Compositions and methods for immunotherapy

Abstract

The present invention relates to methods and compositions for generating and / or increasing immune responses by cellular immunotherapy (e.g., by adoptive transfer of tumor-specific genetically modified lymphocyte subsets). The present invention provides compositions comprising genetically modified lymphocytes expressing at least two transgenes having the ability to modulate the immune system and innate and adaptive immune responses.

Description

[0001] Cross-references

[0002] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62 / 935,308, filed on November 14, 2019. The above application is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention generally relates to compositions and methods for treating cancer or tumors in a subject, and more particularly, to compositions and methods for treating cancer or tumors in a subject by modulating the subject's immune system. Background Art

[0004] Adoptive cell transfer or adoptive cell therapy (ACT) is a promising therapeutic approach for treating cancer patients. However, it faces two major obstacles: the short-term survival of transferred cells in cancer patients and the hostile immunosuppressive tumor microenvironment.

[0005] To overcome these limitations, several options have been proposed. For example, some trials have tested the simultaneous administration of interleukin 2 (IL-2) and ACT. IL-2 is a potent immunostimulant; therefore, it can enhance the immune response and improve the survival of transferred cells. However, this approach was unsuccessful due to the toxicity associated with IL-2. U.S. Patent 7381405 describes a method for preparing IL-2-transduced lymphocytes that can secrete IL-2 for ACT. This method is based on the assumption that lymphocytes will secrete their own growth factors (such as IL-2), thereby reducing their dependence on other exogenous factors for survival in the body. Despite successful results in an in vitro setting, clinical trials determined that this approach is ineffective. IL-2-transduced lymphocytes are no more effective than non-transduced lymphocytes in treating cancer (Heemskerk et al., Human Gene Therapy, 2008).

[0006] The emergence of chimeric antigen receptor (CAR) T cells provides a useful tool for improving ACT. TRUCKs (T cells redirected for universal cytokine killing) (International Publication WO 2017 / 108805) and Armored CAR (US Patent 10124023) are representative examples of CAR T cells, which are also designed to secrete recombinant interleukin-12 (IL-12) and CD40L, respectively. However, these strategies also have disadvantages. For example, in TRUCK, high transgenic IL-12 production limits the expansion of T cells and increases apoptosis, showing limited therapeutic effects. In addition, so far, the clinical application of armored CAR T cells has been limited to liquid tumors.

[0007] Solid tumors and their microenvironment present a number of challenges to the success of ACT therapies. These challenges include effective trafficking and penetration of tumors and overcoming tumor-mediated immunosuppression. Despite many efforts, state-of-the-art ACT therapies do not provide functional durability of long-term efficacy in the immunosuppressive solid tumor microenvironment.

[0008] Therefore, there is an urgent need to identify new ACT therapies that can provide functional persistence to cells and / or can alter the cytokine milieu to overcome the immunosuppressive tumor microenvironment. Summary of the invention

[0009] The present invention addresses the above needs in multiple aspects. In one aspect, the present invention provides a composition comprising a plurality of transgenic lymphocytes expressing at least two transgenes (eg, therapeutic transgenes) for modulating the immune system of a subject.

[0010] In some embodiments, the transgene is selected from the group consisting of an antibody, an antibody fragment, a receptor, a decoy, a checkpoint blockade modulator, a cytokine, a chemokine, a hormone, a cell elimination tag, and combinations thereof.

[0011] In some embodiments, the decoy is selected from PD1, CTLA4, LAG3, VEGFR1, TIM3, TIGIT and SIRPα decoys. In some embodiments, the decoy is a PD1 decoy. In some embodiments, the PD-1 decoy is a PD-1 IgG4 (e.g., PD-1 IgG4 Fc) decoy.

[0012] In some embodiments, the cytokine is selected from LIGHT or a variant / fragment thereof, IL-33 or a variant / fragment thereof, IL-2 or a variant / fragment thereof, IL-15 or a variant / fragment thereof, IL-12 or a variant / fragment thereof, and CD40L or a variant / fragment thereof. In some embodiments, the cytokine is a mutant cytokine.

[0013] In some embodiments, the cell elimination tag is selected from tEGFR, Her2, CD20 and CD19.

[0014] In some embodiments, the at least two transgenes include two or more of PD-1 decoy or variants / fragments thereof, IL-2 variants / fragments, LIGHT or variants / fragments thereof, IL-33 or variants / fragments thereof, and CD40L or variants / fragments thereof. In some embodiments, the at least two transgenes further include truncated EGFR (tEGFR) or variants / fragments thereof, truncated HER2 (tHER2) or variants / fragments thereof, or CD20 or variants / fragments thereof. In some embodiments, the PD-1 decoy or variants / fragments thereof and tEGFR or variants / fragments thereof (or tHER2 or variants / fragments thereof, CD20 or variants / fragments thereof, CD19 or variants / fragments thereof) are included on the same vector.

[0015] In some embodiments, the at least two transgenes include: (a) PD-1 decoy or its variant and tEGFR or its variant; (b) PD-1 decoy or its variant and IL-2 variant; (c) PD-1 decoy or its variant and LIGHT or its variant; (d) PD-1 decoy or its variant and IL-33 or its variant; (e) PD-1 decoy or its variant and CD40L or its variant; (f) PD-1 decoy or its variant, IL-2 variant and IL-33 or its variant; (g) PD-1 decoy or its variant, tEGFR or its variant and IL-2 variant; (h) PD-1 decoy or its variant , tEGFR or its variants and LIGHT or its variants; (i) PD-1 decoy or its variants, tEGFR or its variants and IL-33 or its variants; (j) PD-1 decoy or its variants, tEGFR or its variants and CD40L or its variants; (k) PD-1 decoy or its variants, tEGFR or its variants, IL-2 variants and IL-33 or its variants; (l) PD-1 decoy or its variants, tEGFR or its variants, IL-2 variants and CD40L or its variants; or (m) PD-1 decoy or its variants, tEGFR or its variants, IL-33 variants and CD40L or its variants.

[0016] In some embodiments, the PD-1 decoy comprises an amino acid sequence of any one of SEQ ID NOs: 1-4, 6-17, 42, 44, 47-48, and 51-52, or an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 1-4, 6-17, 42, 44, 47-48, and 51-52.

[0017] In some embodiments, the IL-2 variant comprises the amino acid sequence of any one of SEQ ID NOs: 21-23 or an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 21-23.

[0018] In some embodiments, the IL-33 comprises the amino acid sequence of any one of SEQ ID NOs: 25 and 27, or an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 25 and 27.

[0019] In some embodiments, the LIGHT comprises an amino acid sequence of any one of SEQ ID NOs: 28-29 and 31 or an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 28-29 and 31.

[0020] In some embodiments, the CD40L comprises an amino acid sequence of any one of SEQ ID NOs: 32-34, 36 and 38, or an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 32-34, 36 and 38.

[0021] In some embodiments, the tEGFR comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 40 or an amino acid sequence of SEQ ID NO: 40. In some embodiments, the HER2 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 45 or an amino acid sequence of SEQ ID NO: 45. In some embodiments, the CD20 comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 49 or an amino acid sequence of SEQ ID NO: 49.

[0022] In some embodiments, the transgene comprises an antibody or antibody fragment selected from VEGF, TGF-B, 4-1BB, CD28, CD27, NKG2D, PD1, PDL1 and CTLA4 antibodies. In some embodiments, the antibody is a PD1 antibody.

[0023] In some embodiments, the plurality of lymphocytes comprises at least two lymphocyte subsets. In some embodiments, the plurality of lymphocytes consists of two lymphocyte subsets. In some embodiments, each subset of the plurality of lymphocytes expresses at least one transgene. In some embodiments, the at least two transgenes are different from each other.

[0024] In some embodiments, the plurality of lymphocytes comprises: (i) a first subpopulation expressing at least two transgenes; and (ii) a second subpopulation expressing at least two transgenes, wherein at least one transgene of the first subpopulation is different from a transgene of the second subpopulation, or wherein at least one transgene of the first subpopulation is the same as a transgene of the second subpopulation.

[0025] In some embodiments, (i) the first subpopulation expresses at least PD-1 decoy or its variant and IL-2 variant, and the second subpopulation expresses at least PD-1 decoy or its variant and LIGHT or its variant; (ii) the first subpopulation expresses at least PD-1 decoy or its variant and IL-2 variant, and the second subpopulation expresses at least PD-1 decoy or its variant and IL-33 or its variant; (iii) the first subpopulation expresses at least PD-1 decoy or its variant and IL-2 variant, and the second subpopulation expresses at least PD-1 decoy or its variant and CD40L or its variant; (iv) the first subpopulation expresses at least one PD-1 decoy or its variant and LIGHT or its variant, the second subpopulation expresses at least PD-1 decoy or its variant and LIGHT or its variant, and the second subpopulation expresses at least PD-1 decoy or its variant and CD40L or its variant; or (vi) the first subpopulation expresses at least PD-1 decoy or its variant and IL-33 or its variant, and the second subpopulation expresses at least PD-1 decoy or its variant and CD40L or its variant.

[0026] In some embodiments, the first subpopulation or the second subpopulation further expresses tEGFR or a variant thereof, truncated HER2 (tHER2) or a variant thereof, CD20 or a variant thereof, or CD19 or a variant thereof.

[0027] In some embodiments, (i) the first subpopulation expresses at least PD-1 decoy or its variant, tEGFR or its variant, and IL-2 variant, and the second subpopulation expresses at least PD-1 decoy or its variant, tEGFR or its variant, and LIGHT or its variant; (ii) the first subpopulation expresses at least PD-1 decoy or its variant, tEGFR or its variant, and IL-2 variant, and the second subpopulation expresses at least PD-1 decoy or its variant, tEGFR or its variant, and IL-33 or its variant; (iii) the first subpopulation expresses at least PD-1 decoy or its variant, tEGFR or its variant, and IL-2 variant, and the second subpopulation expresses at least PD-1 decoy or its variant, tEGFR or its variant, and CD40L or its variant; (iv) the first subpopulation at least expresses PD-1 decoy or its variant, tEGFR or its variant and LIGHT or its variant, and the second subpopulation at least expresses PD-1 decoy or its variant, tEGFR or its variant and IL-33 or its variant; (v) the first subpopulation at least expresses PD-1 decoy or its variant, tEGFR or its variant and LIGHT or its variant, and the second subpopulation at least expresses PD-1 decoy or its variant, tEGFR or its variant and CD40L or its variant; or (vi) the first subpopulation at least expresses PD-1 decoy or its variant, tEGFR or its variant and IL-33 or its variant, and the second subpopulation at least expresses PD-1 decoy or its variant, tEGFR or its variant and CD40L or its variant.

[0028] In some embodiments, the first subpopulation or the second subpopulation further expresses tEGFR or a variant thereof, tHER2 or a variant thereof, or CD20 or a variant thereof.

[0029] In some embodiments, the two subpopulations are combined in a ratio of about 1:1 to about 1: 100. In some embodiments, the two subpopulations are combined in a ratio of about 1:1.

[0030] In some embodiments, the lymphocytes are autologous. In some embodiments, the lymphocytes are tumor infiltrating lymphocytes. In some embodiments, the lymphocytes express chimeric antigen receptors (CAR). In some embodiments, the lymphocytes express recombinant T cell receptors (TCR). In some embodiments, the recombinant T cell receptors (TCR) show responsiveness to NY-ESO1, MAGE-A1, MAGE-A3, MAGEA-10, MAGE-C2, SSX2, MAGE-A12, or a combination thereof.

[0031] Also within the scope of the present invention is a pharmaceutical composition comprising an effective amount of the above-described composition and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent.

[0032] The present invention also provides a kit comprising an effective amount of the above composition.

[0033] In another aspect, the present invention provides a method for preparing the above-mentioned composition. The method comprises: (a) providing a plurality of lymphocytes; (b) introducing a nucleic acid molecule encoding at least two transgenes into the plurality of lymphocytes to obtain a plurality of genetically modified lymphocytes; and (c) amplifying the plurality of genetically modified lymphocytes in a cell culture medium.

[0034] Optionally, the method comprises: (a) providing a plurality of lymphocytes; (b) introducing two or more nucleic acid molecules into the plurality of lymphocytes, each of the two or more nucleic acid molecules encoding at least one transgene, thereby obtaining a plurality of genetically modified lymphocytes; and (c) expanding the plurality of genetically modified lymphocytes in a cell culture medium.

[0035] In some embodiments, the at least two transgenes comprise two or more of PD-1 decoy, IL-2 variant / fragment, LIGHT or its variant / fragment, IL-33 or its variant / fragment, and CD40L or its variant / fragment. In some embodiments, the at least two transgenes further comprise tEGFR or its variant / fragment. In some embodiments, the PD-1 decoy or its variant / fragment and the tEGFR or its variant / fragment (or the tHER2 or its variant / fragment, CD20 or its variant / fragment, or CD19 or its variant / fragment) are contained on the same vector.

[0036] In some embodiments, the method comprises: (a) introducing a first nucleic acid molecule encoding at least two transgenes into a first plurality of lymphocytes to obtain a first plurality of genetically modified lymphocytes; and (b) introducing a second nucleic acid molecule encoding at least two transgenes into a second plurality of lymphocytes to obtain a second plurality of genetically modified lymphocytes.

[0037] In some embodiments, the method further comprises expanding the first plurality of lymphocytes in the cell culture medium after the step of introducing the first nucleic acid, or expanding the second plurality of lymphocytes in the cell culture medium after the step of introducing the second nucleic acid.

[0038] In some embodiments, the method further comprises combining the first plurality of genetically engineered lymphocytes with the first plurality of genetically engineered lymphocytes at a predetermined ratio between about 1:1 and about 1:100 (eg, 1:1).

[0039] In some embodiments, the cell culture medium is a defined cell culture medium.In some embodiments, the cell culture medium comprises a neoantigenic peptide.

[0040] In yet another aspect, the present invention also provides a method for treating cancer / tumor or chronic infection in a subject. The method comprises administering to a subject in need thereof an effective therapeutic amount of a composition or pharmaceutical composition, as described above.

[0041] In some embodiments, the cancer is selected from melanoma, sarcoma, ovarian cancer, prostate cancer, lung cancer, bladder cancer, microsatellite instability-high (MSI-high) tumors, head and neck tumors, renal cancer, and breast cancer.

[0042] In some embodiments, the composition is administered by intravenous infusion. In some embodiments, the method further comprises administering a second therapeutic agent to the subject. In some embodiments, the second therapeutic agent is an anticancer agent or an antitumor agent. In some embodiments, the composition or pharmaceutical composition is administered to the subject before, after, or simultaneously with the second therapeutic agent.

[0043] The foregoing summary of the invention is not intended to define every aspect of the present invention, and other aspects are described in other sections, such as the following specific embodiments. The entire document is intended to be associated as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if these combinations of features are not found in the same sentence, paragraph or chapter of this document. Other features and advantages of the present invention will become apparent through the following specific embodiments. However, it should be understood that while indicating specific embodiments of the present invention, the specific embodiments and specific examples are given only by way of illustration, because various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from the described specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figures 1A, 1B, 1C and 1D (collectively referred to as "Figure 1") are a set of graphs showing OT-1CD8 + -T cells can be genetically engineered to secrete PD1 IgG4 decoy and IL-2 variant (called IL-2 V ), LIGHT, IL-33 or CD40L. Figure 1A shows that OT-1CD8 + -T cells were genetically modified to secrete PD1 IgG4 and mutIL2. Transduction efficiency was determined by FACS (Figure 1A; left panel), and secretion was assessed by ELISA (Figure 1A; middle and right panels). Figure 1B shows that OT-1CD8 + -T cells were genetically modified to secrete PD1 IgG4 and LIGHT. Transduction efficiency was determined by FACS (Figure 1B; left and left middle panels), and secretion was assessed by ELISA (Figure 1B; right and right middle panels). Figure 1C shows that OT-1CD8+ -T cells were genetically modified to secrete PD1 IgG4 and IL-33. Transduction efficiency was determined by FACS (Figure 1C; left and middle left panels), and secretion was assessed by ELISA (Figure 1C; middle right and middle right panels). Figure 1D shows that OT-1CD8 + -T cells were genetically engineered to secrete PD1 IgG4 and CD40L. Transduction efficiency was determined by FACS (Figure 1C; left and middle panels), and secretion was assessed by ELISA (Figure 1D; right and middle panels).

[0045] Figures 2A, 2B, 2C, 2D, 2E, 2F, 2G and 2H (collectively referred to as "Figure 2") are a set of figures showing adoptive transfer of genetically modified OT-1CD8 + -T cells, secreting a combination of three immunomodulatory factors, significantly improved tumor control in large B16-OVA tumors without pretreatment. Figures 2A and 2B show that receiving T cells secreting PD-1IgG4, IL-2 V and LIGHT's OT-1CD8 + Figure 2C and 2D show the tumor growth curves (Figure 2A) and overall survival curves (Figure 2B) of mice receiving OT-1CD8 + Figures 2E and 2F show the tumor growth curves (Figure 2C) and overall survival curves (Figure 2D) of mice receiving PD-1 IgG4, IL-33, and light. V and LIGHT's OT-1CD8 + -T cells in mice (Figure 2E) and overall survival curve (Figure 2F). The experiment was performed in a blinded manner, with 6 animals in each group. Figures 2G and 2H show the tumor growth curves of mice receiving PD-1IgG4, IL-2 V OT-1CD8 and CD40L + -T cells (Figure 2G) and overall survival curves of mice. Survival analysis was performed using the log-rank mantel-cox model. The Kruskal-Wallis test was used to compare the groups with those receiving UTOT-1CD8 + Tumor growth in mice with -T cells on day 27. Multiple comparisons were corrected using Dunn's test *p<0.05, **p<0.001, ***p<0.0001.

[0046] FIG. 3A , FIG. 3B , FIG. 3C , FIG. 3D , FIG. 3E , FIG. 3F , FIG. 3G , FIG. 3H , FIG. 3I , FIG. 3J and FIG. 3K are a set of graphs showing orthogonal T cell engineering by expanding adoptively transferred CD8 + T cells and mobilize endogenous anti-tumor immunity to improve the efficacy of ACT in immunocompetent hosts. Figure 3A shows the experimental design. Figure 3B is a waterfall chart showing the changes in tumor volume starting from day 17. The best response (minimum tumor volume) observed for each animal at least 12 days after the first ACT was calculated (*24 days after tumor inoculation, **31 days after tumor inoculation). Tumor objective response rate (ORR) includes complete remission (CR; 100% reduction in tumor volume) and partial remission (PR; tumor change ≤-30%). Figures 3C, 3D, 3E, and 3F show that mice bearing B16-OVA tumors can be treated with engineered or untransformed OT1 cells as indicated; tumors are then collected on days 17 and 24, and cell quantification is performed by flow cytometry. Data are from three independent experiments (n>=5 animals / group). Figure 3C shows the CD8 + Figure 3D shows the total number of TILs on days 17 and 24. + Figure 3E shows the total number of endogenous CD45.1 on days 17 and 24. neg Figure 3F shows the total number of CD8 TILs on day 24 when endogenous and exogenous TCF1 + CD8 + Figure 3G shows representative immunofluorescence micrographs of tumor sections from each experimental group on day 24, showing OT1 and endogenous TCF1 + CD8 + TIL. Filled triangle: TCF1 + OT1, open triangle: TCF1 neg OT1, white arrow: TCF1 + Endogenous CD8 + Figure 3H shows that, as described above, PD1d / 2 V / 33+OT-1 cells were administered to B16 OVA tumor-bearing CD8KO mice or wtC57BL6 mice treated with the drug FTY720 at 100 μg / mouse (administered intraperitoneally every three days starting two days before the first cell transfer). Figure 3I shows that mice bearing B16-OVA tumors were treated as described above, and then tumors were harvested on day 24 and Treg quantification was performed by flow cytometry. Data are from three independent experiments (n>=5 animals / group). CD8 +Figures 3J and 3K show the ratio of PD1d / 2 Tregs in the presence or absence of 250 μg / mouse of PD-1d / 2 Tregs-specific depleting antibodies administered intraperitoneally starting 1 day before the first cell transfer and maintained every 3 days. V Figure 3B shows the relationship between the growth of B16-OVA tumors and the tumor volume in Figure 3C, Figure 3D, Figure 3E, and Figure 3F. Figure 3B shows the relationship between the growth of B16-OVA tumors and the tumor volume in Figure 3C, Figure 3D, Figure 3E, and Figure 3F. Figure 3B shows the relationship between the growth of B16-OVA tumors and the tumor volume in Figure 3H, Figure 3J, and Figure 3K. Figure 3C shows the relationship between the growth of B16-OVA tumors and the tumor volume in Figure 3H, Figure 3J, and Figure 3K. Figure 3D shows the relationship between the growth of B16-OVA tumors and the tumor volume in Figure 3H, Figure 3J, and Figure 3K. Figure 3D shows the relationship between the growth of B16-OVA tumors and the tumor volume in Figure 3H, Figure 3E, and Figure 3F. Figure 3D ...

[0047] Figures 4A, 4B, 4C, 4D, 4E, 4F, and 4G are a set of graphs showing that orthogonal engineering induces a new effector-like CD8 T cell subset that is distinct from terminally exhausted and transient CX3CR1+ effector-like T cells. Figure 4A shows the experimental design. Mice bearing B16-OVA tumors were treated as described above; tumors were then collected on days 17 and 24, CD45+ cell suspensions enriched for CD8+ TILs were obtained by FACS sorting, and single cells were sequenced using 10X Genomics. Figure 4B shows a UMAP plot depicting low-dimensional expression and unsupervised clustering results of cellular heterogeneity, with contour plots depicting areas of high cell density for each treatment. Figure 4C shows the TILPRED-predicted CD8 T ILs states (top) and volcano plots (bottom), depicting GzmC+C5 and GzmC neg Figure 4D shows the genes that are significantly differentially expressed between terminally exhausted cells. V Projection of 33 / 33 (day 24) TILs on a reference TIL map using ProjecTILs. On the right, radar plots show the expression levels of important T cell markers projected and reference exhausted T cell states. Figure 4E shows a dot plot depicting specific marker clusters. Figure 4F shows the expression levels of Gzmc+C5 and Gzmc neg CD8 TIL-Tox knockout gene signature enrichment analysis (GSEA) of C4 cells. Figure 4G shows that mice bearing B16-OVA tumors were treated with engineered or untransformed OT1 cells on days 12 and 15 after tumor cell inoculation. Tumors or spleens were collected on day 24 (PD1d / 2V / 33OT1), and the intracellular expression of granzyme C was detected by flow cytometry. A summary of two independent experiments is shown, (n>=5 animals / group) (UT: untransduced). One-way ANOVA and Dunnet's test were used to correct for multiple comparisons. *p<0.05, **p<0.01, ***p<0.001, ***p<0.0001.

[0048] Figure 5A, Figure 5B, Figure 5C, Figure 5D, Figure 5E, and Figure 5F are a set of graphs showing orthogonal engineering of TOX expression with GzmC + TCF1 neg CD8 + Figure 5A shows the expression of exogenous and endogenous CD8 TILs based on the expression of granzyme C and TCF1 at day 24. + T cell compartment analysis. OT1 TILs were not obtained from tumors after PD1d / 33ACT (UT: untransduced). Figure 5B shows the gating strategy for the evaluation of TOX and phenotypic markers. PD1d / 2 V Not included in statistical analysis because CD8 TILs are predominantly TCF1 + Figure 5C shows the expression of PD-1 in TCF1 neg CD8 + Figure 5D shows the surface expression of TIM-3 in PD-1+TCF1 neg CD8 + Surface expression in TILs shows the expression of these two surface markers from 4 independent experiments (PD1d / 2 V / 33) or 2 (other groups) (n=4 or 5 animals / experiment, day 24 after tumor inoculation). Figure 5E shows PD-1 + TCF1 neg CD8 + The expression of TOX in TILs was shown in 2 independent experiments (PD1d / 2 V / 33, PD1d / 33) or 1 (other groups) (n=4 or 5 animals / experiment, 24 days after tumor inoculation). Figure 5F shows TCF1 neg Surface expression of KLRG1 in CD8 TILs, representative experiments from two independent experiments (n=5 animals / group) are shown. One-way analysis of variance (ANOVA) with Dunnet's test was corrected for multiple comparisons, *p<0.05, **p<0.01, ***p<0.001, **p<0.0001. Primary OT-1 T cells isolated from the spleen of non-tumor-bearing mice were used as an internal negative control for FACS staining.

[0049] FIG6A , FIG6B , FIG6C , FIG6D , FIG6E , FIG6F , FIG6G , FIG6H , and FIG6I are a set of graphs showing GzmC + TCF1 neg CD8 + TILs are multifunctional effector cells that express no significant co-inhibitory receptors. OT1 and endogenous CD8 TILs from animals treated with engineered or untransduced (UT) OT1 cells were analyzed on day 24 to quantify PD-1 + TCF1 neg CD8 + Effector molecules in TILs. OT1 TILs were not collected from tumors after PD1d / 33ACT or UT. PD1d / 2 V Not included in statistical analysis because CD8 TILs are predominantly TCF1 + . Figure 6A shows the surface expression of CD69. Figure 6B shows the intracellular expression of Ki-67. Figure 6C shows the intracellular expression of granzyme B. Figure 6D shows the normalized MFI of granzyme B expression relative to primary OT-1 T cells isolated from non-tumor bearing mice. Figure 6E shows the co-expression of granzyme B, and Figure 6F shows the intracellular expression of TNFα and INFγ after 4 hours of in vitro stimulation with aCD3 and aCD28 antibodies. The data shown in Figures 6A and 6B are from 2 independent experiments (PD1d / 2 V / 33, PD1d / 33) or 1 (other groups) (n=4-6 animals / group). Figures 6C, 6D, 6E and 6F show data from 2 independent experiments (PD1d / 2 V / 33) or 1 (other groups) (n = 4-6 animals / group). One-way analysis of variance (ANOVA) combined with Dunnet's test was used to correct for multiple comparisons, *p<0.05, **p<0.01, ***p<0.001, **p<0.0001. Primary OT-1 T cells isolated from the spleen of non-tumor-bearing mice were used as internal negative controls for FACS staining. Figures 6G and 6H show that PD1d / 2 V Tumor growth control over time in B16-OVA tumor-bearing mice treated with / 33+OT-1 cells in the presence or absence of 250 μg / mouse of antibodies against the indicated surface markers, administered by intraperitoneal injection starting 1 day before cell transfer and maintained every 3 days for a maximum of 6 doses; αPD-L1 (Figure 6G) and αPD-L1+αTIM3 (Figure 6H). Figure 6I shows the effect of PD1d / 2 V / 33 + OT1 cells or secrete IL-2 VTumor growth control over time in B16-OVA tumor-bearing mice treated with genetically engineered OT1 T cells expressing IL-33 (without PD-1 extracellular domain). V Similar to OT-1 cells expressing IgG4 / IL-33, this group was composed of OT-1 cells (without PD-1 decoy production) and OT-1 cells expressing IgG4 / IL-2 V OT-1 cells were mixed at a 1:1 ratio. Expression of IgG4 Fc and IL-33 was not significantly different from PD1d / 33 (n = 8 animals / group) as confirmed by FACS and ELISA. Representative experiments from two independent experiments (n = 6 animals / group) are shown in Figures 6G, 6H, and 6I. Primary OT-1 T cells isolated from the spleen of non-tumor-bearing mice were used as an internal negative control for FACS staining.

[0050] FIG. 7A , FIG. 7B , FIG. 7C , and FIG. 7D are a set of diagrams showing orthogonal engineering driving TOX neg / low G JZ + Figure 7A shows the TCF1 collected on day 24. + CD8 + Analysis of PD-1 expression in TILs. Figure 7B shows the evaluation of PD-1 + TCF1 + Figure 7C shows the gating strategy for TOX expression in cells. V GzmC+PD-1 + TCF1 + CD8 TIL and GzmC neg PD-1 + TCF1 + Analysis of TOX expression in CD8 TIL cells. The data shown in Figures 7A, 7B and 7C are from 2 independent experiments (n = 4-6 animals / group). One-way analysis of variance (ANOVA) combined with Dunnet's test was corrected for multiple comparisons, *p<0.05, **p<0.01, ***p<0.001, **p<0.0001. Figure 7D shows the expression of TOX in CD8 TIL cells from PD1d / 2 on day 24. V TCF1 obtained from mice treated with / 33+OT1 (3 independent experiments, n>=4 animals / group) + CD8 + Granzyme C expression in TILs (MFI normalized to native OT1) compared with endogenous TCF1 obtained at day 12 after tumor inoculation + CD8 TIL (baseline, n = 6 animals) and TCF1 after ex vivo expansion +Comparative analysis of genetically engineered OT1 cells (before ACT, n=14) Primary OT-1 T cells isolated from the spleen of non-tumor bearing mice were used as an internal negative control for FACS staining.

[0051] FIG8A , FIG8B , FIG8C , FIG8D , FIG8E , FIG8F , FIG8G , and FIG8H are a set of graphs showing novel TCF1 induced by orthogonal engineering neg CD8 + TIL effector status correlates with tumor response dynamics. Figure 8A shows the experimental design. Mice bearing B16-OVA tumors were treated on days 12 and 15 after tumor cell inoculation. Tumors were harvested on days 17, 24, and 38, and enriched for CD8 by FACS sorting and single-cell sequencing using 10X Genomics. + CD45+ cell suspension in TIL. Figure 8B shows the UMAP graph, which shows a low-dimensional representation of cellular heterogeneity and only PD1d / 2 at different time points V Figure 8: Unsupervised clustering results for the 1:1 ratio of 1:1 and 1:1 ratio of 1:1. Figure 8: Unsupervised clustering results for the 1:1 ratio of .... Figure 8: Unsupervised clustering results for the 1:1 ratio of 1:1 ratio of 1:1. Figure 8: Unsupervised clustering results for the 1:1 ratio of 1:1 ratio of 1:1. Figure 8: Unsupervised clustering results for the 1:1 ratio of 1:1 ratio of 1:1. + Analysis of the expression of granzyme C in TIL cells. CD8 in the spleen of mice treated with the triple-combination + T cells and CD8 + TIL were included in the control group. V Figure 8E shows the total CD8 collected during tumor control (day 24) and escape (day 38). + Analysis of intratumoral persistence of OT1 (CD45.1+) in TILs. Figure 8F shows the expression of exogenous and endogenous CD8 collected during tumor control (day 24) and escape (day 38) based on PD-1 and TCF1 expression. + Analysis of TILs. After 4 h of in vitro stimulation with anti-CD3 and anti-CD28 antibodies, harvested during tumor control (day 24) or escape (day 38) were analyzed for PD-1 + TCF1 negIntracellular expression of granzyme B (Figure 8G), TNFα and INFγ (Figure 8H) in CD8 TIL. Data obtained from 2 independent experiments (tumor control) or 1 (escape) (n = 4-6 animals / group) are shown. The two groups were compared using a two-tailed Student's t-test. *p<0.05, **p<0.01, ***p<0.001, ***p<0.0001. Primary OT-1 T cells isolated from the spleen of non-tumor-bearing mice were used as internal negative controls for FACS staining.

[0052] Figure 9A, Figure 9B, Fig. 9C and Fig.9D is a set of figures showing the characteristics of PD-1 decoy variants and hCD8+ T cells transduced with PD-1 decoy variants and tEGFR. Figure 9A shows a titration ELISA of soluble monomeric PD1 decoy variants (bacterial products) on plates coated with human PDL1 protein. Bound PD1 decoy molecules were detected with anti-His tag antibodies. The PD-1 decoy variant 4XMUT_M70 binds PD-L1 10 times more strongly than the WT PD1 decoy, while the variant 6XDM binds PD-L1 7.5 times more strongly than the WT PD1 decoy. Figure 9B shows the binding of retrovirally transduced CD8 + Detection of tEGFR and intracellular PD1 decoy on T cells. Fig. 9C NY-TCR(I53F) engineered CD8 co-expressing PD-1 decoy (variant) and tEGFR is shown + T cells produce IFNγ. Engineered T cells were co-cultured with different PD-L1+ target tumor cells (100,000 cells of each type) at a 1:1 ratio for 48 hours. NA8 and HLA / A2+NY-ESO-1-, SAOS2 and A375 are HLA / A2+NY-ESO-1+. Supernatants were collected after 48 hours, diluted 1 / 25, and assessed for the presence of IFNγ using a commercial ELISA kit from Thermo. Data from a representative T cell donor are shown. In all experiments, the variants performed better than the WT PD-1 decoy. Fig.9D ADCC test results of human T cells transduced to express PD1 bait (4XMUT_M70E) and tEGFR are shown. Engineered CD8 T cells with PD1 bait_tEGFR retrovirus were labeled with chromium. Engineered T cells were co-cultured with anti-EGFR antibodies and co-cultured with PBMCs from the same donor in different ratios. The negative control was NT (untransduced) T cell killing evaluated at 4 hours. As a positive control, T cells were treated with hydrochloric acid.

[0053] Figures 10A, 10B, 10C and 10D are a set of graphs showing the results of antibody-dependent cellular cytotoxicity (ADCC) assays in which T cells were engineered to express tEGFR. Figure 10A shows that tEGFR-engineered CD8 + T cells were loaded with chromium and cultured with PBMC at different ratios for 4-5 hours while reducing the concentration of the anti-EGFR antibody cetuximab. As a negative control, tCD30 engineered T cells were used in the experiment with the maximum concentration of cetuximab (100 μg / ml). The released chromium was used to measure the lysed T cells. Figures 10B and 10C show the tHER2 engineered CD8 + ADCC analysis results of T cells (left: Herceptin (Figure 10B); right: Herceptin (Figure 10C)). Figure 10D shows that CD20 engineered CD8 + Results of ADCC analysis of T cells.

[0054] Fig.11A , Fig. 11B and Fig. 11C is a set of figures showing representative constructs carrying transgenes for transducing lymphocytes. The representative constructs carry PD-1 decoy and tEGFR ( Fig.11A ), CD40L variants ( Fig. 11B ) and IL-2 variant (also known as IL-2 V )( Fig. 11C ). DETAILED DESCRIPTION

[0055] The present invention relates to methods and compositions for generating and / or increasing immune responses by cellular immunotherapy (e.g., by adoptive transfer of tumor-specific genetically modified lymphocyte subsets). The present invention provides compositions comprising transgenic lymphocytes that express at least two transgenes capable of modulating the immune system and innate and adaptive immune responses. The methods and compositions disclosed in the present application are embodiments of a platform technology called genetically engineered T cell performance enhancement (GEEP-T). TM ).GEEP-T TM The present invention aims to provide genetically engineered lymphocytes with enhanced anti-tumor function, and methods for developing such lymphocytes.

[0056] A. Compositions and Kits

[0057] In one aspect, the invention provides a composition comprising a plurality of genetically engineered lymphocytes expressing at least two transgenes (eg, therapeutic transgenes) for use in modulating the immune system of a subject.

[0058] In some embodiments, lymphocytes are peripheral blood lymphocytes (PBL). In some embodiments, lymphocytes are tumor infiltrating lymphocytes (TIL). Lymphocytes can include T cells, B cells, NK cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils and basophils. In some embodiments, lymphocytes are derived from CD34 hematopoietic stem cells, embryonic stem cells or induced pluripotent stem cells. Lymphocytes can be autologous, allogeneic, isogenic or xenogeneic. In some embodiments, lymphocytes are autologous. In some embodiments, lymphocytes are human lymphocytes.

[0059] In some embodiments, the lymphocytes may be tumor infiltrating lymphocytes (TIL). In some embodiments, the lymphocytes may express chimeric antigen receptors (CAR). In some embodiments, the lymphocytes may express recombinant T cell receptors (TCR). CAR or TCR may bind to cancer antigens. In some embodiments, CAR or TCR may show reactivity to NY-ESO1, MAGE-A1, MAGE-A3, MAGEA-10, MAGE-C2, SSX2, MAGE-A12, or a combination thereof.

[0060] In some embodiments, the transgenic encoded molecule is selected from: a soluble receptor, a decoy, a dominant negative, a microenvironmental modulator, an enzyme, an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, a translocase, a kinase, a transporter, a modifier, a molecular chaperone, an ion channel, an antibody, a cytokine, a chemokine, a hormone, a DNA, a ribozyme, a biosensor, an epigenetic modifier, a transcription factor, a coding RNA, a non-coding RNA, a small RNA, a long RNA, an IRES element, or an exosome-derived RNA.

[0061] In some embodiments, the transgene encodes at least two molecules selected from: a soluble receptor, a decoy, a dominant negative mutant, a microenvironmental modulator, an enzyme, an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, a translocase, a kinase, a transporter, a modifier, a molecular chaperone, an ion channel, an antibody, a cytokine, a chemokine, a hormone, DNA, a ribozyme, a biosensor, an epigenetic modifier, a transcription factor, a coding RNA, a non-coding RNA, a small RNA, a long RNA, an IRES element, an exosome-derived RNA, or any combination thereof.

[0062] In some embodiments, the two or more molecules encoded by the transgene are linked by a self-cleaving peptide sequence. In some embodiments, the transgene expression is regulated by a constitutively activated promoter. In some embodiments, the transgene expression is regulated by an inducible promoter. In some embodiments, the transgene expression is induced by the activation state of lymphocytes. In some embodiments, the transgene is introduced into lymphocytes by integration-competent gamma retroviruses or lentiviruses, DNA transposition, etc.

[0063] In some embodiments, the transgene is selected from the group consisting of an antibody, an antibody fragment, a receptor, a decoy, a checkpoint blockade modulator, a cytokine, a chemokine, a hormone, a cell elimination tag, and combinations thereof.

[0064] In some embodiments, the antibody or antibody fragment can be a VEGF, TGF-B, 4-1BB, CD28, CD27, NKG2D, PD1, PDL1 or CTLA4 antibody. In some embodiments, the antibody is a PD1 antibody. In some embodiments, the bait can be a PD1, CTLA4, LAG3, VEGFR1, TIM3, TIGIT or SIRPα bait. In some embodiments, the bait is a PD1 bait, such as a PD-1 IgG4 bait.

[0065] In some embodiments, the cytokine is selected from LIGHT or a variant / fragment thereof, IL-33 or a variant / fragment thereof, IL-2 or a variant / fragment thereof, IL-15 or a variant / fragment thereof, IL-12 or a variant / fragment thereof, and CD40L or a variant / fragment thereof. In some embodiments, the cytokine is a mutant cytokine.

[0066] In some embodiments, the cell elimination tag is selected from tEGFR, Her2, CD20 and CD19.

[0067] In some embodiments, the transgene comprises two or more of PD-1 decoy or variants / fragments thereof, IL-2 variants / fragments, LIGHT or variants / fragments thereof, IL-33 or variants / fragments thereof, and CD40L or variants / fragments thereof. In some embodiments, the transgene further comprises tEGFR or variants / fragments thereof, tHER2 or variants / fragments thereof, CD20 or variants / fragments thereof, or CD19 or variants / fragments thereof.

[0068] In some embodiments, the PD-1 decoy or variant / fragment thereof is contained on the same vector as a cell elimination tag (CET), such as tEGFR or variant / fragment thereof, tHER2 or variant / fragment thereof, CD20 or variant / fragment thereof, and CD19 or variant / fragment thereof.

[0069] In some embodiments, the at least two transgenes include: (a) PD-1 decoy or its variant / fragment and tEGFR or its variant / fragment; (b) PD-1 decoy or its variant / fragment and IL-2 variant / fragment; (c) PD-1 decoy or its variant / fragment and LIGHT or its variant / fragment; (d) PD-1 decoy or its variant / fragment and IL-33 or its variant / fragment; (e) PD-1 decoy or its variant / fragment and CD40L or its variant / fragment; (f) PD-1 decoy or its variant / fragment, IL-2 variant / fragment and IL-33 or its variant / fragment; (g) PD-1 decoy or its variant / fragment, tEGFR or its variant / fragment and IL-2 variant / fragment; (h) PD-1 decoy or its variant / fragment, tEGFR or its variants / fragments, and LIGHT or its variants / fragments; (i) PD-1 decoy or its variants / fragments, tEGFR or its variants / fragments and IL-33 or its variants / fragments; (j) PD-1 decoy or its variants / fragments, tEGFR or its variants / fragments, and CD40L or its variants / fragments; (k) PD-1 decoy or its variants / fragments, tEGFR or its variants / fragments, IL-2 variants / fragments and IL-33 or its variants / fragments; (l) PD-1 decoy or its variants / fragments, tEGFR or its variants / fragments, IL-2 variants / fragments and CD40L or its variants / fragments; or (m) PD-1 decoy or its variants / fragments, tEGFR or its variants / fragments, IL-33 variants / fragments and CD40L or its variants / fragments.

[0070] In some embodiments, the PD-1 decoy comprises an amino acid sequence of any one of SEQ ID NOs: 1-4, 6-17, 42, 44, 47-48, and 51-52, or an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) identity to any one of SEQ ID NOs: 1-4, 6-17, 42, 44, 47-48, and 51-52.

[0071] In some embodiments, the IL-2 variant comprises an amino acid sequence of any one of SEQ ID NOs: 21-23, or an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) identity to any one of SEQ ID NOs: 21-23.

[0072] In some embodiments, the IL-33 comprises the amino acid sequence of any one of SEQ ID NOs: 25 and 27, or an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) identity to any one of SEQ ID NOs: 25 and 27.

[0073] In some embodiments, the LIGHT comprises an amino acid sequence of any one of SEQ ID NOs: 28-29 and 31, or an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) identity to any one of SEQ ID NOs: 28-29 and 31.

[0074] In some embodiments, the CD40L comprises an amino acid sequence of any one of SEQ ID NOs: 32-34, 36, and 38, or an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) identity to any one of SEQ ID NOs: 32-34, 36, and 38.

[0075] In some embodiments, the tEGFR comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:40 or an amino acid sequence of SEQ ID NO:40.

[0076] In some embodiments, the HER2 comprises an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) identical to SEQ ID NO:45 or an amino acid sequence of SEQ ID NO:45.

[0077] In some embodiments, the CD20 comprises an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) identical to SEQ ID NO:49 or an amino acid sequence of SEQ ID NO:49.

[0078] Also within the scope of the invention are novel PD-1 decoy variants comprising an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) identity to any one of SEQ ID NOs: 6-17, 42, 44, 47-48 and 51-52 or an amino acid sequence of any one of SEQ ID NOs: 6-17, 42, 44, 47-48 and 51-52.

[0079] In some embodiments, the composition comprises at least two lymphocyte subsets. For example, the composition can include two, three, four, five or more genetically modified lymphocyte subsets. Each genetically modified lymphocyte subset can express at least one transgene. For example, each genetically modified lymphocyte subset can express two, three, four, five or more transgenes.

[0080] In some embodiments, the composition comprises two genetically modified lymphocyte subsets, wherein each subset expresses at least one transgene. In some embodiments, the composition comprises two genetically modified lymphocyte subsets, wherein each subset expresses two transgenes. In some embodiments, the composition comprises three genetically modified lymphocyte subsets, wherein each subset expresses at least one transgene. In some embodiments, the composition comprises four genetically modified lymphocyte subsets, wherein each subset expresses at least one transgene. In some embodiments, the composition comprises five or more genetically modified lymphocyte subsets, wherein each subset expresses at least one transgene.

[0081] In some embodiments, the composition comprises at least two genetically modified lymphocyte subsets, wherein each subset expresses at least two transgenes, and wherein each subset shares one transgene. In some embodiments, the composition comprises at least two genetically modified lymphocyte subsets, wherein each subset expresses at least two transgenes, and wherein each subset expresses a different transgene.

[0082] In some embodiments, the plurality of lymphocytes may include: (i) a first subpopulation expressing at least two transgenes; and (ii) a second subpopulation expressing at least two transgenes, wherein at least one transgene of the first subpopulation is different from a transgene of the second subpopulation, or wherein at least one transgene of the first subpopulation is the same as a transgene of the second subpopulation. In some embodiments, the composition of lymphocytes may express three transgenes upon combining the first subpopulation and the second subpopulation.

[0083] In some embodiments, (i) the first subpopulation expresses at least PD-1 decoy or its variant / fragment and IL-2 variant / fragment, and the second subpopulation expresses at least PD-1 decoy or its variant / fragment and LIGHT or its variant / fragment; (ii) the first subpopulation expresses at least PD-1 decoy or its variant / fragment and IL-2 variant / fragment, and the second subpopulation expresses at least PD-1 decoy or its variant / fragment and IL-33 or its variant / fragment; (iii) the first subpopulation expresses at least PD-1 decoy or its variant / fragment and IL-2 variant / fragment, and the second subpopulation expresses at least PD-1 decoy or its variant / fragment and CD40L or its variant / fragment; iv) the first subpopulation expresses at least one PD-1 decoy or its variant / fragment and LIGHT or its variant / fragment, and the second subpopulation expresses at least PD-1 decoy or its variant / fragment and IL-33 or its variant / fragment; or (v) the first subpopulation expresses at least PD-1 decoy or its variant / fragment and LIGHT or its variant / fragment, and the second subpopulation expresses at least PD-1 decoy or its variant / fragment and CD40L or its variant / fragment; or (vi) the first subpopulation expresses at least PD-1 decoy or its variant / fragment and IL-33 or its variant / fragment, and the second subpopulation expresses at least PD-1 decoy or its variant / fragment and CD40L or its variant / fragment.

[0084] In some embodiments, the first subpopulation or the second subpopulation further expresses tEGFR or a variant thereof, tHER2 or a variant thereof, CD20 or a variant thereof, or CD19 or a variant thereof.

[0085] In some embodiments, (i) the first subpopulation expresses at least PD-1 decoy or variant / fragment thereof, tEGFR or variant / fragment thereof, and IL-2 variant / fragment, and the second subpopulation expresses at least PD-1 decoy or variant / fragment thereof, tEGFR or variant / fragment thereof, and LIGHT or variant / fragment thereof; (ii) the first subpopulation expresses at least PD-1 decoy or variant / fragment thereof, tEGFR or variant / fragment thereof, and IL-2 variant / fragment, and the second subpopulation expresses at least PD-1 decoy or variant / fragment thereof, tEGFR or variant / fragment thereof, and IL-33 or variant / fragment thereof; (iii) the first subpopulation expresses at least PD-1 decoy or variant / fragment thereof, tEGFR or variant / fragment thereof, and IL-2 variant / fragment, and the second subpopulation expresses at least PD-1 decoy or variant / fragment thereof, tEGFR or variant / fragment thereof, and CD40L or variant / fragment thereof; (iv) the first subpopulation at least expresses PD-1 decoy or its variant / fragment, tEGFR or its variant / fragment and LIGHT or its variant / fragment, and the second subpopulation at least expresses PD-1 decoy or its variant / fragment, tEGFR or its variant / fragment and IL-33 or its variant / fragment; (v) the first subpopulation at least expresses PD-1 decoy or its variant / fragment, tEGFR or its variant / fragment and LIGHT or its variant / fragment, and the second subpopulation at least expresses PD-1 decoy or its variant / fragment, tEGFR or its variant / fragment and CD40L or its variant / fragment; or (vi) the first subpopulation at least expresses PD-1 decoy or its variant / fragment, tEGFR or its variant / fragment and IL-33 or its variant / fragment, and the second subpopulation at least expresses PD-1 decoy or its variant / fragment, tEGFR or its variant / fragment and CD40L or its variant / fragment.

[0086] As used herein, the term "variant" refers to a first molecule related to a second molecule (also referred to as a "parent" molecule). The variant molecule can be derived from, separated from, based on, or homologous to the parent molecule. As used herein, a "functional variant" of a protein refers to a variant of the protein that at least partially retains the activity of the protein. Functional variants may include mutants (which may be insertion, deletion, or substitution mutants), including polymorphs, etc. Functional variants also include fusion products of such proteins with another, generally unrelated nucleic acid, protein, polypeptide, or peptide. Functional variations may be naturally occurring or artificial.

[0087] In some embodiments, transgenic variants may include one or more conservative modifications. Transgenic variants with one or more conservative modifications may retain desired functional properties, which can be tested using functional assays known in the art.

[0088] As used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or change the binding properties of the protein containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitution refers to replacing an amino acid residue with an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include: amino acids with basic side chains (such as lysine, arginine, histidine); acidic side chains (such as aspartic acid, glutamic acid); uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan); non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine); β-branched side chains (e.g., threonine, valine, isoleucine); aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine) include one or more conservative modifications. Cas proteins with one or more conservative modifications may retain desired functional properties, which can be tested using functional assays known in the art.

[0089] As used herein, the homology percentage between two amino acid sequences is equal to the percent identity between the two sequences. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity of the position = number of identical positions / total number of positions × 100), taking into account the number of gaps and the length of each gap, gaps need to be introduced to achieve optimal alignment of the two sequences. The comparison of sequences and the determination of the percent identity between the two sequences can be accomplished using a mathematical algorithm, as described in the following non-limiting examples.

[0090] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program of the GCG software package (available at www.gcg.com), using a Blossum62 matrix or a PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6.

[0091] The term "homolog" or "homologous", when applied to a polypeptide, refers to a high degree of sequence identity between two polypeptides, or a high degree of similarity between three-dimensional structures, or a high degree of similarity between active sites and mechanisms of action. In some embodiments, a homolog has greater than 60% sequence identity, more preferably greater than 75% sequence identity, and still more preferably greater than 90% sequence identity to a reference sequence. The term "substantial identity" applied to polypeptides means that two peptide sequences share at least 75% sequence identity when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights.

[0092] As used herein, a peptide or polypeptide "fragment" refers to a peptide, polypeptide or protein that is less than the full length. For example, a peptide or polypeptide fragment can have a length of at least about 3, at least about 4, at least about 5, at least about 10, at least about 20, at least about 30, at least about 40 amino acids or a single unit length thereof. For example, a fragment length can be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more amino acids. There is no upper limit to the size of a peptide fragment. However, in some embodiments, a peptide fragment length can be less than about 500 amino acids, less than about 400 amino acids, less than about 300 amino acids or less than about 250 amino acids.

[0093] Variants, mutants and homologues having significant identity to the transgene are also within the scope of the invention. For example, such variants and homologues may have at least about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to the transgene sequences described herein.

[0094] In some embodiments, the transgenic variant is a fusion polypeptide comprising a transgenic sequence fused to a fusion partner (e.g., N-terminal or C-terminal fusion). In some embodiments, the fusion partner comprises a fragment of a human immunoglobulin polypeptide sequence (e.g., a CH3 domain; or a partial or complete Fc region, such as IgG4Fc). For example, PD-1 or a variant / fragment thereof, IL-2 or a variant / fragment thereof, IL-33 or a variant / fragment thereof, CD40L or a variant / fragment thereof, or LIGHT or a variant / fragment thereof can be directly or indirectly fused or connected to a fusion partner at the N-terminus or C-terminus through a linker, such as IgG4Fc or a variant / fragment thereof.

[0095] The term "fusion polypeptide" or "fusion protein" refers to a protein produced by linking two or more polypeptide sequences together. The fusion polypeptides included in the present invention include the translation product of a chimeric gene construct that links a nucleic acid sequence encoding a first polypeptide to a nucleic acid sequence encoding a second polypeptide to form a single open reading frame. In other words, a "fusion polypeptide" or "fusion protein" is a recombinant protein composed of two or more proteins that are linked together by a peptide bond or several peptides. The fusion protein may also include a peptide connector between the two domains.

[0096] It is known that immunosuppressive polypeptides that inhibit or reduce immune responses by binding thereof include CD47, PD-1, CTLA-4 and their corresponding ligands, including SIRPα, PD-L1, PD-L2, B7-1 and B7-2. This polypeptide is present in the tumor microenvironment and inhibits the immune response to tumor cells. In various embodiments, the immune response of immune response cells is enhanced by transgenic inhibition, blocking or antagonizing the interaction of immunosuppressive polypeptides and / or their ligands. In one aspect, transgenes can be used as knockouts of inhibitory / checkpoint molecules, including but not limited to PD-1, CTLA-4, LAG-3, TIGIT, VISTA, TIM-3 and CBL-B.

[0097] It is known that co-stimulatory polypeptides that stimulate or increase immune responses by binding include CD28, OX-40, 4-1BB, CD27 and NKG2D and their corresponding ligands, including B7-1, B7-2, OX-40L, 4-1BBL, CD70 and NKG2D ligands. This polypeptide is present in the tumor microenvironment and activates the immune response to tumor cells. In various embodiments, the immune response of immune response cells can be enhanced by transgenic promotion, stimulation or excitement of proinflammatory polypeptides and / or their ligands.

[0098] In some embodiments, the transgenic is a cytokine or a growth factor. The terms "growth factor" and "cytokine" refer to signal molecules that control cell activity in an autocrine, paracrine or endocrine manner. They exert their biological functions by binding to specific receptors and activating related downstream signaling pathways, thereby regulating gene transcription in the nucleus and ultimately stimulating biological responses (Nicola N.Oxford; New York: Oxford University Press; 1994). Growth factors and cytokines affect a variety of physiological processes, such as cell proliferation, differentiation, apoptosis, immune or hematopoietic responses, morphogenesis, angiogenesis, metabolism, wound healing, and maintaining tissue homeostasis in adult organisms. Historically, growth factors have been considered to be biological parts that have a positive effect on cell growth and proliferation, while cytokines are generally considered to have immune or hematopoietic responses. However, with the fusion of different research fields, it has been found that "cytokines" and "growth factors" can have similar functions, and therefore, these terms can be used interchangeably in this article.

[0099] TGF-β superfamily: TGF-β superfamily includes TGF-β proteins, bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), glial-derived neurotrophic factor (GDNF), activins, inhibins, Nodal, Lefty, and Müllerian inhibitory factor (MIS). TGF-β superfamily members are multifunctional regulators of various biological processes, such as morphogenesis, embryonic development, adult stem cell differentiation, immune regulation, wound healing, inflammation, and cancer.

[0100] (1) BMP-like family: (BMP (i.e. BMP1-10, BMP-15), GDF (i.e. GDF1-15), AMH

[0101] (2) GDNF-like family: GDNF, Artemin, Neuturin and Persephone

[0102] (3) TGF-β-like family: TGF-β (i.e., TGF-β-1, TGF-β-2, TGF-β-3), activins (i.e., activin A / AB / B, inhibin A / B), Nodal

[0103] Epidermal growth factor (EGF): EGF family members include EGF, TGF-α, neuregulin, amphiregulin, betacellulin, etc. The members of the EGF family are known for their ability to stimulate cell proliferation, differentiation and survival. Downregulation of this family member and its receptor is closely related to tumorigenesis (Herbst RS. International Journal of Radiation Oncology, Biology, Physics 2004, 59 (2 Suppl): 21-26).

[0104] Platelet-derived growth factor (PDGF): Platelet-derived growth factor (PDGF) is a potent mitogenic and chemotactic protein. There are four known PDGF proteins encoded by four genes (PDGFA, PDGFB, PDGFC, and PDGFD). PDGF is secreted as disulfide-linked homodimers or heterodimers, including PDGF-AA, PDGF-BB, PDGF-CC, PDGF-DD, and PDGF-AB. There are two known PDGF receptors with intrinsic tyrosine kinase activity; PDGFRα and PDGFRβ, both of which can form heterodimers and homodimers. Ligand binding promotes receptor dimerization, autophosphorylation, and subsequent activation of multiple downstream intracellular signaling cascades. Signaling through PDGFRα is essential for the development of facial bones, hair follicles, spermatogenesis, oligodendrocytes and astrocytes, and the development of lung and intestinal villi, while signaling through PDGFRβ is essential for the development of blood vessels, kidneys, and white adipocytes (Heldin CH. Cell Commun Signal 2013, 11:97).

[0105] Fibroblast growth factor (FGF) family: In humans, 22 members of the FGF family have been identified, all of which are heparin-binding proteins. High-affinity interactions with cell surface-associated heparan sulfate proteoglycans are essential for receptor tyrosine kinase-mediated FGF signal transduction (Ornitz DM, Itoh N. Genome Biology 2001, 2 (3): REVIEWS 3005). FGF is a multifunctional protein that is primarily mitogenic, but also has regulatory, morphological, and endocrine effects. FGF is involved in embryonic development (Heldin CH: Targeting the PDGF signaling pathway in tumor treatment. Cell Commun Signal 2013, 11: 97), mature tissue / system angiogenesis (Kim BS et al. Biochemical and biophysical research communications 2014, 450(4): 1333-1338), keratinocyte organization (Tsuboi R, et al. The Journal of Investigative Dermatology 1993, 101(1): 49-53) and wound healing (Lee JG, Kay EP. Investigative Ophthalmology & Visual Science 2006, 47(4): 1376-1386).

[0106] Insulin-like growth factor (IGF): Insulin-like growth factor (IGF) is a protein with a high sequence similarity to insulin. The IGF receptor is a disulfide-linked heterotetrameric transmembrane protein with a cytoplasmic tyrosine kinase domain. There are two types of insulin-like growth factor receptors, IGFII-R and IGFII-R. The availability of IGF can be regulated by IGF binding proteins 1-6 (Griffeth RJ et al., Basic and clinical andrology 2014, 24: 12). The main effect of IGF is to promote cell growth. In fact, most of the effects of pituitary growth hormone are mediated by IGF, mainly IGF-1. Growth hormone stimulates many tissues, especially the liver, to synthesize and secrete IGF-1, which in turn stimulates hypertrophy (increase in cell size) and hyperplasia (increase in cell number) of most tissues, including bones. IGF can also induce neuronal survival, protect chondrocytes, and activate osteocytes (Brahmkhatri VP et al., BioMed research international 2015, 2015: 538019).

[0107] Vascular endothelial growth factor (VEGF): VEGF is a homodimeric glycoprotein growth factor specific to endothelial cells (Ferrara N, Gerber HP, LeCouter. Nature Medicine 2003, 9 (6): 669-676). They regulate angiogenesis and vascular permeability, especially during embryogenesis, bone growth and reproductive function. They also play an important role in hematopoiesis. VEGF signals mainly through tyrosine kinases VEGFR1 and VEGFR2, and stimulate cell survival, proliferation, migration and / or adhesion (Ferrara N. Endocrine Reviews 2004, 25 (4): 581-611). Downregulation of vascular endothelial growth factor is associated with tumors, intraocular neovascularization and other diseases (Ferrara N et al., Nature Medicine 2003, 9 (6): 669-676). Members of the VEGF gene family include VEGF / VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F and placental growth factor (PlGF) (P1GF) (Holmes DI, Zachary I. Genome Biology 2005, 6(2):209).

[0108] Hepatocyte growth factor (HGF): HGF is secreted by mesenchymal cells and acts as a multifunctional cytokine on cells mainly derived from epithelial and endothelial cells. It regulates cell growth, cell motility and morphogenesis by activating the tyrosine kinase signaling cascade through HGFR (Okada M et al., Pediatric Research 2004, 56 (3): 336-344). HGF has been shown to play an important role in embryonic organ development, adult organ regeneration and wound healing. In addition, its ability to stimulate mitosis, cell motility and matrix invasion makes it play a central role in angiogenesis and tumorigenesis (Sharma NS et al., FASEB 2010, 24 (7): 2364-2374).

[0109] Tumor necrosis factor (TNF): Cytokines known to be involved in tumor cell apoptosis were initially classified under the tumor necrosis factor (or TNF family). All TNF family members share a trimeric, conserved C-terminal domain called the "TNF homology domain" or THD. The THD, which is responsible for receptor binding, shares approximately 20–30% sequence identity among family members. Although most ligands are synthesized as membrane-bound proteins, soluble forms can be produced by restriction enzyme digestion (Bodmer JL et al., Trends in Biochemical Sciences 2002, 27(1): 19-26). The first two members of the family are TNFα and TNFβ. To date, 19 TNF superfamily ligands and 32 TNF superfamily receptors have been identified. Although many TNF superfamily members promote or inhibit apoptosis, they also regulate key functions of the innate and adaptive immune systems, including natural killer cell activation, T cell co-stimulation, and B cell homeostasis and activation (Croft M. Nature Reviews Immunology 2009, 9 (4): 271-285). LIGHT (homologous to lymphotoxin, exhibits inducible expression, and competes with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes) is a type II transmembrane glycoprotein of the TNF ligand superfamily. LIGHT is expressed on immature DCs and activated T cells and binds to three different receptors, namely herpes virus entry mediator (HVEM), lymphotoxin-β receptor (LTβR), and decoy receptor 3 / TR6. After binding to HVEM, LIGHT co-stimulates T cells, accelerating proliferation and cytokine production. Another example is CD154, also known as CD40 ligand or CD40L. It is a protein primarily expressed on activated T cells and is a member of the TNF superfamily of molecules. It binds to CD40 on antigen presenting cells, resulting in a variety of effects depending on the target cell type. Another example is Fas ligand (FasL or CD95L or CD178). Fas ligand / receptor interactions play an important role in the regulation of the immune system and the progression of cancer.

[0110] Interleukins (ILs): Interleukins are a large class of immunomodulatory proteins that regulate the growth, differentiation, and activation of cells in the immune or hematopoietic system during immune responses. Based on their distinguishing structural characteristics, known ILs can be divided into four major categories, including: IL1-like cytokines, class I helical cytokines (IL4-like, γ-chain, and IL-6 / 12-like), class II helical cytokines (IL-10-like and IL-28-like), and IL-17-like cytokines (Table 1).

[0111]

[0112]

[0113] Interferons (IFNs): IFNs are a group of signaling proteins that are produced and released by host cells in response to the presence of pathogens such as viruses, bacteria, parasites, or tumor cells. Interferons also have immunomodulatory functions; they inhibit B-cell activation, enhance T-cell activity, and increase the cell-destructive capacity of natural killer cells. More than 20 different IFN genes and proteins have been identified in animals, including humans. They are generally divided into two categories: type I IFNs and type II IFNs. Type I IFNs are also called viral IFNs and include IFN-α, IFN-β, and IFN-ω. Type II IFNs are also called immune interferons (IFN-γ). Viral IFNs are induced by viral infection, while type II IFNs are induced by mitosis or antigenic stimulation. Most types of virus-infected cells are able to synthesize type I IFNs in cell culture. In contrast, IFN-γ is synthesized only by certain cells of the immune system, including natural killer cells, CD4 Th1 cells, and CD8 cytotoxicity suppressor cells (Samuel CE. Clinical Microbiology Reviews 2001, 14(4):778-809, table of contents).

[0114] In some embodiments, the transgene is a decoy receptor. A "decoy receptor" is a receptor that can effectively recognize and bind to a specific growth factor or cytokine, but is structurally incapable of signaling or activating the intended receptor complex. It acts as an inhibitor, binding to the ligand and preventing it from binding to the conventional receptor.

[0115] In some embodiments, the transgene is a soluble bait. "Soluble bait" refers to a polypeptide expressed and secreted from a cell that binds to a specific receptor on different cells, thereby inhibiting the binding of its natural ligand to the receptor. Non-limiting examples of soluble baits include PD1 bait, CTLA-4 bait, LAG3 bait, VEGFR1 bait, TIM3 bait, TIGIT bait, and SIRPα bait. In one embodiment, the PD-1 bait is expressed and secreted by lymphocytes, and this PD-1 bait inhibits the binding of natural PD-1 on T cells to PDL-1 on antigen presenting cells (APCs) by occupying the binding site of PD-L1 on antigen presenting cells (APCs), thereby inhibiting the immunosuppressive signal of T cells, thereby enhancing the immune response of T cells.

[0116] PD-1 decoy: PD-1 is a strong negative regulator of T lymphocytes in the tumor microenvironment. In one embodiment, T cells expressing a dominant negative deletion mutant of PD-1 (a non-limiting example of a PD-1 decoy) are generated by retroviral transduction. This PD-1 decoy increases the secretion of IFN-γ by antigen-specific T cells in response to tumor cells expressing a cognate antigen. In another embodiment, a soluble fragment of a PD-1 extracellular domain (a non-limiting example of a PD-1 decoy) with a higher binding affinity to PDL-1 is administered as a competitive antagonist of PDL-1. Non-limiting examples of soluble PD-1 extracellular domain variants are disclosed in Maute et al. (Maute et al., PNAS 2015 Nov 24; 112 (47): E6506-E6514). In yet another embodiment, a PD-1 decoy molecule comprising a PD1 extracellular domain fused to the Fc region of human IgG4 (PD-1IgG4) can be used to enhance tumor control in vivo. In another embodiment, this PD-1 decoy can be expressed and secreted by TILs.

[0117] As described herein, PD-1 decoys can also be generated by computationally based rational design to develop binding and / or solubility enhancing variants of the PD-1 extracellular domain. For example, single and multiple amino acid substitutions predicted to increase the binding affinity of PD-1 to PD-L1 are evaluated in recombinant soluble proteins produced in bacterial expression systems. The variants can be evaluated by direct titration ELISA binding to plate-captured PD-L1 variants of interest, and then the variants can be cloned into retroviral vectors to evaluate T cell secretion. PD-1 decoys that exhibit low solubility during bacterial production are discarded because low solubility generally corresponds to no or low yield of T cells.

[0118] PD-1 decoys produced by engineered human T cells also contain an Fc portion (e.g., IgG4Fc) to increase the affinity and stability of the protein. PD1-Fc decoys produced by human primary T cells can be evaluated using ELISA. To evaluate functionality, a co-culture assay was established in which human primary T cells were co-engineered to express the A2 / NY-ESO-1 T cell receptor (TCR) to allow tumor cell recognition (via lentiviral transduction), and to express PD1-Fc decoy and cell surface tEGFR (encoded in a bicistronic retroviral vector). These co-transduced T cells (or control T cells containing only the TCR or PD1 decoy) were then transfected with PD-1. posCo-culture of target tumor cells. The level of IFNγ present in the co-culture supernatant was evaluated to determine the optimal PD-1 decoy variant (i.e., the higher the level of IFNγ, the better the PD1 decoy blocked PD-L1 on the surface of target tumor cells). 4XMUT_M70 and 6XDM are PD1-Fc decoy variants that showed high binding affinity to PD-L1 and high solubility (Figure 9A-D).

[0119] Cell Elimination Tag (CET): The transgene, such as a PD1-Fc decoy, can be constitutively expressed from a bicistronic retroviral vector that also encodes a CET, such as tEGFR, tHER2, CD20, or CD19 (Figure 10A-D). The purpose of the CET is fourfold. First, it can be used as a means to assess transduction efficiency, and second, it can be used to enrich engineered cells (on anti-EGFR coated beads) if necessary. Third, it can be used as a means to track engineered T cells in the patient after implantation (via FACs from drawn blood samples or tumor biopsies). Finally, if toxicity occurs in patients on cetuximab, it can be used as an elimination tag via ADCC. A truncated human EGFR polypeptide (huEGFRt) lacking the extracellular N-terminal ligand binding domain and intracellular receptor tyrosine kinase activity, but retaining the native amino acid sequence, type I transmembrane cell surface localization and conformational integrity of the binding epitope of the pharmaceutical-grade anti-EGFR monoclonal antibody cetuximab (Erbitux), was described by Want et al. (Wang X et al., Blood. 2011 Aug 4; 118(5): 1255-63. Epub 2011 Jun 7). Other examples of CETADCC may include tHER2 (containing Herceptin or Herceptin), CD20 (containing Rituximab) and CD19. Griffioen et al. (Griffioen et al., Haematologica. 2009 Sep; 94(9): 1316-20) described CD20 as a CET. Budde et al. (Budde et al., Blood 2013; 122(21): 1660) and Annesley et al. (Annesley et al., Blood 2019; 134(Supplement_1): 223) described CD19 as a CET.

[0120] LIGHT: LIGHT is a type II transmembrane glycoprotein of the TNF ligand superfamily (Mauri et al., Immunition 1998 January; 8(1): 21-30). It is expressed on immature dendritic cells and activated T cells (Tamada K et al., J Immunol. 2000 Apr 15; 164(8): 4105-10) and binds to three different receptors, namely herpes virus entry mediator (HVEM), lymphotoxin-β receptor (LTβR) and decoy receptor 3 / TR6. After binding to HVEM, light co-stimulates T cells and accelerates proliferation and cytokine production (Tamada et al., Nat Med. 2000 March; 6(3): 283-9). In one embodiment, the LIGHT protein can be engineered to be expressed and secreted from TILs.

[0121] IL-33: Cytokines are central intercellular mediators in the inflammatory tumor microenvironment, among which interleukin-33 (IL-33) is considered to be an alarmin released after cell damage. IL-33 was found to be a member of the IL-1 cytokine family. The IL-1 gene family contains 11 members (IL-1α, IL-1β, IL-1RA, IL-18, IL-36RA, IL-36α, IL-37, IL-36β, IL-36γ, IL-38, IL-33), which induce a complex network of proinflammatory cytokines and regulate and initiate inflammatory responses by expressing integrins on leukocytes and endothelial cells (Role of interleukin-1 in the pathogenesis and treatment of inflammatory diseases) (Dinarello CA., Blood. 2011 Apr 7; 117(14): 3720-32). The development of tumors can trigger anti-tumor immune responses. Type 1 immune response is a key component of cell-mediated immunity, including tumor-induced IFN-γ-producing Th1 cells, cytotoxic T lymphocytes, NKT cells, and γδT cells to limit tumor growth and metastasis (Galon J et al., Science. 2006 Sep 29; 313(5795): 1960-4.). Since inflammation is another important component of malignant tumors, IL-33 can play a role in improving cancer surveillance and anti-tumor immunity. In one embodiment of the present invention, IL-33 can be engineered to be expressed and secreted from TIL.

[0122] IL-2: Interleukin-2 (IL-2) was one of the first cytokines to be molecularly characterized. It has primarily been shown to support the growth and expansion of T cells and NK cells. IL-2 was approved for clinical use in 1992, but the precise description of its receptor biology is still under investigation. Systemic high-dose (HD) IL-2 treatment produces durable responses in patients with melanoma and renal cancer, but only in a relatively small subset of patients. In addition, systemic HD IL-2 treatment can induce significant toxicity, also limiting its clinical relevance. IL-2 promotes the activation and expansion of T cells and NK cells in vitro. In one embodiment, IL-2 or a functional variant thereof can be engineered to be expressed and secreted from TILs. Such TILs can also be engineered to secrete additional transgenes.

[0123] CD40L: As an immune co-stimulatory molecule, CD40 and its ligand CD40L can complement each other. Previous studies have shown that CD40 and CD40L play a key role in humoral and cellular immunity, and the expression of CD40 and CD40L is closely related to the occurrence and development of various diseases (Elgueta et al., Immunol Rev 2009; 229: 152-172). CD40 has been found to be highly expressed in bladder cancer, breast cancer, ovarian cancer and other tumors (Hussain et al., Br J cancer 2011; 88: 586). CD40L, as the main ligand of CD40, is mainly expressed on the surface of activated CD4+T cells. When CD40 binds to CD40L, CD40L can activate T lymphocytes and Fas-mediated tumor cell apoptosis pathways.

[0124] On the other hand, the above-mentioned genetically modified lymphocytes can be incorporated into a pharmaceutical composition suitable for administration. The pharmaceutical composition generally comprises substantially isolated / purified lymphocytes and a pharmaceutically acceptable carrier in a form suitable for administration to a subject. The pharmaceutically acceptable carrier is determined in part by the specific composition being administered and the specific method for administering the composition. The pharmaceutical composition is generally manufactured in full accordance with all good manufacturing practices (GMP) prescribed by the U.S. Food and Drug Administration.

[0125] The terms "pharmaceutically acceptable" and "physiologically tolerable" refer to compositions, carriers, diluents and agents that can be used interchangeably, including materials that can be applied to or administered to a subject without causing adverse physiological effects to an extent that prohibits the administration of the composition. For example, "pharmaceutically acceptable excipients" include excipients that can be used to prepare generally safe, non-toxic and desirable pharmaceutical compositions, and include excipients that are acceptable for veterinary use and human medical use.

[0126] Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. The use of such media and compounds for pharmaceutically active substances is well known in the art. Unless any conventional media or compounds are incompatible with the disclosed compositions, their use in the compositions is contemplated. In some embodiments, a second therapeutic agent such as an anticancer or antitumor agent may also be incorporated into the pharmaceutical composition.

[0127] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be a liquid that is easy to inject. It must remain stable under manufacturing and storage conditions and must prevent contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.) and a suitable mixture thereof. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, maintaining the desired particle size in the case of dispersion, and by using a surfactant.

[0128] In some embodiments, the composition includes the genetically modified lymphocytes described above and optionally a cryoprotectant (eg, glycerol, DMSO, polyethylene glycol).

[0129] The composition or pharmaceutical composition described herein can be provided in a kit. In one embodiment, the kit includes (a) a container comprising the composition and optionally (b) information material. The information material can be descriptive, instructive, marketing or other materials related to the methods described herein and / or preparations used for treatment benefits. For example, the kit may include production instructions, treatment regimens used, and administration cycles. In one embodiment, the kit also includes additional therapeutic agents (e.g., checkpoint regulators). The kit may include one or more containers, each of which contains different reagents. For example, the kit includes a first container containing the composition and a second container for additional therapeutic agents.

[0130] The container may include a unit dose of the pharmaceutical composition.In addition to the composition, the kit may also include other ingredients, such as solvents or buffers, adjuvants, stabilizers or preservatives.

[0131] The kit optionally includes a device suitable for administering the composition, such as a syringe or other suitable delivery device. The device may be pre-loaded with one or both agents, or may be empty but suitable for loading.

[0132] B. Methods of Making the Compositions

[0133] In another aspect, the present invention also provides a method for preparing the above composition, comprising: (a) providing a plurality of lymphocytes; (b) introducing a nucleic acid molecule encoding at least two transgenes into the plurality of lymphocytes to obtain a plurality of genetically modified lymphocytes; and (c) amplifying the plurality of genetically modified lymphocytes in a cell culture medium.

[0134] In some embodiments, the method may include: (a) providing a plurality of lymphocytes; (b) introducing two or more nucleic acid molecules into the plurality of lymphocytes, each of the two or more nucleic acid molecules encoding at least one transgene, thereby obtaining a plurality of genetically modified lymphocytes; and (c) expanding the plurality of genetically modified lymphocytes in a cell culture medium.

[0135] In some embodiments, the transgene comprises two or more of PD-1 decoy, IL-2 variant / fragment, LIGHT or its variant / fragment, IL-33 or its variant / fragment, and CD40L or its variant / fragment. In some embodiments, the transgene further comprises tEGFR or its variant / fragment. In some embodiments, PD-1 decoy or its variant / fragment and tEGFR or its variant / fragment (or tHER2 or its variant / fragment, CD20 or its variant / fragment, or CD19 or its variant / fragment) are contained on the same vector.

[0136] In some embodiments, the at least two transgenes include: (a) PD-1 decoy or a variant thereof and tEGFR or a variant thereof; (b) PD-1 decoy or a variant thereof and IL-2 variant; (c) PD-1 decoy or a variant thereof and LIGHT or a variant thereof; (d) PD-1 decoy or a variant thereof and IL-33 or a variant thereof; (e) PD-1 decoy or a variant thereof and CD40L or a variant thereof; (f) PD-1 decoy or a variant thereof, IL-2 variant and IL-33 or a variant thereof; (g) PD-1 decoy or a variant thereof, tEGFR or a variant thereof and IL-2 variant; (h) PD-1 decoy or a variant thereof, tEGFR or its variants, and LIGHT or its variants; (i) PD-1 decoy or its variants, tEGFR or its variants, IL-33 or its variants; (j) PD-1 decoy or its variants, tEGFR or its variants, CD40L or its variants; (k) PD-1 decoy or its variants, tEGFR or its variants, IL-2 variants and IL-33 or its variants; (l) PD-1 decoy or its variants, tEGFR or its variants, IL-2 variants and CD40L or its variants; or (m) PD-1 decoy or its variants, tEGFR or its variants, IL-33 variants and CD40L or its variants.

[0137] In some embodiments, the method may include: (a) introducing a first nucleic acid molecule encoding at least two transgenes into a first plurality of lymphocytes to obtain a first plurality of genetically engineered lymphocytes; and (b) introducing a second nucleic acid molecule encoding at least two transgenes into a second plurality of lymphocytes to obtain a second plurality of genetically engineered lymphocytes. In some embodiments, the method further includes combining the first plurality of genetically engineered lymphocytes with the first plurality of genetically engineered lymphocytes at a predetermined ratio between about 1:1 and about 1:100 (e.g., 1:1, 1:2, 1:5, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100).

[0138] In some embodiments, the method comprises: a) introducing transgenes in different lymphocyte subsets, wherein each subset expresses at least one transgene, and b) combining at least two lymphocyte subsets. In some embodiments, each subset expresses at least two transgenes according to the above embodiments. In some embodiments, the lymphocyte composition expresses at least three different transgenes.

[0139] In some embodiments, the method for obtaining the above-mentioned tumor-specific gene-modified lymphocyte subset composition can be performed in vitro or ex vivo. A more specific form of the method can be disclosed as in PCT / EP2018 / 080343, the entire contents of which are incorporated herein by reference.

[0140] In some embodiments, the method can further include expanding the first plurality of lymphocytes in the cell culture medium after the step of introducing the first nucleic acid, or expanding the second plurality of lymphocytes in the cell culture medium after the step of introducing the second nucleic acid.

[0141] The term "culture" or "expansion" refers to maintaining or culturing cells under conditions where the cells can proliferate while avoiding senescence. For example, cells can be cultured in a culture medium that optionally contains one or more growth factors (i.e., a growth factor cocktail). In some embodiments, the cell culture medium is a defined cell culture medium. The cell culture medium may include a new antigenic peptide. A stable cell line may be established to allow the cells to continue to propagate.

[0142] a. Lymphocytes

[0143] Prior to the amplification and genetic modification of lymphocytes as described herein, lymphocytes from a subject source are obtained. Lymphocytes can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. As described herein, any number of lymphocyte lines available in the art can be used. Lymphocytes can be obtained using any number of techniques known to those skilled in the art, such as Ficoll. TM Separation, obtained from a unit of blood collected from a subject. Circulating blood cells of an individual are obtained by apheresis. Apheresis products usually contain lymphocytes, including T lymphocytes, monocytes, granulocytes, B lymphocytes, other nucleated leukocytes, red blood cells and platelets. The cells obtained by apheresis can be washed to remove the plasma portion, and the cells are placed in a suitable buffer or culture medium for subsequent processing steps. The cells can be washed with phosphate buffered saline (PBS). Optionally, the washing solution may lack calcium, may lack magnesium, or may lack many (not all) divalent cations. As will be readily appreciated by those of ordinary skill in the art, the washing step can be achieved by methods known to those skilled in the art, such as using a semi-continuous automatic flow centrifuge (e.g., Cobe 2991 cell processo, Baxter CytoMate or e1Haemonetics cell Saver 5) according to the manufacturer's instructions. After washing, the cells can be resuspended in a variety of biocompatible buffers, for example, Ca-free 2+ , Mg-free 2+ Alternatively, the undesired components of the aliquot can be removed and the cells can be resuspended directly in culture medium.

[0144] As described herein, lymphocytes can be isolated from peripheral blood by lysis of erythrocytes and depletion of monocytes, for example by PERCOLL TM Gradient centrifugation or counter-flow centrifugation elution. If desired, specific subpopulations of lymphocytes, such as T lymphocytes (i.e., Cd3+, CD28+, CD4+, CD8+, CD45RA+, or CD45RO+ T lymphocytes) can also be isolated by positive or negative selection techniques. For example, T lymphocytes can be isolated by incubation with anti-CD3 / anti-CD28 coupled magnetic beads (i.e., 3x28), e.g. M-450 CD3 / CD28 T, the incubation time is long enough (i.e., 30 minutes to 24 hours) to positively select the desired T lymphocytes. For the isolation of T lymphocytes from leukemia patients, using a longer incubation time (e.g., 24 hours) can increase cell performance. Any situation where there are fewer T lymphocytes than other cell types, such as the isolation of tumor infiltrating lymphocytes (TIL) from tumor tissue or immunocompromised individuals, can use a longer culture time to isolate T lymphocytes. Those skilled in the art recognize that multiple rounds of selection can also be used. It may be desirable to perform a selection procedure and use 'unselected' cells during activation and amplification. The "unselected" cells can also be subjected to a new round of selection.

[0145] Lymphocyte populations (e.g., T lymphocytes) enriched by negative selection can be performed using a combination of antibodies for unique surface markers of negatively selected cells. One method is to sort and / or select cells using a cocktail of monoclonal antibodies for cell surface markers present in negatively selected cells by negative magnetic immunoadhesion or flow cytometry. For example, in order to enrich CD4+ cells by negative selection, monoclonal antibodies typically include antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. Alternatively, regulatory T lymphocytes are consumed by anti-C25 coupled magnetic beads or other similar selection methods.

[0146] The lymphocytes used for stimulation may also be frozen after the washing step. Without wishing to be bound by theory, the freezing and subsequent thawing steps provide a more homogeneous product by eliminating granulocytes and to some extent monocytes from the cell population. After the washing steps remove plasma and platelets, the cells may be suspended in a freezing solution. Although many solutions and freezing parameters are known in the art and would be useful in this context, one method involves the use of PBS containing 20% ​​DMSO and 8% human serum albumin, or medium containing 10% dextran 40 and 5% glucose human albumin and 7.5% DMSO or 31.25% Plasmalyte A, 31.25% 5% glucose, 0.45% NaCl, 10% dextran 40 and 5% glucose, medium containing 20% ​​human albumin serum and 7.5% DMSO or other suitable cell freezing medium containing, for example, Hespan and PlasmaLyte A. The cells can then be frozen at -80°C at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen tank. Other controlled freezing methods can be used, as can immediate uncontrolled freezing at -20°C or in liquid nitrogen.

[0147] Cryopreserved cells can be thawed and washed as described herein, and left to stand for one hour at room temperature before activation using the method described in the present invention. As described herein, lymphocytes can be amplified, frozen and used later. As described herein, samples can be collected from patients soon after being diagnosed as a specific disease as described herein, but before any treatment. Before any number of related treatment modes, cells can be isolated from a blood sample or a single collection of a subject, including but not limited to using such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiotherapy, immunosuppressive agents (immunosuppressiveagents) such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil and FK506, antibodies or other immunoablative agents (immunoablatories) such as CAMPATH, anti-CD3 antibodies, cyclophosphamide, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228 and radiation. These drugs inhibit calcium-dependent calcineurin (e.g., cyclosporine and FK506) or inhibit p70S6 kinase, which is important for growth factor (rapamycin)-induced signaling (Liu et al., Cell 66:807-815 1991; Henderson et al., Immun 73:316-321, 1991, Bierer et al., Curr. Opin. Immun., 5:763-773, 1993). These cells can be isolated from the patient and frozen for later use with bone marrow or stem cell transplantation, T lymphocyte ablative therapy using chemotherapy drugs (e.g., fludarabine), external beam radiation therapy (XRT), cyclophosphamide or antibodies (e.g., OKT3 or CAMPATH) (e.g., before, at the same time, or after transplantation). As described herein, cells can be isolated prior to treatment and can be frozen for use in treatment after B lymphocyte ablative therapy (e.g., agents reactive with CD20 (e.g., rituximab)).

[0148] Before or after genetically modifying lymphocytes (e.g., T lymphocytes) to express a desired transgene, lymphocytes can generally be activated and expanded using methods such as those described in U.S. Patents 6352694, 6534055, 6905680, 6692964, 5858358, 6887466, 6905681, 7144575, 7067318, 7172869, 7232566, 7175843, 5883223, 6905874, 6797514, 6867041, and U.S. Patent Application Publication No. 20060121005.

[0149] b. Carrier

[0150] Transgenes can be introduced into lymphocytes by a variety of methods, including but not limited to cell transduction using integration-competent gamma-retroviruses or lentiviruses, and DNA transposition.

[0151] A variety of vectors can be used for transgenic expression. Certain viruses infect cells or enter cells through receptor-mediated endocytosis, and the ability to integrate into the host cell genome and stably and efficiently express viral genes makes them attractive candidate viruses for transferring exogenous nucleic acids into cells. Therefore, in certain embodiments, a nucleotide sequence encoding one or more transgenics or fragments thereof is introduced into a host cell for expression using a viral vector. The viral vector may include a nucleotide sequence encoding one or more transgenics or fragments thereof that are operably connected to one or more control sequences (e.g., promoters). Alternatively, the viral vector may not include a control sequence, but relies on a control sequence in the host cell to drive the expression of a transgenic or fragment thereof. Non-limiting examples of viral vectors that can be used to deliver nucleic acids include adenoviral vectors, AAV vectors, and retroviral vectors.

[0152] For example, adeno-associated virus (AAV) can be used to introduce nucleotide sequences encoding one or more transgenes or fragments thereof into host cells for expression. The AAV system has been described previously and is well known in the art (Kelleher and Vos, Biotechniques, 17(6): 1110-71994; Cotten et al., Proc Natl Acad Sci USA, 89(13): 6094-6098, 1992; Curiel, Nat Immun, 13(2-3): 141-64, 1994; Muzyczka, Curr-Top Microbiol Immunol, 158: 97-1291992). For example, details on the generation and use of rAAV vectors are described in U.S. Pat. Nos. 5,139,941 and 4,797,368, each of which is incorporated herein by reference in its entirety for all purposes.

[0153] In some embodiments, retroviral expression vectors can be used to introduce nucleotide sequences encoding one or more transgenes or fragments thereof into host cells for expression. These systems have been described previously and are well known in the art (Nicolas and Rubinstein, In, Rodriguez and Denhardt, eds., Stoneham: Butterworth, pp. 494-513, 1988; Temin, In: Gene Transfer, Kucherlapati (ed.), New York: Plenum Press, pp. 149-188, 1986). Vectors for eukaryotic expression in mammalian cells include AD5, pSVL, pCMV, pRc / RSV, pcDNA3, pBPV, and the like, and vectors derived from viral systems such as vaccinia virus, adeno-associated virus, herpes virus, retrovirus, etc. using promoters such as CMV, SV40, EF-1, UbC, RSV, ADV, BPV, and β-actin.

[0154] A combination of retrovirus and a suitable packaging system can also be used, and the capsid protein will play a role in infecting the target cells. Typically, cells and viruses will be incubated in culture medium for at least 24 hours. Then, in some applications, cells are allowed to grow in culture medium for a short time before analysis, such as 24-73 hours, or at least two weeks, and cells are also allowed to grow for five weeks or more. Commonly used retroviral vectors have "defects", that is, they cannot produce the viral proteins required for productive infection. The replication of the vector needs to be grown in a packaging cell line. The host cell specificity of the retrovirus is determined by the envelope protein env (pl20). The envelope protein is provided by the packaging cell line. There are at least three types of envelope proteins, namely ecotropic, amphoteric and heterotropic. Retroviruses packaged with ecotropic envelope proteins, such as MMLV, can infect most cell types of mice and rats. Ecotropic packaging cell lines include BOSC23. Retroviruses carrying amphoteric envelope proteins, such as 4070A, can infect most mammals, including cell types of humans, dogs and mice. Amphotropic packaging cell lines include PA12 and PA317. Retroviruses packaged with heterotropic envelope proteins, such as AKR env, are able to infect most mammalian cell types except mouse cells. The vector can include genes that must be removed later, such as using a recombinase system such as Cre / Lox, or genes that are destroyed by the cells expressing them (e.g., by including genes that allow selective toxicity, such as herpes virus TK, BCL-xs, etc.). Suitable inducible promoters are activated in the desired target cell type, whether it is a transfected cell or its progeny.

[0155] Non-limiting examples of vectors for use in the present invention include retroviral vector SFG.MCS and helper plasmids RD114, Peg-Pam3 (Arber et al., J Clin Invest 2015 Jan 2; 125(1): 157-168), lentiviral vector pRRL, and helper plasmids R8.74 and pMD2G (eg, Addgene Plasmid #12259), lentiviral vector pRRL and helper plasmids R8.74 and pMD2G (eg, Addgene Plasmid #12259). In some embodiments, the Sleeping Beauty transposon system can be used (Deniger et al., 2016 Mol Ther. Jun; 24(6): 1078-1089). In some embodiments, transgenes can be introduced into cells by deforming the cell as it passes through a small opening, disrupting the cell membrane and allowing the material to be inserted into the cell, such as electroporation (Xiaojun et al., 2017 Protein Cell, 8(7): 514-526) or Cell This electroporation method of RNA encoding a transgene allows for transient expression of the transgene in cells, thereby limiting toxicity and other adverse effects of the engineered cells (Barrett et al., 2011 Hum Gene Ther. Dec; 22(12): 1575-1586).

[0156] In some embodiments, genome editing techniques, such as CRISPR / Cas9 systems, designer zinc fingers, transcription activation-like effects (TALEs) or homing meganuclease, can be used to induce transgenic expression in immune cells. In general, "CRISPR / Cas9 systems" are collectively referred to as transcripts and other elements involved in expressing or guiding CRISPR-related ("Cas") gene activity, including sequences encoding Cas genes, tracr (trans-activation CRISPR) sequences (such as tracrRNA or tracrRNA active parts), tracr pairing sequences (including "direct repeats" and direct repeats of the parts processed by tracrRNA in endogenous CRISPR systems), guide sequences (also referred to as "intervals" in the context of endogenous CRISPR systems) or other sequences and transcripts from CRISPR loci. One or more elements of a CRISPR system can be derived from a type I, type II or type III CRISPR system. Alternatively, one or more elements of a CRISPR system can be from a specific organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes. In general, CRISPR systems are characterized by promoting the formation of a CRISPR complex at the site of a target sequence (also called a protospacer sequence in the context of endogenous CRISPR systems).

[0157] In some embodiments, genetic modification is introduced by transfecting lymphocytes with a vector (e.g., a lentiviral vector) encoding one or more transgenes or functional fragments thereof and CA9 or functional fragments thereof. In some embodiments, one, two, or more vectors can be used to introduce one or more transgenes or functional fragments thereof and CA9 or functional fragments thereof into immune cells.

[0158] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, liposome transfection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing exogenous vectors and / or nucleic acids are well known in the art. For example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0159] Chemical methods for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, microbeads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles and liposomes. An exemplary colloidal system used as a release carrier in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0160] In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. Lipid preparations are used to introduce nucleic acids into host cells (in vitro, in vitro or in vivo). On the other hand, nucleic acids can be associated with lipids. Nucleic acids associated with lipids can be encapsulated in the aqueous interior of liposomes, dispersed in the lipid bilayer of liposomes, combined with liposomes by binding molecules associated with liposomes and oligonucleotides, embedded in liposomes, forming complexes with liposomes, dispersed in solutions containing lipids, mixed with lipids, combined with lipids, included in lipids, contents or in complexes formed with micelles in the form of suspensions, or otherwise combined with lipids. Compositions associated with lipids, lipid / DNA or lipid / expression vectors are not limited to any specific structure in solution. For example, they can be present in a double-layer structure, such as micelles or have a "folded" structure. They can also be simply dispersed in solution, possibly forming aggregates of uneven size or shape. Lipids are fatty substances, which can be natural or synthetic lipids. For example, lipids include fat droplets naturally present in the cytoplasm, and compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0161] Lipids suitable for use can be obtained from commercial sources. For example, distyrylphosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, Missouri; dihexadecyl phosphate ("DCP") can be obtained from K&K Laboratories (Plainview, New York); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristoylphosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids (Birmingham, Alabama). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the only solvent because it evaporates more easily than methanol. "Liposome" is a general term that includes a variety of unique multilayer lipid carriers formed by bilayers or closed lipid aggregates. Liposomes can be described as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes are formed by multilayer lipids separated by an aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. Before forming a closed structure, the lipid components undergo self-rearrangement and capture dissolved water and solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5:505-10). However, compositions having different structures from normal vesicles in solution are also included. For example, lipids can present micellar structures or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also considered.

[0162] Regardless of the method used to introduce exogenous nucleic acid into the host cell, the presence of the recombinant DNA sequence in the host cell can be confirmed by a series of tests. Such tests include, for example, "molecular biology" tests well known to those skilled in the art, such as Southern and Northern hybridizations, RT-PCR and PCR; biochemical tests, such as determining the presence or absence of specific peptides, such as by immunological means (ELISA and Western blot) or by the tests described herein to identify agents within the scope of the present invention.

[0163] C. Treatment methods

[0164] The present invention also provides a method for treating cancer or tumor, which comprises administering an effective therapeutic amount of the above composition or pharmaceutical composition to a subject in need.

[0165] As used herein, the terms "subject" and "patient" are used interchangeably, regardless of whether the subject has or is receiving any form of treatment. As used herein, the terms "subject" and "subjects" may refer to any vertebrate, including but not limited to mammals (e.g., cattle, pigs, camels, llamas, alpacas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats and mice, non-human primates (e.g., monkeys, such as cynomolgus monkeys, chimpanzees, etc.) and humans). The subject may be human or non-human. In more exemplary aspects, the mammal is a human.

[0166] In some embodiments, the subject is human. In some embodiments, the subject has cancer. In some embodiments, the subject is immunodepleted.

[0167] As used to describe the present invention, "cancer", "tumor" and "malignant tumor" are all equivalent to the proliferation of a tissue or organ. If the tissue is part of the lymphatic or immune system, the malignant cells may include non-solid tumors of circulating cells. Malignant tumors of other tissues or organs may give rise to solid tumors. The methods of the present invention may be used to treat lymphocytes, circulating immune cells and solid tumors.

[0168] Cancers that can be treated include non-vascularized or substantially non-vascularized tumors, and vascularized tumors. Cancers can include non-solid tumors (such as blood tumors, such as leukemias and lymphomas) or can include solid tumors. The types of cancers that can be treated with the compositions of the present invention include, but are not limited to, carcinomas, blastomas, and mesenchymal cell carcinomas, and certain leukemias or malignant lymphoid tumors, benign and malignant tumors, and malignant tumors, such as sarcomas, carcinomas, and melanomas. Also included are adult tumors / cancers and childhood tumors / cancers.

[0169] Hematological cancer is a cancer of the blood or bone marrow. Examples of hematological (or blood-borne) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myeloid (myelocytic) leukemia, acute myelogenous (myelogenous) leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroid leukemia), chronic leukemias (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and advanced forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0170] Solid tumors are abnormal masses of tissue that usually do not contain cysts or areas of fluid. Solid tumors can be either benign or malignant. Different types of solid tumors are named after the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Solid tumors, such as sarcomas and carcinomas, include fibrosarcomas, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovial, mesothelioma, Ewing sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, pancreatic cancer, breast cancer, stomach cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, endometrial cancer, cervical cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, branchial carcinoma. Tracheal cancer, renal cell carcinoma, liver cancer, bile duct cancer, choriocarcinoma, Wilms tumor, cervical cancer, testicular tumors, seminoma, bladder cancer, melanoma, and central nervous system tumors (such as gliomas) (such as brain stem gliomas and mixed gliomas), glioblastoma (also includes astrocytomas, central nervous system lymphomas, germ cell tumors, medulloblastomas, schwannomas, craniopharyngiomas, ependymomas, pinealomas, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, neuroblastomas, retinoblastomas, and brain metastases).

[0171] In some embodiments, the cancer is selected from melanoma, sarcoma, ovarian cancer, prostate cancer, lung cancer, bladder cancer, microsatellite instability-high tumors, head and neck tumors, kidney cancer, and breast cancer.

[0172] The pharmaceutical composition can be administered in a manner suitable for the disease to be treated (or prevented) as described above. The amount and frequency of administration will be determined by factors such as the patient's condition, the type and severity of the patient's disease, although the appropriate dose can be determined through clinical trials.

[0173] When an "immunologically effective amount", "effective anti-tumor amount", "effective tumor inhibition amount" or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by the physician taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and condition of the patient (subject). It can generally be stated that the pharmaceutical composition comprising the lymphocytes described herein can be administered in an amount of 10 4 Up to 10 9 cells / kg body weight (e.g. 10 5 Up to 10 6 The lymphocyte composition can be administered at a dosage of 10 cells / kg body weight, including all integer values ​​within these intervals. At these dosages, the lymphocyte composition can also be administered multiple times. The cells can be administered using common infusion techniques in immunotherapy (e.g., see Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). Those skilled in the medical field can easily determine the optimal dosage and treatment regimen for a particular patient by monitoring the patient's disease signs and adjusting the treatment accordingly.

[0174] The administration of the composition of the present invention can be carried out in any convenient manner, including infusion or injection (i.e., intravenous injection, intrathecal injection, intramuscular injection, intraluminal injection, intratracheal injection, intraperitoneal injection or subcutaneous injection), transdermal administration or other methods known in the art. It can be administered once every two weeks, once a week, or more frequently, but the frequency of administration may be reduced during the maintenance stage of the disease or disorder. In some embodiments, the composition is administered by intravenous infusion.

[0175] In some cases, cells activated and expanded using the methods described herein or other methods known in the art (wherein lymphocytes are expanded to therapeutic levels) are administered to a patient together with (e.g., before, simultaneously with, or after) any number of related therapeutic modalities. Lymphocytes can also be used in combination with chemotherapy, radiotherapy, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies or other immunoablative agents, such as CAMPATH, anti-cancer antibody CD3 or other antibody therapy, cytotoxins, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and radiation.

[0176] The compositions of the present invention can also be administered to patients in conjunction with T lymphocyte ablation therapy using chemotherapeutic agents (e.g., fludarabine), external beam radiation therapy (XRT), cyclophosphamide, or antibodies (e.g., OKT3 or CAMPATH) prior to, during, or after bone marrow transplantation. It is also described herein that the compositions can be administered after ablation therapy of B lymphocytes, such as agents that react with CD20, such as rituximab. For example, the subject can receive standard treatment with high-dose chemotherapy followed by a peripheral blood stem cell transplant. In some cases, after transplantation, the subject receives an infusion of expanded lymphocytes, or the expanded lymphocytes are administered before or after surgery.

[0177] In some embodiments, the method may further include administering a second therapeutic agent to the subject. The second therapeutic agent is an anticancer or antitumor agent. In some embodiments, the composition is administered to the subject before, after, or concomitantly with the second therapeutic agent (including chemotherapeutic agents and immunotherapeutic agents).

[0178] In some embodiments, the method further comprises administering an effective therapeutic amount of an immune checkpoint regulator. Examples of immune checkpoint regulators may include PD1, PDL1, CTLA4, TIM3, LAG3, and TRAIL. The checkpoint modulator may be administered simultaneously, separately, or concomitantly with the composition of the present invention.

[0179] A "chemotherapeutic agent" is a compound used to treat cancer. Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide (CYTOXAN™); alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzotepa, carboquinone, methyldopa, and uredepa; ethyleneimines and methylamines, including hexamethylmelamine, triethylmelamine, tripolyphosphamide, triethylenethiophosphamide, and trihydroxymethylmelamine; acetospermines (particularly bratacin and latacinone); camptothecins (including the synthetic analog topotecan); bryostatin; callyst atin; CC-1065 (including its synthetic analogs adolesin, kazelesin and biszelesin); cryptophycin (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including its synthetic analogs KW-2189 and CBI-TMI); cypermethrin; cypermethrin; cypermethrin; sponge inhibitors; nitrogen mustards, such as chlorambucil, naphthyl mustard, cyclophosphamide, estramustine, ifosfamide, nitrogen mustard, mechlorethamine hydrochloride, oxide hydrochloride), melphalan, nebixin, phenylephrine, prednimustine, trofosfamide, uracil nitrogen mustard; such as nitrosureas, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, etc.; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, see, e.g., Agnew Chem. Intl. Ed. Engl. 33: 183-186 (1994); danemycins, including danemycin A; esperamicins;and neocarcinostatisin chromophores and related chromophores (enediyne antibiotic chromophores), aclarubicin, actinomycin, anthramycin, azaserine, bleomycin, actinomycin C, calabikine, carminomycin, carmomycin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrroline-doxorubicin), cin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs, such as Fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens, such as calusterone, dromostanolone propionate propionate, epitiostanol, mepitiostane, testolactone; antiadreners, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as frolinic acid; aceglucuronolide; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elfornithine; elacetamide; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitrarine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-acetylhydrazine; procarbazine; razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannitol mustard; dibromomannitol; dibromodulanol; pipobroman; gacytosine; cytarabine ("Ara-C"); cyclophosphamide; thiotepa; taxanes, such as paclitaxel ( Bristol-Myers Squibb Oncology, Princeton, NJ) and doxetaxel ( Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; norvinblastine (Navelbine); mitoxantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronic acid; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Also included in this definition are anti-hormones used to regulate or inhibit the effects of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen, raloxifene, aromatase inhibitor 4 (5) -imidazole, 4-hydroxytamoxifen, troxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide and xeloda, gemcitabine, KRAS mutant covalent inhibitors and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Other examples include irinotecan, oxaliplatin and other standard colon cancer regimens.

[0180] "Immunotherapeutic agent" may include a biological agent for treating cancer. In some embodiments, the immunotherapeutic agent may include an immune checkpoint inhibitor (e.g., PD-1, PD-L1, TIM-3, LAG-3, VISTA, DKG-α, B7-H3, B7-H4, TIGIT, CTLA-4, BTLA, CD160, TIM1, IDO, LAIR1, IL-12, or an inhibitor of a combination thereof). Examples of the immunotherapeutic agent include atezolizumab, avelumab, blinatumomab, daratumumab, cimiprilimab, durvalumab, elotuzumab, laherparepvec, ipilimumab, nivolumab, obitutuzumab, ofatumumab, pembrolizumab, cetuximab, and talimogene.

[0181] D. Definition

[0182] In order to help understand the specific embodiments of the compositions and methods according to the present invention, some clear definitions are provided so as to disclose various aspects of the present invention without ambiguity. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0183] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the invention belongs. The following references provide general definitions of many terms in the present invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); Cambridge Dictionary of Science and Technology (Walker Publishing House, 1988); Glossary of Genetics, Fifth Edition, R. Rieger et al. (Editors), Springer Verlag (1991); Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, unless otherwise specified, the following terms have the meanings given below.

[0184] As used herein, "expression" refers to the process of transcribing a polynucleotide from a DNA template (e.g., into mRNA or other RNA transcripts) and / or the process of subsequently translating the transcribed mRNA into a peptide, polypeptide, or protein. Transcripts and encoded polypeptides may be collectively referred to as "gene products." If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells.

[0185] As used herein, the term "recombinant" refers to a cell, microorganism, nucleic acid molecule or vector modified by the introduction of an exogenous nucleic acid molecule, or a cell, microorganism, nucleic acid molecule or vector with controlled expression of an endogenous nucleic acid molecule or gene. Downregulation or change to a constitutive change, such a change or modification can be introduced by genetic engineering. For example, genetic alteration includes modification by the introduction of a nucleic acid molecule encoding one or more proteins or enzymes (which may include expression control elements, such as promoters), or addition, deletion, replacement of another nucleic acid molecule, or other functional destruction or functional increase of the cell's genetic material. Exemplary modifications include modifications in the coding region of a heterologous or homologous polypeptide derived from a reference molecule or a parent molecule or its functional fragment.

[0186] The "transgene" or "therapeutic transgene" refers to RNA derived from a soluble receptor, a decoy, a decoy receptor, a dominant negative mutant, a microenvironment regulator, an enzyme, an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, a translocase, a kinase, a transporter, a modifier, a molecular chaperone, an ion channel, an antibody, a cytokine, a growth factor, a chemokine, a hormone, DNA, a ribozyme, a biosensor, an epigenetic modifier, a transcription factor, a coding RNA, a non-coding RNA, a small RNA, a long RNA, an IRES element or an exosome.

[0187] As used herein, the term "functional variant" refers to a modified transgene having substantial or significant sequence identity or similarity to a wild-type transgene, such a functional variant retains the biological activity of the wild-type transgene of which it is a variant. In some embodiments, functional variants of transgenes are used.

[0188] The term "antigen recognition receptor" as used herein refers to a receptor that can activate immune cells (e.g., T cells) in response to antigen binding. Exemplary antigen recognition receptors can be natural or genetically engineered TCRs, or genetically engineered TCR-like mAbs (Hoydahl et al., Antibodies 2019 8:32) or a CAR in which the tumor antigen binding domain is fused to an intracellular signaling domain that can activate immune cells (e.g., T cells). T cell clones expressing natural TCRs against specific cancer antigens have been previously published (Traversari et al., J Exp Med, 1992 176:1453-7; Ottaviani et al., Cancer Immunol Immunother, 2005 54:1214-20; Chaux et al., J Immunol, 1999 163:2928-36; Luiten and van der Bruggen, Tissue Antigens, 2000 55:149-52; van der Bruggen et al., Eur J Immunol, 1994 24:3038-43; Huang et al., J Immunol, 1999 162:6849-54; Ma et al., Int J Cancer, 2004 109:698-702; Ebert et al., Cancer Immunol, 2005 110:694-703; Res, 2009: 1046-54; Ayyoub et al., J Immunol 2002 168: 1717-22; Chaux et al., European Journal of Immunology, 2001 31: 1910-16; Wang et al., Cancer Immunol Immunother, 2007 56: 807-18; Schultz et al., Cancer Research, 2000 60: 6272-75; Cesson et al., Cancer Immunol Immunother, 2010 60: 23-25; Zhang et al., Journal of Immunology, 2003 171: 219-25; Gnjatic et al., PNAS, 2003 100: 8862-67; Chen et al., PNAS, 2004). In one embodiment, such TCRs can be sequenced and genetically engineered into TILs for adoptive cell therapy. In certain aspects, TCRs that recognize MAGE-A1 antigen, MAGE-A3 antigen, MAGE A-10 antigen, MAGE-C2 antigen, NY-ESO-1 antigen, SSX2 antigen, and MAGE-A12 antigen can be genetically engineered into TILs for adoptive cell therapy.In other embodiments, the genetically engineered TIL with TCR is also engineered to secrete a transgene. In other embodiments, CAR is used. In other embodiments, CAR is also modified to secrete a transgene.

[0189] As used herein, the term "antibody" refers not only to complete antibody molecules, but also to antibody molecule fragments that retain immunogen binding ability. Such fragments are also well-known in the art and are often used both in vitro and in vivo. Therefore, as used herein, the term "antibody" refers not only to complete immunoglobulin molecules, but also to well-known active fragments f(ab')2 and fab.F(ab')2 and fab fragments lacking the Fc fragments of complete antibodies are cleared from the circulation faster and can bind less to nonspecific tissues of complete antibodies (Wahl et al., J.Nucl.Med.24:316-325 (1983). The antibodies of the present invention include all-natural antibodies, bispecific antibodies; chimeric antibodies; fab, fab', single-chain v region fragments (scFv), fusion polypeptides and unconventional antibodies.

[0190] As used herein, the term "single-chain variable fragment" or "scFv" is a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin, covalently linked to form a VH::VL heterodimer. The heavy chain (VH) and the light chain (VL) are either directly linked or linked by a peptide-encoded linker (e.g., 10, 15, 20, 25 amino acids) to link the n-terminus of VH and the C-terminus of VL, or the C-terminus of VH and the n-terminus of VL. The linker is usually rich in glycine to increase flexibility, and serine or threonine to increase solubility. Despite the removal of the constant region and the introduction of the linker, the scFv protein retains the specificity of the original immunoglobulin. As described by Huston et al., a single-chain Fv polypeptide antibody can be expressed from a nucleic acid comprising VH and VL coding sequences (Proc. Nat. Acad. Sci., 85: 5879-58831988). See also U.S. Patent Nos. 5,091,513, 5,132,405, and 4,956,778; and U.S. Patent Publication Nos. 20050196754 and 20050196754. Antagonistic single chain antibodies with inhibitory activity have been described (see, e.g., Zhao et al., Hybridoma (Larchmont) 2008 27(6):455-51; Peter et al., J cachexia sarcopenia muscle 2012 Aug. 12; Shieh et al., J Immunol 2009 183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife et al., J Clin Invst 2006 116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3):173-84; Moosmayer et al., Ther Immunol 1995 2(10:31-40). Agonistic single chain antibodies with stimulatory activity have been described (see Peter et al., J Biol Chern 2003 25278(38):36740-7; Xie et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit Rev Immunol 1997 17(5-6):427-55; Ho et al., Biochim Biophys Acta 2003 1638(3):257-66).

[0191] As used herein, "treating" or "treatment" refers to the administration of a compound or drug to a subject suffering from a disorder for the purpose of curing, alleviating, alleviating, remedying, delaying the onset of, preventing or ameliorating the disorder, a symptom of the disorder, a disease state secondary to the disorder, or a predisposition to the disorder.

[0192] In the context of an immune response, the terms "stimulate" or "enhance" refer to triggering or increasing an immune response, such as an increase in the ability of immune cells to target and / or kill cancer cells or to target and / or kill pathogens and pathogen-infected cells (e.g., EBV-positive cancer cells).

[0193] As used herein, the term "immune response" refers to any type of immune response, including, but not limited to, innate immune responses (e.g., activation of the Toll receptor signaling cascade), cell-mediated immune responses (e.g., responses mediated by T cells (e.g., antigen-specific T cells) and non-specific cells of the immune system), and humoral immune responses (e.g., responses mediated by B cells (e.g., by the production and secretion of antibodies into plasma, lymph and / or tissue fluids). The term "immune response" is meant to include all aspects of the ability of a subject's immune system to respond to an antigen and / or immunogen (e.g., both the initial response to an immunogen (e.g., a pathogen) and the acquired (e.g., memory) response generated by the adaptive immune response).

[0194] As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as a test tube or reaction vessel, cell culture, etc., rather than within a multicellular organism.

[0195] As used herein, the term "in vivo" refers to events that occur within a multicellular organism, such as a non-human animal.

[0196] As used herein, the term "disease" is generally synonymous with the terms "disorder" and "condition" (as in medical condition) and are used interchangeably therewith because they all reflect an abnormal condition of the human or animal body or a part thereof that impairs normal function, usually manifests itself by distinctive signs and symptoms, and results in a decrease in the life span or quality of life of a human or animal.

[0197] As used herein, the terms "decrease", "reduced", "reduction", "decrease" or "inhibit" generally refer to a decrease in a statistically significant amount. However, for the avoidance of doubt, "reduced", "reduction" or "reduction" or "inhibit" refers to a decrease of at least 10% compared to a control level, for example, a decrease of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g., a level of absence compared to a control sample), or any decrease between 10-100% compared to a control level.

[0198] As used herein, the term "modulate" means any change in a biological state, ie, increase, decrease, etc.

[0199] As used herein, the terms "increased", "increase" or "enhance" or "activate" generally refer to an increase in a statically significant amount; for the avoidance of any doubt, the terms "increased", "increase" or "enhance" or "activate" refer to an increase of at least 10% compared to a control level, such as an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including an increase of 100% or any increase between 10-100% compared to a control level, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold compared to a control level, or any increase between 2-fold and 10-fold or more than 10-fold.

[0200] The terms "effective amount", "effective dose" or "effective dosage" are defined as an amount sufficient to achieve or at least partially achieve the desired effect. An "effective therapeutic amount" or "effective therapeutic dose" of a drug or therapeutic agent refers to any amount of the drug that, when used alone or in combination with another therapeutic agent, promotes regression of a disease by reducing the severity of disease symptoms, increasing the frequency and duration of disease symptom-free periods, or prevents damage or disability due to susceptibility to the disease. An "effective prophylactic amount" or "effective prophylactic dose" of a drug refers to an amount of the drug that, when administered alone or in combination with another therapeutic agent to a subject at risk of developing the disease or recurring the disease, inhibits the progression or recurrence of the disease. The ability of a therapeutic or prophylactic agent to promote regression of a disease or inhibit the progression or recurrence of a disease can be assessed using a variety of methods known to those skilled in the art, such as evaluation in human subjects during clinical trials, evaluation in animal model systems predictive of human efficacy, or by testing the activity of the agent in in vitro assays.

[0201] Doses are usually expressed in relation to body weight. Thus, a dose expressed in [g, mg, or other unit] / kg (or g, mg, etc.) usually refers to [g, mg, or other unit] "per kilogram (or g, mg, etc.) of body weight", even if the word "body weight" is not explicitly mentioned.

[0202] The term "agent" is used herein to refer to a compound, a mixture of compounds, a biomacromolecule (e.g., a nucleic acid, an antibody, a protein or a portion thereof, e.g., a peptide), or an extract made from a biological material, e.g., a bacterial, plant, fungal, or animal (especially mammalian) cell or tissue. The activity of such an agent may make it suitable as a "therapeutic agent," i.e., a biologically, physiologically, or pharmacologically active substance that acts locally or systemically in a subject.

[0203] The terms "therapeutic agent," "agent having therapeutic ability," or "therapeutic agent" are used interchangeably and refer to a molecule or compound that confers some beneficial effect upon administration to a subject. Such beneficial effects include enabling a diagnostic assay; amelioration of a disease, symptom, disorder, or pathological condition; reducing or preventing the occurrence of a disease, symptom, disorder, or condition; and generally combating a disease, symptom, disorder, or pathological condition.

[0204] Unless the context clearly indicates otherwise, "combination" therapy as used herein is intended to include the administration of two or more therapeutic agents in a coordinated manner, and includes, but is not limited to, concomitant administration. Specifically, combination therapy includes combined administration (e.g., administration of a combined preparation or simultaneous administration of a single therapeutic composition) and continuous or sequential administration, provided that the administration of one therapeutic agent is in some way dependent on the administration of another therapeutic agent. For example, a therapeutic agent can only be administered after a different therapeutic agent has been administered and allowed to act within a specified time period. For example, see Kohrt et al., (2011) Blood 117: 2423.

[0205] "Sample", "test sample" and "patient sample" can be used interchangeably herein. The sample can be a sample of serum, urine, plasma, amniotic fluid, cerebrospinal fluid, cells (e.g., antibody-producing cells) or tissue. Such samples can be used directly after being obtained from the patient, or can be pre-treated by filtering, distilling, extracting, concentrating, centrifuging, inactivating interfering components, adding reagents, etc., to change the properties of the sample in a certain manner described herein or in other ways known in the art. The terms "sample" and "biological sample" used herein generally refer to biological materials that are tested and / or suspected of containing an analyte of interest (e.g., an antibody). The sample can be any tissue sample from a subject. The sample can contain proteins from a subject.

[0206] As used herein, the terms "inhibit" and "antagonize" mean to reduce or completely prevent the expression, stability, function or activity of a molecule, reaction, interaction, gene, mRNA and / or protein by a measurable amount. An inhibitor is a compound that, for example, binds to proteins, genes and mRNAs, partially or completely blocks stimulation, reduces, prevents, delays activation, inactivates, desensitizes or downregulates protein, gene and mRNA stability, expression, function and activity, such as an antagonist.

[0207] "Parenteral" administration of the compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im) or intrasternal injection, or infusion techniques.

[0208] As used herein, the term "pharmaceutical composition" refers to a mixture of at least one compound used in the present invention and other chemical components, such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners and / or excipients. The pharmaceutical composition facilitates administration of the compound to an organism.

[0209] As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, that does not abrogate the biological activity or properties of the composition and is relatively non-toxic, i.e., the material can be administered to a subject without causing adverse biological effects or interacting in a deleterious manner with any components of the composition.

[0210] The term "pharmaceutically acceptable carrier" includes pharmaceutically acceptable salts, pharmaceutically acceptable materials, compositions or vehicles, such as liquid or solid fillers, diluents, excipients, solvents or encapsulating materials, involved in carrying or delivering the compounds of the invention into or to a subject so that they can perform their intended function. Typically, these compounds are carried or transported from one organ or part of the body to another. Each salt or carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not causing harm to the subject. Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; tragacanth powder; malt; gelatin; talc; excipients, such as cocoa butter and suppositories; peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and Ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffered saline; diluents; granulating agents; lubricants; binders; disintegrants; wetting agents; emulsifiers; colorants; release agents; coating agents; sweeteners; flavoring agents; spices; preservatives; antioxidants, plasticizers; gelling agents; thickeners; hardeners; setting agents; suspending agents; surfactants; humectants; carriers; stabilizers and other non-toxic compatible substances used in pharmaceutical formulations, or any combination thereof. As used herein, "pharmaceutically acceptable carriers" also include any and all coatings, antibacterial and antifungal agents, absorption delaying agents, etc. that are compatible with the activity of the compound and are physiologically acceptable to the subject. Supplementary active compounds can also be incorporated into the composition.

[0211] It is noted herein that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0212] Unless otherwise stated, the terms "including," "comprising," "containing," or "having" and variations thereof are meant to encompass the items listed thereafter and equivalents thereof as well as additional subject matter.

[0213] The phrases "in one embodiment," "in various embodiments," "in some embodiments," etc. are used repeatedly. These phrases do not necessarily refer to the same embodiment, but they may unless the context dictates otherwise.

[0214] The term "and / or" or " / " means any item, any combination of the items, or all terms related to the items.

[0215] The word "substantially" does not exclude "completely", for example, a component "substantially free" from Y may be completely free from Y. If necessary, the word "substantially" may be omitted from the definition of the present invention.

[0216] As used herein, the term "approximately" or "about" when applied to one or more values ​​of interest refers to a value similar to the reference value. In some embodiments, the term "approximately" or "about" refers to a range of values ​​that fall within (greater or less than) 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction of the reference value, unless otherwise specified or obvious from the context (unless the number would exceed 100% of the possible value). Unless otherwise indicated herein, the term "approximately" is intended to include values ​​close to the range, such as weight percentages, that are equivalent in terms of their functionality in a single component, composition, or embodiment.

[0217] It should be understood that the values ​​and ranges provided herein, all values ​​and ranges included in these values ​​and ranges are meant to be included within the scope of the present invention. In addition, this application also contemplates all values ​​falling within these ranges and the upper or lower limit of a value range.

[0218] As used herein, the term "each" when used to refer to a collection of items is intended to identify a single item in the collection, but does not necessarily refer to each item in the collection. Exceptions may occur if express disclosure or context clearly requires.

[0219] The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present invention and is not intended to limit the scope of the present invention unless otherwise required. No language in the specification should be construed as indicating that any non-claimed element is essential to the practice of the present invention.

[0220] Unless otherwise specified herein or the context clearly contradicts otherwise, all methods described herein are performed in any suitable order. For any method provided, the steps of the method can occur simultaneously or sequentially. When the steps of the method occur sequentially, unless otherwise specified, these steps can occur in any order.

[0221] Where the method comprises a combination of steps, every combination or subcombination of steps is included within the scope of the invention unless otherwise indicated herein.

[0222] Each publication, patent application, patent, and other reference cited herein is hereby incorporated by reference in its entirety to the extent that it does not conflict with the present disclosure. The publications disclosed herein are intended to be disclosed only prior to the filing date of the present invention. Nothing herein shall be construed as an admission that the present invention is not entitled to be published in advance by virtue of prior inventions. In addition, the publication date provided may be different from the actual publication date, which may require independent confirmation.

[0223] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes thereto are suggested to those skilled in the art and are to be included within the spirit and purview of the present application and the scope of the appended claims.

[0224] E. Examples

[0225] Example 1

[0226] This example describes the materials and methods used in the subsequent examples.

[0227] Mice and cell lines

[0228] Female C57BL / 6 mice aged 6 weeks were purchased from Harlan (Harlan, the Netherlands) and housed in the animal facility of the University of Lausanne (UNIL, Epalinges, Switzerland) according to the guidelines. C57BL / 6 OT-1 CD45.1+ and C57BL / 6CD8a- / - mice were described by Hogquist KA et al. (Hogquist KA et al., Cell 76(1):17-27 PubMed:8287475 MGI:J:92867) and Fung-Leung WP et al. (Fung-Leung et al., Cell 65(3):443-9 PubMed:1673361 MGI:J:68956). All in vivo experiments were performed in accordance with the approval of the Consumer and Veterinary Affairs Agency (SCAV) of the Canton of Vaud, Switzerland.

[0229] The ovalbumin-expressing B16 melanoma cell line (B16-OVA) was previously generated by retroviral transduction of B16. The F10 cell line was purchased from ATCC and grown in monolayers in DMEM supplemented with 10% fetal calf serum (FCS), 100 U / ml penicillin, and 100 μg / ml streptomycin sulfate. Cells were passaged twice a week to maintain them under exponential growth conditions and routinely tested for mycoplasma contamination. The retroviral eco packaging cell line Phoenix Eco, derived from immortalized normal human embryonic kidney (HEK) cells, was maintained in RPMI 1640 glutamine medium supplemented with 10% heat-inactivated FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin sulfate.

[0230] Human fetal kidney (HEK) 293T cells were purchased from ATCC (CRL-3216) and cultured in RPMI 1640 glutamine medium (Invitrogen), 10% FBS (heat inactivated at 56°C for 30 min; Gibco), 1% penicillin / streptomycin (ThermoFisher Scientific). HEK 293T cells were used to produce retroviral and lentiviral particles. HLA-A2.1 pos / NY-ESO pos Melanoma cell lines Me275 and A375, and HLA-A2.1 pos / NY-ESO neg The cell line NA8 (obtained from UNIL Department of Oncology) was cultured in IMDM supplemented with 10% FBS and 1% penicillin / streptomycin.

[0231] Design of bicistronic expression cassette

[0232] The retroviral vector pMSGV1 (a spliced ​​gag vector based on mouse stem cell virus (MSCV)) containing the MSCV long terminal repeat (LTR) was used as the backbone for all constructs. The expression cassette usually encodes the signal peptide of the mouse IgG-κ chain region V-IIIMOPC 321 (e.g., Uniprot ID: P01650) (SEQ ID NO: 20), and then encodes the N-terminal extracellular domain of mouse PD-1 (e.g., Uniprot ID: Q02242 residues S21-Q167, C83S) (SEQ ID NO: 1) fused with human IgG4_Fc (e.g., Uniprot ID: P01861.1, residues P104-K327) (SEQ ID NO: 19), referred to herein as PD-1.IgG4 bait. The restriction points AgeI and EcoRI are located at the 5' and 3' ends of the first part, respectively. The second part follows the T2A sequence and consists of the signal peptide of mouse interferon β (e.g. Uniprot ID: P01575.1) (SEQ ID NO: 39) and a gene string encoding one of the following molecules: mouse IL-33 (e.g. Uniprot ID: Q8BVZ5.1, residues S109-I266) (SEQ ID NO: 27), mouse LIGHT (e.g. Uniprot ID: Q9QYH9.1, residues D72-V239) (SEQ ID NO: 31), mouse CD40L (e.g., Uniprot ID: P27548, residues M112-L260) (SEQ ID NO: 33) and α-mutant IL-2 (e.g., Uniprot ID: P60568.1, residues A21-T153, mutants: R58A, F62A, Y65A, E82A and C145S) (SEQ ID NO: 23). The restriction sites MluI and SalI were located at the 5' and 3' ends of the second part, respectively. Thus, after respective cloning, the following constructs were obtained: PD-1.IgG4_T2A_IL-2 V , PD-1.IgG4_T2A_IL-33, PD-1.IgG4_T2A_LIGHT, PD-1.IgG4_T2A_CD40L and IgG4_T2A_CD40L. All gene strings were mouse codon optimized and synthesized by GeneArtAG, and all constructs were fully sequenced by Microsynth AG after cloning in MSGV vector.

[0233] like Fig.11A-C, codon-optimized gene strings separated by picornavirus-derived 2A sequences encoding PD1 decoy and truncated EGFR, CD40 ligand decoy and IL-2 variants were ordered from GeneArt (ThermoFisher Scientific) and cloned into retroviral vectors pSFG (for constitutive expression) or pSFG-SIN (self-inactivation) for activation-based gene expression under the NFAT promoter. The vectors were amplified in Stellar competent cells (E. coli HST08, #636763, Takara) and purified with a plasmid mini / maxi prep kit (Genomed) after sequence confirmation (Microsynth AG).

[0234] The gene string encoding the HLA / A2:NY-ESO-1 peptide T cell receptor (TCR), including TCRα23 and TCRβ13.1, was ordered from GeneArt (ThermoFisher Scientific). The TCRα and TCRβ chains were codon optimized and separated by a picornavirus-derived 2A sequence. The gene string was integrated into the lentiviral vector pRRL, in which most of the U3 region of the 3′ long terminal repeat was deleted, resulting in self-inactivation of the 3′ long terminal repeat (SIN).

[0235] Lentivirus production

[0236] 10×10 6 HEK 293T cells were seeded in T150 cell culture flasks with RPMI complete medium (RPMI 1640 glutamine medium (Invitrogen), 10% FBS (Gibco), 1% penicillin / streptomycin). Approximately 24 hours later (70-80% confluency), 107 μl Turbofect and 2 mL The cells were transfected with 7 μg of pVSV-g (VSV glycoprotein expression plasmid), 18 μg of R874 (Rev and Gag / Pol expression plasmid) and 15 μg of pRRL transgenic plasmid with a mixture of Optimem medium (51985026, Invitrogen). After incubation for 30 minutes at room temperature, the DNA mixture was added on top of the cells and the volume was adjusted to a total of 30 ml. After 24 hours, the medium was renewed and the viral supernatant was collected 48 hours after transfection. The viral particles were concentrated by ultracentrifugation and resuspended in 400 μl of RPMI complete medium. Several aliquots of 100-200 μl of virus per Eppendorf tube were prepared and stored at -80°C.

[0237] Retroviral production

[0238] Phoenix Eco cells were cultured at 1 × 10 cells / mL 24 h before being transfected with 14.4 μg of pCL Eco retroviral packaging vector and 21.4 μg of pMSGV transfer plasmid using Turbofect (Thermo Fisher Scientific). 7 Each T-150 tissue culture flask was inoculated in 25 ml of culture medium. All plasmids were purified using the JETSTAR 2.0 Plasmid Maxiprep Kit (Genomed). For the transfection mixture, a 3:1 ratio of Turbofect: plasmid was prepared in Optimem 2 ml and incubated for 30 min at RT. The culture medium was then removed from a T-150 cell culture flask containing 80-90% fused Phoenix Eco cells, the transfection mixture was applied and incubated for 1 minute, and then 25 ml of fresh culture medium was added. The viral supernatant was collected 48 hours after transfection, and then 25 ml of fresh culture medium was added. A second collection was performed again 24 hours later. The viral particles in the two SNs were concentrated for 2 hours at 24000g ultracentrifugation at 4°C with a Beckman JS-24 rotor (BeckmanCoulter), and suspended in 0.5 mL of mouse T cell culture medium, and then the viral titer was determined. Finally, the retrovirus was aliquoted, frozen on dry ice, and stored at -80°C.

[0239] In another embodiment, 10×10 6 HEK 293T cells were seeded in 17 ml of RPMI, 10% fetal bovine serum (FBS, Gibco), 1% penicillin / streptomycin (ThermoFisher Scientific) in T150 cell culture flasks at 37°C overnight. The next day (at 85-95% confluency of 293T cells), a mixture of 120 μl of turbofect (LifeTechnologies) and 3 μl of OptiMem (per T150 cell culture flask) was prepared and then combined with retroviral plasmids: 22 μg of PamPeg, 7 μg of RDF-RD114, 18 μg of SFG or SFG-SIN encoding the gene of interest. The medium was gently removed from the 293T cells and the retroviral plasmid mixture was pipetted onto the 293T cells. After a 5-minute rest, 16 ml of medium was gently added. Incubate overnight at 37°C. The next day, the culture medium was renewed and the next day (48 hours), the virus was collected from the filtered supernatant by ultracentrifugation (24000 x g, 2 hours). Fresh culture medium was added to 293T cells and the virus was collected for the second time after 72 hours. In these two days, several aliquots of 100-200 μl of virus were prepared for each Eppendorf tube and stored at -80°C.

[0240] Mouse T cell transduction

[0241] Primary mouse OT-1 cells were isolated from single cell suspensions of dissociated spleens of 6-10 week old CD45.1+ positive OT-1 C57BL / 6 mice using the Pan T Cell Isolation Kit II for Mouse (Miltenyi Biotec cat#130-095-130) and cultured in RPMI 1640 glutamine medium supplemented with 10% heat-inactivated FBS, 100 U / ml penicillin, 100 μg / ml streptomycin sulfate, 1 mM pyruvate, 50 μM BME, and 10 mM non-essential amino acids (T cell medium).

[0242] The cell density of the culture was maintained at 0.5-1 × 10 6 The cells were cultured at 4 °C for 24 h and 100 μg / ml, and were refreshed every other day with fresh T cell culture medium until day 15 (culture medium was supplemented with only 10 IU / ml human IL-2 without α mutation until day 3, and then with 10 ng / ml of hIL-7 / IL-15). On day 7, cellular expression of the molecules was assessed by intracellular flow cytometry analysis and their presence in the supernatant was assessed by ELISA. Finally, engineered OT-1 T cells were adjusted according to the transduction efficiency of the PD-1.IgG4 bait before cell transfer. Recombinant human IL-7 and human IL-15 were obtained from Miltenyi Biotec.

[0243] The isolated primary OT-1T cells were cultured at 1×10 6 / ml were seeded in T cell culture medium in 24-well culture plates and stimulated with αCD-3 / αCD-28Ab-coated microbeads (Invitrogen) and 10 IU / ml human α-mutant-free IL-2. 24 hours after activation, T cells were first transduced with retrovirus at a 10-fold infection rate (MOI). The transduction was performed in non-tissue culture grade 24-well culture plates (Becton Dickinson Labware), which were pre-coated with 20 mg / ml recombinant retronectin (retronectin; Takara) overnight at 4°C, washed, blocked with 2% bovine serum albumin (BSA) in PBS for 30 minutes at RT, and then a final wash was performed. After adding retrovirus (250 μl), the culture plate was centrifuged at 2000×g for 1.5 hours at 32°C. 125 μl of the supernatant was aspirated and 1×10 6 The plate was centrifuged at 1200 rpm for 10 min and incubated at 37°C with 5% CO. 2Incubate overnight at 4 °C. A second transduction was performed 48 h after activation, following the protocol described above. On day 7, cellular expression of the molecules was assessed by intracellular flow cytometry analysis and their presence in the supernatant by ELISA. Finally, engineered OT-1 T cells were adjusted based on the expression of PD-1.IgG4 bait before cell transfer.

[0244] The cell density of the culture was maintained at 0.5-1 × 10 6 T cells were cultured at 10 cells / ml and refreshed with fresh T cell medium every other day until day 15, when CD44+CD62L+TCF1+ central memory CD8 T cells were generated according to the optimized in vitro expansion protocol. T cell culture medium was supplemented with only 10 IU / ml of human α-mutant-free IL-2 until day 3, and then with 10 ng / ml of hIL-7 / IL-15 until the end of the culture. Recombinant human IL-7 and human IL-15 were obtained from Miltenyi Biotec.

[0245] Purification and activation of human T cells

[0246] Healthy donor apheresis and buffy coats were purchased from Transfusion Interrégionale CRS SA (Epalinges, Switzerland) with written consent under approved university institutional review board protocols. Peripheral blood mononuclear cells (PBMCs) were prepared by density gradient centrifugation with Lymphoprep (Stem Cell Technologies), and CD8 + or CD4 + T cells were negatively isolated using CD8 or CD4 magnetic microspheres (Miltenyi) according to the manufacturer's protocol. Isolated CD8 were stimulated with anti-CD3 / CD28 microspheres (Invitrogen) at a 2:1 ratio of microspheres:T cells in the presence of human IL-2 (GlaxoSmithKline). + and CD4 + T cells.

[0247] Lentiviral and retroviral transduction of human T cells

[0248] 24 hours after activation, lentiviral transduction of T cells was performed by adding viral particles directly to the culture medium (MOI 20) and amplified by the concomitant addition of Lentiboost (Sirion Biotech). Retroviral transduction of T cells was performed 48 hours after activation. T cells were transferred to retronectin-coated plates that had been previously seeded with retroviral particles and spun at 2000×g for 1.5 hours. The next day, T cells were removed from the retronectin-coated plates. Anti-CD3 / anti-CD28 microbeads (Thermo Fisher Scientific) were removed 5 days after activation, and T cells were maintained in culture medium supplemented with 10% heat-inactivated FBS (Gibco), 1% penicillin / streptomycin, 10 ng / ml human IL-7 (Miltenyi) and 10 ng / ml IL-15 (Miltenyi) at a concentration of 0.5-1×10 6 T cells / ml in RPMI 1640 glutamine (Thermo Fisher).

[0249] Co-transduction of human T cells with lentivirus and retrovirus

[0250] For co-transduction, human T cells were purified and bead activated (0.5 × 10 per 48 wells) before adding concentrated lentivirus (100 μl) and optionally 1 μl Lentiboost (Sirion Biotech) to increase transduction efficiency. 6 T cells + 1×10 6 Anti-CD3 / anti-CD28 beads + 50IU / ml IL-2) for 18-22 hours. The next day, the transduced T cells are transferred to a retronectin-coated plate pre-spun with retroviral particles at 2000×g for 1.5 hours. The next day, the T cells are transferred to a tissue culture dish. On the fifth day, the beads are removed, the T cells are transferred to a larger well, and fresh culture medium supplemented with 10ng / ml IL-15 and 10ng / ml IL-7 is provided. Fresh culture medium plus cytokines are provided every 2-3 days. From the 7th to 10th day, the co-transduction efficiency can be determined by flow cytometry.

[0251] Retroviral transduction of tumor infiltrating lymphocytes (TIL)

[0252] Thawed TILs were previously expanded from isolated patient tumor fragments. 0.5 × 10 WT TILs were stimulated in 48-well plates in 500 μl RPMI, 10% FBS, 25 μl GMP-grade TransAct (1:20, Miltenyi Biotech) and 6000 IU / ml IL-2. 6TIL. Non-tissue culture plates were coated with retronectin (Takara Bio, 1mg / ml diluted 50 times, 250μl per 48 wells) at 4°C overnight. The next day, retronectin was removed and blocked with 500μl RPMI, 10% FBS (Gibco), 1% penicillin / streptomycin for 30 minutes at 37°C. Subsequently, the culture medium was removed, and 50-100μl concentrated retrovirus was added to 50μl culture medium, and then rotated at 2000g for 1 hour at 25°C. The supernatant was then removed, TIL was added, and rotated at 1000g for 10 minutes at 25°C. Incubate overnight at 37°C, then transfer to a 48-well tissue culture plate and add fresh culture medium. On the 5th day, TIL was transferred to a plate with larger wells and fresh culture medium (RPMI, 10% FBS (Gibco), 1% penicillin / streptomycin, 6000IU / ml IL-2) was added. Assess transduction efficiency on days 7-10. Provide fresh medium every 2-3 days starting on day 5.

[0253] Flow cytometric analysis

[0254] All FACS data were acquired on an LCRII flow cytometer (BD) and analyzed using FlowJo software. Fixable aqua-dead dyes L34965 or L34975 (Invitrogen) were used according to the manufacturer's instructions for exclusion of dead cells. The following antibodies were used for T cell staining: anti-Vb13.1: PE (IM2292, BD Bioscience), anti-IFNγ: PeCy7 (502527, Biolegend). Tetramer (A2 / NY-ESO-1157-165; produced in-house) staining was used to assess TCR transduction efficiency.

[0255] Flow cytometric analysis to evaluate the expression of immunomodulatory factors by genetically engineered T cells

[0256] Genetically engineered OT-1T cells one week after transduction were incubated with 50 μl Live / Dead Fixable aqua dead in PBS at room temperature for 30 minutes, washed, and then incubated again for 30 minutes at 4°C with 50 μl FCR blocking reagent (clone 2.4G2 BD Pharmingen). The cells were washed again and incubated for another 30 minutes at 4°C with surface markers for anti-CD3 (145-2C11, Invitrogen), CD8α (53-6.7, BioLegend) and CD45.1 (A20, BioLegend) antibodies. For intracellular staining, the following antibodies were used: anti-human hIgG4-Fc (Abcam, clone number: HP6025) and anti-mouse IL-33 (eBioscience, clone number: 396118) for detecting PD-1.IgG4 bait. After surface staining, genetically engineered OT-1 cells were washed twice and fixed / permeabilized using the FoxP3 transcription factor staining buffer kit (Invitrogen) according to the manufacturer's recommendations. For detection of each molecule, cells were also washed and incubated with the respective antibodies for 30 minutes at room temperature. Cells were washed and resuspended in PBS supplemented with 2% BSA and 0.01% azide (FACS buffer), obtained by BD flow cytometer LSRII flow cytometer, and analyzed using FlowJo software v11 (Tree Star Corporation).

[0257] Flow cytometric analysis to assess intracellular cytokine or PD1-Fc decoy or CD40L decoy production

[0258] To assess intracellular cytokine production or production of PD1 bait or CD40L bait by FACS, 50,000 live T cells per well were activated for 7 h with a combination of plate-coated anti-CD3 (5 μg / ml) and soluble anti-CD28 (2 μg / ml) antibodies in a round-bottom 96-well plate (or with anti-CD3 / anti-CD28 beads). To prevent protein secretion, Golgi stop (BD Biosciences) was added to the wells at a dilution of 1:400 1.5 h after the start of the assay. T cells were fixed and permeabilized using a standard fixation / permeabilization kit (BD Biosciences) according to the manufacturer's instructions and then assessed for transduction efficiency or the ability to produce molecules of interest. Anti-Fc antibodies were used to detect baits. Specific antibodies against the cytokines of interest (IL-2, IFN-γ) were used.

[0259] ELISA to evaluate the secretion of immunomodulatory factors by genetically engineered T cells

[0260] One week after activation and transduction, 106 Genetically engineered OT-1T cells were inoculated in 1 mL of serum-free RPMI medium for 72 hours. SNs were then collected and tested for each molecule. For PD1.IgG4, a modified ELISA with the following settings was used. Plates were coated with anti-mouse PD1 antibody (R&D, AF1021, 2 μg / m) and incubated with SNs. PD1.IgG4 was detected with anti-hIgG4-HRP antibody (Abcam, ab99817, dilution 1:1000).

[0261] For IL-2 V A modified ELISA with the following settings was used. Plates were coated with anti-human IL-2 antibody (R&D, AF-202-NA, 3 μg / ml), incubated with supernatants, and IL-2 was detected with biotinylated polyclonal anti-human IL-2 antibody (Invitrogen, 13-7028-81, dilution 1:500) followed by streptavidin HRP (BioLegend, dilution 1:1000). V SN from OT-1 T cells were transduced to express the fusion molecules TIM-3.IgG4 or IL-2 V As a negative control. To detect LIGHT, IL-33 and CD40L, three commercial ELISA kits were used: Mouse LIGHT / TNFSF14 DuoSet ELISA (DY1794-05) developed by R&D, LEGEND MAXTM Mouse IL-33 ELISA Kit (436407) developed by BioLegend, and Mouse CD40Ligand / TNFSF5 ELISA Kit (NBP1-92662) developed by NovusBiological.

[0262] Adoptive cell transplantation in tumor-bearing mice

[0263] B16-OVA tumor cells were collected with 0.05% trypsin, washed, and resuspended in PBS for injection. 5 Tumor cells were injected subcutaneously into the right flank of 7-week-old C57BL / 6 mice. On day 11 (average tumor volume 100-200 mm 3 ), the mice were regrouped to obtain the relative tumor mean volume between experimental groups, where n ≥ 5 mice / group. On days 12 and 15, mice were transferred intravenously with 5×10 6Genetically engineered CD44+CD62L+TCF1+OT-1T cells or control non-transduced OT-1 were treated. Mice were monitored three times a week, and tumor length (L; maximum longitudinal measurement) and width (W; maximum transverse measurement) were measured by calipers by independent researchers in a blinded manner. Tumor volume (V) was calculated using the formula: V = (L x W 2 ) / 2. The mean tumor volume / group ± SD was plotted. Once the tumor reached 1000 mm 3 , or, depending on the protocol, if the mice were distressed or moribund, they were killed.

[0264] The secretion of immunomodulatory factors by genetically engineered T cells was assessed by ELISA.

[0265] One week after transduction, 1×10 6 Genetically engineered OT-1T cells were seeded in 24-well plates in 1 mL of serum-free RPMI medium for 72 hours. SNs were then collected and tested for each molecule by ELISA. PD1.IgG4 Domestic-ELISA: Coating antibody: anti-mouse PD-1 (R&D, AF1021, 2 μg / ml), detection antibody: anti-hIgG4-HRP (Abcam, ab99817, dilution 1:1000). IL-2 V Domestic ELISA: Coating antibody: anti-human IL-2 (R&D, AF-202-NA, 3 μg / m), secondary antibody biotinylated polyclonal anti-human IL-2 (Invitrogen, 13-7028-81, dilution 1:500), streptavidin HRP (BioLegend, dilution 1:1000). SN from OT-1 T cells were transduced to express the fusion molecule TIM-3.IgG4 or IL-2 V As a negative control, for the detection of IL-33, the commercial LEGEND MAXTM Mouse IL-33 ELISA Kit developed by BioLegend (436407) was used.

[0266] Chromium release assay to measure ADCC

[0267] Autologous PBMCs were thawed and cultured at 1×10 6 / ml and added to 6-well plates at a concentration of 3 ml per well in the presence of 10 ng / ml GM-CSF. The next day, 0.5 × 10 6EGFR+ T cells, resuspended, and placed in a 37°C water bath for about 1 hour. The T cells were then washed twice and suspended at a concentration of 400,000 cells / ml, and 50 μl of T cells (=2000 cells) were transferred to each well. 300 μg / ml or 30 μg / ml of cetuximab (anti-EGFR antibody) were prepared. 50 μl of cetuximab was added to the T cells and incubated at 37°C for 30 minutes. 1.2×10 6 PBMCs (effector cells) were harvested at a concentration of 1.2×10 cells / ml. 1 / 3 dilution of effector PBMCs (1.2×10 6 , 0.4×10 6 , 1.33×10 6 and 0.42×10 6 PBMC / ml), and 50μl was added to the wells containing tEGFR+T cells and anti-EGFR antibodies. Different ratios of effector cells to target cells were set up (30:1, 10:1, 3:1, 1:1, in triplicate). As a positive control, 1x TritonX (=maximum chromium release) was added to the T cells. All negative controls were set up (medium only, T cells without PBMC, T cells plus PBMC but no antibodies, etc.). The plate was rotated at 1500rpm and placed at 37°C for 4-5 hours. 50μl of supernatant was transferred to the lumaplate wells and allowed to dry overnight. The next day, chromium levels were assessed using a topCounter.

[0268] Co-culture assay and ELISA to measure cytokine production

[0269] 1×10 6 TCR + Co-engineered TCR-T cells expressing PD1 decoy plus truncated EGFR (and all control T cell conditions) were prepared at a concentration of 1 × 10 T cells / ml. 6 Tumor cells were prepared at a concentration of 100 μl of each T cell and tumor cell. 100 μl of each T cell and tumor cell were combined in a 96-well round bottom plate. The plate was rotated at 1500 rpm for 1 minute and incubated at 37°C for 48-72 hours. IFN-γ levels in the supernatant were assessed by ELISA (Invitrogen) according to the manufacturer's recommendations.

[0270] Co-culture assay and ELISA to assess secretion of PD1 and CD40L decoy

[0271] In a final volume of 200 μL complete RPMI medium, place 1 × 10 6Primary UTD and co-transduced T cells (engineered to express NYTCR and secrete PD1 decoy plus tEGFR) were incubated with 1 × 10 6 Target cells were co-cultured in duplicate in 96-well round-bottom plates. Plates were spun at 1500 rpm for 1 min and incubated at 37°C. After 24 h, co-culture supernatants were collected and tested for the presence of PD1 Fc fusion bait molecules by capturing plate-bound anti-PD1 antibodies or plate-bound human PD-L1 protein. Bound PD1 Fc bait molecules were detected with anti-IgG Fc antibodies. Secreted CD40L bait in supernatants was assessed using the same conditions except that a commercial ELISA kit (Invitrogen) was used.

[0272] Immune subset depletion, checkpoint blockade, and FTY720 treatment.

[0273] Starting 1 day before treatment, 250 μg / dose of depletion antibodies were administered intraperitoneally every three days to deplete specific cell subsets: CD4 T cells with α-mouse CD4 (clone GK1.5, BioXCell), NK cells with α-mouse NK1.1 (clone PK136, BioXCell), neutrophils with α-mouse Ly6G (clone 1A8, BioXCell). For checkpoint blockade, mice were injected intraperitoneally with 250 μg / dose of α-mouse PD-L1 (BioXcell, 10F.962) and α-mouse TIM-3 (BioXcell, RMT3-23) every three days. To block lymphocyte migration to secondary lymphoid organs, a stock solution of FTY720 obtained from SIGMA was prepared (10 mg / ml in DMSO) and then diluted to 1 mg / ml in water before administration. Finally, 100 μg of the drug was injected intraperitoneally every three days starting 2 days before treatment. PBMC flow cytometry confirmed the depletion and sequestration of immune cells (FTY720).

[0274] Preparation of single tumor cell suspensions, antibodies for flow cytometry and in vitro restimulation of cytokine production.

[0275] Tumors were excised 5 and 12 days after the first adoptive cell transfer and isolated into single-cell suspensions using a commercial mouse tumor isolation kit (Miltenyi Biotec, 130-096-730) combining mechanical dissociation with enzymatic degradation of the extracellular matrix. 6Live cells were seeded in 96-well plates and incubated with 50 μl Live / Dead Fixable aqua dead in PBS for 30 min at room temperature, followed by blocking Fc receptors by incubation with 50 μl purified anti-CD16 / CD32 monoclonal antibody (clone 2.4G2 BD Pharmingen) for 30 min at 4°C. Cells were then stained with 50 μl of the fluorochrome-conjugated monoclonal antibody of interest in FACS buffer for 30 min at 4°C. Subsequently, cells were washed twice and fixed / permeabilized for intracellular staining using the FoxP3 transcription factor staining buffer kit (Invitrogen). Stained cells were analyzed using an LSRII cytometer and FlowJo software.

[0276] The following antibodies were used: CD45.1 (cloneA20, BioLegend); CD3 (clone 145-2C11, Invitrogen), CD4 (clone GK1.5, BioLegend); CD8 (clone 53.6.7, BioLegend), FOXP3 (clone FJK-16S, Invitrogen), NK1.1 (clone PK136, BioLegend), CD44 (clone IM7, BioLegend), PD-1 (clone 29F.1A12, BioLegend), LY6C (clone HK1.4, BioLegend), granzyme C (clone SFC1D8, BioLegend), TCF1 (clone C63D9, Cell Signaling Technology), anti-rabbit IgG (H+L), F(ab') 2Fragment (AF488 or PE coupling (Cell Signaling Technology)), granzyme B (cloneGB11, Novul BioLegend), CD69 (clone H1.2F3, BioLegend), TIM-3 (clone RMT3-23, BioLegend), CD137 / 4-1BB (clone 17B5, Invitrogen), KLRG1 (clone 2F1 / KLRG1, BioLegend), KI67 (clone 16A8, BioLegend), IFNg (clone XM61.L, Invitrogen), TNFa (clone MP6-XT22, BioLegend), TOX (clone TXRX10, Invitrogen), CD45 (clone 30-F11, BioLegend),

[0277] Fluorescence minus one (FMO) controls were performed in parallel using panels of antibodies with one antibody omitted in sequence. FMO staining was used as a control for the following antibodies: TCF1, Ki67, 4-1BB, Granzyme B, TNFa, IFNg, PD-1, and TIM-3. Isotype controls were used for Granzyme C staining (clone HTK888, BioLegend). Precision Count Beads TM (BioLegend) was used to obtain absolute cell counts during flow cytometric acquisition.

[0278] To detect cytokine production, single tumor cell suspensions (2.5 × 10 6 1 μg / ml live cells) were restimulated in vitro for 4 h in the presence of Brefeldin A (5 μg / ml) in 24-well plates coated with 1 μg / ml anti-mouse CD3 (clone 17A2, Invitrogen) and 2 μg / ml soluble anti-mouse CD28 (clone 37.51, Invitrogen). Cells were surface stained before fixation and permeabilization as described above, followed by intracellular staining.

[0279] Immunofluorescence labeling and microscopy

[0280] For immunohistochemical analysis, tumor tissues were isolated and fixed in PBS containing 1% PFA overnight, infiltrated with 30% sucrose the next day (overnight), and then embedded and frozen in OCT compound. Low frozen sections were collected on SuperfrostPlus slides (Fisher Scientific), air-dried, and pre-incubated with blocking solution containing BSA, normal mouse serum, normal donkey serum (Sigma), and 0.1% triton. Then, primary antibodies were diluted in PBS containing 0.1% triton and labeled overnight at 4°C. After washing with PBS containing 0.1% triton, secondary reagents were diluted in PBS containing 0.1% triton and applied for 45 minutes at room temperature. Finally, after additional washing with PBS and 0.1% triton, cell nuclei were stained with DAPI (Sigma), followed by washing with PBS and mounting in DABCO (domestic). Images were acquired with a Zeiss AxioImager Z1 microscope and an AxioCam MRC5 camera. Images were processed using Fiji (NIH) or Adobe Photoshop. Exposure and image processing were identical for groups of mice to allow direct comparison.

[0281] Antibodies (ab) used for CD8 / CD45.1 / CD105 labeling: 1° ab: rat-mouse CD8a (clone53-6.7), rabbit-mouse CD105 (clone MJ7 / 18), mouse-mouse CD45.1 Biotin (clone A20.1). 2° Reagents: donkey-rat Alexa 488 (Invitrogen, #A21208), donkey-rabbit Cy3 (Jackson ImmunoResearch #711-165-152), streptavidin APC (Biolegend, #405207).

[0282] Antibodies (ab) used for CD8-CD45.1-TCF1 labeling: 1° ab: rat-mouse CD8a (53-6.7), rabbit-mouse TCF-1 (Cellsignalling, clone C63D9), #2203), mouse-mouse CD45.1 Biotin (clone A20.1). 2° ab: donkey-rat Alexa 488 (Invitrogen, #A21208), donkey-rabbit Cy3 (Jackson ImmunoResearch #711-165-152), streptavidin APC (Biolegend, #405207).

[0283] Single-cell RNA-seq analysis.

[0284] The aggregated UMI count matrix generated by CellRanger was screened to select high-quality CD8 TIL transcriptomes. First, cells with 500 to 5000 genes, 2000 to 30000 UMI counts, mitochondrial content less than 5%, and ribosomal protein content percentage less than 50% were retained. Next, CD8 T cells were screened as cells expressing Cd2, Cd8a, and CD8b1 (>=1UMI) but not Cd4 (0UMI). Cells expressing Cd14, Csf1r, Cd19, Spi1, Foxp3, H2-Aa, and H2-Ab1 were also removed, and 1788 high-quality CD8 TIL transcriptomes were obtained.

[0285] For dimensionality reduction, first use Seurat 3.1.1vst method (Stuart et al., Cell, vol.177, issue 7, p1888-1902.e21, June 13, 2019) with default parameters to identify high variability genes (HVG). Next, mitochondria, ribosomal protein encoding genes and cell cycle genes (genes carrying Gene Ontology term GO: 0007049) are removed from a series of HVGs, and the remaining HVGs (1649) are scaled to mean = 0 and variance = 1. Standardized HVGs are used for the first step of dimensionality reduction using PCA, and the second setting uses UMAP (implemented in Seurat v3.1.1) to reduce the first 10 principal components (other parameters are default). Clustering is performed using Seurat's shared nearest neighbor method, using FindNeighbor and FindClusters (resolution = 0.2) with default parameters. TILPRED was used with default parameters to supervise the classification of CD8 TIL status (https: / / github.com / carmonalab / TILPRED; Santiago J.Carmona et al., OncoImmunology, 9:1 (2020)). Cluster differentially expressed genes were identified using FindAllMarkers and MAST v1.10 (Finak, G et al., Genome Biol 16278 (2015)), with parameters of min.pct=0.25, logfc.threshold=0.25. In order to compare Gzmc cluster and traditional exhaustion cluster, the initial "exhausted" cluster (origin'exhausted'cluster) was sub-clustered by increasing the "resolution" parameter to 0.3. Differential expression analysis between the refined exhaustion cluster and Gzmc cluster was evaluated using FindAllMarkers and MAST v1.10, with parameters of min.pct=0.1 and logfc.threshold=0.25. Gene set enrichment analysis of these clusters with the TOX-KO signature (Scott, AC et al., Nature 571, 270–274 (2019)) was calculated using the GSEA function in the clusterProfiler package v3.12 (Guangchuang Yu et al., OMICS: A Journal of Integrative Biology. May 2012. 284-287), using default parameters, and using the top 200 differentially expressed cluster genes with p-values ​​< 0.01, sorted by decreasing fold change.

[0286] Statistical analysis

[0287] Normal distribution of data was assessed using the Shapiro-Will normality test. Two groups were compared using the two-tailed Student's t-test (if normally distributed and homoscedastic), or the Welch-corrected t-test (if normally distributed but not homoscedastic), or the nonparametric Mann-Whitney test if data were not normally distributed. A similar strategy was used to compare more than two groups. If normal distribution was not present, the Kruskal-Wallis test was used. If normal distribution and homoscedasticity were present, the one-way ANOVA test was used; if normal distribution but not homoscedasticity were present, the Brown-Forsythe and Welch ANOVA tests were used. Multiple comparison corrections were performed using the Dunn test (Kruskal Wallis test), the Dunnet test (one-way ANOVA test), and the Tukey test (Brown Forsythe test). Survival analysis was performed using the log-rank Mantel-Cox model. Pearson's correlation test was used to calculate the correlation between the number of CD8 T cells within TCF1+OT-1 tumors and the total number of tumor-infiltrating OT-1. All these statistical analyses were performed using GraphPad Prism 8.0, *p<0.05, **p<0.01, ***p<0.001, ***p<0.0001.

[0288] Statistical analysis of tumor control was performed using the change (%) in tumor volume on day 17 after tumor inoculation. The best response (minimum tumor volume) observed at least 12 days after the first effect was calculated for each animal. The objective response rate and clinical benefit rate of the treatment group were calculated based on the total number of mice in each group, because (1) objective response included complete response (CR; tumor volume reduction of 100%) and partial response (PR; ≤ tumor change -30%; (2) clinical benefit included CR, PR, and stable disease (-30% < tumor change ≤ +20%).

[0289] The predicted probabilities of the variables “objective response” and “clinical response” were calculated using exact logistic regression. Tumor change values ​​as continuous variables were also analyzed using linear regression. P values ​​less than 0.05 were considered statistically significant.

[0290] Example 2

[0291] PD1 decoy molecule cloned into IL-2 V , LIGHT or interleukin 33 (IL-33) sequences in retroviral constructs: each construct was codon-optimized to encode only two molecules separated by the self-cleaving peptide T2A. Thus, at least four different constructs are available: i) PD1.IgG4_T2A_IL-2V , expressing both PD1 decoy and IL-2 V ; ii) PD1.IgG4_T2A_LIGHT, expressing PD1 decoy and LIGHT; iii) PD1.IgG4_T2A_IL-33, expressing PD1 decoy and IL-33; and iv) PD1.IgG4_T2A_CD40L, expressing PD1 decoy and CD40L.

[0292] Figure 1 shows the efficiency of transfection and transduction of OT-1 T cells with the designed constructs. In each tested component, the cells showed efficient transduction and high secretion levels of each expressed secretory protein.

[0293] Using specific ELISA, it was also confirmed that the engineered OT-1T cells could secrete each specific immunomodulatory factor combination PD1.IgG4_T2A_IL-2 V , PD1.IgG4_T2A_LIGHT; and PD1.IgG4_T2A_IL-33 and PD1.IgG4_T2A_CD40L.

[0294] The results showed that T cells could be successfully engineered to express two different exogenous secretory proteins. These cells exhibited advanced properties by persistently expressing and secreting the respective exogenous secretory proteins in high amounts.

[0295] Example 3

[0296] As depicted in Figures 2A and 2B, ACT with OT-1T cells secreting PD1.IgG4, LIGHT, and IL-2V significantly improved the control of large B16-OVA tumors. This combined strategy induced tumor regression after ACT and improved overall survival compared to untransformed OT-1T cells. As depicted in Figures 2C and 2D, the use of OT-1T cells secreting PD1.IgG4, LIGHT, and IL-33 also significantly improved the control of large B16-OVA tumors. This combined strategy showed better anti-tumor activity than the PD1.IgG4, LIGHT combined strategy. Compared with untransfected OT-1T cells, the IgG4, LIGHT, and IL-33 combination strategy improved overall survival.

[0297] Adoptive transfer of OT-1 T cells secreting PD1.IgG4, IL-2V, and IL-33 nearly doubled overall survival compared to untransfected OT-1 T cells, and extended overall survival by 10 days or more compared to other combination strategies (Figure 2F). As described in Figures 2G and 2H, ACT using OT-1 T cells secreting PD1.IgG4, IL-2V, and CD40L significantly improved control of large B16-OVA tumors. This combination strategy induced tumor regression after ACT and improved overall survival compared to untransfected OT-1 T cells.

[0298] In summary, T cells can be genetically engineered to secrete combinations of immunomodulatory factors to control advanced tumors. The above examples also highlight the therapeutic feasibility of mixing T cell populations with the same antigen specificity but different secretory properties to obtain high-order combinations of immunomodulatory factors. An important advantage of this approach is increased safety, as these molecules are secreted in large quantities in the tumor microenvironment (that is, they are not administered systemically).

[0299] Example 4

[0300] Adoptive immunotherapy provides an opportunity to reprogram T cells and the tumor microenvironment. As shown in this example, orthogonally engineered adoptively transferred T cells with IL-2Rβγ-binding IL-2 variants, PD1 decoy, and IL-33 have a significant effect on transferred and endogenous CD8 + Cellular reprogramming enabled cell-autonomous T cell expansion, engraftment, and tumor control in immunocompetent hosts. Tumor-infiltrating lymphocytes (TILs) adopted a novel effector state that was distinct from typical TOX-driven exhaustion and characterized by TOX inhibition, abundant granzyme C, effector molecules, survival, and pro-cell markers. TILs in this state, driven dynamically by the interplay between IL-2 variants and IL-33, uncoupled persistence from TOX-driven exhaustion and successfully controlled tumors. Thus, rational T cell engineering in the absence of host immune cell clearance could both optimize reprogramming of adoptively transferred T cells and mobilize endogenous immunity to a novel state compatible with tumor control.

[0301] Presumably, T cells could be endowed with intrinsic properties that would allow them to autonomously achieve the necessary expansion in the absence of immune cell clearance and to reach a desired functional state compatible with implantation and rejection of moderately immunogenic tumors. In the present invention, an attempt was made to modify T cells by orthogonal combinatorial engineering, i.e., introducing genes whose products could produce favorable perturbations, thereby reprogramming T cells and enabling them to reprogram adaptive and innate immunity in the TME. The PD-1 / PD-L1 inhibitory pathway targets secreted PD-1 decoy (PD1d), a fusion molecule comprising the extracellular domain of mouse PD-1 linked to the Fc region of human IgG4. To support T cell expansion, a human IL-2 variant (IL-2 V )(T. Carmenate et al., Journal of immunology 190, 6230-6238 (2013); G. Rojas et al., Scientific Reports 9, 800 (2019)). Important advantages of this molecule compared to wild-type IL-2 include reduced toxicity and reduced sequestration of regulatory T cells (Tregs). It has also been hypothesized that, unlike wild-type IL-2, which drives terminal effector differentiation, IL-2 V It promotes CD8 + T cell stemness, which is a favorable feature of ACT (JG Crompton et al., Immunol Rev 257, 264-276 (2014)). Finally, in order to generate favorable inflammatory signals in tumors, IL-33 was used. Two retroviral vectors were constructed, one encoding soluble PD1d and IL-2 V (PD1d / 2 V module), and another encoding soluble PD1d and mouse IL-33 (PD1d / 33 module). CD8 + T cells were transduced with retrovirus carrying one or the other module and then pooled at a 1:1 ratio to generate the triple combination (PD1d / 2 V / 33) ACT cocktail. In addition, OT1 T cells were used to treat advanced B16-OVA melanoma in immunocompetent recipient mice. As shown in the figure below, this example investigates the potential for these interventions to induce CD8 + T cell status and the desired CD8 T cell engraftment and tumor regression in the absence of preconditioning immune cell depletion or exogenous cytokine support + T cell status.

[0302] Orthogonal T cell engineering improves ACT efficacy in a cell-autonomous manner

[0303] As the initial engineering module, the antitumor potential of the PD-1 decoy was first evaluated. The molecule was well expressed and secreted from engineered OT1 cells and bound to plates immobilized with PD-L1 in vitro (competed by a saturating PD-L1 neutralizing antibody). ACT with PD1d-engineered OT1 cells showed significant antitumor activity in immune cell-depleted (irradiated) mice. Next, we examined the PD1d / 2 V Or whether the PD1d / 33 module can effectively transduce OT1 cells to secrete PD1d and IL-2 respectively V , or PD1d and IL-33. The expression of PD1d was assessed, a transduction efficiency of greater than 75% was obtained, and simultaneous secretion of all molecules was confirmed by ELISA.

[0304] Next, orthogonal engineered ACT was performed without pre-treatment immune cell depletion (Figure 3A). In the absence of immune cell depletion or exogenous cytokines, approximately 70% of T CM and about 30% of T EM (Effect memory) phenotype 5×10 6 Untransduced OT1 cells were administered as two infusions to patients with palpable (100 m 3 ) tumors in mice that were unable to control tumor growth (Figure 3B). V Transduction of the same dose of OT1 cells had a small but non-significant effect on tumor growth (Figure 3B; Tables 2 and 3). Under the same conditions, no dual-engineered PD-1d / 2 V Systemic administration of anti-(α)PD-L1 antibodies with untransduced OT1 cells produced results comparable to those with PD1d-OT1 cells. Similarly, OT1 cells transduced with IL-33 had minimal effects, as did PD1d / 33-OT1 cells. Strikingly, PD1d / 2 V / 33-OT1 cells (i.e., 1:1 mixed PD1d / 2 V The treatment with PD1d / 33 cells was statistically significantly superior to the other treatments (Figure 3B; Tables 2 and 3). The objective response rate (ORR) of this treatment was 85.7% with a predicted probability of 83.3% (Tables 2 and 3), while the objective response rate (ORR) of all other treatments ranged from 0% to 9%. V The effectiveness of / 33-OT1 cells in the absence of immune cell clearance and exogenous cytokine support, early treatment of mice (starting from day 6) resulted in complete tumor eradication and cure.

[0305] Orthogonal engineering in immunocompetent hosts leads to adoptive transfer of CD8 + Cell-autonomous proliferation of cells and residual endogenous anti-tumor immunity

[0306] Then the dual engineered PD1d / 2 V The anti-tumor effect of PD1d / 33 and triple engineered PD1d / 2 V At baseline (day 12), untreated B16-OVA tumors showed a modest spontaneous CD8 + T cell infiltration, expressing CD44 + Phenotype of cells stimulated by antigen. + T cells began to accumulate in tumors within 4 days, and by day 5 after ACT (day 17), tumors in mice treated with the triple-engineered cells already showed significantly higher levels of CD8 + T cells were implanted. One week later (day 24), PD1d / 2 V CD8 in tumors of mice treated with OT1 cells + TILs were significantly more in mice treated with dual engineered cells than in mice treated with dual engineered cells, and CD8 + The number of TILs in mice treated with OT1 cells was significantly higher than that in mice treated with untransduced OT1 cells (Figure 3C). + )TIL, PD1d / 2 in tumors was observed two weeks after ACT V / 33-OT1 cells expanded significantly (Figure 3D), while PD1d / 2 V -OT1 and PD1d / 33-OT1 cells showed modest or minimal levels of engraftment, respectively. Thus, the efficacy of the triple-engineered combination ACT was associated with the unique expansion of adoptively transferred T cells in tumors and tumor regression.

[0307] Given that ACT was performed in fully immunocompetent hosts, we investigated whether endogenous immune effector cells were involved. Strikingly, the total CD8 + About 50% or more of TILs are endogenous (CD45.1 neg CD45.2 + )(Figure 3E). V Some expansion of endogenous TILs was observed in tumors treated with either -OT1 or PD1d / 33-OT1 cells, but these expansions were particularly prominent in tumors treated with triple-engineered cells.

[0308] In CD8 +The presence of a stem-like pool of cells in the T cell compartment that express the TCF1 transcription factor has previously been implicated in the ability to mobilize anti-tumor (and chronic viral infection) immunity after PD-1 blockade, and the use of such precursor cells for ACT may improve efficacy. + CD8 + The presence or persistence of TILs. Notably, in metastatic PD-1d / 2 V / 33 or dual engineered PD1d / 2 V After OT1 cells, TCF1 + OT1 + The significant expansion of TILs (Figures 3F and 3G) suggests that only these two shared IL-2 V Importantly, the stem cell-like TCF1 + CD8 + Expansion of OT1 and CD8 T cells also occurred in endogenous TILs (Figures 3F and 3G). In these tumors, 10-20% of OT1 and 30-50% of endogenous CD8 + TIL in PD1d / 2 V / 33-ACT and TCF1 was observed + There is a strong direct correlation between the presence of OT1 cells and the total number of OT1 TILs. V Our engineering approach achieves conditions that promote stemness and thereby facilitate transfer and persistence of endogenous T cells.

[0309] Importantly, effective tumor control in genetically engineered OT1 cells is not only related to TCF1 + CD8 + The increase of TILs was also associated with a large amount of TCF1 neg Effector-like CD8 + TIL related——PD1d / 2 V / 33-ACT alone fulfilled this condition (Figure 3G). In fact, in these tumors, 80-90% of OT1 TILs and 50-70% of endogenous CD8 + TIL is TCF1 neg Importantly, although in PD1d / 2 V -High frequency of TCF1 also appeared after ACT + CD8 + TILs, but TCF1 in these tumors neg CD8 + TILs were much less, suggesting that IL-2 V Drives TCF1 + CD8 +TILs expanded, but in the absence of IL-33 co-expression, these cells were less likely to transform into TCF1 neg status, confirming that Tcf1 inhibition is associated with effector differentiation. In contrast, PD1d / 33-ACT is associated with low TCF1 + CD8 + and high TCF1 neg CD8 + TIL frequency was associated with poor overall TIL expansion (Figures 3F and 3G).

[0310] To understand the role of endogenous T cells in tumor control after ACT, CD8 knockout tumor-bearing mice were treated with PD1d / 2 V The antitumor effect of ACT was observed in the absence of endogenous CD8 + T cells were lost (Figure 3H). + Engagement of T cells is essential for effective tumor control. Strikingly, tumor control was not dependent on the recruitment of endogenous T cells from lymph nodes, as demonstrated by co-administration of FTY720, a drug that impairs lymphocytes egress from lymph nodes (Figure 3H). Thus, rather than recruiting systemic CD8 + T cells are recruited into the tumor.

[0311] Next, the interaction of ACT with tumor Tregs was assessed. V Preferential expansion of CD8 + Rather than Treg, consistent with the maximum expansion of CD8+ cells, the CD8 / Treg ratio was at PD1d / 2 V / 33-ACT was the highest (Figure 3I). + TIL total expansion is more complete than CD8 + TIL should be less, before PD1d / 2V / 33-ACT, antibody-mediated CD4 + T cell depletion did not impair but significantly improved tumor control and mouse survival (Figure 3J).

[0312] Finally, we investigated whether the three engineered ACTs mobilized innate immunity. V Increased after ACT, especially PD1d / 2 V / 33-ACT, but they failed to activate, and they were dispensable. Importantly, however, tumor control is closely related to the mobilization and activation of neutrophils (Figure 3K). Therefore, orthogonal engineering achieves tumor regression in immunocompetent hosts by mobilizing adaptive and innate immunity. Although there are limited examples of ACT-mediated tumor control in lymphoreplete mice carrying hematological tumors, this is the first time that ACT has been demonstrated to be successful in advanced, poorly immunogenic solid tumors without any supportive treatment, SK Vodnala et al., Science 363, eaau0135 (2019).

[0313] Intratumoral GzmC induced by orthogonal engineering + TCF1 neg Effector CD8 + New subsets of T cells persist independently of TOX

[0314] To further understand the molecular status of TILs associated with tumor control in the triple-engineered ACT and the impact of individual dual-engineered modules, TILs were analyzed by single-cell (sc) RNA-Seq (Figure 4A). + Unsupervised cluster analysis of TILs revealed five distinct transcriptomic states (clusters C1 to C5) (Figure 4B). To interpret the results, TILPRED, a machine learning tool that assigns cells to previously described molecular TIL states in untreated mouse tumors, was used (SJ Carmona et al., OncoImmunology 9, 1737369 (2020)). The study found that TILs from tumors treated with non-engineered OT1 cells presented similar characteristics to TILs from untreated tumors, with a predominant pool of progenitors and terminally exhausted cells (C4) and few circulating (C3), effector memory (C2), and naive cells (C1) (Figures 4B and 4C). Therefore, in the absence of engineering and host conditions, ACT does not affect TIL status. PD1d / 2 V After -ACT, TILs exhibited a predominantly naive-like pool (C1), which is consistent with the above-mentioned TCF1 + In contrast, after PD1 / 33-ACT, TILs exhibited a predominantly effector memory state (C2) with some circulating cells (C3). Thus, only IL-2 VLocal expression of IL-33, or IL-33 alone, redirected TILs to naive-like and effector-memory states, respectively. Moreover, the combination of these two cytokines resulted in a completely new state (C5) implicated in tumor control that was separate from any of the states supported by each cytokine, respectively. C5 was found in three engineered ACT TILs exclusively during the response phase and not in any other tumor condition (Figure 4B). The novelty of this state was also supported by ProjecTIL, a tool that projects (sc)RNAseq data onto a reference TIL atlas, which revealed that while cells in clusters C1-C4 were consistent with previously described reference states, C5 emerged as a novel state that had never been described before, characterized by upregulation of a unique effector-like transcriptional program (Figure 4D).

[0315] C5 containing TILs were primarily identified by TILPRED and ProjecTIL and are broadly classified as “terminally exhausted” CD8 + cells, identical to TILs in the C4 cluster. In fact, cells in both clusters shared relatively high gene expression of co-inhibitory receptors, including Pdcd1, Lag3, Tigit, Havcr2 / TIM3, and Entpd1 / CD39, and co-stimulatory receptors and activation markers Tnfsfr9 / 4-1BB (Figure 4E). Given their separation by UMAP and the upregulation of the ProjecTIL effector-like transcriptional program, differential expression analysis was used to determine the distinct molecular signatures of C5 and C4 terminally exhausted TILs. Relative to the typical terminally exhausted cell C4, C5 TILs exhibited a unique effector signature, with significant downregulation of exhaustion-related transcription factors Tox, bhlhe40, and Batf and multiple inhibitory receptors. Notably, it also downregulated Cx3cr1, a unique marker of transient effector-like exhausted cells (Figure 4C, lower half; Table 3). In addition, C5 TILs significantly upregulated effector cell markers, including multiple granzymes, most notably Gzmc, which constitutes a C5-specific marker (Figure 4E), and the anti-apoptotic genes Bcl2 and Ly6c2, which are related to the precursor CD8 + T cell-related markers, CX3CR1 + Compared with C4 cells, C5 cells were consistently enriched in Tox knockout CD8 TIL signals (Figure 4F). Thus, FACS confirmed that the majority of OT1 (approximately 80%) and endogenous (approximately 70%) CD8 + TILs expressed granzyme C during the reactive phase (Figure 4G). GzmC was not detected in the spleen. + CD8 +cells, suggesting that local cues in the reprogrammed TME drive T cells specifically into this state in the tumor. Importantly, at baseline, the remaining experimental groups (Figure 4G) and endogenous CD8 + GzmC was found in TIL or OT1 cells after in vitro expansion. + CD8 + The frequency of T cells was negligible, suggesting that PDd1 / 2 V / 33-ACT specifically led to profound intratumoral reprogramming of TILs (including endogenous TILs), generating a new CD8 + T cell effector phenotype, which clearly requires IL-2 V Local interaction between IL-33 and IL-33.

[0316] TOX neg / low G JZ + TCF1 neg Effector CD8 + TILs are multifunctional cells with meaningless expression of co-inhibitory receptors

[0317] Further characterization of PD1d / 2 V / 33-ACT after GzmC + Effector CD8 + Effect of TIL status on tumor rejection. Gating strategy was used to identify TCF1 in OT1 and endogenous compartments neg Effector CD8 + Cells. Studies have found that PD1 / 2 V After 33-ACT, most OT1 and two-thirds of endogenous GzmC + CD8 + TIL is indeed TCF1 neg These cells were then co-cultured with TCF1 from other groups. neg Effector CD8 + TILs were compared (Figure 5B) (analyzed from PD1d / 33-OT1, untransduced OT1, and untransduced OT1 ACT plus αPD-L1), and importantly, all of these cells were GzmC neg (Figure 5A). From PD1d / 2 V / 33-ACT and GzmC neg TCF1 neg CD8 + Most GzmCs of effector OT1 + TCF1 neg CD8 + TIL and endogenous TIL from other groups were PD-1 +(Figure 5C), and a significant proportion of these cells were also TIM3 + (Figure 5D). Notably, consistent with the (sc) RNA-seq data, almost no OT1 cells expressed TOX, and only about 40% of endogenous PD-1 + G JZ + TCF1 neg cells expressed TOX (Figure 5E). + ) and C4(GzmC neg )-depleted cells, most of which were differentially expressed in PD1 + G JZ + TCF1 neg OT1 and endogenous TILs showed high expression of LY6C, which is a terminally exhausted CD8 + A marker missing from TILs. GzmC + TCF1 neg OT1 or endogenous TILs also lowly expressed / no KLRG1, a marker for short-lived effector cells (Figure 5F). V / 33-ACT TOX low / neg G JZ + TCF1 neg CD8 + TILs were not typical terminally exhausted cells, and most OT1 and approximately half of endogenous cells were found to express CD69, suggesting recent TCR-induced activation (Figure 6A). neg / low G JZ + PD-1 + TCF1 neg CD8 + T cells expressed more Ki-67 than TILs in other groups (Figure 6B). Finally, according to (sc) RNAseq analysis (Figures 6C and 6D), V Most OT1 and endogenous CD8 + TIL for GrzmB + In these PD-1 + In OT1 cells, an important part was identified, namely the double GzmC high and GzmB high (Figure 6E), which was not detected in endogenous cells. V PD-1 collected during tumor regression in 33-ACT + CD8 +TILs were tested for cytokine responses to in vitro CD3 / CD28 stimulation. Approximately half of OT1 and endogenous TILs produced TNFα upon stimulation, and a portion of cells produced both TNFα and IFNγ (Figure 6F), demonstrating multifunctional effector properties. Thus, the triple engineered ACT produced a unique and potent tumor rejection phenotype, namely CD8 + Effector TILs do not receive the TOX program.

[0318] To test whether co-inhibitory receptors such as PD-1 or TIM-3 are uncoupled from the TOX exhaustion program and their inhibitory functions are irrelevant in the unique TIL state, PD1d / 2 V / 33-ACT combined with αPD-L1 or dual αPD-L1 / αTIM3 antibodies. However, no improvement in tumor control was observed (Figure 6G and 6H). V In the context of the engineered ACT with IL-33 / PD-1 combination, PD-1 blockade is completely unnecessary. In fact, removal of the PD-1 ectodomain from the PD-1_IgG4 decoy does not inhibit the activation of PD-1 by only activating 2 V ACT of the / 33 module impacted tumor control (Figure 6I). Together, these data suggest that in immunocompetent hosts, orthogonal engineering of T cells with βγ-binding IL-2 and IL-33 can generate a new effector TIL state that is endowed with tumor control capacity, in which TOX remains suppressed and co-inhibitory receptors are insignificantly expressed.

[0319] Orthogonal Engineering Drives TOX neg / low G JZ + Precursor differentiation

[0320] Similar to chronic viral infection, CD8 + T cell-mediated antitumor responses (also after PD-1 blockade) may be mediated by intratumoral TCF1 + PD-1 + Precursor-depleted CD8 + T cells maintain, these cells have stem cell-like properties and express TOX, a transcription factor that is essential for the generation and persistence of these cells. V Inhibition of TCF1 in the ACT background neg The role of TOX in cells was studied by IL-2 V Does it also inhibit TCF1 + TOX program in prostate. V In the response phase after / 33-ACT, a large amount of TCF1 was detected + CD8 +All of these cells expressed GzmC (Figure 5A), and most also expressed PD-1. + ( Figure 7A ), but most of these (OT1 and endogenous) are TOX neg (Figures 7B and 7C). Therefore, under orthogonal engineered ACT, tumor-reactive TCF1 + The precursors already inactivate the TOX program and upregulate GzmC (Figure 7D). Importantly, PD1d / 2 V - TCF1 after ACT + PD-1 + CD8 + Downregulation of TOX was also found in TILs, whereas significant expression in the same TIL subsets after PD1 / 33-ACT indicated that IL-2 V In TCF1 + CD8 + Inhibits TOX at the level of the precursor state.

[0321] In PD1d / IL-2 V -After ACT, PD-1 was determined neg TCF1 + precursors (OT1 and endogenous) (Figure 7A). These are also TOX neg , and showed antigen-stimulated T CM or T EM Therefore, in the presence of IL-2 V In the presence of tumor-specific TCF1 + TIL is not entirely PD-1 + However, in the presence of IL-33 alone, 40% of endogenous TCF1 + PD-1 neg CD8 + TIL is TOX + cells, most of which are T EM Therefore, it can be concluded that orthogonal engineering reprograms TCF1 + The stem cell-like compartment suppresses the TOX program and upregulates a transcriptional program predicted by GzmC expression. Importantly, these two programs appear to be independent. V is a key factor supporting stemness and persistence in a TOX-independent manner, and binding to IL-33 is also required to trigger pro-CD8 + GzmC in TIL + Activation of the differentiation program.

[0322] G JZ + Dynamic evolution of effector states

[0323] Finally, the GzmC after three engineered ACTs was evaluated. + Dynamic evolution of TIL status. Comparison of the dynamic evolution of TIL status from day 5 after ACT (day 17) to tumor response (day 24) and PD1d / 2 V CD8 T cells were collected from tumors that escaped after the initial response to 33-ACT (day 38). + (sc)RNA-seq data of TILs (Fig. 8A). Adding the escape time point to all previous TILs did not change the annotation and cluster distribution of cells described previously. Thus, early after ACT, TILs were mainly distributed in the proliferating (C3), effector memory (C2), and exhausted pools (C4) (Fig. 8B). At day 12 after ACT, cells migrated in the new C5 state, associated with tumor regression. Interestingly, subsequent progression was associated with the migration of TILs from C5 to the new C6 state (Fig. 8B). The new adaptive state upon tumor escape is indeed different from the C5 GzmC associated with rejection + Effector state (Figures 8B and 8C). As expected, blockade of PD-1 (or TIM-3) failed to control tumors (Figures 6G, 6H, and 6I), and escape cells were distant from C4-depleted terminally differentiated cells and expressed significantly less TOX than these cells (Figure 8B). V Clustering of the 33 / 33 samples allowed for increased resolution, indicating that C6 cells were closer to but distinct from the canonical effector memory C2 state observed during early or post-ACT responses, and presented lower levels of Fosl2, Bcl2, Gzma, Gzmb, and Tnfrsf9 (CD137) (Figure 8B). Consistently, ProjecTIL analysis revealed that the escape-stage C6 state deviated from the reference map effector memory state, downregulating the Fosl2-driven effector gene program. Thus, escape was not mediated by canonical depletion, and cells retained PD1d / 2 at least to a large extent. V / 33-ACT displayed TOX repression features but lacked expression of GzmC (and GzmB).

[0324] Flow cytometry confirmed the above observations. After three engineered ACTs, from day 5 to day 12, the landmark GzmC of cluster 5 + CD8 + The TIL population increased significantly in tumors, consistent with tumor regression (Figure 8D), whereas these cells were lost during tumor escape. In addition, a significant decrease in OT1 cells was observed during tumor progression (Figure 8E), consistent with the TCF1 expression of OT1 and endogenous TILs. neg Consistent with the C6 state, the residual CD8 + TIL presents PD-1 neg / lo T EM CD8+ Phenotype, and in vitro expanded T EM CD8 + Granzyme B was significantly downregulated compared to T cells. Finally, compared to TCF1 cells harvested during tumor control neg PD-1 + CD8 + Compared to T cells harvested during the escape period, TCF1 neg PD-1 + CD8 + T cells showed loss of granzyme B expression and polyfunctionality (Figures 8G and 8H). Therefore, it can be concluded that the optimal TIL effector state is associated with the dynamics of tumor response.

[0325] discuss

[0326] This example demonstrates that in the context of solid tumor ACT, orthogonal combinatorial T cell engineering can successfully overcome homeostatic barriers in the host and lead to profound reprogramming of TILs and the tumor microenvironment and tumor regression in the absence of immune cell depletion or exogenous cytokine support. Performing ACT in an immunocompetent host has a unique advantage not only because it can significantly reduce the toxicity and cost of current ACT, but also because it can take advantage of the full spectrum of host immunity. This example also demonstrates that in these cases, endogenous CD8 + Cells (especially pre-existing CD8 + Both TILs and neutrophils are mobilized to fight tumors and are critical for achieving tumor regression.

[0327] Recent studies using high-dimensional computational analysis have found that multiple types of CD8 + States - naive, effector memory, cytotoxic and exhausted (SJ Carmona et al., OncoImmunology 9, 1737369 (2020)). While the cytotoxic TIL state observed primarily in human samples is primarily enriched in bystander cells, there is ample evidence that the exhausted compartment is enriched in tumor-specific CD8 TILs (AM van der Leun et al., Nature Reviews Cancer 20218-232 (2020)). Notably, this compartment is highly heterogeneous, formed by a continuum of cell states arranged hierarchically along the differentiation axis, as precursor or terminally exhausted CD8 +T cells. Although immune checkpoint blockade (ICB) has achieved important clinical response levels to date, its effect is mainly based on inducing changes in the exhausted CD8 T cell state that already exists before treatment, rather than inducing a new, non-exhausted effector-like state (J.-C. Beltra et al., Immunition 52825-841.e828 (2020)). Therefore, pharmacological reprogramming of CD8 TILs to this "desired" effector state represents an effective strategy to improve the clinical response of current immunotherapy.

[0328] T cell engineering offers unlimited opportunities to rationally reprogram TILs and reprogram the TME in a paracrine manner. This example demonstrates the use of IL-2 variants that bind only to the βγ chain receptor, in combination with stimulation of CD8 + The orthogonal engineering of PD-1 blockade and IL-33, a potent activator of innate immunity, of T cells was used together to reprogram the TME. This combination resulted in both exogenous and endogenous TILs adopting a new effector state characterized by the unique expression of multiple granzymes (most notably granzyme-C) and the inhibition of TOX, a transcription factor that is required for exhausted CD8 T cells in chronic viral infections and cancer. + The generation and maintenance of T cell populations is essential (ACScott et al., Nature 571, 270-274 (2019)). This state is consistent with the significant local CD8 + T cell expansion, potent effector function, and effective tumor control were associated. Under this new program, PD-1 and other co-inhibitory receptors were still expressed, indicating that their upregulation in the context of sustained antigenic stimulation is not strictly dependent on TOX. In addition, pharmacological blockade of the PD-1 and TIM3 pathways confirmed that their expression is functionally insignificant.

[0329] CD8 + The T cell depletion program was implemented by stable expression of TOX in the TCF1 progenitor compartment. However, TCF1 expanded by orthogonal engineering + The CD8 state remained TOX-negative and upregulated granzyme-C, a marker never detected in typical progenitor-depleted compartments. Thus, the progenitor-like cell state induced by this therapy is consistent with the TCF1 that has been described in chronic viral infections and cancer. + TOX + In a similar manner, the multifunctional GzmC was expanded by orthogonal engineering. + TCF1 neg PD-1 + TOX low / neg The effector-like state is not only different from the typical terminally exhausted cell state (TOX +PD-1 + CX3CR1 neg Gz neg ) and also from a transient effector-like depletion state (CX3CR1 + TIM3 + In fact, although TOX is downregulated in this state, it is derived from the typical GzmC neg TCF1 + Progenitor-depleted cells do not significantly upregulate Gzmc (J.-C. Beltra et al., Immunity 52, 825-841.e828 (2020)). In addition, it expresses Cx3cr1 and Klrg1, which are induced by orthogonal engineering of GzmC + In the effector state, they are significantly downregulated. + T-helper cells form CX3CR1 + However, in the context of this approach, these cells are deleterious. Therefore, we hypothesized that TCF1 + and TCF1 neg G JZ + CD8 + TILs represent a novel TOX-independent CD8 + Precursor and effector states of the T cell differentiation program.

[0330] The therapeutic manipulation of TOX has become a promising strategy to eliminate T cell exhaustion in the context of cancer patients. Recently, it has been shown that TOX gene knockout or TOX2 gene deletion can improve the function of CAR-T cells, and TOX gene heterozygous deletion can enhance anti-tumor T cell response (H. Seo et al., Proceedings of the National Academy of Sciences 116, 12410-12415 (2019); O. Khan et al., Nature 571, 211-218 (2019)). This example demonstrates an alternative, new approach to therapeutically target TOX to induce non-exhausted, highly functional CD8 + effector state. In fact, studies have shown that IL-2 V Not only in transferred T cells but also in endogenous CD8 + In the T cell tumor compartment, it promotes the stemness of CD8 T cells and inhibits TOX. IL-33 may indirectly drive Gzmc upregulation and Tcf1 inhibition through TME reprogramming, leading to CD8 + T cells differentiate into multifunctional effector cells. This optimal TIL state is lost upon tumor escape, and the resulting EM-like state is not only different from the effector GzmC+ , and also differs from the typical effector memory CD8 TIL state. Therefore, tumor escape to orthogonal engineered T cells is not mediated by reactivation of the TOX-driven exhaustion program but rather by the lack of intratumoral CD8 + T cells move toward optimal GzmC + Effector CD8 + This is consistent with the fact that PD-1 blockade depletes CD8 + A key distinction is the rejuvenation of T cells, which reacquire an exhausted phenotype during tumor escape.

[0331] In summary, this example shows that CD8 + Orthogonal co-engineering of T cells, specifically secreting IL-2 variants that do not bind to CD25 and the alarmin IL-33, can control advanced melanoma. Although combination T cell therapy has no efficacy, CD4 depletion has been shown to result in long-term survival, suggesting that Tregs play a role in disease progression, providing opportunities for additional co-interventions. Thus, the present invention demonstrates the potential for clinical translation of co-engineered T cells to reprogram the TME and induce highly functional CD8 + state.

[0332] Table 2: Observed responses and predicted probabilities of objective response and clinical benefit by treatment group.

[0333]

[0334]

[0335] Note: Objective response includes complete response (CR; 100% reduction in tumor volume) and partial response (PR; ≤ tumor change -30%). Clinical benefit includes CR, PR and stable disease (-30% < tumor change ≤ +20%). The probability of occurrence was calculated using exact logistic regression.

[0336] Table 3: Prediction of change in tumor size from baseline for each group using linear regression.

[0337]

[0338] *: -100% is the minimum plausible value for tumor size change. Note: p value compares the effect of each treatment with triple combination (PD1d+IL-2V+IL-33). Model adjusted R 2 :43.8%.

[0339] Example 5

[0340] CD40L decoy and IL-2 variants and combined GEEP therapy

[0341] The SIN retroviral vector was constructed to encode a trimeric CD40L decoy and an IL-2 variant that does not bind to CD25. These molecules are expressed under NFAT and are therefore only produced in activated T cells, which should only occur in the tumor microenvironment. Preclinical models have shown that the IL-2 variant promotes a less differentiated phenotype and supports in vivo engraftment (i.e., persistence of T cells). In preclinical studies, CD40L has also been shown to promote tumor control. It can act on antigen-presenting cells, such as dendritic cells, to activate them and thus provide better T cell support. Therefore, the CD40L decoy is a tumor microenvironment reprogramming.

[0342] T cells were first engineered with PD1 decoy-tEGFR and then transduced by co-transduction or mixing of different engineered T cell populations with CD40L decoy and IL2 V Combination. If necessary, tEGFR (or cell elimination tag (CET)) can be used to assess transduction efficiency and for enrichment of engineered cells (on anti-EGFR coated beads). It can be used as a method to track engineered T cells in patients after implantation (by collecting FACs from blood samples or tumor biopsies). In addition, it can be used as an elimination tag via ADCC in patients who experience toxicity from cetuximab.

[0343] Table 4 Representative sequences of illustrative transgenes

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366] Sequence Listing <110> Ludwig Institute for Cancer Research Coukos, George Osorio, de Jesus Corria Zoete, Vincent Irving, Melita <120> Compositions and methods for immunotherapy <130> 084276.00282 <150> 62 / 935,308 <151> 2019-11-14 <160> 52 <170> PatentIn version 3.5 <210> 1 <211> 147 <212> PRT <213> House mouse <400> 1 Ser Gly Trp Leu Leu Glu Val Pro Asn Gly Pro Trp Arg Ser Leu Thr 1 5 10 15 Phe Tyr Pro Ala Trp Leu Thr Val Ser Glu Gly Ala Asn Ala Thr Phe 20 25 30 Thr Cys Ser Leu Ser Asn Trp Ser Glu Asp Leu Met Leu Asn Trp Asn 35 40 45 Arg Leu Ser Pro Ser Asn Gln Thr Glu Lys Gln Ala Ala Phe Ser Asn 50 55 60 Gly Leu Ser Gln Pro Val Gln Asp Ala Arg Phe Gln Ile Ile Gln Leu 65 70 75 80 Pro Asn Arg His Asp Phe His Met Asn Ile Leu Asp Thr Arg Arg Asn 85 90 95 Asp Ser Gly Ile Tyr Leu Cys Gly Ala Ile Ser Leu His Pro Lys Ala 100 105 110 Lys Ile Glu Glu Ser Pro Gly Ala Glu Leu Val Val Thr Glu Arg Ile 115 120 125 Leu Glu Thr Ser Thr Arg Tyr Pro Ser Pro Ser Pro Lys Pro Glu Gly 130 135 140 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Ile Gly Ala Arg Arg Thr Gly Gln Pro 195 200 205 Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly 210 215 220 Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro 225 230 235 240 Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser Gly 245 250 255 Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg 260 265 270 Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu 275 280 285 <210> 3 <211> 147 <212> PRT <213> Homo sapiens <400> 3 Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr 1 5 10 15 Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe 20 25 30 Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr 35 40 45 Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu 50 55 60 Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu 65 70 75 80 Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn 85 90 95 Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala 100 105 110 Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg 115 120 125 Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly 130 135 140 Gln Phe Gln 145 <210> 4 <211> 371 <212> PRT <213> artificial sequence <220> <223> synthetic substances <400> 4 Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr 1 5 10 15 Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe 20 25 30 Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr 35 40 45 Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu 50 55 60 Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu 65 70 75 80 Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn 85 90 95 Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala 100 105 110 Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg 115 120 125 Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly 130 135 140 Gln Phe Gln Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly 145 150 155 160 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 165 170 175 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln 180 185 190 Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val 195 200 205 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr 210 215 220 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 225 230 235 240 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile 245 250 255 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 260 265 270 Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser 275 280 285 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 290 295 300 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 305 310 315 320 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val 325 330 335 Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met 340 345 350 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 355 360 365 Pro Gly Lys 370 <210> 5 <211> 1113 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Substance <400> 5 cctggctggt ttctggacag ccctgacaga ccctggaatc ctccaacatt cagccccgct 60 ctgctggtgg ttaccgaggg cgataatgcc accttcacct gtagcttcag caacaccagc 120 gagagcttcg tgctgaactg gtacagaatg agccccagca accagaccga caagctggcc 180 gcctttcctg aggatagatc tcagcccggc caggactgcc ggttcagagt tacacagctg 240 cccaacggcc gggacttcca catgtctgtc gtccgggcca gaagaaacga cagcggcaca 300 tatctgtgcg gcgccatttc tctggcccct aaggctcaga tcaaagagag cctgagagcc 360 gagctgagag tgacagaaag acgggccgaa gtgcccacag ctcacccttc accttctcca 420 agacctgccg gccagtttca gcctccttgt cctagctgtc ctgctcctga gtttctcggc 480 ggaccctccg tgttcctgtt tcctccaaag cctaaggaca ccctgatgat cagcagaacc 540 cctgaagtga cctgcgtggt ggtggacgtg tcccaagagg accctgaggt gcagttcaat 600 tggtacgtgg acggcgtgga agtgcacaac gccaagacca agcctagaga ggaacagttc 660 aacagcacct acagagtggt gtccgtgctg accgtgctgc accaggattg gctgaacggc 720 aaagagtaca agtgcaaggt gtccaacaag ggcctgccta gcagcatcga gaaaccatc 780 agcaaggcca agggccagcc aagagaaccc caggtgtaca cactgcctcc aagccaagag 840 gaaatgacca agaaccaggt gtccctgacc tgcctggtca agggcttcta cccttccgat 900 atcgccgtgg aatgggagag caatggccag cctgagaaca actacaagac cacacctcct 960 gtgctggaca gcgacggctc attcttcctg tacagcagac tgaccgtgga caagagcaga 1020 tggcaagagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1080 acccagaagt ccctgagcct gtctcctggc aaa 1113 <210> 6 <211> 129 <212> PRT <213> artificial sequence <220> <223> synthetic substances <400> 6 Met Ala Ser Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser 1 5 10 15 Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys 20 25 30 Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Met 35 40 45 Ser Pro Ser Asn Gln Asp Asp Arg Leu Ala Ala Phe Pro Glu Asp Arg 50 55 60 Ser Gln Pro Gly Gln Asp Ala Arg Phe Arg Val Thr Gln Leu Pro Asn 65 70 75 80 Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser 85 90 95 Gly Thr Tyr Phe Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Lys Ile 100 105 110 Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Leu 115 120 125 Glu <210> 7 <211> 135 <212> PRT <213> artificial sequence <220> <223> synthetic substances <400> 7 Met Ala Ser Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser 1 5 10 15 Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys 20 25 30 Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Met 35 40 45 Ser Pro Ser Asn Gln Asp Asp Arg Leu Ala Ala Phe Pro Glu Asp Arg 50 55 60 Ser Gln Pro Gly Gln Asp Ala Arg Phe Arg Val Thr Gln Leu Pro Asn 65 70 75 80 Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser 85 90 95 Gly Thr Tyr Phe Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Lys Ile 100 105 110 Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Leu 115 120 125 Glu His His His His His His 130 135 <210> 8 <211> 367 <212> PRT <213> artificial sequence <220> <223> synthetic substances <400> 8 Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser Pro Ala Leu 1 5 10 15 Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys Ser Phe Ser 20 25 30 Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Met Ser Pro Ser 35 40 45 Asn Gln Asp Asp Arg Leu Ala Ala Phe Pro Glu Asp Arg Ser Gln Pro 50 55 60 Gly Gln Asp Ala Arg Phe Arg Val Thr Gln Leu Pro Asn Gly Arg Asp 65 70 75 80 Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser Gly Thr Tyr 85 90 95 Phe Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Lys Ile Lys Glu Ser 100 105 110 Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val Pro Thr 115 120 125 Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln Phe Gln Thr Pro 130 135 140 Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 145 150 155 160 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 165 170 175 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 180 185 190 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 195 200 205 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 210 215 220 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 225 230 235 240 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 245 250 255 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 260 265 270 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 275 280 285 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 290 295 300 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 305 310 315 320 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 325 330 335 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 340 345 350 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 355 360 365 <210> 9 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Substance <400> 9 Met Ala Ser Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser 1 5 10 15 Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys 20 25 30 Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Met 35 40 45 Ser Pro Ser Asn Gln Val Asp Arg Leu Ala Ala Phe Pro Glu Asp Arg 50 55 60 Ser Gln Pro Gly Gln Asp Ala Arg Phe Arg Val Thr Gln Leu Pro Asn 65 70 75 80 Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser 85 90 95 Gly Thr Tyr Phe Cys Gly Ala Ile Ser Leu Ala Pro Lys Trp Lys Ile 100 105 110 Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Leu 115 120 125 Glu <210> 10 <211> 135 <212> PRT <213> artificial sequence <220> <223> synthetic substances <400> 10 Met Ala Ser Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser 1 5 10 15 Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys 20 25 30 Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Met 35 40 45 Ser Pro Ser Asn Gln Val Asp Arg Leu Ala Ala Phe Pro Glu Asp Arg 50 55 60 Ser Gln Pro Gly Gln Asp Ala Arg Phe Arg Val Thr Gln Leu Pro Asn 65 70 75 80 Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser 85 90 95 Gly Thr Tyr Phe Cys Gly Ala Ile Ser Leu Ala Pro Lys Trp Lys Ile 100 105 110 Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Leu 115 120 125 Glu His His His His His His 130 135 <210> 11 <211> 367 <212> PRT <213> artificial sequence <220> <223> synthetic substances <400> 11 Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser Pro Ala Leu 1 5 10 15 Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys Ser Phe Ser 20 25 30 Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Met Ser Pro Ser 35 40 45 Asn Gln Val Asp Arg Leu Ala Ala Phe Pro Glu Asp Arg Ser Gln Pro 50 55 60 Gly Gln Asp Ala Arg Phe Arg Val Thr Gln Leu Pro Asn Gly Arg Asp 65 70 75 80 Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser Gly Thr Tyr 85 90 95 Phe Cys Gly Ala Ile Ser Leu Ala Pro Lys Trp Lys Ile Lys Glu Ser 100 105 110 Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val Pro Thr 115 120 125 Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln Phe Gln Thr Pro 130 135 140 Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 145 150 155 160 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 165 170 175 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 180 185 190 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 195 200 205 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 210 215 220 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 225 230 235 240 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 245 250 255 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 260 265 270 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 275 280 285 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 290 295 300 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 305 310 315 320 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 325 330 335 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 340 345 350 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 355 360 365 <210> 12 <211> 129 <212> PRT <213> artificial sequence <220> <223> synthetic substances <400> 12 Met Ala Ser Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser 1 5 10 15 Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys 20 25 30 Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Glu 35 40 45 Ser Pro Ser Asn Gln Asp Asp Arg Leu Ala Ala Phe Pro Glu Asp Arg 50 55 60 Ser Gln Pro Gly Gln Asp Ala Arg Phe Arg Val Thr Gln Leu Pro Asn 65 70 75 80 Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser 85 90 95 Gly Thr Tyr Phe Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Lys Ile 100 105 110 Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Leu 115 120 125 Glu <210> 13 <211> 135 <212> PRT <213> artificial sequence <220> <223> synthetic substances <400> 13 Met Ala Ser Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser 1 5 10 15 Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys 20 25 30 Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Glu 35 40 45 Ser Pro Ser Asn Gln Asp Asp Arg Leu Ala Ala Phe Pro Glu Asp Arg 50 55 60 Ser Gln Pro Gly Gln Asp Ala Arg Phe Arg Val Thr Gln Leu Pro Asn 65 70 75 80 Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser 85 90 95 Gly Thr Tyr Phe Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Lys Ile 100 105 110 Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Leu 115 120 125 Glu His His His His His His 130 135 <210> 14 <211> 370 <212> PRT <213> artificial sequence <220> <223> synthetic substances <400> 14 Met Ala Ser Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser 1 5 10 15 Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys 20 25 30 Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Glu 35 40 45 Ser Pro Ser Asn Gln Asp Asp Arg Leu Ala Ala Phe Pro Glu Asp Arg 50 55 60 Ser Gln Pro Gly Gln Asp Ala Arg Phe Arg Val Thr Gln Leu Pro Asn 65 70 75 80 Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser 85 90 95 Gly Thr Tyr Phe Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Lys Ile 100 105 110 Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu 115 120 125 Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln Phe 130 135 140 Gln Thr Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly 145 150 155 160 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 165 170 175 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 180 185 190 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 195 200 205 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg 210 215 220 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 225 230 235 240 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 245 250 255 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 260 265 270 Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu 275 280 285 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 290 295 300 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 305 310 315 320 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 325 330 335 Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 340 345 350 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 355 360 365 Gly Lys 370 <210> 15 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Substance <400> 15 Met Ala Ser Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser 1 5 10 15 Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys 20 25 30 Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Glu 35 40 45 Ser Pro Ser Asn Gln Val Asp Arg Leu Ala Ala Phe Pro Glu Asp Arg 50 55 60 Ser Gln Pro Gly Gln Asp Ala Arg Phe Arg Val Thr Gln Leu Pro Asn 65 70 75 80 Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser 85 90 95 Gly Thr Tyr Phe Cys Gly Ala Ile Ser Leu Ala Pro Lys Trp Lys Ile 100 105 110 Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Leu 115 120 125 Glu <210> 16 <211> 135 <212> PRT <213> artificial sequence <220> <223> synthetic substances <400> 16 Met Ala Ser Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser 1 5 10 15 Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys 20 25 30 Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Glu 35 40 45 Ser Pro Ser Asn Gln Val Asp Arg Leu Ala Ala Phe Pro Glu Asp Arg 50 55 60 Ser Gln Pro Gly Gln Asp Ala Arg Phe Arg Val Thr Gln Leu Pro Asn 65 70 75 80 Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser 85 90 95 Gly Thr Tyr Phe Cys Gly Ala Ile Ser Leu Ala Pro Lys Trp Lys Ile 100 105 110 Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Leu 115 120 125 Glu His His His His His His 130 135 <210> 17 <211> 370 <212> PRT <213> artificial sequence <220> <223> synthetic substances <400> 17 Met Ala Ser Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser 1 5 10 15 Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys 20 25 30 Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Glu 35 40 45 Ser Pro Ser Asn Gln Val Asp Arg Leu Ala Ala Phe Pro Glu Asp Arg 50 55 60 Ser Gln Pro Gly Gln Asp Ala Arg Phe Arg Val Thr Gln Leu Pro Asn 65 70 75 80 Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser 85 90 95 Gly Thr Tyr Phe Cys Gly Ala Ile Ser Leu Ala Pro Lys Trp Lys Ile 100 105 110 Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu 115 120 125 Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln Phe 130 135 140 Gln Thr Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly 145 150 155 160 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 165 170 175 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 180 185 190 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 195 200 205 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg 210 215 220 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 225 230 235 240 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 245 250 255 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 260 265 270 Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu 275 280 285 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 290 295 300 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 305 310 315 320 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 325 330 335 Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 340 345 350 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 355 360 365 Gly Lys 370 <210> 18 <211> 224 <212> PRT <213> Homo sapiens <400> 18 Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser 1 5 10 15 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 20 25 30 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro 35 40 45 Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 50 55 60 Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val 65 70 75 80 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 85 90 95 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr 100 105 110 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 115 120 125 Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 130 135 140 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 145 150 155 160 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 165 170 175 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 180 185 190 Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 195 200 205 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220 <210> 19 <211> 327 <212> PRT <213> Homo sapiens <400> 19 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 20 <211> 108 <212> PRT <213> Mus musculus <400> 20 Asp Val Gln Met Ile Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Ile Val Thr Met Thr Cys Gln Ala Ser Gln Gly Thr Ser Ile Asn 20 25 30 Leu Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Ile Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Asp 65 70 75 80 Glu Asp Met Ala Thr Tyr Phe Cys Leu Gln His Ser Tyr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 <210> 21 <211> 153 <212> PRT <213> Homo sapiens <400> 21 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu 20 25 30 Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile 35 40 45 Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe 50 55 60 Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu 65 70 75 80 Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys 85 90 95 Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile 100 105 110 Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala 115 120 125 Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe 130 135 140 Cys Gln Ser Ile Ile Ser Thr Leu Thr 145 150 <210> 22 <211> 133 <212> PRT <213> artificial sequence <220> <223> synthetic substances <400> 22 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 23 <211> 133 <212> PRT <213> artificial sequence <220> <223> synthetic substances <400> 23 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Ala Met Leu Thr Ala Lys Phe Ala Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Ala Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 24 <211> 402 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Substance <400> 24 gcccctacca gcagcagcac caaaaagaca cagctgcaac tggaacacct cctgctggac 60 ctgcagatga tcctgaacgg catcaacaac tacaagaacc ccaagctgac cgccatgctg 120 acagccaagt tcgccatgcc taagaaggcc accgagctga agcacctcca gtgcctggaa 180 gaggccctga agcctctgga agaagtgctg aatctggccc agagcaagaa cttccacctg 240 aggcctaggg acctgatcag caacatcaac gtgatcgtgc tggaactgaa gggcagcgag 300 acaaccttca tgtgcgagta cgccgacgag acagctacca tcgtggaatt tctgaaccgg 360 tggatcacct tcagccagag catcatcagc accctgacct ga 402 <210> 25 <211> 159 <212> PRT <213> Homo sapiens <400> 25 Ser Ile Thr Gly Ile Ser Pro Ile Thr Glu Tyr Leu Ala Ser Leu Ser 1 5 10 15 Thr Tyr Asn Asp Gln Ser Ile Thr Phe Ala Leu Glu Asp Glu Ser Tyr 20 25 30 Glu Ile Tyr Val Glu Asp Leu Lys Lys Asp Glu Lys Lys Asp Lys Val 35 40 45 Leu Leu Ser Tyr Tyr Glu Ser Gln His Pro Ser Asn Glu Ser Gly Asp 50 55 60 Gly Val Asp Gly Lys Met Leu Met Val Thr Leu Ser Pro Thr Lys Asp 65 70 75 80 Phe Trp Leu His Ala Asn Asn Lys Glu His Ser Val Glu Leu His Lys 85 90 95 Cys Glu Lys Pro Leu Pro Asp Gln Ala Phe Phe Val Leu His Asn Met 100 105 110 His Ser Asn Cys Val Ser Phe Glu Cys Lys Thr Asp Pro Gly Val Phe 115 120 125 Ile Gly Val Lys Asp Asn His Leu Ala Leu Ile Lys Val Asp Ser Ser 130 135 140 Glu Asn Leu Cys Thr Glu Asn Ile Leu Phe Lys Leu Ser Glu Thr 145 150 155 <210> 26 <211> 477 <212> DNA <213> Homo sapiens <400> 26 agcatcaccg gcatcagccc catcacagag tatctggcca gcctgagcac ctacaacgac 60 cagagcatca cattcgccct ggaagatgag agctacgaga tctacgtgga agatctgaag 120 aaggacgaga agaaagacaa ggtgctgctg agctactacg agtctcagca ccccagcaat 180 gagtctggcg acggcgtgga cggaaagatg ctgatggtta cactgagccc caccaaggat 240 ttctggctgc acgccaacaa caaagagcac agcgtcgagc tgcacaagtg cgagaagcct 300 ctgcctgacc aggccttctt cgtgctgcac aacatgcaca gcaactgcgt gtccttcgag 360 tgcaagaccg atcctggcgt gttcatcggc gtgaaggaca accatctggc cctgatcaag 420 gtggacagca gcgagaatct gtgcaccgag aacatcctgt tcaagctgag cgagaca 477 <210> 27 <211> 158 <212> PRT <213> Mus musculus <400> 27 Ser Ile Gln Gly Thr Ser Leu Leu Thr Gln Ser Pro Ala Ser Leu Ser 1 5 10 15 Thr Tyr Asn Asp Gln Ser Val Ser Phe Val Leu Glu Asn Gly Cys Tyr 20 25 30 Val Ile Asn Val Asp Asp Ser Gly Lys Asp Gln Glu Gln Asp Gln Val 35 40 45 Leu Leu Arg Tyr Tyr Glu Ser Pro Cys Pro Ala Ser Gln Ser Gly Asp 50 55 60 Gly Val Asp Gly Lys Lys Leu Met Val Asn Met Ser Pro Ile Lys Asp 65 70 75 80 Thr Asp Ile Trp Leu His Ala Asn Asp Lys Asp Tyr Ser Val Glu Leu 85 90 95 Gln Arg Gly Asp Val Ser Pro Pro Glu Gln Ala Phe Phe Val Leu His 100 105 110 Lys Lys Ser Ser Asp Phe Val Ser Phe Glu Cys Lys Asn Leu Pro Gly 115 120 125 Thr Tyr Ile Gly Val Lys Asp Asn Gln Leu Ala Leu Val Glu Glu Lys 130 135 140 Asp Glu Ser Cys Asn Asn Ile Met Phe Lys Leu Ser Lys Ile 145 150 155 <210> 28 <211> 240 <212> PRT <213> Homo sapiens <400> 28 Met Glu Glu Ser Val Val Arg Pro Ser Val Phe Val Val Asp Gly Gln 1 5 10 15 Thr Asp Ile Pro Phe Thr Arg Leu Gly Arg Ser His Arg Arg Gln Ser 20 25 30 Cys Ser Val Ala Arg Val Gly Leu Gly Leu Leu Leu Leu Leu Met Gly 35 40 45 Ala Gly Leu Ala Val Gln Gly Trp Phe Leu Leu Gln Leu His Trp Arg 50 55 60 Leu Gly Glu Met Val Thr Arg Leu Pro Asp Gly Pro Ala Gly Ser Trp 65 70 75 80 Glu Gln Leu Ile Gln Glu Arg Arg Ser His Glu Val Asn Pro Ala Ala 85 90 95 His Leu Thr Gly Ala Asn Ser Ser Leu Thr Gly Ser Gly Gly Pro Leu 100 105 110 Leu Trp Glu Thr Gln Leu Gly Leu Ala Phe Leu Arg Gly Leu Ser Tyr 115 120 125 His Asp Gly Ala Leu Val Val Thr Lys Ala Gly Tyr Tyr Tyr Ile Tyr 130 135 140 Ser Lys Val Gln Leu Gly Gly Val Gly Cys Pro Leu Gly Leu Ala Ser 145 150 155 160 Thr Ile Thr His Gly Leu Tyr Lys Arg Thr Pro Arg Tyr Pro Glu Glu 165 170 175 Leu Glu Leu Leu Val Ser Gln Gln Ser Pro Cys Gly Arg Ala Thr Ser 180 185 190 Ser Ser Arg Val Trp Trp Asp Ser Ser Phe Leu Gly Gly Val Val His 195 200 205 Leu Glu Ala Gly Glu Lys Val Val Val Arg Val Leu Asp Glu Arg Leu 210 215 220 Val Arg Leu Arg Asp Gly Thr Arg Ser Tyr Phe Gly Ala Phe Met Val 225 230 235 240 <210> 29 <211> 167 <212> PRT <213> Homo sapiens <400> 29 Asp Gly Pro Ala Gly Ser Trp Glu Gln Leu Ile Gln Glu Arg Arg Ser 1 5 10 15 His Glu Val Asn Pro Ala Ala His Leu Thr Gly Ala Asn Ser Ser Leu 20 25 30 Thr Gly Ser Gly Gly Pro Leu Leu Trp Glu Thr Gln Leu Gly Leu Ala 35 40 45 Phe Leu Arg Gly Leu Ser Tyr His Asp Gly Ala Leu Val Val Thr Lys 50 55 60 Ala Gly Tyr Tyr Tyr Ile Tyr Ser Lys Val Gln Leu Gly Gly Val Gly 65 70 75 80 Cys Pro Leu Gly Leu Ala Ser Thr Ile Thr His Gly Leu Tyr Lys Arg 85 90 95 Thr Pro Arg Tyr Pro Glu Glu Leu Glu Leu Leu Val Ser Gln Gln Ser 100 105 110 Pro Cys Gly Arg Ala Thr Ser Ser Ser Arg Val Trp Trp Asp Ser Ser 115 120 125 Phe Leu Gly Gly Val Val His Leu Glu Ala Gly Glu Lys Val Val Val 130 135 140 Arg Val Leu Asp Glu Arg Leu Val Arg Leu Arg Asp Gly Thr Arg Ser 145 150 155 160 Tyr Phe Gly Ala Phe Met Val 165 <210> 30 <211> 501 <212> DNA <213> Homo sapiens <400> 30 gatggacctg ccggatcttg ggagcagctg atccaagagc ggagaagcca cgaagtgaac 60 cctgccgcac atctgacagg cgccaattct agcctgacag gctctggtgg acccctgctg 120 tgggaaactc aactgggact cgccttcctg agaggcctga gctatcatga tggcgccctg 180 gtggttacca aggccggcta ctactacatc tacagcaagg tgcagctcgg cggcgtggga 240 tgtcctcttg gactggcctc tacaatcacc cacggcctgt acaagcggac ccctagatac 300 cccgaggaac tggaactgct ggtgtcccag cagagccctt gtggcagagc cacaagcagc 360 agcagagttt ggtgggacag cagctttctc ggcggagtgg tgcatctgga agccggcgaa 420 aaggtggtcg tcagagtgct ggatgagaga ctcgtgcggc tgagagatgg caccagaagc 480 tacttcggcg ccttcatggt t 501 <210> 31 <211> 168 <212> PRT <213> Mus musculus <400> 31 Asp Gly Gly Lys Gly Ser Trp Glu Lys Leu Ile Gln Asp Gln Arg Ser 1 5 10 15 His Gln Ala Asn Pro Ala Ala His Leu Thr Gly Ala Asn Ala Ser Leu 20 25 30 Ile Gly Ile Gly Gly Pro Leu Leu Trp Glu Thr Arg Leu Gly Leu Ala 35 40 45 Phe Leu Arg Gly Leu Thr Tyr His Asp Gly Ala Leu Val Thr Met Glu 50 55 60 Pro Gly Tyr Tyr Tyr Val Tyr Ser Lys Val Gln Leu Ser Gly Val Gly 65 70 75 80 Cys Pro Gln Gly Leu Ala Asn Gly Leu Pro Ile Thr His Gly Leu Tyr 85 90 95 Lys Arg Thr Ser Arg Tyr Pro Lys Glu Leu Glu Leu Leu Val Ser Arg 100 105 110 Arg Ser Pro Cys Gly Arg Ala Asn Ser Ser Arg Val Trp Trp Asp Ser 115 120 125 Ser Phe Leu Gly Gly Val Val His Leu Glu Ala Gly Glu Glu Val Val 130 135 140 Val Arg Val Pro Gly Asn Arg Leu Val Arg Pro Arg Asp Gly Thr Arg 145 150 155 160 Ser Tyr Phe Gly Ala Phe Met Val 165 <210> 32 <211> 261 <212> PRT <213> Homo sapiens <400> 32 Met Ile Glu Thr Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly 1 5 10 15 Leu Pro Ile Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu 20 25 30 Ile Thr Gln Met Ile Gly Ser Ala Leu Phe Ala Val Tyr Leu His Arg 35 40 45 Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp Phe Val 50 55 60 Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser Leu Ser 65 70 75 80 Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe Val Lys 85 90 95 Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser Phe Glu 100 105 110 Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val Ile Ser 115 120 125 Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly 130 135 140 Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln 145 150 155 160 Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr 165 170 175 Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser 180 185 190 Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala 195 200 205 Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His 210 215 220 Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn 225 230 235 240 Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe 245 250 255 Gly Leu Leu Lys Leu 260 <210> 33 <211> 149 <212> PRT <213> Mus musculus <400> 33 Met Gln Arg Gly Asp Glu Asp Pro Gln Ile Ala Ala His Val Val Ser 1 5 10 15 Glu Ala Asn Ser Asn Ala Ala Ser Val Leu Gln Trp Ala Lys Lys Gly 20 25 30 Tyr Tyr Thr Met Lys Ser Asn Leu Val Met Leu Glu Asn Gly Lys Gln 35 40 45 Leu Thr Val Lys Arg Glu Gly Leu Tyr Tyr Val Tyr Thr Gln Val Thr 50 55 60 Phe Cys Ser Asn Arg Glu Pro Ser Ser Gln Arg Pro Phe Ile Val Gly 65 70 75 80 Leu Trp Leu Lys Pro Ser Ser Gly Ser Glu Arg Ile Leu Leu Lys Ala 85 90 95 Ala Asn Thr His Ser Ser Ser Gln Leu Cys Glu Gln Gln Ser Val His 100 105 110 Leu Gly Gly Val Phe Glu Leu Gln Ala Gly Ala Ser Val Phe Val Asn 115 120 125 Val Thr Glu Ala Ser Gln Val Ile His Arg Val Gly Phe Ser Ser Phe 130 135 140 Gly Leu Leu Lys Leu 145 <210> 34 <211> 149 <212> PRT <213> Homo sapiens <400> 34 Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val Ile Ser 1 5 10 15 Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly 20 25 30 Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln 35 40 45 Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr 50 55 60 Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser 65 70 75 80 Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala 85 90 95 Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His 100 105 110 Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn 115 120 125 Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe 130 135 140 Gly Leu Leu Lys Leu 145 <210> 35 <211> 447 <212> DNA <213> Homo sapiens <400> 35 atgcagaagg gcgaccagaa tcctcagatc gccgctcacg tgatcagcga ggccagcagc 60 aagacaacaa gcgtgctgca gtgggccgag aagggctact acaccatgag caacaacctg 120 gtcaccctgg aaaacggcaa gcagctgacc gtgaagagac agggcctgta ctacatctac 180 gcccaagtga ccttctgcag caacagagag gccagctctc aggccccttt tatcgccagc 240 ctgtgcctga agtcccctgg cagattcgag cggattctgc tgagagccgc caacacacac 300 agcagcgcca aaccttgtgg ccagcagtct attcacctcg gcggagtgtt tgagctgcag 360 cctggcgcaa gcgtgttcgt gaatgtgaca gaccctagcc aggtgtccca cggcaccggc 420 tttacatctt tcggcctgct gaagctg 447 <210> 36 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Substance <400> 36 Met Gly Thr Asn Lys Cys Leu Leu Gln Ile Ala Leu Leu Leu Cys Phe 1 5 10 15 Ser Thr Thr Ala Leu Ser Arg Met Lys Gln Ile Glu Asp Lys Ile Glu 20 25 30 Glu Ile Leu Ser Lys Ile Tyr His Ile Glu Asn Glu Ile Ala Arg Ile 35 40 45 Lys Lys Leu Ile Gly Glu Val Gly Gly Gly Ser Gly Gly Gly Ser Gly 50 55 60 Gly Gly Ser Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His 65 70 75 80 Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala 85 90 95 Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn 100 105 110 Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala 115 120 125 Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe 130 135 140 Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu 145 150 155 160 Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln 165 170 175 Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val 180 185 190 Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe 195 200 205 Thr Ser Phe Gly Leu Leu Lys Leu 210 215 <210> 37 <211> 651 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Substance <400> 37 atgggaacaa acaaatgcct gctgcagatc gccctgctgc tgtgctttag cacaacagcc 60 ctgagccgga tgaagcagat cgaggacaag atcgaagaga tcctgagcaa gatctaccac 120 atcgagaacg agatcgcccg gatcaagaaa ctgatcggcg aagtcggcgg aggctctggt 180 ggtggatctg gcggaggatc tatgcagaaa ggcgaccaga atcctcagat cgccgctcac 240 gtgatcagcg aggccagcag caagacaaca agcgtgctgc agtgggccga gaagggctac 300 tacaccatga gcaacaacct ggtcaccctg gaaaacggca agcagctgac cgtgaagaga 360 cagggcctgt actacatcta cgcccaagtg accttctgca gcaacagaga ggccagctct 420 caggcccctt ttatcgccag cctgtgcctg aagtcccctg gcagattcga gcggattctg 480 ctgagagccg ccaacacaca cagcagcgcc aaaccttgtg gccagcagtc tattcacctc 540 ggcggagtgt ttgagctgca gcctggcgca agcgtgttcg tgaatgtgac agaccctagc 600 caggtgtccc acggcaccgg ctttacatct ttcggcctgc tgaagctctg a 651 <210> 38 <211> 261 <212> PRT <213> Homo sapiens <400> 38 Met Ile Glu Thr Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly 1 5 10 15 Leu Pro Ile Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu 20 25 30 Ile Thr Gln Met Ile Gly Ser Ala Leu Phe Ala Val Tyr Leu His Arg 35 40 45 Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp Phe Val 50 55 60 Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser Leu Ser 65 70 75 80 Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe Val Lys 85 90 95 Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser Phe Glu 100 105 110 Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val Ile Ser 115 120 125 Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly 130 135 140 Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln 145 150 155 160 Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr 165 170 175 Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser 180 185 190 Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala 195 200 205 Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His 210 215 220 Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn 225 230 235 240 Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe 245 250 255 Gly Leu Leu Lys Leu 260 <210> 39 <211> 182 <212> PRT <213> Mus musculus <400> 39 Met Asn Asn Arg Trp Ile Leu His Ala Ala Phe Leu Leu Cys Phe Ser 1 5 10 15 Thr Thr Ala Leu Ser Ile Asn Tyr Lys Gln Leu Gln Leu Gln Glu Arg 20 25 30 Thr Asn Ile Arg Lys Cys Gln Glu Leu Leu Glu Gln Leu Asn Gly Lys 35 40 45 Ile Asn Leu Thr Tyr Arg Ala Asp Phe Lys Ile Pro Met Glu Met Thr 50 55 60 Glu Lys Met Gln Lys Ser Tyr Thr Ala Phe Ala Ile Gln Glu Met Leu 65 70 75 80 Gln Asn Val Phe Leu Val Phe Arg Asn Asn Phe Ser Ser Thr Gly Trp 85 90 95 Asn Glu Thr Ile Val Val Arg Leu Leu Asp Glu Leu His Gln Gln Thr 100 105 110 Val Phe Leu Lys Thr Val Leu Glu Glu Lys Gln Glu Glu Arg Leu Thr 115 120 125 Trp Glu Met Ser Ser Thr Ala Leu His Leu Lys Ser Tyr Tyr Trp Arg 130 135 140 Val Gln Arg Tyr Leu Lys Leu Met Lys Tyr Asn Ser Tyr Ala Trp Met 145 150 155 160 Val Val Arg Ala Glu Ile Phe Arg Asn Phe Leu Ile Ile Arg Arg Leu 165 170 175 Thr Arg Asn Phe Gln Asn 180 <210> 40 <211> 357 <212> PRT <213> Homo sapiens <400> 40 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly 20 25 30 Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe 35 40 45 Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala 50 55 60 Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu 65 70 75 80 Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile 85 90 95 Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu 100 105 110 Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala 115 120 125 Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu 130 135 140 Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr 145 150 155 160 Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys 165 170 175 Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly 180 185 190 Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu 195 200 205 Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys 210 215 220 Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu 225 230 235 240 Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met 245 250 255 Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala 260 265 270 His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val 275 280 285 Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His 290 295 300 Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro 305 310 315 320 Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala 325 330 335 Thr Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly 340 345 350 Ile Gly Leu Phe Met 355 <210> 41 <211> 1074 <212> DNA <213> Homo sapiens <400> 41 atgcttctgc tggttacatc tctgctgctg tgcgagctgc cccatcctgc ctttctgctg 60 atccccagaa aagtgtgcaa cggcatcggc atcggagagt tcaaggacag cctgagcatc 120 aacgccacca acatcaagca cttcaagaac tgcaccagca tcagcggcga cctgcacatt 180 ctgcctgtgg cctttagagg cgacagcttc acccacacac ctccactcga tccccaagag 240 ctggacatcc tgaaaaccgt gaaagagatc accggatttc tgttgatcca ggcttggccc 300 gagaaccgga cagatctgca cgccttcgag aacctggaaa tcatcagagg ccggaccaag 360 cagcacggcc agttttctct ggctgtggtg tccctgaaca tcaccagcct gggcctgaga 420 agcctgaaag aaatcagcga cggcgacgtg atcatctccg gcaacaagaa cctgtgctac 480 gccaacacca tcaactggaa gaagctgttc ggcaccagcg gccagaaaac aaagatcatc 540 agcaaccggg gcgagaacag ctgcaaggct acaggccaag tgtgccacgc tctgtgtagc 600 cctgaaggct gttggggacc cgagcctaga gattgcgtgt cctgcagaaa cgtgtcccgg 660 ggcagagaat gcgtggacaa gtgcaatctg ctggaaggcg agccccgcga gttcgtggaa 720 aacagcgagt gcatccagtg tcaccccgag tgtctgcccc aggccatgaa cattacctgt 780 accggcagag gccccgacaa ctgtattcag tgcgcccact acatcgacgg ccctcactgc 840 gtgaaaacat gtcctgctgg cgtgatggga gagaacaaca ccctcgtgtg gaagtatgcc 900 gacgccggac atgtgtgcca cctgtgtcac cctaattgca cctacggctg tacaggccct 960 ggcctggaag gctgtccaac aaacggacct aagatcccct ctatcgccac cggcatggtt 1020 ggagccctgc tgctgcttct ggttgtggcc cttggcatcg gcctgtttat gtag 1074 <210> 42 <211> 752 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Substance <400> 42 Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser Pro Ala Leu 1 5 10 15 Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys Ser Phe Ser 20 25 30 Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Met Ser Pro Ser 35 40 45 Asn Gln Asp Asp Arg Leu Ala Ala Phe Pro Glu Asp Arg Ser Gln Pro 50 55 60 Gly Gln Asp Ala Arg Phe Arg Val Thr Gln Leu Pro Asn Gly Arg Asp 65 70 75 80 Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser Gly Thr Tyr 85 90 95 Phe Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Lys Ile Lys Glu Ser 100 105 110 Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val Pro Thr 115 120 125 Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln Phe Gln Thr Pro 130 135 140 Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 145 150 155 160 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 165 170 175 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 180 185 190 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 195 200 205 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 210 215 220 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 225 230 235 240 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 245 250 255 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 260 265 270 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 275 280 285 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 290 295 300 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 305 310 315 320 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 325 330 335 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 340 345 350 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Ala 355 360 365 Ser Arg Lys Arg Arg Ser Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu 370 375 380 Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Leu Leu Leu Val 385 390 395 400 Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe Leu Leu Ile 405 410 415 Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser 420 425 430 Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser 435 440 445 Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser 450 455 460 Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys 465 470 475 480 Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu 485 490 495 Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly 500 505 510 Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn 515 520 525 Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp 530 535 540 Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn 545 550 555 560 Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser 565 570 575 Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala 580 585 590 Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val 595 600 605 Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn 610 615 620 Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile 625 630 635 640 Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr 645 650 655 Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly 660 665 670 Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn 675 680 685 Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys 690 695 700 His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys 705 710 715 720 Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly 725 730 735 Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met 740 745 750 <210> 43 <211> 2259 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Substance <400> 43 gacagccctg acagaccctg gaatcctcca acattcagcc ccgctctgct ggtggttacc 60 gagggcgata atgccacctt cacctgtagc ttcagcaaca ccagcgagag cttcgtgctg 120 aactggtaca gaatgagccc cagcaaccag gacgacagac tggccgcctt tcctgaggat 180 agatctcagc ccggccagga cgcccggttc agagttacac agctgcccaa cggccgggac 240 ttccacatgt ctgtcgtccg ggccagaaga aacgacagcg gcacatattt ttgcggcgcc 300 atttctctgg cccctaaggc taaaatcaaa gagagcctga gagccgagct gagagtgaca 360 gaaagacggg ccgaagtgcc cacagctcac ccttcacctt ctccaagacc tgccggccag 420 tttcagactc ctccttgtcc tagctgtcct gctcctgagt ttctcggcgg accctccgtg 480 ttcctgtttc ctccaaagcc taggcacc ctgatgatca gcagaacccc tgaagtgacc 540 tgcgtggtgg tggacgtgtc ccaagaggac cctgaggtgc agttcaattg gtacgtggac 600 ggcgtggaag tgcacaacgc caagaccaag cctagagagg aacagttcaa cagcacctac 660 agagtggtgt ccgtgctgac cgtgctgcac caggattggc tgaacggcaa agatcaag 720 780 ggccagccaa gagaacccca ggtgtacaca ctgcctccaa gccaagagga aatgaccaag 840 aaccaggtgt ccctgacctg cctggtcaag ggcttctacc cttccgatat cgccgtggaa 900 tgggagagca atggccagcc tgagaacaac tacaagacca cacctcctgt gctggacagc 960 gacggctcat tcttcctgta cagcagactg accgtggaca agagcagatg gcaagagggc 1020 aacgtgttca gctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagtcc 1080 ctgagcctgt ctctgggcaa agccagccgg aagagaagat ctggatctgg cgaaggcaga 1140 ggctccctgc tgacttgcgg agatgtggaa gagaaccccg gacctatgct tctgctggtt 1200 acatctctgc tgctgtgcga gctgccccat cctgcctttc tgctgatccc cagaaaagtg 1260 tgcaacggca tcggcatcgg agagttcaag gacagcctga gcatcaacgc caccaacatc 1320 aagcacttca agaactgcac cagcatcagc ggcgacctgc acattctgcc tgtggccttt 1380 agaggcgaca gcttcaccca cacacctcca ctcgatcccc aagagctgga catcctgaaa 1440 accgtgaaag agatcaccgg atttctgttg atccaggctt ggccccgagaa ccggacagat 1500 ctgcacgcct tcgagaacct ggaaatcatc agaggccgga ccaagcagca cggccagttt 1560 tctctggctg tggtgtccct gaacatcacc agcctgggcc tgagaagcct gaagaaatc 1620 agcgacggcg acgtgatcat ctccggcaac aagaacctgt gctacgccaa caccatcaac 1680 tggagaagc tgttcggcac cagcggccag aaaacaaaga tcatcagcaa ccggggcgag 1740 aacagctca aggctacagg ccaagtgtgc cacgctctgt gtagccctga aggctgttgg 1800 ggacccgagc ctagagattg cgtgtcctgc agaaacgtgt cccggggcag agaatgcgtg 1860 gacaagtgca atctgctgga aggcgagccc cgcgagttcg tggaaaacag cgagtgcatc 1920 cagtgtcacc ccgagtgtct gccccaggcc atgaacatta cctgtaccgg cagaggcccc 1980 gacaactgta ttcagtgcgc ccactacatc gacggccctc actgcgtgaa aacatgtcct 2040 gctggcgtga tgggagagaa caacaccctc gtgtggaagt atgccgacgc cggacatgtg 2100 tgccacctgt gtcaccctaa ttgcacctac ggctgtacag gccctggcct ggaaggctgt 2160 ccaacaaacg gacctaagat cccctctatc gccaccggca tggttggagc cctgctgctg 2220 cttctggttg tggcccttgg catcggcctg tttatgtag 2259 <210> 44 <211> 732 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Substance <400> 44 Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser Pro Ala Leu 1 5 10 15 Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys Ser Phe Ser 20 25 30 Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Met Ser Pro Ser 35 40 45 Asn Gln Val Asp Arg Leu Ala Ala Phe Pro Glu Asp Arg Ser Gln Pro 50 55 60 Gly Gln Asp Ala Arg Phe Arg Val Thr Gln Leu Pro Asn Gly Arg Asp 65 70 75 80 Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser Gly Thr Tyr 85 90 95 Phe Cys Gly Ala Ile Ser Leu Ala Pro Lys Trp Lys Ile Lys Glu Ser 100 105 110 Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Pro Pro Cys Pro Ser 115 120 125 Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 130 135 140 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 145 150 155 160 Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn 165 170 175 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 180 185 190 Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 195 200 205 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 210 215 220 Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys 225 230 235 240 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu 245 250 255 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 260 265 270 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 275 280 285 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 290 295 300 Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly 305 310 315 320 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 325 330 335 Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Ala Ser Arg Lys Arg 340 345 350 Arg Ser Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp 355 360 365 Val Glu Glu Asn Pro Gly Pro Met Leu Leu Leu Val Thr Ser Leu Leu 370 375 380 Leu Cys Glu Leu Pro His Pro Ala Phe Leu Leu Ile Pro Arg Lys Val 385 390 395 400 Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn 405 410 415 Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp 420 425 430 Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr 435 440 445 Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu 450 455 460 Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp 465 470 475 480 Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln 485 490 495 His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu 500 505 510 Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser 515 520 525 Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu 530 535 540 Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu 545 550 555 560 Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro 565 570 575 Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn 580 585 590 Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly 595 600 605 Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro 610 615 620 Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro 625 630 635 640 Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val 645 650 655 Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp 660 665 670 Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys 675 680 685 Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly 690 695 700 Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu 705 710 715 720 Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met 725 730 <210> 45 <211> 675 <212> PRT <213> Homo sapiens <400> 45 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Thr Gln Val Cys Thr Gly Thr Asp Met Lys 20 25 30 Leu Arg Leu Pro Ala Ser Pro Glu Thr His Leu Asp Met Leu Arg His 35 40 45 Leu Tyr Gln Gly Cys Gln Val Val Gln Gly Asn Leu Glu Leu Thr Tyr 50 55 60 Leu Pro Thr Asn Ala Ser Leu Ser Phe Leu Gln Asp Ile Gln Glu Val 65 70 75 80 Gln Gly Tyr Val Leu Ile Ala His Asn Gln Val Arg Gln Val Pro Leu 85 90 95 Gln Arg Leu Arg Ile Val Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr 100 105 110 Ala Leu Ala Val Leu Asp Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro 115 120 125 Val Thr Gly Ala Ser Pro Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser 130 135 140 Leu Thr Glu Ile Leu Lys Gly Gly Val Leu Ile Gln Arg Asn Pro Gln 145 150 155 160 Leu Cys Tyr Gln Asp Thr Ile Leu Trp Lys Asp Ile Phe His Lys Asn 165 170 175 Asn Gln Leu Ala Leu Thr Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys 180 185 190 His Pro Cys Ser Pro Met Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser 195 200 205 Ser Glu Asp Cys Gln Ser Leu Thr Arg Thr Val Cys Ala Gly Gly Cys 210 215 220 Ala Arg Cys Lys Gly Pro Leu Pro Thr Asp Cys Cys His Glu Gln Cys 225 230 235 240 Ala Ala Gly Cys Thr Gly Pro Lys His Ser Asp Cys Leu Ala Cys Leu 245 250 255 His Phe Asn His Ser Gly Ile Cys Glu Leu His Cys Pro Ala Leu Val 260 265 270 Thr Tyr Asn Thr Asp Thr Phe Glu Ser Met Pro Asn Pro Glu Gly Arg 275 280 285 Tyr Thr Phe Gly Ala Ser Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu 290 295 300 Ser Thr Asp Val Gly Ser Cys Thr Leu Val Cys Pro Leu His Asn Gln 305 310 315 320 Glu Val Thr Ala Glu Asp Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys 325 330 335 Pro Cys Ala Arg Val Cys Tyr Gly Leu Gly Met Glu His Leu Arg Glu 340 345 350 Val Arg Ala Val Thr Ser Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys 355 360 365 Lys Ile Phe Gly Ser Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp 370 375 380 Pro Ala Ser Asn Thr Ala Pro Leu Gln Pro Glu Gln Leu Gln Val Phe 385 390 395 400 Glu Thr Leu Glu Glu Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro 405 410 415 Asp Ser Leu Pro Asp Leu Ser Val Phe Gln Asn Leu Gln Val Ile Arg 420 425 430 Gly Arg Ile Leu His Asn Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu 435 440 445 Gly Ile Ser Trp Leu Gly Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly 450 455 460 Leu Ala Leu Ile His His Asn Thr His Leu Cys Phe Val His Thr Val 465 470 475 480 Pro Trp Asp Gln Leu Phe Arg Asn Pro His Gln Ala Leu Leu His Thr 485 490 495 Ala Asn Arg Pro Glu Asp Glu Cys Val Gly Glu Gly Leu Ala Cys His 500 505 510 Gln Leu Cys Ala Arg Gly His Cys Trp Gly Pro Gly Pro Thr Gln Cys 515 520 525 Val Asn Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys Val Glu Glu Cys 530 535 540 Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn Ala Arg His Cys 545 550 555 560 Leu Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly Ser Val Thr Cys 565 570 575 Phe Gly Pro Glu Ala Asp Gln Cys Val Ala Cys Ala His Tyr Lys Asp 580 585 590 Pro Pro Phe Cys Val Ala Arg Cys Pro Ser Gly Val Lys Pro Asp Leu 595 600 605 Ser Tyr Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln 610 615 620 Pro Cys Pro Ile Asn Cys Thr His Ser Cys Val Asp Leu Asp Asp Lys 625 630 635 640 Gly Cys Pro Ala Glu Gln Arg Ala Ser Pro Leu Thr Ser Ile Ile Ser 645 650 655 Ala Val Val Gly Ile Leu Leu Val Val Val Leu Gly Val Val Phe Gly 660 665 670 Ile Leu Ile 675 <210> 46 <211> 2028 <212> DNA <213> Homo sapiens <400> 46 atgcttctgc tggttacatc tctgctgctg tgcgagctgc cccatcctgc ctttctgctg 60 atccccacac aagtgtgcac cggcaccgac atgaagctga gactgcctgc ctctcctgag 120 acacacctgg acatgctgag acacctgtac cagggctgtc aggtggtgca gggcaatctg 180 gaactgacct acctgcctac caacgccagc ctgagctttc tgcaggacat ccaagaggtg 240 cagggatacg tgctgattgc ccacaatcaa gtgcgccagg tgccactgca gcggctgaga 300 atcgttagag gcacccagct gttcgaggac aactatgccc tggccgtgct ggacaatggc 360 gaccctctga acaacaccac acctgtgaca ggcgcttctc ctggcggact gagagaactg 420 cagctgagaa gcctgaccga gatcctgaaa ggcggcgtgc tgatccagag aaaccctcag 480 ctgtgctacc aggacaccat cctgtggaag gacatcttcc acaagaacaa ccagctggcc 540 ctgacactga tcgacaccaa cagaagcaga gcctgccatc cttgcagccc catgtgcaag 600 ggctctagat gttggggcga gagcagcgag gattgccaga gcctgaccag aacagtgtgt 660 gccggcggat gtgccagatg caaaggacct ctgcctaccg actgctgcca cgagcaatgt 720 gccgctggat gtacaggccc caagcactct gattgcctgg cctgcctgca cttcaaccac 780 tctggaatct gcgaactgca ctgccccgct ctggtcacct aaacaccga taccttcag 840 agcatgccca atcctgaggg cagatacacc ttcggcgcca gctgtgtgac agcctgtcct 900 tacaactacc tgagcaccga cgtgggcagc tgtacccttg tgtgccctct gcataatcaa 960 1020 gtgtgttacg gcctcggcat ggaacacctg agaaagtgc gggccgtgac cagcgccaat 1080 atccaagaat ttgccggctg caagaagatc tttggcagcc tcgcttcct gcctgagagc 1140 ttcgatggcg atcctgccag caatactgcc cctctgcagc ctgaacagct ccaggtgttc 1200 gagacactgg aagagatcac cggctacctg tatatcagcg cctggccaga cagcctgcct 1260 gacctgtccg tgttccagaa cctgcaagtg atccggggca gaatcctgca caacggggcc 1320 tattctctga ccctgcaagg cctgggaatc agctggctgg gactgagatc cctgagagag 1380 cttggatctg gcctggctct gatccaccac aatacccacc tgtgcttcgt gcacaccgtg 1440 ccttgggacc agctgtttcg gaatcctcat caggccctgc tgcacaccgc caacagacct 1500 gaggatgagt gtgttggcga aggcctggct tgtcaccagc tctgtgctag aggacactgt 1560 tggggccctg gacctacaca gtgcgtgaac tgtagccagt tcctgcgggg ccaagagtgc 1620 gtggaagagt gtagagttct gcagggactg ccccgggaat acgtgaacgc cagacactgt 1680 ctgccttgtc accctgagtg ccagcctcag aatggcagcg tgacctgttt tggccctgag 1740 gccgatcagt gcgtggcctg tgctcactac aaggaccctc cattctgcgt ggccagatgt 1800 cctagcggcg tgaagcctga tctgagctac atgcccatct ggaagttccc cgatgaggaa 1860 ggcgcttgcc agccttgtcc tatcaactgc acccacagct gcgtggacct ggacgataag 1920 ggatgtccag ccgagcagag agcctctcca ctgacctcta tcatctctgc cgtcgtgggc 1980 atcctgctgg tggtggttct gggcgttgtg ttcggcatcc tgatttga 2028 <210> 47 <211> 1070 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Substance <400> 47 Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser Pro Ala Leu 1 5 10 15 Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys Ser Phe Ser 20 25 30 Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Met Ser Pro Ser 35 40 45 Asn Gln Asp Asp Arg Leu Ala Ala Phe Pro Glu Asp Arg Ser Gln Pro 50 55 60 Gly Gln Asp Ala Arg Phe Arg Val Thr Gln Leu Pro Asn Gly Arg Asp 65 70 75 80 Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser Gly Thr Tyr 85 90 95 Phe Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Lys Ile Lys Glu Ser 100 105 110 Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val Pro Thr 115 120 125 Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln Phe Gln Thr Pro 130 135 140 Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 145 150 155 160 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 165 170 175 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 180 185 190 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 195 200 205 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 210 215 220 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 225 230 235 240 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 245 250 255 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 260 265 270 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 275 280 285 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 290 295 300 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 305 310 315 320 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 325 330 335 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 340 345 350 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Ala 355 360 365 Ser Arg Lys Arg Arg Ser Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu 370 375 380 Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Leu Leu Leu Val 385 390 395 400 Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe Leu Leu Ile 405 410 415 Pro Thr Gln Val Cys Thr Gly Thr Asp Met Lys Leu Arg Leu Pro Ala 420 425 430 Ser Pro Glu Thr His Leu Asp Met Leu Arg His Leu Tyr Gln Gly Cys 435 440 445 Gln Val Val Gln Gly Asn Leu Glu Leu Thr Tyr Leu Pro Thr Asn Ala 450 455 460 Ser Leu Ser Phe Leu Gln Asp Ile Gln Glu Val Gln Gly Tyr Val Leu 465 470 475 480 Ile Ala His Asn Gln Val Arg Gln Val Pro Leu Gln Arg Leu Arg Ile 485 490 495 Val Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr Ala Leu Ala Val Leu 500 505 510 Asp Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro Val Thr Gly Ala Ser 515 520 525 Pro Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser Leu Thr Glu Ile Leu 530 535 540 Lys Gly Gly Val Leu Ile Gln Arg Asn Pro Gln Leu Cys Tyr Gln Asp 545 550 555 560 Thr Ile Leu Trp Lys Asp Ile Phe His Lys Asn Asn Gln Leu Ala Leu 565 570 575 Thr Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys His Pro Cys Ser Pro 580 585 590 Met Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser Ser Glu Asp Cys Gln 595 600 605 Ser Leu Thr Arg Thr Val Cys Ala Gly Gly Cys Ala Arg Cys Lys Gly 610 615 620 Pro Leu Pro Thr Asp Cys Cys His Glu Gln Cys Ala Ala Gly Cys Thr 625 630 635 640 Gly Pro Lys His Ser Asp Cys Leu Ala Cys Leu His Phe Asn His Ser 645 650 655 Gly Ile Cys Glu Leu His Cys Pro Ala Leu Val Thr Tyr Asn Thr Asp 660 665 670 Thr Phe Glu Ser Met Pro Asn Pro Glu Gly Arg Tyr Thr Phe Gly Ala 675 680 685 Ser Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu Ser Thr Asp Val Gly 690 695 700 Ser Cys Thr Leu Val Cys Pro Leu His Asn Gln Glu Val Thr Ala Glu 705 710 715 720 Asp Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys Pro Cys Ala Arg Val 725 730 735 Cys Tyr Gly Leu Gly Met Glu His Leu Arg Glu Val Arg Ala Val Thr 740 745 750 Ser Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys Lys Ile Phe Gly Ser 755 760 765 Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp Pro Ala Ser Asn Thr 770 775 780 Ala Pro Leu Gln Pro Glu Gln Leu Gln Val Phe Glu Thr Leu Glu Glu 785 790 795 800 Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro Asp Ser Leu Pro Asp 805 810 815 Leu Ser Val Phe Gln Asn Leu Gln Val Ile Arg Gly Arg Ile Leu His 820 825 830 Asn Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu Gly Ile Ser Trp Leu 835 840 845 Gly Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly Leu Ala Leu Ile His 850 855 860 His Asn Thr His Leu Cys Phe Val His Thr Val Pro Trp Asp Gln Leu 865 870 875 880 Phe Arg Asn Pro His Gln Ala Leu Leu His Thr Ala Asn Arg Pro Glu 885 890 895 Asp Glu Cys Val Gly Glu Gly Leu Ala Cys His Gln Leu Cys Ala Arg 900 905 910 Gly His Cys Trp Gly Pro Gly Pro Thr Gln Cys Val Asn Cys Ser Gln 915 920 925 Phe Leu Arg Gly Gln Glu Cys Val Glu Glu Cys Arg Val Leu Gln Gly 930 935 940 Leu Pro Arg Glu Tyr Val Asn Ala Arg His Cys Leu Pro Cys His Pro 945 950 955 960 Glu Cys Gln Pro Gln Asn Gly Ser Val Thr Cys Phe Gly Pro Glu Ala 965 970 975 Asp Gln Cys Val Ala Cys Ala His Tyr Lys Asp Pro Pro Phe Cys Val 980 985 990 Ala Arg Cys Pro Ser Gly Val Lys Pro Asp Leu Ser Tyr Met Pro Ile 995 1000 1005 Trp Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln Pro Cys Pro Ile 1010 1015 1020 Asn Cys Thr His Ser Cys Val Asp Leu Asp Asp Lys Gly Cys Pro 1025 1030 1035 Ala Glu Gln Arg Ala Ser Pro Leu Thr Ser Ile Ile Ser Ala Val 1040 1045 1050 Val Gly Ile Leu Leu Val Val Val Leu Gly Val Val Phe Gly Ile 1055 1060 1065 Leu Ile 1070 <210> 48 <211> 1050 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Substance <400> 48 Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser Pro Ala Leu 1 5 10 15 Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys Ser Phe Ser 20 25 30 Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Met Ser Pro Ser 35 40 45 Asn Gln Val Asp Arg Leu Ala Ala Phe Pro Glu Asp Arg Ser Gln Pro 50 55 60 Gly Gln Asp Ala Arg Phe Arg Val Thr Gln Leu Pro Asn Gly Arg Asp 65 70 75 80 Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser Gly Thr Tyr 85 90 95 Phe Cys Gly Ala Ile Ser Leu Ala Pro Lys Trp Lys Ile Lys Glu Ser 100 105 110 Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Pro Pro Cys Pro Ser 115 120 125 Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 130 135 140 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 145 150 155 160 Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn 165 170 175 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 180 185 190 Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 195 200 205 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 210 215 220 Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys 225 230 235 240 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu 245 250 255 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 260 265 270 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 275 280 285 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 290 295 300 Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly 305 310 315 320 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 325 330 335 Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Ala Ser Arg Lys Arg 340 345 350 Arg Ser Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp 355 360 365 Val Glu Glu Asn Pro Gly Pro Met Leu Leu Leu Val Thr Ser Leu Leu 370 375 380 Leu Cys Glu Leu Pro His Pro Ala Phe Leu Leu Ile Pro Thr Gln Val 385 390 395 400 Cys Thr Gly Thr Asp Met Lys Leu Arg Leu Pro Ala Ser Pro Glu Thr 405 410 415 His Leu Asp Met Leu Arg His Leu Tyr Gln Gly Cys Gln Val Val Gln 420 425 430 Gly Asn Leu Glu Leu Thr Tyr Leu Pro Thr Asn Ala Ser Leu Ser Phe 435 440 445 Leu Gln Asp Ile Gln Glu Val Gln Gly Tyr Val Leu Ile Ala His Asn 450 455 460 Gln Val Arg Gln Val Pro Leu Gln Arg Leu Arg Ile Val Arg Gly Thr 465 470 475 480 Gln Leu Phe Glu Asp Asn Tyr Ala Leu Ala Val Leu Asp Asn Gly Asp 485 490 495 Pro Leu Asn Asn Thr Thr Pro Val Thr Gly Ala Ser Pro Gly Gly Leu 500 505 510 Arg Glu Leu Gln Leu Arg Ser Leu Thr Glu Ile Leu Lys Gly Gly Val 515 520 525 Leu Ile Gln Arg Asn Pro Gln Leu Cys Tyr Gln Asp Thr Ile Leu Trp 530 535 540 Lys Asp Ile Phe His Lys Asn Asn Gln Leu Ala Leu Thr Leu Ile Asp 545 550 555 560 Thr Asn Arg Ser Arg Ala Cys His Pro Cys Ser Pro Met Cys Lys Gly 565 570 575 Ser Arg Cys Trp Gly Glu Ser Ser Glu Asp Cys Gln Ser Leu Thr Arg 580 585 590 Thr Val Cys Ala Gly Gly Cys Ala Arg Cys Lys Gly Pro Leu Pro Thr 595 600 605 Asp Cys Cys His Glu Gln Cys Ala Ala Gly Cys Thr Gly Pro Lys His 610 615 620 Ser Asp Cys Leu Ala Cys Leu His Phe Asn His Ser Gly Ile Cys Glu 625 630 635 640 Leu His Cys Pro Ala Leu Val Thr Tyr Asn Thr Asp Thr Phe Glu Ser 645 650 655 Met Pro Asn Pro Glu Gly Arg Tyr Thr Phe Gly Ala Ser Cys Val Thr 660 665 670 Ala Cys Pro Tyr Asn Tyr Leu Ser Thr Asp Val Gly Ser Cys Thr Leu 675 680 685 Val Cys Pro Leu His Asn Gln Glu Val Thr Ala Glu Asp Gly Thr Gln 690 695 700 Arg Cys Glu Lys Cys Ser Lys Pro Cys Ala Arg Val Cys Tyr Gly Leu 705 710 715 720 Gly Met Glu His Leu Arg Glu Val Arg Ala Val Thr Ser Ala Asn Ile 725 730 735 Gln Glu Phe Ala Gly Cys Lys Lys Ile Phe Gly Ser Leu Ala Phe Leu 740 745 750 Pro Glu Ser Phe Asp Gly Asp Pro Ala Ser Asn Thr Ala Pro Leu Gln 755 760 765 Pro Glu Gln Leu Gln Val Phe Glu Thr Leu Glu Glu Ile Thr Gly Tyr 770 775 780 Leu Tyr Ile Ser Ala Trp Pro Asp Ser Leu Pro Asp Leu Ser Val Phe 785 790 795 800 Gln Asn Leu Gln Val Ile Arg Gly Arg Ile Leu His Asn Gly Ala Tyr 805 810 815 Ser Leu Thr Leu Gln Gly Leu Gly Ile Ser Trp Leu Gly Leu Arg Ser 820 825 830 Leu Arg Glu Leu Gly Ser Gly Leu Ala Leu Ile His His Asn Thr His 835 840 845 Leu Cys Phe Val His Thr Val Pro Trp Asp Gln Leu Phe Arg Asn Pro 850 855 860 His Gln Ala Leu Leu His Thr Ala Asn Arg Pro Glu Asp Glu Cys Val 865 870 875 880 Gly Glu Gly Leu Ala Cys His Gln Leu Cys Ala Arg Gly His Cys Trp 885 890 895 Gly Pro Gly Pro Thr Gln Cys Val Asn Cys Ser Gln Phe Leu Arg Gly 900 905 910 Gln Glu Cys Val Glu Glu Cys Arg Val Leu Gln Gly Leu Pro Arg Glu 915 920 925 Tyr Val Asn Ala Arg His Cys Leu Pro Cys His Pro Glu Cys Gln Pro 930 935 940 Gln Asn Gly Ser Val Thr Cys Phe Gly Pro Glu Ala Asp Gln Cys Val 945 950 955 960 Ala Cys Ala His Tyr Lys Asp Pro Pro Phe Cys Val Ala Arg Cys Pro 965 970 975 Ser Gly Val Lys Pro Asp Leu Ser Tyr Met Pro Ile Trp Lys Phe Pro 980 985 990 Asp Glu Glu Gly Ala Cys Gln Pro Cys Pro Ile Asn Cys Thr His Ser 995 1000 1005 Cys Val Asp Leu Asp Asp Lys Gly Cys Pro Ala Glu Gln Arg Ala 1010 1015 1020 Ser Pro Leu Thr Ser Ile Ile Ser Ala Val Val Gly Ile Leu Leu 1025 1030 1035 Val Val Val Leu Gly Val Val Phe Gly Ile Leu Ile 1040 1045 1050 <210> 49 <211> 297 <212> PRT <213> Homo sapiens <400> 49 Met Thr Thr Pro Arg Asn Ser Val Asn Gly Thr Phe Pro Ala Glu Pro 1 5 10 15 Met Lys Gly Pro Ile Ala Met Gln Ser Gly Pro Lys Pro Leu Phe Arg 20 25 30 Arg Met Ser Ser Leu Val Gly Pro Thr Gln Ser Phe Phe Met Arg Glu 35 40 45 Ser Lys Thr Leu Gly Ala Val Gln Ile Met Asn Gly Leu Phe His Ile 50 55 60 Ala Leu Gly Gly Leu Leu Met Ile Pro Ala Gly Ile Tyr Ala Pro Ile 65 70 75 80 Cys Val Thr Val Trp Tyr Pro Leu Trp Gly Gly Ile Met Tyr Ile Ile 85 90 95 Ser Gly Ser Leu Leu Ala Ala Thr Glu Lys Asn Ser Arg Lys Cys Leu 100 105 110 Val Lys Gly Lys Met Ile Met Asn Ser Leu Ser Leu Phe Ala Ala Ile 115 120 125 Ser Gly Met Ile Leu Ser Ile Met Asp Ile Leu Asn Ile Lys Ile Ser 130 135 140 His Phe Leu Lys Met Glu Ser Leu Asn Phe Ile Arg Ala His Thr Pro 145 150 155 160 Tyr Ile Asn Ile Tyr Asn Cys Glu Pro Ala Asn Pro Ser Glu Lys Asn 165 170 175 Ser Pro Ser Thr Gln Tyr Cys Tyr Ser Ile Gln Ser Leu Phe Leu Gly 180 185 190 Ile Leu Ser Val Met Leu Ile Phe Ala Phe Phe Gln Glu Leu Val Ile 195 200 205 Ala Gly Ile Val Glu Asn Glu Trp Lys Arg Thr Cys Ser Arg Pro Lys 210 215 220 Ser Asn Ile Val Leu Leu Ser Ala Glu Glu Lys Lys Glu Gln Thr Ile 225 230 235 240 Glu Ile Lys Glu Glu Val Val Gly Leu Thr Glu Thr Ser Ser Gln Pro 245 250 255 Lys Asn Glu Glu Asp Ile Glu Ile Ile Pro Ile Gln Glu Glu Glu Glu 260 265 270 Glu Glu Thr Glu Thr Asn Phe Pro Glu Pro Pro Gln Asp Gln Glu Ser 275 280 285 Ser Pro Ile Glu Asn Asp Ser Ser Pro 290 295 <210> 50 <211> 894 <212> DNA <213> Homo sapiens <400> 50 atgacaacac ctagaaatag cgtgaacggc acattccccg ccgagcctat gaagggacct 60 atcgccatgc agagcggccc caagcctctg tttagacgga tgtctagcct cgtgggcccc 120 acacagagct ttttcatgag agagagcaag accctgggcg ccgtgcagat catgaacggc 180 ctgtttcaca ttgccctcgg cggcctgctg atgatccctg ccggaatcta tgcccctatc 240 tgcgtgaccg tgtggtatcc tctgtggggc ggcatcatgt acatcatctc tggatctctg 300 ctggccgcca ccgaagaa cagcaagaaag tgtctggtca agggcaagat gatcatgaat 360 agcctgagcc tgttcgccgc catcagcggc atgatcctga gcatcatgga tatcctgaat 420 atcaagatca gccacttcct gaagatggaa agcctgaact tcatcagggc ccacacacct 480 tacatcaaca tctacaactg cgagcccgcc aatcctagcg aagaataag ccccagcaca 540 footctgct actctatcca gagcctgttt ctgggcatcc tgagcgtgat gctgatcttc 600 gcattcttcc aagagctggt tatcgccggc atcgtggaaa acgagtgaa gcggacctgc 660 agcagaccca agagcaacat cgtgctgctg agcgccgagg aaaagaaaga gcagaccatc 720 gagatcaaag aggaagtcgt cggcctgacc gagacaagca gccagcctaa gaacgaag 780 gacattgaga tcatccccat ccaagagaag gaagagaag agactgagac aaacttcccc 840 gagcctcctc aggaccaaga gagcagcccc attgagaacg acagcagccc ttga 894 <210> 51 <211> 692 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Substance <400> 51 Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser Pro Ala Leu 1 5 10 15 Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys Ser Phe Ser 20 25 30 Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Met Ser Pro Ser 35 40 45 Asn Gln Asp Asp Arg Leu Ala Ala Phe Pro Glu Asp Arg Ser Gln Pro 50 55 60 Gly Gln Asp Ala Arg Phe Arg Val Thr Gln Leu Pro Asn Gly Arg Asp 65 70 75 80 Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser Gly Thr Tyr 85 90 95 Phe Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Lys Ile Lys Glu Ser 100 105 110 Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val Pro Thr 115 120 125 Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln Phe Gln Thr Pro 130 135 140 Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 145 150 155 160 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 165 170 175 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 180 185 190 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 195 200 205 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 210 215 220 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 225 230 235 240 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 245 250 255 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 260 265 270 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 275 280 285 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 290 295 300 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 305 310 315 320 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 325 330 335 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 340 345 350 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Ala 355 360 365 Ser Arg Lys Arg Arg Ser Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu 370 375 380 Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Thr Thr Pro Arg 385 390 395 400 Asn Ser Val Asn Gly Thr Phe Pro Ala Glu Pro Met Lys Gly Pro Ile 405 410 415 Ala Met Gln Ser Gly Pro Lys Pro Leu Phe Arg Arg Met Ser Ser Leu 420 425 430 Val Gly Pro Thr Gln Ser Phe Phe Met Arg Glu Ser Lys Thr Leu Gly 435 440 445 Ala Val Gln Ile Met Asn Gly Leu Phe His Ile Ala Leu Gly Gly Leu 450 455 460 Leu Met Ile Pro Ala Gly Ile Tyr Ala Pro Ile Cys Val Thr Val Trp 465 470 475 480 Tyr Pro Leu Trp Gly Gly Ile Met Tyr Ile Ile Ser Gly Ser Leu Leu 485 490 495 Ala Ala Thr Glu Lys Asn Ser Arg Lys Cys Leu Val Lys Gly Lys Met 500 505 510 Ile Met Asn Ser Leu Ser Leu Phe Ala Ala Ile Ser Gly Met Ile Leu 515 520 525 Ser Ile Met Asp Ile Leu Asn Ile Lys Ile Ser His Phe Leu Lys Met 530 535 540 Glu Ser Leu Asn Phe Ile Arg Ala His Thr Pro Tyr Ile Asn Ile Tyr 545 550 555 560 Asn Cys Glu Pro Ala Asn Pro Ser Glu Lys Asn Ser Pro Ser Thr Gln 565 570 575 Tyr Cys Tyr Ser Ile Gln Ser Leu Phe Leu Gly Ile Leu Ser Val Met 580 585 590 Leu Ile Phe Ala Phe Phe Gln Glu Leu Val Ile Ala Gly Ile Val Glu 595 600 605 Asn Glu Trp Lys Arg Thr Cys Ser Arg Pro Lys Ser Asn Ile Val Leu 610 615 620 Leu Ser Ala Glu Glu Lys Lys Glu Gln Thr Ile Glu Ile Lys Glu Glu 625 630 635 640 Val Val Gly Leu Thr Glu Thr Ser Ser Gln Pro Lys Asn Glu Glu Asp 645 650 655 Ile Glu Ile Ile Pro Ile Gln Glu Glu Glu Glu Glu Glu Thr Glu Thr 660 665 670 Asn Phe Pro Glu Pro Pro Gln Asp Gln Glu Ser Ser Pro Ile Glu Asn 675 680 685 Asp Ser Ser Pro 690 <210> 52 <211> 692 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Substance <400> 52 Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser Pro Ala Leu 1 5 10 15 Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys Ser Phe Ser 20 25 30 Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Met Ser Pro Ser 35 40 45 Asn Gln Asp Asp Arg Leu Ala Ala Phe Pro Glu Asp Arg Ser Gln Pro 50 55 60 Gly Gln Asp Ala Arg Phe Arg Val Thr Gln Leu Pro Asn Gly Arg Asp 65 70 75 80 Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser Gly Thr Tyr 85 90 95 Phe Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Lys Ile Lys Glu Ser 100 105 110 Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val Pro Thr 115 120 125 Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln Phe Gln Thr Pro 130 135 140 Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 145 150 155 160 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 165 170 175 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 180 185 190 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 195 200 205 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 210 215 220 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 225 230 235 240 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 245 ...

Claims

1. A composition comprising a plurality of genetically modified lymphocytes expressing a transgene for regulating a subject's immune system; wherein the transgene comprises a PD1-Fc fusion decoy and a CD40L having any one of the amino acid sequences of SEQ ID NOs: 32-34, 36 and 38.

2. The composition of claim 1, wherein the PD1-Fc fusion decoy is a PD-1.IgG4 decoy.

3. The composition of claim 1, wherein the transgene further comprises tEGFR, HER2, truncated HER2 (tHER2), CD20, or CD19.

4. The composition of claim 1, wherein the transgenes are contained on the same vector.

5. The composition of claim 1, wherein the PD1-Fc fusion bait comprises an amino acid sequence of any one of SEQ ID NOs: 1-4, 6-17, 42, 44, 47-48 and 51-52.

6. The composition of claim 3, wherein: The tEGFR comprises the amino acid sequence of SEQ ID NO: 40; the HER2 comprises the amino acid sequence of SEQ ID NO: 45; and the CD20 comprises the amino acid sequence of SEQ ID NO:

49.

7. The composition of claim 1, wherein the plurality of genetically modified lymphocytes comprises at least two lymphocyte subsets.

8. The composition of claim 1, wherein the plurality of genetically modified lymphocytes consists of two lymphocyte subsets.

9. The composition of claim 7, wherein each subpopulation of the plurality of genetically modified lymphocytes expresses at least one transgene.

10. The composition of claim 7, wherein the transgenes are different from each other.

11. The composition of claim 7, wherein the plurality of genetically modified lymphocytes comprises: (i) a first subpopulation expressing at least two transgenes; and (ii) a second subpopulation expressing at least two transgenes, wherein at least one transgene of the first subpopulation is different from a transgene of the second subpopulation, or wherein at least one transgene of the first subpopulation is the same as a transgene of the second subpopulation.

12. The composition of claim 11, wherein the first or second subpopulation expresses a PD1-Fc fusion decoy and a CD40L having any one of the amino acid sequences of SEQ ID NOs: 32-34, 36 and 38.

13. The composition of claim 11, wherein the first subpopulation expresses a PD1-Fc fusion decoy and a CD40L having any one of the amino acid sequences of SEQ ID NOs: 32-34, 36 and 38; wherein the second subpopulation expresses a PD1-Fc fusion decoy and an IL-2 variant having an amino acid sequence of SEQ ID NOs: 21-23.

14. The composition of claim 11, wherein the first subpopulation or the second subpopulation further expresses tEGFR; tHER2; CD20; or CD19.

15. The composition of claim 7, wherein the two subpopulations are combined in a ratio of about 1:1 to about 1:

100.

16. The composition of claim 15, wherein the two subpopulations are combined in a ratio of about 1:

1.

17. The composition of claim 1, wherein the lymphocytes are autologous lymphocytes.

18. The composition of claim 1, wherein the lymphocytes are tumor infiltrating lymphocytes.

19. The composition of claim 1, wherein the lymphocytes express a chimeric antigen receptor (CAR).

20. The composition of claim 1, wherein the lymphocytes express a recombinant T cell receptor (TCR).

21. The composition of claim 20, wherein the recombinant T cell receptor (TCR) exhibits reactivity against NY-ESO1, MAGE-A1, MAGE-A3, MAGE A-10, MAGE-C2, SSX2, MAGE-A12, or a combination thereof.

22. A pharmaceutical composition comprising an effective amount of the composition according to any one of the preceding claims and a pharmaceutically acceptable carrier.

23. The pharmaceutical composition of claim 22, further comprising a second therapeutic agent.

24. A kit comprising an effective amount of the composition of any one of claims 1-21 or the pharmaceutical composition of any one of claims 22-23.

25. A method for preparing the composition of any one of claims 1 to 21, comprising: Provide multiple lymphocytes; introducing a nucleic acid molecule encoding a transgene into the plurality of lymphocytes to obtain a plurality of genetically modified lymphocytes; The genetically modified multiple lymphocytes express PD1-Fc fusion bait and CD40L having any one of the amino acid sequences of SEQ ID NOs: 32-34, 36 and 38; The genetically modified plurality of lymphocytes are expanded in a cell culture medium.

26. The method of claim 25, wherein the cell culture medium is a defined cell culture medium.

27. The method of claim 26, wherein the cell culture medium comprises a neoantigenic peptide.

28. Use of a composition as described in any one of claims 1-21 or a pharmaceutical composition as described in any one of claims 22-23 in the preparation of a medicament for treating cancer / tumor or chronic infection in a subject, comprising administering an effective therapeutic amount of a composition as described in any one of claims 1-21 or a pharmaceutical composition as described in any one of claims 22-23 to a subject in need thereof.

29. The use of claim 28, wherein the cancer is selected from melanoma, sarcoma, ovarian cancer, prostate cancer, lung cancer, bladder cancer, microsatellite high instability tumors, head and neck tumors, kidney cancer and breast cancer.

30. The use of claim 28, wherein the composition is administered by intravenous infusion.

31. The use of claim 28, further comprising administering a second therapeutic agent to the subject.

32. The use of claim 31, wherein the second therapeutic agent is an anticancer agent or an antitumor agent.

33. The use of claim 31, wherein the composition or pharmaceutical composition is administered to the subject before, after or concomitantly with the second therapeutic agent.

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