RU2847583C1 - Nanoparticle systems for stimulating and maintaining immune system responsiveness in treatment areas - Google Patents

Abstract

FIELD: biotechnology; medicine.

SUBSTANCE: invention discloses nanoparticles capable of genetically modifying monocytes/macrophages in vivo and activating said immune cells.

EFFECT: immune cells genetically modified by nanoparticles according to the present invention can be used in cancer immunotherapy.

10 cl, 12 dwg, 2 tbl, 3 ex

Description

CROSS-REFERENCE TO A RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application No. 62/956,033, filed December 31, 2019, the contents of which are incorporated herein by reference in their entirety as if fully set forth herein.

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[0002] The invention relates to nanoparticle systems that genetically modify monocytes/macrophages in vivo to (1) recruit additional immune cells to the treatment area; (2) maintain an activated state at the treatment area, providing a continuous stimulatory signal to other immune cells; and (3) secrete multispecific immune cell binding molecules that bind antigens on target cells at the treatment area and bind and activate recruited immune cells to kill the bound cells. The systems can also inhibit transforming growth factor beta (TGFβ) activity.

PREREQUISITES FOR THE CREATION OF THE INVENTION

[0003] Macrophages are key immune effector cells that infiltrate cancer tissue in large numbers. However, in the tumor microenvironment, macrophages transition from an activated tumoricidal state to an immunosuppressive phenotype that actually promotes tumor growth and metastasis. Pollard, Nat Rev Cancer 4, 71–78 (2004); Mantovani, et al., Nat Rev Clin Oncol (2017).

[0004] With the understanding that immunosuppressive macrophages in the tumor microenvironment promote cancer growth and metastasis, much effort has been directed toward developing therapies that target immunosuppressive tumor-associated macrophages (TAMs). Many anti-TAM efforts have focused on killing TAMs to relieve immunosuppression in the tumor microenvironment. However, with this approach, TAMs are simply replaced by newly recruited macrophages within the tumor environment. Furthermore, most therapeutic agents developed to date, although capable of successfully killing some TAMs, fail to sufficiently penetrate the tumor microenvironment. Although some small molecule drugs and antibodies have shown some success, these approaches result in the suppression of all macrophages in the body, causing dangerous side effects. Bowman & Joyce, Immunotherapy 6, 663–666 (2014). Thus, more effective treatment strategies with fewer side effects are urgently needed for all cancer sufferers.

[0005] Significant progress has been made in genetically modifying T cells of the immune system to target and kill cell types of interest, such as cancer cells. Many of these T cells have been genetically modified to express a chimeric antigen receptor (CAR). CARs are proteins containing several distinct subcomponents that allow genetically modified T cells to recognize and kill cancer cells. The subcomponents include at least an extracellular component and an intracellular component. The extracellular component contains a binding domain that specifically binds a marker predominantly present on the surface of cells of interest. When the binding domain binds such markers, the intracellular component signals the T cell to kill the bound cell. CARs further contain a transmembrane domain that can link the extracellular component to the intracellular component, as well as other subcomponents that can enhance CAR function. For example, the inclusion of one or more linker sequences, such as a spacer region, can allow a CAR to have additional conformational flexibility, often increasing the ability of the binding domain to bind a target cellular marker.

[0006] Clinical trials with CAR-expressing T cells (CAR-T) have demonstrated a positive response in patients with refractory large B-cell lymphoma when traditional treatments failed. (Neelapu, et al. 2017 N Engl J Med 377:2531-2544). However, although genetically modified CAR-T cells lead to the destruction of cancer cells, they failed to provide long-term anti-cancer activity in vivo in some diseases. One reason for this failure may be based on the immunosuppressive effects of the tumor microenvironment.

[0007] Bispecific T-cell binding antibodies bind both a cancer cell antigen and a T-cell activating epitope to target T cells to the cancer cells for cancer cell destruction. See, for example, US 2008/0145362. Most current bispecific T-cell binding antibodies contain paired monospecific binding domains from antibodies. Some work has investigated the use of combinations of such antibodies targeting two different T-cell activating epitopes (e.g., CD3 and CD28). However, many of these antibodies have short half-lives in vivo , so dosing remains challenging.

[0008] Several research groups have explored ways to overcome some of the challenges associated with the administration of bispecific T-cell-binding antibodies. For example, Stadler et al. (Nature Medicine 23, 815–817) described the injection of nanocarriers that deliver nucleic acids encoding bispecific T-cell-binding antibodies. By expressing these antibodies in vivo , this approach achieved sustained levels of circulating bispecific T-cell-binding antibodies, eliminating the need for continuous delivery infusion pumps. However, circulating bispecific T-cell-binding antibodies do not effectively penetrate solid tumors, and the activity of T cells recruited to and infiltrating the tumor microenvironment is suppressed by myeloid-derived suppressor cells.

[0009] Choi et al. (Nature Biotechnology, 37, 1049–1058, 2019) investigated the genetic modification of T cells to produce and secrete bispecific T cell-binding antibodies. In fact, Choi et al. investigated the genetic modification of T cells to express CARs and also to secrete bispecific T cell-binding antibodies. However, these T cells required ex vivo genetic modification. Furthermore, CAR T cells also inefficiently penetrate solid tumors and proliferate within the tumor site (often as a result of exposure to myeloid-derived suppressor cells). Thus, despite significant advances in cancer treatment strategies, significant unmet needs remain.

[0010] Furthermore, protein factors of the transforming growth factor β (TGFβ) family are present at high levels in solid tumors and contribute to immune dysfunction in the tumor microenvironment.

ESSENCE OF THE INVENTION

[0011] The present invention relates to systems and methods for reversing the immunosuppressive, tumor-promoting state of tumor-associated macrophages (TAMs) and converting these TAMs into highly tumor-killing macrophages. Accordingly, the systems and methods disclosed herein are directed not simply to killing TAMs, but to redirecting their activity from tumor maintenance to tumor killing. In specific embodiments, the systems and methods are used as a therapeutic approach to induce cancer cell killing and/or to reduce or prevent the growth or development of new cancer cells. The results described herein demonstrate that these systems and methods result in the complete eradication and suppression of ovarian cancer, which is notoriously difficult to control.

[0012] The use of the TAM activation strategies disclosed herein has been shown to recruit immune cells to the tumor site. However, many of the recruited immune cells do not bind cancer antigens expressed by tumor cells, and thus these recruited cells provide less benefit to the anti-cancer response than might otherwise be achieved. To address this problem, the present invention provides for the genetic modification of activated TAMs to express multispecific immune cell binding molecules. The activated TAMs then provide three critical aspects for the success of the anti-cancer therapy described herein. They (1) recruit immune cells to the tumor site; (2) remain activated at the tumor site, providing a continuous stimulatory signal for other immune cells; and (3) secrete multispecific immune cell binding molecules that bind cancer antigens at the tumor site and bind and activate the recruited immune cells to kill the bound cancer cells. The approach described for killing cancer cells can also be applied to other cell types of interest, such as disease-associated cells, autoreactive cells, infected cells, and microbial cells, and so on.

[0013] In specific embodiments, altering or maintaining activated states of macrophages in vivo is accomplished by using a nanoparticle system to deliver nucleotides encoding activation regulators, such as transcription factors. Particularly useful nanoparticles have a positively charged core and a neutral or negatively charged surface, and deliver nucleotides encoding (i) a transcription factor that creates or maintains an activated state of a macrophage; (ii) a kinase and/or (iii) a multispecific immune cell binding molecule. In preferred embodiments, the systems will include nanoparticles that deliver nucleotides encoding each of these three components. A nanoparticle size of <130 nm ensures tumor penetration. The nanoparticles may optionally include a ligand targeting the TAM to more selectively uptake the nanoparticles by the TAM. As one example, OAMs express CD206, a cell surface receptor that can be targeted by the addition of mannose to the surface of nanoparticles.

[0014] Specific embodiments include a nanoparticle with a diameter <130 nm having a positively charged polymer core and a neutral or negatively charged coating. Nucleotides encoding interferon-regulatory factor 5 (IRF5); a kinase, IKKβ; a multispecific antibody, and, optionally, a TGFβ inhibitor are encapsulated in the positively charged polymer core. In this example, the bispecific antibody binds a cancer antigen selected from EpCam or tyrosinase-related protein 1 (TYRP1/gp75) and an immune cell activating epitope selected from CD3, CD28, or 4-1BB.

[0015] The systems disclosed herein also include a transforming growth factor beta (TGFβ) inhibitor.

BRIEF DESCRIPTION OF DRAWINGS

[0016] Some of the drawings presented herein may be better understood in color. The inventors consider color versions of the drawings to be part of the initially filed application and reserve the right to submit color versions of the drawings later during consideration of the application.

[0017] FIGS. 1A-1D. Schematic of genetic transformation of tumor-associated macrophages (TAMs) into tumoricidal cells using targeted mRNA nanoparticles. (FIG. 1A) An injectable nanocarrier was developed to deliver in vitro transcribed mRNA encoding M1-polarizing transcription factors as a novel way to rationally reprogram TAMs for therapeutic purposes without causing systemic toxicity. The first planned clinical application is illustrated, intended for the treatment of patients suffering from ovarian cancer using repeated intraperitoneal infusions of mRNA nanoparticles. (FIG. 1B) Schematic of genetic reprogramming of intracranial TAMs into tumoricidal macrophages using targeted mRNA nanoparticles. (FIG. 1C) Schematic of genetic transformation of tumor-associated macrophages (TAMs) into tumoricidal, bispecific antibody-secreting cells using targeted mRNA nanoparticles. An injectable nanocarrier co-delivering in vitro -transcribed mRNA encoding M1-polarizing transcription factors and antibodies that redirect T cells to a tumor antigen provides a novel approach to rationally reprogram TAMs and activate the host adaptive immune response for therapeutic purposes without causing systemic toxicity. (FIG. 1D) Illustrative formats of bispecific binding molecules in Fc and non-Fc formats.

[0018] FIGS. 2A-2K. Nanoparticles carrying mRNA encoding IRF5 and IKKβ are capable of conferring a proinflammatory M1-like phenotype. (FIG. 2A) Design of macrophage-targeted polymeric NPs formulated with mRNA molecules encoding key regulators of macrophage polarization. The nanoparticles are composed of a PbAE-mRNA polyplex core coated with a PGA-dimannose layer that targets the nanoparticles to mannose receptors (CD206) expressed by M2-like macrophages. Also shown is synthetic mRNA encapsulated within the NPs, which is designed to encode reprogramming transcription factors. (FIG. 2B) Transmission electron microscopy images of a population of NPs (scale bar 200 nm) and a single NP (inset, scale bar 50 nm). (FIG. 2C) Size distribution of NPs measured using a NanoSight NS300 instrument. (FIG. 2D) NPs demonstrated a high transfection rate (46%) of bone marrow-derived macrophages (BMDMs) after 1 h of exposure. (FIG. 2E) Gene transfer efficiency into bone marrow-derived macrophages (BMDMs) measured by flow cytometry 24 h after nanoparticle transfection. (FIG. 2F) Relative viability of NP-transfected and non-transfected macrophages (assessed by annexin V and PI staining); n/s, not significant. (FIG. 2G) Expression kinetics of codon-optimized IRF5 mRNA (blue, left y-axis) and endogenous IRF5 mRNA (black, right y-axis) measured by qRT-PCR, n=3 for each time point. (FIG. 2H) Timeline of NP transfection protocols and culture conditions for BMDMs described for FIGS. 2I-2K. (FIG. 2I) Gene expression profiles for IRF5/IKKβ NP-transfected macrophages compared to the M1 signature of cells stimulated with the Toll-like receptor 6 agonist MPLA. Results are presented as a scatter plot of the large data set showing the distribution of fold changes in gene expression. M1 signature genes are shown. The p-value for agreement between IRF5/IKKβ NP-transfected macrophages and the M1 signature gene set was determined using GSEA. (FIG. 2J) Heatmap of M1 signature gene expression in IL-4-cultured macrophages compared to IL-4-cultured cells transfected with IRF5/IKKβ NPs. (FIG. 2K) Boxplots show the mean counts for the indicated genes and SEM.

[0019] FIG. 3. In vitro screening of the effect of different members of the interferon-regulating factor (IRF) family (delivered with or without their activating kinase) on the phenotype of mouse macrophages. BMDMs from C57BL/6 mice were incubated in M-CSF-conditioned medium and transfected with mRNA-PBAE NPs carrying synthetic mRNA encoding (1) control GFP, (2) mouse IRF5, (3) mouse IRF5 and IKKβ kinase, which phosphorylates IRF5, (4) mouse IRF8 and IKKβ kinase, (5) mouse IRF8 K310R, which is a mutant IRF8 with Lys-310 replaced by Arg (K310R) ( White et al ., J Biol Chem. 2016 Jun 24), or (6) mouse IRF7/3 (5D). This fusion protein contains the DNA-binding domain (DBD) and constitutively active domain (CAD) of IRF-7, as well as the nuclear export signal (NES) and IRF binding domain of IRF3 ( Lin et al ., Molecular and Cellular Biology. 18, 5, 1998). Two days after NP transfection, cells were harvested for flow cytometric analysis of the TAM-associated macrophage marker Egr2 and the activated macrophage marker CD38. Based on this in vitro screening, NP-based co-delivery with mRNA encoding mIRF5 and IKKβ kinase was selected for the remaining in vitro and in vivo therapeutic experiments described herein.

[0020] FIGS. 4A-4J. Repeated intraperitoneal injections of mRNA nanocarriers delivering IRF5 and IKKβ genes to macrophages more than double the median survival of mice bearing disseminated ovarian cancer. (FIG. 4A) Timelines and dosing regimens. Arrows indicate the time of i.p. injection. (FIG. 4B) Serial bioluminescence imaging of tumor growth in control and treated mice. (FIG. 4C) Kaplan-Meier survival curves for treated mice compared to control animals. Statistical analysis was performed using the log-rank test. (FIG. 4D) Flow cytometric quantification of the extent of in vivo transfection in different immune cell subsets 48 hours after a single i.p. dose of D-mannose-coated NPs carrying GFP mRNA as a control: measurements were performed for macrophages (CD45+, CD11b+, MHCII+, CD11c-, Ly6C-/low, Ly6G-), monocytes (CD45+, CD11b+, MHCII+, CD11c-, Ly6C+, Ly6G-), neutrophils (CD45+, CD11b+, MHCII+, CD11c-, Ly6G+), CD4+ T cells (CD45+, TCR-β chain+, CD4+, CD8-), CD8+ T cells (CD45+, TCR-β chain+, CD4-, CD8+) and natural killer cells (CD45+, TCR-β chain-, CD49b+). (FIG. 4E) Flow cytometric analysis of macrophage phenotypes in the mesentery of mice bearing disseminated ovarian cancer ID8. Animals received either 4 doses of IRF5/IKKβ NPs or PBS. (FIG. 4F) Boxplots show the relative percentages (left panel) and absolute numbers (right panel) of Ly6C-, F4/80+, and CD206+ (M2-like) macrophages. (FIG. 4G) Corresponding numbers for Ly6C-, F4/80+, and CD206- (M1-like) macrophages. (FIG. 4H) Representative hematoxylin and eosin-stained sections of ovarian tumor cell-infiltrated mesenteries from control PBS (upper panel) or IRF5/IKKβ NP-treated animals (lower panel; scale bar, 100 μm). Representative tumor lesions at 10x magnification are shown in the right panel (scale bar, 50 μm). (FIG. 4I) Results of Luminex assay measurement of cytokines produced by isolated peritoneal macrophages in animals from each treatment group. CD11b+, F4/80+ peritoneal macrophages were isolated by fluorescence-activated cell sorting and cultured ex vivo . Cell culture supernatants were collected after 24 h. In parallel experiments, FACS-sorted CD11b+, F4/80+ peritoneal macrophages were directly analyzed by qRT-PCR to determine the expression levels of four major regulators of macrophage phenotypes (serpin B2, Retn1A, Ccl11, and Ccl5). The results are presented as boxplots in FIG. 4J.

[0021] FIGS. 5A-5F. Macrophage-programming mRNA nanocarriers were highly biocompatible and safe for repeated administration. (FIG. 5A) In vivo biodistribution of macrophage-targeted IRF5/IKKβ NPs after i.p. administration. NP-delivered (codon-optimized) mRNA was detected by qPCR 24 hours after a single injection of nanoparticles containing 50 μg mRNA. (FIG. 5B) Schematic representation of the time course of the experiment. *Twenty-four hours after the last dose, mice were sacrificed by _CO2 inhalation. Blood was collected from the retro-orbital sinus into heparin-coated tubes for serum biochemistry and complete blood count. Autopsies were performed for histologic analysis of the liver, spleen, pancreas, mesentery and omentum, stomach, and bladder. (FIG. 5C) Representative hematoxylin and eosin-stained sections of various organs from control or NP-treated animals. Scale bars, 100 μm. Tumor lesions found in NP-treated animals are shown and described based on the pathologist's review. The pertinent findings for each numbered image are as follows: [1] Discrete foci of cellular infiltrates composed primarily of mononuclear cells admixed with few granulocytes; moderate extramedullary hematopoiesis. [2] In a few focally extensive areas, hepatocytes are mildly to moderately swollen. [3] Moderate myeloid (predominant), erythroid, and megakaryocytic hyperplasia in the red pulp. [4] Moderate hypocellularity of the white pulp. [5] The mesentery shows moderate, multifocal infiltrates of macrophages, lymphocytes, plasma cells, and granulocytes. [6] Mild to moderate infiltrates of macrophages admixed with lymphocytes, plasma cells, and granulocytes; moderate dissociation of acini and loss of acinars; moderate diffuse loss of zymogen granules from acinar cells. [7] Dense collections of lymphocytes admixed with macrophages around the fat pad. [8] Moderate multifocal vacuolar degeneration of chief and parietal cells of the gastric mucosa. (FIG. 5D) Serum biochemistry and blood count. (FIG. 5E, 5F) Measurement of serum IL-6 (FIG. 5E) and TNFα (FIG. 5F) cytokine levels in the Luminex assay 4 or 8 days after a single i.p. injection of IRF5/IKKβ NPs.

[0022] FIGS. 6A-6I. Intravenously infused IRF5/IKKβ NPs can control lung tumor metastasis. (FIG. 6A) In vivo biodistribution of macrophage-targeted IRF5/IKKβ NPs after intravenous administration. Codon-optimized mRNA was quantified by qPCR 24 hours after a single intravenous injection of nanoparticles containing 50 μg of mRNA. (FIG. 6B-6H) C57BL/6 albino mice were injected with 1 x 10 ⁶ firefly luciferase-expressing B16F10 melanoma cells via tail vein to initiate lung metastasis. After 7 days, animals were randomly assigned to an IRF5/IKKβ NPs group, a control GFP NPs group, or a control PBS group. (FIG. 6B) Time courses and dosing regimens. (FIG. 6C) Confocal microscopy images of normal lungs (left panel) and B16F10 tumor-infiltrated lungs (right panel). Infiltrating macrophage populations fluoresce green. (FIG. 6D) Sequential bioluminescence imaging of tumors. (FIG. 6E) Kaplan-Meier survival curves for each treatment group, “m” denotes median survival. Statistical analysis was performed using the log-rank test, and P<0.05 was considered statistically significant. (FIG. 6F) Representative photographs (top row) and photomicrographs of lungs containing B16F10 melanoma metastases from each group after 2 weeks of treatment. (FIG. 6G) Number of tumor lesions in the lungs. (FIG. 6H) Phenotypic characterization of monocyte/macrophage populations in bronchoalveolar lavage of animals from each treatment group. (FIG. 6I) Summary of the relative percentages of suppressor and activated macrophages.

[0023] FIGS. 7A-7F. Macrophage reprogramming improves radiation therapy outcomes in glioma. (FIG. 7A) T2 MRI scan and histological staining results after PDGFβ-driven glioma induction in RCAS-PDGF-B/Nestin-Tv-a; Ink4a/Arf-/- ; Pten-/- transgenic mice at day 21 after induction. (FIG. 7B) Confocal microscopy results of CD68+ TAMs infiltrating the glioma margin. Scale bar, 300 μm. (FIG. 7C) Flow cytometric analysis of macrophage populations (F4/80+, CD11b+) in healthy brain tissue compared to glioma. (FIGS. 7D-7E) Kaplan-Meier survival curves for mice bearing established gliomas receiving IRF5/IKKβ alone (FIG. 7D) or in combination with brain tumor radiotherapy (FIG. 7E). Time courses and dosing regimens are shown at the top. M, median survival. Statistical analysis was performed using the log-rank test, and P<0.05 was considered statistically significant. (FIG. 7F) Serial bioluminescence imaging of tumor progression.

[0024] FIGS. 8A-8E. Nanoparticles carrying IVT mRNA encoding human IRF5/IKKβ efficiently reprogram human macrophages. (FIG. 8A) Time course and culture conditions for differentiation of human monocytic THP-1 cells into M2-like suppressor macrophages. (FIG. 8B) Bioluminescence imaging of M2-differentiated THP1-Lucia cells cultured in 24-well plates and transfected with NPs carrying human IRF5/IKKβ mRNA at the indicated concentrations compared to GFP mRNA. IRF-induced Lucia luciferase levels were determined 24 hours post-transfection using Quanty-Luc. (FIG. 8C) Summary of bioluminescence results. (FIG. 8D-8E) Differences in IL-1β cytokine secretion (FIG. 8D) and surface expression (FIG. 8E) of the macrophage M1 marker CD80.

[0025] FIG. 9. List of antibodies used in the myeloid and lymphoid tissue immunophenotyping panels described above in Example 1.

[0026] FIG. 10A, 10B. T cells contribute to the antitumor effects achieved using macrophage-programming nanoparticles. (10A) Nanoparticle-mediated macrophage programming enhances T cell recruitment to tumor lesions. Representative confocal images of peritoneal metastases of ID8 ovarian cancer cells to the mesentery are shown. Tissues were collected after 6 twice-weekly i.p. injections of PBS or IRF5/IKKβ NPs (50 μg mRNA/dose) and stained for the indicated lymphocyte and myeloid cell markers (a, c). Tumor, Mesentery. Scale bar: 100 μm. (10B) Boxplots showing fluorescent signals for each phenotypic marker, constructed using Halo™ image analysis software. N=5. Boxes represent mean values, and the line within the box represents the median. Bars within the boxes show minimum and maximum values. Whiskers represent 95% confidence intervals. N=5 biologically independent samples.

[0027] FIG. 11. Illustrative sequences of the invention.

[0028] FIG. 12. Protein/coding sequence pairs with associated annotations.

DETAILED DESCRIPTION OF THE INVENTION

[0029] Macrophages are key effector immune cells that infiltrate cancer tissue in large numbers. However, in an immunosuppressive tumor environment, they transition from an activated tumoricidal state to an immunosuppressive phenotype, which promotes tumor growth and metastasis. These tumor-associated immunosuppressive macrophages (TAIMs) are associated with poor prognosis (Komohara Y et al . (2014) Cancer science 105(1): 1-8). They induce angiogenesis, lymphogenesis, and stromal remodeling. They also play a key role in promoting tumor invasion and metastasis by secreting the enzymes plasmin, uPA, matrix metalloproteinases (MMPs), and cathepsin B (Komohara, Y et al . (2016) Advanced drug delivery reviews 99: 180–185; Gocheva V et al . (2010) Genes Dev 24: 241–255; Wang R et al . (2011) Lung Cancer 74: 188–196). In addition to promoting tumor growth and progression, TAMs can also interact with other immune cells and suppress innate and adaptive antitumor immune responses.

[0030] Some small molecule drugs aim to block the localization of TAM progenitor cells to the tumor by targeting pathways involved in cell recruitment or expansion (i.e., inhibitors of the CSF-1/CSF-1R pathway (Pyon; teck et al. Nat Med 19, 1264-1272 (2013); Tap et al. N Engl J Med 373, 428-437 (2015)), or the CCL2 pathway (Nywening, et al. Lancet Oncol 17, 651-662 (2016)). These approaches require repeated systemic exposure to high doses of small molecule drugs. Furthermore, clinical trials of these drugs have demonstrated low response rates unless they are used in combination with cytoreductive therapy. Nywening, et al . Lancet Oncol 17, 651-662 (2016); Butowski et al . Neuro Oncol 18, 557–564 (2016). Furthermore, these small-molecule approaches do not effectively stimulate macrophage antitumor activity.

[0031] Conventional nanocarriers such as liposomes have been formulated with bisphosphonates or other antiproliferative agents to systemically kill tumor macrophages (i.e., liposomal clodronate) (Fritz et al., Front Immunol 5, 587 (2014)). Oncolytic viruses have also been used to deliver siRNA to suppress tumor immune evasion pathways and indirectly stimulate TAM phagocytosis (Chao et al., Curr Opin Immunol 24, 225–232 (2012)). However, macrophages killed by these approaches are naturally replaced by newly arriving macrophages, which also become immunosuppressive.

[0032] Antibodies that induce functional activation of TAMs have been developed. These approaches use antibodies directed to specific tumor antigen types. Mantovani et al., Nat Rev Clin Oncol (2017). However, the successful use of these antibodies is limited by their low tumor penetrating ability and heterogeneous distribution. Thurber et al., Adv Drug Deliv Rev 60, 1421–1434 (2008). They also do not address the problem of immune evasion in tumor variants that lack the antigen targeted by the antibody.

[0033] None of the described approaches directly and effectively programs or reprograms TAMs to remain or become activated tumoricidal macrophages as described herein. The systems and methods disclosed herein are significantly innovative because they allow TAMs to be reprogrammed into tumor-killing macrophages while simultaneously reducing the tumor-promoting TAM load. Currently, no other methods exist that allow clinicians to rationally reprogram TAMs for these therapeutic purposes. Mantovani, et al ., Nat Rev Clin Oncol (2017); Gabrilovich & Nagaraj, Nat Rev Immunol 9, 162-174 (2009). This in itself may provide a therapeutic effect in the treatment of tumors. In fact, the efficacy of the approaches disclosed herein has been demonstrated in ovarian cancer, melanoma, and glioblastoma models. Specifically, infusion of nanoparticles formulated with nucleotides encoding interferon-regulatory factor 5 (IRF5) in combination with its activating kinase IKKβ reversed the immunosuppressive, tumor-promoting state of TAMs and reprogrammed them to a phenotype that induced antitumor immunity and promoted tumor regression.

[0034] One interesting observation was that T cells contribute to the antitumor effect achieved by the macrophage-programming nanoparticles. In fact, all animals treated with IRF5/IKKβ nanoparticles showed multifocal dense accumulation of host T cells around the tumors, indicating that genetic programming of immunostimulatory macrophages can restore lymphocyte migration and infiltration into solid tumors (NPs increased T cell infiltration into tumors by an average of 10.6-fold (CD8) and 3.5-fold (CD4); see FIGS. 10A, 10B).

[0035] Since most T cells that are recruited to a tumor lack a therapeutically significant T cell receptor that would bind cancer antigens at the tumor site, the present invention provides for the use of nanoparticles that deliver nucleotides encoding macrophage-programming transcription factors and T cell redirecting macromolecules (such as bispecific antibodies) to further activate innate and adaptive immune cells (illustrated in FIG. 1C).

[0036] One of the key advantages of bispecific molecules over existing technologies is that these molecules are directly secreted by the TAM and, therefore, reach the highest concentration at the tumor site (with minimal systemic exposure). Given the rapid clearance rate of bispecific antibodies (e.g., 2 hours in human serum), traditional bispecific antibody therapy must be administered by continuous intravenous infusion and is associated with dose-limiting toxicity. This approach has had little clinical success in the treatment of solid tumors, which are protected from T cell attack by myeloid-derived suppressor cells. Using the approaches described herein, clinicians can genetically modify monocytes/macrophages in vivo to (1) recruit additional immune cells to the tumor site; (2) maintain an activated state at the tumor site, providing a continuous stimulatory signal for other immune cells; and (3) the secretion of multispecific immune cell-binding molecules that bind cancer antigens within the tumor and bind and activate recruited immune cells to kill bound cells. Importantly, this therapy acts from within the tumor, which contrasts with existing combination therapies, which can disrupt immune homeostasis.

[0037] In specific embodiments, nanoparticles are used to provide cells with nucleotides encoding genes encoding activation regulators, such as transcription factors (e.g., interferon-regulating factors (IRF)) and/or kinases (e.g., IKKβ). These activation regulators regulate macrophage polarization. Macrophage polarization is a highly dynamic process that alters the physiological activity of macrophages. As noted above, in most tumors, TAMs exhibit an immunosuppressive phenotype, which may be an "M2" phenotype. In contrast, activated macrophages may exhibit an "M1" phenotype, which leads to the destruction of tumor cells. Specific embodiments disclosed herein alter cell polarization, converting tumor-promoting TAMs into tumor-killing macrophages. Specific embodiments disclosed herein enable genetically modifying monocytes to maintain an activated state after subsequent differentiation into macrophages, so that the macrophages do not become immunosuppressive in the tumor region. These effects attenuate the immunosuppressive environment within tumors, with the induction of inflammatory cytokines, activation of other immune cells, and phagocytosis of tumor cells.

[0038] "Macrophage activation" means the process of changing the phenotype or function of a macrophage from (i) an inactivated state to an activated state; (ii) a non-activated state to an activated state; (iii) an activated state to a more activated state; or (iv) an inactivated state to a non-activated state. "Inactivated state" means an immunosuppressive phenotype that promotes tumor growth and metastasis. Non-activated state means that the macrophage neither promotes tumor growth or metastasis nor stimulates tumor cell killing. "Activated" means that the macrophage exhibits tumoricidal activity. In particular embodiments, the activated state results in an M1 phenotype, as described in more detail below. In particular embodiments, the inactivated state results in an M2 phenotype, also as described in more detail below.

[0039] In specific embodiments, one advantage of the disclosed systems and methods is that patients can be spared systemic toxicity because treatment-induced inflammation remains localized to the treatment area. To achieve this advantage, locally administered nanoparticles target TAMs in the tumor environment, deliver nucleotides that selectively reprogram signaling pathways that control macrophage polarization, and can be completely disrupted locally by physiological pathways (Sahin et al ., Nat Rev Drug Discov 13, 759-780 (2014)). The nanoparticles described herein can also be administered intravenously, where they can be taken up by monocytes in the bloodstream.

[0040] Achieving high expression of exogenous nucleotides in solid tumors in vivo is a challenging task. Prior to the present invention, nucleotide delivery systems based on viruses or conventional nanocarriers such as liposomes have been limited due to their limited diffusion in tumor tissue. Jain & Stylianopoulos, Nat Rev Clin Oncol 7, 653-664 (2010). To overcome this barrier, certain embodiments of the invention utilize nanoparticles (also referred to herein as NPs) with enhanced diffusion capacity, allowing the NPs to deliver nucleotides to a large population of TAMs within the tumor. Certain embodiments of the invention utilize NPs <130 nm in size and having a neutral surface charge.

[0041] Certain embodiments may further include a targeting ligand attached to the surface of the NP. For example, macrophage mannose receptor 1 (MRC1), also known as CD206, is a type I transmembrane protein that is expressed by macrophages. CD206 also exhibits high levels of expression in OAMs. Accordingly, in certain embodiments, dimannose may be attached to the surface of the NP to provide more selective targeting of the mannose receptor (CD206) expressed on the cell surface of OAMs. For more information on CD206 binding and targeting ligands, see Zhang et al., Nature Communications, 10, 3974 (2019). Other TAM cell surface receptors that may be targeted include early growth response protein 2 (Egr2), CD163, CD23, interleukin (IL)27RA, CLEC4A, CD1a, CD1b, CD11b, CD14, CD16, CD31, CD93, CD115, CD192, CD226, IL13-Ra1, IL-4r, IL-1R type II, IL-1R type II decoy, IL-10r, macrophage scavenger receptors A and B, Ym-1, Ym-2, low-density lipoprotein receptor-like protein 1 (LRP1), IL-6r, CXCR1/2, CX3CR1, CXCR3, CXCR4, and PD-L1.

[0042] In specific embodiments, the systems and methods disclosed herein comprise administering nanoparticles to a subject in need thereof. The nanoparticles are targeted to monocytes in the bloodstream and/or macrophages present in tumors in the subject and are engineered to be internalized by the monocytes/macrophages. Once internalized, the nanoparticles deliver one or more nucleotide sequences encoding IRF5 and IKKβ. The one or more nucleotide sequences modify the monocytes/macrophages to express IRF5 and IKKβ. Without wishing to be bound by a particular theory, IKKβ kinase activates the transcription factor IRF5 by phosphorylation. Activated IRF5 then induces the expression of type I interferon (IFN) genes, inflammatory cytokines including tumor necrosis factor (TNF), IL-6, IL-12, and IL-23, and tumor suppressors. In M2 macrophages that have internalized one or more nucleotide sequences encoding IRF5 and IKKβ, expression of the aforementioned genes, through the action of IRF5, results in a phenotypic or functional switch of the macrophages from the M2 phenotype to the M1 phenotype, which enables the macrophages to kill or otherwise trigger the destruction of tumor cells, thereby treating cancer. In specific embodiments, the nanoparticles are internalized by monocytes/macrophages via phagocytosis. In specific embodiments, the nanoparticles are internalized by monocytes/macrophages via ligand-mediated endocytosis (e.g., CD-206-mediated endocytosis). In particular embodiments, delivery of nanoparticles containing IRF5 and IKKβ genes to macrophages may include, for example, (1) binding to macrophages, (2) internalization of the nanoparticles by macrophages, (3) release from endocytic vesicles into the cytoplasm after internalization, (4) release of one or more nucleotide sequences that (5) can be transferred to the nucleus of macrophages and (6) transcribed to deliver genes for expression of IRF5 and IKKβ.

[0043] As previously noted, the nanoparticles in the systems disclosed herein further genetically modify monocytes/macrophages to produce and secrete bispecific molecules that activate immune cells. This approach is depicted in FIG. 1C, where a nanoparticle containing nucleotides encoding transcription factors and a bispecific antibody is encapsulated in a positively charged core. In the approach depicted in FIG. 1C, the nanoparticles are taken up by monocytes in the bloodstream. These monocytes then exit the bloodstream and enter the tumor site. As a result of the uptake of the nanoparticles, the cells express transcription factors that enter the nucleus and allow the creation or maintenance of an activated state of macrophages. The activated state of macrophages attracts immune cells to the tumor site, where the macrophages also secrete bispecific antibodies. Bispecific antibodies bind cancer antigens in the tumor site, as well as activating epitopes on recruited immune cells.

[0044] Transforming growth factor β (TGFβ) family proteins are involved in a wide range of regulatory pathways in a wide variety of cell and tissue types and at various stages of normal and pathological processes. In cancer, TGFβ is a pleiotropic cytokine that is found at high levels in solid tumors. TGFβ induces regulatory T cells (Treg) and inhibits CD8+ and TH1 cells in the tumor microenvironment, causing immune dysfunction. See, for example, Ravi et al., Nature Communications 9, 741 (2018). Accordingly, certain embodiments disclosed herein result in a reduction in the level of TGFβ or neutralization of TGFβ in the tumor microenvironment. For example, the nanoparticles described herein can deliver nucleotides encoding a TGFβ inhibitor, such as an anti-TGFβ antibody.

[0045] Aspects of the present invention are now described in more detail in the following sections: (1) Macrophages and Macrophage Phenotypes; (2) Cellular Pathways Affecting Macrophage Polarization; (3) Target Antigens and Associated Binding Domains; (4) Immune Cell Activating Epitopes and Associated Binding Domains; (5) Bispecific Molecule Formats; (6) TGFβ Inhibitors; (7) Nucleotide Sequences; (8) Nanoparticles; (9) Compositions for Administration; (10) Methods of Use; (11) Illustrative Embodiments; (12) Experimental Examples; and (13) Concluding Paragraphs. These headings do not limit the interpretation of the invention and are provided for organizational purposes only.

[0046] (1) Macrophages and Macrophage Phenotypes. As used herein, the term "macrophage" refers to a white blood cell of the immune system differentiated from bone marrow monocytes. Macrophages are characterized by their phagocytic activity and their ability to present antigen. Macrophages are key participants in both innate and adaptive immune responses. Phenotypically, macrophages express the surface marker F4/80 (Ly71) and may also express other surface markers such as CD11b (Macl), CD11c, CD14, CD40, or CD68.

[0047] Macrophages play an important role in both innate and adaptive immunity by activating T lymphocytes. In cancer, macrophages are one of the major populations of infiltrating leukocytes associated with solid tumors (Gordon S & Taylor PR (2005) Nature Reviews Immunology 5(12): 953-964). They can be recruited to the tumor site from surrounding tissues or by the tumor itself through the secretion of chemotactic molecules. Macrophages participate in immune responses to tumors in a polarized manner depending on their phenotype. The term "phenotype" is used herein to refer to the physical traits or biochemical characteristics of a cell resulting from the interaction of its genotype and environment, and may include cellular functions.

[0048] Macrophages that activate Th1 T cells mediate the inflammatory response and are often referred to as having an M1-polarized or "classically activated" phenotype. Activated macrophages (i.e., M1 macrophages or macrophages with an M1 phenotype), also referred to as "killer macrophages," inhibit cell proliferation, cause tissue injury, mediate resistance to pathogens, and possess potent tumoricidal activity. These macrophages can upregulate the expression of mediators responsible for antigen presentation and costimulation, stimulating neutrophil infiltration into the tumor site, leading to neutrophil-mediated tumor regression. The M1 phenotype may also be indicated by increased antigen presentation compared to the corresponding control state. In specific embodiments, the M1 phenotype may be indicated by the production of reactive oxygen species (ROS) and nitric oxide (NO) by M1 macrophages. NO has an antiproliferative effect, which is necessary for protection against pathogens and aberrant cells, such as tumor cells. In specific embodiments, the M1 phenotype may be indicated by a proinflammatory state that induces Th1 immunity by producing cytokines, such as IL-12. In specific embodiments, activated macrophages are classically activated macrophages, which can phagocytose pathogens.

[0049] In addition to function, the M1 phenotype may also be evidenced by surface markers expressed by macrophages; factors, proteins, or compounds produced by macrophages upon polarization; or genes induced by macrophages upon polarization. M1 polarization may result in a phenotype evidenced by the expression of CD80, CD86, iNOS, suppressor of cytokine signaling 3 (SOCS3), TNFα, IL-1, IL-6, IL-12, IL-23, type I IFN, CXCL1, CXCL2, CXCL3, CXCL5, CXCL8, CXCL9, and CXCL10. In particular embodiments, the M1 phenotype includes increased CD80 expression. In particular embodiments, the M1 phenotype includes CD206-, MHCII+, CD11c-, and CD11b+.

[0050] On the other hand, macrophages that activate Th2 T cells mediate an anti-inflammatory response and are often referred to as having an "M2" phenotype. Macrophages in an inactivated state (i.e., M2 macrophages or macrophages with an M2 phenotype), also called "repairing macrophages," are involved in metazoan parasite control, cell proliferation, tissue repair, tumor progression, anti-inflammatory pathways, and immunosuppression. The M2 phenotype can reduce antigen presentation and phagocytosis compared to the corresponding control state. An M2 phenotype may also be indicated by, for example, the expression of one or more of arginase 1 (Arg1 (arginase activity is associated with proliferative effects and tissue repair responses)), IL-10, TGFβ, PPArγ, KLF4, CD206 (MRC1), dectin-1 (a non-TLR pattern recognition receptor), DC-SIGN (a C-type lectin), scavenger receptor A, scavenger receptor B-1, CD163 (high-affinity scavenger receptor for hemoglobin-haptoglobin complex), the chemokine receptors CCR2, CXCR1 and CXCR2, YM1 (chitinase 3-like 3), and Fizz1; and the secretion of the chemokines CCL17, CCL22, and CCL24. In specific embodiments, inactivated macrophages promote metastasis and/or chemotherapy resistance. In specific embodiments, the M2 phenotype comprises CD206+, MHCII-, CD11c+, and ^CD11blow .

[0051] Table 1 presents specific combinations of criteria that can be used to distinguish the M1 phenotype from the M2 phenotype (including subphenotypes designated M2a, M2b, M2c, and M2d).

Table 1. Illustrative criteria for classification of macrophage phenotypes.

M1 M2a M2b M2c M2d Stimulation/Activation IFNγ
LPS
GM-CSF IL-4
IL-13
Fungal and helminthic infection IC
IL-1R IL-10
TGFβ
GC IL-6
LIF
Adenosine Marker expression CD86
CD80
CD68
MHC II
IL-1R
TLR2
TLR4
iNOS
SOCS3
CD28
Gpr18
Fpr2
CD64 CD163
CD23
MHC II
SR
MMR/CD206
CD200R
TGM2
False R
IL-1R II
In mice only:
Ym1/2
Fizz1
Arg-1 CD86
MHC II CD163
TLR1
TLR8 VEGF Cytokine secretion TNF
IL-1β
IL-6
IL-12
IL-23 IL-10
TGFβ
IL-1ra IL-1
IL-6
IL-10 TNFα IL-10
TGFβ IL-10
IL-12
TNFα
TGFβ Secretion of chemokines CCL10
CCL11
CCL5
CCL8
CCL9
CCL2
CCL3
CCL4 CCL17
CCL22
CCL24 CCL1 CCR2 CCL5
CXCL10
CXCL16

Adapted from: Röszer T (2015) Mediators Inflamm 2015, 816460 and Duluc D et al . (2007) Blood 110: 4319–4330. Arg-1, arginase-1; Fizz1, resistin-like molecule alpha (Retnl-alpha); GC, glucocorticoids; IC, immune complexes; IL1-ra, IL-1 receptor antagonist; LIF, leukocyte inhibitory factor; TGM2, transglutaminase 2; TGFβ, transforming growth factor beta; TNFα, tumor necrosis factor alpha; TLR, Toll-like receptor; MMR (CD206), macrophage mannose receptor; iNOS, inducible nitric oxide synthase; SR, scavenger receptor; SOCS3, suppressor of cytokine signaling 3; VEGF, vascular endothelial growth factor; Ym1 (also known as chitinase-3-like protein 3 (Chi3l3)).

[0052] Assays for assessing the phenotype of macrophages can take advantage of the different molecular signatures characteristic of the M1 or M2 phenotype. A commonly accepted marker profile for M1 macrophages is CD80+, while M2 macrophages can be characterized as CD163+. Thus, flow cytometry can be used to assess the presence of these markers. The direction of macrophages to the M1 type and the departure from the M2 type can also be assessed by measuring an increase in the IL-12/IL-10 ratio or the CD163-/CD163+ macrophage ratio. In particular embodiments, the morphology of M1 versus M2 can be assessed by light microscopy. In particular embodiments, a phagocytosis assay can be used in combination with other assays to assess whether a macrophage has the M1 phenotype or the M2 phenotype. Phagocytosis assays for various macrophage populations can be performed by incubating the target cell with macrophages at a concentration corresponding to their normalized total surface area per cell. The target cell can be added to macrophage cultures. The target cell can be labeled, for example, with a fluorescent tag. The phagocytosis index can be determined by the median total fluorescence intensity measured per macrophage. Quantitative assessment of phagocytosis can be performed, for example, by flow cytometry. Tumor cell killing assays can also be used. In specific embodiments, the M1 phenotype comprises reduced expression of M2 macrophage signature genes, including the serpin B2 gene (urokinase-type plasminogen activator inhibitor), CCL2 (C-C motif chemokine ligand 2), CCL11 (C-C motif chemokine ligand 11), and Retn1a (resistin-like alpha; Fizz1). In specific embodiments, the M1 phenotype comprises increased expression of M1 differentiation genes, including CCL5 (C-C motif chemokine ligand 5).

[0053] Gene expression (e.g., expression in M1 of CD80, CD86, and/or other genes noted above) can be measured in assays well known to those skilled in the art. Methods for quantifying gene expression include NanoString ^nCounter® expression assays (NanoString Technologies, Inc., Seattle, WA), Northern blotting, dot blotting, microarrays, serial analysis of gene expression (SAGE), RNA-seq, and quantitative RT-PCR. Methods for quantifying gene product expression, such as protein levels, include ELISA (enzyme-linked immunosorbent assay), Western blotting, FACS, radioimmunoassay (RIA), sandwich assay, fluorescence in situ hybridization (FISH), immunohistological staining, immunoelectrophoresis, immunoprecipitation, and immunofluorescence using detection reagents such as antibody or protein binding reagents.

[0054] Embodiments disclosed herein can be used to genetically modify phagocytic cells such as polymorphonuclear neutrophils, monocytes, monocyte-derived macrophages, tissue macrophages, epithelial cells, fibroblasts, and dendritic cells. Phagocytic cells can be professional or non-professional. Professional phagocytes include polymorphonuclear neutrophils, monocytes, monocyte-derived macrophages, and tissue macrophages. In specific embodiments, the primary function of a professional phagocytic cell is phagocytosis. Non-professional phagocytes include all other cell types that can perform phagocytosis, but it is not considered the primary function of the cell. Examples of non-professional phagocytes include epithelial cells, fibroblasts, and dendritic cells. For more information on professional and non-professional phagocytes, see Lim, Grinstein, and Roth, Frontiers in Cellular and Infection Microbiology, May 2017, Vol. 7, Article 191.

[0055] (2) Cellular pathways influencing macrophage polarization. Macrophage polarization toward an activated or inactivated phenotype occurs as a result of the macrophage's interaction with a number of different molecules or environments. For example, polarization of M1 macrophages is triggered by stimuli including Toll-like receptor (TLR) ligands (e.g., lipopolysaccharide (LPS), muramyl dipeptide, lipoteichoic acid, imiquimod, CpG), IFNγ, TNFα, and macrophage colony-stimulating factor (GM-CSF). Polarized M2 macrophages can be divided into subsets depending on the stimuli that initiate polarization: the M2a subtype is initiated by IL-4, IL-13, or fungal and helminthic infections; M2b is initiated by IL-1 receptor ligands, immune complexes, and LPS; M2c is initiated by IL-10, TGFβ, and glucocorticoids; and M2d is initiated by IL-6 and adenosine. M2 macrophage polarization can also be initiated by IL-21, GM-CSF, complement components, and apoptotic cells. Macrophage polarization is also modulated by local microenvironmental conditions, such as hypoxia.

[0056] The aforementioned molecules and environment influence macrophage polarization by triggering various intracellular signaling pathways involving transcription factors. Transcription factors involved in both M1 and M2 polarization include IRFs, signal transducers and activators of transcription (STATs), SOCS3 proteins, and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB). Mitogen-activated protein kinases (MAPKs) also play a role in directing macrophage function toward the M1 or M2 phenotype.

[0057] The IRF/STAT pathways, activated by stimuli such as IFN and TLR signaling described above, polarize macrophages to the M1 activation state via STAT1. Conversely, stimuli such as IL-4 and IL-13 direct macrophages to the M2 activation state via STAT6 (Sica A & Bronte V (2007) J Clin Invest 117: 1155-1166). Thus, these signaling events lead either to the development of an inflammatory immune response and tumoricidal activity, as in the case of M1 macrophage polarization, or to the development of an immunosuppressive tumor-promoting response, as in the case of M2 macrophage polarization.

[0058] Some intracellular molecules involved in the induction of the M1 phenotype include the G protein-coupled receptor P2Y(2)R, which plays a role in the induction of NO via NOS2 (Eun SY et al. (2014) Int Immunopharmacol 18: 270-276); SOCS3, which activates the NFκB/PI-3 kinase pathway to produce NO (Arnold CE et al. (2014) Immunology 141: 96-110); and the growth and differentiation factor activin A, which stimulates the appearance of M1 markers and reduces IL-10 expression (Sierra-Filardi E et al. (2011) Blood 117: 5092-5101).

[0059] Other intracellular molecules involved in the induction of the M1 phenotype include IRFs. IRFs are a group of transcription factors with diverse functions, including virus-mediated IFN activation, as well as modulation of cell growth, differentiation, apoptosis, and immune system activity. IRF family members are characterized by a conserved N-terminal DNA-binding domain containing tryptophan (W) repeats.

[0060] IRF5 is a transcription factor with a helix-loop-helix DNA-binding motif that mediates viral- and IFN-induced signaling pathways. It acts as a molecular switch that controls whether macrophages promote or inhibit inflammation. IRF5 activates type I IFN genes, inflammatory cytokines including TNF, IL-6, IL-12, and IL-23, tumor suppressors, and Th1 and Th17 responses. It is encoded by the human IRF5 gene located on chromosome 7q32 (OMIM ID 607218). Several isoforms/transcriptional variants of IRF5 are known to exist. In particular embodiments, human IRF5 isoforms include isoform 1 (UniProt Accession No. Q13568-1, SEQ ID NO: 1), isoform 2 (UniProt Accession No. Q13568-2, SEQ ID NO: 2), isoform 3 (UniProt Accession No. Q13568-3, SEQ ID NO: 3), isoform 4 (UniProt Accession No. Q13568-4, SEQ ID NO: 4), isoform 5 (UniProt Accession No. Q13568-5, SEQ ID NO: 5), and isoform 6 (UniProt Accession No. Q13568-6, SEQ ID NO: 6). In particular embodiments, human IRF5 isoforms include isoform 1 encoded by the nucleotide sequence set forth in SEQ ID NO: 23, isoform 2 encoded by the nucleotide sequence set forth in SEQ ID NO: 24, isoform 3 encoded by the nucleotide sequence set forth in SEQ ID NO: 25, isoform 4 encoded by the nucleotide sequence set forth in SEQ ID NO: 26, isoform 5 encoded by the nucleotide sequence set forth in SEQ ID NO: 27, and isoform 6 encoded by the nucleotide sequence set forth in SEQ ID NO: 28. In particular embodiments, mouse IRF5 comprises the amino acid sequence set forth in SEQ ID NO: 7. In particular embodiments, mouse IRF5 is encoded by the nucleotide sequence set forth in SEQ ID NO: 29. It has been shown that M1 Macrophages increase IRF5 expression.

[0061] IRF1 and IRF8 also play important roles in myeloid cell development and function, including macrophage activation by proinflammatory signals such as IFNγ. Dror N et al. (2007) Mol Immunol. 44(4):338–346. In particular embodiments, human IRF1 comprises the amino acid sequence set forth in SEQ ID NO: 8. In particular embodiments, human IRF1 is encoded by the nucleotide sequence set forth in SEQ ID NO: 30. In particular embodiments, mouse IRF1 comprises the amino acid sequence set forth in SEQ ID NO: 12. In particular embodiments, mouse IRF1 is encoded by the nucleotide sequence set forth in SEQ ID NO: 34. In particular embodiments, human IRF8 comprises the amino acid sequence set forth in SEQ ID NO: 11. In particular embodiments, human IRF8 is encoded by the nucleotide sequence set forth in SEQ ID NO: 33. In particular embodiments, mouse IRF8 comprises the amino acid sequence set forth in SEQ ID NO: 16. In particular embodiments, mouse IRF8 is encoded by the nucleotide sequence set forth in SEQ ID NO: 38.

[0062] IRF3 is a homolog of IRF1 and IRF2. It contains several functional domains, including the NES, DBD, C-terminal IRF binding domain, and several regulatory phosphorylation sites. IRF3 exists in an inactive cytoplasmic form that, upon serine/threonine phosphorylation, forms a complex with the CREB binding protein, a transcriptional coactivator. This complex is translocated to the nucleus and activates the transcription of IFNα and β, as well as other interferon-inducible genes. In particular embodiments, human IRF3 isoforms include isoform 1 (UniProt Accession No. Q14653-1), isoform 2 (UniProt Accession No. Q14653-2), isoform 3 (UniProt Accession No. Q14653-3), isoform 4 (UniProt Accession No. Q14653-4), and isoform 5 (UniProt Accession No. Q14653-5). In specific embodiments, human IRF3 isoform 1 comprises the amino acid sequence set forth in SEQ ID NO: 9. In specific embodiments, human IRF3 isoform 1 is encoded by the nucleotide sequence set forth in SEQ ID NO: 31. In specific embodiments, mouse IRF3 comprises the amino acid sequence set forth in SEQ ID NO: 13. In specific embodiments, mouse IRF3 is encoded by the nucleotide sequence set forth in SEQ ID NO: 35.

[0063] IRF7 has been shown to play a role in the transcriptional activation of type I IFN genes. In particular embodiments, human IRF7 isoforms include isoform A (UniProt Accession No. Q92985-1), isoform B (UniProt Accession No. Q92985-2), isoform C (UniProt Accession No. Q92985-3), and isoform D (UniProt Accession No. Q92985-4). In specific embodiments, human IRF7 isoform A comprises the amino acid sequence set forth in SEQ ID NO: 10. In specific embodiments, human IRF7 isoform A is encoded by the nucleotide sequence set forth in SEQ ID NO: 32. In specific embodiments, mouse IRF7 comprises the amino acid sequence set forth in SEQ ID NO: 14. In specific embodiments, mouse IRF7 is encoded by the nucleotide sequence set forth in SEQ ID NO: 36.

[0064] One or more IRF mutants that promote IRF activation can also be used. For example: phosphomimetic mutants of human IRF5 variant 3/variant 4 (isoform 4, SEQ ID NO: 4) in which amino acid residues S425, S427, S430, S436 are replaced with phosphorylation mimic residues such as aspartic acid residues (Chen W et al. (2008) Nat Struct Mol Biol. 15(11): 1213-1220); Phosphomimetic mutants of human IRF5 variant 5 (isoform 2, SEQ ID NO: 2) in which amino acid residues T10, S158, S309, S317, S451, and/or S462 are replaced with phosphorylation mimic residues such as aspartic acid residues (Chang Foreman HC et al. , infra); mutation of residues S156, S158, and T160 of human IRF5 isoform a (variant 1, isoform 3, SEQ ID NO: 3) and isoform b (variant 2, isoform 1, SEQ ID NO: 1) with phosphorylation mimic residues such as aspartic acid residues to constitutively accumulate IRF5 in the nucleus (Lin R et al. (2005) J Biol Chem 280(4): 3088–3095); and IRF3 phosphomimetic mutants in which the S396 amino acid residue of IRF3 is replaced with phosphorylation-mimicking residues such as aspartic acid (Chen W et al. , below). In specific embodiments, the mouse IRF7/IRF3 fusion protein has mutations at Asp(D) in four serine residues and one threonine residue in the IRF3 binding domains (SEQ ID NO: 15), which confers constitutive activation and translocation of the fusion protein (Lin R et al. (1998) supra; Lin et al. (2000) Molecular and Cellular Biology 20: 6342-6353). In specific embodiments, a mouse IRF7/IRF3 fusion protein having mutations to D at four serine residues and one threonine residue in the IRF3 binding domains is encoded by the nucleotide sequence set forth in SEQ ID NO: 37. In specific embodiments, a mouse IRF8 mutant has a lysine (K) substitution at amino acid position 310 to arginine (R) (SEQ ID NO: 17). In specific embodiments, a mouse IRF8 mutant having a K substitution at amino acid position 310 to R is encoded by the nucleotide sequence set forth in SEQ ID NO: 39. Small ubiquitin-like modifiers (SUMOs), which bind to IRF8 primarily at position K310, inhibit the activation of IRF8 responsive genes. Sentrin-specific protease 1 (SENP1) targets SUMO 2/3. SENP1 activity "deprives" IRF8 (and other proteins) of SUMO and induces the transition of IRF8 from a repressor of M1 macrophage differentiation to an activator (directly and through transactivation activity). Preventing SUMO binding to IRF8 by mutating the K310 residue increases specific transcription of the IRF8 gene by 2-5-fold (see Chang TH et al. (2012), above).

[0065] Specific embodiments of the present invention include genetically modified IRF transcription factors. In specific embodiments, the genetically modified IRF transcription factors include IRF that lacks a functional autoinhibitory domain and is therefore insensitive to feedback inactivation (Thompson et al. (2018) Front Immunol 9: 2622). For example, human IRF5 with 2-3-fold increased activity can be produced by deleting aa 489-539 in the human IRF5 protein (Barnes et al. (2002) Mol Cell Biol 22: 5721-5740). In specific embodiments, the autoinhibitory domain of IRF4, a transcription factor that promotes the M2 phenotype, can be deleted or mutated to create a more active IRF4 in the context of treating an autoimmune disease. In specific embodiments, the autoinhibitory domain of IRF is at the carboxyl terminus of the IRF protein. In specific embodiments, genetically modified IRF transcription factors include IRFs lacking one or more functional nuclear export signals (NESs) to retain IRFs in the nucleus and thereby increase transcription levels. For example, nuclear accumulation of human IRF5 can be achieved by mutating the NES in human IRF5 by replacing two leucine residues with alanine (L157A/L159A) (Lin et al. (2000) Molecular and Cellular Biology 20: 6342-6353). In specific embodiments, genetically modified IRF transcription factors include fusion proteins of one or more IRFs, fusion proteins of fragments of one or more IRFs, and fusion proteins of mutant IRFs.

[0066] NFκB is also a key transcription factor associated with M1 macrophage activation. NFκB regulates the expression of a large number of inflammation-related genes, including TNFα, IL1B, cyclooxygenase 2 (COX-2), IL-6, and IL12p40. NFκB activity is modulated by activation of the inhibitor of kappa B kinase (IKK) ternary complex (two kinases, IKKα, IKKβ, and a regulatory protein, IKKγ). When incoming signals converge at the IKK complex, they first activate IKKβ by phosphorylation, and activated IKKβ further phosphorylates an inhibitory molecule, inhibitor of kappa B (I-κB). This leads to proteasomal degradation of I-κB and the release of the NFκB p65/p50 heterodimer from the NFκB/I-κB complex. The NFκB p65/p50 heterodimer is then translocated to the nucleus and binds to the promoters of inflammation-related genes.

[0067] IKKβ is an activating kinase for NFκB, as well as other transcription factors such as IRF5. IKKβ also phosphorylates several other components of the signaling pathway, including FOXO3, NCOA3, BCL10, IRS1, NEMO/IKBKG, the NFκB subunits RELA and NFKB1, and the IKK-related kinases TBK1 and IKBKE. In particular embodiments, human IKKβ isoforms include isoform 1 (UniProt Accession No. O14920-1, SEQ ID NO: 18), isoform 2 (UniProt Accession No. O14920-2, SEQ ID NO: 19), isoform 3 (UniProt Accession No. O14920-3, SEQ ID NO: 20), and isoform 4 (UniProt Accession No. O14920-4, SEQ ID NO: 21). In specific embodiments, human IKKβ isoforms include isoform 1 encoded by the nucleotide sequence set forth in SEQ ID NO: 40, isoform 2 encoded by the nucleotide sequence set forth in SEQ ID NO: 41, isoform 3 encoded by the nucleotide sequence set forth in SEQ ID NO: 42, and isoform 4 encoded by the nucleotide sequence set forth in SEQ ID NO: 43. In specific embodiments, mouse IKKβ comprises the amino acid sequence set forth in SEQ ID NO: 22. In specific embodiments, mouse IKKβ is encoded by the nucleotide sequence set forth in SEQ ID NO: 44.

[0068] As indicated, hypoxia also influences macrophage polarization through the hypoxia-inducible factors HIF-1α and HIF-2α. HIF-1α regulates NOS2 expression and supports the emergence of the M1 phenotype, while HIF-2α regulates Arg1 expression and supports the emergence of the M2 phenotype (Takeda N et al. (2010) Genes Dev 24: 491-501).

Table 2. Signaling molecules and genes involved in macrophage polarization. M1 M2 Signaling molecules STAT1 alpha/beta
IRF5
Btk
P2Y(2)R
SOCS3
Activin A
HIF1-α STAT6
KLF-4
NFκB p50 homodimers
PPARγ
HIF-2α
IL-21
BMP-7
FABP4
LXRα Genes TNFα, Cox-2, CCL5, NOS2 Arg-1, Mrc-1, Fizz1, PPARγ

Adapted from Sica A and Mantovani A 2012 (above) and Chávez-Galán L et al. (2015) Front Immunol 6, 253. Arg-1, arginase-1; Fizz1, resistin-like molecule alpha (Retnl-alpha); STATs, signal transducers and activators of transcription; IRF, interferon-regulatory factor; SOCS3, suppressor of cytokine signaling 3; Btk, Bruton's tyrosine kinase; HIF-1α, hypoxia-inducible factor 1; KLF-4, Krüppel-like factor 4; TNFα, tumor necrosis factor alpha; BMP-7, bone morphogenetic protein 7; P2Y(2)R, P2Y purinergic receptor 2; PPARγ, peroxisome proliferator-activated receptor γ; NFκB, nuclear factor kappa B; FABP4, fatty acid-binding protein 4; LXRα; liver X receptor alpha.

[0069] The present invention relates to the co-expression of IRF transcription factors with one or more molecules capable of activating IRF to reprogram a TAM to an activated state to kill a tumor. In particular embodiments, co-expression strategies include: co-expressing IRF5 and IKKβ; co-expressing IRF5 and TANK-binding kinase 1 (TBK-1), TNF receptor-associated factor adaptor 6 (TRAF6), receptor-interacting protein kinase 2 (RIP2), and/or NFκB kinase-ε (IKKε) (Chang Foreman HC et al . (2012) PLoS One 7(3): e33098); co-expression of IRF5 and DNA-PK protein kinase (Ryzhakov G et al. (2015) J of Interferon & Cytokine Res 35 (2):71–78); co-expression of IRF5 and protein tyrosine kinase BCR-ABL (Massimo M et al . (2014) Carcinogenesis 35(5):1132–1143); and co-expression of IRF5 or IRF8 with one or more components of the COP9 signalosome (Korczeniewska J et al . (2013) Mol Cell Biol 33(6):1124–1138; Cohen H et al . (2000) J Biol Chem 275(50):39081–39089).

[0070] (3) Target Antigens and Associated Binding Domains. The antigens described herein refer to proteins expressed by cell types of interest. Cells or cell types of interest include any predetermined cell type that can be recognized and destroyed by the immune system. In some embodiments, cell types of interest for the present invention are cell types that have an adverse, detrimental, or otherwise undesirable effect (or are predisposed to such an effect) on the health, viability, or well-being of a subject. Cells of interest can include, for example, (i) eukaryotic cells that are cancerous or infected with a pathogen such as a virus, and (ii) prokaryotic cells such as certain bacteria, fungi, or yeast. Cells of interest also include autoreactive cells that can be harmful and/or cause autoimmunity. Such autoreactive cells of interest include, for example, autoreactive immune cells, autoreactive lymphocytes, autoreactive T cells, and autoreactive B cells. Autoreactive cells of interest may also be self-reactive cells programmed during development to control the immune response, such as regulatory T cells. Autoreactive cells contribute to the development of autoimmune conditions in subjects, for example, by inappropriately recognizing and binding self-antigens. In some embodiments, a cell type that can be harmful if overrepresented in a localized or circulating cell population may be a cell type of interest for the invention. For example, an inflammatory response may result in overrepresentation of immune cells, in which case the cells of interest may include, for example, neutrophils or mast cells. Additionally, in some cases, the cells of interest may be cells previously administered as part of a treatment, such as genetically modified cells (e.g., cells expressing a chimeric antigen receptor (CAR)).

[0071] In particular embodiments, antigens are preferentially expressed by cells of interest. The phrase "preferentially expressed" means that the antigen is detected at higher levels on cells of interest compared to other cell types. In some cases, the antigen is expressed only by cells of interest. In other cases, the antigen is expressed on cells of interest at least 25%, 35%, 45%, 55%, 65%, 75%, 85%, 95%, 96%, 97%, 98%, 99%, or 100% more than on other cell types.

[0072] The following are examples of cancer cell antigens associated with various types of cancer:

Type of cancer Associated cancer antigens Ovarian cancer EpCam, L1-CAM, MUC16, folate receptor (FOLR), Lewis Y, ROR1, mesothelin, WT-1, PD-L1, EGFR, CD56 Melanoma Tyrosinase-related protein 1 (TYRP1/gp75); GD2, PD-L1, EGFR Glioblastoma EGFR variant III (EGFRvIII), IL13Ra2 Multiple myeloma B-cell maturation antigen (BCMA), PD-L1, EGFR Prostate cancer PSMA, WT1, prostate stem cell antigen (PSCA), SV40 T, PD-L1, EGFR Breast cancer HER2, ERBB2, ROR1, PD-L1, EGFR, MUC16, FOLR, CEA Cancer from stem cells CD133, PD-L1, EGFR Mesothelioma mesothelin, PD-L1, EGFR Renal cell carcinoma Carboxyanhydrase IX (CAIX); PD-L1, EGFR Pancreatic cancer mesothelin, CEA, CD24, ROR1, PD-L1, EGFR, MUC16 Lung cancer ROR1, PD-L1, EGFR, mesothelin, MUC16, FOLR, CEA, CD56 Cholangiocarcinoma mesothelin, PD-L1, EGFR Bladder cancer MUC16, PD-L1, EGFR Neuroblastoma ROR1, glypican-2, CD56, disialoganglioside, PD-L1, EGFR Colorectal cancer CEA, PD-L1, EGFR Merkel cell carcinoma CD56, PD-L1, EGFR

[0073] Exemplary binding domains for cancer cell antigens may be generated de novo or derived from known antibodies or binding domains specific for the selected cancer antigen.

[0074] Epithelial cell adhesion molecule (EpCam; also called EGP-40, Trop-1, 17-1A, KSA, KS1/4, AUA1, GA733-2, and CD326) is overexpressed in several cancers, including ovarian cancer. It is a 40-kDa surface glycoprotein that has an extracellular domain with two EGF-like repeats. Antibodies directed against EpCam are commercially available (Richter et al., Am. J. Obstet. Gynecol. 2010, 203(6): 582.e1-582e7). Exemplary antibodies that bind EpCam include MT201 (adecatumumab) and edrecolomab.

[0075] Tyrosinase-related protein 1, or glycoprotein gp75 (TYRP1/gp75), is a melanosomal protein implicated in melanocyte malignancy and melanoma progression (Ghanem et al., Mol. Oncol. 2011 April; 5(2): 150-155). Illustrative antibodies that bind TYRP1/gp75 include TA99 (Saenger, et al., Cancer Research, 68(23): 9884–9891, 2008), 20D7 (Patel, et al., IOS Press, 16(3–4): 127–1036, 2007), and flanvotumab (IMC-20D7S) (Khalil, et al., Clinical Cancer Research, 22(21): 5204–5210, 2016.

[0076] In specific embodiments, an antibody that binds TYRP1/gp75 is described in US7951370. In specific embodiments, an antibody that binds TYRP1/gp75 has a CDRL1 sequence comprising RASQSVSSYLA (SEQ ID NO: 84), a CDRL2 sequence comprising DASNRAT (SEQ ID NO: 85), a CDRL3 sequence comprising QQRSNWLMYT (SEQ ID NO: 253), a CDRH1 sequence comprising GYTFTSYAMN (SEQ ID NO: 254), a CDRH2 sequence comprising WINTNTGNPTYAQGFTG (SEQ ID NO: 255), and a CDRH3 sequence comprising RYSSSWYLDY (SEQ ID NO: 256).

[0077] In particular embodiments, an antibody that binds TYRP1/gp75 has a CDRL1 sequence comprising RASGNIYNYLA (SEQ ID NO: 257), a CDRL2 sequence comprising DAKTLAD (SEQ ID NO: 258), a CDRL3 sequence comprising QHFWSLPFT (SEQ ID NO: 259), a CDRH1 sequence comprising GFNIKDYFLH (SEQ ID NO: 260), a CDRH2 sequence comprising WINPDNGNTVYDPKFQG (SEQ ID NO: 261), and a CDRH3 sequence comprising DYTYEKAALDY (SEQ ID NO: 262).

[0078] In specific embodiments, the TYRP1/gp75-binding antibodies have a light chain variable region comprising the sequence:

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWLMYTFGQGTKLEIK (SEQ ID NO: 263), and a heavy chain variable region comprising the sequence

QVQLVQSGSELKKPGASVKISCKASGYTFTSYAMNWVRQAPGQGLECMGWINTNTGNPTYAQGFTGRFVFSMDTSVSTAYLQISSLKAEDTAIYYCAPRYSSSWYLDYWGQGTLVTVSS (SEQ ID NO: 264), or a heavy chain variable region comprising the sequence

QVQLVQSGSELKKPGASVKISCKASGYTFTSYAMNWVRQAPGQGLESMGWINTNTGNPTYAQGFTGRFVFSMDTSVSTAYLQISSLKAEDTAIYYCAPRYSSSWYLDYWGQGTLVTVSS (SEQ ID NO: 265).

[0079] Illustrative antibodies with binding domains that bind mesothelin include anetumab ravtansine, amatuximab, and HN1.

[0080] In particular embodiments, the HN1 antibody has a CDRL1 sequence comprising RASEGIYHWLA (SEQ ID NO: 55), a CDRL2 sequence comprising KASSLAS (SEQ ID NO: 58), a CDRL3 sequence comprising QQYSNYPLT (SEQ ID NO: 61), a CDRH1 sequence comprising TYYMQ (SEQ ID NO: 64), a CDRH2 sequence comprising VINPSGVTSYAQKFQG (SEQ ID NO: 71), and a CDRH3 sequence comprising WALWGDFGMDV (SEQ ID NO: 73).

[0081] US8206710 describes mesothelin-binding antibodies having: a light chain variable region comprising the sequence

MGWSCIILFLVATATGVHSDIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTF GSGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 79)

and a heavy chain variable region containing the sequence

MGWSCIILFLVATATGVHSQVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCAR GGYDGRGFDYWGSGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* (SEQ ID NO: 112);

and also an antibody having a light chain variable region containing the sequence

MGWSCIILFLVATATGVHSEIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLIYDTSKLASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWSKHPLTF GSGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 113)

and a heavy chain variable region containing the sequence

MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLITPYNGASSYNQKFRGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR GGYDGRGFDYWGSGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 114).

[0082] Additional mesothelin-binding antibodies are described in US8911732, US7081518, US8357783 and US8425904.

[0083] MUC16-binding domains can be derived from the antibodies oregovomab, ovarex, and abagovomab. US 7,723,485 describes a MUC16-binding antibody having a light chain variable region comprising the sequence

DIQMTQSPSSLSASVGDRVTITGRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 115)

and a heavy chain variable region containing the sequence

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVHQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIGNVNHKPSNTKVDKKVEPKSCDK THTGPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 116).

[0084] WO2016149368 describes a MUC16-binding antibody having a light chain variable region comprising a CDRL1 sequence comprising SEDIYSG (SEQ ID NO: 117), a CDRL2 sequence comprising GAS, a CDRL3 sequence comprising GYSYSSTL (SEQ ID NO: 118), a CDRH1 sequence comprising TLGMGVG (SEQ ID NO: 119), a CDRH2 sequence comprising HIWWDDDKYYNPALKS (SEQ ID NO: 120), and a CDRH3 sequence comprising IGTAQATDALDY (SEQ ID NO: 121).

[0085] The folate receptor binding antibody includes farletuzumab. In specific embodiments, farletuzumab is described in US9133275. In particular embodiments, farletuzumab has a light chain variable region comprising a CDRL1 sequence comprising KASQSVSFAGTSLMH (SEQ ID NO: 122), a CDRL2 sequence comprising RASNLEA (SEQ ID NO: 123), and a CDRL3 sequence comprising QQSREYPYT (SEQ ID NO: 124), and a heavy chain variable region comprising a CDRH1 sequence comprising GYFMN (SEQ ID NO: 125), a CDRH2 sequence comprising RIHPYDGDTFYNQKFQG (SEQ ID NO: 126), and a CDRH3 sequence comprising YDGSRAMDY (SEQ ID NO: 127). Additional FOLR-binding antibodies are described in US10101343B2, US8388972, and US8709432.

[0086] An exemplary anti-EGFR antibody is cetuximab. In particular embodiments, cetuximab is described in US7598350. In particular embodiments, cetuximab has a light chain variable region comprising a CDRL1 sequence comprising RASQSVSSYLA (SEQ ID NO: 84), a CDRL2 sequence comprising DASNRAT (SEQ ID NO: 85), a CDRL3 sequence comprising HQYGSTPLT (SEQ ID NO: 130), a CDRH1 sequence comprising SGDYYWS (SEQ ID NO: 131), a CDRH2 sequence comprising YIYYSGSTDYNPSLKS (SEQ ID NO: 132), and a CDRH3 sequence comprising VSIFGVGTFDY (SEQ ID NO: 133).

[0087] Additional EGFR-binding domains are described in US7247301, US7723484, US7132511 and US5844093. US7723484, in particular, describes an EGFR-binding antibody having a light chain variable region comprising the sequence

EIVLTQSPDFQSVTPKEKVTITCRASYSIGTNIHWYQQKPDQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQNNNWPTTFGGGTKVEIK (SEQ ID NO: 134),

and a heavy chain variable region containing the sequence

QVTLKESGPVLVKPTETLTLTCTVSGFSLSNWDVHWIRQPPGKALEWLAVIWSGGATDYNTPFNSRLTISKDTSKSQVVLTMTNMDPVDTATYYCARALDYYDYNFAYWGQGTMVTVSS (SEQ ID NO: 135).

[0088] CD19-binding domains are found in the antibodies FMC63, SJ25C1, and HD37. (SJ25C1: Bejcek et al. Cancer Res 2005, PMID 7538901; HD37: Pezutto et al. JI 1987, PMID 2437199). In particular embodiments, the FMC63 CDRs comprise a CDRL1 sequence comprising RASQDISKYLN (SEQ ID NO: 136), a CDRL2 sequence comprising SRLHSGV (SEQ ID NO: 137), a CDRL3 sequence comprising GNTLPYTFG (SEQ ID NO: 138), a CDRH1 sequence comprising DYGVS (SEQ ID NO: 139), a CDRH2 sequence comprising VTWGSETTYYNSALKS (SEQ ID NO: 140), and a CDRH3 sequence comprising YAMDYWG (SEQ ID NO: 141).

[0089] A number of antibodies specific for RORI are also known to those skilled in the art and can be readily characterized with respect to sequence, epitope binding, and affinity. See, for example, WO2008076868, WO/2008103849, WO201008069, WO2010124188, WO2011079902, WO2011054007, WO2011159847, WO2012076066, WO2012076727, WO2012045085, and WO2012097313.

[0090] Specific examples of antibodies that bind ROR1 include R11, R12, 2A2, and Y31.

[0091] Antibody R11 has a CDRL1 sequence including QASQSIDSNLA (SEQ ID NO: 142), a CDRL2 sequence including RASNLAS (SEQ ID NO: 143), a CDRL3 sequence including LGGVGNVSYRTS (SEQ ID NO: 144), a CDRH1 sequence including DYPIS (SEQ ID NO: 145), a CDRH2 sequence including FINSGGSTWYASWVKG (SEQ ID NO: 146), and a CDRH3 sequence including GYSTYYCDFNI (SEQ ID NO: 147).

[0092] Antibody R12 has a CDRL1 sequence including TLSSAHKTDTID (SEQ ID NO: 148), a CDRL2 sequence including GSYTKRP (SEQ ID NO: 149), a CDRL3 sequence including GADYIGGYV (SEQ ID NO: 150), a CDRH1 sequence including AYYMS (SEQ ID NO: 151), a CDRH2 sequence including TIYPSSGKTYYATWVNG (SEQ ID NO: 152), and a CDRH3 sequence including DSYADDGALFNI (SEQ ID NO: 153).

[0093] Antibody 2A2 has a CDRL1 sequence including KASQNVDAAVA (SEQ ID NO: 154), a CDRL2 sequence including SASNRYT (SEQ ID NO: 155), a CDRL3 sequence including QQYDIYPYT (SEQ ID NO: 156), a CDRH1 sequence including DYEMH (SEQ ID NO: 157), a CDRH2 sequence including AIDPETGGTAYNQKFKG (SEQ ID NO: 158), and a CDRH3 sequence including YYDYDSFTY (SEQ ID NO: 159).

[0094] Antibody Y31 has a CDRL1 sequence including QASQSIGSYLA (SEQ ID NO: 160), a CDRL2 sequence including YASNLAS (SEQ ID NO: 161), a CDRL3 sequence including LGSLSNSDNV (SEQ ID NO: 162), a CDRH1 sequence including SHWMS (SEQ ID NO: 163), a CDRH2 sequence including IIAASGSTYYANWAKG (SEQ ID NO: 164), and a CDRH3 sequence including DYGDYRLVTFNI (SEQ ID NO: 165).

[0095] The Her2 binding domain can be obtained from the 4D5 antibody. The 4D5 antibody has a CDRL1 sequence comprising RASQDVNTAVAW (SEQ ID NO: 166), a CDRL2 sequence comprising YSASFLES (SEQ ID NO: 167), a CDRL3 sequence comprising QQHYTTPT (SEQ ID NO: 168), a CDRH1 sequence comprising SGFNTKDTYIHW (SEQ ID NO: 169), a CDRH2 sequence comprising RIYPTNGYTRYADSVKGR (SEQ ID NO: 170), and a CDRH3 sequence comprising WGGDGFYAMDV (SEQ ID NO: 171).

[0096] PD-L1-binding antibodies include antibody 3G10 and the antibodies described in US 2016/0222117. In particular embodiments, the binding domains derived from antibody 3G10 comprise a CDRL1 sequence comprising RASQSVSSYL (SEQ ID NO: 172), a CDRL2 sequence comprising DASNRAT (SEQ ID NO: 85), a CDRL3 sequence comprising QQRSNWPRT (SEQ ID NO: 173), a CDRH1 sequence comprising DYGFS (SEQ ID NO: 174), a CDRH2 sequence comprising WITAYNGNTNYAQKLQG (SEQ ID NO: 175), and a CDRH3 sequence comprising DYFYGMDY (SEQ ID NO: 176).

[0097] The PD-L1 binding domains may also comprise a CDRL1 sequence comprising RASQDVSTAVA (SEQ ID NO: 177), a CDRL2 sequence comprising SASFLYS (SEQ ID NO: 178), a CDRL3 sequence comprising QQYLYHPAT (SEQ ID NO: 179), a CDRH1 sequence comprising SGFTFSDSWIH (SEQ ID NO: 180), a CDRH2 sequence comprising WISPYGGSTYYADSVKG (SEQ ID NO: 181), and a CDRH3 sequence comprising RHWPGGFDY (SEQ ID NO: 182), or (ii) a CDRL1 sequence comprising TGTSSDVGGYNYVS (SEQ ID NO: 183), a CDRL2 sequence comprising DVSNRPS (SEQ ID NO: 184), a CDRL3 sequence including SSYTSSSTRV (SEQ ID NO: 185), a CDRH1 sequence including SGFTFSSYIMM (SEQ ID NO: 186), a CDRH2 sequence including SIYPSGGITFYADTVKG (SEQ ID NO: 187), and a CDRH3 sequence including IKLGTVTTVDY (SEQ ID NO: 188).

[0098] Additional antibodies with PD-L1-binding domains include atezolizumab, avelumab, and durvalumab.

[0099] In particular embodiments, antigens are expressed by virus-infected cells. Exemplary viruses include adenoviruses, arenaviruses, bunyaviruses, coronaviruses, flaviviruses, hantaviruses, hepadnaviruses, herpesviruses, papillomaviruses, paramyxoviruses, parvoviruses, picornaviruses, poxviruses, orthomyxoviruses, retroviruses, reoviruses, rhabdoviruses, rotaviruses, spongiform viruses, or togaviruses. In additional embodiments, the viral antigens include peptides expressed by CMV, cold viruses, Epstein-Barr, influenza viruses, hepatitis A, B and C viruses, herpes simplex, HIV, influenza, Japanese encephalitis, measles, polio, rabies, respiratory syncytial viruses, rubella viruses, smallpox, varicella, or West Nile fever virus.

[0100] As further specific examples, coronavirus antigens include the spike (S) protein, cytomegalovirus antigens include envelope glycoprotein B and CMV pp65; Epstein-Barr virus antigens include EBV EBNAI, EBV P18, and EBV P23; hepatitis antigens include hepatitis B virus S, M, and L proteins, hepatitis B virus pre-S antigen, HBCAG DELTA, HBV HBE, hepatitis C viral RNA, HCV NS3, and HCV NS4; herpes simplex virus antigens include immediate early proteins and glycoprotein D; HIV antigens include the gene products of the gag, pol, and env genes, such as HIV gp32, HIV gp41, HIV gp120, HIV gp160, HIV P17/24, HIV P24, HIV P55 GAG, HIV P66 POL, HIV TAT, HIV GP36, Nef protein, and reverse transcriptase; Influenza antigens include hemagglutinin and neuraminidase; Japanese encephalitis virus antigens include the E, ME, ME-NS1, NS1, NS1-NS2A, and 80% E proteins; Measles virus antigens include the measles virus fusion protein; Rabies antigens include the rabies glycoprotein and rabies nucleoprotein; Respiratory syncytial virus antigens include the RSV fusion protein and the M2 protein; Rotavirus antigens include VP7sc; Rubella antigens include the E1 and E2 proteins; and varicella-zoster virus antigens include gpI and gpII. See Fundamental Virology, 3rd edition, eds. Fields, BN and Knipe, DM (Raven Press, New York, 1991) for additional examples of viral antigens.

[0101] In particular embodiments, the antigens are expressed by cells associated with bacterial infections. Illustrative bacteria include anthrax; gram-negative bacilli, chlamydia, diphtheria, Haemophilus influenzae, Helicobacter pylori , malaria, mycobacterium tuberculosis, whooping cough, pneumococci, rickettsia, staphylococci, streptococci, and tetanus.

[0102] As specific examples of bacterial antigens, anthrax antigens include anthrax protective antigen; Gram-negative bacilli antigens include lipopolysaccharides; Haemophilus influenzae antigens include capsular polysaccharides; diphtheria antigens include diphtheria toxin; Mycobacterium tuberculosis antigens include mycolic acid, heat shock protein 65 (HSP65), major secreted 30-kDa protein, and antigen 85A; pertussis antigens include hemagglutinin, pertactin, FIM2, FIM3, and adenylate cyclase; pneumococcal antigens include pneumolysin and pneumococcal capsular polysaccharides; rickettsia antigens include rompA; streptococcal antigens include M proteins; and tetanus antigens include tetanus toxin.

[0103] Superbugs. In particular embodiments, lymphocytes are modified to target multidrug-resistant "superbugs." Examples of superbugs include Enterococcus faecium, Clostridium difficile, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae (including Escherichia coli, Klebsiella pneumoniae, Enterobacter spp.).

[0104] In particular embodiments, the antigens are expressed by cells associated with fungal infections. Illustrative fungi include Candida, Coccidioides, Cryptococci, Histoplasma, Leishmania, Plasmodium, Protozoa, Parasites, Schistosomes, Herpes Zoster, Toxoplasma, and Trypanosoma cruzi .

[0105] As further specific examples of fungal antigens, coccidioides antigens include spherule antigens; cryptococcal antigens include capsular polysaccharides; histoplasma antigens include heat shock protein 60 (HSP60); leishmania antigens include gp63 and lipophosphoglycan; Plasmodium falciparum antigens include merozoite surface antigens, sporozoite surface antigens, circumsporozoite antigens, gametocyte/gamete surface antigens, protozoan antigens, and other parasitic antigens including the hemostage antigen pf 155/RESA; schistosome antigens include glutathione S-transferase and paramyosin; ringworm antigens include trichophytin; toxoplasma antigens include SAG-1 and p30; and Trypanosoma cruzi antigens include the 75-77 kDa antigen and the 56 kDa antigen.

[0106] In specific embodiments, the antigens are expressed by cells associated with autoimmune or allergic conditions. Illustrative autoimmune conditions include acute necrotizing hemorrhagic encephalopathy, allergic asthma, alopecia areata, anemia, aphthous ulcer, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), asthma, autoimmune thyroiditis, celiac disease, conjunctivitis, Crohn's disease, cutaneous lupus erythematosus, dermatitis (including atopic dermatitis and eczematous dermatitis), diabetes, diabetes mellitus, erythema nodosum leprosy, keratoconjunctivitis, multiple sclerosis, myasthenia gravis, psoriasis, scleroderma, Sjogren's syndrome including keratoconjunctivitis sicca due to Sjogren's syndrome, Stevens-Johnson syndrome, systemic lupus erythematosus, ulcerative colitis, vaginitis and Wegener's granulomatosis.

[0107] Examples of autoimmune antigens include glutamic acid decarboxylase 65 (GAD 65), native DNA, myelin basic protein, myelin proteolipid protein, acetylcholine receptor components, thyroglobulin, and the thyroid-stimulating hormone (TSH) receptor. Examples of allergic antigens include pollen antigens such as Japanese cedar pollen antigens, ragweed pollen antigens, perennial ryegrass pollen antigens, animal-derived antigens (such as dust mite antigens and cat antigens), histocompatibility antigens, and penicillin and other therapeutic drugs.

[0108] Binding domains for antigens expressed by cells of interest can also be derived from T cell receptors (TCRs). There are numerous ways to identify and select specific TCRs for use as binding domains. For example, the sequences of numerous TCRs that bind specific antigen fragments are known and publicly available.

[0109] Useful TCRs can also be identified by isolating T cells that bind a specific antigen and sequencing the TCR chains. As examples, antigen-specific T cells can be induced by in vitro culturing of isolated human T cells in the presence of an antigen/MHC complex. TCR genes encoding TCRs that bind the antigen/MHC complex can be readily cloned, for example, by the 5' RACE method using primers corresponding to sequences specific for the TCR α-chain gene and the TCR β-chain gene.

[0110] In certain embodiments, pairing of the TCR chains after sequencing may be required (i.e., to perform paired chain analysis). Various methods can be used to pair individual α and β chains. In certain embodiments, pairing after sequencing may be unnecessary or relatively simple, such as in embodiments in which information about the pairing of the α and β chains is not lost during the procedure, such as during sequencing from single cells. Chain pairing can also be performed using multi-well sequencing. Assays such as ^PairSEQ® (Adaptive Biotechnologies Corp., Seattle, WA) have also been developed.

[0111] For specific examples of TCRs that can be used in the context of the present invention, see, for example, WO2018/129270; WO2017/112944; WO2011/039507; US 8008438; US2016/0083449; US2015/0246959; Stromnes, et al. (2015) Cancer cell 28(5): 638-652; Kobayashi, et al. (2013) Nature Medicine 19: 1542-1546); Varela-Rohena, et al. (2008) Nature Medicine. 14(12): 1390-1395); and Robbins et al. (2008) The Journal of Immunology 180(9): 6116-6131.

[0112] (4) Epitopes that activate immune cells and associated binding domains. Immune cells that can serve as targets for localized activation include, for example, T cells and natural killer (NK) cells.

[0113] T cell activation can be mediated by two distinct signals: those that initiate antigen-dependent primary activation and provide a T cell receptor-like signal (primary cytoplasmic signal sequences), and those that act in an antigen-independent manner, providing a secondary or costimulatory signal (secondary cytoplasmic signal sequences). A combination of bispecific antibodies can be directed to any combination of T cell activating epitopes that, upon binding, induce T cell activation. Examples of such T-cell activating epitopes are found on T-cell markers, including CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, 4-1BB (CD 137), OX40, lymphocyte function-associated antigen 1 (LFA-1), LIGHT, NKG2C, and B7-H3. T-cell suppressor receptors that can be blocked include PD-1, LAG3, TIM-3, BTLA, CTLA-4, and CD200. Antibodies with PD-1-binding domains include pembrolizumab and nivolumab, while CTLA-4 blocking antibodies include ipilimumab.

[0114] CD3 is a core element of T cell receptor signal transduction and is expressed on all mature T cells. Binding domains for CD3 can be derived from, for example, OKT3, 20G6-F3, 4B4-D7, 4E7-C9, and 18F5-H10.

[0115] OKT3 is described in U.S. Patent No. 5,929,212. The OKT3 antibody has a CDRL1 sequence comprising SASSSVSYMN (SEQ ID NO: 189), a CDRL2 sequence comprising RWIYDTSKLAS (SEQ ID NO: 190), a CDRL3 sequence comprising QQWSSNPFT (SEQ ID NO: 191), a CDRH1 sequence comprising KASGYTFTRYTMH (SEQ ID NO: 192), a CDRH2 sequence comprising INPSRGYTNYNQKFKD (SEQ ID NO: 193), and a CDRH3 sequence comprising YYDDHYCLDY (SEQ ID NO: 194).

[0116] The following sequence represents an OKT3-derived scFv that retains the ability to bind CD3:

QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINR (SEQ ID NO: 195). It can be used as a CD3-binding domain.

[0117] Antibody 20G6-F3 has a CDRL1 sequence including QSLVHNNGNTY (SEQ ID NO: 196), a CDRL2 sequence including KVS, a CDRL3 sequence including GQGTQYPFT (SEQ ID NO: 197), a CDRH1 sequence including GFTFTKAW (SEQ ID NO: 198), a CDRH2 sequence including IKDKSNSYAT (SEQ ID NO: 199), and a CDRH3 sequence including RGVYYALSPFDY (SEQ ID NO: 200).

[0118] Antibody 4B4-D7 has a CDRL1 sequence including QSLVHDNGNTY (SEQ ID NO: 201), a CDRL2 sequence including KVS, a CDRL3 sequence including GQGTQYPFT (SEQ ID NO: 197), a CDRH1 sequence including GFTFSNAW (SEQ ID NO: 202), a CDRH2 sequence including IKARSNNYAT (SEQ ID NO: 203), and a CDRH3 sequence including RGTYYASKPFDY (SEQ ID NO: 204).

[0119] Antibody 4E7-C9 has a CDRL1 sequence including QSLEHNNGNTY (SEQ ID NO: 205), a CDRL2 sequence including KVS, a CDRL3 sequence including GQGTQYPFT (SEQ ID NO: 197), a CDRH1 sequence including GFTFSNAW (SEQ ID NO: 202), a CDRH2 sequence including IKDKSNNYAT (SEQ ID NO: 206), and a CDRH3 sequence including RYVHYGIGYAMDA (SEQ ID NO: 207).

[0120] Antibody 18F5-H10 has a CDRL1 sequence including QSLVHTNGNTY (SEQ ID NO: 208), a CDRL2 sequence including KVS, a CDRL3 sequence including GQGTHYPFT (SEQ ID NO: 209), a CDRH1 sequence including GFTFTNAW (SEQ ID NO: 210), a CDRH2 sequence including KDKSNNYAT (SEQ ID NO: 211), and a CDRH3 sequence including RYVHYRFAYALDA (SEQ ID NO: 212).

[0121] Further examples of anti-CD3 antibodies, binding domains and CDR regions can be found in WO2016/116626. TR66 can also be used.

[0122] CD28 is a surface glycoprotein present on 80% of peripheral T cells in humans and is present on both resting and activated T cells. CD28 binds B7-1 (CD80) and B7-2 (CD86) and is the most potent known costimulatory molecule (June et al., Immunol. Today 15:321 (1994); Linsley et al., Ann. Rev. Immunol. 11:191 (1993)).

[0123] In specific embodiments, the CD28 binding domain may be derived from CD80, CD86, or the antibodies TGN1412, 9D7, 9.3, KOLT-2, 15E8, 248.23.2, and EX5.3D10.

[0124] In particular embodiments, the binding domain of TGN1412 comprises a CDRL1 sequence comprising HASQNIYVWLN (SEQ ID NO: 213), a CDRL2 sequence comprising KASNLHT (SEQ ID NO: 214), a CDRL3 sequence comprising QQGQTYPYT (SEQ ID NO: 215), a CDRH1 sequence comprising SYYIH (SEQ ID NO: 216), a CDRH2 sequence comprising CIYPGNVNTNYNEKFKD (SEQ ID NO: 217), and a CDRH3 sequence comprising SHYGLDWNFDV (SEQ ID NO: 218).

[0125] In particular embodiments, the CD80/CD86 binding domain is derived from one or more monoclonal antibodies described in U.S. Patent No. 7,531,175. In particular embodiments, the CD80/CD86 binding domain comprises a CDRL1 sequence comprising SVSSSISSSNLH (SEQ ID NO: 219), a CDRL2 sequence comprising GTSNLAS (SEQ ID NO: 220), a CDRL3 sequence comprising QQWSSYPLT (SEQ ID NO: 221), a CDRH1 sequence comprising DYYMH (SEQ ID NO: 222), a CDRH2 sequence comprising WIDPENGNTLYDPKFQG (SEQ ID NO: 223), and a CDRH3 sequence comprising EGLFFAY (SEQ ID NO: 224).

[0126] Activated T cells express 4-1BB (CD137). 4-1BB, also called CD137 or TNFSF9 (UniProt accession no. Q07011), is a T-cell costimulatory receptor.

[0127] The 4-1BB binding domains can be obtained from the monoclonal antibody described in U.S. Patent No. 9,382,328B2.

[0128] In particular embodiments, the 4-1BB binding domain comprises a CDRL1 sequence comprising RASQSVS (SEQ ID NO: 225), a CDRL2 sequence comprising ASNRAT (SEQ ID NO: 226), a CDRL3 sequence comprising QRSNWPPALT (SEQ ID NO: 227), a CDRH1 sequence comprising YYWS (SEQ ID NO: 228), a CDRH2 sequence comprising INH, and a CDRH3 sequence comprising YGPGNYDWYFDL (SEQ ID NO: 229).

[0129] In particular embodiments, the 4-1BB binding domain comprises a CDRL1 sequence comprising SGDNIGDQYAH (SEQ ID NO: 230), a CDRL2 sequence comprising QDKNRPS (SEQ ID NO: 231), a CDRL3 sequence comprising ATYTGFGSLAV (SEQ ID NO: 232), a CDRH1 sequence comprising GYSFSTYWIS (SEQ ID NO: 233), a CDRH2 sequence comprising KIYPGDSYTNYSPS (SEQ ID NO: 234), and a CDRH3 sequence comprising GYGIFDY (SEQ ID NO: 235).

[0130] Cytotoxic T cells destroy tumor cells. These cells are also known as CD8+ T cells because they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to MHC class I-associated antigen, which is present on the surface of almost every cell in the body.

[0131] In particular embodiments, the CD8 binding domain can be obtained from the OKT8 antibody. The OKT8 antibody comprises a CDRL1 sequence comprising RTSRSISQYLA (SEQ ID NO: 236), a CDRL2 sequence comprising SGSTLQS (SEQ ID NO: 237), a CDRL3 sequence comprising QQHNENPLT (SEQ ID NO: 238), a CDRH1 sequence comprising GFNIKD (SEQ ID NO: 239), a CDRH2 sequence comprising RIDPANDNT (SEQ ID NO: 240), and a CDRH3 sequence comprising GYGYYVFDH (SEQ ID NO: 241).

[0132] In specific embodiments, immune cells can be activated by suppressing the activity of inhibitory epitopes, such as PD-1, LAG3, TIM-3, BTLA, CTLA-4, VISTA and/or CD200.

[0133] PD-1, also referred to as CD279 (UniProt Accession No. Q15116), is an inhibitory cell surface receptor involved in the regulation of T-cell immune responses. In specific embodiments, the PD-1 binding domain may be derived from the monoclonal antibody described in U.S. Patent Publication 2011/0271358. In particular embodiments, the PD-1 binding domain comprises a CDRL1 sequence comprising RASQSVSTSGYSYMH (SEQ ID NO: 242), a CDRL2 sequence comprising FGSNLES (SEQ ID NO: 243), a CDRL3 sequence comprising QHSWEIPYT (SEQ ID NO: 244), a CDRH1 sequence comprising SSWIH (SEQ ID NO: 245), a CDRH2 sequence comprising YIYPSTGFTEYNQKFKD (SEQ ID NO: 246), and a CDRH3 sequence comprising WRDSSGYHAMDY (SEQ ID NO: 247).

[0134] In particular embodiments, the PD-1 binding domain may be derived from the monoclonal antibody described in U.S. Patent Application 20090217401A1. In particular embodiments, the PD-1 binding domain comprises a CDRL1 sequence comprising RASQSVSSYLA (SEQ ID NO: 84), a CDRL2 sequence comprising DASNRAT (SEQ ID NO: 85), a CDRL3 sequence comprising QQSSNWPRT (SEQ ID NO: 248), a CDRH1 sequence comprising NSGMH (SEQ ID NO: 249), a CDRH2 sequence comprising VLWYDGSKRYYADSVKG (SEQ ID NO: 250), and a CDRH3 sequence comprising NDDY (SEQ ID NO: 251).

[0135] LAG3, also called CD223 (UniProt Accession No. P18627), binds HLA class II antigens and is involved in lymphocyte activation. In particular embodiments, the LAG3-binding domain may be derived from the monoclonal antibody described in WO/2014/008218.

[0136] TIM-3, also known as HAVcr-2 or TIMD-3 (UniProt Accession No. Q9TDQ0), is a cell surface receptor that plays an inhibitory role in innate and adaptive immune responses. In specific embodiments, the TIM-3 binding domain can be derived from the monoclonal antibody described in U.S. Patent Publication 2015/0218274.

[0137] BTLA, also known as CD272 (UniProt Accession No. Q7Z6A9), is an inhibitory receptor that inhibits the immune response of lymphocytes. In particular embodiments, the BTLA-binding domain (e.g., scFv) can be derived from one or more monoclonal antibodies described in U.S. Patent Publication 2012/0288500.

[0138] CTLA-4, also known as CD152 (UniProt Accession No. P16410), is an inhibitory receptor that is a major negative regulator of T cell responses. In specific embodiments, the CTLA-4 binding domain can be derived from the monoclonal antibody described in U.S. Patent No. 6,984,720.

[0139] CD200 (also known as membrane glycoprotein ox-2, UniProt Accession No. P41217) is a protein capable of delivering inhibitory signals to immune cells. In specific embodiments, the CD200 binding domain can be derived from one or more monoclonal antibodies described in U.S. Patent Publication 2013/0189258.

[0140] In specific embodiments, natural killer cells (also known as NK cells, K cells, and killer cells) are targeted for localized activation by bispecific molecules. NK cells can induce apoptosis or cell lysis by releasing granules that rupture cell membranes and can secrete cytokines to recruit other immune cells.

[0141] Examples of activating proteins expressed on the surface of NK cells include NKG2D, CD8, CD16, KIR2DL4, KIR2DS1, KIR2DS2, KIR3DS1, NKG2C, NKG2E, NKG2D, and several members of the natural cytotoxicity receptor (NCR) family. Examples of NCRs that activate NK cells upon ligand binding include NKp30, NKp44, NKp46, NKp80, and DNAM-1.

[0142] Examples of commercially available antibodies that bind the NK cell receptor and induce and/or enhance NK cell activation include: 5C6 and 1D11, which bind and activate NKG2D (available from BioLegend ^® San Diego, CA); mAb 33, which binds and activates KIR2DL4 (available from BioLegend ^® ); P44-8, which binds and activates NKp44 (available from BioLegend ^® ); SK1, which binds and activates CD8; and 3G8, which binds and activates CD16. Additional NK cell activating antibodies are described in WO/2005/0003172 and U.S. Patent No. 9,415,104.

[0143] With respect to CDR sequences and segments, the structural units of natural antibodies include a tetramer. Each tetramer includes two pairs of polypeptide chains, with each pair consisting of one light chain and one heavy chain. The amino-terminal portion of each chain includes a variable region responsible for antigen recognition and epitope binding. The variable regions have the same general structure, consisting of relatively conserved framework regions (FRs) linked by three hypervariable regions, also called complementarity determining regions (CDRs). The CDRs from the two chains of each pair are arranged in a row with the framework regions, allowing for binding of a specific epitope. From N-terminus to C-terminus, the variable regions of both light and heavy chains include the FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 domains. The assignment of amino acids to each domain is typically made according to the definitions given in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk, J. Mol. Biol., 196:901–917 (1987); Chothia et al., Nature, 342:878–883 (1989).

[0144] (5) Multispecific Molecule Formats. As indicated, multispecific immune cell binding molecules bind both an antigen at the treatment site (e.g., a cancer antigen at the tumor site) and an immune cell activating epitope to target the immune cells to the cells of interest for destruction. Bispecific antibodies bind an antigen on the cell of interest and one immune cell activating epitope. Trispecific antibodies can bind two antigens on the cell of interest and one immune cell activating epitope, or one antigen on the cell of interest and two immune cell activating epitopes (e.g., for a primary activation signal (e.g., CD3) and a costimulatory activation signal (e.g., CD28 or 4-1BB). Quadraspecific antibodies can bind four different binding partners, divided in different combinations between antigens on the cells of interest and immune cell activating epitopes. In particular embodiments, the immune cells are T cells or natural killer (NK) cells.

[0145] Exemplary bispecific antibody formats are described, for example, in WO2009/080251, WO2009/080252, WO2009/080253, WO2009/080254, WO2010/112193, WO2010/115589, WO2010/136172, WO2010/145792, and WO2010/145793. For a review of additional bispecific formats that can be used, see Brinkmann & Kontermann, mAbs, 2017. 9:2, 182-212, DOI: 10.1080/19420862.2016.1268307. In Yu et al. (Journal of Hematology & Oncology (2017) 10, 155) additional formats have been described that are particularly useful in the treatment of solid tumors, such as, for example, Fc-format (quadromes, knobs-in-holes, scFv-IgG, (IgG)2, nanobodies, and scFv-Fc) and non-Fc-format (F(ab') ₂ , scFv-HAS-scFv, tand-scFv, diabodies, DART, ImmTAC, dock-and-lock antibodies, and tand-Ab).

[0146] In the portion not covered in sections (3) or (4) above, various additional binding domains can be obtained from many sources, such as antibodies, TCRs, fibronectin, affibodies, natural ligands (e.g., CD80 and CD86 for CD28), and so on. In specific embodiments, binding domains can be obtained from whole antibodies or antibody binding fragments, such as Fv, Fab, Fab', F(ab') ₂ , Fc, as well as single-chain Fv fragments (scFv), or any biologically active fragments of immunoglobulin that specifically bind a cancer antigen or an epitope that activates immune cells (e.g., a T cell receptor). Antibodies or antigen-binding fragments include all or a portion of polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, bispecific antibodies, minibodies, and linear antibodies.

[0147] Multispecific molecules containing binding domains of human origin, or humanized antibodies, have reduced immunogenicity in humans and have a reduced number of non-immunogenic epitopes compared to non-human antibodies. The binding domains will typically be selected from those that have reduced antigenicity in human subjects. The binding domains may, in particular, include any peptide that specifically binds a selected cancer antigen or an epitope that activates immune cells. Sources of binding domains include variable regions of antibodies from different biological species (which may be in the form of antibodies, sFv, scFv, Fab, scFv-based grabatoles or soluble VH domains, or domain antibodies). In these antibodies, the antigen-binding regions can be formed using only the variable region of the heavy chain, i.e., these functional antibodies are homodimers of heavy chains alone (they are called “heavy chain antibodies”) (Jespers et al ., Nat. Biotechnol. 22:1161, 2004; Cortez-Retamozo et al . , Cancer Res. 64:2853, 2004; Baral et al . , Nature Med. 12:580, 2006; and Barthelemy et al ., J. Biol. Chem. 283:3639, 2008).

[0148] Phage display libraries of partially or fully synthetic antibodies are available and can be screened for an antibody, or fragment thereof, that can bind a selected epitope. For example, binding domains can be identified by screening a Fab phage library for Fab fragments that specifically bind the target of interest (see Hoet et al ., Nat. Biotechnol. 23 :344, 2005). Phage display libraries of human antibodies are also available. In addition, traditional hybridoma strategies can be used to develop binding domains using the target of interest as an immunogen in convenient systems (such as, for example, mice, HuMAb ^mouse® , TC mouse ^™ , KM ^mouse® , llamas, chickens, rats, hamsters, rabbits, etc.). In specific embodiments, the binding domains specifically bind selected epitopes expressed by target cancer cells and/or T cells and do not cross-react with non-specific components or irrelevant targets. Once identified, the amino acid sequence or polynucleotide sequence encoding the CDRs in the binding domain can be isolated and/or determined.

[0149] An alternative source of binding domains includes sequences encoding random peptide libraries or sequences encoding engineered amino acid diversity in the loop regions of alternative, non-antibody scaffolds such as scTCR (see, e.g., Lake et al ., Int. Immunol. 11:745, 1999; Maynard et al ., J. Immunol. Methods 306:51, 2005; U.S. Patent No. 8,361,794), mAb ² or Fcab™ (see, e.g., PCT Patent Application Publication Nos. WO 2007/098934; WO 2006/072620), affibodies, avimers, phynomers, cytotoxic T lymphocyte-associated protein 4 (Weidle et al., ., Cancer Gen. Proteo. 10:155, 2013), and the like (Nord et al. , Protein Eng. 8:601, 1995; Nord et al ., Nat. Biotechnol. 15:772, 1997; Nord et al ., Euro. J. Biochem. 268:4269, 2001; Binz et al. , Nat. Biotechnol. 23:1257, 2005; Boersma and Plückthun, Curr. Opin. Biotechnol. 22:849, 2011).

[0150] In specific embodiments, an antibody fragment is used as one or more binding domains in a multispecific molecule. The term "antibody fragment" means a portion of a complete or full-length antibody that retains the ability to bind an epitope. Examples of antibody fragments include Fv, scFv, Fab, Fab', Fab'-SH, F(ab') ₂ ; diabodies and linear antibodies.

[0151] A single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy and light chains of immunoglobulins linked by a short peptide linker. Fv fragments comprise the VL and VH domains of one arm of an antibody. Although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be joined, for example, using recombinant methods, by a synthetic linker, allowing them to be produced as a single protein chain in which the VL and VH regions are paired to form monovalent molecules (single-chain Fv (scFv)). For further information regarding Fv and scFv, see, for example, Bird, et al., Science 242 (1988) 423-426; Huston, et al., Proc. Natl. Acad. Sci. USA 85 (1988) 5879–5883; Plueckthun, in: The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York (1994) 269–315; WO1993/16185; U.S. Patent 5,571,894 and U.S. Patent 5,587,458.

[0152] A Fab fragment is a monovalent fragment of an antibody containing VL, VH, CL, and CH1 domains. An F(ab') ₂ fragment is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region. For information regarding Fab and F(ab') ₂ fragments having an increased half-life in vivo , see U.S. Patent 5,869,046. Diabodies contain two epitope-binding sites, which may be bivalent. See, for example, EP 0404097; WO1993/01161; and Holliger, et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448. Dual-valence redirecting antibodies (DART™; based on the diabody format but with a C-terminal disulfide bridge for additional stabilization (Moore et al., Blood 117, 4542-51 (2011))) can also be used. Antibody fragments can also include dedicated CDR regions. For a review of antibody fragments, see Hudson et al., Nat. Med. 9 (2003) 129-134.

[0153] In specific embodiments, multispecific antibodies may also comprise a naturally occurring receptor or ligand for the epitope as a binding domain. For example, if the binding target is PD-L1, the binding domains may comprise PD-1 (including, for example, a PD-1/anti-CD3 fusion protein). One example of a fusion receptor for binding is ^Enbrel® (Amgen). Natural receptors or ligands may also be modified to enhance binding. For example, betalacept is a modified version of abatacept. In specific embodiments, multispecific molecules may include a naturally occurring receptor or ligand that induces phagocytosis. Calreticulin (UniProt accession number P27797) is a protein localized to the endoplasmic reticulum of healthy cells. In dying cells, it translocates to the cell surface and induces phagocytosis by immune cells such as macrophages. In specific embodiments, the binding domains may comprise calreticulin or a calreticulin fragment capable of inducing phagocytosis.

[0154] In particular embodiments, a multispecific molecule may include a single-chain antibody fused to the C-terminus of a light chain (see, e.g., Oncoimmunology, 2017; 6(3): e1267891). This format may be useful because the presence of an Fc region may help preserve the half-life of the protein. The presence of an Fc region may also be useful because Fc interacts with multiple receptors and may contribute to the immune response. Antibody-scFv fusion proteins may also be useful because the antibody portion binds its epitope in a dimeric format, which increases avidity, and the scFv portion binds its epitope in a monomeric format, which may be useful, for example, for binding T-cell epitopes, and allows multimerization only in the presence of a target (e.g., a cancer cell). Such embodiments may be "trispecific."

[0155] As indicated, the binding domains of the multispecific molecules can be connected by a linker. A linker is an amino acid sequence that can provide flexibility and room for conformational movement between the binding domains of the multispecific molecule. Any suitable linker can be used. Examples of linkers can be found in Chen et al., Adv Drug Deliv Rev. 2013 Oct 15; 65(10): 1357-1369. Linkers can be flexible, rigid, or semi-rigid, depending on the desired presentation of the functional domain for the target. Frequently used flexible linkers include Gly-Ser linkers such as GGSGGGSGGSG (SEQ ID NO: 252), GGSGGGSGSG (SEQ ID NO: 63), and GGSGGGSG (SEQ ID NO: 65). Additional examples include: GGGGSGGGGS (SEQ ID NO: 90); GGGSGGGS (SEQ ID NO: 128), and GGSGGS (SEQ ID NO: 129). Linkers that include one or more antibody hinge regions and/or immunoglobulin heavy chain constant regions, such as only the CH3 or CH2CH3 sequence, can also be used.

[0156] In some situations, flexible linkers may be unable to maintain the distance or arrangement of the binding domains required for a particular use. In these cases, rigid or semi-rigid linkers may be useful. Examples of rigid or semi-rigid linkers include proline-rich linkers. In particular embodiments, a proline-rich linker is a peptide sequence containing more proline residues than would be expected based on chance alone. In particular embodiments, a proline-rich linker is a linker containing at least 30%, at least 35%, at least 36%, at least 39%, at least 40%, at least 48%, at least 50%, or at least 51% proline residues. Specific examples of proline-rich linkers include fragments of proline-rich salivary proteins (PRPs).

[0157] Specific examples of bispecific antibodies are described in Yu et al., (Journal of Hematology & Oncology (2017) 10, 155) and include catumaxomab (EpCAM/CD3; in triomat format, evaluated in clinical trial NCT00189345); MT110 (EpCAM/CD3; in BiTE format (Amgen), evaluated in clinical trial NCT00635596); ertumaxomab (HER2/CD3; in triomat format, evaluated in clinical trial NCT00452140); MDX-447 (EGFR/CD64; in 2(Fab') format, evaluated in clinical trial NCT00005813); MM-141 (HER3/IGF-IR; in scFv-IgG format, being evaluated in clinical trial NCT01733004); AMG211 (CEA/CD3; in BiTE format, being evaluated in clinical trial NCT02760199); RO6958688 (CEA/CD3; in IgG format, being evaluated in clinical trial NCT02324257); RO6895882 (CEA/IL2; in scFv-IgG format, being evaluated in clinical trial NCT02004106); TF2 (CEA/HSG; in Dock-and-lock format, being evaluated in clinical trial NCT00860860); anti-CEA x anti-DTPA (CEA/di-DTPA-131; in scFv-IgG format, evaluated in clinical trial NCT00467506); BAY2010112 (PSMA/CD3; in BiTE format, evaluated in clinical trial NCT01723475) and MOR209/ES414 (PSMA/CD3; in scFv-IgG-Fc-scFv format, evaluated in clinical trial NCT02262910). AMG701 (BCMA-targeted antibody) and solitomab (EpCam/CD3-targeted antibody) can also be used. For more information on EpCam-binding bispecific molecules, see Brischwein et al., Mol. Immunol. 2006; 43: 1129–1143 and Schlereth et al., Cancer Research, 2005; 65: 2882–2889. The bispecific anti-PD-L1/CD3 antibody described in Horn et al., Oncotarget, Aug. 29, 2017 8(35) can also be used. Additional specific examples of bispecific antibodies are described in WO2014/167022; US2016/0208001; US2014/0302037 and US2014/0308285. One recent example is blinatumomab. See Ellerman, Methods, 154 (2019) 102–117 for more information regarding bispecific T-cell binding molecules.

[0158] (6) TGFβ inhibitors. In humans, there are three highly homologous isoforms of TGFβ: TGFβ1, TGFβ2, and TGFβ3. A variety of TGFβ inhibitory peptides and antibodies exist. In particular embodiments, monocytes/macrophages can be reprogrammed to express a TGFβ inhibitory peptide or antibody. Examples of TGFβ inhibitors include trabedersen (AP12009; an antisense oligonucleotide evaluated in clinical trials NCT00431561, NCT00844064, and NCT00761280); disittertide (a peptide evaluated in clinical trials NCT00574613 and NCT00781053); lerdelimumab (a humanized antibody); Metelimumab (a humanized antibody being evaluated in clinical trial NCT00043706); Frezolimumab (a humanized antibody being evaluated in clinical trials NCT00464321, NCT01284322, and NCT01291784); LY2382770 (a humanized antibody being evaluated in clinical trial NCT01113801); SIX-100 (an antibody being evaluated in clinical trial NCT01371305); Avotermin (a recombinant protein being evaluated in clinical trials NCT004322111 and NCT00656227); and IMC-TR1 (a humanized antibody being evaluated in clinical trial NCT01646203).

[0159] Ravi et al. (Nature Communications 9, 741 (2018)) describe bifunctional antibody-ligand decoys (Y-traps) comprising an antibody targeting CTLA-4 or PD-L1 fused to a TGFβ receptor II ectodomain sequence that simultaneously shuts down autocrine/paracrine TGFβ in the target cell microenvironment (anti-CTLA4-TGFβRII ecd and anti-PDL1-TGFβRII ecd ). Figure 2B of Ravi et al. shows the amino acid sequences of the heavy chain and light chain of anti-CTLA4-TGFβRII, including the ligand-binding sequence of the extracellular domain of TGFβRII. Cuende et al., Science Translational Medicine, April 2015, 7(284) further describe the production of antibodies that inhibit TGFβ in vivo ; for example, the anti-GARP monoclonal antibodies MHG-8 and LHG-10 block the production of active TGFβ1.

[0160] Examples of additional TGFβ inhibitors include tranilast, pirfenidone, Lefty-1 (1105I Accession Nos.: NM_010094 (mouse) and NM_020997 (human)), SB-431542, SB-202190, and SB-505124 (Lindemann et al., Mol. Cancer, 2003, 2: 20; GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), SM16 (Biogen Idec), LY2109761, LY364947, LY580276, LY2157299 (Lilly Research Laboratories), A-83-01 (WO 2009/146408), ALK5 inhibitor II (2-[3-[6-methylpyridin-2-yl]-1H-pyrazol-4-yl]-1,5-naphthyridine), TGFβRI kinase inhibitor VIII (6-[2-tert-butyl-5-[6-methylpyridin-2-yl]-1H-imidazol-4-yl]-quinoxaline) and its derivatives.

[0161] (7) Nucleotide Sequences. According to the present invention, nucleotides encoding genes that regulate activation states and genes that lead to the expression of multispecific molecules and, optionally, a TGFβ inhibitor, are delivered to immune cells such as monocytes and/or macrophages. The term "gene" means a nucleotide sequence that encodes the encoded molecule. This definition encompasses various polymorphisms, mutant sequences and/or variant sequences, if such changes do not affect the function of the encoded molecule. The term "gene" may encompass not only coding sequences, but also regulatory regions such as promoters, enhancers and termination regions. The term may also encompass all introns and other DNA sequences spliced from the mRNA transcript, along with variants resulting from alternative splicing sites. Nucleotide sequences encoding the encoded molecule may be RNA that directs the expression of the encoded molecule. These nucleotide sequences include RNA sequences that, in certain embodiments, are translated into protein. One skilled in the art understands that, in certain embodiments, DNA sequences containing the base thymine (T) may be equivalent to mRNA sequences having the same sequence, except that the base T is replaced by the base uracil (U). Nucleotide sequences include both full-length nucleotide sequences and non-full-length sequences derived from full-length sequences. Sequences may also include naturally occurring sequences with degenerate codons or sequences that can be introduced to ensure codon preference in a particular immune cell. Gene sequences encoding the molecules described herein are provided in publicly available databases and publications. "Encoding" means the property of nucleotide sequences, such as a plasmid, gene, cDNA, mRNA, to serve as templates for the synthesis of an activation regulator, a multispecific antibody and/or a TGFβ inhibitor.

[0162] In particular embodiments, the nucleotide sequences comprise synthetic mRNAs. In particular embodiments, the synthetic mRNA is genetically modified to enhance intracellular stability through 5' capping. A variety of different 5' cap structures can be used to generate the 5' cap of a synthetic mRNA molecule. For example, an anti-reversible cap analog (ARCA) comprises a 5'-5'-guanine-guanine triphosphate bond, where one guanine contains an N7 methyl group as well as a 3'-O-methyl group. Caps can also be added to synthetic mRNA molecules after transcription using enzymes responsible for creating 5' cap structures. For example, recombinant vaccinia virus capping enzyme and recombinant 2'-O-methyltransferase can form a canonical 5'-5'-triphosphate bond between the last 5'-terminal nucleotide of mRNA and the nucleotide guanine, where guanine has N7 methylation and the terminal 5'-nucleotide contains 2'-O-methyl, yielding the Cap1 structure. This results in the formation of a cap with higher translational competence and cellular stability, as well as reduced activation of cellular proinflammatory cytokines.

[0163] In particular embodiments, other modifications of the synthetic mRNA to reduce immunogenicity, increase mRNA stability, and/or facilitate mRNA translation may include the 5' and 3' untranslated regions (UTRs), a Kozak translation initiation sequence in the 5'UTR, modified ribonucleosides, and/or a polyA tail. In particular embodiments, the modified ribonucleosides may include pseudouridine (Ψ), 5-methylcytidine (5mC), N6-methyladenosine (m6A), 2-thiouridine (2sU), 5-methoxyuridine (5moU), and N-1-methylpseudouridine (m1Ψ). In particular embodiments, the UTRs may include the UTRs of alpha and/or beta globin.

[0164] Specific embodiments for producing synthetic mRNA include creating a DNA template containing the coding sequence of the DNA of the desired protein with a _T100-250 sticky 5' end by PCR amplification from an appropriate DNA plasmid. The DNA template can then be used to produce mRNA by an in vitro transcription reaction. During in vitro transcription, a 5' cap structure (e.g., ARCA), modified ribonucleosides, and/or a 3' poly(A) tail can be included. A number of in vitro transcription systems are commercially available, including, for example, the MEGAscript T7 Transcription Kit (ThermoFisher Scientific, Waltham, MA), the Riboprobe™ T7 System (Promega, Madison, WI), the AmpliScribe™ T7 High-Throughput Transcription Kit (Epicentre, Madison, WI), and the HiScribe™ T7 In Vitro Transcription Kit (New England Biolabs, Ipswich, MA). In particular embodiments, synthetic mRNA can be synthesized by companies that synthesize nucleotides (e.g., TriLink Biotechnologies, San Diego, CA).

[0165] Synthetic mRNA or other nucleotide sequences can be produced in a cyclic form. Such nucleotide sequences can be cyclized or concatemerized to create a translationally competent molecule that facilitates interactions between poly-A-binding proteins and 5'-end-binding proteins. The cyclization or concatemerization mechanism can occur in at least 3 different ways: 1) chemical, 2) enzymatic, or 3) ribozyme-catalyzed. The newly formed 5'-/3'-linkage can be intramolecular or intermolecular.

[0166] In the first pathway, the 5'-end and 3'-end of the nucleotide sequence may contain chemically reactive groups that, when placed next to each other, form a new covalent bond between the 5'-end and 3'-end of the molecule. The 5'-end may contain an NHS-ester reactive group, and the 3'-end may contain a 3'-amino-terminal nucleotide, so that in an organic solvent, the 3'-amino-terminal nucleotide at the 3'-end of the nucleotide molecule will undergo nucleophilic attack on the 5'-NHS-ester group, forming a new 5'-/3'-amide bond.

[0167] In a second pathway, T4 RNA ligase can be used to enzymatically couple a 5'-phosphorylated nucleotide molecule to the 3'-hydroxyl group of the nucleotide molecule, forming a new phosphodiester bond. In an illustrative reaction, 1 μg of the nucleotide molecule can be incubated at 37°C for 1 hour with 1-10 units of T4 RNA ligase (New England Biolabs, Ipswich, MA) according to the manufacturer's protocol. The ligation reaction can occur in the presence of a cleaved oligonucleotide capable of base pairing with the 5' and 3' regions adjacent to each other, facilitating the enzymatic ligation reaction.

[0168] In the third pathway, the 5' or 3' end of the cDNA template encodes a ribozyme ligase sequence such that, during in vitro transcription, the resulting nucleotide molecule may contain an active ribozyme sequence capable of ligating the 5' end of a nucleotide molecule to the 3' end of a nucleotide molecule. The ribozyme ligase may be derived from a group I intron, hepatitis delta virus, a hairpin ribozyme, or may be selected by the SELEX (systematic evolution of ligands by exponential enrichment) method. The ribozyme ligase reaction may last from 1 to 24 hours at a temperature of 0 to 37°C.

[0169] In specific embodiments, the nucleotide sequences include a plasmid, cDNA, or mRNA that may contain, for example, a sequence (e.g., a gene) for expressing the encoded molecule. Suitable plasmids include standard plasmid vectors and minicircular plasmids that can be used to transfer genes into a monocyte/macrophage. The nucleotide sequences (e.g., minicircular plasmids) may also contain any additional sequence that facilitates transient expression in the modified cell. For example, the nucleotide sequences may include promoters, such as general promoters, tissue-specific promoters, cell-specific promoters, and/or cytoplasm-specific promoters. As indicated, promoters and plasmids (e.g., minicircular plasmids) are generally well known in the art and can be prepared using conventional methods.

[0170] For additional information regarding nucleotide sequences that can be used in embodiments of the present invention, see Hardee et al., Genes (2017), 8, 65. Hardee et al. discusses methods using non-viral DNA vectors for gene therapy, including plasmids, minicircles, and minivectors. Plasmid vectors and minimized DNA vectors have been successfully used to deliver gene therapeutics for the treatment of cancer. More recently, minicircles have been used to genetically modify T cells to deliver bispecific antibodies that allow the T cells to kill B-cell lymphomas (Hardee et al., Genes (2017), 8, 65). Specific embodiments include the use of double-stranded DNA (integrating and/or non-integrating), conventional plasmids, mini-circle plasmids, and/or closed linear cDNA (see Li et al., PLoS One, Aug. 1, 2013 doi.org/10.1371/journal.pone. 0069879). cDNA is non-viral, AAV-derived vector DNA with covalently closed ends (Li et al., PLoS One, 2013, doi.org/10.1371/journal.pone. 0069879).

[0171] In specific embodiments, a nucleotide sequence encoding a macrophage activation regulator is used in combination with one or more additional nucleotide sequences encoding other activation regulators (e.g., combinations of IRFs, multispecific antibodies, and/or TGFβ inhibitors). In specific embodiments, a nucleotide sequence encoding an IRF is used in combination with one or more additional nucleotide sequences encoding other IRFs and with a nucleotide sequence encoding IKKβ. In specific embodiments, a nucleotide sequence encoding an IRF is used in combination with nucleotide sequences encoding IKKβ, multispecific antibodies, and/or TGFβ inhibitors in a ratio of 0.5:1, 1:1, 2:1, 3:1, 4:1, or 5:1. In specific embodiments, a nucleotide sequence encoding IRF is used in combination with a nucleotide sequence encoding IKKβ in a 3:1 ratio.

[0172] In specific embodiments, nucleotide sequences can be delivered as part of a gene editing system. Gene editing systems modify or affect specific sequences of the endogenous genome of a cell. Gene editing systems are useful for targeted gene editing, such as gene disruption, gene editing by homologous recombination, and gene therapy by inserting therapeutic genes into appropriate chromosomal target sites of the human genome.

[0173] In specific embodiments, transcription activator-like effector nucleases (TALENs) are used as gene editing systems. TALENs are fusion proteins that include a transcription activator-like effector (TALE) DNA-binding protein and a DNA cleavage domain. TALENs are used to edit genes and genomes by introducing double-strand breaks (DSBs) into DNA, which trigger repair mechanisms in cells. Typically, two TALENs must bind and flank a target DNA region on either side for the DNA cleavage domain to dimerize and induce a DSB. The DSB is repaired in the cell by non-homologous end joining (NHEJ) or by homologous recombination (HR) with an exogenous double-stranded fragment of donor DNA.

[0174] As noted above, TALENs were designed to bind a target sequence, such as an endogenous genome, and cleave DNA at the location of the target sequence. The TALE fragments in TALENs are DNA-binding proteins secreted by Xanthomonas bacteria. The TALE DNA-binding domain contains a highly conserved repeat of 33 or 34 amino acids, with different residues at the 12th and 13th positions of each repeat. These two positions, called the repeat variable two residues (RVD), show a strong correlation with specific nucleotide recognition. Accordingly, targeting specificity can be improved by varying the amino acids in the RVD and incorporating unconventional amino acids into the RVD.

[0175] Examples of DNA cleavage domains that can be used in TALEN fusion proteins are wild-type and variant FokI endonucleases. The FokI domain functions as a dimer, requiring two constructs with unique DNA-binding domains for sites on the target sequence. The FokI cleavage domain cleaves a five- or six-base pair spacer sequence separating two inverted half-sites.

[0176] In specific embodiments, MegaTALs are used as gene editing systems. MegaTALs have a rare-cutting single-strand nuclease structure in which a TALE is fused to the DNA-cleaving domain of a meganuclease. Meganucleases, also known as homing endonucleases, are single peptide chains that combine DNA recognition and nuclease functions in a single domain. Unlike TALENs, MegaTALs require delivery of only a single peptide chain for functional activity.

[0177] In specific embodiments, zinc finger nucleases (ZFNs) are used as gene editing systems. ZFNs are a class of site-specific nucleases designed to bind and cleave DNA at specific positions. ZFNs are used to introduce a DSB at a specific site in a DNA sequence, allowing the ZFNs to target unique sequences in the genome in different cells. Furthermore, after the double-strand break, homologous recombination or non-homologous end joining occurs to repair the DSB, allowing for genome editing.

[0178] ZFNs are synthesized by fusing a zinc finger DNA-binding domain with a DNA-cleaving domain. The DNA-binding domain contains three to six zinc finger proteins, which are transcription factors. The DNA-cleaving domain contains a catalytic domain, such as that of the FokI endonuclease.

[0179] Guide RNA, for example, can be used with gene editing systems such as CRISPR-Cas systems. CRISPR-Cas systems include CRISPR repeats and a set of CRISPR-associated genes (Cas).

[0180] In general, any system capable of resulting in functional expression of the delivered nucleotide sequences can be used in accordance with the present invention. However, in specific embodiments, delivery using viral vectors is excluded.

[0181] (8) Nanoparticles. In some examples, nanoparticles used in the systems and methods disclosed herein can function to condense and protect nucleotide sequences from enzymatic degradation. Particularly useful materials for use in nanoparticles for this purpose include positively charged lipids and/or polymers, including poly(β-amino ester) (PbAE).

[0182] Examples of positively charged polymers that can be used in the nanoparticles of the present invention include polyamines; polyorganic amines (e.g., polyethyleneimine (PEI), polyethyleneimine cellulose); poly(amidoamines) (PAMAM); polyamino acids (e.g., polylysine (PLL), polyarginine); polysaccharides (e.g., cellulose, dextran, DEAE-dextran, starch); spermine, spermidine, poly(vinylbenzyltrialkylammonium), poly(4-vinyl-N-alkylpyridinium), poly(acryloyltrialkylammonium), and Tat proteins.

[0183] Examples of positively charged lipids include esters of phosphatidic acid with an amino alcohol, such as the ester of dipalmitoylphosphatidic acid or distearoylphosphatidic acid with hydroxyethylenediamine. More specific examples of positively charged lipids include 3β-[N--(N',N'-dimethylaminoethyl)carbamoyl)cholesterol (DC-chol); N,N'-dimethyl-N,N'-dioctacylammonium bromide (DDAB); N,N'-dimethyl-N,N'-dioctacylammonium chloride (DDAC); 1,2-dioloyloxypropyl-3-dimethylhydroxyethylammonium chloride (DORI); 1,2-dioleoyloxy-3-[trimethylammonio]propane (DOTAP); N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); dipalmitoylphosphatidylcholine (DPPC); 1,2-dioctadecyloxy-3-[trimethylammonio]propane (DSTAP); as well as cationic lipids described, for example, in Martin et al., Current Pharmaceutical Design 2005, 11, 375-394.

[0184] Blends of lipids and polymers in any concentration and ratio can also be used. Blending different types of polymers in varying ratios using different grades can result in characteristics borrowed from each of the polymers included in the blend. Various chemical variations of end groups can also be used.

[0185] In specific embodiments described herein, porous nanoparticles created from any material capable of forming a porous network may also be used. Illustrative materials include metals, transition metals, and metalloids. Illustrative metals, transition metals, and metalloids include lithium, magnesium, zinc, aluminum, and silica. In specific embodiments, the porous nanoparticles comprise silica. The exceptionally high surface area of mesoporous silica (greater than 1000 ^m2 /g) allows for nucleotide loading at levels exceeding those of conventional DNA carriers such as liposomes.

[0186] The particles can be formed in a variety of shapes, including spheroidal, cuboidal, pyramidal, oblong, cylindrical, toroidal, and the like. Nucleotides can be incorporated into the pores of the nanoparticles in a variety of ways. For example, nucleotides can be encapsulated in porous nanoparticles. In other aspects, nucleotides can be bound (e.g., covalently and/or non-covalently) to the surface, or in close proximity to the surface, of the porous nanoparticles. In particular embodiments, nucleotides can be incorporated into the porous nanoparticles, such as integrated into the material of the porous nanoparticles. For example, nucleotides can be incorporated into the polymer matrix of the polymeric nanoparticles.

[0187] In particular embodiments, the nanoparticles disclosed herein are coated. The coating may serve to protect the encapsulated nucleotides and/or reduce or prevent off-target binding. Off-target binding is reduced or prevented by reducing the surface charge of the nanoparticles to neutral or negative. As described in more detail elsewhere herein, coatings may include neutrally or negatively charged polymer-based and/or liposome-based coatings. In particular embodiments, the coating is a dense surface coating of a hydrophilic and/or neutrally charged hydrophilic polymer sufficient to prevent exposure of the encapsulated nucleotides to the environment prior to their release into the immune cell. In particular embodiments, the coating covers at least 80% or at least 90% of the surface of the nanoparticle. In particular embodiments, the coating comprises polyglutamic acid (PGA). In particular embodiments, PGA may serve as a linker for attaching a targeting ligand to the nanoparticle. In certain embodiments, PGA can serve as a linker for attaching dimannose to the nanoparticle. In certain embodiments, the coating comprises hyaluronic acid.

[0188] Examples of neutrally charged polymers that can be used as a coating in embodiments of the invention include polyethylene glycol (PEG); poly(propylene glycol); and polyalkylene oxide copolymers ( ^Pluronic® , BASF Corp., Mount Olive, NJ).

[0189] Neutral charged polymers also include zwitterionic polymers. Zwitterionicity is the property of overall charge neutrality in the presence of both positive and negative electrical charges. Zwitterionic polymers can behave like regions of cell membranes that resist cell and protein adhesion.

[0190] Zwitterionic polymers include zwitterionic constituent units having pendant groups (i.e., groups extending from the polymer backbone) with zwitterionic groups. Exemplary zwitterionic pendant groups include carboxybetaine groups (e.g., -Ra-N+(Rb)(Rc)-Rd- _CO2- , where Ra is a linker group covalently linking the polymer backbone to the cationic nitrogen center of the carboxybetaine groups, Rb and Rc are nitrogen substituents, and Rd is a linker group covalently linking the cationic nitrogen center to the carboxyl group of the carboxybetaine group).

[0191] Examples of negatively charged polymers include alginic acids; carboxylic acid polysaccharides; carboxymethyl cellulose; carboxymethyl cellulose-cysteine; carrageenan (e.g., ^Gelcarin® 209, ^Gelcarin® 379, FMC Corporation, Philadelphia, PA); chondroitin sulfate; glycosaminoglycans; mucopolysaccharides; negatively charged polysaccharides (e.g., dextran sulfate); poly(acrylic acid); poly(D-aspartic acid); poly(L-aspartic acid); poly(L-aspartic acid), sodium salt; poly(D-glutamic acid); poly(L-glutamic acid); poly(L-glutamic acid), sodium salt; poly(methacrylic acid); sodium alginate (e.g., ^Protanal® LF 120M, ^Protanal® LF 200M, Protanal® ^LF 200D, FMC Biopolymer Corp., Drammen, Norway); sodium carboxymethylcellulose (CMC); sulfated polysaccharides (heparins, agaropectins); pectin, gelatin, and hyaluronic acid.

[0192] In particular embodiments, the polymers disclosed herein may include "star polymers," which are branched polymers in which two or more polymer branches extend from a core. The core is a group of atoms having two or more functional groups from which branches can be extended by polymerization. In particular embodiments, the nanoparticles of the present invention comprise star polymers. In particular embodiments, the nanoparticles of the present invention comprise star polymers and a coating. In particular embodiments, the nanoparticles of the present invention comprise star polymers and a coating containing PGA. In particular embodiments, the nanoparticles of the present invention comprise star polymers and a coating containing hyaluronic acid.

[0193] In particular embodiments, the branches are zwitterionic or negatively charged polymer branches. For star polymers, branch precursors can be converted into zwitterionic or negatively charged polymers by hydrolysis, ultraviolet irradiation, or heating. The polymers can also be prepared by any polymerization method effective for polymerizing unsaturated monomers, including atom transfer radical polymerization (ATRP), reversible addition-fragmentation polymerization (RAFT), photopolymerization, ring-opening polymerization (ROP), condensation, Michael addition reactions, branch formation/propagation reactions, or other reactions.

[0194] Liposomes are microscopic vesicles comprising at least one concentric lipid bilayer. The lipids that form the vesicles are selected to achieve a desired degree of fluidity or rigidity of the final complex. In certain embodiments, liposomes are a lipid composition that surrounds an aqueous core. In some examples, the liposome structure can be used to encapsulate a nanoparticle within its core (i.e., a liposomal nanoparticle). In certain embodiments, the nanoparticles of the present invention are used as nanoparticles within liposomal nanoparticles. Lipid nanoparticles (LNPs) are liposome-like structures that lack the continuous lipid bilayer characteristic of liposomes. Solid lipid nanoparticles (SLNPs) are LNPs that are solid at room temperature and body temperature.

[0195] The liposomes and similar structures described in the preceding paragraph may be neutral (cholesterol) or bipolar, and include phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and sphingomyelin (SM), as well as other types of bipolar lipids, including dioleoylphosphatidylethanolamine (DOPE), with a hydrocarbon chain length in the range of 14-22, and saturated or having one or more C=C double bonds. Examples of lipids capable of forming a stable liposome, alone or in combination with other lipid components, are phospholipids such as hydrogenated soy phosphatidylcholine (HSPC), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebrosides, distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (DOPE-mal). Additional phosphorus-free lipids that can be incorporated into liposomes include stearylamine, dodecylamine, hexadecylamine, isopropyl myristate, triethanolamine lauryl sulfate, alkyl aryl sulfate, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, amphoteric acrylic polymers, polyethyloxylated fatty acid amides, DDAB, dioctadecyl dimethyl ammonium chloride (DODAC), 1,2-dimyristoyl-3-trimethyl ammonium propane (DMTAP), DOTAP, DOTMA, DC-chol, phosphatidic acid (PA), dipalmitoyl phosphatidyl glycerol (DPPG), dioleoyl phosphatidyl glycerol, DOPG, and dicetyl phosphate. In specific embodiments, lipids used to create the liposomes described herein include cholesterol, hydrogenated soy phosphatidylcholine (HSPC), and derivatized vesicle-forming lipid PEG-DSPE.

[0196] Methods for producing liposomes are described, for example, in U.S. Patent Nos. 4,229,360; 4,224,179; 4,241,046; 4,737,323; 4,078,052; 4,235,871; 4,501,728; and 4,837,028, as well as in Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980) and Hope et al., Chem. Phys. Lip. 40:89 (1986). For more information on nanoparticles, see Yetisgin et al., Molecules 2020, 25, 2193.

[0197] Particle size can vary over a wide range and can be measured in a variety of ways. As indicated, in preferred embodiments, the particles are nanoparticles <130 nm in size. However, the NPs of the present invention can also have a minimum size of equal to or less than 500 nm, less than 150 nm, less than 140 nm, less than 120 nm, less than 110 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 40 nm, less than 30 nm, less than 20 nm, or less than 10 nm. In particular embodiments, the nanoparticles are NPs between 90 and 130 nm in size.

[0198] In particular embodiments, the NPs may have a minimum size in the range of 5 nm to 500 nm, 10 nm to 100 nm, 20 nm to 90 nm, 30 nm to 80 nm, 40 nm to 70 nm, and 40 nm to 60 nm. In particular embodiments, the size is the diameter of the NPs or coated NPs. In particular embodiments, a population of nanoparticles of the present invention may have an average minimum size of equal to or less than 500 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 40 nm, less than 30 nm, less than 20 nm, or less than 10 nm. In particular embodiments, the population of NPs in the composition of the present invention may have an average diameter in the range of 5 nm to 500 nm, 10 nm to 100 nm, 20 nm to 90 nm, 30 nm to 80 nm, 40 nm to 70 nm, 40 nm to 60 nm, 70 nm to 130 nm, or 75 nm to 125 nm. Nanoparticle sizes can be determined using, for example, conventional techniques such as dynamic light scattering and/or electron microscopy. Although not preferred, microparticles may also be used in particular embodiments.

[0199] In particular embodiments, PbAE polymers are mixed with nucleotides (e.g., in vitro transcribed mRNA) in a ratio of 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or more to form PbAE-nucleotide polyplexes. In particular embodiments, PbAE polymers are mixed with nucleotides (e.g., in vitro transcribed mRNA) in a ratio of 60:1 to form PbAE-nucleotide polyplexes. In particular embodiments, PbAE-nucleotide polyplexes can be combined with PGA/dimannose to form final NPs.

[0200] In particular embodiments, the positively charged polymer cores are PbAE formed by combining 1,4-butanediol diacrylate with 4-amino-1-butanol in a 1:1 molar ratio of diacrylate to amine monomers. The polymer can be piperazine-capped polymer 447. When conjugated with dimannose, α-D-mannopyranosyl-(1→2)-α-D-mannopyranose (dimannose, Omicron Biochemicals Inc.) can be modified to glycosylamine prior to conjugation with PGA.

[0201] Codon-optimized mRNAs can be capped with the anti-reverse cap analog 3'-O-Me-m7G(5')ppp(5')G (ARCA) with complete substitution by the modified ribonucleotides pseudouridine (Ψ) and 5-methylcytidine (m5C).

[0202] To generate nanoparticles, PbAE-447 polymers can be added to mRNA at a 60:1 (w/w) ratio and immediately vortexed for 15 s at medium speed. The mixture can then be incubated at room temperature for 5 min to form PbAE-mRNA polyplexes. In the next step, 100 μg/mL PGA/dimannose in NaOAc buffer can be added to the polyplex solution, vortexed for 15 s at medium speed, and incubated for 5 min at room temperature. In this process, PGA/dimannose coats the surfaces of the PbAE-mRNA polyplexes, forming the final NPs. For long-term storage, D-sucrose (60 mg/mL) can be added to the NP solutions as a cryoprotectant. The nanoparticles can be quickly frozen in dry ice and then lyophilized. Dried NPs can be stored at -20°C or -80°C until use. For in vivo use, lyophilized NPs can be resuspended in water at a 1:20 (weight:volume) ratio.

[0203] In certain embodiments, the NPs have a size of 99.8 ± SE/24.5, a polydispersity of 0.183, and a near-neutral surface charge (3.40 ± SE/2.15 mV ζ-potential). These physicochemical properties of the NPs can be characterized using a Zetapals instrument (Brookhaven Instrument Corporation) at 25°C. To measure the hydrodynamic radius and polydispersity using dynamic light scattering, the NPs can be diluted fivefold in 25 mM NaOAc (pH = 5.2). To measure the ζ-potential, the NPs can be diluted 10-fold in 10 mM PBS (pH = 7.0). To assess the stability of the NPs, freshly prepared nanoparticles can be diluted in 10 mM PBS buffer (pH = 7.4). The hydrodynamic radius and polydispersity of NPs were measured every 10 min for 5 h, and their particle sizes and concentrations were determined by particle tracking analysis using a Nanosite 300 instrument (Malvern). Freshly prepared NPs (25 μL, containing 0.83 μg mRNA) were loaded onto glow-discharge-treated 200-mesh carbon/formvar-coated copper grids. After 30 s, the grids were sequentially treated with 50% Karnovsky's fixative, 0.1 M cacodylate buffer, _dH2O , and then 1% (w/v) uranyl acetate. Samples were visualized using a JEOL JEM-1400 transmission electron microscope operating at 120 kV (JEOL USA).

[0204] In particular embodiments, the nanoparticles may optionally include a binding domain directed to ligands that bind cellular markers present on the surface of monocytes and/or macrophages.

[0205] M2-binding domains. Egr2 is expressed by M2 macrophages. Commercially available antibodies to Egr2 can be obtained from Thermo Fisher, Waltham, MA; Abcam, Cambridge, MA; Millipore Sigma, Burlington, MA; Miltenyi Biotec, Bergisch Gladbach, Germany; LifeSpan Biosciences, Inc., Seattle, WA; and Novus Biologicals, Littleton, CO. The production of anti-Egr2 antibodies is described, for example, in Murakami K et al. (1993) Oncogene 8(6): 1559-1566. Anti-Egr2 antibodies include: rabbit monoclonal anti-Egr2 antibody clone EPR4004; mouse monoclonal anti-Egr2 antibody clone 1G5; mouse monoclonal anti-Egr2 antibody clone OTI1B12; a rabbit polyclonal anti-Egr2 antibody recognizing amino acid residues 200-300 of human Egr2; a rabbit polyclonal anti-Egr2 antibody recognizing amino acid residues 340-420 of human Egr2; and a rabbit polyclonal anti-Egr2 antibody recognizing amino acid residues 370-420 of human Egr2. Binding domains can be derived from these antibodies and other antibodies disclosed herein.

[0206] In particular embodiments, the targeting ligand may be a nanobody comprising a binding domain comprising a CDR1 sequence comprising SGNIFSINAIG (SEQ ID NO: 45), a CDR2 sequence comprising TITLSGSTN (SEQ ID NO: 46), a CDR3 sequence comprising NTYSDSDVYGY (SEQ ID NO: 47). These are CDR sequences that bind CD206.

[0207] In particular embodiments, the targeting ligand may be a nanobody comprising a CDR1 sequence comprising PGFKLDYYAIA (SEQ ID NO: 48), a CDR2 sequence comprising SINSSGGST (SEQ ID NO: 49), and a CDR3 sequence comprising LRRYYGLNLDPGSYDY (SEQ ID NO: 50). These are CDR sequences that bind CD206.

[0208] In particular embodiments, the targeting ligand comprises a human or humanized binding domain (e.g., a nanobody) comprising a CDR1 sequence comprising GFPFNIYPMS (SEQ ID NO: 51), a CDR2 sequence comprising YISHGGTTT (SEQ ID NO: 52), and a CDRH3 sequence comprising GYARLMTDSELV (SEQ ID NO: 53). These are the CDR sequences that bind CD206.

[0209] Many additional antibodies specific for CD206 are known to those skilled in the art and can be readily characterized for sequence, epitope binding, and affinity. See, for example, WO 2014/140376, WO 2013/174537, and US 7,560534. Commercially available antibodies to CD206 can be obtained from Thermo Fisher, Waltham, MA; Proteintech, Rosemont, IL; BioLegend, San Diego, CA; R & D Systems, Minneapolis, MN; LifeSpan Biosciences, Inc., Seattle, WA; Novus Biologicals, Littleton, CO, and Bio-Rad, Hercules, CA. In specific embodiments, the anti-CD206 antibody comprises rat anti-mouse CD206 monoclonal antibody clone C068C2 (Catalog No. 141732, Biolegend, San Diego, CA).

[0210] In particular embodiments, the targeting ligand comprises a binding domain comprising a CDRL1 sequence comprising ASQSVSHDV (SEQ ID NO: 54), a CDRL2 sequence comprising YTS, a CDRL3 sequence comprising QDYSSPRT (SEQ ID NO: 56), a CDRH1 sequence comprising GYSITSDY (SEQ ID NO: 57), a CDRH2 sequence comprising YSG, and a CDRH3 sequence comprising CVSGTYYFDYWG (SEQ ID NO: 59). These are the CDR sequences of the Mac2-48 antibody, which binds CD163.

[0211] In particular embodiments, the targeting ligand comprises a binding domain comprising a CDRL1 sequence comprising ASQSVSSDV (SEQ ID NO: 60), a CDRL2 sequence comprising YAS, a CDRL3 sequence comprising QDYTSPRT (SEQ ID NO: 62), a CDRH1 sequence comprising GYSITSDY (SEQ ID NO: 57), a CDRH2 sequence comprising YSG, and a CDRH3 sequence comprising CVSGTYYFDYWG (SEQ ID NO: 59). These are the CDR sequences of the Mac2-158 antibody, which binds CD163.

[0212] Many additional antibodies or binding domains specific for CD163 are known to those skilled in the art and can be readily characterized for sequence, epitope binding, and affinity. See, for example, WO 2011/039510, WO 2002/032941, WO 2002/076501, and US 2005/0214871. Commercially available antibodies for CD163 can be obtained from Thermo Fisher, Waltham, MA; Enzo Life Sciences, Inc., Farmingdale, NY; BioLegend, San Diego, CA; R & D Systems, Minneapolis, MN; LifeSpan Biosciences, Inc., Seattle, WA; and RDI Research Diagnostics, Flanders, NJ. In specific embodiments, anti-CD163 antibodies may include: mouse monoclonal anti-CD163 antibody clone 3D4; mouse monoclonal anti-CD163 antibody clone Ber-Mac3; mouse monoclonal anti-CD163 antibody clone EDHu-1; and mouse monoclonal anti-CD163 antibody clone GHI/61.

[0213] In particular embodiments, the targeting ligand comprises a binding domain comprising a CDRL1 sequence comprising RSSKSLLYKDGKTYLN (SEQ ID NO: 66), a CDRL2 sequence comprising LMSTRAS (SEQ ID NO: 67), a CDRL3 sequence comprising QQLVEYPFT (SEQ ID NO: 68), a CDRH1 sequence comprising GYWMS (SEQ ID NO: 69), a CDRH2 sequence comprising EIRLKSDNYATHYAESVKG (SEQ ID NO: 70), and a CDRH3 sequence comprising FID. These are the CDR sequences that bind CD23.

[0214]Many antibodies or binding domains specific for CD23 are known to those skilled in the art and can be readily characterized for sequence, epitope binding, and affinity. See, for example, US 7,008,623, US 6,011,138 A (antibodies including 5E8, 6G5, 2C8, B3B1, and 3G12), US 2009/0252725, Rectoret al. (1985) J. Immunol. 55: 481-488; Flores-Rumeoet al. (1993) Science 241: 1038-1046; Sherret al. (1989) J. Immunol. 142: 481-489; and Peneet al., (1988) PNAS 85: 6820-6824. Commercially available antibodies to CD23 can be obtained from Thermo Fisher, Waltham, MA; Abcam, Cambridge, MA; Bioss Antibodies, Inc., Woburn, MA; Bio-Rad, Hercules, CA; LifeSpan Biosciences, Inc., Seattle, WA; and Boster Biological Technology, Pleasanton, CA. In particular embodiments, anti-CD23 antibodies can include: mouse monoclonal anti-CD23 antibody clone Tu 1; rabbit monoclonal anti-CD23 antibody clone SP23; rabbit monoclonal anti-CD23 antibody clone EPR3617; mouse monoclonal anti-CD23 antibody clone 5B5; mouse monoclonal anti-CD23 antibody clone 1B12; mouse monoclonal anti-CD23 antibody clone M-L23.4 and mouse monoclonal anti-CD23 antibody clone 3A2.

[0215] M1 binding domains. In particular embodiments, the targeting ligand comprises a binding domain comprising a CDRL1 sequence comprising SSNIGDNY (SEQ ID NO: 72), a CDRL2 sequence comprising RDS, a CDRL3 sequence comprising QSYDSSLSGS (SEQ ID NO: 74), a CDRH1 sequence comprising GFTFDDYG (SEQ ID NO: 75), a CDRH2 sequence comprising ISWNGGKT (SEQ ID NO: 76), and a CDRH3 sequence comprising ARGSLFHDSSGFYFGH (SEQ ID NO: 77). These are the CDR sequences of the Ab79 antibody, which binds CD38.

[0216] In particular embodiments, the targeting ligand comprises a binding domain comprising a CDRL1 sequence comprising NSNIGSNT (SEQ ID NO: 78), a CDRL2 sequence comprising SDS, a CDRL3 sequence comprising QSYDSSLSGSR (SEQ ID NO: 80), a CDRH1 sequence comprising GFTFNNYG (SEQ ID NO: 81), a CDRH2 sequence comprising ISYDGSDK (SEQ ID NO: 82), and a CDRH3 sequence comprising ARVYYYGFSGPSMDV (SEQ ID NO: 83). These are the CDR sequences of the Ab19 antibody, which binds CD38.

[0217] In particular embodiments, the targeting ligand comprises a binding domain comprising a CDRL1 sequence comprising RASQSVSSYLA (SEQ ID NO: 84), a CDRL2 sequence comprising DASNRAT (SEQ ID NO: 85), a CDRL3 sequence comprising QQRSNWPPTF (SEQ ID NO: 86), a CDRH1 sequence comprising SFAMS (SEQ ID NO: 87), a CDRH2 sequence comprising AISGSGGGTYYADSVKG (SEQ ID NO: 88), and a CDRH3 sequence comprising DKILWFGEPVFDY (SEQ ID NO: 89). These are the CDR sequences of the CD38-binding antibody daratumumab, which is described in U.S. Patent No. 7,829,693.

[0218] Many antibodies specific for CD38 are known to those skilled in the art and can be readily characterized for sequence, epitope binding, and affinity. See, for example, WO 2005/103083, WO 2006/125640, WO 2007/042309, WO 2008/047242, WO 2012/092612, WO 2006/099875, WO 2011/154453, WO 2015/130728, US 7829693, and US 2016/0200828. Commercially available antibodies for CD38 can be obtained from Thermo Fisher, Waltham, MA; Abcam, Cambridge, MA; and Millipore Sigma, Burlington, MA. In particular embodiments, the anti-CD23 antibodies may include: rabbit monoclonal anti-CD38 antibody clone GAD-3; mouse monoclonal anti-CD38 antibody clone HIT2; mouse monoclonal anti-CD38 antibody clone AT1; mouse monoclonal anti-CD38 antibody clone AT13/5; rat monoclonal anti-CD38 antibody clone NIMR-5; and rat monoclonal IgG2a, κ, anti-CD38 antibody clone 90/CD38 (catalog # BD Biosciences, San Jose, CA).

[0219] In particular embodiments, G protein-coupled receptor 18 (Gpr18) is targeted on M1 macrophages. Commercially available antibodies for Gpr18 can be obtained from Assay Biotechnology Company Inc., Sunnyvale, CA; Thermo Fisher, Waltham, MA; Abcam, Cambridge, MA; GeneTex, Inc., Irvine, CA; and Novus Biologicals, Littleton, CO. In particular embodiments, anti-Gpr18 antibodies include: a rabbit polyclonal anti-Gpr18 antibody that recognizes a portion of amino acids 1-50 of human Gpr18; a rabbit polyclonal anti-Gpr18 antibody that recognizes a region comprising amino acids 160-240 of human Gpr18; a rabbit polyclonal anti-Gpr18 antibody that recognizes a region comprising amino acids 100-180 of human Gpr18; rabbit monoclonal anti-Gpr18 antibody clone EPR12359 and rabbit polyclonal anti-Gpr18 antibody recognizing the region including amino acids 140-190 of human Gpr18.

[0220] In specific embodiments, formyl peptide receptor 2 (Fpr2) is a target on M1 macrophages. Commercially available antibodies to Fpr2 can be obtained from Atlas Antibodies, Bromma, Sweden; Biorbyt, LLC, San Francisco, CA; Cloud-Clone Corp., Katy, TX; US Biological Life Sciences, Salem, MA, and Novus Biologicals, Littleton, CO. In specific embodiments, anti-fpr2 antibodies include: mouse monoclonal anti-fpr2 antibody clone GM1D6; mouse monoclonal anti-fpr2 antibody clone 304405; recombinant anti-fpr2 antibody clone REA663; and rabbit polyclonal anti-fpr2 antibody recognizing a region comprising amino acids 300-350 of fpr2.

[0221] In particular embodiments, the targeting ligand comprises a binding domain comprising a CDRL1 sequence comprising RASQSVSSYLA (SEQ ID NO: 84), a CDRL2 sequence comprising DASSRAT (SEQ ID NO: 91), a CDRL3 sequence comprising QLRSNWPPYT (SEQ ID NO: 92), a CDRH1 sequence comprising GYGMH (SEQ ID NO: 93), a CDRH2 sequence comprising VIWYDGSNKYYADSVKG (SEQ ID NO: 94), and a CDRH3 sequence comprising DTGDRFFDY (SEQ ID NO: 95). These are CDR sequences that bind CD64.

(9) Compositions for administration. The nanoparticles disclosed herein may be provided in a composition intended for administration to subjects. The compositions comprise a nanoparticle disclosed herein and a pharmaceutically acceptable carrier.

[0222] Illustrative commonly used pharmaceutically acceptable carriers include any and all bulking agents or fillers, solvents or co-solvents, dispersion media, coatings, surfactants, antioxidants (e.g., ascorbic acid, methionine, vitamin E), preservatives, isotonic agents, absorption delaying agents, salts, stabilizers, buffering agents, chelating agents (e.g., EDTA), gels, binders, dispersing and/or lubricants.

[0223] Illustrative buffering agents include citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and/or trimethylamine salts.

[0224] Illustrative preservatives include phenol, benzyl alcohol, meta-cresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium halides, hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0225] Illustrative isotonic agents include polyhydric sugar alcohols, including trihydric or higher order sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol, or mannitol.

[0226] Illustrative stabilizers include organic sugars, polyhydric sugar alcohols, polyethylene glycol, sulfur-containing reducing agents, amino acids, low molecular weight polypeptides, proteins, immunoglobulins, hydrophilic polymers, or polysaccharides.

[0227] In particular embodiments, the compositions are formulated for intraperitoneal, intravenous, or intracranial injection. The compositions described herein may also be formulated for intra-arterial, intranodal, intralymphatic, intratumoral, intramuscular, oral, and/or subcutaneous administration, and more particularly, for administration by intra-arterial, intranodal, intralymphatic, intratumoral, intramuscular, and/or subcutaneous injection. The compositions described herein may be formulated for administration by infusion, perfusion, or ingestion.

[0228] Injectable compositions may be formulated as aqueous solutions, for example, in buffers including Hank's solution, Ringer's solution, or saline. Aqueous solutions may contain auxiliary substances such as suspending, stabilizing, and/or dispersing agents. Alternatively, the preparation may be in lyophilized and/or powder form for reconstitution with a suitable vehicle, for example, sterile, pyrogen-free water, before use.

[0229] The compositions may also be formulated as depot preparations. Depot preparations may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil), or ion exchange resins, or as sparingly soluble derivatives, such as sparingly soluble salts.

[0230] In addition, the compositions can be formulated as sustained-release systems using semipermeable matrices of solid polymers containing nanoparticles. Various sustained-release materials have been created and are well known to those skilled in the art. Sustained-release systems can, depending on their chemical nature, release nanoparticles after administration for periods ranging from several weeks to more than 100 days.

[0231] Compositions for oral administration may be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like.

[0232] When formulated for the treatment of cancer, the disclosed compositions may also include nucleotides carrying one or more anti-cancer genes selected from the p53, RB, BRCA1, E1A, bcl-2, MDR-1, p21, p16, bax, bcl-xs, E2F, IGF-I VEGF, angiostatin, oncostatin, endostatin, GM-CSF, IL-12, IL-2, IL-4, IL-7, IFNγ, TNFα and/or HSV-tk genes.

[0233] Any formulation of the composition described herein may preferably contain any other pharmaceutically acceptable carriers, which include carriers that do not cause significant adverse allergic or other untoward reactions that outweigh the benefit of administration, whether for research, prophylactic and/or therapeutic treatment. Illustrative pharmaceutically acceptable carriers and formulations are described in Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990. In addition, the formulations can be manufactured in accordance with standards of sterility, pyrogenicity, general safety and purity as required by the U.S. FDA Office of Biological Standards and/or other appropriate foreign regulatory authorities.

[0234] In particular embodiments, the nanoparticles are provided in a composition that can comprise, for example, at least 0.1% w/v or w/w nanoparticles; at least 1% w/v or w/w nanoparticles; at least 10% w/v or w/w nanoparticles; at least 20% w/v or w/w nanoparticles; at least 30% w/v or w/w nanoparticles; at least 40% w/v or w/w nanoparticles; at least 50% w/v or w/w nanoparticles; at least 60% w/v or w/w nanoparticles; at least 70% w/v or w/w nanoparticles; at least 80% w/v or w/w nanoparticles; at least 90% w/v or w/w nanoparticles; at least 95% w/v or w/w nanoparticles; or at least 99% w/v or w/w nanoparticles.

[0235] (10) Methods of Use. The methods disclosed herein involve altering the activation state of macrophages from an inactivated state to an activated state by introducing nanoparticles containing nucleotides encoding one or more IRFs and IKKβ into the macrophages. In particular embodiments, the alteration results in a decrease in the percentage of macrophages in an inactivated state (e.g., M2 macrophages) in a population of macrophages treated with nanoparticles containing nucleotides encoding one or more IRFs and IKKβ by 5 times, 10 times, 15 times, 20 times, or more, compared to the percentage of macrophages in an inactivated state that were not treated with nanoparticles containing nucleotides encoding one or more IRFs and IKKβ. In specific embodiments, the change results in a reduction in the number of inactivated macrophages (e.g., M2 macrophages) in a population of macrophages treated with nanoparticles containing nucleotides encoding one or more IRFs and IKKβ by 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, or more, compared to the number of inactivated macrophages that were not treated with nanoparticles containing nucleotides encoding one or more IRFs and IKKβ.

[0236] In specific embodiments, altering the activation state of macrophages from an inactivated state to an activated state by introducing nanoparticles comprising nucleotides encoding one or more IRFs and IKKβ into macrophages results in: restoration of lymphocyte migration and infiltration into treatment areas, such as solid tumors, or areas of infection or inflammation; an increase in the release of proinflammatory (anti-tumor) cytokines, including IL-1β, IL-12, IFNγ and/or TNFα by 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, or more; a decrease in the release of cytokines associated with the M2 macrophage phenotype, including IL-6, by 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, or more.

[0237] In particular embodiments, changing the activation state of macrophages from an inactivated state to an activated state comprises introducing into the macrophages nanoparticles containing nucleotides encoding IRF5 and IRF8. In particular embodiments, changing the activation state of macrophages from an inactivated state to an activated state comprises introducing into the macrophages nanoparticles containing nucleotides encoding mutant IRFs that are constitutively active or more active than their wild-type IRF counterpart.

[0238] The methods described herein also result in the secretion of multispecific molecules (e.g., bispecific molecules) from genetically modified monocytes/macrophages, and optionally TGFβ inhibitors.

[0239] The methods described herein include treating subjects (humans, pets, livestock, and laboratory animals) with the compositions described herein. Treating subjects includes delivering a therapeutically effective amount. Therapeutically effective amounts are amounts effective for prophylactic treatment and/or therapeutic treatment.

[0240] An "effective amount" is the amount of a compound necessary to achieve a desired physiological change in a subject. Effective amounts are often administered for research purposes. The effective amounts disclosed herein result in immunomodulation of cells in a subject. In specific embodiments, the cells to be immunomodulated are immunosuppressive cells. In specific embodiments, the cells to be immunomodulated are macrophages. In specific embodiments, immunomodulation of macrophages comprises switching immunosuppressive macrophages to activated macrophages. In specific embodiments, immunomodulation of macrophages comprises switching M2 macrophages to M1 macrophages. In specific embodiments, the cells to be immunomodulated include immunosuppressive cells, including MDSCs, Tregs, DCregs, neutrophils, Th17s, Bregs, and/or MSCs. In specific embodiments, immunomodulation of immunosuppressive cells involves phenotypic and/or functional switching of immunosuppressive cells from pro-tumor to anti-tumor.

[0241] "Prophylactic treatment" is treatment administered to a subject who has no signs or symptoms of a disease or condition, or who exhibits only early signs or symptoms of a disease or condition, so that the treatment is administered to reduce, prevent, or lower the risk of developing the disease or condition in the future. Thus, prophylactic treatment is a preventative measure against a disease or disorder. For example, in specific embodiments, prophylactic treatment includes administering the compositions disclosed herein to a subject who has had cancer but is in remission, so that the treatment is administered to reduce the likelihood of or delay the occurrence of a relapse.

[0242] "Therapeutic treatment" is treatment administered to a subject exhibiting symptoms or signs of a disease or condition and is administered to the subject for the purpose of reducing or eliminating these signs or symptoms of the disease or condition. For example, in specific embodiments, therapeutic treatment includes administering the compositions disclosed herein to a subject who has cancer to reduce or eliminate tumors and/or metastases.

[0243] In specific embodiments, therapeutically effective amounts provide an anti-cancer effect in a subject. Cancer (medical term: malignancy) refers to a class of diseases in which a group of cells exhibit uncontrolled growth (dividing beyond the norm), invasion (penetration and destruction of adjacent tissues), and sometimes metastasis. The term "metastasis" refers to the spread of cancer cells from their original site of proliferation to another part of the body. The formation of metastases is a very complex process and depends on the detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membrane into body cavities and vessels, and then, after transport by blood, infiltration of target organs. Finally, the growth of a new tumor, i.e., a secondary tumor or metastatic tumor, at the target site depends on angiogenesis. Tumor metastasis often occurs even after removal of the primary tumor, since tumor cells or components can persist and develop metastatic potential.

[0244] In specific embodiments, the therapeutically effective amount provides an anti-tumor effect in the subject. A "tumor" is a swelling or lesion resulting from the abnormal growth of cells (called neoplastic cells or tumor cells). A "tumor cell" is an abnormal cell that divides by a mechanism of rapid, uncontrolled cellular proliferation and continues to divide after the stimuli that initiated the new division cease. Tumors exhibit a partial or complete lack of structural organization and functional coordination with normal tissue and typically form a distinct mass of tissue that may be benign, precancerous, or malignant.

[0245] The term "antitumor effect" means a biological effect that can be manifested by a decrease in the number of tumor cells, a decrease in the number of metastases, a decrease in tumor volume, an increase in survival, induction of apoptosis of cancer cells, induction of death of cancer cells, induction of chemo- or radiosensitivity in cancer cells, inhibition of angiogenesis near cancer cells, inhibition of proliferation of cancer cells, inhibition of tumor growth, prevention of metastasis, prolongation of the life of a subject, a decrease in pain associated with cancer, a decrease in the number of metastases and/or a decrease in the rate of relapse or recurrence of cancer after treatment. Accordingly, the compositions disclosed herein can be used to treat various types of cancer, they can prevent or significantly delay metastasis and/or can prevent or significantly delay relapse. In specific embodiments, the overall survival of a subject with cancer treated with the nanoparticle composition described herein is increased by 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, or more, compared to a control subject with the same cancer not treated with the nanoparticle composition. In specific embodiments, the number of metastases in a subject with cancer treated with the nanoparticle composition described herein is reduced by 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, or more, compared to a control subject with the same cancer not treated with the nanoparticle composition.

[0246] In particular embodiments, the therapeutic treatment comprises administering the compositions disclosed herein to a subject suffering from cancer in combination with another therapy to reduce or eliminate tumors. In particular embodiments, therapies for use in combination with the compositions disclosed herein include cancer vaccines, CAR immunotherapy (e.g., CAR-T immunotherapy), chemotherapy, radiation therapy, hormonal therapy, signal transduction inhibitors, gene expression modulators, apoptosis inducers, angiogenesis inhibitors, and monoclonal antibodies that deliver toxic molecules. In specific embodiments, administration of the nanoparticle composition disclosed herein to a subject suffering from cancer in combination with radiation therapy increases overall survival by 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, or more, compared to a control subject with the same cancer who was not administered the nanoparticle composition in combination with radiation therapy.

[0247] Cancer that can be treated using the systems and methods disclosed herein includes ovarian cancer, breast cancer, brain cancer, melanomas, lung metastases, seminomas, teratomas, neuroblastomas, gliomas, colorectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, skin cancer, cervical cancer, bowel cancer, liver cancer, colon cancer, stomach cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophageal cancer, colorectal cancer, pancreatic cancer, ear, nose and throat (ENT) cancer, prostate cancer, uterine cancer, lung cancer and metastases thereof.

[0248] In specific embodiments, therapeutically effective amounts provide antipathogenic effects. Antipathogenic effects may include anti-infective effects. Anti-infective effects may include a reduction in the frequency of infections, a reduction in the severity of infections, a reduction in the duration of infections, a reduction in the number of infected cells, a reduction in the volume of infected tissue, an increase in life expectancy, induction of sensitivity of infected cells to immune clearance, a reduction in pain associated with an infection, and/or a reduction or elimination of a symptom associated with the infection being treated.

[0249] In specific embodiments, therapeutically effective amounts provide anti-inflammatory effects. Anti-inflammatory effects may include a reduction in inflammation-related pain, fever, redness, swelling, and/or loss of function.

[0250] In specific embodiments, therapeutically effective amounts provide anti-Crohn's disease effects or anti-ulcerative colitis effects. Anti-Crohn's disease effects or anti-ulcerative colitis effects may include a reduction in diarrhea, a reduction in rectal bleeding, a reduction in unexplained weight loss, a reduction in fever, a reduction in abdominal pain and cramps, a reduction in fatigue and loss of energy, and/or a restoration of appetite.

[0251] In specific embodiments, therapeutically effective amounts provide anti-arthritic effects. Anti-arthritic effects may include reduction of pain, stiffness, swelling, redness in joints, and/or restoration of range of motion. Types of arthritis include rheumatoid arthritis (RA), ankylosing spondylitis, and psoriatic arthritis.

[0252] In specific embodiments, therapeutically effective amounts provide anti-psoriatic plaque effects. Anti-psoriatic plaque effects may include a reduction in redness, scaling, itching, burning, soreness, nail bed abnormalities, and/or joint swelling or numbness.

[0253] Therapeutically effective amounts (also referred to herein as doses) for administration can be initially estimated based on the results of in vitro assays and/or studies in animal models. For example, in animal models, a dose can be adjusted to achieve a range of circulating concentrations that includes the _IC50 determined in cell culture for a particular target. Such information can be used to more accurately determine useful doses for subjects of interest.

[0254] The actual dose administered to a particular subject may be determined by a physician, veterinarian, or researcher taking into account such parameters as physical and physiological factors, including the target, body weight, severity of the condition, type of disease, previous or concomitant therapeutic interventions, idiopathic status of the subject, and route of administration.

[0255] Useful doses are often in the range of 0.1 to 5 mcg/kg or 0.5 to 1 mcg/kg. In particular embodiments, the dose may comprise 1 mcg/kg, 5 mcg/kg, 10 mcg/kg, 15 mcg/kg, 20 mcg/kg, 25 mcg/kg, 30 mcg/kg, 35 mcg/kg, 40 mcg/kg, 45 mcg/kg, 50 mcg/kg, 55 mcg/kg, 60 mcg/kg, 65 mcg/kg, 70 mcg/kg, 75 mcg/kg, 80 mcg/kg, 85 mcg/kg, 90 mcg/kg, 95 mcg/kg, 100 mcg/kg, 150 mcg/kg, 200 mcg/kg, 250 mcg/kg, 350 mcg/kg, 400 µg/kg, 450 µg/kg, 500 µg/kg, 550 µg/kg, 600 µg/kg, 650 µg/kg, 700 µg/kg, 750 µg/kg, 800 µg/kg, 850 µg/kg, 900 µg/kg, 950 µg/kg, 1000 µg/kg, from 0.1 to 5 mg/kg or 0.5 to 1 mg/kg. In particular embodiments, the dose may comprise 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 85 mg/kg, 90 mg/kg, 95 mg/kg, 100 mg/kg, 150 mg/kg, 200 mg/kg, 250 mg/kg, 350 mg/kg, 400 mg/kg, 450 mg/kg, 500 mg/kg, 550 mg/kg, 600 mg/kg, 650 mg/kg, 700 mg/kg, 750 mg/kg, 800 mg/kg, 850 mg/kg, 900 mg/kg, 950 mg/kg, 1000 mg/kg and more.

[0256] Therapeutically effective amounts can be achieved by administering one or more doses over a course of treatment (e.g., daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, or annually. In particular embodiments, therapeutically effective amounts can be achieved by administering repeated doses over a course of treatment.

[0257] The nanoparticle compositions described herein can be administered by injection, inhalation, infusion, perfusion, lavage, or ingestion. Routes of administration can include intravenous, intradermal, intraarterial, parenteral, intranasal, intranodal, intralymphatic, intraperitoneal, intracranial, intralesional, intraprostatic, intravaginal, intrarectal, local, intrathecal, intratumoral, intramuscular, intravesicular, oral, subcutaneous, and/or sublingual; and more particularly, by intravenous, intratumoral, intraperitoneal, and/or intracranial injection. Local administration includes administering a therapeutically effective amount of the composition disclosed herein to a specific area, organ, or body cavity. For example, intraperitoneal injection can be used to deliver a therapeutic agent for the treatment of ovarian cancer, or intracranial injection can be used to deliver a therapeutic agent for the treatment of glioma. Administration of a therapeutic agent to the tumor site can involve ligand-mediated targeting of the therapeutic agent (e.g., nanoparticle compositions) to tumor cells and/or tumor-supporting cells, but not to healthy tissue, using the targeting ligands described above. Administration of a therapeutic agent to the tumor site can involve passive targeting of the therapeutic agent (e.g., nanoparticle compositions) to tumor cells and/or tumor-supporting cells, but not to healthy tissue. Particular embodiments of passive targeting can include the phenomenon of enhanced permeability and retention (EPR), based on the size range of the nanoparticles, as well as leaky vasculature and impaired lymphatic drainage of tumor tissues. Systemic administration, in contrast, is general administration to the body and is accomplished by intravenous injection of a composition or therapeutic agent into the bloodstream. Systemic administration of a therapeutic agent may be beneficial for less localized forms of cancer, such as metastatic cancer.

[0258] (11) Illustrative Embodiments

1. A nanoparticle comprising:

a targeting ligand that binds to a professional phagocyte; and

a nucleic acid that encodes a protein molecule having at least a first binding domain and a second binding domain,

wherein the first binding domain is specific for a cell surface protein expressed by an immune cell, and

wherein the second binding domain is specific for a cell surface protein expressed by the cell of interest.

2. The nanoparticle of embodiment 1, wherein the cell of interest is a cancer cell, an infected cell, an autoreactive cell, or a prokaryotic cell.

3. The nanoparticle of embodiment 1 or 2, wherein the targeting ligand binds a cell surface protein expressed by a monocyte, a macrophage, or both.

4. The nanoparticle of any one of embodiments 1-3, wherein the targeting ligand is dimannose.

5. The nanoparticle of any one of embodiments 1-4, wherein the nucleic acid is ribonucleic acid (RNA).

6. The nanoparticle of embodiment 5, wherein the RNA is messenger RNA (mRNA).

7. The nanoparticle of embodiment 6, wherein the mRNA is synthetic RNA or in vitro transcribed RNA (IVT RNA).

8. The nanoparticle of any one of embodiments 1-7, wherein the first binding domain is specific for a cell surface protein of a lymphocyte.

9. The nanoparticle of embodiment 8, wherein the lymphocyte is selected from the group consisting of a T cell, a B cell, a natural killer (NK) cell, and a tumor infiltrating lymphocyte (TIL).

10. The nanoparticle of any one of embodiments 1-9, wherein the first binding domain is specific for a cell surface protein of a T cell selected from the group consisting of a CD8+ T cell, a CD4+ T cell, a gamma/delta T cell, and an NK T cell.

11. The nanoparticle of any one of embodiments 1-10, wherein the first binding domain is specific for CD3.

12. The nanoparticle of any one of embodiments 1-11, wherein the protein molecule is a multispecific T-cell binding molecule.

13. The nanoparticle of any one of embodiments 1-12, wherein the protein molecule is an EpCAM-CD3 bispecific T cell binding molecule.

14. The nanoparticle of any one of embodiments 1-13, wherein the second binding domain is specific for an antigen expressed by the cell of interest.

15. The nanoparticle of any one of embodiments 1-14, further comprising a second nucleic acid encoding one or more interferon regulatory factors (IRFs).

16. The nanoparticle of any one of embodiments 1-15, further comprising a tumor cell proliferation inhibitor or a nucleic acid encoding a tumor cell proliferation inhibitor.

17. The nanoparticle of any one of embodiments 1-16, wherein the nucleic acid encodes an antibody or an antigen-binding fragment of an antibody.

18. The nanoparticle of any one of embodiments 1-17, comprising a nucleic acid encoding a CD40-CD40L inhibitor or a TGFβ inhibitor.

19. The nanoparticle of any one of embodiments 1-18, which comprises a liposome, a liposomal nanoparticle, a lipid nanoparticle, or a solid lipid nanoparticle.

20. A composition comprising:

a first plurality of nanoparticles, wherein each of the first plurality of nanoparticles includes:

a targeting ligand that binds to a professional phagocyte; and

a nucleic acid encoding a protein molecule having a first binding domain specific for a cell surface protein expressed by an immune cell and a second binding domain specific for a cell surface protein expressed by a cell of interest.

21. The composition of embodiment 20, wherein the cell of interest is a cancer cell, an infected cell, an autoreactive cell, or a prokaryotic cell.

22. The composition of embodiment 20 or 21, wherein the targeting ligand binds a cell surface protein expressed by a monocyte, a macrophage, or both.

23. The composition of any one of embodiments 20-22, wherein the targeting ligand is dimannose.

24. The composition of any one of embodiments 20-23, wherein the nucleic acid is RNA.

25. The composition of embodiment 24, wherein the RNA is mRNA.

26. The composition of embodiment 25, wherein the mRNA is synthetic RNA or IVT RNA.

27. The composition of any one of embodiments 20-26, wherein the first binding domain is specific for a lymphocyte cell surface protein.

28. The composition of embodiment 27, wherein the lymphocyte is selected from the group consisting of a T cell, a B cell, a NK cell, and a TIL.

29. The composition of any one of embodiments 20-28, wherein the first binding domain is specific for a cell surface protein of a T cell selected from the group consisting of a CD8+ T cell, a CD4+ T cell, a gamma/delta T cell, and an NK T cell.

30. The composition of any one of embodiments 20-29, wherein the first binding domain is specific for CD3.

31. The composition of any one of embodiments 20-30, wherein the protein molecule is a bispecific T cell binding molecule.

32. The composition of any one of embodiments 20-31, wherein the protein molecule is an EpCAM-CD3 bispecific T cell binding molecule.

33. The composition of any one of embodiments 20-32, wherein the second binding domain is specific for an antigen expressed by the cell of interest.

34. The composition according to any one of embodiments 20-33, further comprising a pharmaceutically acceptable carrier.

35. The composition of any one of embodiments 20-34, wherein at least the nanoparticles of a subset of the first plurality of nanoparticles further comprise one or more of (a) a nucleic acid encoding one or more interferon-regulating factors (IRFs) and (b) a nucleic acid encoding IKKβ.

36. The composition of any one of embodiments 20-35, wherein the nanoparticles of the first plurality comprise a liposome, a liposomal nanoparticle, a lipid nanoparticle, or a solid lipid nanoparticle.

37. The composition according to any one of embodiments 20-36, further comprising:

a second plurality of nanoparticles, wherein at least nanoparticles of a subset of the second plurality of nanoparticles comprise one or more of (a) a nucleic acid encoding one or more interferon-regulating factors (IRFs), and (b) a nucleic acid encoding IKKβ.

38. The composition according to any one of embodiments 20-37, further comprising a tumor cell proliferation inhibitor.

39. The composition of any one of embodiments 20-38, wherein at least the nanoparticles of a subset of the first or second plurality of nanoparticles further comprise a nucleic acid encoding a tumor cell proliferation inhibitor.

40. The composition of any one of embodiments 20-39, wherein at least the nanoparticles of a subset of the first or second plurality of nanoparticles further comprise a nucleic acid encoding an antigen-binding fragment of a tumor cell proliferation inhibitor antibody.

41. The composition of any one of embodiments 37-40, wherein the second plurality of nanoparticles comprises a liposome, a liposomal nanoparticle, a lipid nanoparticle, or a solid lipid nanoparticle.

42. The composition of any one of embodiments 20-41, further comprising a third plurality of nanoparticles, wherein at least the nanoparticles of a subset of the third plurality of nanoparticles comprise a nucleic acid encoding an antigen-binding fragment of a tumor cell proliferation inhibitor antibody.

43. The composition of any one of embodiments 38-42, wherein the tumor cell proliferation inhibitor is a CD40-CD40L inhibitor or a TGFβ inhibitor.

44. The composition of embodiment 42 or 43, comprising a first plurality of nanoparticles and a third plurality of nanoparticles without a second plurality of nanoparticles.

45. The composition of any one of embodiments 42-44, wherein the third plurality of nanoparticles comprises a liposome, a liposomal nanoparticle, a lipid nanoparticle, or a solid lipid nanoparticle.

46. A composition for treating a condition in a human subject, comprising:

a first plurality of nanoparticles, where each of the plurality of nanoparticles includes

(i) a targeting ligand that binds a monocyte, a macrophage, or both; and

(ii) an mRNA encoding a protein molecule having at least a first binding domain specific for a cell surface protein expressed by a lymphocyte and a second binding domain specific for a cell surface protein expressed by a cell of interest;

wherein the first plurality of nanoparticles stimulate or enhance the immune response in a human subject and thereby treat the condition.

47. The composition of embodiment 46, wherein the cell of interest is a cancer cell, an infected cell, an autoreactive cell, or a prokaryotic cell.

48. The composition of any one of embodiments 46 or 47, wherein the targeting ligand is dimannose.

49. The composition of any one of embodiments 46-48, wherein the mRNA is synthetic RNA or IVT RNA.

50. The composition of any one of embodiments 46-49, wherein the first binding domain is specific for a cell surface protein of a lymphocyte.

51. The composition of embodiment 50, wherein the lymphocyte is selected from the group consisting of a T cell, a B cell, a NK cell, and a TIL.

52. The composition of any one of embodiments 46-51, wherein the first binding domain is specific for a cell surface protein of a T cell selected from the group consisting of a CD8+ T cell, a CD4+ T cell, a gamma/delta T cell, and an NK T cell.

53. The composition of any one of embodiments 46-52, wherein the first binding domain is specific for CD3.

54. The composition of any one of embodiments 46-53, wherein the protein molecule is a bispecific T cell binding molecule.

55. The composition of any one of embodiments 46-54, wherein the protein molecule is an EpCAM-CD3 bispecific T cell binding molecule.

56. The composition of any one of embodiments 46-55, wherein the second binding domain is specific for an antigen expressed by the cell of interest.

57. The composition of any one of embodiments 46-56, further comprising a pharmaceutically acceptable carrier.

58. The composition of any one of embodiments 46-57, wherein at least the nanoparticles of a subset of the first plurality of nanoparticles further comprise one or more of (a) mRNA encoding one or more interferon-regulating factors (IRFs), (b) mRNA encoding IKKβ, or (c) mRNA encoding one or more IRFs and mRNA encoding IKKβ, and (c) mRNA encoding a tumor cell proliferation inhibitor.

59. The composition of any one of embodiments 46-58, wherein the nanoparticles of the first plurality comprise a liposome, a liposomal nanoparticle, a lipid nanoparticle, or a solid lipid nanoparticle.

60. The composition according to any one of embodiments 46-59, further comprising:

a second plurality of nanoparticles, where each of the second plurality of nanoparticles includes

a targeting ligand that binds a monocyte, a macrophage, or both, and

one or more of (a) mRNA encoding one or more interferon regulatory factors (IRFs), (b) mRNA encoding IKKβ, and (c) mRNA encoding a tumor cell proliferation inhibitor.

61. The composition of embodiment 60, wherein the second plurality of nanoparticles comprise mRNA encoding an antigen-binding fragment of a tumor cell proliferation inhibitor antibody.

62. The composition of embodiment 60 or 61, wherein the tumor cell proliferation inhibitor is a CD40-CD40L inhibitor or a TGFβ inhibitor.

63. The composition of any one of embodiments 60-62, wherein the second plurality of nanoparticles comprises a liposome, a liposomal nanoparticle, a lipid nanoparticle, or a solid lipid nanoparticle.

64. A method of treating a condition in a human subject, comprising:

administering to a human subject a composition comprising a first plurality of nanoparticles, wherein each of the first plurality of nanoparticles comprises:

(i) a targeting ligand that binds a monocyte, a macrophage, or both; and

(ii) an mRNA encoding a protein molecule having at least a first binding domain specific for a cell surface protein expressed by a lymphocyte and a second binding domain specific for a cell surface protein expressed by a cell of interest;

where a plurality of nanoparticles stimulate or enhance the immune response in a human subject and thereby treat the condition.

65. The method of embodiment 64, wherein the cell of interest is a cancer cell, an infected cell, an autoreactive cell, or a prokaryotic cell.

66. The method of embodiment 64 or 65, wherein the targeting ligand is dimannose.

67. The method according to any one of embodiments 64-66, wherein the mRNA is synthetic RNA or IVT RNA.

68. The method according to any one of embodiments 64-67, wherein the lymphocyte is selected from the group consisting of a T cell, a B cell, a NK cell, and a TIL.

69. The method according to any one of embodiments 64-68, wherein the first binding domain is specific for a cell surface protein of a T cell selected from the group consisting of a CD8+ T cell, a CD4+ T cell, a gamma/delta T cell, and an NK T cell.

70. The method according to any one of embodiments 64-69, wherein the first binding domain is specific for CD3.

71. The method according to any one of embodiments 64-70, wherein the protein molecule is a bispecific T cell binding molecule.

72. The method according to any one of embodiments 64-71, wherein the protein molecule is an EpCAM-CD3 bispecific T cell binding molecule.

73. The method of any one of embodiments 64-72, wherein the second binding domain is specific for an antigen expressed by the cell of interest.

74. The method according to any one of embodiments 64-73, wherein the composition further comprises a pharmaceutically acceptable carrier.

75. The method of any one of embodiments 64-74, wherein at least the nanoparticles of a subset of the first plurality of nanoparticles further comprise one or more of (a) a nucleic acid encoding one or more interferon-regulating factors (IRFs), (b) a nucleic acid encoding IKKβ, and (c) a nucleic acid encoding a tumor cell proliferation inhibitor.

76. The composition of any one of embodiments 64-75, wherein the nanoparticles of the first plurality comprise a liposome, a liposomal nanoparticle, a lipid nanoparticle, or a solid lipid nanoparticle.

77. The method according to any one of embodiments 64-76, further comprising:

administering to a human subject a composition comprising a second plurality of nanoparticles, wherein each of the second plurality of nanoparticles comprises

a targeting ligand that binds a monocyte, a macrophage, or both, and

one or more of (a) mRNA encoding one or more interferon regulatory factors (IRFs) and (b) mRNA encoding IKKβ.

78. The method according to any one of embodiments 75-77, wherein at least the nanoparticles of a subset of the first or second plurality of nanoparticles further comprise mRNA encoding a tumor cell proliferation inhibitor.

79. The composition of embodiment 77 or 78, wherein the second plurality of nanoparticles comprises a liposome, a liposomal nanoparticle, a lipid nanoparticle, or a solid lipid nanoparticle.

80. The method according to any one of embodiments 64-79, further comprising:

administering to a human subject a composition comprising a third plurality of nanoparticles, wherein each of the third plurality of nanoparticles comprises

a targeting ligand that binds a monocyte, a macrophage, or both, and

mRNA encoding a tumor cell proliferation inhibitor.

81. The method according to any one of embodiments 75-80, wherein the mRNA encoding the tumor cell proliferation inhibitor encodes an antigen-binding fragment of an antibody of the tumor cell proliferation inhibitor.

82. The method according to any one of embodiments 75-81, wherein the tumor cell proliferation inhibitor is a CD40-CD40L inhibitor or a TGFβ inhibitor.

83. The composition of any one of embodiments 80-82, wherein the third plurality of nanoparticles comprises a liposome, a liposomal nanoparticle, a lipid nanoparticle, or a solid lipid nanoparticle.

84. The method according to any one of embodiments 77-83, wherein the step of introducing a composition comprising a first plurality of nanoparticles and the step of introducing a composition comprising a second plurality of nanoparticles are performed simultaneously or sequentially.

85. The method according to any one of embodiments 77-83, wherein the step of introducing a composition comprising a first plurality of nanoparticles is performed after the step of introducing a composition comprising a second plurality of nanoparticles.

86. The method according to any one of embodiments 80-85, wherein the step of introducing the composition comprising the third plurality of nanoparticles is performed simultaneously or sequentially with the step of introducing the first plurality of nanoparticles.

87. The method according to any one of embodiments 80-85, wherein the step of introducing the composition comprising the third plurality of nanoparticles is performed simultaneously or sequentially with the step of introducing the second plurality of nanoparticles.

88. The method according to any one of embodiments 80-85, comprising the steps of administering a composition comprising a first plurality of nanoparticles and administering a composition comprising a third plurality of nanoparticles, without the step of administering a composition comprising a second plurality of nanoparticles.

89. Modified professional phagocyte containing:

a nanoparticle loaded with a nucleic acid encoding a protein molecule having at least a first binding domain specific for a cell surface protein expressed by an immune cell and a second binding domain specific for a cell surface protein expressed by a cell of interest,

where the nanoparticle is attached to the surface of a phagocyte or has been internalized by a phagocyte.

90. The modified professional phagocyte of embodiment 89, wherein the cell of interest is a cancer cell, an infected cell, an autoreactive cell, or a prokaryotic cell.

91. A modified professional phagocyte according to any one of embodiments 89 or 90, which is a monocyte or a macrophage.

92. A modified professional phagocyte according to any one of embodiments 89-91, which is a tumor-associated macrophage.

93. The modified professional phagocyte of any one of embodiments 89-92, wherein the nucleic acid is ribonucleic acid (RNA).

94. The modified professional phagocyte of embodiment 93, wherein the RNA is messenger RNA (mRNA).

95. The modified professional phagocyte of embodiment 94, wherein the mRNA is synthetic RNA or in vitro transcribed RNA (IVT RNA).

96. The modified professional phagocyte of any one of embodiments 89-95, wherein the first binding domain is specific for a cell surface protein of a lymphocyte.

97. The modified professional phagocyte of embodiment 96, wherein the lymphocyte is selected from the group consisting of a T cell, a B cell, a NK cell, and a TIL.

98. The modified professional phagocyte of any one of embodiments 89-97, wherein the first binding domain is specific for a cell surface protein of a T cell selected from the group consisting of a CD8+ T cell, a CD4+ T cell, a gamma/delta T cell, and an NK T cell.

99. The modified professional phagocyte of any one of embodiments 89-98, wherein the first binding domain is specific for CD3.

100. The modified professional phagocyte of any one of embodiments 89-99, wherein the protein molecule is a bispecific T-cell binding molecule.

101. The modified professional phagocyte of any one of embodiments 89-100, wherein the protein molecule is an EpCAM-CD3 bispecific T cell binding molecule.

102. The modified professional phagocyte of any one of embodiments 89-101, wherein the nanoparticle is further loaded with one or more of (a) a nucleic acid encoding one or more interferon-regulating factors (IRFs), (b) a nucleic acid encoding IKKβ, and (c) a nucleic acid encoding a tumor cell proliferation inhibitor.

103. The modified professional phagocyte of any one of embodiments 89-102, wherein the nanoparticle comprises a liposome, a liposomal nanoparticle, a lipid nanoparticle, or a solid lipid nanoparticle.

104. A modified professional phagocyte according to any one of embodiments 89-103, further comprising:

a second nanoparticle loaded with one or more of (a) a nucleic acid encoding one or more interferon-regulating factors (IRFs), (b) a nucleic acid encoding IKKβ, and (c) a nucleic acid encoding a tumor cell proliferation inhibitor,

where the second nanoparticle is attached to the surface of the phagocyte or has been internalized by the phagocyte.

105. The modified professional phagocyte of embodiment 104, wherein the second nanoparticle comprises a liposome, a liposomal nanoparticle, a lipid nanoparticle, or a solid lipid nanoparticle.

106. A modified professional phagocyte according to embodiment 104 or 105, wherein the first or second nanoparticle is loaded with a nucleic acid encoding an antibody or an antigen-binding fragment of an antibody of a tumor cell proliferation inhibitor.

107. The modified professional phagocyte of any one of embodiments 102-106, wherein the tumor cell proliferation inhibitor is a CD40-CD40L inhibitor or a TGFβ inhibitor.

108. The modified professional phagocyte of any one of embodiments 89-107, further comprising at least one of

a second nanoparticle loaded with one or more of (a) a nucleic acid encoding one or more interferon-regulating factors (IRFs), (b) a nucleic acid encoding IKKβ, or (c) a nucleic acid encoding a tumor cell proliferation inhibitor; and

a third nanoparticle loaded with a nucleic acid encoding a tumor cell proliferation inhibitor,

where each of the second and third nanoparticles is attached to the surface of the phagocyte or has been internalized by the phagocyte.

109. The modified professional phagocyte of embodiment 108, wherein the third nanoparticle comprises a liposome, a liposomal nanoparticle, a lipid nanoparticle, or a solid lipid nanoparticle.

110. A nanoparticle having a positively charged polymer core and a neutral or negatively charged coating around the polymer core, wherein the positively charged polymer core encapsulates nucleotides encoding at least one binding domain that binds an epitope that activates immune cells and/or at least one binding domain that binds an antigen on a cell of interest.

111. A nanoparticle according to embodiment 110 having a size <130 nm.

112. The nanoparticle of embodiment 110 or 111, wherein the positively charged polymer comprises poly(β-amino ester), poly(L-lysine), poly(ethyleneimine) (PEI), poly(amidoamine) dendrimers (PAMAM), poly(amino esters), poly(dimethylaminoethyl methacrylate) (PDMAEMA), chitosan, poly-(L-lactide-co-L-lysine), poly[α-(4-aminobutyl)-L-glycolic acid] (PAGA), or poly(4-hydroxy-L-proline ether) (PHP).

113. The nanoparticle of any one of embodiments 110-112, wherein the positively charged polymer is poly(β-amino ester).

114. The nanoparticle of any one of embodiments 110-113, wherein the neutral or negatively charged coating comprises polyglutamic acid (PGA), poly(acrylic acid), alginic acid, or cholesterol hemisuccinate/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine.

115. The nanoparticle of any one of embodiments 110-114, wherein the neutral or negatively charged coating comprises polyglutamic acid (PGA).

116. The nanoparticle of any one of embodiments 110-115, wherein the neutral or negatively charged coating comprises a zwitterionic polymer.

117. The nanoparticle of any one of embodiments 110-116, wherein the neutral or negatively charged coating is a liposome.

118. The nanoparticle of embodiment 117, wherein the liposome comprises 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-chol), dioctadecylamidoglycylspermine (DOGS), cholesterol, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

119. The nanoparticle of any one of embodiments 110-118, wherein the nucleotides are ribonucleic acid (RNA).

120. The nanoparticle of embodiment 119, wherein the RNA is synthetic RNA.

121. The nanoparticle of embodiment 119 or 120, wherein the RNA is in vitro transcribed mRNA.

122. The nanoparticle of any one of embodiments 110-121, wherein the nucleotides are integrating or non-integrating double-stranded DNA.

123. The nanoparticle of any one of embodiments 110-122, wherein the nucleotides are in the form of a plasmid, a minicircle plasmid, or a closed linear cDNA.

124. The nanoparticle of any one of embodiments 110-123, wherein the antigen on the cell of interest is a cancer antigen expressed by an ovarian cancer cell, a melanoma cell, a glioblastoma cell, a multiple myeloma cell, a melanoma cell, a prostate cancer cell, a breast cancer cell, a stem cell cancer cell, a mesothelioma cell, a renal cell cancer cell, a pancreatic cancer cell, a lung cancer cell, a cholangiocarcinoma cell, a bladder cancer cell, a neuroblastoma cell, a colorectal cancer cell, or a Merkel cell carcinoma cell.

125. The nanoparticle of any one of embodiments 110-124, wherein the cancer antigen comprises B cell maturation antigen (BCMA), carbonic anhydrase IX (CAIX), CD19, CD24, CD56, CD133, CEA, disialoganglioside, EpCam, EGFR, EGFR variant III (EGFRvIII), ERBB2, folate receptor (FOLR), GD2, glypican-2, HER2, Lewis Y, L1-CAM, mesothelin, MUC16, PD-L1, PSMA, prostate stem cell antigen (PSCA), ROR1, TYRP1/gp75, SV40 T, or WT-1.

126. The nanoparticle of any one of embodiments 110-125, wherein the binding domain that binds a cancer antigen comprises the complementarity determining regions (CDRs) of the antibody adecatumumab, anetumab ravtansine, amatuximab, HN1, oregovomab, ovarex, abagovomab, edrecolomab, farletuzumab, flanvotumab, TA99, 20D7, cetuximab, FMC63, SJ25C1, HD37, R11, R12, 2A2, Y31, 4D5, 3G10, atezolizumab, avelumab, or durvalumab, or is derived from a T cell receptor (TCR).

127. The nanoparticle of any one of embodiments 110-126, wherein the binding domain that binds the cancer antigen is a protein molecule.

128. The nanoparticle of embodiment 127, wherein the nucleotides in the nanoparticle encode various protein molecules, including binding domains, that bind various cancer antigens.

129. The nanoparticle of embodiment 128, wherein different cancer antigens are expressed by the same type of cancer cells.

130. The nanoparticle of embodiment 129, wherein the cancer type is ovarian cancer, melanoma, or glioblastoma.

131. The nanoparticle of any one of embodiments 128-130, wherein the various cancer antigens comprise

at least two cancer antigens selected from EpCam, L1-CAM, MUC16, folate receptor (FOLR), Lewis Y, ROR1, mesothelin, WT-1, PD-L1, EGFR and CD56;

at least two cancer antigens selected from tyrosinase-related protein 1 (TYRP1/gp75); GD2, PD-L1 and EGFR; or two cancer antigens selected from EGFR variant III (EGFRvIII) and IL13Ra2.

132. The nanoparticle of any one of embodiments 110-131, wherein at least one binding domain of the protein molecule binds a viral antigen, a bacterial antigen, a superbug antigen, a fungal antigen, or an autoimmune or allergic antigen.

133. The nanoparticle of embodiment 132, wherein

viral antigen is expressed by adenoviruses, arenaviruses, bunyaviruses, coronaviruses, flaviviruses, hantaviruses, hepadnaviruses, herpesviruses, papillomaviruses, paramyxoviruses, parvoviruses, picornaviruses, poxviruses, orthomyxoviruses, retroviruses, reoviruses, rhabdoviruses, rotaviruses, spongiform viruses, or togaviruses;

The bacterial antigen is expressed by the causative agents of anthrax; gram-negative bacilli, chlamydia, diphtheria pathogens, Haemophilus influenzae, Helicobacter pylori , malaria pathogens, mycobacterium tuberculosis, whooping cough pathogens, pneumococci, rickettsia, staphylococci, streptococci and tetanus pathogens;

superbug antigen is expressed by Enterococcus faecium, Clostridium difficile, Acinetobacter baumannii, Pseudomonas aeruginosa , or Enterobacteriaceae ;

the fungal antigen is expressed by Candida, Coccidioides, Cryptococcus, Histoplasma, Leishmania, Plasmodium, protozoa, parasites, Schistosomes, Herpes zoster, Toxoplasma, or Trypanosoma cruzi ; or

an autoimmune or allergic antigen is expressed in a subject suffering from acute necrotizing hemorrhagic encephalopathy, allergic asthma, focal alopecia, anemia, aphthous ulcer, arthritis, asthma, autoimmune thyroiditis, conjunctivitis, Crohn's disease, cutaneous lupus erythematosus, dermatitis, diabetes, diabetes mellitus, erythema nodosum leprosum, keratoconjunctivitis, multiple sclerosis, myasthenia gravis, psoriasis, scleroderma, Sjogren's syndrome including keratoconjunctivitis sicca due to Sjogren's syndrome, Stevens-Johnson syndrome, systemic lupus erythematosus, ulcerative colitis, vaginitis and/or Wegener's granulomatosis; and/or

where the binding domain for the antigen is derived from the TCR.

134. The nanoparticle of any one of embodiments 110-133, wherein at least one binding domain of the protein molecule binds an immune cell activating epitope expressed by a T cell or a natural killer (NK) cell.

135. The nanoparticle of any one of embodiments 110-134, wherein the immune cell activating epitope is expressed by a T cell.

136. The nanoparticle of embodiment 135, wherein the immune cell activating epitope expressed by the T cell comprises CD2, CD3, CD7, CD8, CD27, CD28, CD30, CD40, CD83, 4-1BB, OX40, lymphocyte function-associated antigen 1 (LFA-1), LIGHT, NKG2C, or B7-H3.

137. The nanoparticle of embodiment 135, wherein the immune cell activating epitope expressed by the T cell comprises CD3, CD28, or 4-1BB.

138. The nanoparticle of any one of embodiments 110-137, wherein the binding domains that bind the immune cell activating epitope are a protein molecule.

139. The nanoparticle of embodiment 138, wherein the nucleotides in the nanoparticle encode various protein molecules comprising binding domains that bind various epitopes that activate immune cells.

140. The nanoparticle of embodiment 139, wherein the various immune cell activating epitopes include CD3 and CD28 or CD3 and 4-1BB.

141. The nanoparticle of any one of embodiments 110-140, wherein the at least one binding domain comprises the CDR regions of the antibody OKT3, 20G6-F3, 4B4-D7, 4E7-C9, 18F5-H10, TGN1412, 9D7, 9.3, KOLT-2, 15E8, 248.23.2, EX5.3D10, OKT8 or SK1.

142. The nanoparticle of any one of embodiments 110-134, wherein the immune cell activating epitope is expressed by an NK cell.

143. The nanoparticle of embodiment 142, wherein the immune cell activating epitope expressed by the NK cell comprises NKG2D, CD8, CD16, KIR2DL4, KIR2DS1, KIR2DS2, KIR3DS1, NKG2C, NKG2E, NKG2D, NKp30, NKp44, NKp46, NKp80, or DNAM-1.

144. The nanoparticle of any one of embodiments 110-143, wherein at least one binding domain comprises the CDR regions of antibody 5C6, 1D11, mAb 33, P44-8, SK1, or 3G8.

145. The nanoparticle of any one of embodiments 110-144, wherein the binding domains are linked via a protein linker.

146. The nanoparticle of embodiment 145, wherein the protein linker is a Gly-Ser linker.

147. The nanoparticle of embodiment 145 or 146, wherein the protein linker is a proline-rich linker.

148. The nanoparticle of any one of embodiments 110-147, wherein the protein molecule is a single chain variable fragment (scFv).

149. A nanoparticle according to any one of embodiments 110-148, wherein in the protein molecule

at least one binding domain binds CEA, and at least one binding domain binds CD3, CD28, or 4-1BB;

at least one binding domain binds EGFR, and at least one binding domain binds CD3, CD28, or 4-1BB;

at least one binding domain binds EpCam, and at least one binding domain binds CD3, CD28, or 4-1BB;

at least one binding domain binds HER2, and at least one binding domain binds CD3, CD28, or 4-1BB;

at least one binding domain binds PD-L1, and at least one binding domain binds CD3, CD28, or 4-1BB;

at least one binding domain binds PSMA, and at least one binding domain binds CD3, CD28, or 4-1BB; or

at least one binding domain binds [TYRP1/gp75], and at least one binding domain binds CD3, CD28, or 4-1BB.

150. The nanoparticle of embodiment 149, wherein the protein molecule comprises catumaxomab, MT110, ertumaxomab, MDX-447, MM-141, AMG211, RO6958688, RO6895882, TF2, BAY2010112, AMG701, solitomab, or blinatumomab.

151. The nanoparticle of any one of embodiments 110-150, wherein the positively charged polymer core further encapsulates nucleotides encoding one or more interferon regulatory factors (IRFs).

152. The nanoparticle of embodiment 151, wherein one or more IRFs lack a functional autoinhibitory domain.

153. The nanoparticle of embodiment 151 or 152, wherein one or more IRFs lack a functional nuclear export signal.

154. The nanoparticle of any one of embodiments 151-153, wherein the one or more IRFs are selected from IRF1, IRF3, IRF5, IRF7, IRF8 and/or a fusion protein of IRF7 and IRF3.

155. The nanoparticle of any one of embodiments 151-154, wherein the one or more IRFs are selected from a sequence having >90%, >95%, or greater than 98% identity to the sequence set forth in SEQ ID NO: 1-17.

156. The nanoparticle of any one of embodiments 151-155, wherein the one or more IRFs comprise IRF5 selected from the sequences set forth in SEQ ID NO: 1-7.

157. The nanoparticle of any one of embodiments 154-156, wherein IRF5 comprises the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3, with one or more mutations selected from S156D, S158D and T160D.

158. The nanoparticle of any one of embodiments 154-157, wherein IRF5 comprises the sequence set forth in SEQ ID NO: 2, with one or more mutations selected from T10D, S158D, S309D, S317D, S451D and S462D.

159. The nanoparticle of any one of embodiments 154-158, wherein IRF5 comprises the sequence set forth in SEQ ID NO: 4, with one or more mutations selected from S425D, S427D, S430D and S436D.

160. The nanoparticle of any one of embodiments 151-159, wherein the one or more IRFs comprise IRF1 comprising the sequence set forth in SEQ ID NO: 8 or 12.

161. The nanoparticle of any one of embodiments 151-160, wherein the one or more IRFs comprise IRF8 comprising the sequence set forth in SEQ ID NO: 11, 16, or 17.

162. The nanoparticle of any one of embodiments 154-161, wherein IRF8 comprises the sequence set forth in SEQ ID NO: 11 with the K310R mutation.

163. The nanoparticle of any one of embodiments 151-162, wherein the one or more IRFs comprise an IRF7/IRF3 fusion protein comprising an N-terminal DNA-binding domain of IRF7, a constitutively active domain, and a C-terminal nuclear export signal of IRF3.

164. The nanoparticle of embodiment 163, wherein the IRF7/IRF3 fusion protein comprises the sequence set forth in SEQ ID NO: 15.

165. The nanoparticle of any one of embodiments 151-164, wherein the one or more IRFs comprise IRF4.

166. The nanoparticle of any one of embodiments 110-165, wherein at least a subset of the nanoparticles comprise nucleotides encoding IKKβ.

167. The nanoparticle of embodiment 166, wherein IKKβ is selected from a sequence having >90%, >95%, or >98% identity to a sequence shown in a sequence selected from SEQ ID NO: 18-22.

168. The nanoparticle of embodiment 166 or 167, wherein IKKβ comprises a sequence shown in a sequence selected from SEQ ID NO: 18-22.

169. The nanoparticle of any one of embodiments 166-168, wherein the nucleotides comprise a sequence set forth in a sequence selected from SEQ ID NO: 23-44.

170. The nanoparticle of any one of embodiments 166-169, wherein the nucleotides encoding one or more IRFs and the nucleotides encoding IKKβ are encapsulated in the same nanoparticle.

171. The nanoparticle of any one of embodiments 151-170, wherein the nucleotides encoding one or more IRFs and the nucleotides encoding IKKβ are encapsulated in the same core of the nanoparticle.

172. The nanoparticle of any one of embodiments 151-170, wherein the nucleotides encoding one or more IRFs and the nucleotides encoding IKKβ are encapsulated in different nanoparticles.

173. The nanoparticle of any one of embodiments 151-172, wherein the nucleotides encoding at least one or more binding domains are encapsulated in the same nanoparticle as nucleotides encoding one or more IRFs and/or IKKβ.

174. The nanoparticle of any one of embodiments 151-172, wherein the nucleotides encoding at least one or more binding domains are encapsulated in different nanoparticles than those in which the nucleotides encoding one or more IRFs and/or IKKβ are encapsulated.

175. The nanoparticle of any one of embodiments 110-174, further comprising a transforming growth factor beta (TGFβ) inhibitor.

176. The nanoparticle of embodiment 175, wherein the TGFβ inhibitor comprises nucleotides encoding a TGFβ inhibitor.

177. The nanoparticle of embodiment 175 or 176, wherein the TGFβ inhibitor comprises CDR regions of an antibody that suppresses TGFβ activity.

178. The nanoparticle of any one of embodiments 175-177, wherein the TGFβ inhibitor is an antibody that suppresses TGFβ activity.

179. The nanoparticle of embodiment 177 or 178, wherein the antibody comprises trabedersen, disittertide, metelimumab, frezolimumab, LY2382770, SIX-100, avotermin and/or IMC-TR1.

180. The nanoparticle of any one of embodiments 110-179, further comprising nucleotides encoding a glucocorticoid-induced leucine zipper (GILZ).

181. The nanoparticle of any one of embodiments 110-180, further comprising nucleotides representing an anti-cancer gene selected from p53, RB, BRCA1, E1A, bcl-2, MDR-1, p21, p16, bax, bcl-xs, E2F, IGF-I VEGF, angiostatin, oncostatin, endostatin, GM-CSF, IL-12, IL-2, IL-4, IL-7, IFNγ, TNFα and/or HSV-tk.

182. A system comprising:

nanoparticles,

wherein at least a subset of the nanoparticles comprise nucleotides encoding one or more interferon-regulating factors (IRFs), and

wherein at least a subset of the nanoparticles comprise nucleotides encoding a protein molecule having at least two binding domains,

wherein one binding domain binds an antigen expressed by a cell of interest in the treatment area, and

where one binding domain binds an epitope that activates immune cells.

183. The system of embodiment 182, wherein the cell of interest is a cancer cell, an infected cell, an autoreactive cell, or a prokaryotic cell.

184. The system of embodiment 182 or 183, wherein the cell of interest is a cancer cell and the treatment area is a tumor area.

185. The system of any one of embodiments 182-184, wherein the nanoparticles have a size <130 nm.

186. The system of any one of embodiments 182-185, wherein the nanoparticles have a positively charged core and a neutral or negatively charged coating on the outer surface of the core.

187. The system of embodiment 186, wherein the positively charged core comprises a positively charged lipid and/or a positively charged polymer.

188. The system of embodiment 186 or 187, wherein the positively charged polymer comprises poly(β-amino ester), poly(L-lysine), poly(ethyleneimine) (PEI), poly(amidoamine) dendrimers (PAMAM), poly(amino esters), poly(dimethylaminoethyl methacrylate) (PDMAEMA), chitosan, poly-(L-lactide-co-L-lysine), poly[α-(4-aminobutyl)-L-glycolic acid] (PAGA), or poly(4-hydroxy-L-proline ether) (PHP).

189. The system of any one of embodiments 186-188, wherein the positively charged polymer is a poly(β-amino ester).

190. The system of any one of embodiments 186-189, wherein the neutral or negatively charged coating comprises polyglutamic acid (PGA), poly(acrylic acid), alginic acid, or cholesterol hemisuccinate/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine.

191. The system of any one of embodiments 186-190, wherein the neutral or negatively charged coating comprises polyglutamic acid (PGA).

192. The system of any one of embodiments 186-191, wherein the neutral or negatively charged coating comprises a zwitterionic polymer.

193. The system of any one of embodiments 186-192, wherein the neutral or negatively charged coating is a liposome.

194. The system of embodiment 193, wherein the liposome comprises 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-chol), dioctadecylamidoglycylspermine (DOGS), cholesterol, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

195. The system of any one of embodiments 182-194, wherein the nucleotides are ribonucleic acid (RNA).

196. The system of embodiment 195, wherein the RNA is synthetic RNA.

197. The system of embodiment 195 or 196, wherein the RNA is in vitro transcribed mRNA.

198. The system of any one of embodiments 182-197, wherein the nucleotides are integrating or non-integrating double-stranded DNA.

199. The system of any one of embodiments 182-198, wherein the nucleotides are in the form of a plasmid, a minicircle plasmid, or a closed linear cDNA.

200. The system of any one of embodiments 182-199, wherein the nucleotides are encapsulated in a positively charged core.

201. The system of any one of embodiments 182-200, wherein one or more IRFs lack a functional autoinhibitory domain.

202. The system of any one of embodiments 182-201, wherein one or more IRFs lack a functional nuclear export signal.

203. The system of any one of embodiments 182-202, wherein the one or more IRFs are selected from IRF1, IRF3, IRF5, IRF7, IRF8 and/or a fusion protein of IRF7 and IRF3.

204. The system of any one of embodiments 182-203, wherein the one or more IRFs are selected from a sequence having >90%, >95%, or greater than 98% identity to the sequence set forth in SEQ ID NO: 1-17.

205. The system of any one of embodiments 182-204, wherein the one or more IRFs comprise IRF5 selected from the sequences set forth in SEQ ID NO: 1-7.

206. The system of embodiment 205, wherein IRF5 comprises the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3, with one or more mutations selected from S156D, S158D, and T160D.

207. The system of embodiment 205 or 206, wherein IRF5 comprises the sequence set forth in SEQ ID NO: 2 with one or more mutations selected from T10D, S158D, S309D, S317D, S451D and S462D.

208. The system of any one of embodiments 205-207, wherein IRF5 comprises the sequence set forth in SEQ ID NO: 4, with one or more mutations selected from S425D, S427D, S430D and S436D.

209. The system of any one of embodiments 182-208, wherein the one or more IRFs comprise IRF1 comprising the sequence set forth in SEQ ID NO: 8 or 12.

210. The system of any one of embodiments 182-209, wherein the one or more IRFs comprise IRF8 comprising the sequence set forth in SEQ ID NO: 11, 16, or 17.

211. The system of any one of embodiments 182-210, wherein IRF8 comprises the sequence set forth in SEQ ID NO: 11 with the K310R mutation.

212. The system of any one of embodiments 182-211, wherein the one or more IRFs comprise an IRF7/IRF3 fusion protein comprising an N-terminal DNA-binding domain of IRF7, a constitutively active domain, and a C-terminal nuclear export signal of IRF3.

213. The system of embodiment 212, wherein the IRF7/IRF3 fusion protein comprises the sequence set forth in SEQ ID NO: 15.

214. The system of any one of embodiments 182-213, wherein the one or more IRFs comprise IRF4.

215. The system of any one of embodiments 182-214, wherein at least a subset of the nanoparticles comprise nucleotides encoding IKKβ.

216. The system of embodiment 215, wherein IKKβ is selected from a sequence having >90%, >95%, or >98% identity to a sequence shown in a sequence selected from SEQ ID NO: 18-22.

217. The system of embodiment 215 or 216, wherein IKKβ comprises a sequence shown in a sequence selected from SEQ ID NO: 18-22.

218. The system of any one of embodiments 182-217, wherein the nucleotides comprise a sequence set forth in a sequence selected from SEQ ID NO: 23-44.

219. The system of any one of embodiments 182-218, wherein the nucleotides encoding one or more IRFs and the nucleotides encoding IKKβ are encapsulated in the same nanoparticle.

220. The system of embodiment 219, wherein the nucleotides encoding one or more IRFs and the nucleotides encoding IKKβ are encapsulated in the same nanoparticle core.

221. The system of any one of embodiments 182-218, wherein the nucleotides encoding one or more IRFs and the nucleotides encoding IKKβ are encapsulated in different nanoparticles.

222. The system of any one of embodiments 182-221, wherein at least one binding domain of the protein molecule binds a cancer antigen expressed by an ovarian cancer cell, a melanoma cell, a glioblastoma cell, a multiple myeloma cell, a melanoma cell, a prostate cancer cell, a breast cancer cell, a stem cell cancer cell, a mesothelioma cell, a renal cell cancer cell, a pancreatic cancer cell, a lung cancer cell, a cholangiocarcinoma cell, a bladder cancer cell, a neuroblastoma cell, a colorectal cancer cell, or a Merkel cell carcinoma cell.

223. The system of embodiment 222, wherein the cancer antigen comprises B cell maturation antigen (BCMA), carbonic anhydrase IX (CAIX), CD19, CD24, CD56, CD133, CEA, disialoganglioside, EpCam, EGFR, EGFR variant III (EGFRvIII), ERBB2, folate receptor (FOLR), GD2, glypican-2, HER2, Lewis Y, L1-CAM, mesothelin, MUC16, PD-L1, PSMA, prostate stem cell antigen (PSCA), ROR1, TYRP1/gp75, SV40 T, or WT-1.

224. The system of any one of embodiments 182-223, wherein at least one binding domain of the protein molecule comprises the complementarity determining regions (CDRs) of an antibody of adecatumumab, anetumab ravtansine, amatuximab, HN1, oregovomab, ovarex, abagovomab, edrecolomab, farletuzumab, flanvotumab, TA99, 20D7, cetuximab, FMC63, SJ25C1, HD37, R11, R12, 2A2, Y31, 4D5, 3G10, atezolizumab, avelumab or durvalumab, or a TCR.

225. The system of any one of embodiments 182-224, wherein the various protein molecules in the system comprise binding domains that bind different cancer antigens.

226. The system of embodiment 225, wherein different cancer antigens are expressed by the same type of cancer cells.

227. The system of embodiment 226, wherein the cancer type is ovarian cancer, melanoma, or glioblastoma.

228. The system of any one of embodiments 225-227, wherein the various cancer antigens comprise

at least two cancer antigens selected from EpCam, L1-CAM, MUC16, folate receptor (FOLR), Lewis Y, ROR1, mesothelin, WT-1, PD-L1, EGFR and CD56;

at least two cancer antigens selected from tyrosinase-related protein 1 (TYRP1/gp75); GD2, PD-L1 and EGFR; or

two cancer antigens selected from EGFR variant III (EGFRvIII) and IL13Ra2.

229. The system of any one of embodiments 182-228, wherein at least one binding domain of the protein molecule binds a viral antigen, a bacterial antigen, a superbug antigen, a fungal antigen, or an autoimmune or allergic antigen.

230. The system of embodiment 229, wherein:

viral antigen is expressed by adenoviruses, arenaviruses, bunyaviruses, coronaviruses, flaviviruses, hantaviruses, hepadnaviruses, herpesviruses, papillomaviruses, paramyxoviruses, parvoviruses, picornaviruses, poxviruses, orthomyxoviruses, retroviruses, reoviruses, rhabdoviruses, rotaviruses, spongiform viruses, or togaviruses;

The bacterial antigen is expressed by the causative agents of anthrax; gram-negative bacilli, chlamydia, diphtheria pathogens, Haemophilus influenzae, Helicobacter pylori , malaria pathogens, mycobacterium tuberculosis, whooping cough pathogens, pneumococci, rickettsia, staphylococci, streptococci and tetanus pathogens;

superbug antigen is expressed by Enterococcus faecium, Clostridium difficile, Acinetobacter baumannii, Pseudomonas aeruginosa , or Enterobacteriaceae ;

the fungal antigen is expressed by Candida, Coccidioides, Cryptococcus, Histoplasma, Leishmania, Plasmodium, protozoa, parasites, Schistosomes, Herpes zoster, Toxoplasma, or Trypanosoma cruzi ; or

an autoimmune or allergic antigen is expressed in a subject suffering from acute necrotizing hemorrhagic encephalopathy, allergic asthma, focal alopecia, anemia, aphthous ulcer, arthritis, asthma, autoimmune thyroiditis, conjunctivitis, Crohn's disease, cutaneous lupus erythematosus, dermatitis, diabetes, diabetes mellitus, erythema nodosum leprosum, keratoconjunctivitis, multiple sclerosis, myasthenia gravis, psoriasis, scleroderma, Sjogren's syndrome, including keratoconjunctivitis sicca associated with Sjogren's syndrome, Stevens-Johnson syndrome, systemic lupus erythematosus, ulcerative colitis, vaginitis and/or Wegener's granulomatosis.

231. The system of any one of embodiments 182-230, wherein at least one binding domain of the protein molecule binds an immune cell activating epitope expressed by a T cell or a natural killer cell.

232. The system of embodiment 231, wherein the immune cell activating epitope is expressed by a T cell.

233. The system of embodiment 232, wherein the immune cell activating epitope expressed by the T cell comprises CD2, CD3, CD7, CD8, CD27, CD28, CD30, CD40, CD83, 4-1BB, OX40, lymphocyte function-associated antigen 1 (LFA-1), LIGHT, NKG2C, or B7-H3.

234. The system of embodiment 233, wherein the immune cell activating epitope expressed by the T cell comprises CD3, CD28, or 4-1BB.

235. The system of any one of embodiments 182-234, wherein the various protein molecules in the system comprise binding domains that bind different epitopes that activate immune cells.

236. The system of embodiment 235, wherein the various immune cell activating epitopes include CD3 and CD28 or CD3 and 4-1BB.

237. The system of any one of embodiments 182-236, wherein at least one binding domain comprises the CDR regions of the antibody OKT3, 20G6-F3, 4B4-D7, 4E7-C9, 18F5-H10, TGN1412, 9D7, 9.3, KOLT-2, 15E8, 248.23.2, EX5.3D10, OKT8, or SK1.

238. The system of embodiment 231, wherein the immune cell activating epitope is expressed by an NK cell.

239. The system of embodiment 238, wherein the immune cell activating epitope expressed by the NK cell comprises NKG2D, CD8, CD16, KIR2DL4, KIR2DS1, KIR2DS2, KIR3DS1, NKG2C, NKG2E, NKG2D, NKp30, NKp44, NKp46, NKp80, or DNAM-1.

240. The system of any one of embodiments 182-239, wherein at least one binding domain comprises the CDR regions of antibody 5C6, 1D11, mAb 33, P44-8, SK1, or 3G8.

241. The system of any one of embodiments 182-240, wherein the binding domains of the protein molecule are linked via a protein linker.

242. The system of embodiment 241, wherein the protein linker is a Gly-Ser linker.

243. The system of embodiment 241 or 242, wherein the protein linker is a proline-rich linker.

244. The system of any one of embodiments 182-243, wherein the protein molecule is a single chain variable fragment (scFv).

245. The system according to any one of embodiments 182-244, wherein in the protein molecule

at least one binding domain binds CEA and at least one binding domain binds CD3, CD28 or 4-1BB;

at least one binding domain binds EGFR and at least one binding domain binds CD3, CD28, or 4-1BB;

at least one binding domain binds EpCam and at least one binding domain binds CD3, CD28 or 4-1BB;

at least one binding domain binds HER2 and at least one binding domain binds CD3, CD28, or 4-1BB;

at least one binding domain binds PD-L1 and at least one binding domain binds CD3, CD28, or 4-1BB;

at least one binding domain binds PSMA and at least one binding domain binds CD3, CD28, or 4-1BB; or

at least one binding domain binds [TYRP1/gp75] and at least one binding domain binds CD3, CD28, or 4-1BB.

246. The system of any one of embodiments 182-245, wherein the protein molecule comprises catumaxomab, MT110, ertumaxomab, MDX-447, MM-141, AMG211, RO6958688, RO6895882, TF2, BAY2010112, AMG701, solitomab, or blinatumomab.

247. The system of any one of embodiments 182-246, wherein the nucleotides encoding at least two binding domains are encapsulated in the same nanoparticle as nucleotides encoding one or more IRFs and/or IKKβ.

248. The system of embodiment 247, wherein the nucleotides encoding at least two binding domains are encapsulated in the same nanoparticle core as the nucleotides encoding one or more IRFs and/or IKKβ.

249. The system of any one of embodiments 182-246, wherein the nucleotides encoding at least two binding domains are encapsulated in different nanoparticles than those in which the nucleotides encoding one or more IRFs and/or IKKβ are encapsulated.

250. The system of any one of embodiments 182-249, further comprising a transforming growth factor beta (TGFβ) inhibitor.

251. The system of embodiment 250, wherein the TGFβ inhibitor comprises nucleotides encoding a TGFβ inhibitor.

252. The system of embodiment 250 or 251, wherein the TGFβ inhibitor comprises the CDR regions of an antibody that suppresses TGFβ activity.

253. The system of any one of embodiments 250-252, wherein the TGFβ inhibitor is an antibody that suppresses TGFβ activity.

254. The system of embodiment 252 or 253, wherein the antibody comprises trabedersen, disittertide, metelimumab, frezolimumab, LY2382770, SIX-100, avotermin and/or IMC-TR1.

255. The system of any one of embodiments 182-254, wherein the nanoparticles further comprise nucleotides encoding a glucocorticoid-induced leucine zipper (GILZ).

256. The system of any one of embodiments 182-255, wherein the nanoparticles further comprise nucleotides representing an anti-cancer gene selected from p53, RB, BRCA1, E1A, bcl-2, MDR-1, p21, p16, bax, bcl-xs, E2F, IGF-I VEGF, angiostatin, oncostatin, endostatin, GM-CSF, IL-12, IL-2, IL-4, IL-7, IFNγ, TNFα and/or HSV-tk.

257. The system of any one of embodiments 182-256, further comprising a pharmaceutically acceptable carrier.

258. A monocyte or macrophage genetically modified to express the nucleotides of the system of any one of embodiments 182-257.

259. A method for modulating the activation state of macrophages in a treatment area in a subject's body, recruiting immune cells to the treatment area, and activating the recruited immune cells, comprising:

administering to a subject a system according to any one of embodiments 182-257, whereby the activation state of macrophages in the treatment area in the subject's body is modulated, immune cells are recruited in the treatment area, and the recruited immune cells are activated.

260. The method of embodiment 259, wherein the treatment area is a tumor area.

261. The method of embodiment 259 or 260, wherein the administration is intravenous administration and the nanoparticles are taken up by monocytes in the bloodstream.

262. The method of embodiment 261, wherein the monocytes migrate to the tumor site and differentiate into macrophages.

263. The method of embodiment 262, wherein the differentiated macrophages are resistant to the suppressive effect of the tumor.

264. The method according to any one of embodiments 259-263, wherein the administration is local administration to the tumor site and the nanoparticles are taken up by tumor-associated macrophages (TAMs).

265. The method of embodiment 264, wherein the local administration is intraperitoneal administration or intracranial administration.

266. The method according to embodiment 264 or 265, wherein the OAM undergoes a phenotype transformation from a suppressor to an activated state.

267. The method of any one of embodiments 264-266, wherein the tumor area comprises an ovarian cancer tumor area, a glioblastoma tumor area, or a melanoma tumor area.

268. The method according to any one of embodiments 259-267, wherein the recruited and activated immune cells are T cells or NK cells.

269. The method according to any one of embodiments 259-268, comprising administering nanoparticles comprising nucleotides encoding one or more IRFs prior to administering nanoparticles comprising nucleotides encoding at least two binding domains.

270. The method according to any one of embodiments 259-269, comprising administering nanoparticles comprising nucleotides encoding one or more IRFs at least 24 hours prior to administering nanoparticles comprising nucleotides encoding at least two binding domains.

[0259] (12) Experimental Examples. Example 1. Materials and Methods. Synthesis of PbAE. The methods used to synthesize the polymer were described previously (Mangraviti A et al. (2015) ACS Nano 9: 1236–1249). 1,4-Butanediol diacrylate was combined with 4-amino-1-butanol in a 1:1 molar ratio of diacrylate to amine monomers. Acrylate-terminated poly(4-amino-1-butanol-co-1,4-butanediol diacrylate) was prepared by heating the mixture to 90°C with stirring for 24 h. 2.3 g of this polymer was dissolved in 2 mL of tetrahydrofuran (THF). To obtain piperazine-capped polymer 447, 786 mg of 1-(3-aminopropyl)-4-methylpiperazine in 13 mL of THF was added to the polymer/THF solution and stirred at room temperature (RT) for 2 h. The capped polymer was precipitated with 5 volumes of diethyl ether, washed with 2 volumes of fresh ether, and dried under vacuum for 1 day. The pure polymer was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 100 mg/mL and stored at -20°C.

[0260] Conjugation of PGA to dimannose. α-D-mannopyranosyl-(1→2)-α-D-mannopyranose (dimannose, Omicron Biochemicals Inc.) was modified to glycosylamine prior to conjugation with polyglutamic acid (PGA). First, dimannose (157 mg) was dissolved in 10.5 mL of saturated aqueous ammonium carbonate solution and then stirred at RT for 24 h. On the second day, additional solid ammonium carbonate was added until dimannose precipitated from the reaction solution. The mixture was stirred until completion, as determined by TLC, followed by lyophilization to remove excess ammonium carbonate. Complete removal of the volatile salt was accomplished by redissolving the solid in methanol. This method produced an amine on the anomeric carbon for future conjugation with PGA.

[0261] For conjugation of aminated dimannose with PGA, the substrate was dissolved in water to a concentration of 30 mg/L and then sonicated for 10 min. Ethyl N'-(3-dimethylaminopropyl)carbodiimide HCl in water (4 mg/mL, 30 equiv) was added with stirring at RT for 4 min. N-hydroxysulfosuccinimide in water (30 mg/mL, 35 equiv) was incubated with the PGA/EDC solution for 1 min. Aminated dimannose in phosphate-buffered saline (PBS) was combined with the resulting activated PGA at a molar ratio of 44:1 and stirred at RT for 6 h. Excess reagents were removed by dialysis against water for 24 h.

[0262] mRNA synthesis. Codon-optimized mRNAs for eGFP, IRF5, and IKK (TriLink Biotechnologies) were capped with the anti-reverse cap analog 3'-O-Me-m7G(5')ppp(5')G (ARCA), with complete substitution by the modified ribonucleotides pseudouridine (Ψ) and 5-methylcytidine (m5C).

[0263] Nanoparticle preparation. IRF5 and IKKβ mRNAs were combined at a 3:1 (w:w) ratio and diluted to a concentration of 100 μg/mL in 25 mM sodium acetate buffer (NaOAc) (pH 5.2). Poly(β-amino esters)-447 (PbAE-447) in DMSO (prepared as described above) were diluted from 100 μg/μL to 6 μg/μL, also in NaOAc buffer. To form nanoparticles, PbAE-447 polymers were added to mRNA at a 60:1 (w:w) ratio and immediately vortexed for 15 sec at medium speed, then the mixture was incubated at RT for 5 min to form PbAE-mRNA polyplexes. In the next step, 100 μg/mL PGA/dimannose in NaOAc buffer was added to the polyplex solution, vortexed for 15 seconds at medium speed, and incubated for 5 minutes at room temperature. During this process, PGA/dimannose coated the surfaces of the PbAE-mRNA polyplexes, forming the final NPs. For long-term storage, D-sucrose (60 mg/mL) was added to the NP solutions as a cryoprotectant. The nanoparticles were quickly frozen in dry ice and then lyophilized. Dried NPs were stored at -20°C or -80°C until use. For in vivo experiments, lyophilized NPs were resuspended in water at a 1:20 (weight:volume) ratio.

[0264] Determination of nanoparticle size distribution and ζ-potential. The physicochemical properties of NPs (including hydrodynamic radius, polydispersity, ζ-potential, and stability) were determined using a Zetapals instrument (Brookhaven Instrument Corporation) at 25°C. To measure the hydrodynamic radius and polydispersity using dynamic light scattering, NPs were diluted 5-fold with 25 mM NaOAc (pH 5.2). To measure the ζ-potential, NPs were diluted 10-fold in 10 mM PBS (pH 7.0). To assess the stability of NPs, freshly prepared nanoparticles were diluted in 10 mM PBS buffer (pH 7.4). The hydrodynamic radius and polydispersity of the NPs were measured every 10 min for 5 h, and their particle sizes and concentrations were determined by particle tracking analysis using a Nanosite 300 instrument (Malvern). For transmission electron microscopy characterization of the NPs, previously described protocols were followed (Smith TT et al. (2017) Nat Nanotechnol 12: 813–820). Freshly prepared NPs (25 μL, containing 0.83 μg mRNA) were loaded onto glow-discharge-treated 200 mesh carbon/formvar-coated copper grids. After 30 s, the grids were sequentially treated with 50% Karnovsky's fixative, 0.1 M cacodylate buffer, _dH2O , then 1% (w/v) uranyl acetate. Samples were visualized using a JEOL JEM-1400 transmission electron microscope operating at 120 kV (JEOL USA).

[0265] Bone marrow-derived macrophages (BMDM) and other cell lines. To generate BMDM, bone marrow progenitor cells were collected from mouse femurs according to established protocols (Zhang X et al. (2008) Curr Protoc Immunol Chapter 14: Unit 14 11). These cells were cultured in complete medium [DMEM supplemented with 4.5 g/L D-glucose, L-glutamine, 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL Glutamax 50 mL/500 mL, supplemented with 20 ng/mL M-CSF (Peprotech, catalog #315-02)] at a seeding density of 0.5-1.0 e6/mL. Cells were allowed to differentiate into BMDMs ex vivo for 7 days under 5% _CO2 at 37°C. They were then conditioned with macrophage conditioning medium [complete macrophage medium supplemented with 20 ng/mL MPLA (Sigma, catalog #L6895) or 20 ng/mL IL4 (eBioscience, catalog #34-8041)]. BMDMs were used for 7-21 days ex vivo . The mouse ovarian cancer cell line ID8, kindly provided by Dr. Katherine Roby (University of Kansas Medical Center, Kansas City, KS) were cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 5 μg/ml insulin, 5 μg/ml transferrin, and 5 ng/ml sodium selenite (all from Sigma-Aldrich). To generate a more aggressive strain, ID8, expressing vascular endothelial growth factor (VEGF), ID8 tumor cells were transfected with the pUNO1 plasmid (Invivogen), encoding murine VEGF along with the blasticidin resistance gene. To obtain stable transfectants, tumor cells were cultured in complete medium containing 10 μg/ml blasticidin (Invivogen) for 3 weeks. The B16F10 melanoma cell line (American Type Culture Collection) was cultured in complete RPMI 1640 medium supplemented with 10% FBS, 100 U/mL penicillin, 2 mM/L glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, 1.0 mM sodium pyruvate, and 0.05 mM 2-mercaptoethanol. For in vivo bioluminescence imaging, both ID8-VEGF and B16F10 cell lines were transduced with firefly luciferase-enhanced retrovirus. The DF-1 cell line carrying the RACS-PDGFβ or RCAS-cre retrovirus was cultured in complete medium supplemented with 10% FBS and 100 U/ml penicillin under 5% _CO2 conditions at 39°C.

[0266] Transfection of BMDM with mRNA. The day before transfection, BMDM were reseeded in 24-well plates in complete macrophage medium at a concentration of 250,000/well. Before transfection, the complete medium was replaced with 300 μl of DMEM without supplements. To transfect these cells, NPs containing 2 μg of mRNA were added to the basal medium and co-cultured with BMDM at 37°C. After 1 hour, the NP-containing medium was removed, and the cells were cultured for an additional 24 hours before assessing transfection efficiency and cell viability.

[0267] Transfection of BMDMs for Macrophage Signature Gene Analysis. BMDMs were reseeded in 24-well plates in conditioned medium 24 hours prior to transfection, allowing the cells to transform into their phenotypes. M2-like macrophages were then exposed to either IRF5/IKKβ NPs carrying 25% eGFP mRNA as a reporter or eGFP NPs (control) containing 2 μg mRNA, according to the transfection protocol described above. The top 10% of highly transfected BMDMs (based on eGFP expression measurement) were sorted 24 hours post-transfection and re-exposed to a low dose (10 ng/mL) of IL4-containing medium for an additional 48 hours prior to RNA isolation. RNA extracted from these cells was compared with RNA from standard M1- or M2-like macrophages to identify signature genes associated with IRF5-NP processing.

[0268] RNA isolation and preparation. For RNA collection, BMDM were lysed in Trizol reagent (Ambion), and total RNA was extracted and purified using ^RNeasy® Plus Universal Mini Kits (QIAGEN) according to the manufacturer's instructions. The RNA sample was quantified using a NanoDrop Microvolume spectrophotometer (Thermo Fisher) and then subjected to quality control performed by FHCRC Genomics Shared Resource using an Agilent 4200 TapeStation analyzer (Agilent).

[0269] Macrophage signature gene analysis using NanoString technology. Gene expression metrics in stimulated BMDM cultures were measured using the ^nCounter® Myeloid Innate Immunity Panel (NanoString Technologies, Seattle, WA), which analyzes 770 genes across 19 different pathways and processes them for 7 different myeloid cell types. Samples were tested using the nCounter Assay System (NanoString Technologies, Seattle, WA). Raw data were processed and quality checked using the R/Bioconductor NanoStringQCPro software package (Nickles D, Sandmann T, Ziman R and Bourgon R (2018) NanoStringQCPro: Quality metrics and data processing methods for NanoString mRNA gene expression data. R package version 1.10.0.). Expression values were normalized to the geometric mean of housekeeping genes and log-transformed using nSolver 4.0 software (NanoString Technologies, Seattle, WA). False event rates for ratio data were calculated from p-values returned by t-tests using the Benjamini-Yekutieli method.

[0270] Flow cytometry and cell sorting. Cells obtained from the spleen, blood, peritoneal lavage, and bronchoalveolar lavage were analyzed by flow cytometry with myeloid and lymphoid cell immunophenotyping panels using the anti-mouse antibody probes listed in FIG. 9. Data were collected using a BD LSRFortessa analyzer with FACSDIVA software (Beckton Dickinson). Peritoneal CD11b+ and F4/80+ macrophages were sorted using a BD FACS ARIA II. All collected data were analyzed using FlowJo 10.0 software.

[0271] Cytokine analysis. Cytokine levels were assessed using the Luminex 200 System (Luminex) at the FHCRC Immunological Monitoring Shared Resource Center. For ex vivo studies, cell culture supernatant was collected to measure IL-6, IL-12p70, INFγ, and TNFα concentrations. For in vivo studies, plasma concentrations of GM-CSF, INFγ, IL-12p70, IL-2, IL-6, and TNFα were measured.

[0272] qRT-PCR analysis. Gene expression levels were determined by qRT-PCR. To measure the levels of selected macrophage signature genes (serpin B2, Retn1A, Ccl5, Ccl11, codon-optimized IRF5, endogenous IRF5, and GAPD housekeeping genes), total RNA was isolated using RNeasy mini columns (Qiagen) according to the manufacturer's instructions. cDNA was synthesized using the qScript cDNA Synthesis Kit (Quanta). For each sample, qRT-PCR was performed in triplicate with PerfeCTa qPCR SuperMix Low ROX (Quanta) using gene-specific probes from the Roche Universal Probe Library (UPL) and PCR primers optimized with ProbeFinder (Roche): serpin B2, UPL-049, F-ACTGGGGCAGTTATGACAGG (SEQ ID NO: 96), R-GATGATCGGCCACAAACTG (SEQ ID NO: 97); Retnla, UPL-078, F-TTGTTCCCTTCTCATCTGCAT (SEQ ID NO: 98), R-CCTTGACCTTATTCTCCACGA (SEQ ID NO: 99); Ccl5, UPL-105, F-CCTACTCCCACTCGGTCCT (SEQ ID NO: 100), R-CTGATTTCTTGGGTTTGCTGT (SEQ ID NO: 101); Ccl11, UPL-018, F-AGAGCTCCACAGCGCTTC (SEQ ID NO: 102), R-CAGCACCTGGGAGGTGAA (SEQ ID NO: 103); codon-optimized IRF5, UPL-022, F-TCTTAAAGACCACATGGTAGAACAGT (SEQ ID NO: 104), R-AGCTGCTGTTGGGATTGC (SEQ ID NO: 105); endogenous IRF5, UPL-011, F-GCTGTGCCCTTAACAAAAGC (SEQ ID NO: 106), R-GGCTGAGGTGGCATGTCT (SEQ ID NO: 107). Signature gene mRNA levels were normalized based on the amplification results of GAPD, UPL-060, F-AGCCACATCGCTCAGACAC (SEQ ID NO: 108), and R-GCCCAATACGACCAAATCC (SEQ ID NO: 109). All qRT-PCR reactions were performed on Quant Studio5 RT-PCR instruments with QuantStudio6 software (Applied Biosystems). In cases where the amplification trace did not cross the threshold and no Ct value was obtained (“indeterminate”), the Ct value corresponding to the highest number of cycles in the assay (40 cycles) was used for relative expression comparisons.

[0273] Mice and in vivo tumor models. With the exception of experiments involving the brain tumor model, mice used in these experiments were obtained from Jackson Laboratory; the remainder were raised and housed in the FHCRC animal facilities. All mice were used in accordance with a protocol approved by the Institutional Animal Care and Use Committee. To model ovarian tumors, 5 × 10 ⁶ vascular epithelial growth factor (VEGFP)-expressing ID8 cells were injected intraperitoneally (i.p.) into 4- to 6-week-old female albino B6 mice (C57BL/6J-Tyr<c-2J>), and tumors were allowed to develop for 2 weeks. For survival studies, animals were injected intraperitoneally with IRF5/eGFP NPs carrying 50 μg mRNA (two doses per week for 9 weeks or until health status reached the level of euthanasia). To study mechanisms, treatment was used for 1, 2, or 3 weeks, followed by euthanasia 48 hours after the last dose. Peritoneal lavage was performed to collect peritoneal cells. To compare the efficacy of IRF5/IKKβ NPs with existing macrophage-targeted therapies, one group of mice was treated with IRF5/IKKβ NPs carrying 50 μg mRNA for 3 weeks, two doses per week; The second group received 15 mg/kg of the PI3Kγ inhibitor IPI-594 (MedKoo Biosciences Inc) formulated in vehicle (5% 1-methyl-2-pyrrolidinone in polyethyleneglycol 400) by gavage daily for 3 weeks; the third group received 30 mg/kg of the CSF1R inhibitor pexidartinib (PLX3397, MedKoo Biosciences Inc) in the same vehicle intraperitoneally daily for 3 weeks.

[0274] To model metastatic lung cancer, 2.5 x 10 ⁴ F-luc-transduced 16F10 cells suspended in 200 μl RPMI medium were injected into 4- to 6-week-old female albino B6 mice (C57BL/6J-Tyr<c-2J) (Jackson Laboratories) and allowed to form tumors for 1 week. For survival studies, mice were retroorbitally injected with PBS with or without suspended IRF5/IKKβ NPs or eGFP NPs carrying 30 μg mRNA. Mice were given 3 doses per week for 3 weeks or until their health status reached the level of euthanasia. For mechanistic studies, mice received the same treatments for 2 weeks. Bronchoalveolar lavage was performed to collect alveolar cells for analysis.

[0275] Glioma-bearing mice were generated according to published protocols (Uhrbom L et al. (2004) Nat Med 10: 1257-1260). Avian DF-1 cells producing the retroviruses RCAS-PDGFβ and RCAS-cre were injected intracranially into both hemispheres of the brain (coordinates: 1 mm caudal to bregma, 2 mm lateral, 2 mm deep from the dura surface) of Nestin-tv-a /Ink4a-arf -/-; Pten-/- (C57BL/6) mice aged 4-6 weeks. Tumors were allowed to develop for 2 weeks. On day 15, mice received 10 Gy irradiation per hemisphere, with the non-irradiated hemisphere covered with a lead shield. The following day, mice received retroorbital injections of IRF5/IKKβ NPs carrying 30 μg mRNA (3 doses/week for 3 weeks) or were allocated to a PBS control group.

[0276] In vivo bioluminescence imaging. D-luciferin (Xenogen) in PBS (15 mg/mL) was used as a substrate for firefly luciferase imaging. Bioluminescence images were acquired using the Xenogen IVIS Spectral Imaging System (Xenogen). Mice were anesthetized with 2% isoflurane (Forane, Baxter Healthcare) before and during imaging. For ID8-VEGF ovarian tumors, each mouse was injected i.p. with 300 μg of D-luciferin, and images were acquired 10 minutes later. For B16F10 metastatic lung tumors, mice were injected i.p. with 3 mg of D-luciferin, and images were acquired 15 minutes later. For brain tumor models, mice were injected retroorbitally with 75 mg/kg body weight of D-luciferin, and images were acquired 4 minutes later. The exposure time varied from 10 s to 5 min.

[0277] Biodistribution analysis. To determine the biodistribution of IRF5 NPs in the ID8-VEGF ovarian tumor model, mice in groups 7-8 were injected intraperitoneally or retroorbitally with NPs carrying 50 μg mRNA. Whole blood was collected 24 hours after injection, and mice were euthanized with _CO2 for organ harvesting (liver, spleen, lungs, kidneys, heart, intestine, pancreas, and diaphragm). All tissues were stabilized with RNAlater and then frozen on dry ice. Codon-optimized IRF5 mRNA levels in each organ were measured by qRT-PCR.

[0278] Toxicity analysis. To determine the potential in vivo toxicity of repeatedly administered macrophage-targeted NPs, we injected mice (5/group) intravenously with 6 consecutive doses of IRF5/IKKβ or eGFP NPs carrying 50 μg mRNA for 3 weeks. The control group received no treatment. Twenty-four hours after the last infusion, mice were anesthetized and blood was collected via the retro-orbital sinus for a complete blood count. Blood was also collected for serum biochemistry and cytokine profiling (performed by Phoenix Central Laboratories, Mukilteo, WA). Animals were then euthanized with CO ₂ for organ removal, which were rinsed with deionized water and fixed in 4% paraformaldehyde. Tissues were processed routinely, and sections were stained with hematoxylin and eosin. Specimens were analyzed in a blinded fashion by a board-certified staff pathologist, Dr. Smitha Pillai MVSc, PhD, DACVP.

[0279] Cytokine assays. Cytokine levels were assessed using the Luminex 200 System (Luminex) at the FHCRC Immunological Monitoring Shared Resource Center. For ex vivo studies, cell culture supernatant was collected to measure IL-6, IL12p70, INFγ, and TNFα concentrations. For in vivo studies, we measured plasma concentrations of GM-CSF, INFγ, IL-12p70, IL-2, IL-6, and TNFα.

[0280] Statistical analysis. The statistical significance of observed differences was analyzed using an unpaired, two-way, one-way ANOVA test. P-values for each measurement are listed in the figures or figure legends. Survival data were analyzed using the log-rank test. All statistical analyses were performed using either GraphPad Prism version 6.0 or R software.

[0281] Results. Design of NPs for the development of IVT mRNA OAM transfection. A targeted mRNA delivery system capable of robust gene expression in target cells was developed by exploiting electrostatic interactions between cationic poly(β-amino ester) (PbAE) polymers and anionic mRNAs (FIG. 2A). Synthetic transcript variants containing modified pseudouridine (Ψ) (Kariko K et al. (2008) Mol Ther 16: 1833–1840) and 5-methylcytidine (m5C) ribonucleotides and capped with ARCA (anti-reversible cap analog) (Quabius ES et al. (2015) N Biotechnol 32: 229–235) were used to enhance the stability and translation of mRNA encapsulated in the resulting nanocarriers. mRNA is released from the mRNA-PbAE complex inside the cell by hydrolytic cleavage of ester bonds in the PbAE backbone. Efficient in vivo transfection of T cells has been previously demonstrated using this system (Smith TT et al. (2017) Nat Nanotechnol). To target the nanoparticles to TAMs and further stabilize the mRNA-PbAE complexes they contain, dimannose moieties were anchored to their surface using polyglutamic acid (PGA) as a linker (Fig. 2A). The NPs were fabricated using a simple two-step charge-induced self-assembly process. First, synthetic mRNA was complexed with the positively charged polymer PBAE, which condenses the mRNA into nanosized complexes. This step was followed by the addition of dimannose-functionalized PGA, which shields the positive charge of the PBAE-mRNA nanoparticles and ensures macrophage targeting. The resulting mRNA nanocarriers had a size of 99.8 ± 24.5 nm, a polydispersity of 0.183, and a neutral surface charge (3.40 ± 2.15 mV ζ-potential, FIGS. 2B-2C). Transfection efficiency was first tested in murine bone marrow-derived macrophages (BMDMs) using NPs formulated with mRNA encoding green fluorescent protein (GFP-NPs). Briefly, 50,000 BMDMs were exposed to NPs containing 1 μg mRNA for 1 h, followed by measurement of GFP expression by flow cytometry the following day. Following a single administration of NPs, we typically transfected 31.9% (±8.5%) of these primary macrophages without reducing their viability (FIGS. 2E-2F). Surface modification of the nanoparticles with dimannose was beneficial, as the transfection rate of non-targeted (but PGA-coated) nanocarriers was reduced to an average of 25% (±2.1%) in this primarily phagocytic cell type. NPs selectively targeted the CD11b+, F4/80+ macrophage population, with 46% of macrophages transfected and expressing high levels of eGFP (FIG. 2D). This high transfection efficiency demonstrates the effectiveness of the disclosed systems and methods for targeted mRNA delivery to TAMs. Based on the results of an in vitro screening of candidate transcription factors that induce macrophage polarization, two mRNAs were selected for inclusion in the NPs: the first encodes IRF5, a key member of the IRF family that promotes macrophage polarization toward the M1 phenotype, and the second encodes IKKβ, a kinase that phosphorylates and activates IRF5.

[0282] Programming immunosuppressive macrophages into proinflammatory phenotypes. To induce macrophage polarization, two mRNAs were selected for inclusion in NPs: the first encodes IRF5, a key member of the interferon-regulating factor family that promotes macrophage polarization toward the M1 phenotype (Krausgruber T et al. (2011) Nat Immunol 12: 231–238); the second encodes IKKβ, a kinase that phosphorylates and activates IRF5 (Ren J et al. (2014) Proc Natl Acad Sci USA 111: 17438–17443). A ratio of 3 IRF5 mRNAs to 1 IKKβ mRNA was used. Using quantitative real-time PCR specific for NP-delivered (and codon-optimized) IRF5 mRNA, mRNA expression in macrophages was found to be maximal on day 1, resulting in a 1500-fold increase in IRF5 levels compared to endogenous levels (FIG. 2A). As expected, gene expression was transient, but IRF5 levels remained highly elevated until day 3 (581-fold increase) and day 5 (87-fold increase), before returning to baseline.

[0283] To determine whether IRF5/IKKβ-encoding NPs could reprogram M2 macrophages to the therapeutically desirable anti-cancer M1 phenotype, NanoString gene expression analysis was used. BMDM were first cultured in the presence of interleukin-4 (IL-4) to induce the M2 suppressor phenotype (FIG. 2H). Following transfection with either control GFP-mRNA NPs or IRF5/IKKβ mRNA-containing NPs, gene expression profiles were analyzed and compared to those of inflammatory macrophages that were generated separately by exposing BMDM to monophosphoryl lipid A, a TLR4 agonist. Despite culture in suppressive medium containing IL-4, macrophages transfected with IRF5/IKKβ mRNA NPs exhibited gene expression profiles similar to those of inflammatory macrophages (FIG. 2I). Macrophage M2 signature genes such as serpin b2 and Ccl2 (Jablonski K et al. (2015) Plos One 10: e0145342; Varga T et al. (2016) J Immunol 196: 4771–4782) were strongly downregulated, while key M1 differentiation genes such as Ccl5 (Sica A et al. (2012) J Clin Invest 122: 787–795) were upregulated (FIG. 2J, 2K). These data suggest that NF-mediated expression of IRF5 and its kinase leads to the redirection of suppressor macrophages towards a proinflammatory phenotype.

[0284] Example 2. Therapeutic effects of NP-delivered pro-M1 genes in disseminated ovarian cancer. To evaluate this treatment approach in a clinically relevant in vivo test system, a model reproducing inoperable, advanced ovarian tumors was used in C57BL/6 mice; these animals were injected with ID8 ovarian cancer cells labeled with luciferase to allow serial bioluminescent imaging of tumor growth (Liao JB et al. (2015) J Immunother Cancer 3: 16; Stephan SB et al. (2015) Nat Biotechnol 33: 97-101). Tumors were allowed to form for two weeks. By this stage, the mice had developed nodules throughout the peritoneal wall and in the intestinal mesentery. Animals were divided into three groups, each receiving PBS (control), GFP NPs (sham), or IRF5/IKKβ NPs i.p. at a dose of 100 μg mRNA/mouse/week for 9 weeks (FIG. 4A). It was observed that in the IRF5/IKKβ NPs-treated group, the disease regressed and was eventually eradicated in 40% of animals (overall median survival of 142 days compared to 60 days in the control; FIGS. 4B-4C). To understand the underlying mechanisms of the antitumor effects mediated by IRF5/IKKβ NPs, we first examined how exclusive targeting of the mannose receptor limits the interaction of the NPs with phagocytes. Flow cytometry of peritoneal lavage fluid collected 24 h after administration of the first dose of dimannose-targeted NPs revealed preferential gene transfer to macrophages and monocytes (average 37.1% and 15.3%, respectively, FIG. 4D), with low or undetectable transfection of non-target cells. Detailed phenotypic and functional analysis of macrophage/monocyte populations in the peritoneum of mice with established ovarian cancer was then performed following administration of IRF5/IKKβ nanoparticles or PBS over a 3-week period (two weekly injections). Flow cytometric analysis showed that IRF5/IKKβ NPs reduced the population of immunosuppressive macrophages (Ly6C-, F4/80+, CD206+) to an average of 2.6%±2.1% compared to 43%±15.6% in the control (FIGS. 4E-4F). In contrast, the proportion of M1-like macrophages increased from 0.5%±0.2% to 10.2%±4.1% (FIGS. 4E, 4G). IRF5 gene therapy also affected the population of other immune cells. In particular, inflammatory monocytes (CD11b+, Ly6C+, Ly6G-) were more numerous (73.4%±3.6% compared to 4.5%±1.9% in untreated mice). One interesting finding in all IRF5 NP-treated animals was the presence of multifocal dense lymphocyte clusters within or around the tumors (FIG. 4H), suggesting that genetic programming of immunostimulatory macrophages can restore lymphocyte migration and infiltration into solid tumors.

[0285] Peritoneal macrophages were isolated by fluorescence-activated cell sorting to analyze their cytokine secretion and were found to have a strong increase in the release of the proinflammatory (anti-tumor) cytokines IL-12 (3.4-fold increase), IFNγ (8.4-fold increase), and TNFα (1.5-fold increase), while IL-6, a regulatory cytokine associated with differentiation toward alternatively activated (M2-like) macrophages, was reduced by 97-fold; FIG. 4I). Genomic expression profiling confirmed differentiation toward an M1-like phenotype of macrophages in mice treated with IRF5/IKKβ nanoparticles. Gene expression levels of macrophages cultured ex vivo in MPLA or IL-4 were included to provide reference values for classical M1-like or M2-like macrophages, respectively (FIG. 4J).

[0286] Biodistribution and Safety. The distribution of nanoparticles in various organs was then quantified 24 hours after intraperitoneal injection using qRT-PCR assays designed to detect only nanoparticle-delivered (codon-optimized) IRF5. The highest concentrations of IVT mRNA were detected in organs located within the peritoneum, including the liver, spleen, intestine, pancreas, and diaphragm (FIG. 5A). Small amounts of particle-delivered mRNA were detected in organs outside the peritoneum (heart, lungs, kidneys), indicating that the i.p.-administered nanocarriers were partially delivered into the systemic circulation. Using these distribution data, we then assessed whether these nanoreagents were biocompatible and safe for repeated dosing. Mice were injected with a total of 8 doses of IRF5/IKKβ NPs (two doses of 50 μg mRNA/week for 4 weeks, FIG. 5B). Animals were euthanized 24 h after the last dose, body weights were recorded, blood was collected via the retroorbital sinus for serum biochemistry, and a complete pathological examination was performed. There were no differences in body weight between groups. The following tissues were assessed by a board-certified staff pathologist: liver, spleen, mesentery, pancreas, stomach, kidney, heart, and lung. Histopathological evaluation revealed multifocal dense aggregates of lymphocytes within or around tumor lesions in all cases, but no signs of inflammation or overt necrosis were observed in tissues lacking neoplastic cells (FIG. 5C). Furthermore, the serum biochemistry of mice treated with IRF5/IKKβ NPs was comparable to that of PBS-treated controls, indicating the absence of systemic toxicity (FIG. 5D). Because small amounts of IRF5 mRNA were systematically detected in biodistribution studies, parallel experiments were designed to quantify inflammatory cytokines in peripheral blood. Following a single i.p. injection of IRF5/IKKβ NPs, a moderate and transient increase in serum interleukin 6 (IL-6) levels, averaging 26.8 pg/mL (FIG. 5E), and tumor necrosis factor α (TNFα) levels, averaging 94.7 pg/mL (FIG. 5F), was observed. Based on previous reports, these levels are 500-fold lower than levels associated with pathological findings and thus can be considered safe (Tarrant JM (2010) Toxicol Sci 117: 4–16; Copeland S et al . (2005) Clin Diagn Lab Immunol 12: 60–67).

[0287] Control of Systemic Tumor Metastasis with Intravenous Infusions of IRF5/IKKβ Nanoparticles. Based on the therapeutic responses achieved with IRF5/IKKβ NPs administered directly into the peritoneal cavity to treat tumor lesions disseminated throughout the peritoneum, the next question of interest was whether intravenously administered mRNA nanocarriers could systemically program macrophages to control disseminated disease. qRT-PCR biodistribution studies revealed that intravenously administered nanocarriers preferentially delivered their mRNA payload to organs with high levels of resident macrophages/phagocytes, primarily the spleen, liver, and lungs (FIG. 6A). To determine the antitumor response in a clinically relevant in vivo assay, nanoparticles containing IRF5/IKKβ mRNA were administered to mice bearing disseminated melanoma lung metastases (FIG. 6B). Recent work has described the fundamental role of monocytes and macrophages in the metastasis of this disease (Butler KL et al. (2017) Sci Rep 7: 45593; Nielsen SR et al. (2017) Mediators Inflamm 2017: 9624760), and confocal microscopy confirmed that tumor engraftment correlated with phagocyte accumulation in the lungs (FIG. 6C). Tumor burden was determined by bioluminescence imaging, and mice with detectable cancer were stratified into groups with matched levels. The groups were then randomly assigned to receive either no therapy (PBS) or intravenous injections of GFP- or IRF5/IKKβ-encapsulating nanoparticles. Only IRF/IKKβ NP therapy significantly reduced lung tumor burden; in fact, it increased overall survival by an average of 1.3-fold (FIGS. 6D–6E). In parallel experiments, mice were sacrificed 22 days after tumor inoculation to examine tumor bioluminescent signals, count lung metastases, and assess macrophage polarization by flow cytometry. The total number of lung metastases in animals receiving IRF5/IKK NPs was 8.7-fold lower (average 40 ± 16 metastases) compared to the PBS control group (average 419 ± 139 metastases; FIGS. 6F–6G). Flow cytometry of bronchoalveolar lavage fluid cells revealed a strong shift from immunosuppressive (CD206+, MHCII-, CD11c+, CD11b _low ) macrophages to activated (CD206-, MHCII+, CD11c-, CD11b+) phagocytes (FIGS. 6H-6I).

[0288] Programming Tumor-Suppressing Phagocytes for Glioma Treatment. As a third in vivo test system, glioma was investigated, which is a difficult-to-treat cancer type where M2-like macrophages represent the majority of non-tumor cells and promote tumor growth and invasion (Hambardzumyan D et al. (2016) Nat Neurosci 19: 20-27). Currently, the standard of treatment for this disease is radiotherapy, which unfortunately provides only temporary stabilization or reduction of symptoms and increases median survival by 3 months (Mann J et al. (2017) Front Neurol 8: 748). To summarize the genetic events and subsequent molecular evolution of the disease, RCAS-PDGF-B/Nestin-tv-a; Ink4a/Arf-/-; Pten-/- transgenic mouse model of PDGFβ-induced glioma (PDG mice (Hambardzumyan D et al. (2009) Transl Oncol 2: 89–95; Quail DF et al. (2016) Science 352: aad3018)). A mixture of DF-1 cells transfected with RCAS-PDGFβ or RCAS-cre retrovirus (1:1 mixture, 2 μl) was stereotaxically injected into the brain tissue. Overexpression of the PDGFβ oncogene and the absence of the Ink4a-arf and Pten tumor suppressor genes in glioma precursor cells resulted in the formation of 4-5 mm diameter tumors (FIG. 7A) with near complete penetrance within 21 days (as previously reported (Hambardzumyan D et al . (2009) Transl Oncol 2: 89-95)). Immunofluorescence confirmed the presence of tumor-infiltrating (CD68+) macrophages (FIG. 7B, shown in the third panel from the left) in established gliomas (shown in the second panel from the left). Flow cytometry revealed that the F4/80+, CD11b+ macrophage population accounted for 32.8% of the total cells in the tumor, a 9-fold increase compared to age-matched healthy control mice (3.7%) (FIG. 7C). PDG mice experimentally express the firefly luciferase gene linked to the promoter of a key cancer gene. The bioluminescence of this reporter has been shown to positively correlate with tumor grade (Uhrbom L et al. (2004) Nat Med 10: 1257–1260), so it was used to monitor tumor progression every four days after treatment initiation. IRF/IKKβ NPs were initially tested as monotherapy: PDG mice were given 9 doses of IRF5/IKKβ mRNA-loaded NPs by intravenous infusion, or PBS in the control group (3 doses/week for 3 weeks). Administration of IRF/IKKβ NPs resulted in only modest suppression of tumor progression (providing an average survival benefit of only 5 days compared to the untreated control group; FIG. 7D). However, the combination of standard-of-care radiation therapy with IRF5/IKKβ NPs injections significantly reduced tumor growth and more than doubled the survival of treated mice compared to the PBS-treated control group (52 days versus 25 days, respectively; FIGS. 7E-7F).

[0289] In conclusion, in vivo results for three preclinical solid tumor models demonstrate that nanoparticles administered either locally or systemically can deliver genes encoding master regulators of macrophage polarization to reprogram immunosuppressive macrophages into tumor-reducing phenotypes.

[0290] Transfer from Murine to Human Macrophages. To confirm that the mouse data are relevant to the treatment of human diseases, NPs delivering IVT mRNA encoding human IRF5 and IKKβ (huIRF5 NPs) were prepared. The human monocytic cell line THP-1 was used as a well-characterized model of M1 and M2 macrophage polarization to test these nanocarriers (Li C et al. (2016) Sci Rep 6: 21044; Surdziel E et al. (2017) Plos One 12: e0183679). M2-type macrophages were generated by treating THP-1 cells with PMA and polarizing them with IL-4 and IL-13 (FIG. 8A). To confirm that huIRF5 NPs are functional and activate the IRF pathway, THP1-Lucia™ ISG cells were transfected with nanoparticles loaded with huIRF5/IKKβ mRNA or control GFP mRNA. THP1-Lucia™ ISG cells secrete a fluorescent Lucia reporter under the control of an IRF-inducible promoter. This composite promoter includes five IFN-stimulated response elements (ISREs) fused to the minimal ISG54 promoter, which does not respond to NF-κB or AP-1 pathway activators. As a result, THP1-Lucia™ ISG cells enable monitoring of the IRF pathway by detecting Lucia luciferase activity. huIRF5 NPs were found to strongly increase luciferase expression in M2-polarized THP-1 cells, indicating that the mRNA constructs are functional in human cells (FIGS. 8B-8C). To determine whether activation of the IRF5 pathway could reprogram M2-polarized THP-1 cells toward an M1-like phenotype, secretion of the proinflammatory cytokine IL-1β was determined after NP transfection. IL-1β production was significantly increased in THP-1 cells transfected with huIRF5 NPs compared to untransfected controls (mean 21-fold; P<0.0001, FIG. 8D), which correlated with robust upregulation (10.9-fold increase in MIF, P<0.0001) of the M1 macrophage cell surface marker CD80 (FIG. 8E).

[0291] Example 3. Nanoparticles delivering IRF5/IKKβ mRNA and an EpCAM-CD3 bispecific antibody will be administered to a preclinical mouse model of stage 4 disseminated ovarian cancer and a model of 4T1 breast cancer lung metastases. The underlying mechanisms (changes in the tumor microenvironment composition) and serum levels of the bispecific antibody (compared to concentrations of the bispecific antibody directly at the tumor site) will be assessed. A side-by-side comparison of in situ -produced and intravenously administered bispecific antibody proteins will also be conducted.

[0292] (13) Final paragraphs. The nucleotide sequences described herein are shown using standard letter abbreviations for nucleotide bases, as defined in 37 CFR §1.822. Only one strand of each nucleotide sequence is shown, but it is understood that the complementary strand is included in embodiments where appropriate.

[0293] Also included are variants of the sequences disclosed and referenced herein. Guidance for determining which amino acid residues can be substituted, inserted, or deleted without loss of biological activity can be found using computer programs well known in the art, such as DNASTAR™ software (Madison, Wisconsin). Preferably, amino acid substitutions in the protein variants disclosed herein are conservative amino acid substitutions, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid substitution includes a substitution within a family of amino acids that have similar side chains.

[0294] Suitable conservative amino acid substitutions in a peptide or protein are known to those skilled in the art and generally can be made without altering the biological activity of the resulting molecule. Those skilled in the art recognize that, in general, substitutions of individual amino acids in non-essential regions of a polypeptide do not significantly alter biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th ed., 1987, The Benjamin/Cummings Pub. Co., p. 224). Naturally occurring amino acids are generally divided into the following families of conservative substitutions: Group 1: alanine (Ala), glycine (Gly), serine (Ser), and threonine (Thr); Group 2: (acidic): aspartic acid (Asp) and glutamic acid (Glu); Group 3: (acidic; also classified as polar negatively charged residues and their amides): asparagine (Asn), glutamine (Gln), Asp, and Glu; Group 4: Gln and Asn; Group 5: (basic; also classified as polar positively charged residues): arginine (Arg), lysine (Lys), and histidine (His); Group 6 (large aliphatic nonpolar residues): isoleucine (Ile), leucine (Leu), methionine (Met), valine (Val), and cysteine (Cys); Group 7 (uncharged polar): tyrosine (Tyr), Gly, Asn, Gln, Cys, Ser, and Thr; Group 8 (large aromatic residues): phenylalanine (Phe), tryptophan (Trp), and Tyr; Group 9 (nonpolar): proline (Pro), Ala, Val, Leu, Ile, Phe, Met, and Trp; Group 11 (aliphatic): Gly, Ala, Val, Leu, and Ile; Group 10 (small aliphatic nonpolar or slightly polar residues): Ala, Ser, Thr, Pro, and Gly; and Group 12 (sulfur-containing): Met and Cys. Further information can be found in Creighton (1984) Proteins, W. H. Freeman and Company.

[0295] When making such changes, the hydrophobicity index of the amino acids can be taken into account. The importance of the hydrophobicity index of an amino acid in conferring interactive biological function on a protein is well known in the art (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1), 105-32). Each amino acid has been assigned a hydrophobicity index based on its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values are: Ile (+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); glutamate (-3.5); Gln(-3.5); aspartate (-3.5); Asn(-3.5); Lys (-3.9) and Arg (-4.5).

[0296] It is known in the art that certain amino acids can be replaced by other amino acids having a similar hydrophobicity index or value and still result in a protein with similar biological activity, i.e., a biologically functional equivalent protein will still be obtained. When making such substitutions, it is preferable to replace amino acids whose hydrophobicity indices are within ±2, especially those whose indices are within ±1, and even more preferred those whose indices are within ±0.5. It is also known in the art that the substitution of such amino acids can be effectively carried out on the basis of hydrophilicity.

[0297] As described in detail in U.S. Pat. No. 4,554,101, amino acid residues have been assigned the following hydrophilicity values: Arg (+3.0); Lys (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); Ser (+0.3); Asn (+0.2); Gln (+0.2); Gly (0); Thr (-0.4); Pro (-0.5±1); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Val (-1.5); Leu (-1.8); Ile (-1.8); Tyr (-2.3); Phe (-2.5); Trp (-3.4). It is understood that an amino acid can be replaced by another having the same hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent, protein. In such changes, it is preferable to replace amino acids whose hydrophilicity values are within ±2, particularly preferable are those whose hydrophilicity values are within ±1, and even more preferable are those whose hydrophilicity values are within ±0.5.

[0298] As noted above, amino acid substitutions may be based on the relative similarity of the amino acid side chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, and the like. As noted elsewhere, gene sequence variants may include codon-optimized variants, polymorphic forms of sequences, sequences with splice variants and/or mutations that do not affect the function of the encoded product to a statistically significant extent.

[0299] Variants of the protein, nucleotide, and gene sequences disclosed herein also include sequences with at least 70% sequence identity, 80% sequence identity, 85% sequence identity, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity with the protein, nucleotide, and gene sequences disclosed herein.

[0300] As used herein, the term "% sequence identity" refers to the relationship between two or more sequences as determined by sequence comparison. In the art, "identity" also means the degree of relatedness of protein, nucleotide, and gene sequences as determined by the match between the strands of such sequences. "Identity" (often referred to as "similarity") can be readily calculated using known methods, including those described in: Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Oxford University Press, NY (1992). Preferred methods for determining identity are designed to ensure the best match between the tested sequences. Methods for determining identity and similarity are systematized in publicly available computer programs. Sequence alignment and percent identity calculations can be performed using the Megalign program from the LASERGENE suite of bioinformatics programs (DNASTAR, Inc., Madison, Wisconsin). Multiple sequence alignments can also be performed using the Clustal alignment method (Higgins and Sharp CABIOS, 5, 151–153 (1989)) with default parameters (opening deletion penalty=10, extending deletion penalty=10). Appropriate programs also include the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP, BLASTN, BLASTX (Altschul, et al., J. Mol. Biol. 215:403–410 (1990); DNASTAR (DNASTAR, Inc., Madison, Wisconsin); and the FASTA program, which includes the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20). Editor(s): Suhai, Sandor. Publisher: Plenum, New York, NY. In the context of the present invention, it is understood that when a sequence analysis program is used for analysis, the results of the analysis are based on the default values of said program. As used herein, the term "default values" shall mean any set of values or parameters that are initially loaded with the program upon first initialization.

[0301] Variants also include nucleotide molecules that hybridize under stringent hybridization conditions to a sequence disclosed herein and provide the same function as the reference sequence. Exemplary stringent hybridization conditions include overnight incubation at 42°C in a solution containing 50% formamide, 5X SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate, and 20 μg/mL denatured, shear-fragmented salmon sperm DNA, followed by washing the filters in 0.1X SSC at 50°C. Changes in the stringency of hybridization conditions and signal detection are primarily achieved by changing the concentration of formamide (lower percentages of formamide result in lower stringency); salt conditions or temperature. For example, moderately high stringency conditions include overnight incubation at 37°C in a solution containing 6XSSPE (20XSSPE = 3M NaCl; 0.2 M _NaH2PO4 ; 0.02 M _EDTA , pH 7.4), 0.5% SDS, 30% formamide, 100 μg/ml salmon sperm blocking DNA; followed by a wash at 50°C with 1XSSPE, 0.1% SDS. Additionally, to achieve even lower stringency conditions, washes performed after stringent hybridization can be performed at higher salt concentrations (e.g., 5XSSC). Variations on the above conditions can be accomplished by including and/or substituting alternative blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary products. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above due to compatibility issues.

[0302] The term "specifically binds" refers to the binding of a binding domain (e.g., a CAR binding domain or a selected cell-targeting ligand of a nanoparticle) to its corresponding binding molecule with an affinity, or Ka, (i.e., the equilibrium association constant of a particular binding interaction, with units of 1/M) equal to or greater than 10 ⁵ M ^-1 , without significantly binding to any other molecules or components in the relevant environment. As used herein, the term "binds" may also be used instead of "specifically binds." Binding domains may be classified as "high-affinity" or "low-affinity." In particular embodiments, "high-affinity" binding domains are binding domains with a Ka of at least ¹⁰⁷ M ^-1 , at least ¹⁰⁸ M ^-1 , at least ¹⁰⁹ M ^-1 , at least ¹⁰¹⁰ M ^-1 , at least ¹⁰¹¹ M ^-1 , at least ¹⁰¹² M ^-1 , or at least ¹⁰¹³ M ^-1 . In particular embodiments, "low-affinity" binding domains are binding domains with a Ka of up to ¹⁰⁷ M ^-1 , up to ¹⁰⁶ M ^-1 , up to ¹⁰⁵ M ^-1 . Alternatively, affinity can be defined as the equilibrium dissociation constant (Kd) of a particular binding interaction, with units of M (e.g., from ^10-5 M to ^10-13 M). In some embodiments, a binding domain may have "enhanced affinity," which refers to selected or engineered binding domains that exhibit stronger binding to the corresponding binding molecule than the wild-type (or original) binding domain. For example, the enhanced affinity may be due to a Ka (equilibrium association constant) for the corresponding binding molecule that is higher than that of the reference binding domain, or a Kd (dissociation constant) for the corresponding binding molecule that is lower than that of the reference binding domain, or a dissociation rate (Koff) for the corresponding binding molecule that is lower than that of the reference binding domain. A variety of assays are known for identifying binding domains that specifically bind a particular target binding molecule and for determining binding affinity, such as Western blotting, ELISA, and the ^BIACORE® assay (see also, e.g., Scatchard, et al., 1949, Ann. NY Acad. Sci. 51:660 and US 5283173, US 5468614, or equivalent).

[0303] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of immunology, molecular biology, microbiology, cell biology, and recombinant DNA. These techniques are described in the following publications: see, e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd edition (1989); F. M. Ausubel, et al. eds., Current Protocols in Molecular Biology, (1987); Methods in Enzymology series (Academic Press, Inc.); M. MacPherson, et al., PCR: A Practical Approach, IRL Press at Oxford University Press (1991); MacPherson et al., eds. PCR 2: Practical Approach, (1995); Harlow and Lane, eds. Antibodies, A Laboratory Manual, (1988); and R. I. Freshney, ed. Animal Cell Culture (1987).

[0304] As will be understood by one of skill in the art, each embodiment disclosed herein may include, consist essentially of, or consist of a particular stated element, step, ingredient, or component thereof. Thus, the terms "include" or "comprising" should be interpreted to mean "contain, consist of, or consist essentially of." The transitional term "contain" or "comprises" means "has," but is not limiting, and allows for the inclusion of unstated elements, steps, ingredients, or components, even in large amounts. The transitional term "consisting of" excludes any element, step, ingredient, or component that is not stated. The transitional term "consisting essentially of" limits the scope of the embodiment to the stated elements, steps, ingredients, or components, as well as those that do not significantly affect the embodiment. A significant effect would cause a statistically significant decrease in the possibility of obtaining the claimed effect according to the corresponding experimental method described herein.

[0305] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used in the specification and claims are to be understood as modified in all instances by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending on the desired properties to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is needed, the term "about" has the meaning reasonably assigned to it by one of skill in the art when used in conjunction with the stated numerical value or range, that is, it means slightly more than or slightly less than the stated value or range, within ±20% of the stated value; ±19% of declared value; ±18% of declared value; ±17% of declared value; ±16% of declared value; ±15% of declared value; ±14% of declared value; ±13% of declared value; ±12% of declared value; ±11% of declared value; ±10% of declared value; ±9% of declared value; ±8% of declared value; ±7% of declared value; ±6% of declared value; ±5% of declared value; ±4% of declared value; ±3% of declared value; ±2% of declared value or ±1% of the stated value.

[0306] Although the numerical ranges and parameters defining the scope of the invention are approximate, the numerical values given in the specific examples are presented as precisely as possible. However, any numerical value inherently contains certain errors that inevitably arise as a result of the standard deviation occurring in the relevant test measurements.

[0307] When used in the context of describing the invention (especially in the context of the following claims), the singular terms shall encompass both the singular and the plural unless otherwise indicated and the context clearly contradicts it. The recitation of ranges of values herein is intended merely to serve as a shorthand method for reciting each individual value falling within the range. Unless otherwise indicated herein, each individual value is included in the specification as if it were individually recited herein. All methods described herein may be applied in any suitable order unless otherwise indicated herein and the context clearly contradicts it. The use of any and all examples or illustrative expressions (e.g., "such as") herein is intended merely to better illuminate the invention and does not place a limitation on the scope of the claimed invention. No language in the specification shall be construed as indicating any non-claimed element that is essential to the practice of the invention.

[0308] The groups of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each member of a group may be mentioned and claimed alone, or in any combination with other members of the group or other elements appearing herein. It is contemplated that one or more members of a group may be included or excluded from a group for reasons of convenience and/or patentability. When any such inclusion or exclusion occurs, the specification is deemed to contain the group as modified, which corresponds to the written description of all Markush groups used in the appended claims.

[0309] Certain embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations from these described embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors intend that skilled artisans will employ such variations as necessary, and the inventors intend that the invention be practiced otherwise than as specifically described herein. Accordingly, the present invention includes all modifications and equivalents of the subject matter set forth in the appended claims, to the extent permitted by applicable law. Moreover, any combination of the above-described elements in all their possible variations is encompassed by the invention, unless otherwise indicated herein or clearly contradicted by context.

[0310] In addition, numerous references have been made throughout this specification to patents, printed publications, journal articles, and other written sources (the "references" in this document). Each of these references is individually incorporated herein by reference in its entirety for the teachings contained therein.

[0311] In conclusion, it should be understood that the embodiments of the invention disclosed herein illustrate the principles of the present invention. Other modifications that can be used are within the scope of the invention. Thus, by way of example, and not limitation, alternative configurations of the present invention can be used in accordance with the teachings set forth herein. Accordingly, the present invention is not limited to exactly what is shown and described.

[0312] The details presented herein are provided solely by way of example and for the purpose of illustrative discussion of preferred embodiments of the present invention, and are presented for the purpose of providing what is believed to be the most useful and understandable description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show the structural details of the invention in more detail than is necessary for a fundamental understanding of the invention; the description, when accompanied by drawings and/or examples, makes it obvious to those skilled in the art how some forms of the invention can be implemented in practice.

[0313] The definitions and explanations used herein are provided and intended to prevail in any future interpretation, unless they are clearly and unambiguously modified in the examples, or when the application of the meaning makes any interpretation meaningless or essentially meaningless. In cases where the interpretation of a term makes it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 3rd edition, or from a dictionary familiar to those skilled in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology (Eds. Attwood T et al., Oxford University Press, Oxford, 2006).

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Gly Gln Thr Asn Thr Pro Pro Pro Phe Glu Ile Phe Phe Cys Phe Gly

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Asp Ala Val Gln Ser Gly Pro His Met Thr Pro Tyr Ser Leu Leu Lys

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Glu Asp Val Lys Trp Pro Pro Thr Leu Gln Pro Pro Thr Leu Arg Pro

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Ser Pro His Met Leu Pro Leu Thr Asp Leu Glu Ile Lys Phe Gln Tyr

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Arg Gly Arg Pro Pro Arg Ala Leu Thr Ile Ser Asn Pro His Gly Cys

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Arg Leu Phe Tyr Ser Gln Leu Glu Ala Thr Gln Glu Gln Val Glu Leu

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Phe Gly Pro Ile Ser Leu Glu Gln Val Arg Phe Pro Ser Pro Glu Asp

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Ile Pro Ser Asp Lys Gln Arg Phe Tyr Thr Asn Gln Leu Leu Asp Val

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Leu Asp Arg Gly Leu Ile Leu Gln Leu Gln Gly Gln Asp Leu Tyr Ala

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Ile Arg Leu Cys Gln Cys Lys Val Phe Trp Ser Gly Pro Cys Ala Ser

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Ala His Asp Ser Cys Pro Asn Pro Ile Gln Arg Glu Val Lys Thr Lys

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Leu Phe Ser Leu Glu His Phe Leu Asn Glu Leu Ile Leu Phe Gln Lys

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Gly Gln Thr Asn Thr Pro Pro Pro Phe Glu Ile Phe Phe Cys Phe Gly

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Asp Ala Val Gln Ser Gly Pro His Met Thr Pro Tyr Ser Leu Leu Lys

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Glu Asp Val Lys Trp Pro Pro Thr Leu Gln Pro Pro Thr Leu Gln Pro

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Glu Ile Lys Phe Gln Tyr Arg Gly Arg Pro Pro Arg Ala Leu Thr Ile

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Ser Asn Pro His Gly Cys Arg Leu Phe Tyr Ser Gln Leu Glu Ala Thr

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Gln Glu Gln Val Glu Leu Phe Gly Pro Ile Ser Leu Glu Gln Val Arg

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Phe Pro Ser Pro Glu Asp Ile Pro Ser Asp Lys Gln Arg Phe Tyr Thr

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Gly Gln Asp Leu Tyr Ala Ile Arg Leu Cys Gln Cys Lys Val Phe Trp

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Ile Phe Phe Cys Phe Gly Glu Glu Trp Pro Asp Arg Lys Pro Arg Glu

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Ser Arg Asp Phe Arg Leu Ile Tyr Asp Gly Pro Arg Asp Met Pro Pro

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Gln Pro Tyr Lys Ile Tyr Glu Val Cys Ser Asn Gly Pro Ala Pro Thr

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Glu Asp Val Lys Trp Pro Pro Thr Leu Gln Pro Pro Thr Leu Gln Pro

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Pro Val Val Leu Gly Pro Pro Ala Pro Asp Pro Ser Pro Leu Ala Pro

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Leu Pro Asp Leu Leu Ile Ser Pro His Met Leu Pro Leu Thr Asp Leu

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Glu Ile Lys Phe Gln Tyr Arg Gly Arg Pro Pro Arg Ala Leu Thr Ile

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Ser Asn Pro His Gly Cys Arg Leu Phe Tyr Ser Gln Leu Glu Ala Thr

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Gln Glu Gln Val Glu Leu Phe Gly Pro Ile Ser Leu Glu Gln Val Arg

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Phe Pro Ser Pro Glu Asp Ile Pro Ser Asp Lys Gln Arg Phe Tyr Thr

290 295 300

Asn Gln Leu Leu Asp Val Leu Asp Arg Gly Leu Ile Leu Gln Leu Gln

305 310 315 320

Gly Gln Asp Leu Tyr Ala Ile Arg Leu Cys Gln Cys Lys Val Phe Trp

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Ser Gly Pro Cys Ala Ser Ala His Asp Ser Cys Pro Asn Pro Ile Gln

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Leu Ile Leu Phe Gln Lys Gly Gln Thr Asn Thr Pro Pro Pro Phe Glu

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Ile Phe Phe Cys Phe Gly Glu Glu Trp Pro Asp Arg Lys Pro Arg Glu

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Lys Lys Leu Ile Thr Val Gln Val Val Pro Val Ala Ala Arg Leu Leu

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Leu Glu Met Phe Ser Gly Glu Leu Ser Trp Ser Ala Asp Ser Ile Arg

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Phe Leu Asn Glu Leu Ile Leu Phe Gln Lys Gly Gln Thr Asn Thr Pro

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Pro Pro Phe Glu Ile Phe Phe Cys Phe Gly Glu Glu Trp Pro Asp Arg

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Lys Pro Arg Glu Lys Lys Leu Ile Thr Val Gln Val Val Pro Val Ala

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Ala Arg Leu Leu Leu Glu Met Phe Ser Gly Glu Leu Ser Trp Ser Ala

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Asp Ser Ile Arg Leu Gln Ile Ser Asn Pro Asp Leu Lys Asp Arg Met

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Val Glu Gln Phe Lys Glu Leu His His Ile Trp Gln Ser Gln Gln Arg

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Leu Gln Pro Val Ala Gln Ala Pro Pro Gly Ala Gly Leu Gly Val Gly

385 390 395 400

Gln Gly Pro Trp Pro Met His Pro Ala Gly Met Gln

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<213> Mus musculus

<400> 7

Met Asn His Ser Ala Pro Gly Ile Pro Pro Pro Pro Arg Arg Val Arg

1 5 10 15

Leu Lys Pro Trp Leu Val Ala Gln Val Asn Ser Cys Gln Tyr Pro Gly

20 25 30

Leu Gln Trp Val Asn Gly Glu Lys Lys Leu Phe Tyr Ile Pro Trp Arg

35 40 45

His Ala Thr Arg His Gly Pro Ser Gln Asp Gly Asp Asn Thr Ile Phe

50 55 60

Lys Ala Trp Ala Lys Glu Thr Gly Lys Tyr Thr Glu Gly Val Asp Glu

65 70 75 80

Ala Asp Pro Ala Lys Trp Lys Ala Asn Leu Arg Cys Ala Leu Asn Lys

85 90 95

Ser Arg Asp Phe Gln Leu Phe Tyr Asp Gly Pro Arg Asp Met Pro Pro

100 105 110

Gln Pro Tyr Lys Ile Tyr Glu Val Cys Ser Asn Gly Pro Ala Pro Thr

115 120 125

Glu Ser Gln Pro Thr Asp Asp Tyr Val Leu Gly Glu Glu Glu Glu Glu

130 135 140

Glu Glu Glu Glu Leu Gln Arg Met Leu Pro Gly Leu Ser Ile Thr Glu

145 150 155 160

Pro Ala Leu Pro Gly Pro Pro Asn Ala Pro Tyr Ser Leu Pro Lys Glu

165 170 175

Asp Thr Lys Trp Pro Pro Ala Leu Gln Pro Pro Val Gly Leu Gly Pro

180 185 190

Pro Val Pro Asp Pro Asn Leu Leu Ala Pro Pro Ser Gly Asn Pro Ala

195 200 205

Gly Phe Arg Gln Leu Leu Pro Glu Val Leu Glu Pro Gly Pro Leu Ala

210 215 220

Ser Ser Gln Pro Pro Thr Glu Pro Leu Leu Pro Asp Leu Leu Ile Ser

225 230 235 240

Pro His Met Leu Pro Leu Thr Asp Leu Glu Ile Lys Phe Gln Tyr Arg

245 250 255

Gly Arg Ala Pro Arg Thr Leu Thr Ile Ser Asn Pro Gln Gly Cys Arg

260 265 270

Leu Phe Tyr Ser Gln Leu Glu Ala Thr Gln Glu Gln Val Glu Leu Phe

275 280 285

Gly Pro Val Thr Leu Glu Gln Val Arg Phe Pro Ser Pro Glu Asp Ile

290 295 300

Pro Ser Asp Lys Gln Arg Phe Tyr Thr Asn Gln Leu Leu Asp Val Leu

305 310 315 320

Asp Arg Gly Leu Ile Leu Gln Leu Gln Gly Gln Asp Leu Tyr Ala Ile

325 330 335

Arg Leu Cys Gln Cys Lys Val Phe Trp Ser Gly Pro Cys Ala Leu Ala

340 345 350

His Gly Ser Cys Pro Asn Pro Ile Gln Arg Glu Val Lys Thr Lys Leu

355 360 365

Phe Ser Leu Glu Gln Phe Leu Asn Glu Leu Ile Leu Phe Gln Lys Gly

370 375 380

Gln Thr Asn Thr Pro Pro Pro Phe Glu Ile Phe Phe Cys Phe Gly Glu

385 390 395 400

Glu Trp Pro Asp Val Lys Pro Arg Glu Lys Lys Leu Ile Thr Val Gln

405 410 415

Val Val Pro Val Ala Ala Arg Leu Leu Leu Glu Met Phe Ser Gly Glu

420 425 430

Leu Ser Trp Ser Ala Asp Ser Ile Arg Leu Gln Ile Ser Asn Pro Asp

435 440 445

Leu Lys Asp His Met Val Glu Gln Phe Lys Glu Leu His His Leu Trp

450 455 460

Gln Ser Gln Gln Gln Leu Gln Pro Met Val Gln Ala Pro Pro Val Ala

465 470 475 480

Gly Leu Asp Ala Ser Gln Gly Pro Trp Pro Met His Pro Val Gly Met

485 490 495

Gln

<210> 8

<211> 325

<212> PROTEIN

<213> Homo sapiens

<400> 8

Met Pro Ile Thr Arg Met Arg Met Arg Pro Trp Leu Glu Met Gln Ile

1 5 10 15

Asn Ser Asn Gln Ile Pro Gly Leu Ile Trp Ile Asn Lys Glu Glu Met

20 25 30

Ile Phe Gln Ile Pro Trp Lys His Ala Ala Lys His Gly Trp Asp Ile

35 40 45

Asn Lys Asp Ala Cys Leu Phe Arg Ser Trp Ala Ile His Thr Gly Arg

50 55 60

Tyr Lys Ala Gly Glu Lys Glu Pro Asp Pro Lys Thr Trp Lys Ala Asn

65 70 75 80

Phe Arg Cys Ala Met Asn Ser Leu Pro Asp Ile Glu Glu Val Lys Asp

85 90 95

Gln Ser Arg Asn Lys Gly Ser Ser Ala Val Arg Val Tyr Arg Met Leu

100 105 110

Pro Pro Leu Thr Lys Asn Gln Arg Lys Glu Arg Lys Ser Lys Ser Ser

115 120 125

Arg Asp Ala Lys Ser Lys Ala Lys Arg Lys Ser Cys Gly Asp Ser Ser

130 135 140

Pro Asp Thr Phe Ser Asp Gly Leu Ser Ser Ser Thr Leu Pro Asp Asp

145 150 155 160

His Ser Ser Tyr Thr Val Pro Gly Tyr Met Gln Asp Leu Glu Val Glu

165 170 175

Gln Ala Leu Thr Pro Ala Leu Ser Pro Cys Ala Val Ser Ser Thr Leu

180 185 190

Pro Asp Trp His Ile Pro Val Glu Val Val Pro Asp Ser Thr Ser Asp

195 200 205

Leu Tyr Asn Phe Gln Val Ser Pro Met Pro Ser Thr Ser Glu Ala Thr

210 215 220

Thr Asp Glu Asp Glu Glu Gly Lys Leu Pro Glu Asp Ile Met Lys Leu

225 230 235 240

Leu Glu Gln Ser Glu Trp Gln Pro Thr Asn Val Asp Gly Lys Gly Tyr

245 250 255

Leu Leu Asn Glu Pro Gly Val Gln Pro Thr Ser Val Tyr Gly Asp Phe

260 265 270

Ser Cys Lys Glu Glu Pro Glu Ile Asp Ser Pro Gly Gly Asp Ile Gly

275 280 285

Leu Ser Leu Gln Arg Val Phe Thr Asp Leu Lys Asn Met Asp Ala Thr

290 295 300

Trp Leu Asp Ser Leu Leu Thr Pro Val Arg Leu Pro Ser Ile Gln Ala

305 310 315 320

Ile Pro Cys Ala Pro

325

<210> 9

<211> 427

<212> PROTEIN

<213> Homo sapiens

<400> 9

Met Gly Thr Pro Lys Pro Arg Ile Leu Pro Trp Leu Val Ser Gln Leu

1 5 10 15

Asp Leu Gly Gln Leu Glu Gly Val Ala Trp Val Asn Lys Ser Arg Thr

20 25 30

Arg Phe Arg Ile Pro Trp Lys His Gly Leu Arg Gln Asp Ala Gln Gln

35 40 45

Glu Asp Phe Gly Ile Phe Gln Ala Trp Ala Glu Ala Thr Gly Ala Tyr

50 55 60

Val Pro Gly Arg Asp Lys Pro Asp Leu Pro Thr Trp Lys Arg Asn Phe

65 70 75 80

Arg Ser Ala Leu Asn Arg Lys Glu Gly Leu Arg Leu Ala Glu Asp Arg

85 90 95

Ser Lys Asp Pro His Asp Pro His Lys Ile Tyr Glu Phe Val Asn Ser

100 105 110

Gly Val Gly Asp Phe Ser Gln Pro Asp Thr Ser Pro Asp Thr Asn Gly

115 120 125

Gly Gly Ser Thr Ser Asp Thr Gln Glu Asp Ile Leu Asp Glu Leu Leu

130 135 140

Gly Asn Met Val Leu Ala Pro Leu Pro Asp Pro Gly Pro Pro Ser Leu

145 150 155 160

Ala Val Ala Pro Glu Pro Cys Pro Gln Pro Leu Arg Ser Pro Ser Leu

165 170 175

Asp Asn Pro Thr Pro Phe Pro Asn Leu Gly Pro Ser Glu Asn Pro Leu

180 185 190

Lys Arg Leu Leu Val Pro Gly Glu Glu Trp Glu Phe Glu Val Thr Ala

195 200 205

Phe Tyr Arg Gly Arg Gln Val Phe Gln Gln Thr Ile Ser Cys Pro Glu

210 215 220

Gly Leu Arg Leu Val Gly Ser Glu Val Gly Asp Arg Thr Leu Pro Gly

225 230 235 240

Trp Pro Val Thr Leu Pro Asp Pro Gly Met Ser Leu Thr Asp Arg Gly

245 250 255

Val Met Ser Tyr Val Arg His Val Leu Ser Cys Leu Gly Gly Gly Leu

260 265 270

Ala Leu Trp Arg Ala Gly Gln Trp Leu Trp Ala Gln Arg Leu Gly His

275 280 285

Cys His Thr Tyr Trp Ala Val Ser Glu Glu Leu Leu Pro Asn Ser Gly

290 295 300

His Gly Pro Asp Gly Glu Val Pro Lys Asp Lys Glu Gly Gly Val Phe

305 310 315 320

Asp Leu Gly Pro Phe Ile Val Asp Leu Ile Thr Phe Thr Glu Gly Ser

325 330 335

Gly Arg Ser Pro Arg Tyr Ala Leu Trp Phe Cys Val Gly Glu Ser Trp

340 345 350

Pro Gln Asp Gln Pro Trp Thr Lys Arg Leu Val Met Val Lys Val Val

355 360 365

Pro Thr Cys Leu Arg Ala Leu Val Glu Met Ala Arg Val Gly Gly Ala

370 375 380

Ser Ser Leu Glu Asn Thr Val Asp Leu His Ile Ser Asn Ser His Pro

385 390 395 400

Leu Ser Leu Thr Ser Asp Gln Tyr Lys Ala Tyr Leu Gln Asp Leu Val

405 410 415

Glu Gly Met Asp Phe Gln Gly Pro Gly Glu Ser

420 425

<210> 10

<211> 503

<212> PROTEIN

<213> Homo sapiens

<400> 10

Met Ala Leu Ala Pro Glu Arg Ala Ala Pro Arg Val Leu Phe Gly Glu

1 5 10 15

Trp Leu Leu Gly Glu Ile Ser Ser Gly Cys Tyr Glu Gly Leu Gln Trp

20 25 30

Leu Asp Glu Ala Arg Thr Cys Phe Arg Val Pro Trp Lys His Phe Ala

35 40 45

Arg Lys Asp Leu Ser Glu Ala Asp Ala Arg Ile Phe Lys Ala Trp Ala

50 55 60

Val Ala Arg Gly Arg Trp Pro Pro Ser Ser Arg Gly Gly Gly Pro Pro

65 70 75 80

Pro Glu Ala Glu Thr Ala Glu Arg Ala Gly Trp Lys Thr Asn Phe Arg

85 90 95

Cys Ala Leu Arg Ser Thr Arg Arg Phe Val Met Leu Arg Asp Asn Ser

100 105 110

Gly Asp Pro Ala Asp Pro His Lys Val Tyr Ala Leu Ser Arg Glu Leu

115 120 125

Cys Trp Arg Glu Gly Pro Gly Thr Asp Gln Thr Glu Ala Glu Ala Pro

130 135 140

Ala Ala Val Pro Pro Pro Gln Gly Gly Pro Pro Gly Pro Phe Leu Ala

145 150 155 160

His Thr His Ala Gly Leu Gln Ala Pro Gly Pro Leu Pro Ala Pro Ala

165 170 175

Gly Asp Lys Gly Asp Leu Leu Leu Gln Ala Val Gln Gln Ser Cys Leu

180 185 190

Ala Asp His Leu Leu Thr Ala Ser Trp Gly Ala Asp Pro Val Pro Thr

195 200 205

Lys Ala Pro Gly Glu Gly Gln Glu Gly Leu Pro Leu Thr Gly Ala Cys

210 215 220

Ala Gly Gly Pro Gly Leu Pro Ala Gly Glu Leu Tyr Gly Trp Ala Val

225 230 235 240

Glu Thr Thr Pro Ser Pro Gly Pro Gln Pro Ala Ala Leu Thr Thr Gly

245 250 255

Glu Ala Ala Ala Pro Glu Ser Pro His Gln Ala Glu Pro Tyr Leu Ser

260 265 270

Pro Ser Pro Ser Ala Cys Thr Ala Val Gln Glu Pro Ser Pro Gly Ala

275 280 285

Leu Asp Val Thr Ile Met Tyr Lys Gly Arg Thr Val Leu Gln Lys Val

290 295 300

Val Gly His Pro Ser Cys Thr Phe Leu Tyr Gly Pro Pro Asp Pro Ala

305 310 315 320

Val Arg Ala Thr Asp Pro Gln Gln Val Ala Phe Pro Ser Pro Ala Glu

325 330 335

Leu Pro Asp Gln Lys Gln Leu Arg Tyr Thr Glu Glu Leu Leu Arg His

340 345 350

Val Ala Pro Gly Leu His Leu Glu Leu Arg Gly Pro Gln Leu Trp Ala

355 360 365

Arg Arg Met Gly Lys Cys Lys Val Tyr Trp Glu Val Gly Gly Pro Pro

370 375 380

Gly Ser Ala Ser Pro Ser Thr Pro Ala Cys Leu Leu Pro Arg Asn Cys

385 390 395 400

Asp Thr Pro Ile Phe Asp Phe Arg Val Phe Phe Gln Glu Leu Val Glu

405 410 415

Phe Arg Ala Arg Gln Arg Arg Gly Ser Pro Arg Tyr Thr Ile Tyr Leu

420 425 430

Gly Phe Gly Gln Asp Leu Ser Ala Gly Arg Pro Lys Glu Lys Ser Leu

435 440 445

Val Leu Val Lys Leu Glu Pro Trp Leu Cys Arg Val His Leu Glu Gly

450 455 460

Thr Gln Arg Glu Gly Val Ser Ser Leu Asp Ser Ser Ser Leu Ser Leu

465 470 475 480

Cys Leu Ser Ser Ala Asn Ser Leu Tyr Asp Asp Ile Glu Cys Phe Leu

485 490 495

Met Glu Leu Glu Gln Pro Ala

500

<210> 11

<211> 426

<212> PROTEIN

<213> Homo sapiens

<400> 11

Met Cys Asp Arg Asn Gly Gly Arg Arg Leu Arg Gln Trp Leu Ile Glu

1 5 10 15

Gln Ile Asp Ser Ser Met Tyr Pro Gly Leu Ile Trp Glu Asn Glu Glu

20 25 30

Lys Ser Met Phe Arg Ile Pro Trp Lys His Ala Gly Lys Gln Asp Tyr

35 40 45

Asn Gln Glu Val Asp Ala Ser Ile Phe Lys Ala Trp Ala Val Phe Lys

50 55 60

Gly Lys Phe Lys Glu Gly Asp Lys Ala Glu Pro Ala Thr Trp Lys Thr

65 70 75 80

Arg Leu Arg Cys Ala Leu Asn Lys Ser Pro Asp Phe Glu Glu Val Thr

85 90 95

Asp Arg Ser Gln Leu Asp Ile Ser Glu Pro Tyr Lys Val Tyr Arg Ile

100 105 110

Val Pro Glu Glu Glu Gln Lys Cys Lys Leu Gly Val Ala Thr Ala Gly

115 120 125

Cys Val Asn Glu Val Thr Glu Met Glu Cys Gly Arg Ser Glu Ile Asp

130 135 140

Glu Leu Ile Lys Glu Pro Ser Val Asp Asp Tyr Met Gly Met Ile Lys

145 150 155 160

Arg Ser Pro Ser Pro Pro Glu Ala Cys Arg Ser Gln Leu Leu Pro Asp

165 170 175

Trp Trp Ala Gln Gln Pro Ser Thr Gly Val Pro Leu Val Thr Gly Tyr

180 185 190

Thr Thr Tyr Asp Ala His His Ser Ala Phe Ser Gln Met Val Ile Ser

195 200 205

Phe Tyr Tyr Gly Gly Lys Leu Val Gly Gln Ala Thr Thr Thr Cys Pro

210 215 220

Glu Gly Cys Arg Leu Ser Leu Ser Gln Pro Gly Leu Pro Gly Thr Lys

225 230 235 240

Leu Tyr Gly Pro Glu Gly Leu Glu Leu Val Arg Phe Pro Pro Ala Asp

245 250 255

Ala Ile Pro Ser Glu Arg Gln Arg Gln Val Thr Arg Lys Leu Phe Gly

260 265 270

His Leu Glu Arg Gly Val Leu Leu His Ser Ser Arg Gln Gly Val Phe

275 280 285

Val Lys Arg Leu Cys Gln Gly Arg Val Phe Cys Ser Gly Asn Ala Val

290 295 300

Val Cys Lys Gly Arg Pro Asn Lys Leu Glu Arg Asp Glu Val Val Gln

305 310 315 320

Val Phe Asp Thr Ser Gln Phe Phe Arg Glu Leu Gln Gln Phe Tyr Asn

325 330 335

Ser Gln Gly Arg Leu Pro Asp Gly Arg Val Val Leu Cys Phe Gly Glu

340 345 350

Glu Phe Pro Asp Met Ala Pro Leu Arg Ser Lys Leu Ile Leu Val Gln

355 360 365

Ile Glu Gln Leu Tyr Val Arg Gln Leu Ala Glu Glu Ala Gly Lys Ser

370 375 380

Cys Gly Ala Gly Ser Val Met Gln Ala Pro Glu Glu Pro Pro Pro Asp

385 390 395 400

Gln Val Phe Arg Met Phe Pro Asp Ile Cys Ala Ser His Gln Arg Ser

405 410 415

Phe Phe Arg Glu Asn Gln Gln Ile Thr Val

420 425

<210> 12

<211> 329

<212> PROTEIN

<213> Mus musculus

<400> 12

Met Pro Ile Thr Arg Met Arg Met Arg Pro Trp Leu Glu Met Gln Ile

1 5 10 15

Asn Ser Asn Gln Ile Pro Gly Leu Ile Trp Ile Asn Lys Glu Glu Met

20 25 30

Ile Phe Gln Ile Pro Trp Lys His Ala Ala Lys His Gly Trp Asp Ile

35 40 45

Asn Lys Asp Ala Cys Leu Phe Arg Ser Trp Ala Ile His Thr Gly Arg

50 55 60

Tyr Lys Ala Gly Glu Lys Glu Pro Asp Pro Lys Thr Trp Lys Ala Asn

65 70 75 80

Phe Arg Cys Ala Met Asn Ser Leu Pro Asp Ile Glu Glu Val Lys Asp

85 90 95

Gln Ser Arg Asn Lys Gly Ser Ser Ala Val Arg Val Tyr Arg Met Leu

100 105 110

Pro Pro Leu Thr Arg Asn Gln Arg Lys Glu Arg Lys Ser Lys Ser Ser

115 120 125

Arg Asp Thr Lys Ser Lys Thr Lys Arg Lys Leu Cys Gly Asp Val Ser

130 135 140

Pro Asp Thr Phe Ser Asp Gly Leu Ser Ser Ser Thr Leu Pro Asp Asp

145 150 155 160

His Ser Ser Tyr Thr Thr Gln Gly Tyr Leu Gly Gln Asp Leu Asp Met

165 170 175

Glu Arg Asp Ile Thr Pro Ala Leu Ser Pro Cys Val Val Ser Ser Ser

180 185 190

Leu Ser Glu Trp His Met Gln Met Asp Ile Ile Pro Asp Ser Thr Thr

195 200 205

Asp Leu Tyr Asn Leu Gln Val Ser Pro Met Pro Ser Thr Ser Glu Ala

210 215 220

Ala Thr Asp Glu Asp Glu Glu Gly Lys Ile Ala Glu Asp Leu Met Lys

225 230 235 240

Leu Phe Glu Gln Ser Glu Trp Gln Pro Thr His Ile Asp Gly Lys Gly

245 250 255

Tyr Leu Leu Asn Glu Pro Gly Thr Gln Leu Ser Ser Val Tyr Gly Asp

260 265 270

Phe Ser Cys Lys Glu Glu Pro Glu Ile Asp Ser Pro Arg Gly Asp Ile

275 280 285

Gly Ile Gly Ile Gln His Val Phe Thr Glu Met Lys Asn Met Asp Ser

290 295 300

Ile Met Trp Met Asp Ser Leu Leu Gly Asn Ser Val Arg Leu Pro Pro

305 310 315 320

Ser Ile Gln Ala Ile Pro Cys Ala Pro

325

<210> 13

<211> 419

<212> PROTEIN

<213> Mus musculus

<400> 13

Met Glu Thr Pro Lys Pro Arg Ile Leu Pro Trp Leu Val Ser Gln Leu

1 5 10 15

Asp Leu Gly Gln Leu Glu Gly Val Ala Trp Leu Asp Glu Ser Arg Thr

20 25 30

Arg Phe Arg Ile Pro Trp Lys His Gly Leu Arg Gln Asp Ala Gln Met

35 40 45

Ala Asp Phe Gly Ile Phe Gln Ala Trp Ala Glu Ala Ser Gly Ala Tyr

50 55 60

Thr Pro Gly Lys Asp Lys Pro Asp Val Ser Thr Trp Lys Arg Asn Phe

65 70 75 80

Arg Ser Ala Leu Asn Arg Lys Glu Val Leu Arg Leu Ala Ala Asp Asn

85 90 95

Ser Lys Asp Pro Tyr Asp Pro His Lys Val Tyr Glu Phe Val Thr Pro

100 105 110

Gly Ala Arg Asp Phe Val His Leu Gly Ala Ser Pro Asp Thr Asn Gly

115 120 125

Lys Ser Ser Leu Pro His Ser Gln Glu Asn Leu Pro Lys Leu Phe Asp

130 135 140

Gly Leu Ile Leu Gly Pro Leu Lys Asp Glu Gly Ser Ser Asp Leu Ala

145 150 155 160

Ile Val Ser Asp Pro Ser Gln Gln Leu Pro Ser Pro Asn Val Asn Asn

165 170 175

Phe Leu Asn Pro Ala Pro Gln Glu Asn Pro Leu Lys Gln Leu Leu Ala

180 185 190

Glu Glu Gln Trp Glu Phe Glu Val Thr Ala Phe Tyr Arg Gly Arg Gln

195 200 205

Val Phe Gln Gln Thr Leu Phe Cys Pro Gly Gly Leu Arg Leu Val Gly

210 215 220

Ser Thr Ala Asp Met Thr Leu Pro Trp Gln Pro Val Thr Leu Pro Asp

225 230 235 240

Pro Glu Gly Phe Leu Thr Asp Lys Leu Val Lys Glu Tyr Val Gly Gln

245 250 255

Val Leu Lys Gly Leu Gly Asn Gly Leu Ala Leu Trp Gln Ala Gly Gln

260 265 270

Cys Leu Trp Ala Gln Arg Leu Gly His Ser His Ala Phe Trp Ala Leu

275 280 285

Gly Glu Glu Leu Leu Pro Asp Ser Gly Arg Gly Pro Asp Gly Glu Val

290 295 300

His Lys Asp Lys Asp Gly Ala Val Phe Asp Leu Arg Pro Phe Val Ala

305 310 315 320

Asp Leu Ile Ala Phe Met Glu Gly Ser Gly His Ser Pro Arg Tyr Thr

325 330 335

Leu Trp Phe Cys Met Gly Glu Met Trp Pro Gln Asp Gln Pro Trp Val

340 345 350

Lys Arg Leu Val Met Val Lys Val Val Pro Thr Cys Leu Lys Glu Leu

355 360 365

Leu Glu Met Ala Arg Glu Gly Gly Ala Ser Ser Leu Lys Thr Val Asp

370 375 380

Leu His Ile Ser Asn Ser Gln Pro Ile Ser Leu Thr Ser Asp Gln Tyr

385 390 395 400

Lys Ala Tyr Leu Gln Asp Leu Val Glu Asp Met Asp Phe Gln Ala Thr

405 410 415

Gly Asn Ile

<210> 14

<211> 457

<212> PROTEIN

<213> Mus musculus

<400> 14

Met Ala Glu Val Arg Gly Val Gln Arg Val Leu Phe Gly Asp Trp Leu

1 5 10 15

Leu Gly Glu Val Ser Ser Gly Gln Tyr Glu Gly Leu Gln Trp Leu Asn

20 25 30

Glu Ala Arg Thr Val Phe Arg Val Pro Trp Lys His Phe Gly Arg Arg

35 40 45

Asp Leu Asp Glu Glu Asp Ala Gln Ile Phe Lys Ala Trp Ala Val Ala

50 55 60

Arg Gly Arg Trp Pro Pro Ser Gly Val Asn Leu Pro Pro Pro Glu Ala

65 70 75 80

Glu Ala Ala Glu Arg Arg Glu Arg Arg Gly Trp Lys Thr Asn Phe Arg

85 90 95

Cys Ala Leu His Ser Thr Gly Arg Phe Ile Leu Arg Gln Asp Asn Ser

100 105 110

Gly Asp Pro Val Asp Pro His Lys Val Tyr Glu Leu Ser Arg Glu Leu

115 120 125

Gly Ser Thr Val Gly Pro Ala Thr Glu Asn Arg Glu Glu Val Ser Leu

130 135 140

Ser Asn Ala Leu Pro Thr Gln Gly Val Ser Pro Gly Ser Phe Leu Ala

145 150 155 160

Arg Glu Asn Ala Gly Leu Gln Thr Pro Ser Pro Leu Leu Ser Ser Asp

165 170 175

Ala Gly Asp Leu Leu Leu Gln Val Leu Gln Tyr Ser His Ile Leu Glu

180 185 190

Ser Glu Ser Gly Ala Asp Pro Val Pro Pro Gln Ala Pro Gly Gln Glu

195 200 205

Gln Asp Arg Val Tyr Glu Glu Pro Tyr Ala Ala Trp Gln Val Glu Ala

210 215 220

Val Pro Ser Pro Arg Pro Gln Gln Pro Ala Leu Thr Glu Arg Ser Leu

225 230 235 240

Gly Phe Leu Asp Val Thr Ile Met Tyr Lys Gly Arg Thr Val Leu Gln

245 250 255

Ala Val Val Gly His Pro Arg Cys Val Phe Leu Tyr Ser Pro Met Ala

260 265 270

Pro Ala Val Arg Thr Ser Glu Pro Gln Pro Val Ile Phe Pro Ser Pro

275 280 285

Ala Glu Leu Pro Asp Gln Lys Gln Leu His Tyr Thr Glu Thr Leu Leu

290 295 300

Gln His Val Ser Pro Gly Leu Gln Leu Glu Leu Arg Gly Pro Ser Leu

305 310 315 320

Trp Ala Leu Arg Met Gly Lys Cys Lys Val Tyr Trp Glu Val Gly Ser

325 330 335

Pro Met Gly Thr Thr Gly Pro Ser Thr Pro Pro Gln Leu Leu Glu Arg

340 345 350

Asn Arg His Thr Pro Ile Phe Asp Phe Ser Thr Phe Phe Arg Glu Leu

355 360 365

Glu Glu Phe Arg Ala Arg Arg Arg Gln Gly Ser Pro His Tyr Thr Ile

370 375 380

Tyr Leu Gly Phe Gly Gln Asp Leu Ser Ala Gly Arg Pro Lys Glu Lys

385 390 395 400

Thr Leu Ile Leu Val Lys Leu Glu Pro Trp Val Cys Lys Ala Tyr Leu

405 410 415

Glu Gly Val Gln Arg Glu Gly Val Ser Ser Leu Asp Ser Ser Ser Leu

420 425 430

Gly Leu Cys Leu Ser Ser Thr Asn Ser Leu Tyr Glu Asp Ile Glu His

435 440 445

Phe Leu Met Asp Leu Gly Gln Trp Pro

450 455

<210> 15

<211> 525

<212> PROTEIN

<213> Mus musculus

<400> 15

Met Ala Glu Val Arg Gly Val Gln Arg Val Leu Phe Gly Asp Trp Leu

1 5 10 15

Leu Gly Glu Val Ser Ser Gly Gln Tyr Glu Gly Leu Gln Trp Leu Asn

20 25 30

Glu Ala Arg Thr Val Phe Arg Val Pro Trp Lys His Phe Gly Arg Arg

35 40 45

Asp Leu Asp Glu Glu Asp Ala Gln Ile Phe Lys Ala Trp Ala Val Ala

50 55 60

Arg Gly Arg Trp Pro Pro Ser Gly Val Asn Leu Pro Pro Pro Glu Ala

65 70 75 80

Glu Ala Ala Glu Arg Arg Glu Arg Arg Gly Trp Lys Thr Asn Phe Arg

85 90 95

Cys Ala Leu His Ser Thr Gly Arg Phe Ile Leu Arg Gln Asp Asn Ser

100 105 110

Gly Asp Pro Val Asp Pro His Lys Val Tyr Glu Leu Ser Arg Glu Leu

115 120 125

Gly Ser Thr Val Gly Pro Ala Thr Glu Asn Arg Glu Glu Val Ser Leu

130 135 140

Ser Asn Ala Leu Pro Thr Gln Gly Val Ser Pro Gly Ser Phe Leu Ala

145 150 155 160

Arg Glu Asn Ala Gly Leu Gln Thr Pro Ser Pro Leu Leu Ser Ser Asp

165 170 175

Ala Gly Asp Leu Leu Leu Gln Val Leu Gln Tyr Ser His Ile Leu Glu

180 185 190

Ser Glu Ser Gly Ala Asp Pro Val Pro Pro Gln Ala Pro Gly Gln Glu

195 200 205

Gln Asp Arg Val Tyr Glu Glu Pro Tyr Ala Ala Trp Gln Val Glu Ala

210 215 220

Val Pro Ser Pro Arg Pro Gln Gln Pro Ala Leu Thr Glu Arg Ser Leu

225 230 235 240

Gly Phe Leu Asp Val Thr Lys Leu Phe Asp Gly Leu Ile Leu Gly Pro

245 250 255

Leu Lys Asp Glu Gly Ser Ser Asp Leu Ala Ile Val Ser Asp Pro Ser

260 265 270

Gln Gln Leu Pro Ser Pro Asn Val Asn Asn Phe Leu Asn Pro Ala Pro

275 280 285

Gln Glu Asn Pro Leu Lys Gln Leu Leu Ala Glu Glu Gln Trp Glu Phe

290 295 300

Glu Val Thr Ala Phe Tyr Arg Gly Arg Gln Val Phe Gln Gln Thr Leu

305 310 315 320

Phe Cys Pro Gly Gly Leu Arg Leu Val Gly Ser Thr Ala Asp Met Thr

325 330 335

Leu Pro Trp Gln Pro Val Thr Leu Pro Asp Pro Glu Gly Phe Leu Thr

340 345 350

Asp Lys Leu Val Lys Glu Tyr Val Gly Gln Val Leu Lys Gly Leu Gly

355 360 365

Asn Gly Leu Ala Leu Trp Gln Ala Gly Gln Cys Leu Trp Ala Gln Arg

370 375 380

Leu Gly His Ser His Ala Phe Trp Ala Leu Gly Glu Glu Leu Leu Pro

385 390 395 400

Asp Ser Gly Arg Gly Pro Asp Gly Glu Val His Lys Asp Lys Asp Gly

405 410 415

Ala Val Phe Asp Leu Arg Pro Phe Val Ala Asp Leu Ile Ala Phe Met

420 425 430

Glu Gly Ser Gly His Ser Pro Arg Tyr Thr Leu Trp Phe Cys Met Gly

435 440 445

Glu Met Trp Pro Gln Asp Gln Pro Trp Val Lys Arg Leu Val Met Val

450 455 460

Lys Val Val Pro Thr Cys Leu Lys Glu Leu Leu Glu Met Ala Arg Glu

465 470 475 480

Gly Gly Ala Ser Ser Leu Lys Thr Val Asp Leu His Ile Asp Asn Asp

485 490 495

Gln Pro Ile Asp Leu Asp Asp Asp Gln Tyr Lys Ala Tyr Leu Gln Asp

500 505 510

Leu Val Glu Asp Met Asp Phe Gln Ala Thr Gly Asn Ile

515 520 525

<210> 16

<211> 424

<212> PROTEIN

<213> Mus musculus

<400> 16

Met Cys Asp Arg Asn Gly Gly Arg Arg Leu Arg Gln Trp Leu Ile Glu

1 5 10 15

Gln Ile Asp Ser Ser Met Tyr Pro Gly Leu Ile Trp Glu Asn Asp Glu

20 25 30

Lys Thr Met Phe Arg Ile Pro Trp Lys His Ala Gly Lys Gln Asp Tyr

35 40 45

Asn Gln Glu Val Asp Ala Ser Ile Phe Lys Ala Trp Ala Val Phe Lys

50 55 60

Gly Lys Phe Lys Glu Gly Asp Lys Ala Glu Pro Ala Thr Trp Lys Thr

65 70 75 80

Arg Leu Arg Cys Ala Leu Asn Lys Ser Pro Asp Phe Glu Glu Val Thr

85 90 95

Asp Arg Ser Gln Leu Asp Ile Ser Glu Pro Tyr Lys Val Tyr Arg Ile

100 105 110

Val Pro Glu Glu Glu Gln Lys Cys Lys Leu Gly Val Ala Pro Ala Gly

115 120 125

Cys Met Ser Glu Val Pro Glu Met Glu Cys Gly Arg Ser Glu Ile Glu

130 135 140

Glu Leu Ile Lys Glu Pro Ser Val Asp Glu Tyr Met Gly Met Thr Lys

145 150 155 160

Arg Ser Pro Ser Pro Pro Glu Ala Cys Arg Ser Gln Ile Leu Pro Asp

165 170 175

Trp Trp Val Gln Gln Pro Ser Ala Gly Leu Pro Leu Val Thr Gly Tyr

180 185 190

Ala Ala Tyr Asp Thr His Ser Ala Phe Ser Gln Met Val Ile Ser

195 200 205

Phe Tyr Tyr Gly Gly Lys Leu Val Gly Gln Ala Thr Thr Thr Cys Leu

210 215 220

Glu Gly Cys Arg Leu Ser Leu Ser Gln Pro Gly Leu Pro Lys Leu Tyr

225 230 235 240

Gly Pro Asp Gly Leu Glu Pro Val Cys Phe Pro Thr Ala Asp Thr Ile

245 250 255

Pro Ser Glu Arg Gln Arg Gln Val Thr Arg Lys Leu Phe Gly His Leu

260 265 270

Glu Arg Gly Val Leu Leu His Ser Asn Arg Lys Gly Val Phe Val Lys

275 280 285

Arg Leu Cys Gln Gly Arg Val Phe Cys Ser Gly Asn Ala Val Val Cys

290 295 300

Lys Gly Arg Pro Asn Lys Leu Glu Arg Asp Glu Val Val Gln Val Phe

305 310 315 320

Asp Thr Asn Gln Phe Ile Arg Glu Leu Gln Gln Phe Tyr Ala Thr Gln

325 330 335

Ser Arg Leu Pro Asp Ser Arg Val Val Leu Cys Phe Gly Glu Glu Phe

340 345 350

Pro Asp Thr Val Pro Leu Arg Ser Lys Leu Ile Leu Val Gln Val Glu

355 360 365

Gln Leu Tyr Ala Arg Gln Leu Val Glu Glu Ala Gly Lys Ser Cys Gly

370 375 380

Ala Gly Ser Leu Met Pro Ala Leu Glu Glu Pro Gln Pro Asp Gln Ala

385 390 395 400

Phe Arg Met Phe Pro Asp Ile Cys Thr Ser His Gln Arg Pro Phe Phe

405 410 415

Arg Glu Asn Gln Gln Ile Thr Val

420

<210> 17

<211> 424

<212> PROTEIN

<213> Mus musculus

<400> 17

Met Cys Asp Arg Asn Gly Gly Arg Arg Leu Arg Gln Trp Leu Ile Glu

1 5 10 15

Gln Ile Asp Ser Ser Met Tyr Pro Gly Leu Ile Trp Glu Asn Asp Glu

20 25 30

Lys Thr Met Phe Arg Ile Pro Trp Lys His Ala Gly Lys Gln Asp Tyr

35 40 45

Asn Gln Glu Val Asp Ala Ser Ile Phe Lys Ala Trp Ala Val Phe Lys

50 55 60

Gly Lys Phe Lys Glu Gly Asp Lys Ala Glu Pro Ala Thr Trp Lys Thr

65 70 75 80

Arg Leu Arg Cys Ala Leu Asn Lys Ser Pro Asp Phe Glu Glu Val Thr

85 90 95

Asp Arg Ser Gln Leu Asp Ile Ser Glu Pro Tyr Lys Val Tyr Arg Ile

100 105 110

Val Pro Glu Glu Glu Gln Lys Cys Lys Leu Gly Val Ala Pro Ala Gly

115 120 125

Cys Met Ser Glu Val Pro Glu Met Glu Cys Gly Arg Ser Glu Ile Glu

130 135 140

Glu Leu Ile Lys Glu Pro Ser Val Asp Glu Tyr Met Gly Met Thr Lys

145 150 155 160

Arg Ser Pro Ser Pro Pro Glu Ala Cys Arg Ser Gln Ile Leu Pro Asp

165 170 175

Trp Trp Val Gln Gln Pro Ser Ala Gly Leu Pro Leu Val Thr Gly Tyr

180 185 190

Ala Ala Tyr Asp Thr His Ser Ala Phe Ser Gln Met Val Ile Ser

195 200 205

Phe Tyr Tyr Gly Gly Lys Leu Val Gly Gln Ala Thr Thr Thr Cys Leu

210 215 220

Glu Gly Cys Arg Leu Ser Leu Ser Gln Pro Gly Leu Pro Lys Leu Tyr

225 230 235 240

Gly Pro Asp Gly Leu Glu Pro Val Cys Phe Pro Thr Ala Asp Thr Ile

245 250 255

Pro Ser Glu Arg Gln Arg Gln Val Thr Arg Lys Leu Phe Gly His Leu

260 265 270

Glu Arg Gly Val Leu Leu His Ser Asn Arg Lys Gly Val Phe Val Lys

275 280 285

Arg Leu Cys Gln Gly Arg Val Phe Cys Ser Gly Asn Ala Val Val Cys

290 295 300

Lys Gly Arg Pro Asn Arg Leu Glu Arg Asp Glu Val Val Gln Val Phe

305 310 315 320

Asp Thr Asn Gln Phe Ile Arg Glu Leu Gln Gln Phe Tyr Ala Thr Gln

325 330 335

Ser Arg Leu Pro Asp Ser Arg Val Val Leu Cys Phe Gly Glu Glu Phe

340 345 350

Pro Asp Thr Val Pro Leu Arg Ser Lys Leu Ile Leu Val Gln Val Glu

355 360 365

Gln Leu Tyr Ala Arg Gln Leu Val Glu Glu Ala Gly Lys Ser Cys Gly

370 375 380

Ala Gly Ser Leu Met Pro Ala Leu Glu Glu Pro Gln Pro Asp Gln Ala

385 390 395 400

Phe Arg Met Phe Pro Asp Ile Cys Thr Ser His Gln Arg Pro Phe Phe

405 410 415

Arg Glu Asn Gln Gln Ile Thr Val

420

<210> 18

<211> 756

<212> PROTEIN

<213> Homo sapiens

<400> 18

Met Ser Trp Ser Pro Ser Leu Thr Thr Gln Thr Cys Gly Ala Trp Glu

1 5 10 15

Met Lys Glu Arg Leu Gly Thr Gly Gly Phe Gly Asn Val Ile Arg Trp

20 25 30

His Asn Gln Glu Thr Gly Glu Gln Ile Ala Ile Lys Gln Cys Arg Gln

35 40 45

Glu Leu Ser Pro Arg Asn Arg Glu Arg Trp Cys Leu Glu Ile Gln Ile

50 55 60

Met Arg Arg Leu Thr His Pro Asn Val Val Ala Ala Arg Asp Val Pro

65 70 75 80

Glu Gly Met Gln Asn Leu Ala Pro Asn Asp Leu Pro Leu Leu Ala Met

85 90 95

Glu Tyr Cys Gln Gly Gly Asp Leu Arg Lys Tyr Leu Asn Gln Phe Glu

100 105 110

Asn Cys Cys Gly Leu Arg Glu Gly Ala Ile Leu Thr Leu Leu Ser Asp

115 120 125

Ile Ala Ser Ala Leu Arg Tyr Leu His Glu Asn Arg Ile Ile His Arg

130 135 140

Asp Leu Lys Pro Glu Asn Ile Val Leu Gln Gln Gly Glu Gln Arg Leu

145 150 155 160

Ile His Lys Ile Ile Asp Leu Gly Tyr Ala Lys Glu Leu Asp Gln Gly

165 170 175

Ser Leu Cys Thr Ser Phe Val Gly Thr Leu Gln Tyr Leu Ala Pro Glu

180 185 190

Leu Leu Glu Gln Gln Lys Tyr Thr Val Thr Val Asp Tyr Trp Ser Phe

195 200 205

Gly Thr Leu Ala Phe Glu Cys Ile Thr Gly Phe Arg Pro Phe Leu Pro

210 215 220

Asn Trp Gln Pro Val Gln Trp His Ser Lys Val Arg Gln Lys Ser Glu

225 230 235 240

Val Asp Ile Val Val Ser Glu Asp Leu Asn Gly Thr Val Lys Phe Ser

245 250 255

Ser Ser Leu Pro Tyr Pro Asn Asn Leu Asn Ser Val Leu Ala Glu Arg

260 265 270

Leu Glu Lys Trp Leu Gln Leu Met Leu Met Trp His Pro Arg Gln Arg

275 280 285

Gly Thr Asp Pro Thr Tyr Gly Pro Asn Gly Cys Phe Lys Ala Leu Asp

290 295 300

Asp Ile Leu Asn Leu Lys Leu Val His Ile Leu Asn Met Val Thr Gly

305 310 315 320

Thr Ile His Thr Tyr Pro Val Thr Glu Asp Glu Ser Leu Gln Ser Leu

325 330 335

Lys Ala Arg Ile Gln Gln Asp Thr Gly Ile Pro Glu Glu Asp Gln Glu

340 345 350

Leu Leu Gln Glu Ala Gly Leu Ala Leu Ile Pro Asp Lys Pro Ala Thr

355 360 365

Gln Cys Ile Ser Asp Gly Lys Leu Asn Glu Gly His Thr Leu Asp Met

370 375 380

Asp Leu Val Phe Leu Phe Asp Asn Ser Lys Ile Thr Tyr Glu Thr Gln

385 390 395 400

Ile Ser Pro Arg Pro Gln Pro Glu Ser Val Ser Cys Ile Leu Gln Glu

405 410 415

Pro Lys Arg Asn Leu Ala Phe Phe Gln Leu Arg Lys Val Trp Gly Gln

420 425 430

Val Trp His Ser Ile Gln Thr Leu Lys Glu Asp Cys Asn Arg Leu Gln

435 440 445

Gln Gly Gln Arg Ala Ala Met Met Asn Leu Leu Arg Asn Asn Ser Cys

450 455 460

Leu Ser Lys Met Lys Asn Ser Met Ala Ser Met Ser Gln Gln Leu Lys

465 470 475 480

Ala Lys Leu Asp Phe Phe Lys Thr Ser Ile Gln Ile Asp Leu Glu Lys

485 490 495

Tyr Ser Glu Gln Thr Glu Phe Gly Ile Thr Ser Asp Lys Leu Leu Leu

500 505 510

Ala Trp Arg Glu Met Glu Gln Ala Val Glu Leu Cys Gly Arg Glu Asn

515 520 525

Glu Val Lys Leu Leu Val Glu Arg Met Met Ala Leu Gln Thr Asp Ile

530 535 540

Val Asp Leu Gln Arg Ser Pro Met Gly Arg Lys Gln Gly Gly Thr Leu

545 550 555 560

Asp Asp Leu Glu Glu Gln Ala Arg Glu Leu Tyr Arg Arg Leu Arg Glu

565 570 575

Lys Pro Arg Asp Gln Arg Thr Glu Gly Asp Ser Gln Glu Met Val Arg

580 585 590

Leu Leu Leu Gln Ala Ile Gln Ser Phe Glu Lys Lys Val Arg Val Ile

595 600 605

Tyr Thr Gln Leu Ser Lys Thr Val Val Cys Lys Gln Lys Ala Leu Glu

610 615 620

Leu Leu Pro Lys Val Glu Glu Val Val Ser Leu Met Asn Glu Asp Glu

625 630 635 640

Lys Thr Val Val Arg Leu Gln Glu Lys Arg Gln Lys Glu Leu Trp Asn

645 650 655

Leu Leu Lys Ile Ala Cys Ser Lys Val Arg Gly Pro Val Ser Gly Ser

660 665 670

Pro Asp Ser Met Asn Ala Ser Arg Leu Ser Gln Pro Gly Gln Leu Met

675 680 685

Ser Gln Pro Ser Thr Ala Ser Asn Ser Leu Pro Glu Pro Ala Lys Lys

690 695 700

Ser Glu Glu Leu Val Ala Glu Ala His Asn Leu Cys Thr Leu Leu Glu

705 710 715 720

Asn Ala Ile Gln Asp Thr Val Arg Glu Gln Asp Gln Ser Phe Thr Ala

725 730 735

Leu Asp Trp Ser Trp Leu Gln Thr Glu Glu Glu Glu His Ser Cys Leu

740 745 750

Glu Gln Ala Ser

755

<210> 19

<211> 754

<212> PROTEIN

<213> Homo sapiens

<400> 19

Met Phe Ser Gly Gly Cys His Ser Pro Gly Phe Gly Arg Pro Ser Pro

1 5 10 15

Ala Phe Pro Ala Pro Gly Ser Pro Pro Pro Ala Pro Arg Pro Cys Arg

20 25 30

Gln Glu Thr Gly Glu Gln Ile Ala Ile Lys Gln Cys Arg Gln Glu Leu

35 40 45

Ser Pro Arg Asn Arg Glu Arg Trp Cys Leu Glu Ile Gln Ile Met Arg

50 55 60

Arg Leu Thr His Pro Asn Val Val Ala Ala Arg Asp Val Pro Glu Gly

65 70 75 80

Met Gln Asn Leu Ala Pro Asn Asp Leu Pro Leu Leu Ala Met Glu Tyr

85 90 95

Cys Gln Gly Gly Asp Leu Arg Lys Tyr Leu Asn Gln Phe Glu Asn Cys

100 105 110

Cys Gly Leu Arg Glu Gly Ala Ile Leu Thr Leu Leu Ser Asp Ile Ala

115 120 125

Ser Ala Leu Arg Tyr Leu His Glu Asn Arg Ile Ile His Arg Asp Leu

130 135 140

Lys Pro Glu Asn Ile Val Leu Gln Gln Gly Glu Gln Arg Leu Ile His

145 150 155 160

Lys Ile Ile Asp Leu Gly Tyr Ala Lys Glu Leu Asp Gln Gly Ser Leu

165 170 175

Cys Thr Ser Phe Val Gly Thr Leu Gln Tyr Leu Ala Pro Glu Leu Leu

180 185 190

Glu Gln Gln Lys Tyr Thr Val Thr Val Asp Tyr Trp Ser Phe Gly Thr

195 200 205

Leu Ala Phe Glu Cys Ile Thr Gly Phe Arg Pro Phe Leu Pro Asn Trp

210 215 220

Gln Pro Val Gln Trp His Ser Lys Val Arg Gln Lys Ser Glu Val Asp

225 230 235 240

Ile Val Val Ser Glu Asp Leu Asn Gly Thr Val Lys Phe Ser Ser Ser

245 250 255

Leu Pro Tyr Pro Asn Asn Leu Asn Ser Val Leu Ala Glu Arg Leu Glu

260 265 270

Lys Trp Leu Gln Leu Met Leu Met Trp His Pro Arg Gln Arg Gly Thr

275 280 285

Asp Pro Thr Tyr Gly Pro Asn Gly Cys Phe Lys Ala Leu Asp Asp Ile

290 295 300

Leu Asn Leu Lys Leu Val His Ile Leu Asn Met Val Thr Gly Thr Ile

305 310 315 320

His Thr Tyr Pro Val Thr Glu Asp Glu Ser Leu Gln Ser Leu Lys Ala

325 330 335

Arg Ile Gln Gln Asp Thr Gly Ile Pro Glu Glu Asp Gln Glu Leu Leu

340 345 350

Gln Glu Ala Gly Leu Ala Leu Ile Pro Asp Lys Pro Ala Thr Gln Cys

355 360 365

Ile Ser Asp Gly Lys Leu Asn Glu Gly His Thr Leu Asp Met Asp Leu

370 375 380

Val Phe Leu Phe Asp Asn Ser Lys Ile Thr Tyr Glu Thr Gln Ile Ser

385 390 395 400

Pro Arg Pro Gln Pro Glu Ser Val Ser Cys Ile Leu Gln Glu Pro Lys

405 410 415

Arg Asn Leu Ala Phe Phe Gln Leu Arg Lys Val Trp Gly Gln Val Trp

420 425 430

His Ser Ile Gln Thr Leu Lys Glu Asp Cys Asn Arg Leu Gln Gln Gly

435 440 445

Gln Arg Ala Ala Met Met Asn Leu Leu Arg Asn Asn Ser Cys Leu Ser

450 455 460

Lys Met Lys Asn Ser Met Ala Ser Met Ser Gln Gln Leu Lys Ala Lys

465 470 475 480

Leu Asp Phe Phe Lys Thr Ser Ile Gln Ile Asp Leu Glu Lys Tyr Ser

485 490 495

Glu Gln Thr Glu Phe Gly Ile Thr Ser Asp Lys Leu Leu Leu Ala Trp

500 505 510

Arg Glu Met Glu Gln Ala Val Glu Leu Cys Gly Arg Glu Asn Glu Val

515 520 525

Lys Leu Leu Val Glu Arg Met Met Ala Leu Gln Thr Asp Ile Val Asp

530 535 540

Leu Gln Arg Ser Pro Met Gly Arg Lys Gln Gly Gly Thr Leu Asp Asp

545 550 555 560

Leu Glu Glu Gln Ala Arg Glu Leu Tyr Arg Arg Leu Arg Glu Lys Pro

565 570 575

Arg Asp Gln Arg Thr Glu Gly Asp Ser Gln Glu Met Val Arg Leu Leu

580 585 590

Leu Gln Ala Ile Gln Ser Phe Glu Lys Lys Val Arg Val Ile Tyr Thr

595 600 605

Gln Leu Ser Lys Thr Val Val Cys Lys Gln Lys Ala Leu Glu Leu Leu

610 615 620

Pro Lys Val Glu Glu Val Val Ser Leu Met Asn Glu Asp Glu Lys Thr

625 630 635 640

Val Val Arg Leu Gln Glu Lys Arg Gln Lys Glu Leu Trp Asn Leu Leu

645 650 655

Lys Ile Ala Cys Ser Lys Val Arg Gly Pro Val Ser Gly Ser Pro Asp

660 665 670

Ser Met Asn Ala Ser Arg Leu Ser Gln Pro Gly Gln Leu Met Ser Gln

675 680 685

Pro Ser Thr Ala Ser Asn Ser Leu Pro Glu Pro Ala Lys Lys Ser Glu

690 695 700

Glu Leu Val Ala Glu Ala His Asn Leu Cys Thr Leu Leu Glu Asn Ala

705 710 715 720

Ile Gln Asp Thr Val Arg Glu Gln Asp Gln Ser Phe Thr Ala Leu Asp

725 730 735

Trp Ser Trp Leu Gln Thr Glu Glu Glu Glu His Ser Cys Leu Glu Gln

740 745 750

Ala Ser

<210> 20

<211> 256

<212> PROTEIN

<213> Homo sapiens

<400> 20

Met Ser Trp Ser Pro Ser Leu Thr Thr Gln Thr Cys Gly Ala Trp Glu

1 5 10 15

Met Lys Glu Arg Leu Gly Thr Gly Gly Phe Gly Asn Val Ile Arg Trp

20 25 30

His Asn Gln Glu Thr Gly Glu Gln Ile Ala Ile Lys Gln Cys Arg Gln

35 40 45

Glu Leu Ser Pro Arg Asn Arg Glu Arg Trp Cys Leu Glu Ile Gln Ile

50 55 60

Met Arg Arg Leu Thr His Pro Asn Val Val Ala Ala Arg Asp Val Pro

65 70 75 80

Glu Gly Met Gln Asn Leu Ala Pro Asn Asp Leu Pro Leu Leu Ala Met

85 90 95

Glu Tyr Cys Gln Gly Gly Asp Leu Arg Lys Tyr Leu Asn Gln Phe Glu

100 105 110

Asn Cys Cys Gly Leu Arg Glu Gly Ala Ile Leu Thr Leu Leu Ser Asp

115 120 125

Ile Ala Ser Ala Leu Arg Tyr Leu His Glu Asn Arg Ile Ile His Arg

130 135 140

Asp Leu Lys Pro Glu Asn Ile Val Leu Gln Gln Gly Glu Gln Arg Leu

145 150 155 160

Ile His Lys Ile Ile Asp Leu Gly Tyr Ala Lys Glu Leu Asp Gln Gly

165 170 175

Ser Leu Cys Thr Ser Phe Val Gly Thr Leu Gln Tyr Leu Ala Pro Glu

180 185 190

Leu Leu Glu Gln Gln Lys Tyr Thr Val Thr Val Asp Tyr Trp Ser Phe

195 200 205

Gly Thr Leu Ala Phe Glu Cys Ile Thr Gly Phe Arg Pro Phe Leu Pro

210 215 220

Asn Trp Gln Pro Val Gln Cys Val Arg Met Trp Pro Gly Thr Val Ala

225 230 235 240

His Ser Cys Asn Pro Ser Thr Leu Gly Gly Arg Gly Arg Trp Ile Ser

245 250 255

<210> 21

<211> 697

<212> PROTEIN

<213> Homo sapiens

<400> 21

Met Ser Ser Asp Gly Thr Ile Arg Leu Thr His Pro Asn Val Val Ala

1 5 10 15

Ala Arg Asp Val Pro Glu Gly Met Gln Asn Leu Ala Pro Asn Asp Leu

20 25 30

Pro Leu Leu Ala Met Glu Tyr Cys Gln Gly Gly Asp Leu Arg Lys Tyr

35 40 45

Leu Asn Gln Phe Glu Asn Cys Cys Gly Leu Arg Glu Gly Ala Ile Leu

50 55 60

Thr Leu Leu Ser Asp Ile Ala Ser Ala Leu Arg Tyr Leu His Glu Asn

65 70 75 80

Arg Ile Ile His Arg Asp Leu Lys Pro Glu Asn Ile Val Leu Gln Gln

85 90 95

Gly Glu Gln Arg Leu Ile His Lys Ile Ile Asp Leu Gly Tyr Ala Lys

100 105 110

Glu Leu Asp Gln Gly Ser Leu Cys Thr Ser Phe Val Gly Thr Leu Gln

115 120 125

Tyr Leu Ala Pro Glu Leu Leu Glu Gln Gln Lys Tyr Thr Val Thr Val

130 135 140

Asp Tyr Trp Ser Phe Gly Thr Leu Ala Phe Glu Cys Ile Thr Gly Phe

145 150 155 160

Arg Pro Phe Leu Pro Asn Trp Gln Pro Val Gln Trp His Ser Lys Val

165 170 175

Arg Gln Lys Ser Glu Val Asp Ile Val Val Ser Glu Asp Leu Asn Gly

180 185 190

Thr Val Lys Phe Ser Ser Ser Leu Pro Tyr Pro Asn Asn Leu Asn Ser

195 200 205

Val Leu Ala Glu Arg Leu Glu Lys Trp Leu Gln Leu Met Leu Met Trp

210 215 220

His Pro Arg Gln Arg Gly Thr Asp Pro Thr Tyr Gly Pro Asn Gly Cys

225 230 235 240

Phe Lys Ala Leu Asp Asp Ile Leu Asn Leu Lys Leu Val His Ile Leu

245 250 255

Asn Met Val Thr Gly Thr Ile His Thr Tyr Pro Val Thr Glu Asp Glu

260 265 270

Ser Leu Gln Ser Leu Lys Ala Arg Ile Gln Gln Asp Thr Gly Ile Pro

275 280 285

Glu Glu Asp Gln Glu Leu Leu Gln Glu Ala Gly Leu Ala Leu Ile Pro

290 295 300

Asp Lys Pro Ala Thr Gln Cys Ile Ser Asp Gly Lys Leu Asn Glu Gly

305 310 315 320

His Thr Leu Asp Met Asp Leu Val Phe Leu Phe Asp Asn Ser Lys Ile

325 330 335

Thr Tyr Glu Thr Gln Ile Ser Pro Arg Pro Gln Pro Glu Ser Val Ser

340 345 350

Cys Ile Leu Gln Glu Pro Lys Arg Asn Leu Ala Phe Phe Gln Leu Arg

355 360 365

Lys Val Trp Gly Gln Val Trp His Ser Ile Gln Thr Leu Lys Glu Asp

370 375 380

Cys Asn Arg Leu Gln Gln Gly Gln Arg Ala Ala Met Met Asn Leu Leu

385 390 395 400

Arg Asn Asn Ser Cys Leu Ser Lys Met Lys Asn Ser Met Ala Ser Met

405 410 415

Ser Gln Gln Leu Lys Ala Lys Leu Asp Phe Phe Lys Thr Ser Ile Gln

420 425 430

Ile Asp Leu Glu Lys Tyr Ser Glu Gln Thr Glu Phe Gly Ile Thr Ser

435 440 445

Asp Lys Leu Leu Leu Ala Trp Arg Glu Met Glu Gln Ala Val Glu Leu

450 455 460

Cys Gly Arg Glu Asn Glu Val Lys Leu Leu Val Glu Arg Met Met Ala

465 470 475 480

Leu Gln Thr Asp Ile Val Asp Leu Gln Arg Ser Pro Met Gly Arg Lys

485 490 495

Gln Gly Gly Thr Leu Asp Asp Leu Glu Glu Gln Ala Arg Glu Leu Tyr

500 505 510

Arg Arg Leu Arg Glu Lys Pro Arg Asp Gln Arg Thr Glu Gly Asp Ser

515 520 525

Gln Glu Met Val Arg Leu Leu Leu Gln Ala Ile Gln Ser Phe Glu Lys

530 535 540

Lys Val Arg Val Ile Tyr Thr Gln Leu Ser Lys Thr Val Val Cys Lys

545 550 555 560

Gln Lys Ala Leu Glu Leu Leu Pro Lys Val Glu Glu Val Val Ser Leu

565 570 575

Met Asn Glu Asp Glu Lys Thr Val Val Arg Leu Gln Glu Lys Arg Gln

580 585 590

Lys Glu Leu Trp Asn Leu Leu Lys Ile Ala Cys Ser Lys Val Arg Gly

595 600 605

Pro Val Ser Gly Ser Pro Asp Ser Met Asn Ala Ser Arg Leu Ser Gln

610 615 620

Pro Gly Gln Leu Met Ser Gln Pro Ser Thr Ala Ser Asn Ser Leu Pro

625 630 635 640

Glu Pro Ala Lys Lys Ser Glu Glu Leu Val Ala Glu Ala His Asn Leu

645 650 655

Cys Thr Leu Leu Glu Asn Ala Ile Gln Asp Thr Val Arg Glu Gln Asp

660 665 670

Gln Ser Phe Thr Ala Leu Asp Trp Ser Trp Leu Gln Thr Glu Glu Glu

675 680 685

Glu His Ser Cys Leu Glu Gln Ala Ser

690 695

<210> 22

<211> 738

<212> PROTEIN

<213> Mus musculus

<400> 22

Met Ser Trp Ser Pro Ser Leu Pro Thr Gln Thr Cys Gly Ala Trp Glu

1 5 10 15

Met Lys Glu Arg Leu Gly Thr Gly Gly Phe Gly Asn Val Ile Arg Trp

20 25 30

His Asn Gln Ala Thr Gly Glu Gln Ile Ala Ile Lys Gln Cys Arg Gln

35 40 45

Glu Leu Ser Pro Lys Asn Arg Asp Arg Trp Cys Leu Glu Ile Gln Ile

50 55 60

Met Arg Arg Leu Asn His Pro Asn Val Val Ala Ala Arg Asp Val Pro

65 70 75 80

Glu Gly Met Gln Asn Leu Ala Pro Asn Asp Leu Pro Leu Leu Ala Met

85 90 95

Glu Tyr Cys Gln Gly Gly Asp Leu Arg Arg Tyr Leu Asn Gln Phe Glu

100 105 110

Asn Cys Cys Gly Leu Arg Glu Gly Ala Val Leu Thr Leu Leu Ser Asp

115 120 125

Ile Ala Ser Ala Leu Arg Tyr Leu His Glu Asn Arg Ile Ile His Arg

130 135 140

Asp Leu Lys Pro Glu Asn Ile Val Leu Gln Gln Gly Glu Lys Arg Leu

145 150 155 160

Ile His Lys Ile Ile Asp Leu Gly Tyr Ala Lys Glu Leu Asp Gln Gly

165 170 175

Ser Leu Cys Thr Ser Phe Val Gly Thr Leu Gln Tyr Leu Ala Pro Glu

180 185 190

Leu Leu Glu Gln Gln Lys Tyr Thr Val Thr Val Asp Tyr Trp Ser Phe

195 200 205

Gly Thr Leu Ala Phe Glu Cys Ile Thr Gly Phe Arg Pro Phe Leu Pro

210 215 220

Asn Trp Gln Pro Val Gln Trp His Ser Lys Val Arg Gln Lys Ser Glu

225 230 235 240

Val Asp Ile Val Val Ser Glu Asp Leu Asn Gly Ala Val Lys Phe Ser

245 250 255

Ser Ser Leu Pro Phe Pro Asn Asn Leu Asn Ser Val Leu Ala Glu Arg

260 265 270

Leu Glu Lys Trp Leu Gln Leu Met Leu Met Trp His Pro Arg Gln Arg

275 280 285

Gly Thr Asp Pro Gln Tyr Gly Pro Asn Gly Cys Phe Arg Ala Leu Asp

290 295 300

Asp Ile Leu Asn Leu Lys Leu Val His Val Leu Asn Met Val Thr Gly

305 310 315 320

Thr Val His Thr Tyr Pro Val Thr Glu Asp Glu Ser Leu Gln Ser Leu

325 330 335

Lys Thr Arg Ile Gln Glu Asp Thr Gly Ile Leu Glu Thr Asp Gln Glu

340 345 350

Leu Leu Gln Glu Ala Gly Leu Val Leu Leu Pro Asp Lys Pro Ala Thr

355 360 365

Gln Cys Ile Ser Asp Ser Lys Thr Asn Glu Gly Leu Thr Leu Asp Met

370 375 380

Asp Leu Val Phe Leu Phe Asp Asn Ser Lys Ile Asn Tyr Glu Thr Gln

385 390 395 400

Ile Thr Pro Arg Pro Gln Pro Glu Ser Val Ser Cys Ile Leu Gln Glu

405 410 415

Pro Lys Arg Asn Leu Ser Phe Phe Gln Leu Arg Lys Val Trp Gly Gln

420 425 430

Val Trp His Ser Ile Gln Thr Leu Lys Glu Asp Cys Asn Arg Leu Gln

435 440 445

Gln Gly Gln Arg Ala Ala Met Met Ser Leu Leu Arg Asn Asn Ser Cys

450 455 460

Leu Ser Lys Met Lys Asn Ala Met Ala Ser Thr Ala Gln Gln Leu Lys

465 470 475 480

Ala Lys Leu Asp Phe Phe Lys Thr Ser Ile Gln Ile Asp Leu Glu Lys

485 490 495

Tyr Lys Glu Gln Thr Glu Phe Gly Ile Thr Ser Asp Lys Leu Leu Leu

500 505 510

Ala Trp Arg Glu Met Glu Gln Ala Val Glu Gln Cys Gly Arg Glu Asn

515 520 525

Asp Val Lys His Leu Val Glu Arg Met Met Ala Leu Gln Thr Asp Ile

530 535 540

Val Asp Leu Gln Arg Ser Pro Met Gly Arg Lys Gln Gly Gly Thr Leu

545 550 555 560

Asp Asp Leu Glu Glu Gln Ala Arg Glu Leu Tyr Arg Arg Leu Arg Glu

565 570 575

Lys Pro Arg Asp Gln Arg Thr Glu Gly Asp Ser Gln Glu Met Val Arg

580 585 590

Leu Leu Leu Gln Ala Ile Gln Ser Phe Glu Lys Lys Val Arg Val Ile

595 600 605

Tyr Thr Gln Leu Ser Lys Thr Val Val Cys Lys Gln Lys Ala Leu Glu

610 615 620

Leu Leu Pro Lys Val Glu Glu Val Val Ser Leu Met Asn Glu Asp Glu

625 630 635 640

Arg Thr Val Val Arg Leu Gln Glu Lys Arg Gln Lys Glu Leu Trp Asn

645 650 655

Leu Leu Lys Ile Ala Cys Ser Lys Val Arg Gly Pro Val Ser Gly Ser

660 665 670

Pro Asp Ser Met Asn Val Ser Arg Leu Ser His Pro Gly Gln Leu Met

675 680 685

Ser Gln Pro Ser Ser Ala Cys Asp Ser Leu Pro Glu Ser Asp Lys Lys

690 695 700

Ser Glu Glu Leu Val Ala Glu Ala His Ala Leu Cys Ser Arg Leu Glu

705 710 715 720

Ser Ala Leu Gln Asp Thr Val Lys Glu Gln Asp Arg Ser Phe Thr Val

725 730 735

Thr Ala

<210> 23

<211> 1497

<212> DNA

<213> Homo sapiens

<400> 23

atgaaccagt ccatcccagt ggctcccacc ccaccccgcc gcgtgcggct gaagccctgg 60

ctggtggccc aggtgaacag ctgccagtac ccagggcttc aatgggtcaa cggggaaaag 120

aaattattct gcatcccctg gaggcatgcc acaaggcatg gtcccagcca ggacggagat 180

aacaccatct tcaaggcctg ggccaaggag acagggaaat acaccgaagg cgtggatgaa 240

gccgatccgg ccaagtggaa ggccaacctg cgctgtgccc ttaacaagag ccgggacttc 300

cgcctcatct acgacggggcc ccgggacatg ccacctcagc cctacaagat ctacgaggtc 360

tgctccaatg gccctgctcc cacagactcc cagccccctg aggattactc ttttggtgca 420

ggagaggagg aggaagaaga ggaagagctg cagaggatgt tgccaagcct gagcctcaca 480

gaggatgtca agtggccgcc cactctgcag ccgcccactc tgcggccgcc tactctgcag 540

ccgcccactc tgcagccgcc cgtggtgctg ggtccccctg ctccagaccc cagccccctg 600

gctcctcccc ctggcaaccc tgctggcttc agggagcttc tctctgaggt cctggagcct 660

gggcccctgc ctgccagcct gccccctgca ggcgaacagc tcctgccaga cctgctgatc 720

agcccccaca tgctgcctct gaccgacctg gagatcaagt ttcagtaccg ggggcggcca 780

ccccgggccc tcaccatcag caacccccat ggctgccggc tcttctacag ccagctggag 840

gccacccagg agcaggtgga actcttcggc cccataagcc tggagcaagt gcgcttcccc 900

agccctgagg acatccccag tgacaagcag cgcttctaca cgaaccagct gctggatgtc 960

ctggaccgcg ggctcatcct ccagctacag ggccaggacc tttatgccat ccgcctgtgt 1020

cagtgcaagg tgttctggag cggggccttgt gcctcagccc atgactcatg ccccaacccc 1080

atccagcggg aggtcaagac caagcttttc agcctggagc attttctcaa tgagctcatc 1140

ctgttccaaa agggccagac caacacccca ccacccttcg agatcttctt ctgctttggg 1200

gaagaatggc ctgaccgcaa accccgagag aagaagctca ttactgtaca ggtggtgcct 1260

gtagcagctc gactgctgct ggagatgttc tcaggggagc tatcttggtc agctgatagt 1320

atccggctac agatctcaaa cccagacctc aaagaccgca tggtggagca attcaaggag 1380

ctccatcaca tctggcagtc ccagcagcgg ttgcagcctg tggcccaggc ccctcctgga 1440

gcaggccttg gtgttggcca ggggccctgg cctatgcacc cagctggcat gcaataa 1497

<210> 24

<211> 1545

<212> DNA

<213> Homo sapiens

<400> 24

atgaaccagt ccatcccagt ggctcccacc ccaccccgcc gcgtgcggct gaagccctgg 60

ctggtggccc aggtgaacag ctgccagtac ccagggcttc aatgggtcaa cggggaaaag 120

aaattattct gcatcccctg gaggcatgcc acaaggcatg gtcccagcca ggacggagat 180

aacaccatct tcaaggcctg ggccaaggag acagggaaat acaccgaagg cgtggatgaa 240

gccgatccgg ccaagtggaa ggccaacctg cgctgtgccc ttaacaagag ccgggacttc 300

cgcctcatct acgacggggcc ccgggacatg ccacctcagc cctacaagat ctacgaggtc 360

tgctccaatg gccctgctcc cacagactcc cagccccctg aggattactc ttttggtgca 420

ggagaggagg aggaagaaga ggaagagctg cagaggatgt tgccaagcct gagcctcaca 480

gatgcagtgc agtctggccc ccacatgaca ccctattctt tactcaaaga ggatgtcaag 540

tggccgccca ctctgcagcc gcccactctg cggccgccta ctctgcagcc gcccactctg 600

cagccgcccg tggtgctggg tccccctgct ccagacccca gccccctggc tcctccccct 660

ggcaaccctg ctggcttcag ggagcttctc tctgaggtcc tggagcctgg gcccctgcct 720

gccagcctgc cccctgcagg cgaacagctc ctgccagacc tgctgatcag cccccacatg 780

ctgcctctga ccgacctgga gatcaagttt cagtaccggg ggcggccacc ccgggccctc 840

accatcagca acccccatgg ctgccggctc ttctacagcc agctggaggc cacccaggag 900

caggtggaac tcttcggccc cataagcctg gagcaagtgc gcttccccag ccctgaggac 960

atccccagtg acaagcagcg cttctacacg aaccagctgc tggatgtcct ggaccgcggg 1020

ctcatcctcc agctacaggg ccaggacctt tatgccatcc gcctgtgtca gtgcaaggtg 1080

ttctggagcg ggccttgtgc ctcagcccat gactcatgcc ccaaccccat ccagcggggag 1140

gtcaagacca agcttttcag cctggagcat tttctcaatg agctcatcct gttccaaaag 1200

ggccagacca acaccccacc acccttcgag atcttcttct gctttgggga agaatggcct 1260

gaccgcaaac cccgagagaa gaagctcatt actgtacagg tggtgcctgt agcagctcga 1320

ctgctgctgg agatgttctc aggggagcta tcttggtcag ctgatagtat ccggctacag 1380

atctcaaacc cagacctcaa agaccgcatg gtggagcaat tcaaggagct ccatcacatc 1440

tggcagtccc agcagcggtt gcagcctgtg gcccaggccc ctcctggagc aggccttggt 1500

gttggccagg ggccctggcc tatgcaccca gctggcatgc aataa 1545

<210> 25

<211> 1514

<212> DNA

<213> Homo sapiens

<400> 25

atgaaccagt ccatcccagt ggctcccacc ccaccccgcc gcgtgcggct gaagccctgg 60

ctggtggccc aggtgaacag ctgccagtac ccagggcttc aatgggtcaa cggggaaaag 120

aaattattct gcatcccctg gaggcatgcc acaaggcatg gtcccagcca ggacggagat 180

aacaccatct tcaaggcctg ggccaaggag acagggaaat acaccgaagg cgtggatgaa 240

gccgatccgg ccaagggaag gccaacctgc gctgtgccct taacaagagc cgggacttcc 300

gcctcatcta cgacgggccc cgggacatgc cacctcagcc ctacaagatc tacgaggtct 360

gctccaatgg ccctgctccc acagactccc agccccctga ggattactct tttggtgcag 420

gagaggagga ggaagaagag gaagagctgc agaggatgtt gccaagcctg agcctcacag 480

atgcagtgca gtctggcccc cacatgacac cctattcttt actcaaagag gatgtcaagt 540

ggccgcccac tctgcagccg cccactctgc agccgcccgt ggtgctgggt ccccctgctc 600

cagaccccag ccccctggct cctccccctg gcaaccctgc tggcttcagg gagcttctct 660

ctgaggtcct ggagcctggg cccctgcctg ccagcctgcc ccctgcaggc gaacagctcc 720

tgccagacct gctgatcagc ccccacatgc tgcctctgac cgacctggag atcaagtttc 780

agtaccgggg gcggccaccc cgggccctca ccatcagcaa cccccatggc tgccggctct 840

tctacagcca gctggaggcc acccaggagc aggtggaact cttcggcccc ataagcctgg 900

agcaagtgcg cttccccagc cctgaggaca tccccagtga caagcagcgc ttctacacga 960

accagctgct ggatgtcctg gaccgcgggc tcatcctcca gctacagggc caggaccttt 1020

atgccatccg cctgtgtcag tgcaaggtgt tctggagcgg gccttgtgcc tcagcccatg 1080

actcatgccc caaccccatc cagcgggagg tcaagaccaa gcttttcagc ctggagcatt 1140

ttctcaatga gctcatcctg ttccaaaagg gccagaccaa caccccacca cccttcgaga 1200

tcttcttctg ctttggggaa gaatggcctg accgcaaacc ccgagagaag aagctcatta 1260

ctgtacaggt ggtgcctgta gcagctcgac tgctgctgga gatgttctca ggggagctat 1320

cttggtcagc tgatagtatc cggctacaga tctcaaaccc agacctcaaa gaccgcatgg 1380

tggagcaatt caagggagctc catcacatct ggcagtccca gcagcggttg cagcctgtgg 1440

cccaggcccc tcctggagca ggccttggtg ttggccaggg gccctggcct atgcacccag 1500

ctggcatgca ataa 1514

<210> 26

<211> 1467

<212> DNA

<213> Homo sapiens

<400> 26

atgaaccagt ccatcccagt ggctcccacc ccaccccgcc gcgtgcggct gaagccctgg 60

ctggtggccc aggtgaacag ctgccagtac ccagggcttc aatgggtcaa cggggaaaag 120

aaattattct gcatcccctg gaggcatgcc acaaggcatg gtcccagcca ggacggagat 180

aacaccatct tcaaggcctg ggccaaggag acagggaaat acaccgaagg cgtggatgaa 240

gccgatccgg ccaagtggaa ggccaacctg cgctgtgccc ttaacaagag ccgggacttc 300

cgcctcatct acgacggggcc ccgggacatg ccacctcagc cctacaagat ctacgaggtc 360

tgctccaatg gccctgctcc cacagactcc cagccccctg aggattactc ttttggtgca 420

ggagaggagg aggaagaaga ggaagagctg cagaggatgt tgccaagcct gagcctcaca 480

gaggatgtca agtggccgcc cactctgcag ccgcccactc tgcagccgcc cgtggtgctg 540

ggtccccctg ctccagaccc cagccccctg gctcctcccc ctggcaaccc tgctggcttc 600

agggagcttc tctctgaggt cctggagcct gggcccctgc ctgccagcct gccccctgca 660

ggcgaacagc tcctgccaga cctgctgatc agcccccaca tgctgcctct gaccgacctg 720

gagatcaagt ttcagtaccg ggggcggcca ccccggggccc tcaccatcag caacccccat 780

ggctgccggc tcttctacag ccagctggag gcacccagg agcaggtgga actcttcggc 840

cccataagcc tggagcaagt gcgcttcccc agccctgagg acatccccag tgacaagcag 900

cgcttctaca cgaaccagct gctggatgtc ctggaccgcg ggctcatcct ccagctacag 960

ggccaggacc tttatgccat ccgcctgtgt cagtgcaagg tgttctggag cgggccttgt 1020

gcctcagccc atgactcatg ccccaacccc atccagcggg aggtcaagac caagcttttc 1080

agcctggagc attttctcaa tgagctcatc ctgttccaaa agggccagac caacacccca 1140

ccacccttcg agatcttctt ctgctttggg gaagaatggc ctgaccgcaa accccgagag 1200

aagaagctca ttactgtaca ggtggtgcct gtagcagctc gactgctgct ggagatgttc 1260

tcaggggagc tatcttggtc agctgatagt atccggctac agatctcaaa cccagacctc 1320

aaagaccgca tggtggagca attcaaggag ctccatcaca tctggcagtc ccagcagcgg 1380

ttgcagcctg tggcccaggc ccctcctgga gcaggccttg gtgttggcca ggggccctgg 1440

cctatgcacc cagctggcat gcaataa 1467

<210> 27

<211> 1239

<212> DNA

<213> Homo sapiens

<400> 27

atgaaccagt ccatcccagt ggctcccacc ccaccccgcc gcgtgcggct gaagccctgg 60

ctggtggccc aggtgaacag ctgccagtac ccagggcttc aatgggtcaa cggggaaaag 120

aaattattct gcatcccctg gaggcatgcc acaaggcatg gtcccagcca ggacggagat 180

aacaccatct tcaaggcctg ggccaaggag acagggaaat acaccgaagg cgtggatgaa 240

gccgatccgg ccaagtggaa ggccaacctg cgctgtgccc ttaacaagag ccgggacttc 300

cgcctcatct acgacggggcc ccgggacatg ccacctcagc cctacaagat ctacgaggtc 360

tgctccaatg gccctgctcc cacagactcc cagccccctg aggattactc ttttggtgca 420

ggagaggagg aggaagaaga ggaagagctg cagaggatgt tgccaagcct gagcctcaca 480

gtgaccgacc tggagatcaa gtttcagtac cggggggcggc caccccgggc cctcaccatc 540

agcaaccccc atggctgccg gctcttctac agccagctgg aggccaccca ggagcaggtg 600

gaactcttcg gccccataag cctggagcaa gtgcgcttcc ccagccctga ggacatcccc 660

agtgacaagc agcgcttcta cacgaaccag ctgctggatg tcctggaccg cgggctcatc 720

ctccagctac agggccagga cctttatgcc atccgcctgt gtcagtgcaa ggtgttctgg 780

agcgggcctt gtgcctcagc ccatgactca tgccccaacc ccatccagcg ggaggtcaag 840

accaagcttt tcagcctgga gcattttctc aatgagctca tcctgttcca aaagggccag 900

accaacaccc caccaccctt cgagatcttc ttctgctttg gggaagaatg gcctgaccgc 960

aaaccccgag agaagaagct cattactgta caggtggtgc ctgtagcagc tcgactgctg 1020

ctggagatgt tctcagggga gctatcttgg tcagctgata gtatccggct acagatctca 1080

aacccagacc tcaaagaccg catggtggag caattcaagg agctccatca catctggcag 1140

tcccagcagc ggttgcagcc tgtggcccag gcccctcctg gagcaggcct tggtgttggc 1200

caggggccct ggcctatgca cccagctggc atgcaataa 1239

<210> 28

<211> 444

<212> DNA

<213> Homo sapiens

<400> 28

atgaaccagt ccatcccagt ggctcccacc ccaccccgcc gcgtgcggct gaagccctgg 60

ctggtggccc aggtgaacag ctgccagtac ccagggcttc aatgggtcaa cggggaaaag 120

aaattattct gcatcccctg gaggcatgcc acaaggcatg gtcccagcca ggacggagat 180

aacaccatct tcaaggcctg ggccaaggag acagggaaat acaccgaagg cgtggatgaa 240

gccgatccgg ccaagtggaa ggccaacctg cgctgtgccc ttaacaagag ccgggacttc 300

cgcctcatct acgacggggcc ccgggacatg ccacctcagc cctacaagat ctacgagact 360

cccagccccc tgaggattac tcttttggtg caggagagga ggaggaagaa gaggaagagc 420

tgcagaggat gttgccaagc ctga 444

<210> 29

<211> 1494

<212> DNA

<213> Mus musculus

<400> 29

atgaatcata gtgcacccgg gatccctcct cctccaagac gagtacgcct caagccctgg 60

ttggtagctc aagtcaactc atgccaatac cctgggcttc agtgggtgaa cggtgagaag 120

aaattgtttt atatcccatg gcgacacgca acaagacatg gcccatcaca ggatggagat 180

aacaccatat ttaaggcatg ggcaaaggaa acaggaaagt acactgagg cgttgatgag 240

gccgatcctg caaaatggaa agcaaatttg cgatgcgctc tcaataaatc acgagatttc 300

caactctttt acgacggccc aagggacatg ccaccacaac cttataaaat ctacgaggta 360

tgttccaacg gtccagcccc aactgaatcc cagcctactg acgactatgt ccttggagaa 420

gaggaggaag aagaagagga ggaacttcag cggatgttgc ctgggttgtc cataactgag 480

cctgccttgc caggaccccc taatgcacca tactcccttc ccaaagaaga tacaaaatgg 540

ccccccgcat tgcaaccccc cgttggtttg ggaccacctg tgcccgaccc aaatctcttg 600

gccccaccaa gcggtaaccc agccggattt cgacaacttc tgcccgaagt ccttgagcca 660

ggtcccttgg cctcttctca gccccctaca gaacctctgc tccccgatct cttgatatct 720

ccccacatgc ttcccttgac tgatttggag ataaaatttc agtatcgcgg ccgagctccc 780

agaacactga ctatatcaaa tccccaaggt tgccgcctgt tttacagtca gttggaggca 840

actcaggagc aagtagagct ctttggggcca gttactctgg agcaggtgag attccctagt 900

ccagaggaca taccaagcga taagcaaaga ttttacacaa atcaacttct ggatgtactt 960

gatcgaggtt tgatccttca gttgcagggc caagatttgt atgccattcg actctgtcaa 1020

tgcaaggtat tttggagcgg cccatgtgcc cttgctcatg gcagctgccc taatcccatc 1080

caaagagaag taaagactaa acttttcagc ctggaacaat ttctcaacga actcattctg 1140

tttcaaaaag gtcagaccaa cacaccccct cctttcgaga ttttcttctg cttcggcgaa 1200

gagtggcctg atgtgaagcc ccgcgaaaaa aagcttatca ccgttcaagt ggtacccgtc 1260

gcagccaggc tccttcttga aatgtttagc ggtgaactct catggtccgc tgacagtatc 1320

cggctccaaa tatcaaaccc tgatcttaaa gaccacatgg tagaacagtt taaagaactc 1380

caccacctgt ggcaatccca acagcagctc cagccaatgg ttcaagctcc tccagtcgct 1440

gggctggacg cctcacaagg accctggccc atgcaccccg tcgggatgca gtaa 1494

<210> 30

<211> 978

<212> DNA

<213> Homo sapiens

<400> 30

atgcccatca ctcggatgcg catgagaccc tggctagaga tgcagattaa ttccaaccaa 60

atcccggggc tcatctggat taataaagag gagatgatct tccagatccc atggaagcat 120

gctgccaagc atggctggga catcaacaag gatgcctgtt tgttccggag ctgggccatt 180

cacacaggcc gatacaaagc aggggaaaag gagccagatc ccaagacgtg gaaggccaac 240

tttcgctgtg ccatgaactc cctgccagat atcgaggagg tgaaagacca gagcaggaac 300

aagggcagct cagctgtgcg agtgtaccgg atgcttccac ctctcaccaa gaaccagaga 360

aaagaaagaa agtcgaagtc cagccgagat gctaagagca aggccaagag gaagtcatgt 420

ggggattcca gccctgatac cttctctgat ggactcagca gctccactct gcctgatgac 480

cacagcagct acacagttcc aggctacatg caggacttgg aggtggagca ggccctgact 540

ccagcactgt cgccatgtgc tgtcagcagc actctccccg actggcacat cccagtggaa 600

gttgtgccgg acagcaccag tgatctgtac aacttccagg tgtcacccat gccctccacc 660

tctgaagcta caacagatga ggatgaggaa gggaaattac ctgaggacat catgaagctc 720

ttggagcagt cggagtggca gccaacaaac gtggatggga aggggtacct actcaatgaa 780

cctggagtcc agcccacctc tgtctatgga gactttagct gtaaggagga gccagaaatt 840

gacagcccag ggggggatat tgggctgagt ctacagcgtg tcttcacaga tctgaagaac 900

atggatgcca cctggctgga cagcctgctg accccagtcc ggttgccctc catccaggcc 960

attccctgtg caccgtag 978

<210> 31

<211> 1284

<212> DNA

<213> Homo sapiens

<400> 31

atgggaaccc caaagccacg gatcctgccc tggctggtgt cgcagctgga cctggggcaa 60

ctggagggcg tggcctgggt gaacaagagc cgcacgcgct tccgcatccc ttggaagcac 120

ggcctacggc aggatgcaca gcaggaggat ttcggaatct tccaggcctg ggccgaggcc 180

actggtgcat atgttcccgg gagggataag ccagacctgc caacctggaa gaggaatttc 240

cgctctgccc tcaaccgcaa agaagggttg cgtttagcag aggaccggag caaggaccct 300

cacgacccac ataaaatcta cgagtttgtg aactcaggag ttggggactt ttcccagcca 360

gacacctctc cggacaccaa tggtggaggc agtacttctg atacccagga agacattctg 420

gatgagttac tgggtaacat ggtgttggcc ccactcccag atccgggacc cccaagcctg 480

gctgtagccc ctgagccctg ccctcagccc ctgcggagcc ccagcttgga caatcccact 540

cccttcccaa acctggggcc ctctgagaac ccactgaagc ggctgttggt gccgggggaa 600

gagtgggagt tcgaggtgac agccttctac cggggccgcc aagtcttcca gcagaccatc 660

tcctgcccgg agggcctgcg gctggtgggg tccgaagtgg gagacaggac gctgcctgga 720

tggccagtca cactgccaga ccctggcatg tccctgacag acaggggagt gatgagctac 780

gtgaggcatg tgctgagctg cctgggtggg ggactggctc tctggcgggc cgggcagtgg 840

ctctgggccc agcggctggg gcactgccac acatactggg cagtgagcga ggagctgctc 900

cccaacagcg ggcatgggcc tgatggcgag gtccccaagg acaaggaagg aggcgtgttt 960

gacctggggc ccttcattgt agatctgatt accttcacgg aaggaagcgg acgctcacca 1020

cgctatgccc tctggttctg tgtgggggag tcatggcccc aggaccagcc gtggaccaag 1080

aggctcgtga tggtcaaggt tgtgcccacg tgcctcaggg ccttggtaga aatggcccgg 1140

gtagggggtg cctcctccct ggagaatact gtggacctgc acatttccaa cagccaccca 1200

ctctccctca cctccgacca gtacaaggcc tacctgcagg acttggtgga gggcatggat 1260

ttccaggggcc ctggggagag ctga 1284

<210> 32

<211> 1512

<212> DNA

<213> Homo sapiens

<400> 32

atggccttgg ctcctgagag ggcagcccca cgcgtgctgt tcggagagtg gctccttgga 60

gagatcagca gcggctgcta tgaggggctg cagtggctgg acgaggcccg cacctgtttc 120

cgcgtgccct ggaagcactt cgcgcgcaag gacctgagcg aggccgacgc gcgcatcttc 180

aaggcctggg ctgtggcccg cggcaggtgg ccgcctagca gcaggggagg tggcccgccc 240

cccgaggctg agactgcgga gcgcgccggc tggaaaacca acttccgctg cgcactgcgc 300

agcacgcgtc gcttcgtgat gctgcgggat aactcggggg acccggccga cccgcacaag 360

gtgtacgcgc tcagccggga gctgtgctgg cgagaaggcc caggcacgga ccagactgag 420

gcagaggccc ccgcagctgt cccaccacca cagggtgggc ccccagggcc attcctggca 480

cacacacatg ctggactcca agccccaggc cccctccctg ccccagctgg tgacaagggg 540

gacctcctgc tccaggcagt gcaacagagc tgcctggcag accatctgct gacagcgtca 600

tggggggcag atccagtccc aaccaaggct cctggagagg gacaagaagg gcttcccctg 660

actggggcct gtgctggagg cccagggctc cctgctgggg agctgtacgg gtgggcagta 720

gagacgaccc ccagccccgg gccccagccc gcggcactaa cgacaggcga ggccgcggcc 780

ccagagtccc cgcaccaggc agagccgtac ctgtcaccct ccccaagcgc ctgcaccgcg 840

gtgcaagagc ccagcccagg ggcgctggac gtgaccatca tgtacaaggg ccgcacggtg 900

ctgcagaagg tggtgggaca cccgagctgc acgttcctat acggcccccc agacccagct 960

gtccgggcca cagaccccca gcaggtagca ttccccagcc ctgccgagct cccggaccag 1020

aagcagctgc gctacacgga ggaactgctg cggcacgtgg cccctgggtt gcacctggag 1080

cttcggggggc cacagctgtg ggcccggcgc atgggcaagt gcaaggtgta ctgggaggtg 1140

ggcggacccc caggctccgc cagcccctcc accccagcct gcctgctgcc tcggaactgt 1200

gacaccccca tcttcgactt cagagtcttc ttccaagagc tggtggaatt ccgggcacgg 1260

cagcgccgtg gctccccacg ctataccatc tacctgggct tcgggcagga cctgtcagct 1320

gggaggccca aggagaagag cctggtcctg gtgaagctgg aaccctggct gtgccgagtg 1380

cacctagagg gcacgcagcg tgagggtgtg tcttccctgg atagcagcag cctcagcctc 1440

tgcctgtcca gcgccaacag cctctatgac gacatcgagt gcttccttat ggagctggag 1500

cagcccgcct ag 1512

<210> 33

<211> 1281

<212> DNA

<213> Homo sapiens

<400> 33

atgtgtgacc ggaatggtgg tcggcggctt cgacagtggc tgatcgagca gattgacagt 60

agcatgtatc caggactgat ttgggagaat gaggagaaga gcatgttccg gatcccttgg 120

aaacacgctg gcaagcaaga ttataatcag gaagtggatg cctccatttt taaggcctgg 180

gcagttttta aagggaagtt taaagaaggg gacaaagctg aaccagccac ttggaagacg 240

aggttacgct gtgctttgaa taagagccca gattttgagg aagtgacgga ccggtcccaa 300

ctggacattt ccgagccata caaagtttac cgaattgttc ctgaggaaga gcaaaaatgc 360

aaactaggcg tggcaactgc tggctgcgtg aatgaagtta cagagatgga gtgcggtcgc 420

tctgaaatcg acgagctgat caaggagcct tctgtggacg attacatggg gatgatcaaa 480

aggagccctt ccccgccgga ggcctgtcgg agtcagctcc ttccagactg gtgggcgcag 540

cagcccagca caggcgtgcc gctggtgacg gggtacacca cctacgacgc gcaccattca 600

gcattctccc agatggtgat cagcttctac tatggggggca agctggtggg ccaggccacc 660

accacctgcc ccgagggctg ccgcctgtcc ctgagccagc ctgggctgcc cggcaccaag 720

ctgtatgggc ccgagggcct ggagctggtg cgcttcccgc cggccgacgc catccccagc 780

gagcgacaga ggcaggtgac gcggaagctg ttcgggcacc tggagcgcgg ggtgctgctg 840

cacagcagcc ggcagggcgt gttcgtcaag cggctgtgcc agggccgcgt gttctgcagc 900

ggcaacgccg tggtgtgcaa aggcaggccc aacaagctgg agcgtgatga ggtggtccag 960

gtcttcgaca ccagccagtt cttccgagag ctgcagcagt tctataacag ccaggggccgg 1020

cttcctgacg gcagggtggt gctgtgcttt ggggaagagt ttccggatat ggcccccttg 1080

cgctccaaac tcattctcgt gcagattgag cagctgtatg tccggcaact ggcagaagag 1140

gctgggaaga gctgtggagc cggctctgtg atgcaggccc ccgaggagcc gccgccagac 1200

caggtcttcc ggatgtttcc agatatttgt gcctcacacc agagatcatt tttcagagaa 1260

aaccaacaga tcaccgtcta a 1281

<210> 34

<211> 990

<212> DNA

<213> Mus musculus

<400> 34

atgccaatca ctcgaatgcg gatgagaccc tggctagaga tgcagattaa ttccaaccaa 60

atcccagggc tgatctggat caataaagaa gagatgatct tccagattcc atggaagcac 120

gctgctaagc acggctggga catcaacaag gatgcctgtc tgttccggag ctgggccatt 180

cacacaggcc gatacaaagc aggagaaaaa gagccagatc ccaagacatg gaaggcaaac 240

ttccgttgtg ccatgaactc cctgccagac atcgaggaag tgaaggatca gagtaggaac 300

aagggcagct ctgctgtgcg ggtgtaccgg atgctgccac ccctcaccag gaaccagagg 360

aaagagagaa agtccaagtc cagccgagac actaagagca aaaccaagag gaagctgtgt 420

ggagatgtta gcccggacac tttctctgat ggactcagca gctctaccct acctgatgac 480

cacagcagtt acaccactca gggctacctg ggtcaggact tggatatgga aagggacata 540

actccagcac tgtcaccgtg tgtcgtcagc agcagtctct ctgagtggca tatgcagatg 600

gacattatac cagatagcac cactgatctg tataacctac aggtgtcacc catgccttcc 660

acctccgaag ccgcaacaga cgaggatgag gaagggaaga tagccgaaga ccttatgaag 720

ctctttgaac agtctgagtg gcagccgaca cacatcgatg gcaagggata cttgctcaat 780

gagccaggga cccagctctc ttctgtctat ggagacttca gctgcaaaga ggaaccagag 840

attgacagcc ctcgagggga cattgggata ggcatacaac atgtcttcac ggagatgaag 900

aatatggact ccatcatgtg gatggacagc ctgctgggca actctgtgag gctgccgccc 960

tctattcagg ccattccttg tgcaccatag 990

<210> 35

<211> 1260

<212> DNA

<213> Mus musculus

<400> 35

atggaaaccc cgaaaccgcg gattttgccc tggctggtgt cacagctgga cctggggcag 60

ctggaaggcg tggcctggct ggacgagagc cgaacgaggt tcaggatccc gtggaagcat 120

ggcctacggc aggacgcaca gatggctgac tttggcatct tccaggcctg ggcagaagcc 180

agtggtgcct acaccccggg gaaggataag ccggacgtgt caacctggaa gaggaatttc 240

cggtcagccc tgaaccggaa agaagtgttg cggttagctg ctgacaatag caaggaccct 300

tatgaccctc ataaagtgta tgagtttgtg actccagggg cgcgggactt cgtacatctg 360

ggtgcctctc ctgacaccaa tggcaaaagc agcctgcctc actcccagga aaacctaccg 420

aagttatttg atggcctgat cttggggccc ctcaaagatg aggggtcctc agatctggct 480

attgtttctg atccttctca acaactgcca agccccaatg tgaacaactt cctaaaccct 540

gcaccccaag aaaatccact gaagcagctg ctagctgagg aacaatggga gttcgaggtg 600

accgccttct accgaggccg ccaggtcttc cagcagacac tcttttgccc ggggggcctg 660

cggctggtgg gcagcacagc tgacatgaca ctgccctggc agccagtcac cctgcccgat 720

cctgaggggt ttctgacgga caagcttgtg aaggagtacg tggggcaggt gctcaaaggg 780

ctgggcaatg ggctggcact gtggcaggct gggcagtgcc tctgggccca gcgcctaggc 840

cactcccacg ccttctgggc tctgggggag gagctgcttc cagacagtgg gcgaggggcct 900

gatggagagg tccacaagga caaggacgga gccgtgttcg acctcaggcc cttcgtggca 960

gatctgattg ccttcatgga aggaagtgga cactccccac gctacactct gtggttctgc 1020

atgggggaaa tgtggcccca ggaccagcca tgggtcaaga ggcttgtgat ggtcaaggtt 1080

gttcctacat gtcttaagga gctgttagag atggcccggg aagggggagc ctcttcactg 1140

aaaaccgtgg acttgcacat ctccaacagc cagcctatct cccttacctc tgaccagtac 1200

aaggcctacc tccaggactt ggtggaggac atggacttcc aggccactgg aaatatctga 1260

<210> 36

<211> 1374

<212> DNA

<213> Mus musculus

<400> 36

atggctgaag tgaggggggt ccagcgagtg ctgtttggag actggctatt gggggaggtc 60

agcagcggcc agtacgaggg gctgcagtgg ctgaacgagg ctcgcacagt cttccgcgta 120

ccctggaagc atttcggtcg tagggatctg gatgaagaag atgcacagat cttcaaggcc 180

tgggctgtgg cccgagggag gtggccacct agtggagtta acctgccacc cccagaggct 240

gaggctgctg agcgaagaga gcgaagaggc tggaagacca acttccgctg tgcactccac 300

agcacagggc gttttatctt gcgccaagac aattcagggg atccagttga tccgcataag 360

gtgtacgaac ttagccggga gcttggatct actgtgggcc cagccacgga aaatagggaa 420

gaagtgagcc tcagcaatgc tctgcccaca cagggtgtgt ccccaggatc atttctggca 480

agagaaaatg ctgggctcca aaccccaagc cctctgcttt ctagtgatgc cggggacctc 540

ttgcttcagg ttctgcagta cagccacata ctggaatccg agtctggggc agaccccgtc 600

ccaccacagg ctcctggcca ggagcaagac cgtgtttacg aggaacccta tgcagcatgg 660

caggtggaag ctgtccccag tcccaggcct caacagccag ctctcaccga gcgcagcctt 720

gggttcctgg atgtgaccat catgtacaag ggccgcacag tgctacaggc agtggtgggg 780

caccccagat gcgtgttcct gtacagcccc atggccccag cagtaagaac ttcagagccc 840

cagccggtga tctttcccag tcctgctgag ctcccagatc agaagcagct gcactacaca 900

gagacgcttc tccagcatgt gtctcccggc cttcagctgg agcttcgagg accgtcactg 960

tgggccctgc gtatgggcaa gtgcaaggtg tactgggagg taggcagccc tatgggcact 1020

accggcccct ccaccccacc ccagctgctg gagcgcaacc gccacacccc catcttcgac 1080

ttcagcactt tcttccgaga actggaggag tttcgggctc ggaggcggca agggtcacca 1140

cactacacca tctacctggg ttttgggcaa gacttgtcag cagggaggcc caaggagaag 1200

accctgatcc tggtgaagct ggagccatgg gtatgcaagg catacctgga gggcgtgcag 1260

cgtgaggtg tgtcctccct ggacagcagc agtctcggct tgtgcttgtc tagcaccaac 1320

agtctctacg aagacatcga acacttcctc atggacctgg gtcagtggcc ttga 1374

<210> 37

<211> 1578

<212> DNA

<213> Mus musculus

<400> 37

atggccgaag ttcgaggagt acagcgcgtg ctgtttgggg actggttgct tggtgaagtc 60

tcttctggtc agtatgaagg cctgcaatgg cttaatgagg cacgcacagt ttttcgagtg 120

ccatggaaac acttcggtag gcgcgatctc gacgaagagg atgcccagat tttcaaggca 180

tgggcagtcg cacggggcag gtggccccct tcaggcgtaa atttgccccc cccagaggct 240

gaagctgctg aacgcagaga acgccgggga tggaagacta actttcgatg tgcccttcac 300

agtacaggca ggttcatctt gcggcaggat aatagtggcg accctgtaga cccacacaag 360

gtttatgagc tgagccggga gcttggctca acagtcggtc ctgcaaccga gaacagagaa 420

gaggtgtcct tgtctaacgc cctcccaact cagggtgtgt ctcccggtag cttcctggca 480

cgcgaaaacg ctggactcca aaccccctcc ccactgttgt ccagtgatgc cggtgatctt 540

ctccttcagg tgctccaata ctcccatata ctggagagcg agtcaggggc tgatcccgtg 600

ccccctcaag ctcctggaca ggaacaagat cgcgtctacg aggagccata tgctgcctgg 660

caggtcgagg ctgtgccatc acctcggcct caacagcccg ctctcaccga gcgctcactt 720

gggtttttgg atgtcactaa acttttcgac ggcctgatac ttggcccatt gaaggacgag 780

ggatcatccg atcttgccat agtaagtgac ccatcacagc agttgccctc accaaacgtc 840

aacaacttcc tcaatccagc tccccaggag aaccccctca aacagcttct cgcagaagag 900

caatgggagt ttgaggtgac tgctttctat agaggtaggc aggtgttcca acaaactctg 960

ttttgccccg gaggtctgcg ccttgtaggt agcaccgcag acatgacact tccctggcaa 1020

cctgtgacac ttcccgatcc tgagggattt ctcacagata aactcgttaa ggaatatgtg 1080

gggcaagtac tcaaaggtct gggcaatggg ttggcccttt ggcaagctgg tcaatgtctc 1140

tgggctcaac gactcggggca ctcacatgct ttttgggctc ttggcgagga gctgctcccc 1200

gacagcgggc gcggacctga cggggaggtt cataaggaca aagacggcgc cgtatttgat 1260

cttagaccct tcgtggcaga tctgatcgct ttcatggaag gatcaggtca tagccccagg 1320

tacacacttt ggttttgcat gggtgaaatg tggcctcagg accaaccttg ggtcaagcgc 1380

ttggtcatgg ttaaggtggt tcccacttgc ctcaaagagt tgttggagat ggctagggaa 1440

ggtggggctt cctcactgaa aaccgtagat ctccacattg ataatgatca gcctatagat 1500

ttggacgacg accaatacaa agcttatctc caggacctgg ttgaagatat ggactttcag 1560

gctacaggta acatctaa 1578

<210> 38

<211> 1275

<212> DNA

<213> Mus musculus

<400> 38

atgtgtgaca ggaacggcgg tagaagactg agacagtggc tgatcgagca aattgacagc 60

tcaatgtacc ctgggttgat atgggaaaac gatgaaaaga caatgttcag aataccctgg 120

aagcacgctg gaaagcagga ttacaaccag gaagtggacg ccagtatttt taaggcttgg 180

gctgtcttca aagggaagtt taaagagggc gacaaagcag agccagcaac ctggaaaacc 240

cgcttgaggt gtgcactcaa taagtcaccc gacttcgagg aagtcactga ccgcagtcaa 300

ttggacatat cagaaccata caaagtctac aggatagtcc ccgaagaaga gcagaaatgc 360

aaactcggtg tagcacctgc tggctgtatg agtgaagtgc ctgaaatgga atgcggcaga 420

tcagaaatcg aagaactcat aaaagaacca agtgtagatg agtatatggg aatgaccaaa 480

agatccccat cccccccaga agcctgtcgg agccaaatct tgcctgactg gtgggtacag 540

caaccctccg ccggacttcc ccttgtgaca ggctatgccg cttacgatac tcatcacagc 600

gcttttagcc agatggttat ttccttctat tatggaggaa aactggtcgg ccaagccaca 660

accacctgcc tcgaggggtg tcgcttgagt ttgagtcaac ccggtcttcc caaactctat 720

ggccccgatg ggcttgaacc tgtctgcttt cccactgctg atactattcc ctcagagaga 780

caacgacaag tcacccgaaa attgtttggc cacctcgaga ggggagtact cttgcactct 840

aacaggaagg gtgtctttgt gaaacgcctc tgtcaaggta gggtattctg ttctggaaat 900

gcagttgttt gcaaaggcag gcctaacaaa ctggaacggg atgaagtcgt acaagtgttc 960

gataccaatc agtttattcg ggagttgcag cagttttacg ctacacaaag tcgcctccct 1020

gacagtcggg ttgtgttgtg cttcggggag gagtttcccg acactgtacc cctccgaagc 1080

aaactcatac tggtacaggt agaacaactt tatgccaggc aactggtgga agaggccggt 1140

aagtcctgtg gcgcaggatc cctgatgcca gccctggaag agccccagcc tgaccaagca 1200

tttaggatgt ttcccgacat ttgtacctca caccagaggc cttttttccg cgaaaaccag 1260

cagataaccg tgtaa 1275

<210> 39

<211> 1275

<212> DNA

<213> Mus musculus

<400> 39

atgtgtgaca ggaacggcgg tagaagactg agacagtggc tgatcgagca aattgacagc 60

tcaatgtacc ctgggttgat atgggaaaac gatgaaaaga caatgttcag aataccctgg 120

aagcacgctg gaaagcagga ttacaaccag gaagtggacg ccagtatttt taaggcttgg 180

gctgtcttca aagggaagtt taaagagggc gacaaagcag agccagcaac ctggaaaacc 240

cgcttgaggt gtgcactcaa taagtcaccc gacttcgagg aagtcactga ccgcagtcaa 300

ttggacatat cagaaccata caaagtctac aggatagtcc ccgaagaaga gcagaaatgc 360

aaactcggtg tagcacctgc tggctgtatg agtgaagtgc ctgaaatgga atgcggcaga 420

tcagaaatcg aagaactcat aaaagaacca agtgtagatg agtatatggg aatgaccaaa 480

agatccccat cccccccaga agcctgtcgg agccaaatct tgcctgactg gtgggtacag 540

caaccctccg ccggacttcc ccttgtgaca ggctatgccg cttacgatac tcatcacagc 600

gcttttagcc agatggttat ttccttctat tatggaggaa aactggtcgg ccaagccaca 660

accacctgcc tcgaggggtg tcgcttgagt ttgagtcaac ccggtcttcc caaactctat 720

ggccccgatg ggcttgaacc tgtctgcttt cccactgctg atactattcc ctcagagaga 780

caacgacaag tcacccgaaa attgtttggc cacctcgaga ggggagtact cttgcactct 840

aacaggaagg gtgtctttgt gaaacgcctc tgtcaaggta gggtattctg ttctggaaat 900

gcagttgttt gcaaaggcag gcctaacaga ctggaacggg atgaagtcgt acaagtgttc 960

gataccaatc agtttattcg ggagttgcag cagttttacg ctacacaaag tcgcctccct 1020

gacagtcggg ttgtgttgtg cttcggggag gagtttcccg acactgtacc cctccgaagc 1080

aaactcatac tggtacaggt agaacaactt tatgccaggc aactggtgga agaggccggt 1140

aagtcctgtg gcgcaggatc cctgatgcca gccctggaag agccccagcc tgaccaagca 1200

tttaggatgt ttcccgacat ttgtacctca caccagaggc cttttttccg cgaaaaccag 1260

cagataaccg tgtaa 1275

<210> 40

<211> 2271

<212> DNA

<213> Homo sapiens

<400> 40

atgagctggt caccttccct gacaacgcag acatgtgggg cctgggaaat gaaagagcgc 60

cttgggacag ggggatttgg aaatgtcatc cgatggcaca atcaggaaac aggtgagcag 120

attgccatca agcagtgccg gcaggagctc agcccccgga accgagagcg gtggtgcctg 180

gagatccaga tcatgagaag gctgacccac cccaatgtgg tggctgcccg agatgtccct 240

gaggggatgc agaacttggc gcccaatgac ctgcccctgc tggccatgga gtactgccaa 300

ggagaggagatc tccggaagta cctgaaccag tttgagaact gctgtggtct gcgggaaggt 360

gccatcctca ccttgctgag tgacattgcc tctgcgctta gataccttca tgaaaacaga 420

atcatccatc gggatctaaa gccagaaaac atcgtcctgc agcaaggaga acagaggtta 480

atacacaaaa ttattgacct aggatatgcc aaggagctgg atcagggcag tctttgcaca 540

tcattcgtgg ggaccctgca gtacctggcc ccagagctac tggagcagca gaagtacaca 600

gtgaccgtcg actactggag cttcggcacc ctggcctttg agtgcatcac gggcttccgg 660

cccttcctcc ccaactggca gcccgtgcag tggcattcaa aagtgcggca gaagagtgag 720

gtggacattg ttgttagcga agacttgaat ggaacggtga agttttcaag ctctttaccc 780

taccccaata atcttaacag tgtcctggct gagcgactgg agaagtggct gcaactgatg 840

ctgatgtggc acccccgaca gaggggcacg gatcccacgt atgggcccaa tggctgcttc 900

aaggccctgg atgacatctt aaacttaaag ctggttcata tcttgaacat ggtcacgggc 960

accatccaca cctaccctgt gacagaggat gagagtctgc agagcttgaa ggccagaatc 1020

caacaggaca cgggcatccc agaggaggac caggagctgc tgcaggaagc gggcctggcg 1080

ttgatccccg ataagcctgc cactcagtgt atttcagacg gcaagttaaa tgaggccac 1140

acattggaca tggatcttgt ttttctcttt gacaacagta aaatcaccta tgagactcag 1200

atctccccac ggccccaacc tgaaagtgtc agctgtatcc ttcaagagcc caagaggaat 1260

ctcgccttct tccagctgag gaaggtgtgg ggccaggtct ggcacagcat ccagaccctg 1320

aaggaagatt gcaaccggct gcagcaggga cagcgagccg ccatgatgaa tctcctccga 1380

aacaacagct gcctctccaa aatgaagaat tccatggctt ccatgtctca gcagctcaag 1440

gccaagttgg atttcttcaa aaccagcatc cagattgacc tggagaagta cagcgagcaa 1500

accgagtttg ggatcacatc agataaactg ctgctggcct ggaggggaaat ggagcaggct 1560

gtggagctct gtgggcggga gaacgaagtg aaactcctgg tagaacggat gatggctctg 1620

cagaccgaca ttgtggactt acagaggagc cccatgggcc ggaagcaggg gggaacgctg 1680

gacgacctag aggagcaagc aagggagctg tacaggagac taagggaaaa acctcgagac 1740

cagcgaactg agggtgacag tcaggaaatg gtacggctgc tgcttcaggc aattcagagc 1800

ttcgagaaga aagtgcgagt gatctatacg cagctcagta aaactgtggt ttgcaagcag 1860

aaggcgctgg aactgttgcc caaggtggaa gaggtggtga gcttaatgaa tgaggatgag 1920

aagactgttg tccggctgca ggagaagcgg cagaaggagc tctggaatct cctgaagatt 1980

gcttgtagca aggtccgtgg tcctgtcagt ggaagcccgg atagcatgaa tgcctctcga 2040

cttagccagc ctgggcagct gatgtctcag ccctccacgg cctccaacag cttacctgag 2100

ccagccaaga agagtgaaga actggtggct gaagcacata acctctgcac cctgctagaa 2160

aatgccatac aggacactgt gagggaacaa gaccagagtt tcacggccct agactggagc 2220

tggttacaga cggaagaaga agagcacagc tgcctggagc aggcctcatg a 2271

<210> 41

<211> 2265

<212> DNA

<213> Homo sapiens

<400> 41

atgttttcag gggggtgtca tagccccggg tttggccgcc ccagccccgc cttccccgcc 60

ccggggagcc cgccccctgc cccgcgtccc tgccgacagg aaacaggtga gcagattgcc 120

atcaagcagt gccggcagga gctcagcccc cggaaccgag agcggtggtg cctggagatc 180

cagatcatga gaaggctgac ccaccccaat gtggtggctg cccgagatgt ccctgagggg 240

atgcagaact tggcgcccaa tgacctgccc ctgctggcca tggagtactg ccaaggagga 300

gatctccgga agtacctgaa ccagtttgag aactgctgtg gtctgcggga aggtgccatc 360

ctcaccttgc tgagtgacat tgcctctgcg cttagatacc ttcatgaaaa cagaatcatc 420

catcgggatc taaagccaga aaacatcgtc ctgcagcaag gagaacagag gttaatacac 480

aaaattattg acctaggata tgccaaggag ctggatcagg gcagtctttg cacatcattc 540

gtggggaccc tgcagtacct ggccccagag ctactggagc agcagaagta cacagtgacc 600

gtcgactact ggagcttcgg caccctggcc tttgagtgca tcacgggctt ccggcccttc 660

ctccccaact ggcagcccgt gcagtggcat tcaaaagtgc ggcagaagag tgaggtggac 720

attgttgtta gcgaagactt gaatggaacg gtgaagtttt caagctcttt accctacccc 780

aataatctta acagtgtcct ggctgagcga ctggagaagt ggctgcaact gatgctgatg 840

tggcaccccc gacagagggg cacggatccc acgtatgggc ccaatggctg cttcaaggcc 900

ctggatgaca tcttaaactt aaagctggtt catatcttga acatggtcac gggcaccatc 960

cacacctacc ctgtgacaga ggatgagagt ctgcagagct tgaaggccag aatccaacag 1020

gacacgggca tcccagagga ggaccaggag ctgctgcagg aagcgggcct ggcgttgatc 1080

cccgataagc ctgccactca gtgtatttca gacggcaagt taaatgaggg ccacacattg 1140

gacatggatc ttgtttttct ctttgacaac agtaaaatca cctatgagac tcagatctcc 1200

ccacggcccc aacctgaaag tgtcagctgt atccttcaag agcccaagag gaatctcgcc 1260

ttcttccagc tgaggaaggt gtggggccag gtctggcaca gcatccagac cctgaaggaa 1320

gattgcaacc ggctgcagca gggacagcga gccgccatga tgaatctcct ccgaaacaac 1380

agctgcctct ccaaaatgaa gaattccatg gcttccatgt ctcagcagct caaggccaag 1440

ttggatttct tcaaaaccag catccagatt gacctggaga agtacagcga gcaaaccgag 1500

tttgggatca catcagataa actgctgctg gcctggaggg aaatggagca ggctgtggag 1560

ctctgtgggc gggagaacga agtgaaactc ctggtagaac ggatgatggc tctgcagacc 1620

gacattgtgg acttacagag gagccccatg ggccggaagc aggggggaac gctggacgac 1680

ctagaggagc aagcaaggga gctgtacagg agactaaggg aaaaacctcg agaccagcga 1740

actgagggtg acagtcagga aatggtacgg ctgctgcttc aggcaattca gagcttcgag 1800

aagaaagtgc gagtgatcta tacgcagctc agtaaaactg tggtttgcaa gcagaaggcg 1860

ctggaactgt tgcccaaggt ggaagaggtg gtgagcttaa tgaatgagga tgagaagact 1920

gttgtccggc tgcaggagaa gcggcagaag gagctctgga atctcctgaa gattgcttgt 1980

agcaaggtcc gtggtcctgt cagtggaagc ccggatagca tgaatgcctc tcgacttagc 2040

cagcctgggc agctgatgtc tcagccctcc acggcctcca acagcttacc tgagccagcc 2100

aagaagagtg aagaactggt ggctgaagca cataacctct gcaccctgct agaaaatgcc 2160

atacaggaca ctgtgaggga acaagaccag agtttcacgg ccctagactg gagctggtta 2220

cagacggaag aagaagagca cagctgcctg gagcaggcct catga 2265

<210> 42

<211> 771

<212> DNA

<213> Homo sapiens

<400> 42

atgagctggt caccttccct gacaacgcag acatgtgggg cctgggaaat gaaagagcgc 60

cttgggacag ggggatttgg aaatgtcatc cgatggcaca atcaggaaac aggtgagcag 120

attgccatca agcagtgccg gcaggagctc agcccccgga accgagagcg gtggtgcctg 180

gagatccaga tcatgagaag gctgacccac cccaatgtgg tggctgcccg agatgtccct 240

gaggggatgc agaacttggc gcccaatgac ctgcccctgc tggccatgga gtactgccaa 300

ggagaggagatc tccggaagta cctgaaccag tttgagaact gctgtggtct gcgggaaggt 360

gccatcctca ccttgctgag tgacattgcc tctgcgctta gataccttca tgaaaacaga 420

atcatccatc gggatctaaa gccagaaaac atcgtcctgc agcaaggaga acagaggtta 480

atacacaaaa ttattgacct aggatatgcc aaggagctgg atcagggcag tctttgcaca 540

tcattcgtgg ggaccctgca gtacctggcc ccagagctac tggagcagca gaagtacaca 600

gtgaccgtcg actactggag cttcggcacc ctggcctttg agtgcatcac gggcttccgg 660

cccttcctcc ccaactggca gcccgtgcag tgcgtaagaa tgtggccggg tacagtggct 720

cactcctgta atcccagcac tttgggaggc cgaggcaggt ggatcagttg a 771

<210> 43

<211> 2094

<212> DNA

<213> Homo sapiens

<400> 43

atgtcatccg atggcacaat caggctgacc caccccaatg tggtggctgc ccgagatgtc 60

cctgagggga tgcagaactt ggcgcccaat gacctgcccc tgctggccat ggagtactgc 120

caaggaggag atctccggaa gtacctgaac cagtttgaga actgctgtgg tctgcgggaa 180

ggtgccatcc tcaccttgct gagtgacatt gcctctgcgc ttagatacct tcatgaaaac 240

agaatcatcc atcgggatct aaagccagaa aacatcgtcc tgcagcaagg agaacagagg 300

ttaatacaca aaattattga cctaggatat gccaaggagc tggatcaggg cagtctttgc 360

acatcattcg tggggaccct gcagtacctg gccccagagc tactggagca gcagaagtac 420

acagtgaccg tcgactactg gagcttcggc accctggcct ttgagtgcat cacgggcttc 480

cggcccttcc tccccaactg gcagcccgtg cagtggcatt caaaagtgcg gcagaagagt 540

gaggtggaca ttgttgttag cgaagacttg aatggaacgg tgaagttttc aagctcttta 600

ccctacccca ataatcttaa cagtgtcctg gctgagcgac tggagaagtg gctgcaactg 660

atgctgatgt ggcacccccg acagaggggc acggatccca cgtatgggcc caatggctgc 720

ttcaaggccc tggatgacat cttaaactta aagctggttc atatcttgaa catggtcacg 780

ggcaccatcc acacctaccc tgtgacagag gatgagagtc tgcagagctt gaaggccaga 840

atccaacagg acacgggcat cccagaggag gaccaggagc tgctgcagga agcgggcctg 900

gcgttgatcc ccgataagcc tgccactcag tgtatttcag acggcaagtt aaatgaggc 960

cacacattgg acatggatct tgtttttctc tttgacaaca gtaaaatcac ctatgagact 1020

cagatctccc cacggcccca acctgaaagt gtcagctgta tccttcaaga gcccaagagg 1080

aatctcgcct tcttccagct gaggaaggtg tggggccagg tctggcacag catccagacc 1140

ctgaaggaag attgcaaccg gctgcagcag ggacagcgag ccgccatgat gaatctcctc 1200

cgaaacaaca gctgcctctc caaaatgaag aattccatgg cttccatgtc tcagcagctc 1260

aaggccaagt tggatttctt caaaaccagc atccagattg acctggagaa gtacagcgag 1320

caaaccgagt ttgggatcac atcagataaa ctgctgctgg cctggaggga aatggagcag 1380

gctgtggagc tctgtgggcg ggagaacgaa gtgaaactcc tggtagaacg gatgatggct 1440

ctgcagaccg acattgtgga cttacagagg agccccatgg gccggaagca ggggggaacg 1500

ctggacgacc tagaggagca agcaagggag ctgtacagga gactaaggga aaaacctcga 1560

gaccagcgaa ctgagggtga cagtcaggaa atggtacggc tgctgcttca ggcaattcag 1620

agcttcgaga agaaagtgcg agtgatctat acgcagctca gtaaaactgt ggtttgcaag 1680

cagaaggcgc tggaactgtt gcccaaggtg gaagaggtgg tgagcttaat gaatgaggat 1740

gagaagactg ttgtccggct gcaggagaag cggcagaagg agctctggaa tctcctgaag 1800

attgcttgta gcaaggtccg tggtcctgtc agtggaagcc cggatagcat gaatgcctct 1860

cgacttagcc agcctgggca gctgatgtct cagccctcca cggcctccaa cagcttacct 1920

gagccagcca agaagagtga agaactggtg gctgaagcac ataacctctg caccctgcta 1980

gaaaatgcca tacaggacac tgtgagggaa caagaccaga gtttcacggc cctagactgg 2040

agctggttac agacggaaga agaagagcac agctgcctgg agcaggcctc atga 2094

<210> 44

<211> 2220

<212> DNA

<213> Mus musculus

<400> 44

atgagctggt caccgtccct cccaacccag acatgtggag cctgggaaat gaaagaacgc 60

ctggggaccg ggggatttgg aaacgtcatc cggtggcaca atcaggcgac aggtgaacag 120

atcgccatca agcaatgccg acaggagctc agcccaaaga acagaccg ctggtgcctc 180

gaaatccaga tcatgagaag gctgaaccat cccaatgtgg tggctgcccg ggatgtccca 240

gaggggatgc agaacctggc acccaatgat ttgccactgc tggccatgga gtactgccaa 300

ggagggagatc tccgaagata cttgaaccag ttcgagaact gctgtggcct gcgggaagga 360

gctgtcctta ccctgctgag tgacatcgca tcggctctta gataccttca cgaaaacaga 420

atcatccatc gagaacctgaa gccagaaaac atcgttctgc agcaaggaga gaaaagatta 480

atacacaaaa ttattgatct aggatatgcc aaggagctgg atcagggcag tctgtgcacg 540

tcatttgtgg ggactctgca atacctggcg ccagagcttc tggagcagca gaagtacacc 600

gtgaccgttg actactggag cttcggcacc ctggccttcg agtgcatcac tggcttccgg 660

cccttcctcc ctaactggca gcctgtgcag tggcactcca aagtccggca gaagagcgaa 720

gtggacatcg ttgttagtga agacttgaat ggagcagtga agttttcaag ttcgctaccc 780

ttccccaata atcttaacag tgtcttggct gaacggctgg agaagtggct gcagctgatg 840

cttatgtggc accctcggca aaggggcacg gatccccagt atggccccaa cggctgcttc 900

agagccctgg atgacatctt gaacttgaag ctggttcatg tcttgaacat ggtcacaggc 960

accgttcaca cataccccgt gacggaggat gagagtctgc agagcttaaa aaccagaatc 1020

caggaagaca cggggatcct gggagacagac caggagctgc tgcaagaggc agggctggtg 1080

ctgctccctg acaagcctgc tactcagtgc atctcagaca gcaagacaaa cgagggcctc 1140

acgttggaca tggatcttgt ttttctcttt gacaacagta aaatcaacta tgagactcag 1200

atcacccccc gaccccaacc ggaaagtgtc agctgtatcc ttcaggagcc caagcggaac 1260

ctctccttct tccagctgag gaaagtgtgg ggccaagtct ggcacagcat cgacgctg 1320

aaggaagact gtaaccggct gcagcaggga cagcgagcag ccatgatgag tctcctccgg 1380

aataacagct gcctctctaa gatgaagaac gccatggcct ccacggccca gcagctcaag 1440

gccaagctgg acttcttcaa aaccagcatc cagatcgacc tggagaagta taaagagcag 1500

accgagtttg ggatcacctc agataaattg ctgctggctt ggcgggagat ggagcaggct 1560

gtggagcagt gtgggcggga gaatgacgtg aagcatctag tagagcggat gatggcactg 1620

cagactgaca ttgtggacct gcagaggagc ccgatgggtc ggaagcaggg gggcaccctg 1680

gatgacctag aggaacaagc gagggagctc taccgaagac tcagggagaa gccaagagac 1740

caaaggacag aaggtgacag ccaggagatg gtacggctgc tgcttcaggc aatccaaagc 1800

tttgagaaga aagttcgggt gatttataca cagctcagta agaccgtggt ttgtaagcag 1860

aaggcactgg agttgctgcc caaggtagaa gaggtagtga gccttatgaa cgaggacgag 1920

aggaccgtgg tccggcttca ggagaagcgg cagaaggaac tctggaacct cctgaagatc 1980

gcctgtagca aagtccgagg tcccgtgagt ggaagcccag acagcatgaa tgtgtctcga 2040

ctcagtcacc ctggtcagct aatgtcccag ccttccagtg cctgtgacag cttacctgaa 2100

tcagacaaga aaagtgaaga actggtggcc gaagcccacg ccctctgctc ccggctagaa 2160

agtgcgctgc aggacactgt gaaggagcaa gacagaagct tcacggtaac cgcctgataa 2220

<210> 45

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR1 of the nanobody that binds CD206

<400> 45

Ser Gly Asn Ile Phe Ser Ile Asn Ala Ile Gly

1 5 10

<210> 46

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR2 of the nanobody that binds CD206

<400> 46

Thr Ile Thr Leu Ser Gly Ser Thr Asn

1 5

<210> 47

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR3 of the nanobody that binds CD206

<400> 47

Asn Thr Tyr Ser Asp Ser Asp Val Tyr Gly Tyr

1 5 10

<210> 48

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR1 of the nanobody that binds CD206

<400> 48

Pro Gly Phe Lys Leu Asp Tyr Tyr Ala Ile Ala

1 5 10

<210> 49

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR2 of the nanobody that binds CD206

<400> 49

Ser Ile Asn Ser Ser Gly Gly Ser Thr

1 5

<210> 50

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR3 of the nanobody that binds CD206

<400> 50

Leu Arg Arg Tyr Tyr Gly Leu Asn Leu Asp Pro Gly Ser Tyr Asp Tyr

1 5 10 15

<210> 51

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR1 of the nanobody that binds CD206

<400> 51

Gly Phe Pro Phe Asn Ile Tyr Pro Met Ser

1 5 10

<210> 52

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR2 of the nanobody that binds CD206

<400> 52

Tyr Ile Ser His Gly Gly Thr Thr Thr

1 5

<210> 53

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR3 of the nanobody that binds CD206

<400> 53

Gly Tyr Ala Arg Leu Met Thr Asp Ser Glu Leu Val

1 5 10

<210> 54

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL1 binding domain, which binds CD163

<400> 54

Ala Ser Gln Ser Val Ser His Asp Val

1 5

<210> 55

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL1 binding domain that binds mesothelin

<400> 55

Arg Ala Ser Glu Gly Ile Tyr His Trp Leu Ala

1 5 10

<210> 56

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL3 binding domain, which binds CD163

<400> 56

Gln Asp Tyr Ser Ser Pro Arg Thr

1 5

<210> 57

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH1 binding domain that binds CD163

<400> 57

Gly Tyr Ser Ile Thr Ser Asp Tyr

1 5

<210> 58

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL2 binding domain that binds mesothelin

<400> 58

Lys Ala Ser Ser Leu Ala Ser

1 5

<210> 59

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH3 binding domain that binds CD163

<400> 59

Cys Val Ser Gly Thr Tyr Tyr Phe Asp Tyr Trp Gly

1 5 10

<210> 60

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL1 binding domain, which binds CD163

<400> 60

Ala Ser Gln Ser Val Ser Ser Asp Val

1 5

<210> 61

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL3 binding domain, which binds mesothelin

<400> 61

Gln Gln Tyr Ser Asn Tyr Pro Leu Thr

1 5

<210> 62

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL3 binding domain, which binds CD163

<400> 62

Gln Asp Tyr Thr Ser Pro Arg Thr

1 5

<210> 63

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Gly-Ser linker

<400> 63

Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly

1 5 10

<210> 64

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH1 binding domain that binds mesothelin

<400> 64

Thr Tyr Tyr Met Gln

1 5

<210> 65

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Gly-Ser linker

<400> 65

Gly Gly Ser Gly Gly Gly Ser Gly

1 5

<210> 66

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL1 binding domain, which binds CD23

<400> 66

Arg Ser Ser Lys Ser Leu Leu Tyr Lys Asp Gly Lys Thr Tyr Leu Asn

1 5 10 15

<210> 67

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL2 binding domain, which binds CD23

<400> 67

Leu Met Ser Thr Arg Ala Ser

1 5

<210> 68

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL3 binding domain, which binds CD23

<400> 68

Gln Gln Leu Val Glu Tyr Pro Phe Thr

1 5

<210> 69

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH1 binding domain that binds CD23

<400> 69

Gly Tyr Trp Met Ser

1 5

<210> 70

<211> 19

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH2 binding domain that binds CD23

<400> 70

Glu Ile Arg Leu Lys Ser Asp Asn Tyr Ala Thr His Tyr Ala Glu Ser

1 5 10 15

Val Lys Gly

<210> 71

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH2 binding domain that binds mesothelin

<400> 71

Val Ile Asn Pro Ser Gly Val Thr Ser Tyr Ala Gln Lys Phe Gln Gly

1 5 10 15

<210> 72

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL1 binding domain, which binds CD38

<400> 72

Ser Ser Asn Ile Gly Asp Asn Tyr

1 5

<210> 73

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH3 binding domain that binds mesothelin

<400> 73

Trp Ala Leu Trp Gly Asp Phe Gly Met Asp Val

1 5 10

<210> 74

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL3 binding domain, which binds CD38

<400> 74

Gln Ser Tyr Asp Ser Ser Leu Ser Gly Ser

1 5 10

<210> 75

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH1 binding domain that binds CD38

<400> 75

Gly Phe Thr Phe Asp Asp Tyr Gly

1 5

<210> 76

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH2 binding domain that binds CD38

<400> 76

Ile Ser Trp Asn Gly Gly Lys Thr

1 5

<210> 77

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH3 binding domain that binds CD38

<400> 77

Ala Arg Gly Ser Leu Phe His Asp Ser Ser Gly Phe Tyr Phe Gly His

1 5 10 15

<210> 78

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL1 binding domain, which binds CD38

<400> 78

Asn Ser Asn Ile Gly Ser Asn Thr

1 5

<210> 79

<211> 232

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL binding domain that binds mesothelin

<400> 79

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Asp Ile Glu Leu Thr Gln Ser Pro Ala Ile Met Ser Ala

20 25 30

Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val

35 40 45

Ser Tyr Met His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg

50 55 60

Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Gly Arg Phe

65 70 75 80

Ser Gly Ser Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile Ser Ser Val

85 90 95

Glu Ala Glu Asp Asp Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Lys His

100 105 110

Pro Leu Thr Phe Gly Ser Gly Thr Lys Val Glu Ile Lys Arg Thr Val

115 120 125

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

130 135 140

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

145 150 155 160

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

165 170 175

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

180 185 190

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

195 200 205

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

210 215 220

Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<210> 80

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL3 binding domain, which binds CD38

<400> 80

Gln Ser Tyr Asp Ser Ser Leu Ser Gly Ser Arg

1 5 10

<210> 81

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH1 binding domain that binds CD38

<400> 81

Gly Phe Thr Phe Asn Asn Tyr Gly

1 5

<210> 82

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH2 binding domain that binds CD38

<400> 82

Ile Ser Tyr Asp Gly Ser Asp Lys

1 5

<210> 83

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH3 binding domain that binds CD38

<400> 83

Ala Arg Val Tyr Tyr Tyr Gly Phe Ser Gly Pro Ser Met Asp Val

1 5 10 15

<210> 84

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL1 binding domain, which binds CD38, CD64, EGFR, PD-1

and TYRP1/gp75

<400> 84

Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala

1 5 10

<210> 85

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL2 binding domain, which binds CD38, EGFR, PD-L1, PD-

1 and TYRP1/gp75

<400> 85

Asp Ala Ser Asn Arg Ala Thr

1 5

<210> 86

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL3 binding domain, which binds CD38

<400> 86

Gln Gln Arg Ser Asn Trp Pro Pro Thr Phe

1 5 10

<210> 87

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH1 binding domain that binds CD38

<400> 87

Ser Phe Ala Met Ser

1 5

<210> 88

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH2 binding domain that binds CD38

<400> 88

Ala Ile Ser Gly Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 89

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH3 binding domain that binds CD38

<400> 89

Asp Lys Ile Leu Trp Phe Gly Glu Pro Val Phe Asp Tyr

1 5 10

<210> 90

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Gly-Ser linker

<400> 90

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 91

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL2 binding domain, which binds CD64

<400> 91

Asp Ala Ser Ser Arg Ala Thr

1 5

<210> 92

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL3 binding domain, which binds CD64

<400> 92

Gln Leu Arg Ser Asn Trp Pro Pro Tyr Thr

1 5 10

<210> 93

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH1 binding domain that binds CD64

<400> 93

Gly Tyr Gly Met His

1 5

<210> 94

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH2 binding domain that binds CD64

<400> 94

Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 95

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH3 binding domain that binds CD64

<400> 95

Asp Thr Gly Asp Arg Phe Phe Asp Tyr

1 5

<210> 96

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> serpin B2, forward primer

<400> 96

actggggcag ttatgacagg 20

<210> 97

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> serpin 2, reverse primer

<400> 97

gatgatcggc cacaaactg 19

<210> 98

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Retnla, direct primer

<400> 98

ttgttccctt ctcatctgca t 21

<210> 99

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Retnla, reverse primer

<400> 99

ccttgacctt attctccacg a 21

<210> 100

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Ccl5, forward primer

<400> 100

cctactccca ctcggtcct 19

<210> 101

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Ccl5, reverse primer

<400> 101

ctgatttctt gggtttgctg t 21

<210> 102

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Ccl11, forward primer

<400> 102

agagctccac agcgcttc 18

<210> 103

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Ccl11, reverse primer

<400> 103

cagcacctgg gaggtgaa 18

<210> 104

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> codon-optimized IRF5 forward primer

<400> 104

tcttaaagac cacatggtag aacagt 26

<210> 105

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> codon-optimized IRF5 reverse primer

<400> 105

agctgctgtt gggattgc 18

<210> 106

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> endogenous IRF5, forward primer

<400> 106

gctgtgccct taacaaaagc 20

<210> 107

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> endogenous IRF5, reverse primer

<400> 107

ggctgaggtg gcatgtct 18

<210> 108

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> GAPD, forward primer

<400> 108

agccacatcg ctcagacac 19

<210> 109

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> GAPD, reverse primer

<400> 109

gcccaatacg accaaatcc 19

<210> 110

<211> 134

<212> PROTEIN

<213> Homo sapiens

<400> 110

Met Asn Thr Glu Met Tyr Gln Thr Pro Met Glu Val Ala Val Tyr Gln

1 5 10 15

Leu His Asn Phe Ser Ile Ser Phe Phe Ser Ser Leu Leu Gly Gly Asp

20 25 30

Val Val Ser Val Lys Leu Asp Asn Ser Ala Ser Gly Ala Ser Val Val

35 40 45

Ala Ile Asp Asn Lys Ile Glu Gln Ala Met Asp Leu Val Lys Asn His

50 55 60

Leu Met Tyr Ala Val Arg Glu Glu Val Glu Ile Leu Lys Glu Gln Ile

65 70 75 80

Arg Glu Leu Val Glu Lys Asn Ser Gln Leu Glu Arg Glu Asn Thr Leu

85 90 95

Leu Lys Thr Leu Ala Ser Pro Glu Gln Leu Glu Lys Phe Gln Ser Cys

100 105 110

Leu Ser Pro Glu Glu Pro Ala Pro Glu Ser Pro Gln Val Pro Glu Ala

115 120 125

Pro Gly Gly Ser Ala Val

130

<210> 111

<211> 405

<212> DNA

<213> Homo sapiens

<400> 111

atgaacaccg aaatgtatca gacccccatg gaggtggcgg tctaccagct gcacaatttc 60

tccatctcct tcttctcttc tctgcttgga ggggatgtgg tttccgttaa gctggacaac 120

agtgcctccg gagccagcgt ggtggccata gacaacaaga tcgaacaggc catggatctg 180

gtgaagaatc atctgatgta tgctgtgaga gaggaggtgg agatcctgaa ggagcagatc 240

cgagagctgg tggagaagaa ctcccagcta gagcgtgaga acaccctgtt gaagaccctg 300

gcaagcccag agcagctgga gaagttccag tcctgtctga gccctgaaga gccagctccc 360

gaatccccac aagtgcccga ggcccctggt ggttctgcgg tgtaa 405

<210> 112

<211> 468

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH binding domain that binds mesothelin

<400> 112

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys

20 25 30

Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe

35 40 45

Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu

50 55 60

Glu Trp Ile Gly Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn

65 70 75 80

Gln Lys Phe Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Asp Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Phe Cys Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp

115 120 125

Gly Ser Gly Thr Pro Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

130 135 140

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

145 150 155 160

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

165 170 175

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

180 185 190

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

195 200 205

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

210 215 220

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

225 230 235 240

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

245 250 255

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

260 265 270

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

275 280 285

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

290 295 300

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

305 310 315 320

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

325 330 335

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

340 345 350

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

355 360 365

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

370 375 380

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

385 390 395 400

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

405 410 415

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

420 425 430

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

435 440 445

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

450 455 460

Ser Pro Gly Lys

465

<210> 113

<211> 232

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL binding domain that binds mesothelin

<400> 113

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu

20 25 30

Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val

35 40 45

Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu

50 55 60

Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe

65 70 75 80

Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu

85 90 95

Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Lys His

100 105 110

Pro Leu Thr Phe Gly Ser Gly Thr Lys Val Glu Ile Lys Arg Thr Val

115 120 125

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

130 135 140

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

145 150 155 160

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

165 170 175

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

180 185 190

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

195 200 205

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

210 215 220

Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<210> 114

<211> 468

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH binding domain that binds mesothelin

<400> 114

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

20 25 30

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe

35 40 45

Thr Gly Tyr Thr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

50 55 60

Glu Trp Met Gly Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn

65 70 75 80

Gln Lys Phe Arg Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser

85 90 95

Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp

115 120 125

Gly Ser Gly Thr Pro Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

130 135 140

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

145 150 155 160

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

165 170 175

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

180 185 190

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

195 200 205

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

210 215 220

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

225 230 235 240

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

245 250 255

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

260 265 270

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

275 280 285

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

290 295 300

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

305 310 315 320

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

325 330 335

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

340 345 350

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

355 360 365

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

370 375 380

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

385 390 395 400

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

405 410 415

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

420 425 430

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

435 440 445

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

450 455 460

Ser Pro Gly Lys

465

<210> 115

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL binding domain that binds MUC16

<400> 115

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Gly Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 116

<211> 450

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH binding domain that binds MUC16

<400> 116

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val His Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Gly Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Gly Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 117

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL1 binding domain that binds MUC16

<400> 117

Ser Glu Asp Ile Tyr Ser Gly

1 5

<210> 118

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL3 binding domain, which binds MUC16

<400> 118

Gly Tyr Ser Tyr Ser Ser Thr Leu

1 5

<210> 119

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH1 binding domain that binds MUC16

<400> 119

Thr Leu Gly Met Gly Val Gly

1 5

<210> 120

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH2 binding domain that binds MUC16

<400> 120

His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ala Leu Lys Ser

1 5 10 15

<210> 121

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH3 binding domain that binds MUC16

<400> 121

Ile Gly Thr Ala Gln Ala Thr Asp Ala Leu Asp Tyr

1 5 10

<210> 122

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL1 binding domain, which binds the folate receptor

<400> 122

Lys Ala Ser Gln Ser Val Ser Phe Ala Gly Thr Ser Leu Met His

1 5 10 15

<210> 123

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL2 binding domain, which binds the folate receptor

<400> 123

Arg Ala Ser Asn Leu Glu Ala

1 5

<210> 124

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL3 binding domain, which binds the folate receptor

<400> 124

Gln Gln Ser Arg Glu Tyr Pro Tyr Thr

1 5

<210> 125

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH1 binding domain that binds the folate receptor

<400> 125

Gly Tyr Phe Met Asn

1 5

<210> 126

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH2 binding domain that binds the folate receptor

<400> 126

Arg Ile His Pro Tyr Asp Gly Asp Thr Phe Tyr Asn Gln Lys Phe Gln

1 5 10 15

Gly

<210> 127

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH3 binding domain that binds the folate receptor

<400> 127

Tyr Asp Gly Ser Arg Ala Met Asp Tyr

1 5

<210> 128

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Gly-Ser linker

<400> 128

Gly Gly Gly Ser Gly Gly Gly Ser

1 5

<210> 129

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Gly-Ser linker

<400> 129

Gly Gly Ser Gly Gly Ser

1 5

<210> 130

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL3 binding domain, which binds EGFR

<400> 130

His Gln Tyr Gly Ser Thr Pro Leu Thr

1 5

<210> 131

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH1 binding domain that binds EGFR

<400> 131

Ser Gly Asp Tyr Tyr Trp Ser

1 5

<210> 132

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH2 binding domain that binds EGFR

<400> 132

Tyr Ile Tyr Tyr Ser Gly Ser Thr Asp Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 133

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH3 binding domain that binds EGFR

<400> 133

Val Ser Ile Phe Gly Val Gly Thr Phe Asp Tyr

1 5 10

<210> 134

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL binding domain that binds EGFR

<400> 134

Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Tyr Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Glu Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 135

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH binding domain that binds EGFR

<400> 135

Gln Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asn Trp

20 25 30

Asp Val His Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Ala Val Ile Trp Ser Gly Gly Ala Thr Asp Tyr Asn Thr Pro Phe Asn

50 55 60

Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Val Leu

65 70 75 80

Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Arg Ala Leu Asp Tyr Tyr Asp Tyr Asn Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Met Val Thr Val Ser Ser

115

<210> 136

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL1 binding domain, which binds CD19

<400> 136

Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn

1 5 10

<210> 137

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL2 binding domain, which binds CD19

<400> 137

Ser Arg Leu His Ser Gly Val

1 5

<210> 138

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL3 binding domain, which binds CD19

<400> 138

Gly Asn Thr Leu Pro Tyr Thr Phe Gly

1 5

<210> 139

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH1 binding domain that binds CD19

<400> 139

Asp Tyr Gly Val Ser

1 5

<210> 140

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH2 binding domain that binds CD19

<400> 140

Val Thr Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser

1 5 10 15

<210> 141

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH3 binding domain that binds CD19

<400> 141

Tyr Ala Met Asp Tyr Trp Gly

1 5

<210> 142

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL1 binding domain, which binds ROR1

<400> 142

Gln Ala Ser Gln Ser Ile Asp Ser Asn Leu Ala

1 5 10

<210> 143

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL2 binding domain that binds ROR1

<400> 143

Arg Ala Ser Asn Leu Ala Ser

1 5

<210> 144

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL3 binding domain that binds ROR1

<400> 144

Leu Gly Gly Val Gly Asn Val Ser Tyr Arg Thr Ser

1 5 10

<210> 145

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH1 binding domain that binds ROR1

<400> 145

Asp Tyr Pro Ile Ser

1 5

<210> 146

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH2 binding domain that binds ROR1

<400> 146

Phe Ile Asn Ser Gly Gly Ser Thr Trp Tyr Ala Ser Trp Val Lys Gly

1 5 10 15

<210> 147

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH3 binding domain that binds ROR1

<400> 147

Gly Tyr Ser Thr Tyr Tyr Cys Asp Phe Asn Ile

1 5 10

<210> 148

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL1 binding domain, which binds ROR1

<400> 148

Thr Leu Ser Ser Ala His Lys Thr Asp Thr Ile Asp

1 5 10

<210> 149

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL2 binding domain that binds ROR1

<400> 149

Gly Ser Tyr Thr Lys Arg Pro

1 5

<210> 150

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL3 binding domain that binds ROR1

<400> 150

Gly Ala Asp Tyr Ile Gly Gly Tyr Val

1 5

<210> 151

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH1 binding domain that binds ROR1

<400> 151

Ala Tyr Tyr Met Ser

1 5

<210> 152

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH2 binding domain that binds ROR1

<400> 152

Thr Ile Tyr Pro Ser Ser Gly Lys Thr Tyr Tyr Ala Thr Trp Val Asn

1 5 10 15

Gly

<210> 153

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH3 binding domain that binds ROR1

<400> 153

Asp Ser Tyr Ala Asp Asp Gly Ala Leu Phe Asn Ile

1 5 10

<210> 154

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL1 binding domain, which binds ROR1

<400> 154

Lys Ala Ser Gln Asn Val Asp Ala Ala Val Ala

1 5 10

<210> 155

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL2 binding domain that binds ROR1

<400> 155

Ser Ala Ser Asn Arg Tyr Thr

1 5

<210> 156

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL3 binding domain that binds ROR1

<400> 156

Gln Gln Tyr Asp Ile Tyr Pro Tyr Thr

1 5

<210> 157

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH1 binding domain that binds ROR1

<400> 157

Asp Tyr Glu Met His

1 5

<210> 158

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH2 binding domain that binds ROR1

<400> 158

Ala Ile Asp Pro Glu Thr Gly Gly Thr Ala Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 159

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH3 binding domain that binds ROR1

<400> 159

Tyr Tyr Asp Tyr Asp Ser Phe Thr Tyr

1 5

<210> 160

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL1 binding domain, which binds ROR1

<400> 160

Gln Ala Ser Gln Ser Ile Gly Ser Tyr Leu Ala

1 5 10

<210> 161

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL2 binding domain that binds ROR1

<400> 161

Tyr Ala Ser Asn Leu Ala Ser

1 5

<210> 162

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL3 binding domain that binds ROR1

<400> 162

Leu Gly Ser Leu Ser Asn Ser Asp Asn Val

1 5 10

<210> 163

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH1 binding domain that binds ROR1

<400> 163

Ser His Trp Met Ser

1 5

<210> 164

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH2 binding domain that binds ROR1

<400> 164

Ile Ile Ala Ala Ser Gly Ser Thr Tyr Tyr Ala Asn Trp Ala Lys Gly

1 5 10 15

<210> 165

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH3 binding domain that binds ROR1

<400> 165

Asp Tyr Gly Asp Tyr Arg Leu Val Thr Phe Asn Ile

1 5 10

<210> 166

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL1 binding domain, which binds Her2

<400> 166

Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp

1 5 10

<210> 167

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL2 binding domain, which binds Her2

<400> 167

Tyr Ser Ala Ser Phe Leu Glu Ser

1 5

<210> 168

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL3 binding domain, which binds Her2

<400> 168

Gln Gln His Tyr Thr Thr Pro Thr

1 5

<210> 169

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH1 binding domain that binds Her2

<400> 169

Ser Gly Phe Asn Thr Lys Asp Thr Tyr Ile His Trp

1 5 10

<210> 170

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH2 binding domain, which binds Her2

<400> 170

Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly Arg

<210> 171

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH3 binding domain that binds Her2

<400> 171

Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Val

1 5 10

<210> 172

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL1 binding domain, which binds PD-L1

<400> 172

Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu

1 5 10

<210> 173

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL3 binding domain, which binds PD-L1

<400> 173

Gln Gln Arg Ser Asn Trp Pro Arg Thr

1 5

<210> 174

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH1 binding domain that binds PD-L1

<400> 174

Asp Tyr Gly Phe Ser

1 5

<210> 175

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH2 binding domain that binds PD-L1

<400> 175

Trp Ile Thr Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu Gln

1 5 10 15

Gly

<210> 176

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH3 binding domain that binds PD-L1

<400> 176

Asp Tyr Phe Tyr Gly Met Asp Tyr

1 5

<210> 177

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL1 binding domain, which binds PD-L1

<400> 177

Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala

1 5 10

<210> 178

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL2 binding domain that binds PD-L1

<400> 178

Ser Ala Ser Phe Leu Tyr Ser

1 5

<210> 179

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL3 binding domain, which binds PD-L1

<400> 179

Gln Gln Tyr Leu Tyr His Pro Ala Thr

1 5

<210> 180

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH1 binding domain that binds PD-L1

<400> 180

Ser Gly Phe Thr Phe Ser Asp Ser Trp Ile His

1 5 10

<210> 181

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH2 binding domain that binds PD-L1

<400> 181

Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 182

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH3 binding domain that binds PD-L1

<400> 182

Arg His Trp Pro Gly Gly Phe Asp Tyr

1 5

<210> 183

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL1 binding domain, which binds PD-L1

<400> 183

Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser

1 5 10

<210> 184

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL2 binding domain that binds PD-L1

<400> 184

Asp Val Ser Asn Arg Pro Ser

1 5

<210> 185

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL3 binding domain, which binds PD-L1

<400> 185

Ser Ser Tyr Thr Ser Ser Ser Thr Arg Val

1 5 10

<210> 186

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH1 binding domain that binds PD-L1

<400> 186

Ser Gly Phe Thr Phe Ser Ser Tyr Ile Met Met

1 5 10

<210> 187

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH2 binding domain that binds PD-L1

<400> 187

Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val Lys

1 5 10 15

Gly

<210> 188

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH3 binding domain that binds PD-L1

<400> 188

Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr

1 5 10

<210> 189

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL1 binding domain, which binds CD3

<400> 189

Ser Ala Ser Ser Ser Val Ser Tyr Met Asn

1 5 10

<210> 190

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL2 binding domain, which binds CD3

<400> 190

Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser

1 5 10

<210> 191

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL3 binding domain, which binds CD3

<400> 191

Gln Gln Trp Ser Ser Asn Pro Phe Thr

1 5

<210> 192

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH1 binding domain, which binds CD3

<400> 192

Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His

1 5 10

<210> 193

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH2 binding domain, which binds CD3

<400> 193

Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp

1 5 10 15

<210> 194

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH3 binding domain, which binds CD3

<400> 194

Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr

1 5 10

<210> 195

<211> 242

<212> PROTEIN

<213> Artificial sequence

<220>

<223> scFv that binds CD3

<400> 195

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gln Ile Val Leu Thr Gln Ser Pro Ala

130 135 140

Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala

145 150 155 160

Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr

165 170 175

Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val

180 185 190

Pro Ala His Phe Arg Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr

195 200 205

Ile Ser Gly Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln

210 215 220

Trp Ser Ser Asn Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile

225 230 235 240

Asn Arg

<210> 196

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL1 binding domain, which binds CD3

<400> 196

Gln Ser Leu Val His Asn Asn Gly Asn Thr Tyr

1 5 10

<210> 197

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL3 binding domain, which binds CD3

<400> 197

Gly Gln Gly Thr Gln Tyr Pro Phe Thr

1 5

<210> 198

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH1 binding domain, which binds CD3

<400> 198

Gly Phe Thr Phe Thr Lys Ala Trp

1 5

<210> 199

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH2 binding domain, which binds CD3

<400> 199

Ile Lys Asp Lys Ser Asn Ser Tyr Ala Thr

1 5 10

<210> 200

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH3 binding domain, which binds CD3

<400> 200

Arg Gly Val Tyr Tyr Ala Leu Ser Pro Phe Asp Tyr

1 5 10

<210> 201

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL1 binding domain, which binds CD3

<400> 201

Gln Ser Leu Val His Asp Asn Gly Asn Thr Tyr

1 5 10

<210> 202

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH1 binding domain, which binds CD3

<400> 202

Gly Phe Thr Phe Ser Asn Ala Trp

1 5

<210> 203

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH2 binding domain, which binds CD3

<400> 203

Ile Lys Ala Arg Ser Asn Asn Tyr Ala Thr

1 5 10

<210> 204

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH3 binding domain, which binds CD3

<400> 204

Arg Gly Thr Tyr Tyr Ala Ser Lys Pro Phe Asp Tyr

1 5 10

<210> 205

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL1 binding domain, which binds CD3

<400> 205

Gln Ser Leu Glu His Asn Asn Gly Asn Thr Tyr

1 5 10

<210> 206

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH2 binding domain, which binds CD3

<400> 206

Ile Lys Asp Lys Ser Asn Asn Tyr Ala Thr

1 5 10

<210> 207

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH3 binding domain, which binds CD3

<400> 207

Arg Tyr Val His Tyr Gly Ile Gly Tyr Ala Met Asp Ala

1 5 10

<210> 208

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL1 binding domain, which binds CD3

<400> 208

Gln Ser Leu Val His Thr Asn Gly Asn Thr Tyr

1 5 10

<210> 209

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL3 binding domain, which binds CD3

<400> 209

Gly Gln Gly Thr His Tyr Pro Phe Thr

1 5

<210> 210

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH1 binding domain, which binds CD3

<400> 210

Gly Phe Thr Phe Thr Asn Ala Trp

1 5

<210> 211

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH2 binding domain, which binds CD3

<400> 211

Lys Asp Lys Ser Asn Asn Tyr Ala Thr

1 5

<210> 212

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH3 binding domain, which binds CD3

<400> 212

Arg Tyr Val His Tyr Arg Phe Ala Tyr Ala Leu Asp Ala

1 5 10

<210> 213

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL1 binding domain, which binds CD28

<400> 213

His Ala Ser Gln Asn Ile Tyr Val Trp Leu Asn

1 5 10

<210> 214

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL2 binding domain, which binds CD28

<400> 214

Lys Ala Ser Asn Leu His Thr

1 5

<210> 215

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL3 binding domain, which binds CD28

<400> 215

Gln Gln Gly Gln Thr Tyr Pro Tyr Thr

1 5

<210> 216

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH1 binding domain that binds CD28

<400> 216

Ser Tyr Tyr Ile His

1 5

<210> 217

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH2 binding domain that binds CD28

<400> 217

Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe Lys

1 5 10 15

Asp

<210> 218

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH3 binding domain that binds CD28

<400> 218

Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val

1 5 10

<210> 219

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL1 binding domain, which binds CD80/CD86

<400> 219

Ser Val Ser Ser Ser Ile Ser Ser Ser Asn Leu His

1 5 10

<210> 220

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL2 binding domain, which binds CD80/CD86

<400> 220

Gly Thr Ser Asn Leu Ala Ser

1 5

<210> 221

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL3 binding domain, which binds CD80/CD86

<400> 221

Gln Gln Trp Ser Ser Tyr Pro Leu Thr

1 5

<210> 222

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH1 binding domain that binds CD80/CD86

<400> 222

Asp Tyr Tyr Met His

1 5

<210> 223

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH2 binding domain that binds CD80/CD86

<400> 223

Trp Ile Asp Pro Glu Asn Gly Asn Thr Leu Tyr Asp Pro Lys Phe Gln

1 5 10 15

Gly

<210> 224

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH3 binding domain that binds CD80/CD86

<400> 224

Glu Gly Leu Phe Phe Ala Tyr

1 5

<210> 225

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL1 binding domain, which binds 4-1BB

<400> 225

Arg Ala Ser Gln Ser Val Ser

1 5

<210> 226

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL2 binding domain, which binds 4-1BB

<400> 226

Ala Ser Asn Arg Ala Thr

1 5

<210> 227

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL3 binding domain, which binds 4-1BB

<400> 227

Gln Arg Ser Asn Trp Pro Pro Ala Leu Thr

1 5 10

<210> 228

<211> 4

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH1 binding domain that binds 4-1BB

<400> 228

Tyr Tyr Trp Ser

1

<210> 229

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH3 binding domain that binds 4-1BB

<400> 229

Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu

1 5 10

<210> 230

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL1 binding domain, which binds 4-1BB

<400> 230

Ser Gly Asp Asn Ile Gly Asp Gln Tyr Ala His

1 5 10

<210> 231

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL2 binding domain, which binds 4-1BB

<400> 231

Gln Asp Lys Asn Arg Pro Ser

1 5

<210> 232

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL3 binding domain, which binds 4-1BB

<400> 232

Ala Thr Tyr Thr Gly Phe Gly Ser Leu Ala Val

1 5 10

<210> 233

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH1 binding domain that binds 4-1BB

<400> 233

Gly Tyr Ser Phe Ser Thr Tyr Trp Ile Ser

1 5 10

<210> 234

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH2 binding domain that binds 4-1BB

<400> 234

Lys Ile Tyr Pro Gly Asp Ser Tyr Thr Asn Tyr Ser Pro Ser

1 5 10

<210> 235

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH3 binding domain that binds 4-1BB

<400> 235

Gly Tyr Gly Ile Phe Asp Tyr

1 5

<210> 236

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL1 binding domain, which binds CD8

<400> 236

Arg Thr Ser Arg Ser Ile Ser Gln Tyr Leu Ala

1 5 10

<210> 237

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL2 binding domain, which binds CD8

<400> 237

Ser Gly Ser Thr Leu Gln Ser

1 5

<210> 238

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL3 binding domain, which binds CD8

<400> 238

Gln Gln His Asn Glu Asn Pro Leu Thr

1 5

<210> 239

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH1 binding domain that binds CD8

<400> 239

Gly Phe Asn Ile Lys Asp

1 5

<210> 240

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH2 binding domain that binds CD8

<400> 240

Arg Ile Asp Pro Ala Asn Asp Asn Thr

1 5

<210> 241

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH3 binding domain that binds CD8

<400> 241

Gly Tyr Gly Tyr Tyr Val Phe Asp His

1 5

<210> 242

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL1 binding domain, which binds PD-1

<400> 242

Arg Ala Ser Gln Ser Val Ser Thr Ser Gly Tyr Ser Tyr Met His

1 5 10 15

<210> 243

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL2 binding domain that binds PD-1

<400> 243

Phe Gly Ser Asn Leu Glu Ser

1 5

<210> 244

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL3 binding domain, which binds PD-1

<400> 244

Gln His Ser Trp Glu Ile Pro Tyr Thr

1 5

<210> 245

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH1 binding domain that binds PD-1

<400> 245

Ser Ser Trp Ile His

1 5

<210> 246

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH2 binding domain that binds PD-1

<400> 246

Tyr Ile Tyr Pro Ser Thr Gly Phe Thr Glu Tyr Asn Gln Lys Phe Lys

1 5 10 15

Asp

<210> 247

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH3 binding domain that binds PD-1

<400> 247

Trp Arg Asp Ser Ser Gly Tyr His Ala Met Asp Tyr

1 5 10

<210> 248

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL3 binding domain, which binds PD-1

<400> 248

Gln Gln Ser Ser Asn Trp Pro Arg Thr

1 5

<210> 249

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH1 binding domain that binds PD-1

<400> 249

Asn Ser Gly Met His

1 5

<210> 250

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH2 binding domain that binds PD-1

<400> 250

Val Leu Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 251

<211> 4

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH3 binding domain that binds PD-1

<400> 251

Asn Asp Asp Tyr

1

<210> 252

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Gly-Ser linker

<400> 252

Gly Gly Ser Gly Gly Gly Ser Gly Gly Ser Gly

1 5 10

<210> 253

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL3 binding domain, which binds TYRP1/gp75

<400> 253

Gln Gln Arg Ser Asn Trp Leu Met Tyr Thr

1 5 10

<210> 254

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH1 binding domain that binds TYRP1/gp75

<400> 254

Gly Tyr Thr Phe Thr Ser Tyr Ala Met Asn

1 5 10

<210> 255

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH2 binding domain that binds TYRP1/gp75

<400> 255

Trp Ile Asn Thr Asn Thr Gly Asn Pro Thr Tyr Ala Gln Gly Phe Thr

1 5 10 15

Gly

<210> 256

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH3 binding domain that binds TYRP1/gp75

<400> 256

Arg Tyr Ser Ser Ser Trp Tyr Leu Asp Tyr

1 5 10

<210> 257

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL1 binding domain that binds TYRP1/gp75

<400> 257

Arg Ala Ser Gly Asn Ile Tyr Asn Tyr Leu Ala

1 5 10

<210> 258

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL2 binding domain that binds TYRP1/gp75

<400> 258

Asp Ala Lys Thr Leu Ala Asp

1 5

<210> 259

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL3 binding domain, which binds TYRP1/gp75

<400> 259

Gln His Phe Trp Ser Leu Pro Phe Thr

1 5

<210> 260

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH1 binding domain that binds TYRP1/gp75

<400> 260

Gly Phe Asn Ile Lys Asp Tyr Phe Leu His

1 5 10

<210> 261

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH2 binding domain that binds TYRP1/gp75

<400> 261

Trp Ile Asn Pro Asp Asn Gly Asn Thr Val Tyr Asp Pro Lys Phe Gln

1 5 10 15

Gly

<210> 262

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRH3 binding domain that binds TYRP1/gp75

<400> 262

Asp Tyr Thr Tyr Glu Lys Ala Ala Leu Asp Tyr

1 5 10

<210> 263

<211> 108

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL binding domain that binds TYRP1/gp75

<400> 263

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Leu Met

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 264

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH binding domain that binds TYRP1/gp75

<400> 264

Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Cys Met

35 40 45

Gly Trp Ile Asn Thr Asn Thr Gly Asn Pro Thr Tyr Ala Gln Gly Phe

50 55 60

Thr Gly Arg Phe Val Phe Ser Met Asp Thr Ser Val Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Pro Arg Tyr Ser Ser Ser Trp Tyr Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 265

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH binding domain that binds TYRP1/gp75

<400> 265

Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Ser Met

35 40 45

Gly Trp Ile Asn Thr Asn Thr Gly Asn Pro Thr Tyr Ala Gln Gly Phe

50 55 60

Thr Gly Arg Phe Val Phe Ser Met Asp Thr Ser Val Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Pro Arg Tyr Ser Ser Ser Trp Tyr Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<---

Claims (36)

1. A nanoparticle for treating cancer in a human subject, having a positively charged polymer core and a neutral or negatively charged coating, comprising: a targeting ligand that binds to a professional phagocyte; and a nucleic acid that encodes a protein molecule having at least a first binding domain and a second binding domain, wherein the first binding domain is specific for a cell surface protein expressed by an immune cell, and wherein the second binding domain is specific for a cell surface protein expressed by a cancer cell. 2. A nanoparticle according to claim 1, where: – the targeting ligand binds a cell surface protein expressed by a monocyte, macrophage, or both; – nucleic acid is ribonucleic acid (RNA); – the first binding domain is specific to the cell surface protein of the lymphocyte; - the first binding domain is specific for a cell surface protein of a T cell selected from the group consisting of a CD8+ T cell, a CD4+ T cell, a gamma/delta T cell, and a NK T cell; – the protein molecule is a bispecific T-cell binding molecule; – the second binding domain is specific for an antigen expressed by the cancer cell; or – the nanoparticle is a liposome, liposomal nanoparticle, lipid nanoparticle or solid lipid nanoparticle. 3. The nanoparticle of claim 1, further comprising a second nucleic acid encoding one or more interferon-regulating factors (IRFs); or comprising a tumor cell proliferation inhibitor or a nucleic acid encoding a tumor cell proliferation inhibitor. 4. A composition for treating cancer in a human subject, comprising at least one nanoparticle according to any one of claims 1-3. 5. A composition for treating cancer in a human subject, comprising a first plurality of nanoparticles comprising at least one nanoparticle according to any one of claims 1-3, wherein the first plurality of nanoparticles stimulate or enhance the immune response in a human subject, thereby treating cancer. 6. A method of treating cancer in a human subject, comprising administering to a human subject a composition comprising a nanoparticle according to any one of claims 1-3. 7. The method according to paragraph 6, further comprising: administering to a human subject a composition comprising a second plurality of nanoparticles, wherein each of the second plurality of nanoparticles comprises a targeting ligand that binds a monocyte, a macrophage, or both, and one or more of (a) mRNA encoding one or more interferon regulatory factors (IRFs) and (b) mRNA encoding IKKβ; or administering to a human subject a composition comprising a third plurality of nanoparticles, wherein each of the third plurality of nanoparticles comprises a targeting ligand that binds a monocyte, a macrophage, or both, and mRNA encoding a tumor cell proliferation inhibitor. 8. The method of claim 7, wherein at least the nanoparticles of a subset of the first or second plurality of nanoparticles further comprise mRNA encoding a tumor cell proliferation inhibitor. 9. A modified professional phagocyte for the treatment of cancer in a human subject, comprising a nanoparticle loaded with a nucleic acid encoding a protein molecule having at least a first binding domain specific for a cell surface protein expressed by an immune cell and a second binding domain specific for a cell surface protein expressed by a cancer cell, where the nanoparticle is attached to the surface of a phagocyte or has been internalized by a phagocyte. 10. A modified professional phagocyte according to claim 9, further comprising at least one of: a second nanoparticle loaded with one or more of (a) a nucleic acid encoding one or more interferon-regulating factors (IRFs), (b) a nucleic acid encoding IKKβ, or (c) a nucleic acid encoding a tumor cell proliferation inhibitor; and a third nanoparticle loaded with a nucleic acid encoding a tumor cell proliferation inhibitor, where each of the second and third nanoparticles is attached to the surface of the phagocyte or has been internalized by the phagocyte.