Chimeric antigen receptors and methods of use thereof

Abstract

The present invention relates to chimeric antigen receptors and methods of using the same. The present invention provides chimeric antigen receptors, cells expressing the same and methods of using the same to treat various diseases, such as cancer.

Description

[0001] This application is a divisional application of Chinese patent application 201580069023.0, "Chimeric antigen receptor and method of use thereof," filed on December 21, 2015.

[0002] Related applications This application claims priority and interest in U.S. Provisional Application No. 62 / 094,625, filed December 19, 2014, and U.S. Provisional Application No. 62 / 252,083, filed November 6, 2015, the contents of which are incorporated herein in their entirety.

[0003] References to sequence lists are incorporated The contents of a text file created on December 21, 2015, entitled “Synthetic Sequence List for Submission with DFCI-102_001WO_ST25” are incorporated herein by reference in their entirety. Technical Field

[0004] This invention generally relates to chimeric antigen receptor cells for treating cancer and other conditions, and methods of using said chimeric antigen receptor cells to treat cancer and other conditions.

[0005] Government interests This invention was made with government support under the authorization granted by [ ]. The government has certain rights to the invention. Background Technology

[0006] T lymphocytes recognize specific antigens through the interaction of the T cell receptor (TCR) with short peptides presented by major histocompatibility complex (MHC) class I or II molecules. For initial activation and clonal expansion, naive T cells rely on specialized antigen-presenting cells (APCs) that provide additional co-stimulatory signals. Without co-stimulation, TCR activation can lead to unresponsiveness and clonal dysfunction. To circumvent immunity, various methods have been developed to derive cytotoxic effector cells with transplantation recognition specificity. Chimeric antigen receptors (CARs) have been constructed, consisting of binding domains derived from natural ligands or antibodies specific to cell surface components of the TCR-associated CD3 complex. Upon antigen binding, these chimeric antigen receptors connect to endogenous signaling pathways within effector cells, generating activation signals similar to those initiated by the TCR complex. Since the first report of chimeric antigen receptors, this concept has been refined, and the molecular design of chimeric receptors has been optimized. With the aid of advances in recombinant antibody technology, chimeric antigen receptors targeting a variety of antigens on the surface of cancer cells and cells infected with human immunodeficiency virus (HIV) have been generated. Summary of the Invention

[0007] In various respects, the present invention provides a chimeric antigen receptor (CAR) having an intracellular signal transduction domain, a transmembrane domain and an extracellular domain.

[0008] In some respects, the transmembrane domain also includes a stem region located between the extracellular domain and the transmembrane domain. The transmembrane domain includes CD28. On the other hand, the CAR also includes one or more additional co-stimulatory molecules located between the transmembrane domain and the intracellular signaling domain. The co-stimulatory molecules are CD28, 4-1BB, ICOS, or OX40. The intracellular signaling domain is, for example, the CD3ζ chain. The extracellular domain is an antibody such as Fab or scFV. Preferably, the antibody is specific to BCMA, CA-9, CD138, CCR4, or influenza virus.

[0009] The present invention also includes a nucleic acid encoding the CAR of the present invention, which further includes a nucleic acid encoding a polypeptide located after an intracellular signal transduction domain. The polypeptide is an antibody, such as scFV. Preferably, the antibody is specific to CCR4, PD-1, PDL-1, PD-L2, CXCR4, or GITR. The present invention also provides a vector comprising the nucleic acid of the present invention and cells comprising said vector.

[0010] In another aspect, the present invention provides genetically engineered cells that express and load the chimeric antigen receptor of the present invention onto their cell surface membrane. The cells are T cells or NK cells. T cells are CD4+ cells. + or CD8 + The cell is CD4. + and CD8 cells + A mixed population. Cells are further engineered to express and secrete peptides, such as antibodies.

[0011] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although similar or equivalent methods and materials may be used in the practice of this invention, applicable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are expressly incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail. Furthermore, the materials, methods, and embodiments described herein are illustrative only and not restrictive.

[0012] Other features and advantages of the invention will be apparent from and contained therein in the following detailed description and claims. Attached Figure Description

[0013] Figure 1. ADCC of CAIX-specific antibodies. 1 μg / ml of CAIX-specific scFv-Fc microantibody was 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) microantibodies were used as negative controls. A, CAIX + sk-rc-09 cells; B, CAIX + sk-rc-52 cells; C, CAIX-sk-rc-59 cells.

[0014] Figure 2. Construction and expression of CAIX-specific CARs. A. Construction: The first-generation CAR, scFv-CD8-TCRζ (CD8CAR), consists of a specific anti-CAIX scFv coupled to a truncated human CD8α extracellular domain, hinge (H), transmembrane (TM), and intracellular region, and then coupled to the signal transduction domain of human TCRζ. The second-generation CAR, scFv-CD28-TCRζ (CD28CAR), contains an anti-CAIX scFv fused to the extracellular, TM, and intracellular signal transduction domains of human CD28 into TCRζ. Both anti-CAIX CARs were cloned into a bicistronic self-inactivating (SIN) lentiviral vector, and their expression was driven by an internal eF1-< promoter. The CAR control construct contained an unrelated anti-HIV CCR5-specific A8 scFv substitute. B. FACS analysis: The reporter gene ZsGreen was used to quantify primary T cell transduction efficiency using the lentiviral CAR constructs. In addition, anti-CAIX scFv CAR was stained with CAIX-Fc fusion protein, and C9-tag (TETSQVAPA) was stained with 1D4 antibody. Untransduced activated T cells, LAK was used only as an unstained cell control (i) or stained with secondary antibodies (ii. PE-anti-human IgG and iii. APC-anti-mouse IgG) as staining controls. C. Western blot: Indicates the molecular size of monomeric / dimeric structures of anti-CAIX (clone G36) CD28 and anti-CCR5 (clone A8) CD28 CARs, as well as endogenous TCRζ chains in untransduced T cells.

[0015] Figure 3. Effector functions of CAIX-specific CART. A. Cytokine secretion. Anti-CAIX CART, unrelated CART, or activated control T cells (LAK) were co-cultured overnight with renal cancer cell lines sk-rc-52 (CAIX+) and sk-rc-59 (CAIX-) for cytokine production. One representative of 2-3 results is shown. B. ELISPOT. G36 CART or control A8 CART cells were added to tumor cells overnight. IFN-γ or granzyme B secretory T cells were detected by ELISPOT. Similar results were obtained in 2-3 experiments. C. CAIX-specific CART cells, control A8 CART cells, or LAK cells with specific anti-tumor cytotoxicity were incubated in varying amounts of target tumor cells in a 4-hour cytotoxicity assay at the proportions shown. One of two experiments is shown. Clone 4-1 is an in vivo passaged subclone of sk-rc-52.

[0016] Figure 4. Clonal expansion of CAR-T cells after tumor exposure. A. Proliferation. CAR-transduced T cells or untransduced T cells (LAK) were seeded weekly with irradiated tumor cells (CAIX+sk-rc-52 and CAIX-sk-rc-59) at three different ratios of tumor to T cells, as shown. T cell counts were performed every 3–4 days, in triplicate from two separate wells. Similar results were obtained in both experiments. B. Clonal enrichment. In the tumor stimulation experiments, CAR-T and T cell subset expression in CAR-T and LAK cell cultures was detected by flow cytometry at one and two weeks. One result representing the two is shown.

[0017] Figure 5 The regression of human RCC xenografts was achieved through the establishment of CAR-T cells. Thymedetomized nude mice were subcutaneously inoculated with 7.5 × 10⁻⁶ cells on each side. 6 SK-RC-52 and 5×10 6 50 x 10sk-rc-59 RCC tumor cells. Six days after tumor implantation, mice were intravenously injected with 50 x 10-1 SK-RC-59 RCC tumor cells. 6 G36 CD28 CART cells, A8 CD28 CART cells (≥20% CAR+), LAK or PBS alone. Inject high-dose IL-2 (1×10⁻⁶) every 2-3 days. 5 Tumor size was measured with calipers every 2–3 days. Experiment 1, n = 7 and Experiment 2, n = 8. Tumor sizes for these two experiments are shown. In both experiments, +, p < 0.05; *, p < 0.01; **, p < 0.001, G36 Tandem treated mice versus control mice without T cells. Other statistical calculations are reported in the text.

[0018] Figure 6. In vivo antitumor activity of CAR+ T cells. ZsGreen expression in CART cells is shown in the top image. CART cells were pre-stained with Far Red dye, centrifuged, and examined by fluorescence microscopy (bottom image). B. In situ staining of G36CD28 CART cells in degenerating tumors. CART cells were intravenously injected into RCC-established mice, and tumor tissue was collected on days 1–3. Confocal microscopy was used to measure tumor cell apoptosis by TUNNEL assay with PE-Cy5 dye (shown in red). Transduced T cells are shown by ZsGreen. Cell nuclei were counterstained with DPAI. Two representative slides are shown, indicating tumor cell apoptosis at the tumor margin (top image) and inside the tumor bed (middle image), respectively. Magnified view (bottom image) shows CART cells interacting with multiple tumors, while several surrounding tumor cells are near death. C. Granulase B+ T cells and tumor necrosis. After treatment with CART cells, degenerating CAIX+ sk-rc-52 tumors were stained with granase B antibody (brown) and H&E. The higher magnification views (middle and bottom views of sections a and b above) 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 periphery (middle view) and inside the tumor (bottom view).

[0019] Figure 7 CAIX-sk-rc-52 tumors treated with control LAK cells show negative granzyme B staining (left) (top), and the corresponding histology is shown in H&E (right).

[0020] Figure 8 Low background staining of granzyme B in CAIX-sk-rc-59 tumors treated with G36 CD28z CART cells.

[0021] Figure 9 Low background staining of granzyme B in CAIX-sk-rc-59 tumors treated with LAK cells.

[0022] Figure 10 Positive control (left) of sk-rc-52 tumors stained with locally injected G36 CD28z CART cells and tumor morphology shown in H&E (right).

[0023] Figure 11Expression of αCAIX CAR and αPD-L1 scFv-Fc transiently transfected 293T cells. Transfection with lentiviral pHAGE-EF1αG36-C9tag-CD28-CD3zeta-IRES-anti-PDL1 scFv-Fc(IgG4)-WPRE or control pHAGE-EF1α-A716-C9tag-CD28-CD3zeta-IRES-ZsGreen-WPRE plasmid for 10 cells. 6 Two 293T cells were transfected. Cells and supernatant were harvested at 24 and 48 hours post-transfection. Left: Purified CAIX(ECD)-Fc-biotin and streptavidin-ABC were used for cell staining and flow cytometry analysis. Right: Total human IgG in the supernatant was quantified using a human IgG quantification kit.

[0024] Figure 12 (A) Total PBMCs from four donors were stimulated with 50 ng / ml SEB and treated with 10 μg / ml anti-PDL1(#42) sIgG4 or anti-influenza sIgG4. Recovery of IFNγ production (top) or TNFα production (bottom) was measured. (B) Parental anti-CCR4 antibody c1567IgG inhibited the chemotaxis of Tregs to CCL22. (C) CD4 + CD25 - T cells were labeled with CFSE and incubated with Tregs at a ratio of 10:1, then stimulated with anti-CCR4 mAb and control mAb in the presence of anti-CD3 / 28 co-stimulation. Cells were collected after 3 and 7 days and analyzed by flow cytometry. Fluorescent CD4+ cells were observed in T cells. + CD25 - The percentage of cells was calculated from T and counting beads. The proliferation percentage was normalized to CD4 count on day 0. + CD25 - T effector cells. Data shown were calculated from two independent experiments. Bars represent mean ± SD. (D) CD4 cells treated with mAb2-3 and control antibody. + CD25 - IFNγ ELISpot analysis of Teff cells. Data represent quantification from three independent experiments using blood from three independent donors. (E) Left, CFSE-labeled Teffs (5 x 10⁻⁶) 4 ) and unlabeled Treg (5×10 3CFSE-labeled Teffs were co-incubated with 20 μg / ml PHA in 96-well plates for 5 days. CFSE-labeled Teffs were collected, and CFSE intensity was analyzed by flow cytometry. Teff proliferation was only observed when the co-culture was incubated with anti-GITR mAb. IFNγ production in the same culture was further measured by MSD. CFSE-labeled Teffs (5 x 10⁻⁶) were... 4 ) and unlabeled Treg (5×10 3 CFSE-labeled Teffs were co-incubated with 20 μg / ml PHA in 96-well plates for 5 days. CFSE-labeled Teffs were collected, and CFSE intensity was analyzed by flow cytometry. After 5 days of incubation with PHA, Teffs proliferated, but not in Teff / Treg co-culture. Left, IFNγ production in the same culture was measured by MSD.

[0025] Figure 13 This is a diagram illustrating different CART configurations according to the present invention.

[0026] Figure 14 This is an illustration showing different CART configurations according to the present invention.

[0027] Figure 15 This is another illustration showing different CART configurations according to the present invention.

[0028] Figure 16 This is a series of illustrations and diagrams indicating a chimeric antigen receptor (CAR) construct for CD8+ T cell transduction. (A) Schematic diagram of a second-generation pHAGE lentiviral vector containing CD28 as a co-stimulatory domain. An anti-carbonic anhydrase IX (CAIX) or anti-B cell maturation antigen (BCMA) scFv (as a negative control) is inserted after the eIFα promoter to express the CAR-binding domain. The second cassette following 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 generate lentivirus, the pHAGE vector, along with packaging plasmids (Gag, Rev, Tat, and VSVG), was transfected into 293T LentiX cells using polyethyleneimine. Virus was collected two days after transfection using the LentiX Concentrator (Clontech) according to the manufacturer's instructions. TM(A) Purification and concentration were performed. LTR: Long terminal repeat, eIFα: eukaryotic initiation factor α, scFv: single-chain variable fragment, C9 TAG: C9 peptide TETSQVAPA, IRES: internal ribosome entry site, WPRE: post-transcriptional regulatory element of marmot hepatitis virus. (B) T cells were transduced with lentivirus to generate anti-CAIX CART cells capable of recognizing CAIX-positive RCCs and releasing anti-PD-L1 IgG1 or IgG4 in the tumor microenvironment to block PD-1 / PD-L1-induced T cell exhaustion. (C) Percentage of CART cells 14 days post-transduction, representing stable long-term CAR expression in CD8+ T cells integrated with lentivirus. Dynabeads were used. TM The CD8 positive isolation kit (Life Technologies) selects CD8+ T cells and uses Dynabeads in the presence of IL-21 50 U / mL. TM Human T cell activators CD3 / CD28 (Life Technologies) were used for activation. IL-21 was added to the culture medium every 2 days. After 14 days, CAR-T cells were incubated with human CAIX-Fc or BCMA-Fc, followed by incubation with APC-conjugated anti-human Fc IgG and analyzed by FACS. (D) IgG concentration secreted into transduced T cells as assessed by a human IgG ELISA kit (Bethyl Laboratories). (E) Anti-PD-L1 antibody concentration in the supernatant of 293T 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 agarose beads (GE Healthcare) and biotin-enzymes using EZ-Link Sulfo-NHS-LC (Thermo Scientific). TM Biotinylation was performed on these antibodies. These antibodies were then combined with pre-immobilized antibodies in 96-well MaxiSorp plates (Nunc...). TMIncubate with 5 μg / mL human PD-L1. Biotinylated antibodies were detected by incubation with streptoacidin-HRP for 1 h and TMB colorimetric assay. 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 expansion of CD8+ CART cells. (F) CART cell concentration versus days in the presence of skrc-52 CAIX+ / PD-L1 cells. * P < 0.05 compared to all CARS versus anti-BCMA / anti-SARS IgG1. (G) Percentage of transduced T cells versus days in the presence of CAIX-positive cells. * P < 0.05 compared to all CARS versus anti-BCMA / anti-SARS IgG1. After transfection with CD8+ CART cells expressing anti-PD-L1 IgG1 (anti-CAIX / anti-PD-L1 IgG1), IgG4 (anti-CAIX / anti-PD-L1 IgG4), or non-specific anti-SARS Ab (anti-CAIX / anti-SARS IgG1), or anti-BCMA CAR (negative control) / (anti-BCMA / anti-SARS IgG1) expressing non-specific anti-SARS Ab, CD8+ CART cells were pre-cultured for 5 days and administered Dynabeads in the presence of IL-21 50 U / mL. TM Human T activator CD3 / CD28 activation (Life Technologies). After removing the beads, CAR-T cells were cultured with skrc52CAIX + PD-L1 and IL-21 (50 U / mL), with skrc52CAIX + PD-L1 and IL-21 (50 U / mL) added to the culture medium every 2 days for 21 days. Results are expressed as mean ± SD from three donors, in duplicate.

[0029] Figure 17 This is a series of diagrams illustrating the effector function of CART cells. (A) Skrc59 CAIX+ / PD-L1+ cells incubated overnight (ON) with CART cells anti-CAIX / anti-PD-L1 IgG1, anti-CAIX / PD-L1 IgG4, anti-CAIX / anti-SARS IgG1, or anti-BCMA / anti-SARS IgG1, or (B) Skrc52 CAIX- / PD-L1- cells. These CART cells were used 4 days after lentiviral transduction. Viability was measured by MTT (Molecular Probes) assay. TMAssess cell viability. P < 0.05. All CAR-T cells compared to anti-BCMA / anti-SARS IgG1 (C) IL-2 released by CAR-T cells after overnight contact with Skrc59 CAIX+ / PD-L1+ cells or (D) Skrc52 CAIX- / PD-L1 cells. Human IL-2 ELISA Ready-SET-Go kit (eBioscience) was used. TM Assess IL-2 secretion. P < 0.001. All CART cells compared with 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. Human IFNγ ELISA Ready-SET-Go Kit (eBiosciences) was used. TM IFNγ secretion was evaluated. P < 0.05. All CART cells were compared with anti-BCMA / anti-SARS IgG1. (G) After incubation with supernatant (SN) containing 500 ng / mL anti-PD-L1 IgG1, anti-PD-L1 IgG4, or anti-SARS IgG1, Ab-dependent cell-mediated cytotoxicity was observed in skrc59 CAIX+ / PD-L1+ or (H)skrc52 CAIX- / PD-L1-. To obtain antibodies, Dynabeads were used in the presence of IL-21 50 U / mL. TM CAR-T cells were incubated for 6 days with the human T activator CD3 / CD28 (Life Technologies). After 7 days, the culture medium containing mAb was collected and processed using EasySep. TM NK cells were purified using a 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 cultured in 96-well plates at 1.5 × 10⁻⁶ cells / well. 3 / well plated. RCC cells were incubated at 37°C for 1 hour, and then 500 ng / mL of the corresponding anti-PD-L1 IgG1, anti-PD-L1 IgG4, or anti-SARS IgG1 were added to 50 μL of CART cell supernatant. After incubation, the cells were washed with culture medium and incubated with NK cells at 12.5:1, 25:1, or 50:1 ratios at 37°C for 4 hours. The culture supernatant was harvested by centrifugation and analyzed using the CytoTox 96 non-radioactive cytotoxicity assay (Promega). TM LDH was measured in the supernatant at 490 nm. These results are expressed as mean ± SD from the three donors, in duplicate.

[0030] Figure 18 This is a series of graphs depicting the expression of depleted markers in CAR-T 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. Using Dynabeads TM CD8 Positive Isolation Kit (Life Technologies) selects CD8+ CART cells using Dynabeads. TM Human T activators CD3 / CD28 (Life Technologies) were activated and transduced with the following CARs: anti-CAIX / anti-PD-L1 IgG1, 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 activators CD3 / CD28. Subsequently, CAR cells were co-cultured with Skrc-59 CAIX+ PD-L1+ for 2 days to stimulate depletion. CAR 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 depleted CAR 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 mean ± SD for all three donors.

[0031] Figure 19 This is a series of images and graphics depicting the role of CAR-T cells in an in situ model of human RCC. (A) Injection of 5.0 × 10⁻⁶ cells into NSG mice (N = 35). 4 SKRC-59 CAIX-positive, PD-L1-positive, and luciferase-positive RCC cells were collected. One week later, mice were injected with 1.0 × 10⁻⁶ cells. 7One CAR or untransduced T cell IV (day 0). CAR cells were pre-transduced with 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 / PD-L1 IgG4 (N = 6 mice per group). Tumor bioluminescence was quantified using IVIS 5 minutes after fluorescein IP injection. A. Tumor imaging before CAR injection (day 0) and at days 7, 14, 23, and 30 after the first CAR cell injection. A second injection of 2.5 × 10⁻⁶ cells was performed on day 17. 6 (A) Tumor imaging after resection on day 30. Scale bar = 1 cm. (B) Tumor growth curve. P < 0.05 when comparing the anti-PD-L1 IgG1 and IgG4 groups with anti-BCMA / anti-SARS IgG1, and ** P < 0.05 when comparing the anti-PD-L1 IgG1 and IgG4 groups with anti-CAIX / Anti-SARS IgG1. (D) Tumor weight after 30 days of treatment. *P < 0.05 compared with anti-BCMA / anti-SARS IgG1 CAR, ** P < 0.05 compared with anti-CAIX / anti-SARS IgG1.

[0032] Figure 20This is a series of diagrams and histological images depicting the antitumor activity from CART cells. (A) Expression of depletion markers in tumor-infiltrating lymphocytes (TILs). All mouse kidney tumors were divided into two parts, one of which was fragmented into small pieces and digested with collagenase and DNase to extract TILs. The depletion markers of CART cells, PD-1, Tim-3, and Lag3, were analyzed. * P < 0.05, compared with untransduced anti-BCMA / anti-SARS IgG1 CAR and anti-CAIX / anti-SARS IgG1. (B) Detection of Ki67 and granzyme B as markers of tumor cell proliferation to analyze CART cell activity in tissues. Four-micron sections of formalin-fixed paraffin-embedded tissues were dewaxed and rehydrated in a decreasing series of ethanol. Endogenous peroxidase activity was quenched using 3% hydrogen peroxide. Antigen retrieval was performed for 45 seconds at 123°C and 15 PSI in citrate buffer (pH = 6.0) using a pressure cooker. Tissue sections were incubated for 45 minutes with rabbit anti-human Ki67 polyclonal antibody 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 (produced in our lab), rabbit anti-human granzyme B polyclonal antibody (Abcam, ab4059) 1:100, or rabbit anti-human NCAM (CD56) mAb 1:100 (Abcam, ab133345), followed by incubation with second HRP-conjugated anti-rabbit Ab or HRP-antibiotin protein. Slides were developed using 3,3'-diaminobenzidine (DAB) and counterstained with hematoxylin. Images were acquired using a DP71 digital camera (Olympus) on an Olympus BX51 microscope and displayed on the DP controller software (Olympus). TM The analysis was performed in ( ). The scale bar indicates the magnification of the image in each column [500 μm (40X), 100 μm (200X), or 50 μm (400X)]. (C) A series of graphs show the percentage of TIL staining positive for granzyme B, PD-L1-IHC, and Ki67. (C) The number of Ki67-DAB pixels for TIL is also shown.

[0033] Figure 21This is a series of graphs depicting the effects of IL-2 and IL-21 on the proliferation of CD8+ CART cells. A and B. CART transduced cells in the presence of IL-2 or IL-21 were assessed at 48 h, 72 h, and 120 h after lentiviral transduction. (A) Anti-CAIX CART cells or (B) nonspecific anti-BCMA CART cells (both using ZsGreen from kit 2). Dynabeads were used. TM CD8 positive isolation kit (Life Technologies) selects CD8+ T cells and inoculates them with IL-2 or IL-21 50 U / mL (Peprotech). TM Using Dynabeads in the presence of ) TM Human T cell activators CD3 / CD28 (Life Technologies) were used for activation. CAR-T cell transduction was assessed by ZsGreen expression using FACS. Data represent mean ± SD from both donors. * p < 0.05 compared IL-21 with the untreated control (Ctr); ** p < 0.05 compared IL21 with IL-2. C and D. Viability of RCC cells treated with CD8+ CAR-T cells cultured in the presence of IL-2 or IL-21. Viability of CAR-T cells treated with anti-BCMA, anti-CAIX, or untransduced T cells was assessed by MTT assay after overnight incubation with (C) Skrc-59 CAIX+ / PD-L1+ and (F) Skrc-52 CAIX- / PD-L1-RCC cells. CAR-T cells were pre-cultured for 120 h in the presence of IL2 or IL-21 50 U / mL. These results represent mean ± SD from both donors, in triplicate. * p < 0.05 compared anti-CAIX CAR with anti-BCMA CAR or untransduced T cells.

[0034] Figure 22 This is a series of flow cytometry images depicting the expression of PD-1 and CAIX in renal cell carcinoma (RCC) lines. (A) Negative control, (B) Skrc52 CAIX-PD-L1-, (C) Skrc52 CAIX+PD-L1-, (D) Skrc59 CAIX+PD-L1+. Cells were stained using an anti-human CAIX antibody generated with APC-anti-human Fc IgG and a biotinylated anti-human PD-L1 antibody generated with PE-anti-biotin. Analysis was performed by FACS.

[0035] Figure 23This is a series of diagrams depicting CAR-T cell characterization. (A) Proliferation of anti-CD8+ T cells 2 or 4 days after transduction with 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. (Using Dynabeads) TM The CD8 positive isolation kit (Life Technologies) selects CD8+ T cells and uses Dynabeads in the presence of IL-21 50 U / mL. TM Human T cell activators CD3 / CD28 (Life Technologies) were used for activation. IL-21 was added to the culture medium every 2 days. Proliferation was assessed by FACS using molecular probes. (B) T cell concentrations transduced by CAR at 2 and 4 days post-transduction. CAR cells were incubated with human CAIX-Fc or BCMA-Fc, followed by incubation with APC-conjugated anti-human Fc IgG and analyzed by FACS. (C) Percentage of CAR cells at 2 and 4 days post-transduction. Results are expressed as mean ± SD from three donors, in duplicate.

[0036] Figure 24 This is a series of graphs depicting the role of CAR-T cells in an orthotopic model of human RCC. (A) Comparison of tumor size / days by bioluminescence detection in the CAR-T cell group. NSG mice (N = 35) were injected with 5.0 x 10⁻⁶ cells. 4 The renal capsule of skrc-59 CAIX+, PD-L1+, and luciferase+ RCC cells was observed. One week later, mice were injected with 1.0 × 10⁻⁶ cells. 7 CART or untransduced T-cell IVF. CART cells were pre-transduced with 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 / PD-L1 IgG4 (N = 6 mice per group). Tumor bioluminescence was quantified using IVIS 5 minutes after fluorescein IP injection. On day 17, a second injection of 2.5 × 10⁻⁶ cells was administered. 6CAR cells. * P<0.05, compared with anti-BCMA CAR cells. ** P<0.05, compared with untransduced T cells. *** P<0.05, compared with anti-CAIX / anti-SARS IgG1. (B) Percentage of T cells in mouse blood 8 days after treatment. * P<0.05, compared with untransduced T cells. ** P<0.05, compared with all anti-CAIX CARs. Red blood cells were lysed with ACK lysis buffer (Lonza TM (c) Total tumor-infiltrating lymphocytes (TILs) after 30 days of CAR-T cell treatment. All mouse kidney tumors were divided into two parts, one of which was fragmented into smaller pieces and digested with collagenase and DNase to extract TILs. Cells were stained with Pacific Blue-conjugated anti-human CD45 and analyzed by FACS.

[0037] Figure 25 A and 25B are a series of figures and histological images depicting human natural killer (NK) cells in tumors treated with anti-CAIXCART cells releasing anti-PD-L1 IgG1Ab. (A) Percentage of CD56+ cells (NK marker) in the tumor. Two mice in each group were injected with 4.5 x 10⁻⁶ cells the day before euthanasia. 6NK cells. All mouse kidney tumors were divided into two parts, one of which was fragmented into small pieces and digested with collagenase and DNase to extract NK cells. NK cells present in the tumor were stained with APC-anti-CD56 Ab and analyzed by FACS. * P < 0.05. (B) CD56+ cells in excised tumors were detected by IHC and quantified using the IHC Profiler Plugin in ImageJ software. Four-micron sections of formalin-fixed paraffin-embedded tissue were dewaxed and rehydrated in a decreasing series of ethanol. Endogenous peroxidase activity was quenched using 3% hydrogen peroxide. Antigen retrieval was performed for 45 seconds at 123°C and 15 PSI in citrate buffer (pH = 6.0) using a pressure cooker. Tissue sections were incubated with rabbit anti-human CD56 mAb 1:100 (Abcam, ab133345) for 45 minutes, followed by secondary HRP-conjugated anti-rabbit or anti-mouse Ab. Slides were developed using 3,3'-diaminobenzidine (DAB) and counterstained with hematoxylin. Images were acquired using a DP71 digital camera (Olympus) on an Olympus BX51 microscope and analyzed using DP controller software (Olympus). Quantification was performed using the IHC Profiler Plugin (23) of ImageJ software. Scale bars indicate the magnification of the images (400X). * P < 0.05, compared to untransduced, ** P < 0.05, compared to untransduced and anti-BCMA / anti-SARS IgG1. Detailed Implementation

[0038] This invention relates to chimeric antigen receptors (CARs) for immune cells that are particularly suitable for use in immunotherapy.

[0039] In one implementation, a dual immunotherapy strategy is described based on blocking T cell depletion using an anti-PD-L1 antibody targeting anti-CAIX CAR T cell secretion, which is combined in a single lentiviral construct for improving cancer treatment.

[0040] A newly emerging mechanism associated with the progression of RCC and other tumors is the immune checkpoint pathway, which involves preventing overactive T cell interactions under normal conditions, thereby allowing T cell function in a self-limiting manner. As an evasion mechanism, many tumors can stimulate the expression of immune checkpoint molecules, leading to an incompetent phenotype of T cells that cannot suppress tumor progression. Emerging clinical data highlight the importance of an inhibitory ligand and receptor for immune checkpoints: programmed death-ligand 1 (PD-L1; B7-H1 and CD274) and programmed death receptor-1 (PD-1; CD279) prevent cytotoxic T lymphocytes from killing cancer cells. The PD1 receptor is expressed by many cell types, such as T cells, B cells, natural killer (NK) cells, and host tissues. Tumors and antigen-presenting cells (APCs) expressing PD-L1 can block T cell receptor (TCR) signaling in cytotoxic T lymphocytes by binding to the PD-1 receptor, reducing cytokine production and T cell proliferation. PD-L1 overexpression can be found in many tumor types and can also mediate immunosuppressive function by interacting with other proteins, including CD80 (B7.1) and blocking their ability to activate T cells by binding to CD28.

[0041] Genetically engineering human lymphocytes to express tumor-directed chimeric antigen receptors (CARs) can generate anti-tumor effector cells that bypass tumor immune escape mechanisms due to aberrations in protein-antigen processing and presentation. Furthermore, these transgenic receptors can target tumor-associated antigens that are not protein-derived. In some embodiments of the invention, there are lymphocytes (CARTS) modified to contain at least one CAR, and in specific embodiments of the invention, a single CAR targets two or more antigens. In a preferred embodiment, CARTS are further modified to express and secrete one or more peptides, such as antibodies or cytokines. Such CARTS are referred to herein as assembled CARTS. Assembled CARTS allow for the simultaneous local secretion of peptides at the target site (i.e., the tumor site).

[0042] A modified TCR called a chimeric antigen receptor (CAR), containing a single-chain variable antibody fragment (scFv) previously selected by high affinity for specific tumor-associated antigens, represents a powerful new approach to cancer treatment. The scFv presented in the CAR is linked to an intracellular signaling unit including CD3ζ to induce T cell activation, followed by antigen binding. This structure is characteristic of first-generation CARs, which have been modified in second-generation CARs by linking co-stimulatory intracellular domains of CD28, 4-1BB, or OX40 to CD3, or in third-generation CARs by linking the two elements in tandem to CD3. These intracellular domains are required for complete T cell activation during TCR recognition by antigen-presenting cells (APCs), improving cytokine production and proliferation of CAR-T cells. Due to the difficulty in finding unique tumor-associated antigens, ineffective T cell homing to tumor sites, low persistence of T cells in vivo, and the immunosuppressive microenvironment of solid tumors, the role of CAR-T cells has so far been suitable for treating solid tumors.

[0043] In certain contexts, lymphocytes include chimeric, non-natural, and at least partially engineered receptors. In specific contexts, engineered chimeric antigen receptors (CARs) have one, two, three, four, or more components, and in some embodiments, one or more components facilitate the targeting or binding of lymphocytes to one or more cancer cells containing tumor antigens.

[0044] The CAR according to the invention typically comprises at least one transmembrane polypeptide containing at least one extracellular ligand-binding domain, and a transmembrane polypeptide containing at least one intracellular signal transduction domain; such that the polypeptides assemble together to form a chimeric antigen receptor.

[0045] As used herein, the term "extracellular ligand-binding domain" is defined as an oligopeptide or polypeptide capable of binding ligands. Preferably, this domain will be able to interact with cell surface molecules. For example, an extracellular ligand-binding domain may be selected to recognize ligands that serve as cell surface markers on target cells associated with specific disease states.

[0046] In particular, the extracellular ligand-binding domain may include an antigen-binding domain derived from an antibody against the target antigen.

[0047] As a non-limiting example, the target antigen may be a tumor-associated surface antigen, 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, disialotetrahexosylganglioside GD2, ductal epithelial mucin, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, muthsp70-2, M-CSF, prostaglandins, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, prostein, PSMA, survival and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin-like growth factor (IGF1)-I, IGF-II, IGFI receptor, mesothelin, major histocompatibility complex (MHC) molecules that present tumor-specific peptide epitopes, 5T4, ROR1, Nkp30, NKG2D, tumor matrix antigen, extra domain A (EDA) and outer domain B (EDB) of fibronectin, and tendinin C (TnC) A1 domains of A1 and fibroblast-associated proteins (fap); lineage-specific or tissue-specific antigens such as CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, CTLA-4, B7-1 (CD80), B7-2 (CD86), endothelial glycoprotein, a histocompatibility complex (MHC) molecule, BCMA (CD269, TNFRSF17) or virus-specific surface antigens such as HIV-specific antigens (e.g., HIV gp120); EBV-specific antigens, CMV-specific antigens, HPV-specific antigens, Lasse virus-specific antigens, influenza virus-specific antigens, and any derivatives or variants of these surface markers.

[0048] 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).

[0049] In a preferred embodiment, the extracellular ligand-binding domain is a light chain (V) comprising a target antigen-specific monoclonal antibody linked via a flexible linker.L ) and heavy chain (V H ) Variable fragment single-chain antibody fragments (scFv).

[0050] 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.

[0051] Exemplary antibodies that can be used to construct CARs according to the present invention include, for example, 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 contents of which are incorporated herein by reference in their entirety.

[0052] PDL1(68) Exemplary anti-PDL1 antibodies include antibodies containing the following VH and VL nucleotide sequences: having a VH nucleotide sequence of SEQ ID NO:1485 and a VL nucleotide sequence of SEQ ID NO:1487; having a VH nucleotide sequence of SEQ ID NO:1485 and a VL nucleotide sequence of SEQ ID NO:1487; having a VH nucleotide sequence of SEQ ID NO:1489 and a VL nucleotide sequence of SEQ ID NO:1491; having a VH nucleotide sequence of SEQ ID NO:1493 and a VL nucleotide sequence of SEQ ID NO:1495; having a VH nucleotide sequence of SEQ ID NO:1497 and a VL nucleotide sequence of SEQ ID NO:1499; having a VH nucleotide sequence of SEQ ID NO:1501 and a VL nucleotide sequence of SEQ ID NO:1503; having a VH nucleotide sequence of SEQ ID NO:1505 and a VL nucleotide sequence of SEQ ID NO:1507; having a VH nucleotide sequence of SEQ ID NO:1501; having a VL nucleotide sequence of SEQ ID NO:1503; having a VH nucleotide sequence of SEQ ID NO:1505 and a VL nucleotide sequence of SEQ ID NO:1507; having a VH nucleotide sequence of SEQ ID NO:1485; having a VL nucleotide sequence of SEQ ID NO:1487; having a VH nucleotide sequence of SEQ ID NO:1489; having a VH nucleotide sequence of SEQ ID NO:1489; having a VL nucleotide sequence of SEQ ID NO:1481; having a VH nucleotide sequence of SEQ ID NO:1485; having a VL nucleotide sequence of SEQ ID NO:1487; having a VH nucleotide sequence of SEQ ID NO:1489; having a VL nucleotide sequence of SEQ ID NO:1489; having a VH nucleotide sequence of The VH nucleotide sequence with SEQ ID NO:1509 and the VL nucleotide sequence with SEQ ID NO:1511; the VH nucleotide sequence with SEQ ID NO:1513 and the VL nucleotide sequence with SEQ ID NO:1515; the VH nucleotide sequence with SEQ ID NO:1517 and the VL nucleotide sequence with SEQ ID NO:1519; the VH nucleotide sequence with SEQ ID NO:1521 and the VL nucleotide sequence with SEQ ID NO:1523; the VH nucleotide sequence with SEQ ID NO:1525 and the VL nucleotide sequence with SEQ ID NO:1527; the VH nucleotide sequence with SEQ ID NO:1529 and the VL nucleotide sequence with SEQ ID NO:1531; the VH nucleotide sequence with SEQ ID NO:1533 and the VL nucleotide sequence with SEQ ID NO:1535; the VH nucleotide sequence with SEQ ID NO:1537 and the VL nucleotide sequence with SEQ ID NO:1539.

[0053] Exemplary anti-PDL1 antibodies include antibodies containing the following VH amino acid sequences and VL amino acid sequences: having a VH amino acid sequence of SEQ ID NO: 970 and a VL amino acid sequence of SEQ ID NO: 971; having a VH amino acid of SEQ ID NO: 1486 and a VL polypeptide sequence of SEQ ID NO: 1488; having a VH amino acid of SEQ ID NO: 1490 and a VL polypeptide sequence of SEQ ID NO: 1492; having a VH amino acid of SEQ ID NO: 1494 and a VL polypeptide sequence of SEQ ID NO: 1496; having a VH amino acid of SEQ ID NO: 1498 and a VL polypeptide sequence of SEQ ID NO: 1500; having a VH amino acid of SEQ ID NO: 1502 and a VL polypeptide sequence of SEQ ID NO: 1504; having a VH amino acid of SEQ ID NO: 1506 and a VL polypeptide sequence of SEQ ID NO: 1508; having a VH amino acid of SEQ ID NO: 1510 and a VL polypeptide sequence of SEQ ID NO: 971; having a VH amino acid of SEQ ID NO: 970 and a VL polypeptide sequence of SEQ ID NO: 971; having a VH amino acid of SEQ ID NO: 1486 and a VL polypeptide sequence of SEQ ID NO: 1488; having a VH amino acid of SEQ ID NO: 1490 and a VL polypeptide sequence of SEQ ID NO: 1496; having a VH amino acid of SEQ ID NO: 1498 and a VL polypeptide sequence of SEQ ID NO: 1500; having a VH amino acid of SEQ ID NO: 1502 and a VL polypeptide sequence of SEQ ID NO: 1504; having a VH amino acid of SEQ ID NO: 1506 and a VL polypeptide sequence of SEQ ID NO: 15 The VL polypeptide sequence NO:1512, VH amino acid of SEQ ID NO:1514, VL polypeptide sequence of SEQ ID NO:1516, VH amino acid of SEQ ID NO:1518, VL polypeptide sequence of SEQ ID NO:1520, VH amino acid of SEQ ID NO:1522, VL polypeptide sequence of SEQ ID NO:1524, VH amino acid of SEQ ID NO:1526, VL polypeptide sequence of SEQ ID NO:1528, VH amino acid of SEQ ID NO:1530, VL polypeptide sequence of SEQ ID NO:1532, VH amino acid of SEQ ID NO:1534, VL polypeptide sequence of SEQ ID NO:1536, VH amino acid of SEQ ID NO:1538, and VL polypeptide sequence of SEQ ID NO:1540.

[0054] In other embodiments, the anti-PDL1 antibody has a heavy chain and a light chain, the heavy chain having three CDRs and the light chain having three CDRs: the CDRs of the heavy chain respectively contain amino acid sequences SEQ ID NO: 1541, 1554, 1569 and the CDRs of the light chain respectively contain amino acid sequences 1584, 1599, 1610; or the CDRs of the heavy chain contain amino acid sequences 1543, 1556, 1571 and the CDRs of the light chain contain amino acid sequences 1586, 1616, 1612; or the CDRs of the heavy chain contain amino acid sequences 1544, 1557, 1572 and the CDRs of the light chain contain amino acid sequences 1587, 1601, 1613; or the CDRs of the heavy chain contain amino acid sequences 1545, 1558, 1573 and the CDRs of the light chain respectively contain amino acid sequences SEQ ID NO: 1541, 1554, 1569 and SEQ ID NO: 1584, 1558, 1573 and SEQ ID NO: 1584, 1558, 1599 and SEQ ID NO: 1584, 1559 ... R contains amino acid sequences 1588, 1602, 1614; or the CDR of the heavy chain contains amino acid sequences 1546, 1559, 1574 and the CDR of the light chain contains amino acid sequences 1589, 1603, 1615; or the CDR of the heavy chain contains amino acid sequences 1547, 1560, 1575 and the CDR of the light chain contains amino acid sequences 1590, 1604, 1616; or the CDR of the heavy chain contains amino acid sequences 1548, 1561, 1576 and the CDR of the light chain contains amino acid sequences 1591, 1605, 1617; or the heavy chain The CDR of the heavy chain contains amino acid sequences 1541, 1562, 1577 and the CDR of the light chain contains amino acid sequences 1592, 1599, 1618; or the CDR of the heavy chain contains amino acid sequences 1549, 1563, 1578 and the CDR of the light chain contains amino acid sequences 1593, 1606, 1619; or the CDR of the heavy chain contains amino acid sequences 1550, 1564, 1579 and the CDR of the light chain contains amino acid sequences 1594, 1607, 1620; or the CDR of the heavy chain contains amino acid sequences 1551, 1565, 1580 and the CDR of the light ...92, 1599, 1618. The CDR of the light chain contains amino acid sequences 1595, 1599, and 1621; or the CDR of the heavy chain contains amino acid sequences 1542, 1566, and 1581 and the CDR of the light chain contains amino acid sequences 1596, 1599, and 1622; or the CDR of the heavy chain contains amino acid sequences 1552, 1567, and 1582 and the CDR of the light chain contains amino acid sequences 1597, 1608, and 1623; or the CDR of the heavy chain contains amino acid sequences 1553, 1568, and 1583 and the CDR of the light chain contains amino acid sequences 1598, 1609, and 1624.

[0055] SARS(26) Exemplary SARS neutralizing antibodies include antibodies containing the following VH and VL nucleotide sequences: having the VH nucleotide sequence of SEQ ID NO:1626 and the VL nucleotide sequence of SEQ ID NO:1628; having the VH nucleotide sequence of SEQ ID NO:1630 and the VL nucleotide sequence of SEQ ID NO:1639; having the VH nucleotide sequence of SEQ ID NO:1634 and the VL nucleotide sequence of SEQ ID NO:1640; having the VH nucleotide sequence of SEQ ID NO:1632 and the VL nucleotide sequence of SEQ ID NO:1641; having the VH nucleotide sequence of SEQ ID NO:1633 and the VL nucleotide sequence of SEQ ID NO:1642; having the VH nucleotide sequence of SEQ ID NO:1634 and the VL nucleotide sequence of SEQ ID NO:1643; having the VH nucleotide sequence of SEQ ID NO:1635 and the VL nucleotide sequence of SEQ ID NO:1644; having the VH nucleotide sequence of SEQ ID NO:1635 and the VL nucleotide sequence of SEQ ID NO:1644; having the VH nucleotide sequence of SEQ ID NO:1636 ... The VH nucleotide sequence with SEQ ID NO:1636 and the VL nucleotide sequence with SEQ ID NO:1645; the VH nucleotide sequence with SEQ ID NO:1637 and the VL nucleotide sequence with SEQ ID NO:1646.

[0056] CXCR4(33) Exemplary anti-CXCR4 antibodies include antibodies having the following VH and VL amino acid sequences: having the VH amino acid sequence of SEQ ID NO:771 and the VL amino acid sequence of SEQ ID NO:779; having the VH amino acid sequence of SEQ ID NO:772 and the VL amino acid sequence of SEQ ID NO:780; having the VH amino acid sequence of SEQ ID NO:773 and the VL amino acid sequence of SEQ ID NO:781; having the VH amino acid sequence of SEQ ID NO:774 and the VL amino acid sequence of SEQ ID NO:782; having the VH amino acid sequence of SEQ ID NO:775 and the VL amino acid sequence of SEQ ID NO:783; having the VH amino acid sequence of SEQ ID NO:776 and the VL amino acid sequence of SEQ ID NO:784; having the VH amino acid sequence of SEQ ID NO:777 and the VL amino acid sequence of SEQ ID NO:785; or having the VH amino acid sequence of SEQ ID NO:778 and the VL amino acid sequence of SEQ ID NO:784. The VL amino acid sequence of NO:786.

[0057] In other embodiments, the anti-CXCR4 antibody has a heavy chain and a light chain, wherein the heavy chain has three CDRs and the light chain has three CDRs: the CDRs of the heavy chain respectively contain amino acid sequences SEQ ID NO: 803, 804, 805 and the CDRs of the light chain respectively contain amino acid sequences 806, 807, 808; or the CDRs of the heavy chain respectively contain amino acid sequences 809, 810, 811 and the CDRs of the light chain respectively contain amino acid sequences 812, 813, 814; or the CDRs of the heavy chain respectively contain amino acid sequences 815, 816, 817 and the CDRs of the light chain respectively contain amino acid sequences 818, 819, 820; or the CDRs of the heavy chain... The CDRs of the light chain contain amino acid sequences 827, 828, and 829, respectively, and the CDRs of the light chain contain amino acid sequences 830, 831, and 832, respectively; or the CDRs of the heavy chain contain amino acid sequences 833, 834, and 835, respectively, and the CDRs of the light chain contain amino acid sequences 836, 837, and 838, respectively; or the CDRs of the heavy chain contain amino acid sequences 839, 840, and 841, respectively, and the CDRs of the light chain contain amino acid sequences 842, 843, and 844, respectively.

[0058] Carbonic anhydrase IX (40) Exemplary anti-CA IX antibodies include antibodies containing the following VH amino acid sequences and VL amino acid sequences: antibodies having the VH amino acid sequence of SEQ ID NO:845 and the VL amino acid sequence of SEQ ID NO:846; antibodies having the VH amino acid sequence of SEQ ID NO:847 and the VL amino acid sequence of SEQ ID NO:868; antibodies having the VH amino acid sequence of SEQ ID NO:848 and the VL amino acid sequence of SEQ ID NO:869; antibodies having the VH amino acid sequence of SEQ ID NO:849 and the VL amino acid sequence of SEQ ID NO:870; antibodies having the VH amino acid sequence of SEQ ID NO:850 and the VL amino acid sequence of SEQ ID NO:871; antibodies having the VH amino acid sequence of SEQ ID NO:851 and the VL amino acid sequence of SEQ ID NO:872; antibodies having the VH amino acid sequence of SEQ ID NO:852 and the VL amino acid sequence of SEQ ID NO:873; antibodies having the VH amino acid sequence of SEQ ID NO:853 and the VL amino acid sequence of SEQ ID NO:8746. The VL amino acid sequence with SEQ ID NO:874; the VH amino acid sequence with SEQ ID NO:854 and the VL amino acid sequence with SEQ ID NO:875; the VH amino acid sequence with SEQ ID NO:855 and the VL amino acid sequence with SEQ ID NO:876; the VH amino acid sequence with SEQ ID NO:856 and the VL amino acid sequence with SEQ ID NO:877; the VH amino acid sequence with SEQ ID NO:857 and the VL amino acid sequence with SEQ ID NO:878; the VH amino acid sequence with SEQ ID NO:858 and the VL amino acid sequence with SEQ ID NO:879; the VH amino acid sequence with SEQ ID NO:859 and the VL amino acid sequence with SEQ ID NO:880; the VH amino acid sequence with SEQ ID NO:860 and the VL amino acid sequence with SEQ ID NO:881; the VH amino acid sequence with SEQ ID NO:861 and the VL amino acid sequence with SEQ ID NO:882, and the VL amino acid sequence with SEQ ID NO:874; The VH amino acid sequence with SEQ ID NO: 862 and the VL amino acid sequence with SEQ ID NO: 883; the VH amino acid sequence with SEQ ID NO: 863 and the VL amino acid sequence with SEQ ID NO: 884; the VH amino acid sequence with SEQ ID NO: 864 and the VL amino acid sequence with SEQ ID NO: 885; the VH amino acid sequence with SEQ ID NO: 865 and the VL amino acid sequence with SEQ ID NO: 886;The amino acid sequence having the VH amino acid sequence of SEQ ID NO: 866 and the VL amino acid sequence having the VL amino acid sequence of SEQ ID NO: 887; the amino acid sequence having the VH amino acid sequence of SEQ ID NO: 867 and the VL amino acid sequence having the VL amino acid sequence of SEQ ID NO: 888.

[0059] In other embodiments, the anti-CA IX antibody has a heavy chain and a light chain, the heavy chain having three CDRs and the light chain having three CDRs: the CDRs of the heavy chain respectively contain amino acid sequences SEQ ID NO: 803, 804, 805 and the CDRs of the light chain respectively contain amino acid sequences 806, 807, 808; or the CDRs of the heavy chain contain amino acid sequences 899, 915, 909 and the CDRs of the light chain respectively contain amino acid sequences 905, 906, 952; or the CDRs of the heavy chain contain amino acid sequences 899, 915, 909 and the CDRs of the light chain respectively contain amino acid sequences 935, 943, 953; or the CDRs of the heavy chain contain amino acid sequences 899, 915, 909 and the CDRs of the light chain respectively contain amino acid sequences 935, 906, 954; or the CDRs of the heavy chain contain amino acid sequences 910, 916. The CDRs of the heavy chain and the light chain respectively contain amino acid sequences 936, 944, and 955; or the CDRs of the heavy chain contain amino acid sequences 899, 915, and 909, and the CDRs of the light chain respectively contain amino acid sequences 936, 944, and 956; or the CDRs of the heavy chain contain amino acid sequences 911, 917, and 924, and the CDRs of the light chain respectively contain amino acid sequences 937, 945, and 957; or the CDRs of the heavy chain contain amino acid sequences 899, 915, and 909, and the CDRs of the light chain respectively contain amino acid sequences 935, 946, and 958; or the CDRs of the heavy chain contain amino acid sequences 899, 915, and 909, and the CDRs of the light chain respectively contain amino acid sequences 938, 946, and 959; or the CDRs of the heavy chain contain amino acid sequences 899, 915, and 909. The CDRs of the heavy chain and the light chain respectively contain amino acid sequences 905, 946, and 960; or the CDR of the heavy chain contains amino acid sequences 899, 918, and 925, and the CDR of the light chain respectively contains amino acid sequences 937, 947, and 955; or the CDR of the heavy chain contains amino acid sequences 899, 918, and 926, and the CDR of the light chain respectively contains amino acid sequences 937, 945, and 957; or the CDR of the heavy chain contains amino acid sequences 912, 919, and 927, and the CDR of the light chain respectively contains amino acid sequences 937, 943, and 961; or the CDR of the heavy chain contains amino acid sequences 899, 918, and 928, and the CDR of the light chain respectively contains amino acid sequences 937, 906, and 960; or the CDR of the heavy chain contains amino acid sequences 899, 918, and 928. 928 and the CDR of the light chain respectively contain amino acid sequences 937, 906, and 960;Alternatively, the CDR of the heavy chain may contain amino acid sequences 913, 920, 929, and the CDR of the light chain may contain amino acid sequences 939, 948, 962, respectively; or the CDR of the heavy chain may contain amino acid sequences 935, 944, 955, and the CDR of the light chain may contain amino acid sequences 899, 918, 930, respectively; or the CDR of the heavy chain may contain amino acid sequences 899, 921, 931, and the CDR of the light chain may contain amino acid sequences 935, 944, 955, respectively; or the CDR of the heavy chain may contain amino acid sequences 912, 919, 932, and the CDR of the light chain may contain amino acid sequences 940, 949, 963, respectively; or the CDR of the heavy chain may contain amino acid sequences 899, 915, 909, and the CDR of the light chain may contain amino acid sequences 935, 943, respectively. 960; or the CDR of the heavy chain contains amino acid sequences 914, 922, 933 and the CDR of the light chain contains amino acid sequences 941, 950, 964 respectively; or the CDR of the heavy chain contains amino acid sequences 912, 918, 934 and the CDR of the light chain contains amino acid sequences 942, 951, 965 respectively.

[0060] CC-Chemokine receptor 4 (CCR4) (048) An exemplary CC-chemokine receptor 4 (CCR4) antibody includes a VH nucleotide sequence having SEQ ID NO:969 and a VL nucleotide sequence having SEQ ID NO:971; or a VH nucleotide sequence having SEQ ID NO:969 and a VL nucleotide sequence having SEQ ID NO:972.

[0061] An exemplary CCR4 antibody includes an antibody containing the VH amino acid sequence having SEQ ID NO:970 and the VL amino acid sequence having SEQ ID NO:971.

[0062] In other embodiments, the CCR4 antibody has a heavy chain and a light chain, the heavy chain having three CDRs and the light chain having three CDRs: the CDRs of the heavy chain respectively contain amino acid sequences SEQ ID NO: 973, 974, 975 and the CDRs of the light chain respectively contain amino acid sequences 976, 977, 978.

[0063] Middle East Respiratory Syndrome Coronavirus (MERS-CoV). (85) Exemplary antibodies against Middle East Respiratory Syndrome Coronavirus (MERS-CoV) include antibodies having the following VH and VL nucleotide sequences: VH nucleotide sequence having SEQ ID NO:677 and VL nucleotide sequence having SEQ ID NO:679; VH nucleotide sequence having SEQ ID NO:681 and VL nucleotide sequence having SEQ ID NO:683; VH nucleotide sequence having SEQ ID NO:685 and VL nucleotide sequence having SEQ ID NO:687; VH nucleotide sequence having SEQ ID NO:689 and VL nucleotide sequence having SEQ ID NO:692; VH nucleotide sequence having SEQ ID NO:693 and VL nucleotide sequence having SEQ ID NO:695; VH nucleotide sequence having SEQ ID NO:697 and VL nucleotide sequence having SEQ ID NO:699; and VH nucleotide sequence having SEQ ID NO:701 and VL nucleotide sequence having SEQ ID NO:703.

[0064] Exemplary antibodies against Middle East Respiratory Syndrome Coronavirus (MERS-CoV) include antibodies having the following VH and VL amino acid sequences: having the VH amino acid sequence of SEQ ID NO:678 and the VL amino acid sequence of SEQ ID NO:680; having the VH amino acid sequence of SEQ ID NO:682 and the VL amino acid sequence of SEQ ID NO:684; having the VH amino acid sequence of SEQ ID NO:686 and the VL amino acid sequence of SEQ ID NO:688; having the VH amino acid sequence of SEQ ID NO:690 and the VL amino acid sequence of SEQ ID NO:692; having the VH amino acid sequence of SEQ ID NO:694 and the VL amino acid sequence of SEQ ID NO:696; having the VH amino acid sequence of SEQ ID NO:698 and the VL amino acid sequence of SEQ ID NO:700; and having the VH amino acid sequence of SEQ ID NO:702 and the VL amino acid sequence of SEQ ID NO:704.

[0065] In other embodiments, the anti-Middle East Respiratory Syndrome Coronavirus (MERS-CoV) antibody has a heavy chain and a light chain, wherein the heavy chain has three CDRs and the light chain has three CDRs: the CDRs of the heavy chain contain amino acid sequences 705, 706, and 707 and the CDRs of the light chain contain amino acid sequences 722, 723, and 724; the CDRs of the heavy chain contain amino acid sequences 708, 709, and 710 and the CDRs of the light chain contain amino acid sequences 725, 726, and 727; the CDRs of the heavy chain contain amino acid sequences 711, 712, and 713 and the CDRs of the light chain contain amino acid sequences 728 and 723. The heavy chain CDR contains amino acid sequences 711, 735, and 715, and the light chain CDR contains amino acid sequences 731, 732, and 733; the heavy chain CDR contains amino acid sequences 711, 735, and 716, and the light chain CDR contains amino acid sequences 737, 738, and 739; the heavy chain CDR contains amino acid sequences 717, 718, and 719, and the light chain CDR contains amino acid sequences 736, 742, and 743; and the heavy chain CDR contains amino acid sequences 714, 720, and 721, and the light chain CDR contains amino acid sequences 740, 729, and 741.

[0066] GITR(93) Exemplary anti-human GITR antibodies include antibodies having the following VH and VL nucleotide sequences: having the VH nucleotide sequence of SEQ ID NO:1361 and the VL nucleotide sequence of SEQ ID NO:1363; having the VH nucleotide sequence of SEQ ID NO:1365 and the VL nucleotide sequence of SEQ ID NO:1367; having the VH nucleotide sequence of SEQ ID NO:1369 and the VL nucleotide sequence of SEQ ID NO:1371; having the VH nucleotide sequence of SEQ ID NO:1381 and the VL nucleotide sequence of SEQ ID NO:1375; having the VH nucleotide sequence of SEQ ID NO:1377 and the VL nucleotide sequence of SEQ ID NO:1379; having the VH nucleotide sequence of SEQ ID NO:1381 and the VL nucleotide sequence of SEQ ID NO:1383; having the VH nucleotide sequence of SEQ ID NO:1385 and the VL nucleotide sequence of SEQ ID NO:1387; having the VH nucleotide sequence of SEQ ID NO:1381 and the VL nucleotide sequence of SEQ ID NO:1383; having the VH nucleotide sequence of SEQ ID NO:1385 and the VL nucleotide sequence of SEQ ID NO:1387; having the VH nucleotide sequence of SEQ ID NO:136 ... The VH nucleotide sequence with SEQ ID NO:1389 and the VL nucleotide sequence with SEQ ID NO:1391; the VH nucleotide sequence with SEQ ID NO:1393 and the VL nucleotide sequence with SEQ ID NO:1395; the VH nucleotide sequence with SEQ ID NO:1397 and the VL nucleotide sequence with SEQ ID NO:1398; or the VH nucleotide sequence with SEQ ID NO:1401 and the VL nucleotide sequence with SEQ ID NO:1403.

[0067] Exemplary anti-human GITR antibodies include antibodies having the following sequences: having the VH amino acid sequence of SEQ ID NO:1362 and the VL amino acid sequence of SEQ ID NO:1364; having the VH amino acid sequence of SEQ ID NO:1366 and the VL polypeptide sequence of SEQ ID NO:1368; having the VH amino acid sequence of SEQ ID NO:1371 and the VL amino acid sequence of SEQ ID NO:1372; having the VH amino acid sequence of SEQ ID NO:1382 and the VL amino acid sequence of SEQ ID NO:1376; having the VH nucleotide sequence of SEQ ID NO:1378 and the VL nucleotide sequence of SEQ ID NO:1380; having the VH amino acid sequence of SEQ ID NO:1382 and the VL polypeptide sequence of SEQ ID NO:1384; having the VH amino acid sequence of SEQ ID NO:1386 and the VL amino acid sequence of SEQ ID NO:1388; having the VH amino acid sequence of SEQ ID NO:1390 ... The VL amino acid sequence NO:1392; the VH amino acid sequence having SEQ ID NO:1394 and the VL polypeptide sequence having SEQ ID NO:1396; the VH amino acid sequence having SEQ ID NO:1399 and the VL amino acid sequence having SEQ ID NO:1400; or the VH amino acid sequence having SEQ ID NO:1402 and the VL amino acid sequence having SEQ ID NO:1404.

[0068] In other embodiments, the anti-human GITR antibody has a heavy chain and a light chain, the heavy chain having three CDRs and the light chain having three CDRs: the CDRs of the heavy chain respectively contain amino acid sequences 1405, 1406, and 1407 and the CDRs of the light chain respectively contain amino acid sequences 1408, 1409, and 1410; or the CDRs of the heavy chain respectively contain amino acid sequences 1411, 1412, and 1413 and the CDRs of the light chain respectively contain amino acid sequences 1414, 1415, and 1416; or the CDRs of the heavy chain respectively contain... The CDR of the heavy chain contains amino acid sequences 1417, 1418, and 1419, and the CDR of the light chain contains amino acid sequences 1420, 1421, and 1422, respectively; or the CDR of the heavy chain contains amino acid sequences 1423, 1424, and 1425, and the CDR of the light chain contains amino acid sequences 1426, 1427, and 1428, respectively; or the CDR of the heavy chain contains amino acid sequences 1429, 1430, and 1431, and the CDR of the light chain contains amino acid sequences 1432, 1433, and 1434, respectively; or the CDR of the heavy chain contains amino acid sequences 1420, 1421, and 1422, respectively. R contains amino acid sequences 1435, 1436, and 1437 respectively, and the CDR of the light chain contains amino acid sequences 1438, 1439, and 1440 respectively; or the CDR of the heavy chain contains amino acid sequences 1441, 1442, and 1443 respectively, and the CDR of the light chain contains amino acid sequences 1444, 1445, and 1446 respectively; or the CDR of the heavy chain contains amino acid sequences 1447, 1448, and 1449 respectively, and the CDR of the light chain contains amino acid sequences 1450, 1451, and 1452 respectively; or the heavy chain contains amino acid sequences 1435, 1436, and 1437 ...38, 1439, and 1440 respectively. The CDR of the heavy chain contains amino acid sequences 1453, 1454, and 1455, and the CDR of the light chain contains amino acid sequences 1456, 1457, and 1458, respectively; or the CDR of the heavy chain contains amino acid sequences 1459, 1460, and 1461, and the CDR of the light chain contains amino acid sequences 1462, 1463, and 1464, respectively; or the CDR of the heavy chain contains amino acid sequences 1465, 1466, and 1467, and the CDR of the light chain contains amino acid sequences 1468, 1469, and 1470, respectively.

[0069] Flavivivirus (73) An exemplary antibody against West Nile virus envelope protein E (WNE) includes an antibody having a VH nucleotide sequence, a VH amino acid sequence of SEQ ID NO: 1224, and a VL amino acid sequence of SEQ ID NO: 1226.

[0070] An exemplary antibody against West Nile virus envelope protein E (WNE) includes an antibody having a VH nucleotide sequence having SEQ ID NO:1225 and a VL nucleotide sequence having SEQ ID NO:1227.

[0071] In other embodiments, the anti-West Nile virus envelope protein E (WNE) antibody has a heavy chain and a light chain, the heavy chain having three CDRs and the light chain having three CDRs: the CDRs of the heavy chain contain amino acid sequences 1244, 1245 and 1246, respectively, and the CDRs of the light chain contain amino acid sequences 1247, 1248 and 1249, respectively.

[0072] CCR4(65) Exemplary anti-CC chemokine receptor 4 (CCR4) antibodies include antibodies having the following VH nucleotide sequences and VL nucleotide sequences: having the VH nucleotide sequence of SEQ ID NO:1329 and the VL nucleotide sequence of SEQ ID NO:1331; having the VH nucleotide sequence of SEQ ID NO:1333 and the VL nucleotide sequence of SEQ ID NO:1335; having the VH nucleotide sequence of SEQ ID NO:1337 and the VL nucleotide sequence of SEQ ID NO:1192; having the VH nucleotide sequence of SEQ ID NO:1341 and the VL nucleotide sequence of SEQ ID NO:1343; or having the VH nucleotide sequence of SEQ ID NO:1357 and the VL nucleotide sequence of SEQ ID NO:1359.

[0073] Exemplary anti-CC chemokine receptor 4 (CCR4) antibodies include antibodies having the following VH amino acid sequences and VL amino acid sequences: having the VH amino acid sequence of SEQ ID NO:1330 and the VL amino acid sequence of SEQ ID NO:1332; having the VH amino acid sequence of SEQ ID NO:1334 and the VL amino acid sequence of SEQ ID NO:1336; having the VH amino acid sequence of SEQ ID NO:1338 and the VL amino acid sequence of SEQ ID NO:1340; having the VH amino acid sequence of SEQ ID NO:1342 and the VL amino acid sequence of SEQ ID NO:1344; or having the VH amino acid sequence of SEQ ID NO:1358 and the VL amino acid sequence of SEQ ID NO:1360.

[0074] In other embodiments, the anti-CC chemokine receptor 4 (CCR4) antibody has a heavy chain and a light chain, wherein the heavy chain has three CDRs and the light chain has three CDRs: the CDRs of the heavy chain contain amino acid sequences 1203, 1208, and 1211, and the CDRs of the light chain contain amino acid sequences 1207, 1209, and 1216, respectively; or the CDRs of the heavy chain contain amino acid sequences 1204, 1208, and 1212, and the CDRs of the light chain contain amino acid sequences 1207, 1209, and 1217, respectively; or the... The CDR of the heavy chain contains amino acid sequences 1205, 1208, and 1214, and the CDR of the light chain contains amino acid sequences 1207, 1209, and 1218, respectively; or the CDR of the heavy chain contains amino acid sequences 1206, 1208, and 1210, and the CDR of the light chain contains amino acid sequences 1207, 1209, and 1220, respectively; or the CDR of the heavy chain contains amino acid sequences 1202, 1208, and 1210, and the CDR of the light chain contains amino acid sequences 1207, 1209, and 1219, respectively.

[0075] Germline gene VH1-69 (57) for the variable region of human immunoglobulin heavy chain An exemplary antibody against the germline gene VH1-69 of the variable region of human immunoglobulin heavy chain includes an antibody having a VH nucleotide sequence having SEQ ID NO:1153 and a VL nucleotide sequence having SEQ ID NO:1155; or having a VH nucleotide sequence having SEQ ID NO:1163 and a VL nucleotide sequence having SEQ ID NO:1155.

[0076] An exemplary antibody against the germline gene VH1-69 of the variable region of human immunoglobulin heavy chain comprises an antibody having a VH amino acid sequence having SEQ ID NO:1154 and a VL amino acid sequence having SEQ ID NO:1156; or having a VH amino acid sequence having SEQ ID NO:1164 and a VL amino acid sequence having SEQ ID NO:1156.

[0077] In other embodiments, the anti-human immunoglobulin heavy chain variable region germline gene VH1-69 antibody has a heavy chain and a light chain, the heavy chain having three CDRs and the light chain having three CDRs: the CDRs of the heavy chain contain amino acid sequences 1157, 1158 and 1159, respectively, and the CDRs of the light chain contain amino acid sequences 1160, 1161 and 1162, respectively.

[0078] Influenza virus (49) Exemplary anti-influenza antibodies include antibodies having the following VH nucleotide sequences and VL nucleotide sequences: having the VH nucleotide sequence of SEQ ID NO:981 and the VL nucleotide sequence of SEQ ID NO:983; having the VH nucleotide sequence of SEQ ID NO:985 and the VL nucleotide sequence of SEQ ID NO:989; having the VH nucleotide sequence of SEQ ID NO:987 and the VL nucleotide sequence of SEQ ID NO:991; having the VH nucleotide sequence of SEQ ID NO:993 and the VL nucleotide sequence of SEQ ID NO:997; having the VH nucleotide sequence of SEQ ID NO:995 and the VK nucleotide sequence of SEQ ID NO:999; having the VH nucleotide sequence of SEQ ID NO:1001 and the VL nucleotide sequence of SEQ ID NO:1005; having the VH nucleotide sequence of SEQ ID NO:1003 and the VL nucleotide sequence of SEQ ID NO:1007; having the VH nucleotide sequence of SEQ ID NO:1009 and the VL nucleotide sequence of SEQ ID NO:981. VL nucleotide sequence NO:1011; VH nucleotide sequence having SEQ ID NO:1013 and VL nucleotide sequence having SEQ ID NO:1015; VH nucleotide sequence having SEQ ID NO:1017 and VL nucleotide sequence having SEQ ID NO:1019; VH nucleotide sequence having SEQ ID NO:1020 and VL nucleotide sequence having SEQ ID NO:1022.

[0079] Exemplary anti-influenza antibodies include antibodies having the following VH amino acid sequences and VL amino acid sequences: having the VH amino acid sequence of SEQ ID NO:982 and the VL amino acid sequence of SEQ ID NO:984; having the VH amino acid sequence of SEQ ID NO:986 and the VL amino acid sequence of SEQ ID NO:988; having the VH amino acid sequence of SEQ ID NO:986 and the VL amino acid sequence of SEQ ID NO:990; having the VH amino acid sequence of SEQ ID NO:992 and the VL amino acid sequence of SEQ ID NO:994; having the VH amino acid sequence of SEQ ID NO:992 and the VL amino acid sequence of SEQ ID NO:996; having the VH amino acid sequence of SEQ ID NO:998 and the VL amino acid sequence of SEQ ID NO:1000; having the VH amino acid sequence of SEQ ID NO:998 and the VL amino acid sequence of SEQ ID NO:1002; having the VH amino acid sequence of SEQ ID NO:1004 and the VL amino acid sequence of SEQ ID NO:990. The VL amino acid sequence NO:1006; the VH amino acid sequence having SEQ ID NO:1008 and the VL amino acid sequence having SEQ ID NO:1010; the VH amino acid sequence having SEQ ID NO:1012 and the VL amino acid sequence having SEQ ID NO:1014; and the VH amino acid sequence having SEQ ID NO:1016 and the VL amino acid sequence having SEQ ID NO:1018.

[0080] In other embodiments, the anti-influenza antibody has a heavy chain and a light chain, the heavy chain having three CDRs and the light chain having three CDRs: the CDRs of the heavy chain contain amino acid sequences 1023, 1031, and 1039, and the CDRs of the light chain contain amino acid sequences 1047, 1059, and 1071; the CDRs of the heavy chain contain amino acid sequences 1023, 1032, and 1040, and the CDRs of the light chain contain amino acid sequences 1048, 1060, and 1072; the CDRs of the heavy chain contain... The CDR of the heavy chain contains amino acid sequences 1025, 1032, and 1040, and the CDR of the light chain contains amino acid sequences 1057, 1069, and 1081; the CDR of the heavy chain contains amino acid sequences 1026, 1033, and 1041, and the CDR of the light chain contains amino acid sequences 1049, 1061, and 1073; the CDR of the heavy chain contains amino acid sequences 1026, 1033, and 1041, and the CDR of the light chain contains amino acid sequences 1054, 1066, and 1078; the CDR of the heavy chain contains amino acid sequences 1025, 1032, and 1040 ...7, 1069, and 1081; the CDR of the heavy chain contains amino acid sequences 1026, 1033, and 1041, and the CDR of the light chain contains amino acid sequences 1054, 1066, and 1078; the CDR of the heavy chain contains amino acid sequences 1025, 1032, and 1040, and the CDR of the light chain contains amino acid sequences 1057, 1069, and 1081; the CDR of the heavy chain contains amino acid sequences 1025, 1033, and 1041, and the CDR of the light chain contains amino acid sequences 1057, 1069, and 1081; the CDR of the heavy chain contains amino acid sequences 1025, 1033, R contains amino acid sequences 1027, 1034, and 1042, and the CDR of the light chain contains amino acid sequences 1050, 1062, and 1074; the CDR of the heavy chain contains amino acid sequences 1027, 1034, and 1042, and the CDR of the light chain contains amino acid sequences 1056, 1068, and 1080; the CDR of the heavy chain contains amino acid sequences 1028, 1035, and 1043, and the CDR of the light chain contains amino acid sequences 1051, 1063, and 1065; the heavy The CDR of the heavy chain contains amino acid sequences 1028, 1036, and 1044, and the CDR of the light chain contains amino acid sequences 1052, 1064, and 1076; the CDR of the heavy chain contains amino acid sequences 1029, 1037, and 1045, and the CDR of the light chain contains amino acid sequences 1053, 1065, and 1077; the CDR of the heavy chain contains amino acid sequences 1030, 1038, and 1046, and the CDR of the light chain contains amino acid sequences 1058, 1070, and 1082.

[0081] Influenza (78) Exemplary anti-influenza antibodies include antibodies containing the following VH nucleotide sequences and VL nucleotide sequences: having the VH nucleotide sequence of SEQ ID NO:397 and the VL nucleotide sequence of SEQ ID NO:398; having the VH nucleotide sequence of SEQ ID NO:399 and the VL nucleotide sequence of SEQ ID NO:400; having the VH nucleotide sequence of SEQ ID NO:401 and the VL nucleotide sequence of SEQ ID NO:402; having the VH nucleotide sequence of SEQ ID NO:403 and the VL nucleotide sequence of SEQ ID NO:404; or having the VH nucleotide sequence of SEQ ID NO:405 and the VL nucleotide sequence of SEQ ID NO:406; or having the VH nucleotide sequence of SEQ ID NO:407 and the VL nucleotide sequence of SEQ ID NO:408; or having the VH nucleotide sequence of SEQ ID NO:409 and the VL nucleotide sequence of SEQ ID NO:410; or having the VH nucleotide sequence of SEQ ID NO:411 and the VL nucleotide sequence of SEQ ID NO:402. The VL nucleotide sequence of SEQ ID NO:412; or having the VH nucleotide sequence of SEQ ID NO:413 and the VL nucleotide sequence of SEQ ID NO:414; or having the VH nucleotide sequence of SEQ ID NO:415 and the VL nucleotide sequence of SEQ ID NO:416; or having the VH nucleotide sequence of SEQ ID NO:417 and the VL nucleotide sequence of SEQ ID NO:418; or having the VH nucleotide sequence of SEQ ID NO:419 and the VL nucleotide sequence of SEQ ID NO:420; or having the VH nucleotide sequence of SEQ ID NO:421 and the VL nucleotide sequence of SEQ ID NO:422; or having the VH nucleotide sequence of SEQ ID NO:423 and the VL nucleotide sequence of SEQ ID NO:424; or having the VH nucleotide sequence of SEQ ID NO:425 and the VL nucleotide sequence of SEQ ID NO:426; or having the VH nucleotide sequence of SEQ ID NO:427 and the VL nucleotide sequence of SEQ ID NO:428; or having SEQ ID NO:412 and the VL nucleotide sequence of SEQ ID NO:414; or having the VH nucleotide sequence of SEQ ID NO:415 and the VL nucleotide sequence of SEQ ID NO:416; or having the VH nucleotide sequence of SEQ ID NO:417 and the VL nucleotide sequence of SEQ ID NO:418; or having the VH nucleotide sequence of SEQ ID NO:419 and the VL nucleotide sequence of SEQ ID NO:420; or having the VH nucleotide sequence of SEQ ID NO:421 and the VL nucleotide sequence of SEQ ID NO:422; or having the VH nucleotide sequence of SEQ ID NO:423 and the VL nucleotide sequence of SEQ ID NO:424; or having the VH nucleotide sequence of SEQ ID NO:425 and the VL nucleotide sequence of The VH nucleotide sequence of SEQ ID NO:429 and the VL nucleotide sequence of SEQ ID NO:430; or the VH nucleotide sequence of SEQ ID NO:431 and the VL nucleotide sequence of SEQ ID NO:432; or the VH nucleotide sequence of SEQ ID NO:433 and the VL nucleotide sequence of SEQ ID NO:434;Or having the VH nucleotide sequence of SEQ ID NO:435 and the VL nucleotide sequence of SEQ ID NO:436; or having the VH nucleotide sequence of SEQ ID NO:437 and the VL nucleotide sequence of SEQ ID NO:438; or having the VH nucleotide sequence of SEQ ID NO:439 and the VL nucleotide sequence of SEQ ID NO:440; or having the VH nucleotide sequence of SEQ ID NO:441 and the VL nucleotide sequence of SEQ ID NO:442; or having the VH nucleotide sequence of SEQ ID NO:541 and the VL nucleotide sequence of SEQ ID NO:542; or having the VH nucleotide sequence of SEQ ID NO:543 and the VL nucleotide sequence of SEQ ID NO:544; or having the VH nucleotide sequence of SEQ ID NO:545 and the VL nucleotide sequence of SEQ ID NO:546; or having the VH nucleotide sequence of SEQ ID NO:547 and the VL nucleotide sequence of SEQ ID NO:548; or having ... the VH nucleotide sequence of SEQ ID NO:439 and the VL nucleotide sequence of SEQ ID NO:440; or having the VH nucleotide sequence The VH nucleotide sequence of SEQ ID NO:549 and the VL nucleotide sequence of SEQ ID NO:550; or the VH nucleotide sequence of SEQ ID NO:551 and the VL nucleotide sequence of SEQ ID NO:552; or the VH nucleotide sequence of SEQ ID NO:553 and the VL nucleotide sequence of SEQ ID NO:554; or the VH nucleotide sequence of SEQ ID NO:555 and the VL nucleotide sequence of SEQ ID NO:556; or the VH nucleotide sequence of SEQ ID NO:557 and the VL nucleotide sequence of SEQ ID NO:558; or the VH nucleotide sequence of SEQ ID NO:559 and the VL nucleotide sequence of SEQ ID NO:560; or the VH nucleotide sequence of SEQ ID NO:561 and the VL nucleotide sequence of SEQ ID NO:562; or the VH nucleotide sequence of SEQ ID NO:563 and the VL nucleotide sequence of SEQ ID NO:564; or the VH nucleotide sequence of SEQ ID NO:565 and the VL nucleotide sequence of SEQ ID NO:564. VL nucleotide sequence of SEQ ID NO: 566; or VH nucleotide sequence of SEQ ID NO: 567 and VL nucleotide sequence of SEQ ID NO: 568; or VH nucleotide sequence of SEQ ID NO: 569 and VL nucleotide sequence of SEQ ID NO: 570; or VH nucleotide sequence of SEQ ID NO: 571 and VL nucleotide sequence of SEQ ID NO: 572;Or having the VH nucleotide sequence of SEQ ID NO:573 and the VL nucleotide sequence of SEQ ID NO:574; or having the VH nucleotide sequence of SEQ ID NO:575 and the VL nucleotide sequence of SEQ ID NO:576; or having the VH nucleotide sequence of SEQ ID NO:577 and the VL nucleotide sequence of SEQ ID NO:578; or having the VH nucleotide sequence of SEQ ID NO:579 and the VL nucleotide sequence of SEQ ID NO:580; or the VH nucleotide sequence having the nucleotide sequence of SEQ ID NO:581 and the VL nucleotide sequence of SEQ ID NO:582; or having the VH nucleotide sequence of SEQ ID NO:583 and the VL nucleotide sequence of SEQ ID NO:584; or having the VH nucleotide sequence of SEQ ID NO:585 and the VL nucleotide sequence of SEQ ID NO:586; or having the VH nucleotide sequence of SEQ ID NO:587 and the VL nucleotide sequence of SEQ ID NO:588; or having SEQ ID NO:579 and the VL nucleotide sequence of SEQ ID NO:580; or having the VH nucleotide sequence of SEQ ID NO:581 and the VL nucleotide sequence of SEQ ID NO:582; or having the VH nucleotide sequence of SEQ ID NO:583 and the VL nucleotide sequence of SEQ ID NO:584; or having the VH nucleotide sequence of SEQ ID NO:585 and the VL nucleotide sequence of SEQ ID NO:586; or having the VH nucleotide sequence of SEQ ID NO:587 and the VL nucleotide sequence of SEQ ID NO:588; or having the VH nucleotide sequence of SEQ ID NO:589 and the VL nucleotide sequence of SEQ ID NO:580; or having the VH nucleotide sequence of SEQ ID NO:584 and the VL nucleotide sequence of SEQ ID NO:585; or having the V The VH nucleotide sequence of SEQ ID NO:589 and the VL nucleotide sequence of SEQ ID NO:590; or the VH nucleotide sequence of SEQ ID NO:591 and the VL nucleotide sequence of SEQ ID NO:592; or the VH nucleotide sequence of SEQ ID NO:593 and the VL nucleotide sequence of SEQ ID NO:594; or the VH nucleotide sequence of SEQ ID NO:595 and the VL nucleotide sequence of SEQ ID NO:596; or the VH nucleotide sequence of SEQ ID NO:597 and the VL nucleotide sequence of SEQ ID NO:598; or the VH nucleotide sequence of SEQ ID NO:599 and the VL nucleotide sequence of SEQ ID NO:600.

[0082] Exemplary anti-influenza antibodies include antibodies containing the following sequences: having the VH amino acid sequence of SEQ ID NO:469 and the VL amino acid sequence of SEQ ID NO:470; having the VH amino acid sequence of SEQ ID NO:471 and the VL polypeptide sequence of SEQ ID NO:472; having the VH amino acid sequence of SEQ ID NO:473 and the VL amino acid sequence of SEQ ID NO:474; having the VH amino acid sequence of SEQ ID NO:475 and the VL amino acid sequence of SEQ ID NO:476; or having the VH nucleotide sequence of SEQ ID NO:477 and the VL nucleotide sequence of SEQ ID NO:478; having the VH amino acid sequence of SEQ ID NO:479 and the VL amino acid sequence of SEQ ID NO:480; having the VH amino acid sequence of SEQ ID NO:481 and the VL amino acid sequence of SEQ ID NO:482; having the VH amino acid sequence of SEQ ID NO:483 and the VL amino acid sequence of SEQ ID NO:484; having SEQ ID NO:475 and the VL amino acid sequence of SEQ ID NO:476; or having the VH nucleotide sequence of SEQ ID NO:477 and the VL nucleotide sequence of SEQ ID NO:478; having the VH amino acid sequence of SEQ ID NO:479 and the VL amino acid sequence of SEQ ID NO:480; having the VH amino acid sequence of SEQ ID NO:481 and the VL amino acid sequence of SEQ ID NO:482; having the VH amino acid sequence of SEQ ID NO:483 and the VL amino acid sequence of SEQ ID NO:484; or having the VH nucleotide sequence of SEQ ID NO:475 and the VL amino acid sequence of SEQ ID NO:476; or having the VH nucleotide sequence of SEQ ID NO:475 and the VL amino acid sequence of SEQ ID NO:476; or having The VH amino acid sequence with SEQ ID NO:485 and the VL amino acid sequence with SEQ ID NO:486; the VH amino acid sequence with SEQ ID NO:487 and the VL amino acid sequence with SEQ ID NO:488; the VH amino acid sequence with SEQ ID NO:489 and the VL amino acid sequence with SEQ ID NO:490; the VH amino acid sequence with SEQ ID NO:491 and the VL amino acid sequence with SEQ ID NO:492; the VH amino acid sequence with SEQ ID NO:493 and the VL amino acid sequence with SEQ ID NO:494; the VH amino acid sequence with SEQ ID NO:495 and the VL amino acid sequence with SEQ ID NO:496; the VH amino acid sequence with SEQ ID NO:497 and the VL amino acid sequence with SEQ ID NO:498; the VH amino acid sequence with SEQ ID NO:499 and the VL amino acid sequence with SEQ ID NO:500; the VH amino acid sequence with SEQ ID NO:501 and the VL amino acid sequence with SEQ ID NO:485 and the VL amino acid sequence with SEQ ID NO:496; the VH amino acid sequence with SEQ ID NO:497 and the VL amino acid sequence with SEQ ID NO:498; the VH amino acid sequence with SEQ ID NO:499 and the VL amino acid sequence with SEQ ID NO:500; the VH amino acid sequence with SEQ ID NO:501 and the VL amino acid sequence with SEQ ID NO:499; the VH amino acid sequence with SEQ ID NO:490 and the VL amino acid sequence with SEQ ID NO:490; the VH amino acid sequence with SEQ ID NO:495 and the VL amino acid sequence with SEQ ID NO:496; the VH amino acid sequence with SEQ ID NO:497 and the VL amino acid sequence with SEQ ID NO:498; the VH amino acid sequence with SEQ ID NO:499 The VL amino acid sequence of SEQ ID NO:502; the VH amino acid sequence of SEQ ID NO:503 and the VL amino acid sequence of SEQ ID NO:504; the VH amino acid sequence of SEQ ID NO:505 and the VL amino acid sequence of SEQ ID NO:506; the VH amino acid sequence of SEQ ID NO:507 and the VL amino acid sequence of SEQ ID NO:508; the VL amino acid sequence of SEQ ID NO:502;The VH amino acid sequence of SEQ ID NO:509 and the VL amino acid sequence of SEQ ID NO:510; the VH amino acid sequence of SEQ ID NO:511 and the VL amino acid sequence of SEQ ID NO:512; the VH amino acid sequence of SEQ ID NO:513 and the VL amino acid sequence of SEQ ID NO:514; the VH amino acid sequence of SEQ ID NO:515 and the VL amino acid sequence of SEQ ID NO:516; the VH amino acid sequence of SEQ ID NO:517 and the VL amino acid sequence of SEQ ID NO:518; the VH amino acid sequence of SEQ ID NO:519 and the VL amino acid sequence of SEQ ID NO:520; the VH amino acid sequence of SEQ ID NO:521 and the VL amino acid sequence of SEQ ID NO:522; the VH amino acid sequence of SEQ ID NO:523 and the VL amino acid sequence of SEQ ID NO:524; the VH amino acid sequence of SEQ ID NO:525 and the VL amino acid sequence of SEQ ID NO:510; the VH amino acid sequence of SEQ ID NO:511 and the VL amino acid sequence of SEQ ID NO:512; the VH amino acid sequence of SEQ ID NO:513 and the VL amino acid sequence of SEQ ID NO:514; the VH amino acid sequence of SEQ ID NO:515 and the VL amino acid sequence of SEQ ID NO:516; the VH amino acid sequence of SEQ ID NO:517 and the VL amino acid sequence of SEQ ID NO:518; the VH amino acid sequence of SEQ ID NO:519 and the VL amino acid sequence of SEQ ID NO:520; the VH amino acid sequence of SEQ ID NO:521 and the VL amino acid sequence of SEQ ID NO:522; the VH amino acid sequence of SEQ ID NO:523 and the VL amino acid sequence of SEQ ID NO:524 The VL amino acid sequence NO:526; the VH amino acid sequence having SEQ ID NO:527 and the VL amino acid sequence having SEQ ID NO:528; the VH amino acid sequence having SEQ ID NO:529 and the VL amino acid sequence having SEQ ID NO:530; the VH amino acid sequence having SEQ ID NO:531 and the VL amino acid sequence having SEQ ID NO:532; the VH amino acid sequence having SEQ ID NO:533 and the VL amino acid sequence having SEQ ID NO:534; the VH amino acid sequence having SEQ ID NO:535 and the VL amino acid sequence having SEQ ID NO:536; the VH amino acid sequence having SEQ ID NO:537 and the VL amino acid sequence having SEQ ID NO:538; the VH amino acid sequence having SEQ ID NO:539 and the VL amino acid sequence having SEQ ID NO:540; the VH amino acid sequence having SEQ ID NO:601 and the VL amino acid sequence having SEQ ID NO:602; the VH amino acid sequence having SEQ ID NO:526 and the VL amino acid sequence having SEQ ID NO:527 and the VL amino acid sequence having SEQ ID NO:528; the VH amino acid sequence having SEQ ID NO:529 and the VL amino acid sequence having SEQ ID NO:530; the VH amino acid sequence having SEQ ID NO:531 and the VL amino acid sequence having SEQ ID NO:532; the VH amino acid sequence having SEQ ID NO:532 ... The VH amino acid sequence with SEQ ID NO:603 and the VL amino acid sequence with SEQ ID NO:604, the VH amino acid sequence with SEQ ID NO:605 and the VL amino acid sequence with SEQ ID NO:606, the VH amino acid sequence with SEQ ID NO:607 and the VL amino acid sequence with SEQ ID NO:608, the VH amino acid sequence with SEQ ID NO:609 and the VL amino acid sequence with SEQ ID NO:604.The VL amino acid sequence of SEQ ID NO:610, the VH amino acid sequence of SEQ ID NO:611, the VL amino acid sequence of SEQ ID NO:612, the VH amino acid sequence of SEQ ID NO:613, the VL amino acid sequence of SEQ ID NO:614, the VH amino acid sequence of SEQ ID NO:615, the VH amino acid sequence of SEQ ID NO:616, the VH amino acid sequence of SEQ ID NO:617, the VL amino acid sequence of SEQ ID NO:618, the VH amino acid sequence of SEQ ID NO:619, the VL amino acid sequence of SEQ ID NO:620, the VH amino acid sequence of SEQ ID NO:621, the VL amino acid sequence of SEQ ID NO:622, the VH amino acid sequence of SEQ ID NO:623, the VL amino acid sequence of SEQ ID NO:624, the VH amino acid sequence of SEQ ID NO:625, and the VL amino acid sequence of SEQ ID NO:626, and the VL amino acid sequence of SEQ ID NO:626. The VH amino acid sequence with SEQ ID NO:627 and the VL amino acid sequence with SEQ ID NO:628, the VH amino acid sequence with SEQ ID NO:629 and the VL amino acid sequence with SEQ ID NO:630, the VH amino acid sequence with SEQ ID NO:631 and the VH amino acid sequence with SEQ ID NO:632, the VL amino acid sequences with SEQ ID NO:633 and SEQ ID NO:634, the VH amino acid sequence with SEQ ID NO:635 and the VH amino acid sequence with SEQ ID NO:636, the VH amino acid sequence with SEQ ID NO:637 and the VL amino acid sequence with SEQ ID NO:638, the VH amino acid sequence with SEQ ID NO:639 and the VL amino acid sequence with SEQ ID NO:640, the VH amino acid sequence with SEQ ID NO:641 and the VL amino acid sequence with SEQ ID NO:642, the VH amino acid sequence with SEQ ID NO:643 and the VL amino acid sequence with SEQ ID NO:644. The VL amino acid sequence of SEQ ID NO: 644, the VH amino acid sequence of SEQ ID NO: 645, the VL amino acid sequence of SEQ ID NO: 646, the VH amino acid sequence of SEQ ID NO: 647, the VL amino acid sequence of SEQ ID NO: 644, the VH amino acid sequence of SEQ ID NO: 649, the VL amino acid sequence of SEQ ID NO: 650, the VH amino acid sequence of SEQ ID NO: 651, and the VL amino acid sequence of SEQ ID NO: 644.The VL amino acid sequence of SEQ ID NO:652, the VH amino acid sequence of SEQ ID NO:653, the VL amino acid sequence of SEQ ID NO:654, the VH amino acid sequence of SEQ ID NO:655, the VL amino acid sequence of SEQ ID NO:656, the VH amino acid sequence of SEQ ID NO:657, the VL amino acid sequence of SEQ ID NO:658, the VH amino acid sequence of SEQ ID NO:659, and the VL amino acid sequence of SEQ ID NO:660.

[0083] In other embodiments, the anti-influenza antibody has a heavy chain and a light chain, the heavy chain having three CDRs and the light chain having three CDRs: the CDRs of the heavy chain respectively contain amino acid sequences SEQ ID NO: 1, 37, 73 and the CDRs of the light chain respectively contain amino acid sequences 109, 145, 181; or the CDRs of the heavy chain respectively contain amino acid sequences 2, 38, 74 and the CDRs of the light chain respectively contain amino acid sequences 110, 146, 182; or the CDRs of the heavy chain respectively contain amino acid sequences 3, 39, 75 and the CDRs of the light chain respectively contain amino acid sequences 111, 147, 183; or the CDRs of the heavy chain respectively contain amino acid sequences 4, 40, 76 and the CDRs of the light chain respectively contain amino acid sequences 112, 148, 184; or the CDRs of the heavy chain respectively contain amino acid sequences 5, 41, 77 and the CDRs of the light chain respectively contain amino acid sequence 113. 149, 185; or the CDR of the heavy chain contains amino acid sequences 6, 42, 78 respectively, and the CDR of the light chain contains amino acid sequences 114, 150, 186 respectively; or the CDR of the heavy chain contains amino acid sequences 7, 43, 79 respectively, and the CDR of the light chain contains amino acid sequences 115, 151, 187 respectively; or the CDR of the heavy chain contains amino acid sequences 8, 44, 80 respectively, and the CDR of the light chain contains amino acid sequences 116, 152, 188 respectively; or the CDR of the heavy chain contains amino acid sequences 9, 45, 81 respectively, and the CDR of the light chain contains amino acid sequences 117, 153, 189 respectively; or the CDR of the heavy chain contains amino acid sequences 10, 46, 82 respectively, and the CDR of the light chain contains amino acid sequences 118, 154 respectively. 190; or the CDR of the heavy chain contains amino acid sequences 11, 47, 83 and the CDR of the light chain contains amino acid sequences 119, 155, 191; or the CDR of the heavy chain contains amino acid sequences 12, 48, 84 and the CDR of the light chain contains amino acid sequences 120, 156, 192; or the CDR of the heavy chain contains amino acid sequences 13, 49, 85 and the CDR of the light chain contains amino acid sequences 121, 157, 193; or the CDR of the heavy chain contains amino acid sequences 14, 50, 86 and the CDR of the light chain contains amino acid sequences 122, 158, 194; or the CDR of the heavy chain contains amino acid sequences 15, 51, 87 and the CDR of the light chain contains amino acid sequences 123, 159. 195; or the CDR of the heavy chain contains amino acid sequences 16, 52, 88 respectively, and the CDR of the light chain contains amino acid sequences 124, 160 respectively.196; or the CDR of the heavy chain contains amino acid sequences 17, 53, 89 and the CDR of the light chain contains amino acid sequences 125, 161, 197; or the CDR of the heavy chain contains amino acid sequences 18, 54, 90 and the CDR of the light chain contains amino acid sequences 126, 162, 198; or the CDR of the heavy chain contains amino acid sequences 19, 55, 91 and the CDR of the light chain contains amino acid sequences 127, 163, 199; or the CDR of the heavy chain contains amino acid sequences 20, 56, 92 and the CDR of the light chain contains amino acid sequences 128, 164, 200; or the CDR of the heavy chain contains amino acid sequences 21, 57, 93 and the CDR of the light chain contains amino acid sequences 129, 165. 201; or the CDR of the heavy chain contains amino acid sequences 22, 58, 94 and the CDR of the light chain contains amino acid sequences 130, 166, 202; or the CDR of the heavy chain contains amino acid sequences 23, 59, 95 and the CDR of the light chain contains amino acid sequences 131, 167, 203; or the CDR of the heavy chain contains amino acid sequences 24, 60, 96 and the CDR of the light chain contains amino acid sequences 132, 168, 204; or the CDR of the heavy chain contains amino acid sequences 25, 61, 95 and the CDR of the light chain contains amino acid sequences 133, 169, 205; or the CDR of the heavy chain contains amino acid sequences 26, 62, 96 and the CDR of the light chain contains amino acid sequences 134, 170. 206; or the CDR of the heavy chain contains amino acid sequences 27, 63, 97 and the CDR of the light chain contains amino acid sequences 135, 171, 207; or the CDR of the heavy chain contains amino acid sequences 28, 64, 98 and the CDR of the light chain contains amino acid sequences 136, 172, 208; or the CDR of the heavy chain contains amino acid sequences 29, 65, 99 and the CDR of the light chain contains amino acid sequences 137, 173, 209; or the CDR of the heavy chain contains amino acid sequences 30, 66, 100 and the CDR of the light chain contains amino acid sequences 138, 174, 210; or the CDR of the heavy chain contains amino acid sequences 31, 67, 101 and the CDR of the light chain contains amino acid sequences 139, 175, 211; Alternatively, the CDR of the heavy chain may contain amino acid sequences 32, 68, and 102, respectively, and the CDR of the light chain may contain amino acid sequences 140, 176, and 212, respectively; or the CDR of the heavy chain may contain amino acid sequences 33, 69, and 102, respectively.The CDRs of the heavy chain and the light chain respectively contain amino acid sequences 141, 177, and 213; or the CDRs of the heavy chain respectively contain amino acid sequences 34, 70, and 104, and the CDRs of the light chain respectively contain amino acid sequences 142, 178, and 214; or the CDRs of the heavy chain respectively contain amino acid sequences 35, 71, and 105, and the CDRs of the light chain respectively contain amino acid sequences 143, 179, and 215; or the CDRs of the heavy chain respectively contain amino acid sequences 36, 72, and 106, and the CDRs of the light chain respectively contain amino acid sequences 144, 180, and 216; or the CDRs of the heavy chain respectively contain amino acid sequences 217, 247, and 277, and the CDRs of the light chain respectively contain amino acid sequences 307, 337, and 367; or the CDRs of the heavy chain respectively contain amino acid sequences 218 and 248. The CDRs of the heavy chain and the light chain respectively contain amino acid sequences 308, 338, and 368; or the CDRs of the heavy chain respectively contain amino acid sequences 219, 249, and 279, and the CDRs of the light chain respectively contain amino acid sequences 309, 339, and 369; or the CDRs of the heavy chain respectively contain amino acid sequences 220, 250, and 280, and the CDRs of the light chain respectively contain amino acid sequences 310, 340, and 370; or the CDRs of the heavy chain respectively contain amino acid sequences 221, 251, and 281, and the CDRs of the light chain respectively contain amino acid sequences 311, 341, and 371; or the CDRs of the heavy chain respectively contain amino acid sequences 222, 252, and 282, and the CDRs of the light chain respectively contain amino acid sequences 312, 342, and 372; or the CDRs of the heavy chain respectively contain amino acid sequence 223. CDRs of the heavy chain and the light chain respectively contain amino acid sequences 313, 343, and 373; or CDRs of the heavy chain respectively contain amino acid sequences 224, 254, and 284, and CDRs of the light chain respectively contain amino acid sequences 314, 344, and 374; or CDRs of the heavy chain respectively contain amino acid sequences 225, 255, and 285, and CDRs of the light chain respectively contain amino acid sequences 315, 345, and 375; or CDRs of the heavy chain respectively contain amino acid sequences 226, 256, and 286, and CDRs of the light chain respectively contain amino acid sequences 316, 346, and 376; or CDRs of the heavy chain respectively contain amino acid sequences 227, 257, and 287, and CDRs of the light chain respectively contain amino acid sequences 317, 347, and 377. Alternatively, the CDR of the heavy chain may contain amino acid sequences 228, 258, and 288, respectively, and the CDR of the light chain may contain amino acid sequences 318 and 348, respectively.378; or the CDR of the heavy chain contains amino acid sequences 229, 259, 289 respectively, and the CDR of the light chain contains amino acid sequences 319, 349, 379 respectively; or the CDR of the heavy chain contains amino acid sequences 230, 260, 290 respectively, and the CDR of the light chain contains amino acid sequences 320, 350, 380 respectively; or the CDR of the heavy chain contains amino acid sequences 231, 261, 291 respectively, and the CDR of the light chain contains amino acid sequences 321, 351, 381 respectively; or the CDR of the heavy chain contains amino acid sequences 232, 262, 292 respectively, and the CDR of the light chain contains amino acid sequences 322, 352, 382 respectively; or the CDR of the heavy chain contains amino acid sequences 233, 263, 293 respectively, and the CDR of the light chain contains amino acid sequence 323 respectively. 353, 383; or the CDR of the heavy chain contains amino acid sequences 234, 273, 294 and the CDR of the light chain contains amino acid sequences 324, 354, 384; or the CDR of the heavy chain contains amino acid sequences 235, 274, 295 and the CDR of the light chain contains amino acid sequences 325, 355, 385; or the CDR of the heavy chain contains amino acid sequences 236, 275, 296 and the CDR of the light chain contains amino acid sequences 326, 356, 386; or the CDR of the heavy chain contains amino acid sequences 237, 276, 297 and the CDR of the light chain contains amino acid sequences 327, 357, 387; or the CDR of the heavy chain contains amino acid sequences 237, 277, 287. The CDRs of the heavy chain and the light chain respectively contain amino acid sequences 328, 358, and 388; or the CDRs of the heavy chain respectively contain amino acid sequences 238, 278, and 299, and the CDRs of the light chain respectively contain amino acid sequences 329, 359, and 389; or the CDRs of the heavy chain respectively contain amino acid sequences 239, 279, and 300, and the CDRs of the light chain respectively contain amino acid sequences 330, 360, and 390; or the CDRs of the heavy chain respectively contain amino acid sequences 240, 280, and 301, and the CDRs of the light chain respectively contain amino acid sequences 331, 361, and 391; or the CDRs of the heavy chain respectively contain amino acid sequences 241, 281, and 302, and the CDRs of the light chain respectively contain amino acid sequences 332, 362, and 392; or the CDRs of the heavy chain respectively contain amino acid sequence 242. CDRs of amino acids 282, 303, and the light chain respectively contain amino acid sequences 333, 363, and 393; or CDRs of the heavy chain respectively contain amino acid sequences 243, 283, and 293.304 and the CDR of the light chain respectively contain amino acid sequences 334, 364, and 394; or the CDR of the heavy chain respectively contains amino acid sequences 244, 284, and 305, and the CDR of the light chain respectively contains amino acid sequences 335, 365, and 395; or the CDR of the heavy chain respectively contains amino acid sequences 245, 285, and 306, and the CDR of the light chain respectively contains amino acid sequences 336, 366, and 396.

[0084] Other anti-influenza antibodies include those with the amino acid or nucleic acid sequences shown in Table 1 below.

[0085] The amino acid sequences of the complementarity-determining regions of the heavy and light chains of the influenza neutralizing antibodies 3I14 and 3I14VLD94N are shown in Table 2 below.

[0086] Table 2 Other binding domains other than scFv can also be used for predefined lymphocyte targeting, such as camel single-domain antibody fragments or receptor ligands, antibody-binding domains, antibody hypervariable loops, or CDRs as non-limiting examples.

[0087] In a preferred embodiment, the transmembrane domain further comprises a stem region between the extracellular ligand-binding domain and the transmembrane domain. As used herein, the term "stem region" generally refers to any oligonucleotide or polypeptide used to link the transmembrane domain to the extracellular ligand-binding domain. In particular, the stem region provides greater flexibility and accessibility for the extracellular ligand-binding domain. The stem region may contain up to 300 amino acids, preferably 10-100 amino acids, and most preferably 25-50 amino acids. The stem region may be derived from all or part of a naturally occurring molecule, such as all or part of the extracellular region of CD8, CD4, or CD28, or all or part of an antibody constant region. Alternatively, the stem region may be a synthetic sequence corresponding to a naturally occurring stem sequence, or it may be a fully synthetic stem sequence. In a preferred embodiment, the stem region is part of the human CD8α chain.

[0088] The signal transduction domain or intracellular signal transduction domain of the CAR of this invention is responsible for intracellular signal transduction after the extracellular ligand-binding domain binds to the target, thereby activating immune cells and inducing an immune response. In other words, the signal transduction domain is responsible for activating at least one normal effector function of immune cells expressing CAR. For example, the effector function of T cells can be cytolytic activity or helper activity, including the secretion of cytokines. Therefore, the term "signal transduction domain" refers to a protein portion that transduces effector signaling and directs the cell to perform specific functions.

[0089] The signal transduction domain comprises two different types of cytoplasmic signaling sequences: a sequence that initiates antigen-dependent primary activation, and a sequence that functions in an antigen-independent manner to provide secondary or co-stimulatory signals. The primary cytoplasmic signaling sequence may contain a signaling motif called an ITAM, a tyrosine-based activation motif of the immune receptor. ITAMs are well-defined signaling motifs found in the intracytoplasmic tails of various receptors that serve as binding sites for syk / zap70 class tyrosine kinases. Examples of ITAMs used in this invention may include non-limiting examples derived from TCRζ, FcRγ, FcRβ, FcRε, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. In a preferred embodiment, the CAR signal transduction domain may comprise an intracytoplasmic domain of the CD3ζ signaling domain or the FcεRIβ or γ chain. In another preferred embodiment, signal transduction is provided by co-stimulation of CD3ζ, together with CD28 and tumor necrosis factor receptor (TNFr), such as 4-1BB or OX40.

[0090] In specific 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 contains 2, 3, 4 or more co-stimulatory molecules in tandem. The co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands required for an effective immune response.

[0091] "Co-stimulatory ligands" are molecules on antigen-presenting cells that specifically bind to homologous co-stimulatory molecules on T cells, thereby providing signals in addition to the primary signals provided by the binding of the TCR / CD3 complex to peptide-loaded MHC molecules. These signals mediate T cell responses, including but not limited to proliferation, activation, and differentiation. Co-stimulatory ligands can include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible co-stimulatory ligands (ICOS-L), intercellular adhesion molecules (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin β receptor, 3 / TR6, ILT3, ILT4, and Toll-binding molecules). Agonists or antibodies to ligand receptors, and ligands that specifically bind to B7-H3. Co-stimulatory ligands also include, in particular, antibodies that specifically bind to co-stimulatory molecules present on T cells, such as, but not limited 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 ligands that specifically bind to CD83.

[0092] "Co-stimulatory molecules" refer to homologous binding partners on T cells that specifically bind to co-stimulatory ligands, thereby mediating cellular co-stimulatory responses, such as, but not limited to, proliferation. Co-stimulatory molecules include, but are not limited to, MHC class 1 molecules, BTLA, and Toll ligand receptors. Examples of co-stimulatory molecules include 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 ligands that specifically bind to CD83. In another specific embodiment, the signal transduction domain is a TNFR-associated factor 2 (TRAF2) binding motif that co-stimulates the cytoplasmic tail of a member family of TNFR cells. The cytoplasmic tails of co-stimulated TNFR family members contain a TRAF2 binding motif, which consists of a major conserved motif (P / S / A)X(Q / E)E) or a minor motif (PXQXXD), where X is any amino acid. In response to receptor trimerization, TRAF proteins are recruited to the intracellular tails of many TNFRs.

[0093] Distinguishing features of suitable transmembrane peptides include their expression on the surface of immune cells, particularly lymphocytes or natural killer (NK) cells, and their ability to interact together to guide a cellular response of immune cells to predefined target cells. The different transmembrane peptides of the CARs of the present invention, comprising extracellular ligand domains and / or signal transduction domains, interact upon binding to target ligands to participate in signal transduction and induce an immune response. The transmembrane domains can be derived from natural or synthetic sources. The transmembrane domains can be derived from any membrane-bound or transmembrane protein.

[0094] As used herein, the term "part" refers to any subset of a molecule, i.e., a shorter peptide. Alternatively, functional variants of the amino acid sequence of a polypeptide can be prepared by mutations in the DNA encoding the polypeptide. Such variants or functional variants include, for example, deletions or insertions or substitutions of residues within the amino acid sequence. Any combination of deletions, insertions, and substitutions can also be made to obtain the final construct, provided that the final construct has the desired activity, particularly exhibiting specific anti-target cell immune activity. The function of the CAR of the present invention within host cells is detectable in an assay suitable for demonstrating the signal transduction potential of the CAR upon binding to a specific target. Such an assay can be used by those skilled in the art. For example, the assay allows for the detection of signal transduction pathways triggered upon target binding, such as assays involving the measurement of increased calcium ion release, intracellular tyrosine phosphorylation, inositol phosphate conversion, or the resulting production of interleukin (IL)2, interferon-γ, GM-CSF, IL-3, and IL-4.

[0095] cell Embodiments of the present invention include cells expressing CARs (i.e., CARTS). Cells can be of any kind, including immune cells capable of expressing CARs for cancer therapy or cells containing an expression vector encoding a CAR, such as bacterial cells. As used herein, the terms “cell,” “cell line,” and “cell culture” are used interchangeably. All these terms also include their progeny, which are all subsequent generations. It should be understood that all progeny may differ due to intentional or unintentional mutations. In the context of expressing a heterologous nucleic acid sequence, “host cell” refers to a eukaryotic cell capable of replicating the vector and / or expressing the heterologous gene encoded by the vector. Host cells can and have been used as recipients of the vector. Host cells can be “transfected” or “transformed,” which refers to the process of transferring or introducing a foreign nucleic acid into a host cell. Transformed cells include primary test cells and their progeny. As used herein, the terms “engineered” and “recombinant” cells or host cells are intended to indicate cells incorporating a foreign nucleic acid sequence, such as a vector. Thus, recombinant cells are distinguishable from naturally occurring cells that do not contain the recombinantly introduced nucleic acid. In embodiments of the present invention, the host cell is a T cell, including cytotoxic T cells (also known as TC, cytotoxic T lymphocytes, CTL, T-killer cells, cytolytic T cells, CD8+ T cells, or killer T cells); NK cells and NKT cells are also included in the present invention.

[0096] Some vectors may use control sequences that allow them to replicate and / or be expressed in prokaryotic and eukaryotic cells. Those skilled in the art will further understand the conditions under which all of the above-mentioned host cells are incubated to maintain them and allow vector replication. Techniques and conditions that allow for the large-scale production of vectors, as well as the generation of nucleic acids encoded by the vectors and their homologous polypeptides, proteins, or peptides, are also understood and known.

[0097] The cells can be autologous cells, cells of the same gene, allogeneic cells, and even, in some cases, xenogeneic cells.

[0098] In many cases, it may be desirable to kill modified CTLs, where treatment may be terminated, cells become tumor-like, or, in the course of a study, cells may be absent after their presence, or other events may occur. For this purpose, the expression of certain gene products can be provided, where modified cells can be killed under controlled conditions, such as induced suicide genes.

[0099] CARTS assembly The present invention also includes CARTS modified to secrete one or more polypeptides. The polypeptides may be, for example, antibodies or cytokines. Preferably, the antibody is specific to CAIX, GITR, PD-L1, PD-L2, PD-1, or CCR4.

[0100] Assembled CARTS have the advantage of simultaneously secreting peptides at target sites, such as tumor sites.

[0101] CART assembly can be constructed by including a nucleic acid encoding a target polypeptide after the intracellular signal transduction domain. Preferably, an internal ribosome entry site (IRES) is present between the intracellular signal transduction domain and the target polypeptide. Those skilled in the art will understand that more than one polypeptide can be expressed by using multiple IRES sequences in tandem.

[0102] In one embodiment, the methods and compositions presented herein provide second-generation target-specific anti-CAIX CAR T cells with the ability to secrete anti-PD-L1 IgG in an RCC environment to combat T cell depletion. Human anti-CAIX CARs containing the CD28 co-stimulatory domain were selected based on their cytotoxic activity and low immunogenicity in human cRCC xenografts in mice. Anti-CAIX CAR cells secreted anti-PD-L1 IgG1 or IgG4, which were extensively compared with unrelated anti-BCMA CARs or anti-CAIX CARs, all of which secreted unrelated anti-SARS IgG1. We have demonstrated stable expression of the CAR and high proliferation of all CAR cells. Anti-CAIX CAR cells exhibited the ability to undergo clonal expansion upon contact with CAIX+ RCC cells and were also activated, releasing high levels of IFNγ and IL-2, which improved tumor suppression. These anti-CAIX CART cells also secrete high levels of anti-PD-L1 IgG1 or IgG4, and these antibodies can specifically interact with PD-L1, inducing in vitro and in vivo downregulation of depletion markers PD-1, Tim-3, and Lag-3. These results indicate that anti-PD-L1 antibodies secreted in the tumor microenvironment can restore T cell depletion and promote the anti-tumor activity of anti-CAIX CART cells. These anti-CAIX CART cells (primarily those secreting anti-PD-L1) also reduced the proliferation of CAIX+ RCC cells, resulting in slow tumor growth, smaller tumor volume, and smaller weight in an orthotopic NSG mouse model of RCC. Furthermore, anti-CAIX CART cells secreting the anti-PD-L1 IgG1 isotype were also able to induce ADCC in vitro and increase the number of human NK cells infiltrating tumor sites in vivo. Human NK cells were injected into only two mice in each group two days before euthanasia to determine the ability of NK cells to recognize the anti-PD-L1 IgG1 isotype in vivo, which was confirmed. Once our previous experience using this mouse model showed that these cells only lasted a few days in their blood, we did not inject human NK cells at the start of treatment.

[0103] In one implementation, CART cells are injected into mice without the addition of interleukin to avoid their influence on the therapeutic effect of individual CART cells.

[0104] In another implementation, cytokines such as IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 can be used to maintain CART cells.

[0105] Cytokines sharing 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 them, compared with IL-2 or IL-15, IL-21 promotes a less differentiated phenotype in a mouse melanoma model, is associated with the enrichment of tumor-specific CD8 T cells, and is accompanied by enhanced anti-tumor effects.

[0106] In some implementations, IL-21 is used to maintain CAR-T cells.

[0107] CAIX is a stable biomarker for the development of targeted systemic cancer therapies because it is overexpressed in many tumors, is significant in cRCC, but is also physiologically expressed in several tissues.

[0108] In one implementation, the anti-CAIX scFv in the CAR recognizes the catalytic domain of CAIX located in the central part of the protein, which can increase its specificity for higher expression sites of CAIX.

[0109] The proposed choice of CAR28 as the co-stimulatory domain for CARs is based on the fact that CD28 CARs guide active proliferative responses and enhance effector function, while 4-1BB-based CARs induce more progressive T cell accumulation, thus balancing the less rapid effectiveness. In one implementation, CD28 is replaced by 41BB in the CAR construct.

[0110] T cell depletion is common in cancer, and these T cells exhibit low proliferative capacity and cytokine production, 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 depletion include the PD-1 / PD-L1 axis.

[0111] The methods and compositions provided herein offer anti-CAIX CART cell secreted anti-PD-L1 IgG1 or IgG4, which can reduce T cell depletion and improve CART cell efficiency in in vitro and in vivo cRCC treatment.

[0112] Importing the construct into CTL The expression vector encoding CAR can be introduced as one or more DNA molecules or constructs, in which at least one marker that allows selection of host cells containing the construct can be present.

[0113] Constructs can be prepared in a conventional manner, in which genes and regulatory regions are appropriately isolated, ligated, cloned into suitable cloning hosts, and analyzed by restriction or sequencing or other convenient methods. Specifically, using PCR, individual fragments comprising all or part of the functional units can be isolated, where one or more mutations can be introduced appropriately using primer repair, ligation, in vitro mutagenesis, etc. Once completed and proven to have a suitable sequence, the construct can then be introduced into CTLs by any convenient method. Constructs can be integrated and packaged into non-replicating defective viral genomes, such as adenovirus, adeno-associated virus (AAV), or herpes simplex virus (HSV), including retroviral or lentiviral vectors, for infection or transfection into cells. If desired, the construct can include viral sequences for transfection. Alternatively, constructs can be introduced via fusion, electroporation, bio-projectiles, transfection, lipid transfection, etc. Before introducing the construct, host cells can be grown and amplified in a culture, then appropriately treated to introduce and integrate the construct. Cells are then amplified and selected using markers present in the construct. Various biomarkers that can be successfully used include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, etc.

[0114] In some cases, target sites for homologous recombination can be provided, where it is desired to integrate the construct into a specific locus. For example, an endogenous gene can be knocked out and replaced (at the same locus or elsewhere) with a gene encoded by the construct using materials and methods known in the art for homologous recombination. For homologous recombination, .OMEGA or O vectors can be used. 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.

[0115] Constructs can be introduced as a single DNA molecule encoding at least a CAR and optionally another gene, or as a different DNA molecule having one or more genes. Other genes include, for example, genes encoding therapeutic molecules or suicide genes. Constructs can be introduced simultaneously or sequentially, each with the same or different markers.

[0116] Vectors containing useful elements, such as bacterial or yeast origins of replication, selectable and / or amplifiable markers, promoter / enhancer elements for expression in prokaryotes or eukaryotes, which can be used to prepare construct DNA libraries and for transfection, are well known in the art and many are commercially available.

[0117] How to use The cells according to the invention can be used to treat cancer, viral infections, or autoimmune diseases in patients in need. In another embodiment, the isolated cells according to the invention can be used to prepare a medicament for treating cancer, autoimmune diseases, or viral infections in patients in need.

[0118] The present invention relies on a method for treating a patient in need, the method comprising at least one step: (a) providing chimeric antigen receptor cells according to the invention, and (b) administering the cells to the patient.

[0119] The treatment can be amenable, curative, or preventative. It can be part of autologous immunotherapy or allogeneic immunotherapy. Autologous means that the cells, cell lines, or cell populations used to treat the patient are derived from the patient or from a human leukocyte antigen (HLA) compatible donor. Allogeneic means that the cells or cell populations used to treat the patient are not derived from the patient but from a donor.

[0120] This invention is particularly applicable to allogeneic immunotherapy, provided it can convert T cells, typically obtained from a donor, into non-allogeneic reactive cells. This can be performed under standard protocols and can be reproduced multiple times as needed. The resulting modified T cells can be combined and administered to one or more patients, and are available as an "off-the-shelf" therapeutic product.

[0121] Cells that can be used with the disclosed methods are described in the preceding sections. The treatment can be used to treat patients diagnosed with cancer, viral infections, autoimmune diseases, or graft-versus-host disease (GvHD). Treatable cancers include tumors that are not vascularized or have not yet been substantially vascularized, as well as vascularized tumors. Cancers can include non-solid tumors (e.g., hematologic malignancies such as leukemia and lymphoma) or can include solid tumors. Types of cancers treated with the CAR of this invention include, but are not limited to, carcinomas, blastomas, and sarcomas, as well as certain leukemias or lymphomas, benign and malignant tumors, and malignant tumors such as sarcomas, carcinomas, and melanomas. Adult tumors / cancers and pediatric tumors / cancers are also included.

[0122] It can be a treatment in combination with one or more cancer-targeting therapies selected from antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser therapy, and radiotherapy.

[0123] According to a preferred embodiment of the invention, the treatment can be administered to a patient undergoing immunosuppressive therapy. In practice, the invention preferably relies on cells or cell populations that have developed resistance to at least one immunosuppressant due to the inactivation of the gene encoding the receptor for such immunosuppressants. In this regard, immunosuppressive therapy should facilitate the selection and expansion of T cells according to the invention within the patient's body.

[0124] In another embodiment, the cell composition of the present invention is administered to the patient in conjunction with bone marrow transplantation, or T-cell ablation therapy using chemotherapy agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAM PATH (e.g., before, during, or after). In another embodiment, the cell composition of the present invention is administered after B-cell ablation therapy, such as an agent that reacts with CD20, such as Rituxan. For example, in one embodiment, the subject may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, after transplantation, the subject receives an infusion of expanded immune cells of the present invention. In other embodiments, the expanded cells are administered before or after the procedure. The modified cells obtained by any of the methods described herein can be used for specific aspects of the invention to treat patients in need against host-vG rejection and graft-versus-host disease (GvHD); therefore, within the scope of the invention is a method for treating patients in need against host-vG rejection and graft-versus-host disease (GvHD) comprising treating the patient by administering an effective amount of modified cells containing inactivated TCRα and / or TCRβ genes.

[0125] Cellular drug delivery This invention is particularly applicable to allogeneic immunotherapy, provided it can convert T cells, typically obtained from a donor, into non-allogeneic reactive cells. This can be accomplished under standard protocols and can be reproduced multiple times as needed. The resulting modified T cells can be combined and administered to one or more patients, and can be obtained as an "off-the-shelf" therapeutic product.

[0126] Depending on the nature of the cells, they can be introduced into a host organism, such as mammals, in a wide variety of ways. In a specific implementation, cells can be introduced at a tumor site, although in alternative implementations, the cells may return to cancer or be modified to return to cancer. The number of cells used will depend on many factors, including the purpose of introduction, cell lifespan, the protocol used, such as the number of administrations, the cell's proliferative capacity, and the stability of the recombinant construct. Cells can be administered as a dispersion, typically injected at or near the target site. Cells can be in physiologically acceptable culture media.

[0127] In some implementations, cells are encapsulated to inhibit immune recognition and placed at the tumor site.

[0128] Cells can be administered as needed. Various regimens can be employed, depending on the desired response, route of administration, cell lifespan, and the number of cells present. The number of administrations depends at least in part on the factors mentioned above.

[0129] The administration of cells or cell populations according to the invention can be performed in any convenient manner, including by aerosol inhalation, injection, ingestion, infusion, implantation, or transplantation. The compositions described herein can be administered to patients subcutaneously, intradermally, intratumorally, intralymphaticly, intramedullaryly, intramuscularly, intravenously, intralymphaticly, or intraperitoneally. In one embodiment, the cell compositions of the invention are preferably administered by intravenous injection.

[0130] Cells or cell populations can be administered at a rate of 10 per kg of body weight. 4 -10 9 10 cells, preferably 10 5 Up to 10 6 The dosage composition is cells / kg body weight, including integer values ​​for the number of cells within these ranges. Cells or cell populations can be administered in one or more doses. In another embodiment, the effective amount of cells is administered in a single dose. In another embodiment, the effective amount of cells is administered in more than one dose over a period of time. The timing of administration is within the judgment of the attending physician and depends on the patient's clinical condition. Cells or cell populations can be obtained from any source, such as a blood bank or donor. While individual needs vary, the optimal range for determining the effective amount of a given cell type for a particular disease or condition is within the scope of the art. An effective amount is the amount that provides therapeutic or preventative benefit. The dosage administered will depend on the recipient's age, health, and weight, as well as the type of treatment (if any), the frequency of treatment, and the nature of the desired effect.

[0131] It should be understood that this system is subject to many variables, such as cellular response to the ligand, expression efficiency, and, where appropriate, secretion levels, activity of the expressed product, patient-specific needs, which may vary over time and circumstances, and the rate of loss of cell activity due to cell loss or loss of expression activity in individual cells. Therefore, it is anticipated that, for each individual patient, even if universal cells could be administered to a large population, appropriate dosing for each individual patient will be monitored, and such patient monitoring is routine in the art.

[0132] Nucleic acid-based expression systems The CAR of this invention can be expressed by an expression vector. The recombination techniques for generating such expression vectors are well known in the art.

[0133] carrier The term "vector" is used to refer to a vector nucleic acid molecule in which a nucleic acid sequence can be inserted to introduce it into a cell where it can replicate. The nucleic acid sequence can be "exogenous," meaning that the sequence is foreign to the cell to which the vector is introduced, or that the sequence is homologous to a sequence in the cell but located within the host cell's nucleic acid, where the sequence is not normally present. Vectors include plasmids, granules, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YAC). Those skilled in the art are fully capable of constructing vectors using standard recombination techniques (see, for example, Maniatis et al., 1988 and Ausubel et al., 1994, both incorporated herein by reference).

[0134] The term "expression vector" refers to any type of genetic construct containing nucleic acids encoding RNA that can be transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. In others, these sequences are not translated, such as in the production of antisense molecules or ribozymes. Expression vectors can contain a variety of "control sequences," which are nucleic acid sequences required for transcription and, possibly, translation of operable linked coding sequences in a specific host cell. In addition to control sequences that regulate transcription and translation, vectors and expression vectors can contain nucleic acid sequences that are also used for other functions, as described below.

[0135] Promoters and enhancers A promoter is a control sequence, a region of a nucleic acid sequence that controls the initiation and rate of transcription. It may contain genetic elements that regulate the binding of proteins and molecules, such as RNA polymerases and other transcription factors, to initiate specific transcription of the nucleic acid sequence. The terms "operably positioned," "operably linked," "under control," and "under transcriptional control" refer to the promoter being in the correct functional position and / or orientation relative to the nucleic acid sequence to control transcription initiation and / or expression of that sequence.

[0136] Promoters typically contain sequences that locate the site of RNA synthesis initiation. The most well-known example is the TATA box, but in some promoters lacking a TATA box, such as the promoters of mammalian terminal deoxynucleotidyl transferase genes and SV40 late-stage genes, discrete elements covering the start site itself help determine the initiation location. Additional promoter elements regulate the frequency of transcription initiation. They are typically located in a region 30–110 bp upstream of the start site, although many promoters also show functional elements downstream of the start site. To bring the coding sequence “under promoter control,” the 5’ end of the transcription start site of the transcription reading frame is placed “downstream” (i.e., 3’) of the chosen promoter. The “upstream” promoter stimulates DNA transcription and promotes the expression of the coding RNA.

[0137] The spacing between promoter elements is typically flexible, allowing promoter function to be preserved when elements are flipped or moved relative to each other. In the tk promoter, the spacing between promoter elements can increase to 50 bp before activity begins to decline. Depending on the promoter, individual elements appear to act synergistically or independently to activate transcription. Promoters may or may not be used in conjunction with "enhancers," which are cis-regulatory sequences involved in the transcriptional activation of nucleic acid sequences.

[0138] Promoters can be promoters naturally associated with a nucleic acid sequence, such as those obtained by isolating a 5' "non-coding sequence" located upstream of a coding region and / or exon. Such promoters can be referred to as "endogenous." Similarly, enhancers can be enhancers naturally associated with a nucleic acid sequence located downstream or upstream of that sequence. Alternatively, certain advantages can be obtained by placing the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is normally not associated with a nucleic acid sequence in its natural environment. Recombinant or heterologous enhancers also refer to enhancers that are normally not associated with a nucleic acid sequence in their natural environment. Such promoters or enhancers can 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 occurring," i.e., containing different elements of different transcriptional regulatory regions, and / or mutations that alter expression. For example, the promoters most commonly used for recombinant DNA construction include the lactamase (penicillinase), lactose, and tryptophan (trp) promoter systems. In addition to synthetically generating nucleic acid sequences for promoters and enhancers, recombinant cloning and / or nucleic acid amplification technologies (including PCR.TM) can be used in conjunction with the compositions disclosed herein (see U.S. Patent Nos. 4,683,202 and 5,928,906, each incorporated herein by reference). Furthermore, control sequences that direct the transcription and / or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, etc., can also be used.

[0139] Of course, it is important to use effective guides to select promoters and / or enhancers for the expression of DNA fragments in organelles, cell types, tissues, organs, or organisms. Those skilled in the art of molecular biology are generally familiar with the use of combinations of promoters, enhancers, and cell types for protein expression (see, for example, Sambrook et al. 1989, incorporated herein by reference). The promoters used can be constitutive, tissue-specific, inducible, and / or useful under appropriate conditions to guide high-level expression of the introduced DNA fragment, for example, facilitating large-scale production of recombinant proteins and / or peptides. Promoters can be heterologous or endogenous.

[0140] Additionally, any promoter / enhancer combination can be used to drive expression. Using T3, T7, or SP6 cytoplasmic expression systems is another possible implementation. Eukaryotic cells can support cytoplasmic transcription of certain bacterial promoters if appropriate bacterial polymerases are provided, either as part of the delivery complex or as additional genetic expression constructs.

[0141] The identification of tissue-specific promoters or elements and the methods for characterizing their activity are well known to those skilled in the art.

[0142] Effective translation of the encoded sequence may also require specific start signals. These signals include the ATG start codon or adjacent sequences. External translation control signals, including the ATG start codon, may need to be provided. Those skilled in the art will readily be able to determine this and provide the necessary signals.

[0143] In some embodiments of the invention, internal ribosome entry site (IRES) elements are used to generate multigene or polycistronic information, and these can be used in the invention.

[0144] Vectors may include multiple cloning sites (MCS), which are nucleic acid regions containing multiple restriction enzyme sites, any one of which can be used in conjunction with standard recombinant techniques to digest the vector. "Restriction enzyme digestion" refers to the catalytic cleavage of a nucleic acid molecule by an enzyme that functions only at a specific site on the nucleic acid molecule. Many of these restriction enzymes are commercially available. The use of these enzymes is widely understood by those skilled in the art. Typically, restriction enzymes that cleave within the MCS are used to linearize or fragment the vector to allow the foreign sequence to be ligated to the vector. "Lie-off" refers to the process of forming a phosphodiester bond between two nucleic acid fragments, which may or may not be adjacent to each other. Techniques involving restriction enzymes and ligation reactions are well known to those skilled in the art of recombinant technology.

[0145] You can also use splice sites, termination signals, replication start points, and selection markers.

[0146] plasmid vector In some implementations, plasmid vectors are considered for transforming host cells. Typically, plasmid vectors containing replicons and control sequences derived from a species compatible with the host cells are used in conjunction with these hosts. The vectors typically carry a replication site, as well as marker sequences that provide phenotypic selection in the transformed cells. In a non-limiting example, *E. coli* is typically transformed using a derivative of the plasmid pBR322, derived from the *E. coli* species. pBR322 contains ampicillin and tetracycline resistance genes, thus providing a convenient means of identifying transformed cells. The pBR plasmid or other microbial plasmids or phages must also contain, or be modified to contain, promoters that can be used by the microbial organism to express its own proteins.

[0147] Furthermore, phage vectors containing replicons and control sequences compatible with the host microorganism can be used as transformation vectors associated with these hosts. For example, phage λGEM.TM.11 can be used to prepare recombinant phage vectors that can be used to transform host cells such as Escherichia coli LE392.

[0148] Other useful plasmid vectors include the pIN vector (Inouye et al., 1985) and the pGEX vector, used to generate soluble glutathione S-transferase (GST) fusion proteins for subsequent purification and isolation or cleavage. Other suitable fusion proteins are those containing galactosidase, ubiquitin, etc.

[0149] Bacterial host cells containing expression vectors, such as *Escherichia coli*, are grown in any of many suitable culture media, such as LB. As those skilled in the art will understand, expression of recombinant proteins in certain vectors can be induced by contacting the host cells with certain promoter-specific reagents, for example, by adding IPTG to the culture medium or by increasing the incubation temperature. After culturing the bacteria for an additional period, typically 2 to 24 hours, the cells are collected by centrifugation and washed to remove residual culture medium.

[0150] Viral vector The ability of certain viruses to infect or enter cells via receptor-mediated endocytosis and to integrate into the host cell genome and stably and efficiently express viral genes has made them attractive candidates for transferring exogenous nucleic acids into cells (e.g., mammalian cells). Components of the present invention may be viral vectors encoding one or more CARs of the present invention. Non-limiting examples of viral vectors that can be used to deliver the nucleic acids of the present invention are described below.

[0151] Adenovirus vector Specific methods for delivering nucleic acids involve the use of adenoviral expression vectors. Although adenoviral vectors are known to have a low ability to integrate into genomic DNA, this characteristic is offset by the high efficiency of gene transfer provided by these vectors. "Adenoviral expression vector" is intended to include those constructs containing an adenoviral sequence sufficient to (a) support construct packaging and (b) ultimately express the tissue- or cell-specific construct cloned therein. Knowledge of genomic constructs or adenoviruses (a 36 kb linear double-stranded DNA virus) allows for the replacement of large swaths of adenoviral DNA with exogenous sequences up to 7 kb (Grunhaus and Horwitz, 1992).

[0152] AAV carrier Adenovirus-assisted transfection can be used to introduce nucleic acids into cells. Improved transfection efficiency has been reported in cell systems using adenovirus-coupled systems (Kelleher and Vos, 1994; Cotten et al., 1992; Curiel, 1994). Adeno-associated virus (AAV) is an attractive vector system for use in the cells of this invention because of its high integration frequency and ability to infect non-dividing cells, making it suitable for gene delivery into mammalian cells, for example, in tissue culture (Muzyczka, 1992) or in vivo. AAV has a broad range of infectious hosts (Tratschin et al., 1984; Laughlin et al., 1986; Lebkowski et al., 1988; McLaughlin et al., 1988). Details regarding the production and use of rAAV vectors are described in U.S. Patent Nos. 5,139,941 and 4,797,368, each of which is incorporated herein by reference.

[0153] Retroviral vector Retroviruses can be used as delivery vectors because they are able to integrate their genes into the host genome, transfer large amounts of foreign genetic material, infect a wide range of species and cell types, and be packaged in specialized cell lines (Miller, 1992).

[0154] To construct retroviral vectors, nucleic acids (e.g., those encoding the desired sequence) are inserted into the viral genome, replacing certain viral sequences to produce replication-defective viruses. To generate viral particles, packaging cell lines containing the gag, pol, and env genes but lacking the LTR and packaging components are constructed (Mann et al., 1983). When a recombinant plasmid containing cDNA is introduced into a specialized cell line along with the retroviral LTR and packaging sequence (e.g., by calcium phosphate precipitation), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture medium (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The culture medium containing the recombinant retrovirus is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are capable of infecting a variety of cell types. However, integration and stable expression require host cell division (Paskind et al., 1975).

[0155] Lentivirals are complex retroviruses that contain other genes with regulatory or structural functions in addition to the common retroviral genes gag, pol, and env. Lentiviral vectors are well known in the art (see, for example, Naldini et al., 1996; Zufferey et al., 1997; Blomer et al., 1997; US 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 produced through replication, and HIV virulence-attenuating genes, such as env, vif, vpr, vpu, and nef genes, are deleted, making the vector biologically safe.

[0156] Recombinant lentiviral vectors can infect non-dividing cells and can be used for in vivo and in vitro gene transfer and nucleic acid sequence expression. For example, recombinant lentiviruses capable of infecting non-dividing cells, wherein suitable host cells are transfected with two or more vectors carrying packaging functions, namely gag, pol, and env, and rev and tat, are described in U.S. Patent 5,994,136, which is incorporated herein by reference. Recombinant viruses can be targeted by linking envelope proteins to antibodies or specific ligands to target receptors on specific cell types. By inserting the target sequence (including regulatory regions) and, for example, another gene encoding a ligand on a specific target cell into the viral vector, the vector becomes target-specific.

[0157] Other viral vectors Other viral vectors can be used as vaccine constructs in this invention. Vectors derived from viruses 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 provide several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990).

[0158] Using modified virus delivery The nucleic acid to be delivered can be placed in an infectious virus engineered to express a specific binding ligand. Thus, the viral particle will specifically bind to the homologous receptor of the target cell and deliver its contents into the cell. A novel method for designing retroviral vectors to allow specific targeting is developed based on the chemical modification of retroviruses by chemically adding lactose residues to the viral envelope. This modification can allow specific infection of hepatocytes via the sialic acid glycoprotein receptor.

[0159] Another approach to targeting recombinant retroviruses was devised, employing biotinylated antibodies against retroviral envelope proteins and specific cellular receptors. This was achieved by using streptavidin conjugated with a biotinylated component of an antibody (Roux et al., 1989). Using antibodies against major histocompatibility complex class I and II antigens, they demonstrated in vitro infection of various human cells carrying those surface antigens with isotropic viruses (Roux et al., 1989).

[0160] Vector delivery and cell transformation Suitable methods for nucleic acid delivery for cell transfection or transformation are known to those skilled in the art. These methods include, but are not limited to, direct delivery of DNA, such as by in vitro transfection, by injection, etc. Cells can be stably or transiently transformed by applying techniques known in the art.

[0161] In vitro transformation In vitro methods for transfecting eukaryotic cells and tissues removed from a living organism are known to those skilled in the art. Therefore, it is contemplated that the nucleic acids of the present invention can be used for in vitro removal and transfection of cells or tissues. In a particular aspect, transplanted cells or tissues can be placed in a living organism. In a preferred aspect, the nucleic acids are expressed in the transplanted cells.

[0162] The reagent kit of the present invention Any of the compositions described herein may be included in a kit. In a non-limiting example, one or more cells for cell therapy and / or reagents for generating one or more cells for cell therapy containing a recombinant expression vector may be included in the kit. Kit components are provided in suitable container devices.

[0163] Some components of the kit may be packaged in an aqueous medium or in lyophilized form. The container device of the kit typically includes at least one vial, test tube, flask, bottle, syringe, or other container device in which components can be placed, preferably appropriately aliquoted. If the kit contains multiple components, it will typically also include a second, third, or other additional container in which the additional components can be placed separately. However, various combinations of components can be included in vials. The kit of the present invention will also typically include means for containing commercially marketable, sealed-containment components. Such a container may include an injection-molded or blow-molded plastic container in which the desired vial is held.

[0164] When the kit components are provided in one and / or more liquid solutions, the liquid solutions are aqueous solutions, and sterile aqueous solutions are particularly useful. In some cases, the container device itself may be a syringe, pipette, and / or other similar device from which the preparation can be applied to the infected area of ​​the body, injected into an animal, and / or even applied to and / or mixed with other components of the kit.

[0165] However, the kit components can be provided as dry powders. When reagents and / or components are provided as dry powders, the powder can be reconstituted by adding a suitable solvent. It is conceivable that the solvent may also be provided in a separate container. The kit may also include a second container for containing sterile, pharmaceutically acceptable buffers and / or other diluents.

[0166] In specific embodiments of the invention, cells for cell therapy are provided in the kit, and in some cases, cells are essentially the only component of the kit. The kit may include reagents and materials for preparing the desired cells. In specific embodiments, the reagents and materials include primers, nucleotides, suitable buffers or buffering agents, salts, etc., for amplifying the desired sequence; in some cases, the reagents include vectors and / or DNA encoding CARs and / or their regulatory elements as described herein.

[0167] In a specific implementation, the kit includes one or more devices suitable for extracting one or more samples from an individual. These devices may be syringes, scalpels, etc.

[0168] In some embodiments of the present invention, in addition to the cell therapy implementation, the kit also includes a second cancer therapy, such as chemotherapy, hormone therapy, and / or immunotherapy. The kit can be tailored to an individual's specific cancer and includes a corresponding second cancer therapy for that individual.

[0169] Combination therapy In some embodiments of the invention, the clinically applicable method of the invention is combined with other pharmaceuticals, such as anticancer agents, that are effective in treating hyperproliferative diseases. These "anticancer" agents can negatively affect a subject's cancer, for example, by 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 increasing the lifespan of a subject with cancer. More generally, these other compositions will be provided in combined amounts that effectively kill or inhibit cell proliferation. The method may include simultaneously contacting cancer cells with an expression construct and a reagent or multiple factors. This can be done by contacting cells with a single composition or pharmaceutical formulation comprising two reagents or by simultaneously contacting cells with two different compositions or formulations, one composition comprising an expression construct and the other comprising a second reagent.

[0170] Tumor cell resistance to chemotherapy and radiotherapy agents is a major problem in clinical oncology. A current goal of cancer research is to seek ways to improve the efficacy of chemotherapy and radiotherapy by combining them with other therapies. In the context of this invention, it is contemplated that cell therapy can be used similarly in combination with chemotherapy, radiotherapy, or immunotherapy interventions, as well as with pro-apoptotic agents or cell cycle regulators.

[0171] Alternatively, the therapy of the present invention can be administered before or after other drug treatments at intervals ranging from minutes to weeks. In embodiments where other reagents and the present invention are applied to individuals, it is generally ensured that there is not a considerably long period between each delivery such that the reagent and the therapy of the present invention can still exert a beneficial combined effect on the cells. In this case, it is considered that the cells may be contacted in two ways within approximately 12-24 hours of each other, more preferably within approximately 6-12 hours of each other. However, in some cases, it may be necessary to significantly extend the treatment time, with intervals between administrations ranging from several days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks).

[0172] Treatment cycles are expected to be repeated as needed. It is also considered that the cell therapy of this invention can be used in combination with various standard therapies and surgical interventions.

[0173] Chemotherapy Cancer treatment also includes various combination therapies that combine chemotherapy and radiation therapy. Combination chemotherapy therapies include, for example: abraxane, altretamine, docetaxel, Herceptin, methotrexate, novoflurane, zoledronic acid, cisplatin (CDDP), carboplatin, procarbazine, nitrogen mustard, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosamine, daunorubicin, doxorubicin, bleomycin, propram, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binders, paclitaxel, gemcitabine, novibenten, farnesyltransferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine, and methotrexate, or any of the foregoing analogues or derivatives thereof, or combinations thereof.

[0174] In specific implementations, an individual's chemotherapy is used in conjunction with the present invention, for example, before, during, and / or after administration of the present invention.

[0175] Radiation therapy Other factors that cause DNA damage and are widely used include those commonly referred to as gamma rays, X-rays, and / or the targeted delivery of radioactive isotopes to tumor cells. Other forms of DNA damage factors, such as microwave and UV irradiation, are also considered. All of these factors are most likely to cause extensive damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray doses range from 50–200 roentgens per day for extended periods (3 to 4 weeks) to 2000–6000 roentgens per single dose. Radioactive isotope doses vary considerably depending on the isotope's half-life, the intensity and type of radiation, and tumor cell uptake.

[0176] The terms “contact” and “exposed” used in this article, when applied to cells, describe methods of delivering a therapeutic construct and a chemotherapy or radiotherapy agent to or placing them directly alongside target cells. To achieve cell killing or arrest, two agents are delivered to the cells in combined amounts that effectively kill or prevent cell division.

[0177] Immunotherapy Immunotherapy typically relies on the use of immune effector cells and molecules to target and destroy cancer cells. Immune effectors can be, for example, antibodies specific to certain markers on the surface of tumor cells. An antibody alone can serve as an effector of the therapy, or it can recruit other cells to actually influence cell killing. Antibodies can also be conjugated to drugs or toxins (chemotherapy agents, radionuclides, ricin A chain, cholera toxin, pertussis toxin, etc.) and used solely as a target. Alternatively, effectors can be lymphocytes carrying surface molecules that interact directly or indirectly with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0178] Therefore, immunotherapies other than those described herein can be combined with the cell therapies of this invention as part of a combination therapy. The general approach to combination therapies is discussed below. Typically, tumor cells must possess some suitable markers for targeting, i.e., not present on most other cells. Many tumor markers exist, and any one of these may be suitable for targeting in the context of this invention. Common tumor markers include PD-1, PD-L1, CTLA4, carcinoembryonic antigen, prostate-specific antigen, urinary tumor-associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialic acid Lewis antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B, and p155.

[0179] Gene In another embodiment, the second treatment is gene therapy, wherein the therapeutic polynucleotide is administered before, after, or simultaneously with the clinical implementation of the invention. Various expression products are included in the invention, including cell proliferation inducers, cell proliferation inhibitors, or programmed cell death regulators.

[0180] Operation Approximately 60% of cancer patients will undergo some type of surgery, including preventative, diagnostic or staging, curative, and palliative surgeries. Curative surgeries are cancer treatments that can be used in combination with other therapies, such as the treatments of this invention, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies.

[0181] Curative surgery includes resection in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed. Tumor resection refers to the physical removal of at least part of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microsurgical manipulation (Mohs surgery). Furthermore, the present invention can be used in conjunction with the removal of surface cancer, precancerous tissue, or incidental amounts of normal tissue.

[0182] After all cancerous cells, tissue, or part of a tumor are removed, a cavity may form in the body. Treatment can be administered via perfusion, direct injection, or local application of additional anticancer therapies to the area. This treatment can 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 can also be administered at different doses.

[0183] Other reagents Other agents can be combined with this invention to improve therapeutic efficacy. These additional agents include immunomodulators, agents affecting the upregulation of cell surface receptors and GAP linkages, cell inhibitors and differentiation agents, cell adhesion inhibitors, or agents that increase the sensitivity of overproliferating cells to apoptosis inducers. Immunomodulators include tumor necrosis factor; interferon α, β, and γ; IL-2 and other cytokines; F42K and other cytokine analogs; or MIP-1, MIP-1β, MCP-1, RANTES, and other chemokines. Further consideration is that upregulation of cell surface receptors or their ligands, such as Fas / Fas ligands, DR4, or DR5 / TRAIL, will enhance the apoptosis-inducing capacity of this invention by establishing autocrine or paracrine effects on overproliferating cells. Increasing intercellular signaling by increasing GAP linkage number will increase the anti-overproliferative effect on adjacent overproliferating cell populations. In other embodiments, cell growth inhibitors or differentiation agents can be combined with this invention to improve the anti-proliferative efficacy of the treatment. Cell adhesion inhibitors are considered to improve the efficacy of this invention. Examples of cell adhesion inhibitors include focal adhesion kinase (FAK) inhibitors and lovastatin. Furthermore, other agents that increase the sensitivity of overproliferating cells to apoptosis, such as antibody C225, can be used in combination with this invention to improve therapeutic efficacy.

[0184] The present invention also relates to the following embodiments: 1. A chimeric antigen receptor (CAR) comprising an intracellular signal transduction domain, a transmembrane domain, and an extracellular domain.

[0185] 2. The CAR of implementation scheme 1, wherein the transmembrane domain further comprises a stem region located between the extracellular domain and the transmembrane domain.

[0186] 3. The CAR of embodiment 1, wherein the transmembrane domain comprises CD28.

[0187] 4. The CAR of embodiment 1, further comprising one or more additional co-stimulatory molecules located between the transmembrane domain and the intracellular signal transduction domain.

[0188] 5. The CAR of implementation scheme 4, wherein the co-stimulatory molecule is CD28, 4-1BB, ICOS or OX40.

[0189] 6. The CAR of implementation scheme 1, wherein the intracellular signal transduction domain comprises the CD3ζ chain.

[0190] 7. The CAR of implementation scheme 1, wherein the extracellular domain is an antibody.

[0191] 8. The CAR of implementation scheme 7, wherein the antibody is Fab or scFV.

[0192] 9. The CAR of embodiment 7, 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.

[0193] 10. A nucleic acid encoding a CAR of any of the foregoing embodiments, further comprising a nucleic acid encoding a polypeptide located after an intracellular signal transduction domain.

[0194] 11. The nucleic acid of implementation scheme 10, wherein the polypeptide is an antibody.

[0195] 12. The nucleic acid of implementation scheme 11, wherein the antibody is scFV.

[0196] 13. The nucleic acid of implementation scheme 12, wherein the antibody is specific for BCMA, CD138, CCR4, PD-1, PDL-1, PD-L2, CXCR4, GITR, SARS virus, flavivirus, MERS virus, influenza virus, or CAIX.

[0197] 14. A vector comprising a nucleic acid of any one of embodiments 11-13.

[0198] 15. A cell comprising a vector according to embodiment 14.

[0199] 16. A genetically engineered cell that expresses and loads a chimeric antigen receptor of any one of embodiments 1-9 onto its cell surface membrane.

[0200] 17. The genetically engineered cell of embodiment 16, wherein the cell is a T cell or an NK cell.

[0201] 18. The genetically engineered cells of embodiment 17, wherein the T cells are CD4+ cells. + or CD8 + .

[0202] 19. The genetically engineered cell of implementation scheme 18, which contains CD4 + and CD8 cells + A mixed group.

[0203] 20. The cells of embodiment 16, wherein the cells are further engineered to express and secrete peptides.

[0204] 21. The cells of embodiment 20, wherein the expressed and secreted polypeptide is anti-PDL-1. Example

[0205] Example 1: Materials and Methods Cells, culture media, and reagents. Human CAIX+ renal cell carcinoma lines sk-rc-52, sk-rc-09, and CAIX-sk-rc-59 were obtained from Dr. Gerd Ritter at Memorial Sloan-Kettering Cancer Center, New York. They were cultured in R-10 complete medium (Life Technologies) supplemented with 10% FCS, 2 mmol / L L-glutamine, 100 U / ml penicillin, and 100 μg / ml streptomycin (Sigma) at 37°C and 5% CO2. Primary human T cells were maintained in R-10 cells with 10% human serum and 100 IU / ml recombinant human interleukin-2 (IL-2) (Chiron). Human embryonic kidney cell line 293T (ATCC) and mouse fibroblast NIH3T3 cells (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 were obtained from the Boston Children's Hospital Blood Bank from healthy volunteers who had given written informed consent.

[0206] In one implementation, the human ccRCC cell lines Skrc52, initially CAIX+ / PD-L1-, and Skrc59, initially CAIX- / PD-L1+, 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 with 5% CO2. Skrc52 cells were selected from CAIX- / PD-L1 and CAIX+ / PD-L1- cell populations using fluorescence activated cell sorting (FACS). Skrc59 cells were engineered to express high levels of human CAIX and CAIX+ / PD-L1+ cells were selected using FACS.

[0207] Anti-PD-L1 scFv-Fc IgG1 and IgG4 were cloned into a bicis-trans lentiviral vector encoding anti-CAIX second-generation CAR. The anti-PD-L1 antibody (Ab) used to construct CART cells was previously selected in paramagnetic protein liposomes using a human scFv phage display library targeting 27 billion members of full-length PD-L1 (manuscript in preparation). The DNA sequence encoding anti-PD-L1 scFv (clone 42)-Fc IgG1 or IgG4 was codon-optimized and synthesized (Genewiz) with 5'NdeI and 3'ClaI restriction sites to allow for further scFv-Fc cloning, replacing ZsGreen in the lentiviral vector pHAGE-eIFα signal scFvG36 (anti-CAIX)-C9TAG-linker-CD28-CD3ζ-IRES-ZsGreen (e.g., CD28z). This original anti-CAIX vector was previously constructed and published (21). For the negative control clone, firstly, the anti-PD-L1 scFv was replaced with the anti-SARS scFv (clone 11A) in the IgG1-Fc lentiviral vector containing the anti-CAIX G36scFv CAR. The DNA sequence encoding the anti-SARS scFv was amplified by PCR from the plasmid pHAGE CMV-Anti-SARS(11A)scFv-Fc-CD28-gp41-IRES-ZsGreen, and 5' MluI and 3' XbaI restriction sites were inserted. The clone was further cloned using the MluI forward primer 5'TCG ACG CGT GAG GTG CAG CTG GTG CAG T 3' and the XbaI reverse primer 5'TCC TCTAGA CAG GAC GGT GAC CTT GGT CC 3'. We used the final pHAGE-eIFα-scFv G36 (anti-CAIX)-C9TAG-linker-CD28-CD3ζ-IRES-anti-SARS(11A)IgG1 as one of the negative controls. We also replaced the anti-CAIX scFv in the CAR structure containing anti-SARS IgG1 with anti-B cell maturation antigen (BCMA) scFv by double digesting pHAGE-eIFα-A716scFv (anti-BCMA)-C9TAG-linker-CD28-CD3ζ-IRES-ZsGreen and pHAGE-eIFα-G36scFv (anti-CAIX)-C9TAG-linker-CD28-CD3ζ-IRES-anti-SARS(11A)IgG1 with anti-BCMA scFv by double digesting pHAGE-eIFα-A716scFv (anti-BCMA)-C9TAG-linker-CD28-CD3ζ-IRES-anti-SARS(11A)IgG1 with NcoI and NotI.Following the cloning process, we obtained four major plasmids to initiate lentivirus production: an anti-CAIX CAR (anti-CAIX / anti-PD-L1 IgG1) expressing anti-PD-L1 IgG1, an anti-CAIX CAR (anti-CAIX / anti-PD-L1 IgG4) expressing anti-PD-L1 IgG4, an anti-CAIX CAR (anti-CAIX / anti-SARS IgG1) expressing irrelevant anti-SARS Ab, and an anti-BCMA CAR (anti-BCMA / anti-SARS IgG1) expressing irrelevant anti-SARS Ab.

[0208] scFv isolation and conversion of scFv into scFv-Fc. As previously reported, CAIX-specific scFv antibodies were isolated from non-immune human scFv phage libraries and submitted to GenBank, accession numbers GQ903548-GQ903561. 23 The scFv-encoding DNA fragment from the pFarber phage was digested using the SfiI / NotI site and subcloned into the mammalian expression vector pcDNA3.1-F105L hinge filler containing the human IgG1 F105 leader sequence and the human IgG1 hinge-CH2-CH3 Fc portion to express the scFv-Fc antibody. The scFv-Fc plasmid was transiently transfected into 293T cells using Lipofectamine 2000 (Invitrogen), and the expressed antibody was purified using Sepharose Protein A beads (Amersham Bioscience). Specific binding to CAIX was tested by staining with phage scFv antibody or antibodies converted from scFv to the scFv-Fc form after incubation with CAIX-expressing 293T and sk-rc-52 cell lines, and CAIX-negative 293T and sk-rc-59 cell lines. Unrelated anti-HIV CCR5 antibody (clone A8) was also tested in these experiments. 25 Or anti-SARS antibody (11A) 24 A separate fluorescently conjugated second antibody was used as a negative control.

[0209] Constructs of scFv-CD8-TCRζ and scFv-CD28-TCRζ were created. The DNA constructs of Pz1, scFv-CD8-TCRζ, and P28z, scFv-CD28-TCRζ, in the phage vector pSL1180, were obtained from Dr. Michel Sadelain of Memorial Sloan-Kettering Cancer Center, New York. In Pz1, the intracellular domains of scFv and TCRζ were attached to the N- and C-termini of the human CD8α chain, respectively. Similarly, in P28z, the scFv and TCRζ sequences were attached to the N- and C-termini of human CD28, respectively. The amino acid sequence of human CD8α is 71 residues long, consisting of 47 (aa137–183), 23 (aa184–206), and 2 (aa207–208) residues in the extracellular and hinge, transmembrane, and cytoplasmic domains of CD8α. The CD28 sequence in 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 of CD28. The intracellular domain of human CD3ζ, common to both CARs, consists of 112 amino acids (aa 52-163).

[0210] The nucleic acid sequence encoding the internal C9-tag (a nine-amino acid peptide of human rhodopsin, TETSQVAPA) with a GGGGS adapter was amplified by PCR and fused upstream with CD8-TCRζ and CD28-TCRζ sequences having 5' NotI and 3' PacI sites. Primers used to clone the chimeric TCRζ construct were 5' TAG GGC. GCG GCC GC a acc gag accagc cag gtg gcg ccc gcc GGG GGA GGA GGC AGC CCC ACC ACG ACG CCA GCG CCG CGA 3' (forward primer for the CD8 construct, where italics represent NotI sites, uppercase letters represent C9 tag sequences, and underscores indicate GGGGS adapters), 5' TAG GGC GCG GCC GC a acc gag acc agc cag gtg gcg ccc gcc GGC GGA GGA GGC AGC ATT GAA GTT ATG TAT CCT CCT CCT 3' (forward primer for the CD28 construct) and reverse primers for the two constructs CTA GCC T T AAT TAA TTA GCG AGG AGG GGG CAG GGC CTG CAT, with italics indicating PacI sites. These DNA fragments encode functional features arranged in the following order: Not I – C9tag (TETSQVQPQ) – GGGGS – CD8 or CD28 – TCRζ – Pac I. A chimeric TCR construct labeled with an internal C9 peptide was cloned into a pcDNA3.1-F105L hinge-filled vector containing anti-CXCR4 scFv-Fc, clone 48, using NotI and PacI restriction sites. This design allowed us to insert a chimeric TCR receptor construct to replace the Fc fragment. Anti-CAIX scFv (clone G36) and anti-CCR5 scFv (clone A8, as an unrelated scFv control) antibody fragments were then cloned to replace the anti-CXCR4 scFv at the SfiI / NotI site to generate a CAIX-specific chimeric TCR construct.

[0211] The lentiviral vector pHAGE-CMV-DsRed-IRES-ZsGreen and four HIV helper plasmids pHDM-Hgpm2 (HIVgag-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 with high affinity for CAIX+ cells and high ADCC only against CAIX+ tumor cells, was cloned into the pHAGE-EF1α lentiviral vector to replace the first cassette of the DsRed protein.

[0212] Lentiviral production and transduction of human primary T cells. Lentiviral virus was generated by transiently transfecting 293T cells with five plasmids using lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). Cells at 80% confluence were prepared in 15 cm culture dishes (NalgeNunc) 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 20:1:1:2:2. Viral supernatant was collected on day 3 after changing to D-10 medium, filtered through a 0.45 μm filter, and concentrated by ultracentrifugation (Beckman Coulter, Fullerton, CA) at 16,500 rpm (48,960 × g, Beckman SW28 rotor) at 4°C for 90 min. The virus particles were resuspended in R-10 medium and kept frozen at -80°C.

[0213] In one implementation, lentivirus was generated by transiently transfecting five plasmids into 293T cells using polyethyleneimine (PEI). Briefly, 80% confluence 293T cells in 15cm plates were transfected with a total of 30 μg of each of the five plasmids: 5 μg of each structural plasmid pHDH-Hgpm2 (HIV gag-pol), pMD-tat, pRC / CMV-rev, and EnvVSV-G, and 10 μg of the major plasmid encoding the 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 a Lenti-X concentrator (Clontech) according to the manufacturer's instructions and frozen at -80°C.

[0214] Human PBMCs were isolated using a Ficoll density gradient and activated for 4 days with 2 μg / ml PHA (Sigma) and 100 IU / ml human IL-2. Cells were then transduced with lentiviruses with a multiplicity of infection (MOI) of 10-20 in the presence of 10 μg / ml DEAE for 2 or 3 rounds. Three days post-transduction, transduced T cells were collected for in vitro phenotypic and functional analysis, or expanded for in vivo experiments.

[0215] Selection, activation, and lentiviral vector transduction of CD8+ T cells. Blood rings were obtained from healthy volunteers with written informed consent from the blood bank of Brigham and Woman' Hospital (Boston, MA). Human peripheral blood mononuclear cells (PBMCs) were isolated using a Ficoll-Paque PLUS (GE Healthcare, NJ). CD8-positive cells (Life Technologies) were isolated from PBMCs using Dynabeads and cultured in RPMI 1640 medium (Life Technologies) containing 10% heat-inactivated fetal bovine serum, 20 mM HEPES, 100 IU / mL penicillin, and 100 μg / mL streptomycin, with 50 IU / mL IL-21 (Peprotech) added to the medium every 2 days. Assays were performed to determine whether IL-2 or IL-21 was the optimal cytokine for inducing the proliferation of anti-CAIX CART cells, with each cytokine at 50 IU / mL. CD8+ T cells were activated using Dynabeads Human T-Activator CD3 / CD28 (Life Technologies) at a 1:1 ratio. Cells were transduced with lentivirus at 20 and 10 μg / mL of diethylaminoethyl. All assays were performed in triplicate, using T cells from three different healthy donors.

[0216] Flow cytometry analysis. Transduction efficiency of human primary T cells was assessed by reporter gene (ZsGreen) expression. CAIX-Fc protein was expressed from pcDNA3.1 plasmid encoding amino acids 38-397 of CAIX, followed by the human IgG1 hinge, CH2, and CH3 domains, with the CAIX signal peptide (aa 1-37) replaced by an Ig leader sequence. Expression of scFv (G250) on transduced T cells was tested by staining with 1 μg of CAIX-Fc protein followed by APC-conjugated mouse anti-human IgG antibody (Jackson ImmunoResearch). Additionally, expression of the internal rhodopsin nonapeptide (TETSQVAPA) C9 tag within the scFv domain of the TCR construct on 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, human cell subpopulations cultured during clonal expansion experiments were stained with fluorescently conjugated mouse anti-human antibodies (Invitrogen) against CD3 (clone S4.1), CD4 (clone S3.5), or CD8 (clone 3B5). In all cell staining, half a million cells were stained with the recommended antibody concentration according to the company's instructions. Matched isotype control antibodies were used for each sample, and cells were analyzed using a FACSCalibur cytometer (Becton-Dickinson).

[0217] In one implementation, the transduction of 293T cells or CD8+ T cells was confirmed by FACS analysis of anti-CAIX or anti-BCMA expression. Cells were stained with 10 μg / mL human CAIX-Fc or human BCMA-mouse-Fc (ABBioscience) produced in our laboratory, followed by imaging with 1:250 APC-conjugated mouse anti-human IgG Ab (Southern Biotech) or goat anti-mouse IgG Ab (Biolegend). CountBright™ Absoluate counting beads (Molecular Probes) were used for proliferation and clonal expansion assays. All samples were analyzed using LSR Fortessa or FACSCalibur (BD Bioscience), and data were analyzed using FlowJo software. To analyze the T cell depletion 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. Subsequently, CART cells were co-cultured with Skrc-59 CAIX+ PD-L1+ cells for 2 days to stimulate depletion. Using 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, 1×10⁻⁶ antibodies from this assay were analyzed. 6 CART cells and tumor-infiltrating lymphocytes (TILs) collected in vivo were stained and analyzed by FACS. To validate the expression levels of CAIX and PD-L1 in different RCC cell lineages used in this project, we used 10 μg / mL of anti-human CAIX mAb (clone G36) produced in our laboratory and 10 μg / mL of biotinylated mouse anti-human PD-L1 (Biolegend). The primary antibody was detected using 1:250 APC-conjugated anti-human Ab and PE-conjugated avidin, and the results were analyzed by FACS.

[0218] ADCC and cytotoxicity assays of lentivirally transduced T cells. Cytotoxicity assays were performed using the DELFIAEuTDA Cytotoxicity Kit (Perkin Elmer, Boston, MA) according to the manufacturer's instructions. In short, target tumor cells were labeled with the fluorescent ligand (BATDA) at 37°C for 30 min, and 1 × 10⁻⁶ ppm was added to each well of a 96-well U-plate. 4Labeled cells were prepared. For antibody-dependent cytotoxicity (ADCC) assays, anti-CAIX scFv-Fc antibody at concentrations of 1 μg / ml or 5 μg / ml, or an unrelated scFv-Fc antibody group, were added. Assays were established using effector cells (human PBMCs) to target cells (E:T) at ratios of 50:1, 25:1, and 12.5:1. For T cell cytotoxicity assays, effector cells (untransduced or transduced T cells) to target cells (E:T) at different ratios (100:1, 50:1, and 25:1). Cultures were incubated at 37°C in humidified 5% CO2 for 4 hours. After centrifugation at 500×g for 5 minutes, 20 μl of the supernatant was transferred to a flat-bottomed plate. 200 μl of europium solution was added, and the culture was analyzed using a fluorometer (Victor). TM PerkinElmer reads the fluorescence released from the cells. A spontaneous release control was prepared by culturing only labeled cells, and a maximum release control was prepared by adding lysis buffer (provided in the kit) to the labeled cells.

[0219] ELISA, ELISPOT assay, and Western blotting were performed. For cytokine secretion, RCC cell lines sk-rc-52 (CAIX+) or sk-rc-59 (CAIX-) were incubated in 24-well plates at 1 × 10⁻⁶ cells per well. 6 One cell was seeded overnight, followed by 1×10⁶ cells. 6 Untransduced or transduced T cells were used. Before co-culturing with tumor cells, T cells were washed twice with PBS to remove human IL-2. After overnight incubation, the supernatant was collected and IL-2 and IFN-γ were analyzed by ELISA (e-Bioscience). In T cells used for the IFN-γ ELISPOT assay (e-Bioscience), the membrane was visualized using AEC substrate solution, and the number of spots was counted using an ELISPOT plate reader (CTLCellular Technology).

[0220] For Western blotting, the preparation of untransduced and transduced T cells was described. 50One million cells were prepared in non-reducing and reducing buffers (0.1 M dithiothreitol) and run on a 10–20% polyacrylamide gradient gel (Invitrogen). Proteins were transferred to a polyvinylidene fluoride transfer membrane (NEN Life Science Products, Boston, MA) overnight at 100 V, 4 °C. The membrane was incubated with a 1:2000 dilution of the primary antibody, anti-human ζ-chain monoclonal antibody 8D3 (BD ​​Pharmingen, San Diego, CA), followed by a 1:3000 dilution of the secondary antibody, horseradish peroxidase (Caltag). Immunoassay was performed using an ECL Plus Western blot detection system (GE Healthcare, Piscataway, NJ) and X-ray film exposure.

[0221] IgG secreted by CART cells was detected using ELISA. The total level of IgG secreted into the transduced cell culture medium was detected using a Human IgG ELISA Quantitation Set (Bethyl Laboratories). Anti-PD-L1 antibodies secreted by transduced CD8+ CART cells were purified using Protein A agarose beads (GE Healthcare) and biotinylated using EZ-LinkSulfo-NHS-LC-Biotin (Thermo Scientific). These antibodies were incubated with 5 μg / mL of laboratory-produced human PD-L1, which was pre-fixed in MaxiSorp plates (Nunc) at RT for 2 hours. Biotinylated antibodies were detected by incubation with streptoacidin-HRP for 1 hour and developed using SureBlue™ TMB peroxidase substrate and TMB stop solution (KPL). Absorbance was read at λ = 450 nm.

[0222] Tumor cells proliferate, expand their clonal range, and secrete cytokines upon contact. Tumor cells are irradiated (3000 rad) at 2.5 × 10⁶ ppm per well. 5 Inoculation. In a culture medium containing R-10 and 100 IU / ml human IL-2, inoculate at a rate of 1×10⁻⁶. 6 T cells were added and cultured for one week. The T cells were then separated to maintain an appropriate density and restimulated weekly with tumor cells. T cell counts were performed every 3 or 4 days for 2 weeks. The percentage of ZsGreen expression in transduced T cells and T cell subsets was determined weekly using fluorescence activated cell sorting (FACS). For studies of cytokine secretion after tumor cell contact, T cells washed with irradiated tumor cells for one or two weeks were incubated overnight with fresh tumor cells, and the culture supernatant was collected for analysis after 24 hours.

[0223] Clonal expansion of CAIX+ CART cells.

[0224] In one embodiment, Skrc52 CAIX+ / PD-L1- and Skrc52 CAIX- / PD-L1 cells were irradiated with 3,000 rad and spaced at 2.5 × 10⁶ cells per well. 5 Inoculation. Add 1×10⁻⁶ ppm to culture medium containing 50 IU / ml human IL-21 every two days. 6 10 T cells. The T cells were separated to maintain an appropriate density and restimulated weekly with tumor cells. The T cell count was performed weekly by FACS for 3 weeks.

[0225] Effects of anti-CAIX CART cells secreting anti-PD-L1 antibodies on RCC cell viability and antibody-dependent cytotoxicity (ADCC). 2.5 × 10⁻⁶ cells were used. 3 Skrc59 CAIX+ / PD-L1+ and Skrc52 CAIX- / PD-L1- were seeded overnight in 96-well plates. Four days after CAR-T cell transduction, 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. CAR-T cells were removed, and tumor cell viability (MTT) was measured (Life Technologies). For ADCC assays, RCC cells were incubated at 37°C with 50 μL of CAR-T cell supernatant adjusted to contain 500 ng / mL of the corresponding anti-PD-L1 IgG1, anti-PD-L1 IgG4, or anti-SARS IgG1. Cells were then incubated with NK cells at 37°C for 4 hours at a ratio of 12.5:1, 25:1, or 50:1, and lactate dehydrogenase (LDH) was measured in the supernatant using the CytoTox 96® non-radioactive cytotoxicity assay (Promega).

[0226] An ELISA assay was used to detect IL-2 and IFNγ released by functional CART cells. To analyze cytokine secretion, 2.5 × 10⁻⁶ cells were used. 3 RCC cells (Skrc59 CAIX+ / PD-L1+ or Skrc52 CAIX- / PD-L1) were seeded in 96-well plates overnight, followed by incubation overnight with CAR-transduced T cells at ratios of 5:1, 25:1, and 50:1. The supernatant was removed, and IL-2 and IFNγ secretion were analyzed using a human IFNγ or human IL-2 ELISA Ready-SET-Go kit (eBioscience).

[0227] Establishment of an orthotopic renal cell carcinoma model and CAR-T cell therapy. 5×10 4One passage of Skrc59 CAIX+ / PD-L1+ cells was suspended in 10 μL of culture medium and incubated on Matrigel. TM The drug (Life Technologies) was diluted 1:1 and injected subcapsularly into the left kidney of 6-8 week old male NSG mice (N = 35). One week later, tumor implantation was confirmed by bioluminescence (BLI) imaging using the Xenogen IVIS imaging system (Life Technologies). A 1.0 × 10⁻⁶ mcg of the drug was injected into the left kidney of the mice. 7 CAR T cells (anti-BCMACAR / 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 at 7, 14, 23, and 30 days after CAR cell injection. A second injection of 2.5 × 10⁻⁶ cells was given on day 17. 6 CAR or untransfected T cells. Mice were sacrificed 30 days after tumor implantation via standard CO2 inhalation, and tumors were harvested and weighed. Kidney tumors from all mice were divided into two aliquots. One aliquot was fragmented into small pieces and digested to TILs with 0.5 U / mL collagenase and DNase 1.0 mg / mL. The extracted TILs were analyzed by FACS for the expression of depletion markers and the percentage of CAR-T cells. The other aliquot was fixed in 10% buffered formaldehyde and immunohistochemically analyzed for different markers. Two days prior to euthanasia, two mice in each group were injected with 4.5 x 10⁻⁶ T cells. 6 NK cells. NK cells present in the tumor were stained with APC-anti-CD56 Ab and analyzed by FACS. Animal experiments were conducted according to the guidelines of the DFCI Animal Care Committee.

[0228] Tumor establishment and T-cell therapy. In one implementation, due to immune rejection of sk-rc-52 in 6-8 week old female BALB / c nude mice and to accelerate in vivo growth characteristics, 5 million cells were subcutaneously inoculated into mice, harvested, and expanded in vitro. The cell line was then passaged twice more in nude mice, and the passaged cells were expanded for further experiments (subclone 4-1). For the therapeutic experiment, 5 million sk-rc-59 and 7.5 million passaged sk-rc-52 cells were subcutaneously inoculated into nude mice on opposite sides to produce comparable tumor growth rates. After 7 days, when the tumors had grown to approximately 6 mm in size, 50 million untransduced or transduced T cells were intravenously injected. Mice were also treated with 20,000 IU of human IL-2 via peritoneal injection every two days. Tumor size was measured using calipers in two dimensions, and the average of the two tumor diameters is reported in this paper. Animal experiments were conducted according to the guidelines of the Animal Care Committee of the Dana Farber Cancer Institute. When the tumor reached 15 mm in diameter or 2,000 mm... 3 Mice were euthanized and tumors were harvested.

[0229] Immunohistochemistry and immunofluorescence staining. For in vitro examination of transduced T cells, cultured T cells were washed twice with PBS and resuspended in PBS for 15 min at 37°C in 2 μM Far Red DDAO-SE CellTrace dye (Molecular Probe). Cells were then washed twice with culture medium and centrifuged on slides. CART cells pre-stained with Far Red and co-expressing ZsGreen were visualized using confocal microscopy (Zeiss) at the Optical Imaging Core facility, Harvard NeuroDiscovery Center.

[0230] To examine the in situ killing effect of transduced T cells in the tumor bed, frozen sections of tumor cells were prepared for use with the ApopTag peroxidase in situ apoptosis assay 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 the addition of Cy3-conjugated anti-rabbit antibody (Invitrogen) and incubation for another 30 minutes. The sections were fixed with DAPI anti-fading mounting medium, and fluorescence images were examined using confocal microscopy.

[0231] Xenograft tumors and mouse spleens were harvested, fixed in 10% formalin / PBS, and delivered to the Harvard Medical School, Rodent Histopathology Core Facility. Paraffin-embedded sections were dewaxed with xylene and rehydrated with a gradient of alcohols before staining. Immunohistochemical staining was performed by incubation for 1 hour with anti-human granzyme B antibody (Dako, clone GrB-7 (1:200)) as the primary antibody, followed by incubation for 30 minutes with either anti-rabbit antibody (Pierce) or anti-mouse antibody (Dako). Sections were developed using DAB substrate and counterstained with hematoxylin.

[0232] In one embodiment, fixed tumor tissue is paraffin-embedded, sectioned into four-micrometer sections, and placed on a glass slide, ready for IHQ. The tissue is 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-antibiotin staining. The slide is developed using DAB and counterstained with hematoxylin. Images are acquired using a DP71 digital camera (Olympus) on an Olympus BX51 microscope and analyzed in DP controller software (Olympus). Image quantification was performed using the IHC Profiler plugin in ImageJ software, as described below: 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.

[0233] Statistical analysis Use a two-tailed Student's t-test to determine statistical significance.

[0234] Unless otherwise stated, with Figure 16-23 Relevant statistical analyses represented at least three experiments. Statistical significance of the data was assessed using ANOVA and Tukey post-test. A p-value < 0.05 was considered significant. Statistical analyses were performed using IBM SPSS Statistics software version 20.

[0235] Example 2: ADCC-mediated killing of anti-CAIX antibody and selection of CAR targeting moieties We previously reported a group of high-affinity human anti-CAIX antibodies that differed in their epitope mapping, expression levels, and ability to internalize CAIX. 23 Our primary objective was to investigate the antitumor activity of these five anti-CAIX single-chain antibodies as candidates for CAR construction. To test anti-CAIX mAb-mediated ADCC, scFv was converted into scFv-Fc(hIgG1) mini-antibodies. 23 We found that all scFv-Fc cells exhibited antigen-specific tumor lysis. For the sk-rc-09 tumor cell line with high CAIX+ expression, the specific lysis range was 40-57%; for sk-rc-52 with moderate CAIX+ expression, the specific lysis range was 46-60%; and for the CAIX- tumor cell line sk-rc-59, the lysis background was <5%. For negative control scFv-Fc cells, such as anti-CXCR4 48-Fc... 23 and anti-SARS 11A-Fc 24 Only background-level cell lysis was observed (Figure 1). Based on ADCC killing and other published analyses, scFvG36 was selected for further evaluation as a CAR targeting component.

[0236] Construction and expression of CAIX-specific chimeric receptors. Two generations of anti-CAIX CARs were constructed: a first-generation G36 CD8 CAR, in which scFvG36 is coupled with a truncated extracellular, hinge, and transmembrane domain of CD8 plus a TCRζ signaling domain (G36-CD8z). For delivery of co-stimulatory signals, a second-generation CD28 CAR was generated, consisting of a truncated extracellular, transmembrane, and intracellular domain fused to CD28 plus a TCRζ signaling domain (G36-CD28z) (Figure 2A). An unrelated second-generation CD28 CAR was prepared by replacing the scFv with an anti-HIV CCR5 (clone A8) scFv. To detect the expression of these constructs, a human rhodopsin C9 tag was inserted between the scFv and either the CD8 or CD28 domain, and ZsGreen was expressed after the IRES sequence. High concentrations of viral stock were obtained at comparable levels by co-transfecting the vector plasmids into different constructs in 293T cells (data not shown).

[0237] For transduction, peripheral blood lymphocytes were stimulated with PHA mitogen for 3 days. Concentrated lentiviral supernatant was used to infect human primary T cells in the presence of the cationic reagent DEAE, as it showed a 1.5–2-fold increase in transduction rate compared to polybrene (data not shown). In FACS analysis, the transduction rate of primary T cells ranged from 17% to 45% based on ZsGreen expression. A representative experiment showing approximately 25% ZsGreen expression in primary CART cells after lentiviral transduction is shown in Figure 2B, left column. The CAIX-Fc fusion protein bound to G36-CD8z and -CD28z CART cells but not to control A8-CD28z CART cells (Figure 2B, middle column). C9-tag expression was detected at only about one-third of the CAIX-Fc protein level (Figure 2B, right column), which may be related to the following finding: preferential recognition of rhodopsin nonapeptide C9 was found when mAb 1D4 was at the carboxyl terminus, relative to the internal polypeptide sequence (data not shown). ZsGreen expression was maintained in transduced cells cultured in vitro for 6 weeks.

[0238] In Western blots under reducing conditions, G36 and A8 CD28z CARs migrated at a molar weight of approximately 53 kDa, while the endogenous TCRζ migrated at 16 kDa. The G36-CD8z CAR migrated at a molar weight of approximately 48 kDa. Under non-reducing conditions, the two CD28z CARs formed a homodimer (Figure 2C, data for CD8z CAR are not shown).

[0239] Example 3: Transduced T cells enhance cytokine secretion after contact with CAIX+ tumor.

[0240] This study aimed to compare the reported superior effects of using second-generation G36-CD28z CART cells. Compared to first-generation G36-CD8z CART cells, second-generation G36-CD28z CART cells incorporate the signaling component of the co-stimulatory molecule CD28 to bypass MHC presentation and enhance T cell effector function. As shown in Figure 3A, after overnight incubation with CAIX + sk-rc-52 cells, low levels of type I cytokines IL-2, IFNγ, and IL-17 secretion were observed in only control A8 CD28z CART cells or LAK cells alone. In contrast, both first-generation and second-generation G36 CART cells expressing CART showed increased cytokine secretion levels, with second-generation G36-CD28z CART cells secreting significantly more type I cytokines compared to first-generation G36-CD8z CART cells, reflecting their higher activation state. Specifically, G36-CD28z CART cells secreted 6.5 times, 2.3 times, and 4 times more IL-2, IFNγ, and IL-17 than G36-CD8z CART cells, respectively. Minimal stimulation with CAIX-sk-rc-59 cells demonstrated the specificity of cytokine secretion induced by the two G36 CART cell types.

[0241] In the Elispot study, G36-CD28z CART cells became high-yield IFN-γ producing cells after interaction with CAIX+ sk-rc-52 tumor cells (Figure 3B). G36-CD28z CART cells produced 6 times more spots than G36-CD8z CART cells after interaction with CAIX+ sk-rc-52 tumor cells, and 12 times more after interaction with CAIX-sk-rc-59 tumor cells. Similarly, G36-CD28z CART cells exhibited a higher amount of granzyme B-secreting spots after contact with CAIX+ tumors compared to G36-CD8z CART cells and control T cells. PMA and iomycin-stimulated T cells produced the highest amounts of IFN-γ and granzyme B-secreting T cells. These studies demonstrate the specificity and high yield of G36-CD28z CART cell activation through contact with CAIX+ tumor cells.

[0242] Example 4: Specific cytotoxicity through transduced CAR signaling in T cells.

[0243] An in vitro cytotoxicity assay was established to further evaluate the cytotoxic activity of different G36 CART cells. Using different effector-target ratios, G36-CD28z CART cells and their subclones 4-1, which were passaged twice in vivo, showed the highest levels of cell lysis in CAIX+ tumor sk-rc-52 (Fig. 3C). At a high effector-target ratio exceeding 25:1, G36-CD28z CART cells showed 2-3 times higher cytotoxicity compared to G36-CD8z CART cells; while at a low ratio of 5:1, G36-CD28z CART cells showed 8-9 times higher lysis compared to G36-CD8z CART cells. However, even using an E:T ratio of 100:1, G36-CD8z CART cells still exhibited cytotoxicity with tumor lysis exceeding 60%. Unrelated A8-CD28z CART cells and control T cell LAK cells showed background nonspecific tumor lysis, with approximately 20% lysis when using the highest E:T ratio of 100:1. In all cases using CAIX-tumor sk-rc-59, both transduced and untransduced T cells showed background lysis.

[0244] Example 5: Enhancing in vitro proliferation of CAIX+ tumor cells in CART cells using prolonged CAIX+ tumor incubation. In addition to enhanced cytokine secretion and cytotoxicity upon short-term CAIX+ tumor cell contact, CD28 co-stimulatory molecule incorporation into the CAR construct showed increased proliferation upon prolonged contact with antigen-specific tumor cells. Untransduced and transduced (approximately 20%) T cells were mixed weekly with freshly irradiated tumor cells in the presence of 100 units / ml human IL-2. To test different antigen stimulation levels for a fixed number of T cells, we used tumor cell to T cell ratios of 1:8, 1:4, and 1:2. T cell counts were excluded by trypan blue exclusion, and CAR cell fractionation was examined by flow cytometry. The number of transduced and untransduced T cells was maintained in culture with CAIX-sk-rc-59 tumor cells (Figure 4A bottom). The lack of baseline proliferation in control T cells is likely 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 number of G36-CD28z CART cells increased 30-fold, and the number of G36-CD8z CART cells increased 17-fold. At a ratio of 1:4, the number of G36-CD28z CART cells increased 19-fold, and the number of G36-CD8z CART cells increased 4-fold. Neither G36-CD28z nor G36-CD8z CART cells proliferated with increasing tumor cell numbers. Unrelated A8-CD28z CART cells and control T cell LAK cells did not show tumor cell proliferation (Figure 4A top).

[0245] Proliferating T cells were also harvested to examine their enrichment following exposure to CAIX+ tumor cells. The percentage of any CART cells within the population remained unchanged upon exposure to CAIX-tumor cells. However, upon exposure to CAIX+ sk-rc-52 tumor cells, both populations of G36CART cells were enriched. For 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 increased from 19% on day 0 (at the same level only on T cells) to 32% on day 8, reaching 72% on day 16. No expansion of A8-CD28z CART cells was observed during the two-week study (Figure 4B). The percentage of CD8 cells remained constant throughout the 16-day study under all conditions (Figure 4C).

[0246] Example 6: The sustained effector function of CART cells after re-contact with the tumor.

[0247] Cytokine secretion by transduced T cells was also tested 24 hours after exposure to fresh, unirradiated tumor cells, followed by exposure to irradiated tumor cells for one or two weeks. G36-CD28z and G36-CD8z CART cells showed similar IFN-γ secretion levels after one or two weeks of exposure to CAIX+ tumors (sk-rc-52), although G36-CD28z CARs produced 2 to 2.5 times more IFN-γ secretion relative to G36-CD8z CARs via co-stimulatory signaling (Table 1). For IL-2 secretion, both G36-CD28z and G36-CD8z CART cells showed more IL-2 secretion after two weeks of tumor exposure than after one week. G36-CD28z CART cells produced 5 times more IL-2 than G36-CD8z CART cells after one week of exposure, and 2.5 times more after two weeks. Furthermore, G36-CD28z CART cells exposed to tumor cells for two weeks secreted 3.3 times more IL-2 than those exposed to a single tumor exposure, while G36-CD8z CART cells exposed to tumor cells for two weeks secreted 6.8 times more IL-2 than those exposed to tumor cells for one week. These results indicate that the transduced CART cells did not become depleted after a second tumor stimulation and maintained functional activity. When exposed to CAIX-sk-rc59 cells, A8-CD28z, LAK, and G36CART cell treatments showed only background levels of INF-γ and IL-2 secretion.

[0248] Example 7: Suppressing established tumors via CAR-T cells We next tested CAR-T cells to inhibit the growth of established tumor cells in nude mice inoculated with SK-RC-52 tumor cells on the left side and SK-RC-59 tumor cells on the right side, where established cells were used to obtain similar tumor curves. On day 7 post-tumor implantation, with a typical tumor size of approximately 6 × 6 mm, 50 million G36-CD28z CAR-T cells, A8-CD28z CAR-T cells, or untransduced T cells (LAK) were administered intravenously. High-dose IL-2 (2 × 10⁻⁶) was also administered intraperitoneally. 5 In the presence of IU, adoptive T-cell therapy was performed in two separate trials: one with n = 7 tumor sizes in the first trial and the other with n = 8 tumor sizes in the second trial. T-cell therapy was not included to compare tumor growth and the effectiveness of the cell therapy.

[0249] In the first trial, the mean size of treated and untreated CAIX-sk-rc-59 tumors was 6.09 ± 0.02 mm on day 4 and 9.29 ± 0.12 mm on day 25 (across all four test groups). They exhibited the same tumor growth rate in both the control and T-cell therapy groups. Untreated CAIX+ tumors receiving no T-cell therapy showed similar tumor size to CAIX- tumors, with a mean size of 6.09 ± 0.13 mm on day 4 and 9.15 ± 0.11 mm on day 25. However, at each time point examined in the 25-day study, the tumor size in mice treated with G36-CD28z CART cells showed a statistically significant reduction compared to mice without T-cell therapy. Figure 5 On day 7 (p<0.05) and day 25 (p<0.001), G36-CD28z CART treatment also resulted in a greater reduction in tumor size compared to mice treated with A8-CD28zCART cells and LAK, as calculated by a two-tailed t-test. In the second experiment, by day 29, the tumor size in mice treated with G36-CD28z CART cells was significantly smaller than that in mice without T-cell treatment. Mice treated with G36-CD28z CART cells also had smaller tumors than mice treated with A8CD28z CART cells and LAK, p<0.01 on days 8–26 and p<0.001 on day 29. Figure 5 ).

[0250] Partial regression of CAIX+ tumors was considered when the tumor size was less than 30% of the control CAIX- tumor volume in the same mouse that received the same T cells. Partial tumor regression was observed in a high percentage of cases using G36-CD28z CART cells (10 / 15, (67%)), but was not common with unrelated target A8-CD28z CART cells (1 / 15, (7%)) and activated T cell LAK cells (2 / 15, (13%)) (Table 2).

[0251] The frequency of partial regression was found to be statistically significant in mice treated with G36-CD28z CART cells compared to control A8-CD28z CART cells and LAK cells, with p<0.001 and p<0.005 respectively, as determined by Fisher's test.

[0252] Example 8: In situ cytotoxicity of CART cells.

[0253] Samples of the entire transduced T cell population used for in vivo studies were pre-stained with Far Red dye, and CART cells expressing ZsGreen protein within the population were analyzed by confocal microscopy. These results demonstrated a transduction efficiency of approximately 30%, consistent with our FACS analysis (Figure 6A).

[0254] To demonstrate that G36-CD28z CART cell therapy induced apoptotic killing in CAIX+ sk-rc-52 tumor cells in vivo, tumor sections were stained using tunneling assays. On day 3 post-adoptive T-cell therapy, tunneling staining identified apoptotic tumor cells (red) at the tumor periphery (top row, Fig. 6B) and within the tumor bed (middle row, Fig. 6B). Apoptotic tumor cells lost DAPI nuclear staining. A magnified image (bottom row, Fig. 6B) shows ZsGreen-expressing CART cells interacting with two apoptotic tumor cells.

[0255] Due to limitations in fluorescence signal, ZsGreen-expressing CART cells could not be observed from the entire tissue section. Therefore, on day 3 after treatment with G36-CD28z CART cells or LAK, tumors were harvested, and sections were stained with granzyme B antibody to locate activated T cells. In Figure 6C, the dark brown stained area shows granzyme B+ T cells observed infiltrating into the CAIX+ sk-rc-52 tumor section (Figure 6C, top left). These granzyme B+ T cells were observed both around the tumor (Figure 6C, top left (a) and center) and inside the tumor (Figure 6C, top left (b) and bottom). Tumors with necrotic areas are shown on H&E-stained slides (Figure 6C, center and bottom right marked n) and located near the granzyme B+ T cells. In contrast, CAIX+ sk-rc-52 tumors treated with control activated T cells (LAK) ( Figure 7 No granzyme B+ T cells were observed. Similarly, those treated with G36-CD28z CART cells ( Figure 8 ) or treated with LAK ( Figure 9 CAIX-sk-rc-59 showed low background staining during tumor proliferation. For a positive control of granzyme B staining, CART cells were locally injected into sk-rc-52 tumors established in mice. One day later, the mice were sacrificed, and tumor tissue sections were prepared for this staining. Figure 10 ).

[0256] Example 9: In a humanized mouse model, anti-PD-L1 antibody-releasing anti-carbonic anhydrase IX chimeric antigen receptor T cells restored T cell depletion and induced renal cell carcinoma regression. Characterization of anti-CAIX CAR T cells secreting anti-PDL1 IgG1 or IgG4 To develop novel CAR therapies for CAIX + RCC, we engineered a bicistronic lentiviral vector to express anti-CAIX(G36)scFv linked to the CD28 and CD3-ζ signaling domains (G36-CD28z CAR in the first expression cassette and anti-PD-L1 IgG1 or IgG4 in the second expression cassette, following the IRES site). Figure 16 A). By inserting an anti-PD-L1 IgG sequence into a lentiviral vector to prevent T cell depletion, we propose that this may improve the efficacy of G36-CD28z CART cells. Figure 16B). As a control, we used anti-CAIXCAR or anti-BCMA CAR containing unrelated anti-SARS IgG1 mAb in the second box. Lentiviral cells generated from these constructs were transduced into CD8 T cells and cultured in the presence of IL-21, which produced CAR-T cells with a more moderate improvement in proliferation than that seen with IL-2. Figure 21 A and 21B), while maintaining the same specific killing activity against CAIX + RCC (A and 21B). Figure 21 C and 21D). The percentages of CAIX and PD-L1 expression in all RCC lineages used in our experiments are shown in the figure. Figure 22 middle.

[0257] Figure 23 The study demonstrated the functionality of CAR-T cells, showing that CD8 T cells transduced with all CARS were able to proliferate in the presence of IL-21 and anti-CD8 / CD28 antibodies. Figure 23 A and 23B), reaching 65-90% transduction level after four days ( Figure 23 C). Fourteen days after transduction, we assessed the stable long-term expression of CARs by integrating lentiviruses; all CARs remained at approximately 25-50% ( Figure 16 C). The total IgG level secreted by CD8 T cells was also measured, and after 4 days it was approximately 300-650 ng / mL. Figure 16 D). The binding specificity of anti-PD-L1 IgG1 and IgG4 antibodies to human PD-L1 was also confirmed. Figure 16 E). The levels of biotinylated anti-PDL1 IgG1 and IgG4 were significantly lower than total IgG, which can be explained by the fact that some IgG was wasted during purification. The ability of anti-CAIXCART cells to undergo clonal expansion in the presence of CAIX+ RCC cells was determined. Figure 16 (F and 16G). Anti-CAIX CART cells did not expand significantly in the presence of CAIX-RCC cells.

[0258] Effector activity of anti-CAIX CART cells. All anti-CAIX CART cells were able to induce a decrease in Skrc59 CAIX+ / PD-L1+ activity by approximately 50-70%, indicating that the anti-PD-L1 IgG1 and IgG4 secreted by some CART cells did not increase cell death under these assay conditions. Figure 17 A). Anti-CAIX CART cells induced the release of IL-2 and IFNγ in the presence of CAIX+ / PD-L1+ cells, indicating specific activation of anti-CAIX CART cells (respectively). Figure 17 C and 17E).

[0259] Regarding anti-CAIX CART cells secreting anti-PD-L1 IgG, a unique differential effect was observed for the IgG1 isotype. When incubated with natural killer (NK) cells, they induced approximately 60% ADCC in CAIX+ / PD-L1+ RCC cells. Figure 17 G). In the presence of CAIX- / PD-L1 cells, no cell viability, cytokine secretion, or ADCC effect was detected in any CAR-T cells. Figure 17 B, 17D, 17F, and 17H).

[0260] Anti-CAIX CART cells secreting anti-PD-L1 antibodies can reduce T cell depletion in vitro. After induced depletion, compared with parental anti-CAIX or unrelated anti-BCMA CART cells, the anti-PD-L1 IgG1 and IgG4 anti-CAIX CART groups (which were respectively...) Figure 18 In cells A, 18B, and 18C), the depletion markers Lag-3, Tim3, and PD-1 were found to be reduced by approximately 50%. At this point, the cytotoxic activity of anti-CAIX CART cells without anti-PD-L1 was retested on Skrc59CAIX+ / PD-L1+ cells. Figure 18 As shown in Figure D, anti-CAIX CART cells lost their cytotoxic activity against CAIX+ / PD-L1+ RCCs in vitro to levels similar to those of the unrelated CAR group, demonstrating that anti-CAIX CART cells became incompetent. In contrast, the RCC activity of the anti-CAIX CAR anti-PD-L1 IgG1 and IgG4 CART groups was reduced by 25-50%, providing evidence that checkpoint blockade induced by the presence of anti-PD-L1 IgG can lead to reduced T cell depletion.

[0261] Anti-CAIX CART cells secreting anti-PD-L1 antibodies further reduced tumor growth in an orthotopic mouse model of human RCC. NSG mice were used to establish an orthotopic RCC model: Skrc-59 CAIX + PD-L1 + luciferase + positive RCC cells were injected subcapsularly, followed by intravenous injection of 1.0 × 10⁻⁶ cells. 7 One CAR-T or untransduced T cell (day 0), and on day 17 with a lower dose (2.5 × 10⁻⁶). 6 We repeatedly treated mice with the same cells. We did not treat mice with systemic IL-2 to maintain CAR-T cell proliferation, to avoid the potential bias that the molecule might have on tumor growth. Figure 19Data from A-19C indicated that, during the experiment, all three anti-CAEXCART cell groups showed reduced RCC growth compared to unrelated anti-BCMA CART cells or untransduced cells, and the significant antitumor effect of anti-CAEXCART cells secreting anti-PD-L1 IgG1 or IgG4 became apparent at days 23 and 30. Figure 19 A and 19B). However, even one week after CAR-T cell IV treatment, we observed a 2-3 fold reduction in tumor size with anti-PD-L1 secretory CAR-T cells compared to parental anti-CAIX CAR-T cells and two control groups. Figure 24 A). We also analyzed CD45+ T cell survival in mouse blood to measure their survival rate in this passive transfer model. On day 8, we observed that the number of human T cells within PBMCs was only 10-15% ( Figure 24 Therefore, we decided to expand CAR-T cells in vitro for a second injection into mail blood on day 17. One week after the second injection (day 23), the tumors in the anti-PD-L1 IgG1 and IgG4 groups were 1 / 15th the size of the tumors in the control group, and 1 / 5th the size of the anti-CAIX CAR T cells without anti-PD-L1 secretion. Figure 19 C and Figure 24 A). On day 30, the tumor count in mice treated with CAR-T cells secreting anti-PD-L1 antibodies was 1 / 5 that of the control group. Figure 19 C and Figure 24 A). The resected tumor weight was also lower in mice treated with CAR-T cells secreting anti-PD-L1 antibodies, and this was particularly pronounced in the anti-PD-L1 IgG4 antibody group. Figure 19 B and 19D).

[0262] Analysis of CART cell tumor infiltration and evidence that anti-CAIX CART cells secreting anti-PD-L1 antibodies can lead to the reversal of T cell depletion. Analysis of resected tumors showed that approximately 10% of TILs (tumor endothelial cells) in all groups... Figure 24 C).

[0263] One of the most important effects observed in anti-CAIX CART cells that secrete anti-PD-L1 IgG1 or IgG4 antibodies in vivo is their ability to reduce the expression of depletion markers PD-1, Tim-3, and Lag-3. Figure 20 As shown in Figure A, compared with treatment using untransduced control cells on day 30, we observed a reduction of approximately 40-50% in the expression of these markers in TILs. This reduction was also approximately 30-70% compared with anti-CAIX CAR cells that did not secrete anti-PDL-1 antibodies, thus providing evidence for the role of locally secreted antibodies in reducing T cell depletion.

[0264] The effector activity of CART cells and their influence on RCC proliferation in vivo were assessed by immunohistochemical detection of granzyme B, the tumor proliferation marker Ki67, and the tumor immunosuppressive protein PD-L1 in TILs. Figure 20 B and 20C). Granulase B staining showed effector activity of CD8+ cells, particularly in RCC tumors treated with anti-CAIX CART cells secreting anti-PD-L1 IgG4, showing an increased percentage of highly positive staining cells (B and 20C). Figure 20 B and 20C). In tumors treated with anti-CAIX CART cells, PD-L1 expression was significantly decreased, especially in the anti-CAIX / anti-PD-L1 IgG secretion group (B and 20C). Figure 20 B and 20C). Ki67 expression was significantly decreased in the anti-CAIX / anti-PD-L1 IgG secretion group, as can be observed in the total DAB pixel count map; however, when assessing the intensity in positive nuclear staining (Ki67 IHC quantification map), we can note that anti-CAIX / anti-PD-L1 IgG4 exhibited the lowest Ki67 expression intensity. Figure 20 B and 20C). For the quantification of nuclear proteins, such as Ki67, the DAB staining pattern is limited to the cell nucleus, and the threshold features of ImageJ can be used to select positively stained areas for quantification. Therefore, Ki67 IHC quantification map ( Figure 20 C) shows no negative cells, and also displays the number of DAB pixels used to assess total Ki67 staining (including negative cells). Figure 20 C).

[0265] Anti-CAIX CART cells secreting anti-PD-L1 IgG1 antibodies can recruit NK cells to tumors. Tumors in mice treated with anti-CAIX / anti-PD-L1 IgG1 and NK cell injections two days before euthanasia showed 40% more NK cells compared to those treated with anti-BCMA / SARS IgG1, as detected by FACS anti-CD56 staining. Figure 25 A). Increased NK cells in the anti-CAIX / anti-PD-L1 IgG1 group were also detected and quantified by IHC ( Figure 25 B). Few NK cells were also detected in the group treated with CAR-T cells that secrete nonspecific IgG1Ab.

[0266] Other implementation plans Although the invention has been described in detail therein, the foregoing description is intended to illustrate, and not limit, the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

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Claims

1. A chimeric antigen receptor (CAR) comprising an intracellular signal transduction domain, a transmembrane domain, and an extracellular domain.

2. The CAR of claim 1, wherein the transmembrane domain further comprises a stem region located between the extracellular domain and the transmembrane domain.

3. The CAR of claim 1, wherein the transmembrane domain comprises CD28.

4. The CAR of claim 1, further comprising one or more additional co-stimulatory molecules located between the transmembrane domain and the intracellular signal transduction domain.

5. The CAR of claim 4, wherein the co-stimulatory molecule is CD28, 4-1BB, ICOS, or OX40.

6. The CAR of claim 1, wherein the intracellular signal transduction domain comprises the CD3ζ chain.

7. The CAR of claim 1, wherein the extracellular domain is an antibody.

8. The CAR of claim 7, wherein the antibody is Fab or scFV.

9. The CAR of claim 7, 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.

10. A nucleic acid encoding a CAR of any one of the preceding claims, further comprising a nucleic acid encoding a polypeptide located after an intracellular signal transduction domain.

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