Asp Met Asp Val 1 5 10 15 <210> 90 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 90 Ala Arg Gly Asp Ala Tyr Tyr Val Gly Gly Gly Ala Arg Pro Phe Asp 1 5 10 15 Leu <210> 91 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 91 Ala Arg Gly Tyr Ser Tyr Tyr Pro Gly Gly Gly Gly Gly Arg Asn Phe 1 5 10 15 Asp Tyr <210> 92 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 92 Ala Arg Ala Pro Thr Tyr Tyr Ala Ser Arg Asp Ser Tyr Asn Phe Asp 1 5 10 15 Tyr <210> 93 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 93 Ala Arg Asp Thr Thr Tyr Ile Ala Gly Gly His Phe Asp Val 1 5 10 <210> 94 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 94 Ala Arg Ala Ser Gly Tyr Phe Thr Gly Trp Gly Thr Phe Asp Tyr 1 5 10 15 <210> 95 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 95 Ala Arg Gly Arg Tyr Tyr Tyr Thr Val Gly Val Tyr Asp Val 1 5 10 <210> 96 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 96 Ala Arg Gly Gly Gly Tyr Ser Ala Asp Gly Gly Ala Gly Asn Asn Thr 1 5 10 15 Ile Phe Asp Val 20 <210> 97 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 97 Ala Arg Glu Arg Gly Tyr Thr Val Gly Gly Gly Gly Met Asp Val 1 5 10 15 <210> 98 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 98 Ala Arg Glu Tyr Leu Gly Asp Asp Tyr Ser Ser Gly Ser Tyr Phe Asp 1 5 10 15 Val <210> 99 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 99 Ala Arg Glu Ser Gly Tyr Ser Gly Thr Gly Gln Phe Asp Val 1 5 10 <210> 100 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 100 Ala Arg Ser Gly Gly Tyr Tyr Asp Tyr Gly Val Gly Tyr Asp Gln 1 5 10 15 <210> 101 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 101 Ala Arg Ser Gly Gly Tyr Ser Pro Ser Ile Gly Gly Phe Asp Val 1 5 10 15 <210> 102 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 102 Ala Arg Gly Pro Gly Tyr Asp Pro Ser Ser Leu Arg Gly Phe Asp Val 1 5 10 15 <210> 103 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 103 Ala Arg Gly Glu Glu Ala Tyr Tyr Asp Leu 1 5 10 <210> 104 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 104 Ala Arg Gly Thr Ser Tyr Leu Pro Gly Arg Ser Gly Phe Asp Val 1 5 10 15 <210> 105 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 105 Ala Arg Gly Arg Gly Tyr Asp Pro Ser Val Gly Gly Phe Asp Val 1 5 10 15 <210> 106 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 106 Ala Arg Asp Ser Thr Pro Ser Val Thr Ser Ser Leu Tyr Arg Ile Pro 1 5 10 15 Ala Phe Asp Val 20 <210> 107 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 107 Ala Arg Gly Pro Gly Tyr Tyr Pro Asp Ser Asn Asn Tyr Asp Leu 1 5 10 15 <210> 108 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 108 Ala Arg Gly Gly Thr Tyr Ser Pro Gly Gly Thr Tyr Phe Asp Val 1 5 10 15 <210> 109 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 109 Arg Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr 1 5 10 <210> 110 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 110 Asn Ile Gly Ser Lys Ser 1 5 <210> 111 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 111 Gln Thr Val Ser Asn Tyr 1 5 <210> 112 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 112 Ser Ser Asn Ile Gly Ser Asn Thr 1 5 <210> 113 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 113 His Ile Gly Ser Lys Ser 1 5 <210> 114 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 114 Ser Ser Asp Val Gly Gly Tyr Asn His 1 5 <210> 115 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 115 Ser Ser Asp Val Gly Gly Tyr Asn Tyr 1 5 <210> 116 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 116 Ser Ser Asn Met Gly Arg Asn Thr 1 5 <210> 117 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 117 Ser Gly Ser Ile Ala Ser Thr Tyr 1 5 <210> 118 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 118 Ser Gly Ser Ile Ala Ser Asn Tyr 1 5 <210> 119 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 119 Ser Ser Asn Ile Gly Ala Gly Tyr Asp 1 5 <210> 120 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 120 Ser Gly Ser Ile Ala Ser Asn Tyr 1 5 <210> 121 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 121 Asp Ile Gly Ser Lys Ser 1 5 <210> 122 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 122 Ser Gly Ser Val Ser Thr Ser Asn Tyr 1 5 <210> 123 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 123 Ser Asn Asn Val Gly Asn Gln Gly 1 5 <210> 124 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 124 Arg Ser Leu Phe Asp Ser Ser Asp Asn Lys Asn Tyr 1 5 10 <210> 125 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 125 Arg Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr 1 5 10 <210> 126 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 126 Ser Ser Asn Ile Gly Val Asn Tyr 1 5 <210> 127 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 127 Ser Ser Asn Ile Gly Ser Asn Thr 1 5 <210> 128 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 128 Gln Ser Val Asp Arg Gly Tyr 1 5 <210> 129 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 129 Ser Ser Asp Ile Gly Ala Tyr Asn Tyr 1 5 <210> 130 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 130 Ala Leu Pro Lys Gln Tyr 1 5 <210> 131 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 131 Ser Asp Asn Val Gly Asn Gln Gly 1 5 <210> 132 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 132 Asn Asn Asn Val Gly Asn Gln Gly 1 5 <210> 133 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 133 Ser Ser Asp Val Gly Ala His Asn Phe 1 5 <210> 134 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 134 Gln Ser Val Asp Ser His 1 5 <210> 135 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 135 Thr Ser Asn Val Gly Arg Asn Thr 1 5 <210> 136 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 136 Arg Ser Asn Ile Gly Ser Asn Thr 1 5 <210> 137 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 137 Arg Ser Asn Ile Gly Arg Asn Thr 1 5 <210> 138 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 138 Ser Asn Asn Val Gly Asn Gln Gly 1 5 <210> 139 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 139 Gln Ser Val Ser Ser Phe 1 5 <210> 140 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 140 Ala Leu Pro Lys Gln Tyr 1 5 <210> 141 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 141 Ser Gly Ser Ile Ala Ser Asn Tyr 1 5 <210> 142 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 142 Asn Gly Pro Ser Asn Tyr Ile 1 5 <210> 143 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 143 Gln Ser Val Ser Ser Ser Tyr 1 5 <210> 144 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 144 Ser Ser Asn Ile Gly Val Ser Phe 1 5 <210> 145 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 145 Trp Ala Ser 1 <210> 146 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 146 Asp Asp Ser 1 <210> 147 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 147 Ala Ala Ser 1 <210> 148 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 148 Ser Asn Asn 1 <210> 149 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 149 Ser Asn Asn 1 <210> 150 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 150 Asp Val Ser 1 <210> 151 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 151 Glu Val Thr 1 <210> 152 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 152 Asp Asn Asp 1 <210> 153 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 153 Glu Asp His 1 <210> 154 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 154 Glu Asp Asn 1 <210> 155 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 155 Asp Asn Ser 1 <210> 156 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 156 Glu Asp Asn 1 <210> 157 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 157 Asp Asp Ile 1 <210> 158 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 158 Ser Thr Asn 1 <210> 159 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 159 Arg Asn Asn 1 <210> 160 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 160 Trp Ala Ser 1 <210> 161 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 161 Trp Ala Ser 1 <210> 162 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 162 Arg Asn Asn 1 <210> 163 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 163 Ser Asn Asn 1 <210> 164 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 164 Gly Ala Ser 1 <210> 165 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 165 Glu Val Ser 1 <210> 166 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 166 Lys Asp Ser 1 <210> 167 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 167 Arg Asp Asn 1 <210> 168 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 168 Arg Asn Asn 1 <210> 169 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 169 Glu Val Asn 1 <210> 170 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 170 Gly Ala Ser 1 <210> 171 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 171 Asn Asp Asn 1 <210> 172 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 172 Ser Asn Asn 1 <210> 173 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 173 Ser Asn Asn 1 <210> 174 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 174 Lys Asn Asn 1 <210> 175 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 175 Asp Ala Ser 1 <210> 176 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 176 Lys Asp Thr 1 <210> 177 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 177 Glu Asp Asn 1 <210> 178 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 178 Leu Asn Ser Asp Gly Ser His 1 5 <210> 179 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 179 Gly Ala Ser 1 <210> 180 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 180 Arg Asp Asp 1 <210> 181 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 181 Gln Gln Tyr Tyr Ser Gly Ser Trp Thr 1 5 <210> 182 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 182 Gln Val Trp Asp Arg Ser Ser Asp His Val Val 1 5 10 <210> 183 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 183 Gln Gln Tyr Asp Asn Leu Pro Pro Val Thr 1 5 10 <210> 184 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 184 Ser Ala Trp Asp Asp Ser Leu Gly Gly Glu Val 1 5 10 <210> 185 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 185 Gln Val Trp Asp Ser Ser Asn Asp His Pro Val 1 5 10 <210> 186 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 186 Thr Ser Tyr Ala Gly Ser Asn Ser Leu Val 1 5 10 <210> 187 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 187 Ser Ser Tyr Ala Gly Gly Lys Trp Val 1 5 <210> 188 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 188 Ala Ala Trp Asp Asp Ser Leu Asn Gly Pro Val 1 5 10 <210> 189 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 189 Gln Ser Phe Asp Ala Ser Thr Leu Val 1 5 <210> 190 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 190 Gln Ser Tyr Asp Ser Asp Asn His Glu Val Ile 1 5 10 <210> 191 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 191 Gln Ser Tyr Asp Ser Ser Leu Ser Val Val Val 1 5 10 <210> 192 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 192 Gln Ser Tyr Asp Thr Ser Asn Arg Lys Val 1 5 10 <210> 193 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 193 Gln Val Trp Asp Thr Asn Ser Asp Pro Val Phe Val 1 5 10 <210> 194 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 194 Val Leu Tyr Met Gly Ser Gly Ile Ser Met 1 5 10 <210> 195 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 195 Ser Ala Trp Asp Ser Ser Leu Ser Ala Trp Val 1 5 10 <210> 196 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 196 Gln Gln Tyr Phe Ser Ser Pro Pro Ile Phe Thr 1 5 10 <210> 197 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 197 Gln Gln Tyr Tyr Ser Thr Pro Pro Thr 1 5 <210> 198 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 198 Gly Val Trp Asp Asp Ser Leu Asn Gly His Trp Val 1 5 10 <210> 199 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 199 Ala Ala Trp Asp Asp Ser Leu Lys Gly Arg Val 1 5 10 <210> 200 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 200 Gln Gln Tyr Gly Ser Ser Arg Leu Ser 1 5 <210> 201 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 201 Ser Ser Tyr Ala Gly Ser Asn Asn Val Val 1 5 10 <210> 202 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 202 Gln Ala Trp Asp Ser Ser Thr Ala Val 1 5 <210> 203 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 203 Ser Ala Trp Asp Ser Ser Leu Thr Ala Val Val 1 5 10 <210> 204 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 204 Ser Ala Trp Asp Ser Ser Leu Ser Ala Trp Val 1 5 10 <210> 205 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 205 Ala Ala Trp Asp Asp Ser Leu Asp Gly Pro Val 1 5 10 <210> 206 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 206 Gln Gln Arg Ser Met Trp Pro Leu Thr 1 5 <210> 207 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 207 Ser Ser Trp Asp Asp Asp Leu Asn Gly Pro Val 1 5 10 <210> 208 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 208 Gln Ser Tyr Asp Ser Ser Val Val 1 5 <210> 209 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 209 Ala Ala Trp Asp Val Ser Leu Asn Gly Gln Val 1 5 10 <210> 210 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 210 Ser Ala Trp Asp Ser Ser Leu Ser Asp Trp Val 1 5 10 <210> 211 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 211 Gln Gln Arg Phe Asn Trp Pro Pro Thr 1 5 <210> 212 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 212 Gln Ser Ala Asp Ala Ser Glu Asn Ser Val 1 5 10 <210> 213 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 213 Gln Ser Tyr Asp Thr Ser Asn Leu Val 1 5 <210> 214 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 214 Glu Thr Trp Asp Ser Asn Thr His Val Val 1 5 10 <210> 215 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 215 Gln Gln Val Asn Ser Phe Pro Arg Thr 1 5 <210> 216 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 216 Ser Ala Trp Asp Glu Ser Leu Ser Ser Val Leu 1 5 10 <210> 217 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 217 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 218 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 218 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 219 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 219 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 220 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 220 Gly Gly Ile Phe Ser Ser Tyr Ala 1 5 <210> 221 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 221 Gly Val Ile Phe Ser Ser Tyr Ala 1 5 <210> 222 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 222 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 223 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 223 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 224 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 224 Gly Gly Pro Phe Arg Ser Tyr Ala 1 5 <210> 225 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 225 Gly Val Pro Phe Ser Ser Tyr Ala 1 5 <210> 226 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 226 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 227 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 227 Gly Val Ile Phe Ser Ser Tyr Ala 1 5 <210> 228 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 228 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 229 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 229 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 230 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 230 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 231 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 231 Gly Gly Pro Phe Ser Ser Tyr Ala 1 5 <210> 232 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 232 Gly Gly Ile Phe Ser Ser Tyr Ala 1 5 <210> 233 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 233 Gly Gly Ile Phe Arg Ser Tyr Ala 1 5 <210> 234 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 234 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 235 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 235 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 236 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 236 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 237 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 237 Gly Gly Thr Phe Arg Ser Tyr Ala 1 5 <210> 238 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 238 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 239 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 239 Gly Gly Pro Phe Ser Ser Tyr Ala 1 5 <210> 240 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 240 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 241 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 241 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 242 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 242 Gly Val Thr Phe Arg Ser Tyr Ala 1 5 <210> 243 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 243 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 244 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 244 Gly Val Thr Phe Ser Ser Tyr Ala 1 5 <210> 245 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 245 Gly Gly Pro Phe Ser Ser Tyr Ala 1 5 <210> 246 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 246 Gly Gly Ile Phe Ser Ser Tyr Ala 1 5 <210> 247 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 247 Ile Ile Thr Ile Phe Gly Thr Ala 1 5 <210> 248 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 248 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 249 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 249 Ile Ser Ala Ile Phe Gly Thr Ala 1 5 <210> 250 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 250 Ile Thr Pro Ile Phe Gly Thr Ala 1 5 <210> 251 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 251 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 252 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 252 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 253 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 253 Ile Ile Thr Ile Phe Gly Thr Ala 1 5 <210> 254 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 254 Ile Ile Pro Ile Phe Gly Thr Ala 1 5 <210> 255 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 255 Ile Ser Pro Leu Phe Gly Thr Ala 1 5 <210> 256 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 256 Ile Ser Pro Met Phe Gly Thr Ala 1 5 <210> 257 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 257 Ile Ile Pro Ile Phe Gly Thr Ala 1 5 <210> 258 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 258 Ile Ser Pro Val Phe Gly Thr Ala 1 5 <210> 259 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 259 Ile Val Pro Leu Phe Gly Thr Ala 1 5 <210> 260 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 260 Ile Ile Pro Ile Phe Gly Thr Ala 1 5 <210> 261 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 261 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 262 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 262 Ile Ser Ala Ile Phe Gly Thr Ala 1 5 <210> 263 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 263 Ile Ile Pro Met Phe Gly Thr Ala 1 5 <210> 264 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 264 Ile Asn Pro Ile Phe Gly Thr Ala 1 5 <210> 265 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 265 Ile Ser Pro Met Phe Gly Thr Ala 1 5 <210> 266 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 266 Ile Thr Pro Leu Phe Gly Thr Ala 1 5 <210> 267 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 267 Ile Met Pro Ile Phe Gly Thr Ala 1 5 <210> 268 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 268 Ile Ser Pro Met Phe Gly Thr Ala 1 5 <210> 269 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 269 Ile Ile Pro Ile Phe Gly Thr Ala 1 5 <210> 270 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 270 Ile Thr Pro Ile Phe Gly Thr Ala 1 5 <210> 271 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 271 Ile Ser Pro Met Phe Gly Thr Ala 1 5 <210> 272 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 272 Ile Ser Gly Ile Phe Gly Thr Ala 1 5 <210> 273 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 273 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 274 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 274 Ile Ser Pro Met Phe Gly Thr Ala 1 5 <210> 275 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 275 Ile Ser Pro Met Phe Gly Thr Ala 1 5 <210> 276 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 276 Ile Ser Pro Ile Phe Gly Thr Ala 1 5 <210> 277 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 277 Ala Arg Gly Ala Thr Gly Phe Tyr Asp Val 1 5 10 <210> 278 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 278 Ala Arg Gly Arg Glu Tyr Tyr Ala Ser Asn Gly Asp Ser Phe Asp Val 1 5 10 15 <210> 279 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 279 Ala Arg Asp Leu Ser Arg Asp Ser Leu Asn Leu Pro Gly Ser Ser Pro 1 5 10 15 Gly Tyr Asp Leu 20 <210> 280 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 280 Ala Arg Gly Ser Gly Tyr Tyr Val Ala Ala Ser Gly Ala Phe Asp Val 1 5 10 15 <210> 281 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 281 Ala Arg Ser Arg Gly Tyr Ala Pro Gly Thr Ser Phe His Tyr Asp Val 1 5 10 15 <210> 282 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 282 Ala Arg Asp Gln Gly Gly Thr Arg Gly Asn Tyr Phe Asp Val 1 5 10 <210> 283 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 283 Ala Arg Gly Gly Gly Gly Arg Phe Asp Val 1 5 10 <210> 284 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 284 Ala Arg Gly Gly Val Tyr Ser Phe Asp Val 1 5 10 <210> 285 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 285 Ala Arg Gly Leu Gly Thr Tyr Ser Pro Ser Leu Tyr Pro Arg Gly Met 1 5 10 15 Asp Val <210> 286 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 286 Ala Arg Gly Arg Ala Tyr Leu Ser Val Arg Gly Ser Phe Asp Val 1 5 10 15 <210> 287 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 287 Ala Arg Gly Gly Ser Gly Ser Phe Asp Val 1 5 10 <210> 288 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 288 Ala Arg Ser Arg Gly Tyr Thr Val Ser Ser Leu Ala Gly Arg Tyr Phe 1 5 10 15 Asp Gln <210> 289 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 289 Ala Arg Gly Leu Gly Leu Tyr Phe Asp Val 1 5 10 <210> 290 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 290 Ala Arg Val Arg Gly Gly Tyr Gly Pro Tyr Gly Asp Phe Asp Val 1 5 10 15 <210> 291 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 291 Ala Arg Gly Arg Ser Tyr Ile Val Ser Val Ser Pro Gly Phe Asp Val 1 5 10 15 <210> 292 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 292 Ala Arg Asp Ser Gly Ile Ala Ser Gly Tyr Thr Ala Tyr Met Asp Tyr 1 5 10 15 <210> 293 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 293 Ala Arg Gly Ala Gly Ser Thr Phe Asp Val 1 5 10 <210> 294 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 294 Ala Arg Gly Glu Ser Ala Tyr Tyr Ser Arg Asn Tyr Asp Val 1 5 10 <210> 295 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 295 Ala Arg Gly Gly Gly Tyr Tyr Pro Ala Gly Val Gly Arg Tyr Asp Val 1 5 10 15 <210> 296 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 296 Ala Arg Gly Pro Thr Leu Tyr Ser Pro Pro Val Phe Asp Val 1 5 10 <210> 297 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 297 Ala Arg Gly Ala Gly Val Ser Ala Gly Pro Ser Trp Pro Phe Asp Val 1 5 10 15 <210> 298 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 298 Ala Arg Ser Arg Gly Tyr Asn Val Ala Ala Ser Phe Gly Phe Asp Val 1 5 10 15 <210> 299 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 299 Ala Arg Gly Thr Asp Tyr Ser Gly Tyr Arg Gly Phe Asp Val 1 5 10 <210> 300 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 300 Ala Arg Gly Gly Gly Val Phe Asp Val 1 5 <210> 301 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 301 Ala Arg Glu Gly Gly Tyr Ser Pro Gly Gly Val Asp Phe Asp Tyr 1 5 10 15 <210> 302 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 302 Ala Arg Ser Pro Ala Tyr Tyr Phe Gly Pro Asn Met Asp Val 1 5 10 <210> 303 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 303 Ala Arg Gly Pro Gly Tyr His Pro Ala Gly Ala Ser Gly Gln Phe Phe 1 5 10 15 Asp Leu <210> 304 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 304 Ala Arg Gly Arg Gly Tyr Ala Pro Asp Ala Leu Thr Asn Phe Asp Val 1 5 10 15 <210> 305 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 305 Ala Arg Gly Tyr Ser Tyr Tyr Pro Gly Gly Gly Gly Gly Arg Asn Phe 1 5 10 15 Asp Tyr <210> 306 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 306 Ala Arg Ser Gly Gly Tyr Tyr Asp Tyr Gly Val Gly Tyr Asp Gln 1 5 10 15 <210> 307 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 307 Ser Ser Asn Ile Gly Ser Asn Thr 1 5 <210> 308 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 308 Asn Ile Ala Thr Lys Ser 1 5 <210> 309 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 309 Asn Ile Ala Thr Lys Ser 1 5 <210> 310 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 310 Ile Asn Asn Val Gly Asp Gln Gly 1 5 <210> 311 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 311 Asn Ile Gly Ser Lys Ser 1 5 <210> 312 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 312 Ser Asn Asn Val Gly Asn Gln Gly 1 5 <210> 313 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 313 Thr Ser Asn Ile Gly Asn Asn Ala 1 5 <210> 314 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 314 Gly Ser Asn Val Gly Ser Asn Val 1 5 <210> 315 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 315 Ser Ser Asn Ile Gly Arg Asn Asp 1 5 <210> 316 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 316 Ser Ser Asn Ile Gly Ser Asn Thr 1 5 <210> 317 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 317 Ser Ser Asn Leu Gly Ser Asn Tyr 1 5 <210> 318 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 318 Glu Ser Leu Cys Ser Thr Cys 1 5 <210> 319 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 319 Thr Gly Ala Val Thr Ser Gly Tyr Tyr 1 5 <210> 320 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 320 Ser Ser Asn Ile Gly Ser His Ser 1 5 <210> 321 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 321 Ser Ser Asp Val Gly Gly Tyr Asn Tyr 1 5 <210> 322 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 322 Ser Leu Arg Thr Ser Tyr 1 5 <210> 323 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 323 Ser Ser Asn Ile Gly Ser Asn Thr 1 5 <210> 324 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 324 Ser Ser Asn Val Gly Asn Gln Gly 1 5 <210> 325 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 325 Gln Asn Val Leu Tyr Ser Ser Asn Asn Lys Asn Asn 1 5 10 <210> 326 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 326 Ser Gly Ser Val Ser Thr Thr Asn Tyr 1 5 <210> 327 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 327 Ser Asn Asn Val Gly Lys Gln Gly 1 5 <210> 328 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 328 Gln Tyr Ile Asp Arg Ser 1 5 <210> 329 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 329 Ser Gly Ser Val Ser Ser Phe Asn Tyr 1 5 <210> 330 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 330 Ser Ser Asn Ile Gly Asn Asn Ala 1 5 <210> 331 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 331 Ser Gly Ser Ile Ala Ser Thr Tyr 1 5 <210> 332 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 332 Ser Ser Asn Ile Gly Ala Gly Tyr Asp 1 5 <210> 333 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 333 Ser Asn Asn Val Gly Asn Gln Gly 1 5 <210> 334 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 334 Arg Ser Leu Phe Asp Ser Ser Asp Asn Lys Asn Tyr 1 5 10 <210> 335 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 335 Ser Ser Asn Ile Gly Ser Asn Thr 1 5 <210> 336 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 336 Arg Ser Asn Ile Gly Ser Asn Thr 1 5 <210> 337 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 337 Ser Asn Asn 1 <210> 338 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 338 His Asp Ser 1 <210> 339 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 339 Arg Asn Ser 1 <210> 340 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 340 Gly Ala Ser 1 <210> 341 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 341 Tyr Asp Ser 1 <210> 342 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 342 Arg Asn Asn 1 <210> 343 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 343 Ser Leu Asn 1 <210> 344 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 344 Arg Asn Asn 1 <210> 345 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 345 Gly Arg Asp 1 <210> 346 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 346 Ser Asn Asn 1 <210> 347 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 347 Arg Asn Ser 1 <210> 348 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 348 Gly Ala Thr 1 <210> 349 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 349 Ser Thr Ser 1 <210> 350 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 350 Gly Asn Ser 1 <210> 351 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 351 Glu Val Ser 1 <210> 352 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 352 Gln Ser Thr 1 <210> 353 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 353 Ser Asn Asn 1 <210> 354 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 354 Arg Asn Asp 1 <210> 355 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 355 Trp Ala Ser 1 <210> 356 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 356 Asn Thr Asn 1 <210> 357 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 357 Arg Asn Asn 1 <210> 358 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 358 Tyr Ala Ser 1 <210> 359 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 359 Asn Thr Asn 1 <210> 360 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 360 Tyr Asp Asp 1 <210> 361 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 361 Glu Asp His 1 <210> 362 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 362 Asp Asn Ser 1 <210> 363 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 363 Arg Asn Asn 1 <210> 364 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 364 Trp Ala Ser 1 <210> 365 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 365 Ser Asn Asn 1 <210> 366 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 366 Ser Asn Asn 1 <210> 367 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 367 Ala Ala Trp Asp Asp Ser Leu Asn Gly Pro Val 1 5 10 <210> 368 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 368 Ala Ala Trp Asp Asp Ser Leu Ser Gly Pro Trp Val 1 5 10 <210> 369 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 369 Ser Ala Trp Asp Ser Ser Leu Ser Asp Trp Val 1 5 10 <210> 370 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 370 Gln Gln Tyr Ser Ser Ser Pro Tyr Thr 1 5 <210> 371 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 371 Gln Leu Trp Asp His Thr Asn Ser His Val Val 1 5 10 <210> 372 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 372 Ser Ala Trp Asp Asn Thr Val Ser Gly Trp Val 1 5 10 <210> 373 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 373 Glu Ala Trp Asp Asp Ser Leu Ser Gly Pro Val 1 5 10 <210> 374 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 374 Ala Ala Trp Asp Asp Arg Leu Asn Gly Phe Val 1 5 10 <210> 375 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 375 Ala Ala Trp Asp Ala Ser Leu Met Ile Tyr Val 1 5 10 <210> 376 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 376 Ala Ala Trp Asp Asp Ser Leu Asn Gly Tyr Val 1 5 10 <210> 377 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 377 Ala Ala Trp Asp Asp Ser Leu Asn Gly Val Val 1 5 10 <210> 378 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 378 Gln Gln Tyr Gly Ser Ser Pro Gln Thr 1 5 <210> 379 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 379 Leu Leu Tyr Tyr Gly Gly Pro Trp Val 1 5 <210> 380 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 380 Ala Ala Trp Asp Asp Gly Leu Ser Gly Trp Val 1 5 10 <210> 381 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 381 Ala Ser Trp Asp Asp Ser Leu Asn Ala Tyr Val 1 5 10 <210> 382 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 382 Asn Ser Arg Gly Ser Gly Gly Asn Pro Tyr Val 1 5 10 <210> 383 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 383 Ala Ala Trp Asp Asp Ser Leu Asn Gly Arg Val 1 5 10 <210> 384 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 384 Ser Ala Trp Asp Asn Ser Leu Ser Ala Trp Val 1 5 10 <210> 385 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 385 Gln Gln Tyr Tyr Gly Lys Pro Phe Thr 1 5 <210> 386 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 386 Val Leu Tyr Met Gly Arg Gly Ile Tyr Val 1 5 10 <210> 387 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 387 Ser Ala Trp Asp Ser Ser Leu Ser Val Trp Val 1 5 10 <210> 388 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 388 His Gln Thr Ser Ser Leu Pro Trp Thr 1 5 <210> 389 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 389 Ala Leu Tyr Val Gly Gly Gly Ile Ser Val 1 5 10 <210> 390 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 390 Ala Ala Trp Asp Asp Ser Leu Ser Gly Pro Val 1 5 10 <210> 391 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 391 Gln Ser Phe Asp Ala Ser Thr Leu Val 1 5 <210> 392 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 392 Gln Ser Tyr Asp Ser Ser Leu Ser Val Val Val 1 5 10 <210> 393 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 393 Ser Ala Trp Asp Ser Ser Leu Ser Ala Trp Val 1 5 10 <210> 394 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 394 Gln Gln Tyr Phe Ser Ser Pro Pro Ile Phe Thr 1 5 10 <210> 395 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 395 Ala Ala Trp Asp Asp Ser Leu Lys Gly Arg Val 1 5 10 <210> 396 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 396 Gln Dear Tyr Asp Dear Dear Val Val 1 5 <210> 397 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polynucleotide <400> 397 caggttcaat tagtgcagtc tggtgctgaa gtgaaaaagc ccggctcaag tgttaaagta 60 agctgtaagg cgagcggtgg cccattcagc tcat...

Claims

1. A pharmaceutical composition for treating renal cell carcinoma in a subject, comprising a nucleic acid encoding a CAIX-specific chimeric antigen receptor (CAR), comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the nucleic acid further encodes a polypeptide located behind the intracellular signaling domain, and the polypeptide is an anti-PD-L1 scFv-Fc antibody, wherein the Fc of the scFv-Fc antibody is derived from IgG1 or IgG4. Pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, wherein the transmembrane domain further comprises a stalk region located between the extracellular domain and the transmembrane domain, or the transmembrane domain comprises a CD28 transmembrane domain.

3. The pharmaceutical composition according to claim 1 or 2, wherein the nucleic acid further encodes one or more additional costimulatory molecules located between the transmembrane domain and the intracellular signaling domain.

4. The pharmaceutical composition according to claim 3, wherein the co-stimulatory molecule is CD28, 4-1BB, ICOS, or OX40 co-stimulatory molecule.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the intracellular signaling domain comprises a CD3 zeta chain intracellular signaling domain.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the internal ribosome entry site (IRES) is located between a nucleic acid encoding an intracellular signaling domain and a nucleic acid encoding a polypeptide.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the extracellular domain is a Fab antibody or an scFv antibody.

8. A pharmaceutical composition for treating renal cell carcinoma in a subject, comprising genetically engineered cells that express and retain a CAIX-specific chimeric antigen receptor (CAR) on the cell surface membrane, the extracellular domain, the transmembrane domain, and the intracellular signaling domain, wherein the genetically engineered cells are further engineered to express and secrete a polypeptide that is an anti-PD-L1 scFv-Fc antibody, the Fc of the scFv-Fc antibody being derived from IgG1 or IgG4.

9. The pharmaceutical composition according to claim 8, wherein the transmembrane domain further comprises a stalk region located between the extracellular domain and the transmembrane domain, or the transmembrane domain comprises a CD28 transmembrane domain.

10. The pharmaceutical composition according to claim 8 or 9, further comprising one or more additional costimulatory molecules located between the transmembrane domain and the intracellular signaling domain of the CAR.

11. The pharmaceutical composition according to claim 10, wherein the co-stimulatory molecule is CD28, 4-1BB, ICOS, or OX40 co-stimulatory molecule.

12. The pharmaceutical composition according to any one of claims 8 to 11, wherein the intracellular signaling domain comprises a CD3 zeta chain intracellular signaling domain.

13. The pharmaceutical composition according to any one of claims 8 to 12, wherein the extracellular domain is a Fab antibody or an scFv antibody.

14. The pharmaceutical composition according to any one of claims 8 to 13, wherein the cells are T cells or NK cells.

15. T cells CD4 + CD8 + , or CD4 + Cells and CD8 cells + The pharmaceutical composition according to claim 14, which is a mixed group of the following.

16. The pharmaceutical composition according to any one of claims 8 to 15, wherein the extracellular domain is a CAIX-specific scFv antibody.

17. A pharmaceutical composition according to any one of claims 1 to 16, wherein the target is a mammal.

18. The pharmaceutical composition according to claim 17, wherein the subject is a human.

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