KR102508182B1 - Transformed macrophages, chimeric antigen receptors, and related methods - Google Patents

Abstract

Chimeric receptors are described herein. A chimeric receptor has a cytoplasmic domain; transmembrane domain; and an extracellular domain. In an embodiment, the cytoplasmic domain comprises a cytoplasmic portion of a receptor that polarizes a macrophage when activated. In a further embodiment, the wild-type protein comprising a cytoplasmic portion does not comprise the extracellular domain of the chimeric receptor. In an embodiment, binding of the ligand to the extracellular domain of the chimeric receptor activates the intracellular portion of the chimeric receptor. Activation of the intracellular portion of the chimeric receptor can polarize macrophages to either M1 or M2 macrophages.

Description

Transformed macrophages, chimeric antigen receptors, and related methods

The present invention relates generally to biotechnology. More specifically, the invention relates to chimeric antigen receptors, nucleic acids encoding the chimeric antigen receptors, macrophages harboring the chimeric antigen receptors and/or nucleic acids encoding the chimeric antigen receptors, and related methods.

Cancer consists of a group of diseases that include unregulated cell growth and death, genomic instability and mutations, tumor-promoting inflammation, induction of angiogenesis, immune system evasion, deregulation of metabolic pathways, immortal cell replication, and metastatic tissue invasion. [One]. Cancer is the second leading cause of death in the United States after heart disease [2]. More than 1.6 million new cancer cases are projected to be diagnosed each year, and more than 580,000 Americans are projected to die (about 1600 cancer deaths per day), accounting for nearly a quarter of all American deaths [2 ,3].

The immune system plays an important role in the development and progression of cancer. Immune cell infiltration into the tumor site can adversely affect malignant tumor progression and metastasis [4, 5]. Infiltration of macrophages into tumor sites has been shown to account for more than 50% of tumors in certain breast cancer cases, suggesting that macrophages have a significant role in tumor progression [6-8].

Macrophages are cells derived from the myeloid lineage and belong to the innate immune system. They are derived from blood monocytes migrating into tissues. One of their main functions is to phagocytose microorganisms and remove cell debris. They also play important roles in both initiation and resolution of inflammation [9, 10]. Moreover, macrophages can exhibit different responses ranging from pro- and anti-inflammatory depending on the type of stimuli they receive from the surrounding microenvironment [11]. Two major macrophage phenotypes, M1 and M2, have been proposed that correlate with extreme macrophage responses.

M1 pro-inflammatory macrophages are activated upon contact with certain molecules such as lipopolysaccharide (LPS), IFN-γ, Il-1β, TNF-α, and Toll-like receptor binding. M1 macrophages constitute a powerful armament of the immune system deployed to fight infections. They can recognize pathogens directly (pathogen pattern recognition receptors) or indirectly (Fc receptors, complement receptors). They are also armed with the ability to generate reactive oxygen species (ROS) as a means to help kill pathogens. M1 macrophages also secrete pro-inflammatory cytokines and chemokines that attract other types of immune cells and integrate/modulate the immune response. M1 activation is induced by IFN-g, TNFa, GM-CSF, LPS, and other toll-like receptor (TLR) ligands.

In contrast, M2 anti-inflammatory macrophages, also known as alternatively activated macrophages, are activated by anti-inflammatory molecules such as IL-4, IL-13, and IL-10 [12, 13]. M2 macrophages exhibit immunomodulatory, tissue repair, and angiogenic properties, enabling them to recruit regulatory T cells to sites of inflammation. M2 macrophages do not constitute a uniform population and are often further subclassed into M2a, M2b, and M2c categories. The common denominator of all three subpopulations is high IL-10 production accompanied by low production of IL-12. One of their hallmarks is the production of the enzyme arginase-1, which suppresses T cell responses by depleting L-arginine and deprives its substrate of iNOS.

The in vivo molecular mechanisms of macrophage polarization are not well characterized due to the diverse signals that macrophages experience in the cellular microenvironment [10, 14]. Recently, advances have been made in identifying macrophage polarization in vivo under physiological conditions such as ontogeny, pregnancy, and pathological conditions such as allergy, chronic inflammation, and cancer. However, we know that macrophage polarization in vitro is variable and that macrophages can be reciprocally polarized to either phenotype with the help of cytokines [15, 16]. Interferon gamma (IFN-γ) and IL-4 are two cytokines that can polarize macrophages towards M1 and M2 phenotypes, respectively [15].

The presence of macrophages is important for tumor progression and growth and is involved in determining prognosis [17, 18]. Because macrophages can exhibit both pro-inflammatory and anti-inflammatory properties, it is important to understand their polarization and function in tumor progression and metastasis.

macrophage polarization

The tumor microenvironment can affect macrophage polarization. The polarization process can be varied and complex due to the hostile environment of IL-10, glucocorticoid hormones, apoptotic cells, and immune complexes that can interfere with innate immune cell function [11, 19]. The mechanism of polarization is still unclear, but we know that they are involved in transcriptional regulation. For example, macrophages exposed to LPS or IFN-γ will polarize towards an M1 phenotype, whereas macrophages exposed to IL-4 or IL-13 will polarize towards an M2 phenotype. LPS or IFN-γ can interact with Toll-like receptor 4 (TLR4) on the surface of macrophages, inducing the Trif and MyD88 pathways, leading to activation of the transcription factors IRF3, AP-1, and NFκB, thus It activates the TNF gene, interferon gene, CXCL10, NOS2, and IL-12, which are required for the pro-inflammatory M1 macrophage response [20]. Similarly, IL-4 and IL-13 bind to IL-4R and Jak/Stat6, which regulates the expression of genes related to anti-inflammatory response (M2 response), such as CCL17, ARG1, IRF4, IL-10, and SOCS3. activate the pathway.

A further mechanism of macrophage polarization involves microRNA (miRNA) micromanagement. miRNAs are small non-coding RNAs of 22 nucleotides in length, which regulate gene expression after transcription as they affect the rate of mRNA degradation. Several miRNAs, particularly miRNA-155, miRNA-125, miRNA-378 (M1 polarization), and miRNA let-7c, miRNA-9, miRNA-21, miRNA-146, miRNA147, miRNA-187 (M2 polarization) are polarized It has been shown to be highly expressed in macrophages [21].

Macrophage polarization is a complex process, and macrophages behave and elicit different responses to microenvironmental stimuli. Thus, macrophage polarization is better represented by contiguous activation states, with the M1 and M2 phenotypes being the extremes of the spectrum. Recently, there has been much controversy over the definition/explanation of macrophage activation and macrophage polarization. In a recent paper published by Murray et al., they describe a set of standards that must be considered for common definition/explanation of macrophage activation, polarization, activators, and markers. This publication was much needed for the definition and characterization of activated/polarized macrophages [22].

M1 phenotype

M1 pro-inflammatory macrophages or classically activated macrophages are aggressive, highly phagocytic, and produce large amounts of reactive oxygen and nitrogen species, thereby stimulating a Th1 response [11]. M1 macrophages secrete high levels of two important inflammatory cytokines, IL-12 and IL-23. IL-12 induces activation and clonal expansion of Th17 cells, which secrete large amounts of IL-17 contributing to inflammation [23]. These features enable M1 macrophages to control metastasis, inhibit tumor growth, and control microbial infection [24]. Moreover, infiltration and recruitment of M1 macrophages to the tumor site correlates with better prognosis and higher overall survival in patients with solid tumors [17, 18, 25-28].

The polarization of macrophages towards the M1 phenotype is regulated in vitro by transcription factors and miRNAs, as well as inflammatory signals such as IFN-γ, TNF-α, IL-1β, and LPS [29, 30]. Classically activated macrophages initiate induction of CXCL9, CXCL10 (also known as IP-10), the STAT1 transcription factor targeting IFN regulatory factor-1, and inhibitor-1 of cytokine signaling [31]. Cytokine signaling-1 protein functions downstream of cytokine receptors and participates in a negative feedback loop to dampen cytokine signaling. In the tumor microenvironment, Notch signaling plays an important role in the polarization of M1 macrophages, as it enables the transcription factor RBP-J to regulate classical activation. Macrophages deficient in Notch signaling express the M2 phenotype regardless of other exogenous inducers [32]. One important miRNA, miRNA-155, is upregulated when macrophages switch from M2 to M1; M1 macrophages overexpressing miRNA-155 are generally more aggressive and are associated with tumor reduction [33]. Moreover, miRNA-342-5p was found to promote a greater inflammatory response in macrophages by targeting Akt1 in mice. This miRNA also promotes upregulation of Nos2 and IL-6, both of which act as inflammatory signals to macrophages [34]. Other miRNAs, such as miRNA-125 and miRNA-378, have also been shown to be involved in the classical activation pathway of macrophages (M1) [35].

Classically activated macrophages are thought to play an important role in the recognition and destruction of cancer cells because their presence usually indicates a good prognosis. After recognition, malignant cells can be destroyed by M1 macrophages through several mechanisms including contact-dependent phagocytosis and cytotoxicity (ie, release of cytokines such as TNF-α) [24]. However, environmental cues such as the tumor microenvironment or tissue-resident cells can polarize M1 macrophages to M2 macrophages. In vivo studies of murine macrophages have shown that macrophages are variable in their cytokine and surface marker expression and that repolarizing macrophages to an M1 phenotype in the presence of cancer can help the immune system reject tumors [19 ].

M2 phenotype

M2 macrophages are anti-inflammatory and assist in the process of angiogenesis and tissue repair. They express trapper receptors and produce large amounts of IL-10 and other anti-inflammatory cytokines [33, 36]. Expression of IL-10 by M2 macrophages promotes a Th2 response. Th2 cells consequently upregulate the production of IL-3 and IL-4. IL-3 is a protein in the myeloid lineage (granulocytes, monocytes, and dendritic cells), along with other cytokines such as erythropoietin (EPO), granulocyte macrophage colony-stimulating factor (GM-CSF), and IL-6. Stimulates the proliferation of all cells. IL-4 is an important cytokine in the healing process as it contributes to the production of the extracellular matrix [23]. M2 macrophages exhibit functions that can aid tumor progression by enabling blood vessels to supply nutrients to malignant cells to promote their growth. In most solid tumors, the presence of macrophages (thought to be M2) negatively correlates with treatment success and longer survival [37]. Additionally, the presence of M2 macrophages correlates with metastatic potential in breast cancer. Lin and colleagues found that early recruitment of macrophages to mammary tumor sites in mice increased angiogenesis and malignancy rates [38]. The tumor microenvironment is thought to help macrophages maintain the M2 phenotype [23, 39]. Anti-inflammatory signals present in the tumor microenvironment, such as adiponectin and IL-10, can enhance the M2 response [41].

Tumor-Associated Macrophages (TAMs)

Cells exposed to the tumor microenvironment behave differently. For example, tumor-associated macrophages found in the periphery of solid tumors are thought to help promote tumor growth and metastasis and have an M2-like phenotype [42]. Tumor-associated macrophages can be tissue-resident macrophages or bone marrow-derived mobilized macrophages (macrophages that differentiate from monocytes into macrophages and migrate into tissues). A study by Cortez-Retamozo found that a large number of TAM precursors in the spleen migrated to the tumor stroma, suggesting this organ also as a TAM depot [43]. TAM precursors found in the spleen have been shown to initiate migration through their CCR2 chemokine receptors [43]. Recent studies have found CSF-1 as a major factor in attracting macrophages to the tumor periphery, and that CSF-1 production by cancer cells predicts lower survival rates and that it represents poor overall prognosis [44- 46]. Other cytokines, such as TNF-α and IL-6, have also been implicated in the accumulation/recruitment of macrophages to the tumor periphery [45].

The recruitment of macrophages around the tumor border is thought to be regulated by an "angiogenic switch" that is activated in the tumor. The angiogenic switch is defined as the process by which a tumor develops a dense vascular network, potentially enabling a tumor to become metastatic and required for malignant metastasis. In a mouse model of breast cancer, it was observed that the presence of macrophages is required for a complete angiogenic switch. When macrophage maturation, migration, and accumulation around the tumor was delayed, the angiogenic switch was also delayed, suggesting that the angiogenic switch does not occur in the absence of macrophages and that macrophage presence is required for malignant tumor progression. [47]. Moreover, tumor stromal cells produce chemokines such as CSF1, CCL2, CCL3, CCL5, and placental growth factor that will recruit macrophages to the tumor periphery. These chemokines provide an environment for macrophages to activate the angiogenic switch, where macrophages have high levels of IL-10, TGF-β, ARG-1, and low levels of IL-12, TNF-α, and will produce IL-6. Expression levels of these cytokines suggest that macrophages regulate immune evasion. It is important to note that macrophages are attracted to the hypoxic tumor environment and will respond by producing hypoxia-inducible factor-1α (HIF-1α) and HIF-2α, which regulate transcription of genes involved in angiogenesis . During the angiogenic switch, macrophages can also secrete VEGF (stimulated by the NF-κB pathway), which will promote vessel maturation and vascular permeability [48].

It is thought that tumor-associated macrophages may maintain their M2-like phenotype by receiving polarizing signals from malignant cells such as IL-1R and MyD88 mediated through IkB kinase β and the NF-kB signaling cascade. Inhibition of NF-kB in TAMs promotes classical activation [40]. Moreover, other studies have suggested that the p50 NF-kB subunit was involved in suppression of M1 macrophages and reduction of pro-inflammatory tumor growth. p50 NF-κB knock-out mice generated by Saccani et al. suggested that M1 aggressiveness was restored upon p50 NF-κB knockout, reducing tumor survival [49].

Because masses contain large numbers of M2-like macrophages, TAMs can be used as targets for cancer treatment. Reducing the number of TAMs or polarizing them towards the M1 phenotype can help destroy cancer cells and impair tumor growth [50-52]. Luo and colleagues used a vaccine against legumain, a cysteine protease and stress protein that is upregulated in TAMs, which are thought to be potential tumor targets [52]. When mice were vaccinated against legumain, genes controlling angiogenesis were downregulated and tumor growth was stopped [52].

metabolic and activation pathways

Metabolic changes present in tumor cells are controlled by the same genetic mutations that produce cancer [53]. As a result of these metabolic changes, cancer cells can generate signals that can alter the polarization of macrophages and promote tumor growth [54, 55].

M1 and M2 macrophages display distinct metabolic patterns reflecting their dissimilar behavior [56]. The M1 phenotype increases glycolysis and biases glucose metabolism towards the oxidative pentose phosphate pathway, thereby reducing oxygen consumption and consequently increasing the amount of radical oxygen and nitrogen species as well as inflammatory cytokines such as TNF-α, IL-12, and IL-6 [56, 57]. The M2 phenotype increases fatty acid uptake and oxidation, which reduces flux towards the pentose phosphate pathway while increasing the overall cellular redox potential, and consequently captures receptors and immunomodulatory cytokines such as IL-10 and TGF-β. upregulate [56].

Multiple metabolic pathways play an important role in macrophage polarization. Protein kinases such as Akt1 and Akt2 alter macrophage polarization by enabling cancer cells to survive, proliferate, and utilize intermediary metabolism [58]. Other protein kinases can induce macrophage polarization through glucose metabolism by increasing glycolysis and decreasing oxygen consumption [57, 59]. Shu and colleagues first visualized macrophage metabolism and immune responses in vivo using PET scans and glucose analogues [60].

L-arginine metabolism also represents a distinct shift important for cytokine expression in macrophages, and exemplifies distinct metabolic pathways that alter TAM-tumor cell interactions [61]. Classically activated (M1) macrophages prefer inducible nitric oxide synthase (iNOS). The iNOS pathway produces cytotoxic nitric oxide (NO) and consequently exhibits antitumor behavior. Alternatively, activated (M2) macrophages have been shown to favor the arginase pathway and produce ureum and 1-ornithine, which contributes to progressive tumor cell growth [61, 62].

Direct manipulation of metabolic pathways can alter macrophage polarization. Carbohydrate kinase-like protein (CARKL) proteins, which play a role in glucose metabolism, have been used to alter macrophage cytokine signatures [56, 57]. When CARKL is knocked down by RNAi, macrophages tend to adopt an M1-like metabolic pathway (metabolism is biased towards glycolysis and oxygen consumption is reduced). When CARKL is overexpressed, macrophages adopt an M2-like metabolism (reduced glycolytic flux and greater oxygen consumption) (56]. When macrophages adopt an M1-like metabolic state through LPS/TLR4 binding , CARKL levels decrease, genes controlled by the NFκB pathway are activated (TNF-α, IL-12, and IL-6), and increasing concentrations of NADH:NAD+ and GSH:GSSSG complexes increase the cellular redox potential. During an M2-like metabolic state, macrophages upregulate genes regulated by CARKL and STAT6/IL-4 (IL-10 and TGF-β).

Obesity can also affect macrophage polarization. Obesity is associated with a state of chronic inflammation, an environment that activates NKT cells by inducing the IL4/STAT6 pathway, which drives macrophages towards an M2 response. During late-stage diet-induced obesity, macrophages migrate to adipose tissue, where immune cells alter the level of T _H 1 or T _H 2 cytokine expression within adipose tissue, resulting in an M2 phenotype bias and possibly increased insulin. sensitization [63].

M1 phenotype bias by targeting metabolic pathways in TAMs may provide an alternative means of reducing tumor growth and metastasis.

Macrophage Immunotherapy Approaches for Cancer

The role of cancer immunotherapy is to stimulate the immune system to recognize, reject, and destroy cancer cells. Cancer immunotherapy using monocytes/macrophages aims to polarize macrophages towards a pro-inflammatory response (M1), thus enabling macrophages and other immune cells to destroy tumors. Although many cytokine and bacterial compounds can achieve this in vitro, side effects are typically too severe in vivo . The key is to find compounds with minimal or easily managed patient side effects. Immunotherapy using monocytes/macrophages has been used for the past decades, and new approaches are being developed every year [64, 65]. Early immunotherapy established a good basis for better cancer therapy and increased survival rates in patients treated with immunotherapy [66].

Some approaches to cancer immunotherapy involve the use of cytokines or chemokines to recruit activated macrophages and other immune cells to the tumor site, enabling recognition and targeted destruction of the tumor site [67, 68]. IFN-α and IFN-β have been shown to inhibit tumor progression by inducing cell differentiation and apoptosis [69]. In addition, IFN treatment is antiproliferative and can increase S phase time in the cell cycle [70, 71]. Zhang and colleagues performed studies in nude mice using IFN-β gene therapy to target human prostate cancer cells. Their results indicate that adenovirus delivered IFN-β gene therapy engages macrophages and helps to inhibit growth and metastasis [72].

Macrophage inhibitory factor (MIF) is another cytokine that can be used in cancer immunotherapy. MIF is usually found in solid tumors and has a poor prognosis. MIF inhibits aggressive macrophage function and drives macrophages towards an M2 phenotype, which may aid tumor growth and progression. Simpson, Templeton, and Cross (2012) found that MIF induced the differentiation of myeloid cells, macrophage precursors, into an inhibitory population of myeloid cells expressing the M2 phenotype [73 ]. By targeting MIF, they were able to deplete this inhibitory population of macrophages, inhibiting their growth and thus controlling tumor growth and metastasis [73].

The chemokine receptor type 2, CCR2, is critical for the recruitment of monocytes to inflammatory sites, and it has been shown to be a target for the recruitment of macrophages to tumor sites, angiogenesis, and prevention of metastasis. Sanford and colleagues (2013) studied a novel CCR2 inhibitor (PF-04136309) in a pancreatic mouse model, which showed that the CCR2 inhibitor depleted monocyte/macrophage recruitment to the tumor site and inhibited tumor growth and metastasis. and increased antitumor immunity [74]. Another recent study by Schmall et al. showed that macrophages co-cultured with 10 different human lung cancers upregulated CCR2 expression. Moreover, they showed reduced tumor growth and metastasis in a lung mouse model treated with CCR2 antagonists [75].

In another study, liposomes delivering drugs were used to deplete M2 macrophages from tumors and halt angiogenesis. Cancer cells expressing high levels of IL-1β grow faster and induce more angiogenesis in vivo. Kimura and colleagues found that macrophages exposed to tumor cells expressing IL-1β had higher levels of angiogenic factors and chemokines, such as vascular endothelial growth factor A (VEG-A), IL-8, and monocyte chemoattractants. was found to promote tumor growth and angiogenesis [76]. When macrophages were depleted using clodronate liposomes, they found fewer IL-1β-producing tumor cells. They also found that tumor growth and angiogenesis were reduced by inhibiting the NF-κB and AP-1 transcription factors in cancer cells. These findings may suggest that macrophages surrounding tumor sites may be involved in promoting tumor growth and angiogenesis [76].

Compounds such as methionine enkephalin (MENK) have anti-tumor properties in vivo and in vitro. MENK has the ability to polarize M2 macrophages into M1 macrophages by upregulating the production of CD64, MHC-II, and nitric oxide (M1 markers) while downregulating CD206 and arginase-1 (M2 markers). MENK can also upregulate TNF-α and downregulate IL-10 [77].

Recent studies have focused on bisphosphonates as potential inhibitors of M2 macrophages. Bisphosphonates are commonly used to treat patients with metastatic breast cancer to prevent skeletal complications such as bone resorption [78]. While bisphosphonates remain in the body for a short period of time, bisphosphonates can target osteoclasts, the same class of cells as macrophages, due to their high affinity for hydroxyapatite. Once the bisphosphonates are bound to bone, the bone matrix internalizes the bisphosphonates by endocytosis. Once in the cytoplasm, bisphosphonates can inhibit protein prenylation, an event that prevents integrin signaling and endosomal transport, thereby allowing cells to undergo apoptosis [69]. Until recently, it was not known whether bisphosphonates could target tumor-associated macrophages, but a recent study by Junankar et al. showed that macrophages can induce pinocytosis, an event that does not occur in the epithelial cells surrounding the tumor. It has been shown to take up nitrogen-containing bisphosphonate compounds by phagocytosis [79]. Promoting TAMs to apoptosis using bisphosphonates may reduce angiogenesis and metastasis.

A further approach to cancer immunotherapy involves the use of biomaterials capable of eliciting an immune response. Cationic polymers, once dissolved in water, are used in immunotherapy because of their reactivity. Chen et al. generated a potent Th1 immune response using cationic polymers including PEI, polylysine, cationic dextran, and cationic gelatin [77]. They were also able to induce proliferation of CD4+ cells representing M1 macrophages and secretion of IL-12 [77]. Huang and colleagues have also triggered TAMs using biomaterials to generate an anti-tumor response by targeting TLR4 [80]. This study found that TAMs can polarize to the M1 phenotype and express IL-12. They found that these cationic molecules had direct oncolytic activity in mice and showed tumor reduction [80].

TLR4

Toll-like receptor 4 is a protein in humans that is encoded by the TLR4 gene. TLR 4 detects lipopolysaccharide (LPS) on Gram-negative bacteria and thus plays an essential role in risk recognition and activation of the innate immune system (FIG. 7). It cooperates with LY96 (MD-2) and CD14 to mediate signaling when macrophages are induced by LPS. The cytoplasmic domain of TLR4 is responsible for the activation of M1 macrophages when they detect the presence of LPS. This is the functional portion of the receptor that will be coupled to the MOTO-CAR (ie chimeric receptor) to induce activation of monocytes/macrophages when the CAR binds its target protein.

The adapter proteins MyD88 and TIRAP contribute to the activation of several and possibly all pathways through direct interaction with the Toll/interleukin-1 receptor (IL-1R) (TIR) domain of TLR4. However, there may be additional adapters required for activation of a specific subset of pathways, which may contribute to differential regulation of target genes.

thymidine kinase

Human thymidine kinase 1 (TK1) is a well-known nucleotide salvage pathway enzyme that has been primarily studied in connection with its overexpression in tumors. Since TK1 was initially popularized by its expression (sTK) in the serum of cancer patients, its diagnostic and prognostic potential has been extensively studied. For example, several studies have demonstrated that sTK1 rises in a stage-like manner, with higher levels of TK1 indicative of more advanced tumors in many different cancer patients [81].

Other studies have investigated the prognostic potential of TK1. One such study demonstrates that TK1 levels in primary breast tumors can be used to predict recurrence. Another interesting TK1 prognostic study shows a significant decrease in sTK1 levels when patients respond to treatment, whereas sTK1 levels continue to rise in patients who do not appear to respond to treatment. It is also known that sTK1 levels begin to rise before relapse, and in some cases it has been stated that sTK1 levels "at 1 to 6 months before onset of clinical symptoms" can predict relapse. Several other studies confirm the rich potential of TK1 as a diagnostic and prognostic indicator of cancer [82].

Although the diagnostic and prognostic potential of TK1 is well established, the therapeutic potential of TK1 remains relatively unclear. While it is true that HSV-TK has been used in gene therapy and PET imaging utilizes TK1 to identify proliferative cancer cells, few studies have addressed the potential of TK1 immunotherapy. Perhaps this is primarily because TK1 is a known cytosol protein. It has recently been discovered that TK1 is expressed on cancer cells as well as on surface membranes of multiple tumor types, and thus is a highly viable target for tumor immunotherapy.

Chimeric receptors are described herein. A chimeric receptor has a cytoplasmic domain; transmembrane domain; and an extracellular domain. In an embodiment, the cytoplasmic domain comprises a cytoplasmic portion of a receptor that polarizes a macrophage when activated. In a further embodiment, the wild-type protein comprising the cytoplasmic portion does not comprise the extracellular domain of the chimeric receptor (see eg FIG. 21 ). In an embodiment, binding of the ligand to the extracellular domain of the chimeric receptor activates the intracellular portion of the chimeric receptor (see, eg, FIG. 22 ). Activation of the intracellular portion of the chimeric receptor can polarize macrophages to either M1 or M2 macrophages (see, eg, FIGS. 23 and 24A and 25 ).

In certain embodiments, the extracellular domain may include an antibody or fragment thereof that specifically binds a ligand. In embodiments, a chimeric receptor may contain a linker. In an embodiment, a chimeric receptor may contain a hinge region.

A further embodiment includes a cell comprising a chimeric receptor or a nucleic acid encoding a chimeric receptor.

Embodiments include contacting a macrophage comprising a chimeric receptor with a ligand for an extracellular domain of the chimeric receptor; and a method of polarizing macrophages by binding the ligand to the extracellular domain of the chimeric receptor. Binding of the ligand to the extracellular domain of the chimeric receptor activates the cytoplasmic portion, and activation of the cytoplasmic portion polarizes the macrophage.

These and other aspects of the invention will be apparent to those skilled in the art in view of the teachings contained herein.

1A shows a block diagram of the order of elements within the chimeric receptor TK1-MOTO1. 1B shows the sequence of TK1-MOTO1 (SEQ ID NO: 35). Amino acids 1 to 18 are the signal peptide (SP), amino acids 19 to 275 are the anti-TK1 ScFv, amino acids 276 to 290 are the GS linker, amino acids 291 to 313 are the TLR4 transmembrane domain, and amino acids 314 to 496 are the TLR4 cytosol It is a domain.
2A shows a block diagram of the order of elements within the chimeric receptor TK1-MOTO2. 2B shows the sequence of TK1-MOTO2 (SEQ ID NO: 36). Amino acids 1-18 are the signal peptide (SP), amino acids 19-275 are the anti-TK1 ScFv, amino acids 276-290 are the GS linker, amino acids 291-295 are the LRR short hinge, and amino acids 296-318 are the TLR4 transmembrane domain, and amino acids 319 to 500 are the TLR4 cytosol domain.
3A shows a block diagram of the order of elements within the chimeric receptor TK1-MOTO3. 3B shows the sequence of TK1-MOTO3 (SEQ ID NO: 37). Amino acids 1-18 are the signal peptide (SP), amino acids 19-275 are the anti-TK1 ScFv, amino acids 276-290 are the GS linker, amino acids 291-345 are the LRR long hinge, and amino acids 346-368 are the TLR4 transmembrane domain, and amino acids 269 to 501 are the TLR4 cytosol domain.
4A shows a block diagram of the order of elements within the chimeric receptor TK1-MOTO4. 4B shows the sequence of TK1-MOTO4 (SEQ ID NO: 38). Amino acids 1-18 are the signal peptide (SP), amino acids 19-275 are the anti-TK1 ScFv, amino acids 276-290 are the GS linker, amino acids 291-302 are the IgG4 short hinge, and amino acids 303-325 are the TLR4 transmembrane domain, and amino acids 326-508 are the TLR4 cytosol domain.
5A shows a block diagram of the order of elements within the chimeric receptor TK1-MOTO5. 5B shows the sequence of TK1-MOTO5 (SEQ ID NO: 39). Amino acids 1 to 18 are the signal peptide (SP), amino acids 19 to 275 are the anti-TK1 ScFv, amino acids 276 to 290 are the GS linker, amino acids 291 to 409 are the IgG 119 amino acid middle hinge, and amino acids 410 to 432 are the TLR4 transmembrane domain, and amino acids 433 to 615 are the TLR4 cytosol domain.
6A shows a block diagram of the order of elements within the chimeric receptor TK1-MOTO6. 6B shows the sequence of TK1-MOTO6 (SEQ ID NO: 40). Amino acids 1-18 are the signal peptide (SP), amino acids 19-275 are the anti-TK1 ScFv, amino acids 276-290 are the GS linker, amino acids 291-518 are the IgG4 long hinge, and amino acids 519-541 are the TLR4 transmembrane domain, and amino acids 542 to 724 are the TLR4 cytosol domain.
7A shows a block diagram of the order of elements within the chimeric receptor TK1-MOTO7. 7B shows the sequence of TK1-MOTO7 (SEQ ID NO: 41). Amino acids 1-18 are the signal peptide (SP), amino acids 19-275 are the anti-TK1 ScFv, amino acids 276-290 are the GS linker, amino acids 291-358 are the CD8 hinge mutated to C339S and C356S, and amino acid 359 to 381 is the TLR4 transmembrane domain, and amino acids 382 to 564 are the TLR4 cytosol domain.
8A shows a block diagram of the order of elements within the chimeric receptor TK1-MOTO8. 8B shows the sequence of TK1-MOTO8 (SEQ ID NO: 42). Amino acids 1-18 are the signal peptide (SP), amino acids 19-275 are the anti-TK1 ScFv, amino acids 276-290 are the GS linker, amino acids 291-358 are part of the CD8 hinge, and amino acids 359-381 are the TLR4 membrane penetrating domain, and amino acids 382 to 564 are the TLR4 cytosol domain.
9A shows a block diagram of the order of elements within the chimeric receptor TK1-MO-FCGRA-CAR-1. 9B shows the sequence of TK1-MO-FCGRA-CAR-1 (SEQ ID NO: 43). Amino acids 1 to 18 are the signal peptide (SP), amino acids 19 to 275 are the anti-TK1 ScFv, amino acids 276 to 290 are the GS linker, amino acids 291 to 311 are the FCGR3A transmembrane domain, and amino acids 312 to 336 are the FCGR3A cytosol domain, and amino acids 337 to 378 are the FCER1G cytosol domain.
10A shows a block diagram of the order of elements within the chimeric receptor TK1-MO-FCGRA-CAR-2. 10B shows the sequence of TK1-MO-FCGRA-CAR-2 (SEQ ID NO: 44). Amino acids 1-18 are the signal peptide (SP), amino acids 19-275 are the anti-TK1 ScFv, amino acids 276-290 are the GS linker, amino acids 291-358 are the CD8 hinge mutated to C339S and C356S, and amino acid 359 to 379 are the FCGR3A transmembrane domain, amino acids 380 to 404 are the FCGR3A cytosol domain, and amino acids 405 to 446 are the FCER1G cytosol domain.
11A shows a block diagram of the order of elements within the chimeric receptor TK1-MO-FCGRA-CAR-3. 11B shows the sequence of TK1-MO-FCGRA-CAR-3 (SEQ ID NO: 45). Amino acids 1-18 are the signal peptide (SP), amino acids 19-275 are the anti-TK1 ScFv, amino acids 276-290 are the GS linker, amino acids 291-358 are part of the CD8 hinge, and amino acids 359-379 are the FCGR3A membrane penetrating domain, amino acids 380 to 404 are the FCGR3A cytosol domain, and amino acids 405 to 446 are the FCER1G cytosol domain.
12A shows a block diagram of the order of elements within the chimeric receptor TK1-MO-FCGRA-CAR-4. 12B shows the sequence of TK1-MO-FCGRA-CAR-4 (SEQ ID NO: 46). Amino acids 1 to 18 are the signal peptide (SP), amino acids 19 to 275 are the anti-TK1 ScFv, amino acids 276 to 290 are the GS linker, amino acids 291 to 303 are the IgG4 short hinge, and amino acids 304 to 324 are the FCGR3A transmembrane domain, amino acids 325 to 349 are the FCGR3A cytosol domain, and amino acids 350 to 391 are the FCER1G cytosol domain.
13A shows a block diagram of the order of elements within the chimeric receptor TK1-MO-FCGRA-CAR-5. 13B shows the sequence of TK1-MO-FCGRA-CAR-5 (SEQ ID NO: 47). Amino acids 1 to 18 are the signal peptide (SP), amino acids 19 to 275 are the anti-TK1 ScFv, amino acids 276 to 290 are the GS linker, amino acids 291 to 409 are the IgG4 119 amino acid hinge, and amino acids 410 to 430 are the FCGR3A membrane penetrating domain, amino acids 431 to 455 are the FCGR3A cytosol domain, and amino acids 456 to 497 are the FCER1G cytosol domain.
14A shows a block diagram of the order of elements within the chimeric receptor TK1-MO-FCGRA-CAR-6. 14B shows the sequence of TK1-MO-FCGRA-CAR-6 (SEQ ID NO: 48). Amino acids 1 to 18 are the signal peptide (SP), amino acids 19 to 275 are the anti-TK1 ScFv, amino acids 276 to 290 are the GS linker, amino acids 291 to 519 are the IgG4 long hinge, and amino acids 520 to 540 are the FCGR3A transmembrane domain, amino acids 541 to 565 are the FCGR3A cytosol domain, and amino acids 566 to 607 are the FCER1G cytosol domain.
15A shows a block diagram of the order of elements within the chimeric receptor TK1-MO-FCG2A-CAR-1. 15B shows the sequence of TK1-MO-FCG2A-CAR-1 (SEQ ID NO: 49). Amino acids 1 to 18 are the signal peptide (SP), amino acids 19 to 275 are the anti-TK1 ScFv, amino acids 276 to 290 are the GS linker, amino acids 291 to 312 are the FCGR2A transmembrane domain, and amino acids 313 to 390 are the FCGR2A cytosol It is a domain.
16A shows a block diagram of the order of elements within the chimeric receptor TK1-MO-FCG2A-CAR-2. 16B shows the sequence of TK1-MO-FCG2A-CAR-2 (SEQ ID NO: 50). Amino acids 1-18 are the signal peptide (SP), amino acids 19-275 are the anti-TK1 ScFv, amino acids 276-290 are the GS linker, amino acids 291-358 are the CD8 hinge mutated to C339S and C356S, and amino acid 359 to 380 is the FCGR2A transmembrane domain, and amino acids 381 to 458 are the FCGR2A cytosol domain.
17A shows a block diagram of the order of elements within the chimeric receptor TK1-MO-FCG2A-CAR-3. 17B shows the sequence of TK1-MO-FCG2A-CAR-3 (SEQ ID NO: 51). Amino acids 1 to 18 are the signal peptide (SP), amino acids 19 to 275 are the anti-TK1 ScFv, amino acids 276 to 290 are the GS linker, amino acids 291 to 358 are part of the CD8 hinge, and amino acids 359 to 380 are the FCGR2A membrane penetrating domain, and amino acids 381 to 458 are the FCGR2A cytosol domain.
18A shows a block diagram of the order of elements within the chimeric receptor TK1-MO-FCG2A-CAR-4. 18B shows the sequence of TK1-MO-FCG2A-CAR-4 (SEQ ID NO: 52). Amino acids 1-18 are the signal peptide (SP), amino acids 19-275 are the anti-TK1 ScFv, amino acids 276-290 are the GS linker, amino acids 291-303 are the IgG4 short hinge, and amino acids 304-325 are the FCGR2A transmembrane domain, and amino acids 326 to 403 are the FCGR2A cytosol domain.
19A shows a block diagram of the order of elements within the chimeric receptor TK1-MO-FCG2A-CAR-5. 19B shows the sequence of TK1-MO-FCG2A-CAR-5 (SEQ ID NO: 53). Amino acids 1 to 18 are the signal peptide (SP), amino acids 19 to 275 are the anti-TK1 ScFv, amino acids 276 to 290 are the GS linker, amino acids 291 to 409 are the IgG4 119 amino acid hinge, and amino acids 410 to 431 are the FCGR2A membrane penetrating domain, and amino acids 432 to 509 are the FCGR2A cytosol domain.
20A shows a block diagram of the order of elements within the chimeric receptor TK1-MO-FCG2A-CAR-6. 20B shows the sequence of TK1-MO-FCG2A-CAR-6 (SEQ ID NO: 54). Amino acids 1-18 are the signal peptide (SP), amino acids 19-275 are the anti-TK1 ScFv, amino acids 276-290 are the GS linker, amino acids 291-519 are the IgG4 long hinge, and amino acids 520-541 are the FCGR2A transmembrane domain, and amino acids 542 to 619 are the FCGR2A cytosol domain.
21 is a schematic diagram illustrating chimeric receptors.
22 is a schematic diagram showing macrophages expressing chimeric receptors. As shown, the chimeric receptor comprises a cytosol domain of a toll-like receptor, a transmembrane domain, and a ScFv specific for a ligand. Arrows show signaling that polarizes macrophages upon binding of ScFv ligands.
23 is a schematic representation of different macrophage receptors that can be used to construct chimeric receptors.
24a to 24c. 24A is a schematic diagram showing the Fc gamma receptor III signaling cascade leading to cell activation. 24B is a schematic diagram showing the Fc gamma receptor III signaling cascade leading to inhibition of calcium flux and proliferation. 24C is a schematic diagram showing the Fc gamma receptor III signaling cascade leading to apoptosis.
25 is a schematic diagram illustrating the Toll-like receptor signaling cascade.
26 shows a graph illustrating flow cytometry to confirm that the expressed antibody fragment binds to the ligand of interest.
27 shows two images showing phenotypic changes in macrophages after transduction with chimeric receptors.
28 shows two images confirming the expression of chimeric receptors in monocytes.
29 shows three scatter plots of fluorescence activated cell sorting demonstrating the expression of dTomato. The leftmost plot represents a control where only 0.58% of the cells fluoresce to show expression of dTomato. The right two plots show a transduction efficiency of 27.1% after transduction.
30 shows six scatter plots of fluorescence activated cell sorting demonstrating retention of the dye (Alexa 647) and expression of CD80, CD163, CD206, and CD14 in macrophages transduced with the chimeric receptor.
31 shows a histogram demonstrating the relative expression levels of CD80, CD163, CD206, and CD14 in macrophages transduced with the chimeric receptor.
32 shows six images of transduced macrophages expressing chimeric receptors that detect, attack, and induce apoptosis in a lung cancer cell line (NCI-H460).

Chimeric receptors are described herein. A chimeric receptor has a cytoplasmic domain; transmembrane domain; and an extracellular domain. In an embodiment, the cytoplasmic domain comprises a cytoplasmic portion of a receptor that polarizes a macrophage when activated. In a further embodiment, the wild-type protein comprising a cytoplasmic portion does not comprise the extracellular domain of the chimeric receptor. In an embodiment, binding of the ligand to the extracellular domain of the chimeric receptor activates the intracellular portion of the chimeric receptor. Activation of the intracellular portion of the chimeric receptor can polarize macrophages to either M1 or M2 macrophages.

In certain embodiments, the cytoplasmic portion of the chimeric receptor is a toll-like receptor, a myeloid differentiation primary response protein (MYD88) (SEQ ID NO: 19), a toll-like receptor 3 (TLR3) (SEQ ID NO: 1), a toll-like receptor 4 (TLR4) (SEQ ID NO: 3), toll-like receptor 7 (TLR7) (SEQ ID NO: 7), toll-like receptor 8 (TLR8) (SEQ ID NO: 9), toll-like receptor 9 (TLR9) (SEQ ID NO: 11 ), myelin and lymphocyte protein (MAL) (SEQ ID NO: 21), interleukin-1 receptor-associated kinase 1 (IRAK1) (SEQ ID NO: 23), low affinity immunoglobulin gamma Fc region receptor III-A (FCGR3A) (SEQ ID NO: 15), the low affinity immunoglobulin gamma Fc region receptor II-a (FCGR2A) (SEQ ID NO: 13), the cytoplasmic domain from the high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) (SEQ ID NO: 19), or any of the foregoing It may include a sequence having 90% or more sequence identity to any one of the cytoplasmic domains. In certain embodiments, the cytoplasmic portion is not a cytoplasmic domain from a toll-like receptor, FCGR3A, IL-1 receptor, or IFN-gamma receptor. In an embodiment, the cytosol portion can be any polypeptide that, when activated, causes polarization of a macrophage.

In a further embodiment, examples of ligands that bind to the extracellular domain include thymidine kinase (TK1), hypoxanthine-guanine phosphoribosyltransferase (HPRT), receptor tyrosine kinase-like orphan receptor 1 (ROR1) , Mucin-16 (MUC-16), Epidermal Growth Factor Receptor vIII (EGFRvIII), Mesothelin, Human Epidermal Growth Factor Receptor 2 (HER2), Carcinoma Embryonic Antigen (CEA), B-Cell Maturation Antigen ( BCMA ), Gly pecan 3 (GPC3), fibroblast activating protein (FAP), erythropoietin-producing hepatocellular carcinoma A2 (EphA2), natural killer group 2D (NKG2D) ligand, disialoganglioside 2 (GD2), CD19, CD20, CD30, CD33, CD123, CD133, CD138, and CD171, but is not limited thereto. In certain embodiments, the ligand is not TK1 or HPRT.

Antibodies that can be tailored to generate the extracellular domain of a chimeric receptor are well known in the art and commercially available. Examples of commercially available antibodies are anti-HGPRT, clone 13H11.1 (EMD Millipore), anti-ROR1 (ab135669) (Abcam), anti-MUC1[EP1024Y] (ab45167) (Abcam), anti-MUC16[X75] (ab1107) ) (Abcam), anti-EGFRvIII [L8A4] (Absolute antibody), anti-mesothelin [EPR2685 (2)] (ab134109) (Abcam), HER2 [3B5] (ab16901) (Abcam), anti-CEA (LS- C84299-1000) (LifeSpan BioSciences), anti-BCMA (ab5972) (Abcam), anti-glypican 3 [9C2] (ab129381) (Abcam), anti-FAP (ab53066) (Abcam), anti-EphA2 [RM- 0051-8F21] (ab73254) (Abcam), anti-GD2 (LS-C546315) (LifeSpan BioSciences), anti-CD19 [2E2B6B10] (ab31947) (Abcam), anti-CD20 [EP459Y] (ab78237) (Abcam), anti-CD30[EPR4102](ab134080)(Abcam), anti-CD33[SP266](ab199432)(Abcam), anti-CD123(ab53698)(Abcam), anti-CD133(BioLegend), anti-CD123(1A3H4) ab181789 (Abcam), and anti-CD171 (L1.1) (Invitrogen antibodies). Techniques for generating antibody fragments, such as ScFvs, from known antibodies are routine in the art. Additionally, it is also common in the art to generate sequences encoding such fragments and to include them recombinantly as part of a polynucleotide encoding a chimeric protein.

In certain embodiments, the extracellular domain may include an antibody or fragment thereof that specifically binds a ligand. Examples of antibodies and fragments thereof include IgA, IgD, IgE, IgG, IgM, Fab fragments, F(ab') ₂ fragments, monovalent antibodies, ScFv fragments, scRv-Fc fragments, IgNARs, hcIgG, VhH antibodies, nanobodies, and an alphabody, but is not limited thereto. In a further embodiment, the extracellular domain may include any amino acid sequence that enables specific binding of a ligand, including but not limited to dimerization domains, receptors, binding pockets, and the like.

In embodiments, a chimeric receptor may contain a linker. Without limitation, a linker may be positioned between the extracellular and transmembrane domains of the chimeric receptor. Without limitation, the linker can be a G linker, GS linker, G4S linker, EAAAK linker, PAPAP linker, or (Ala-Pro) _n linker. Other examples of linkers are well known in the art.

In an embodiment, a chimeric receptor may contain a hinge region. Without limitation, the hinge region may be located between the extracellular and transmembrane domains of the chimeric receptor. In a further embodiment, a hinge region may be located between the linker and the transmembrane domain. Without limitation, the linker can be a leucine rich repeat (LRR), or a hinge region from a toll-like receptor, IgG, IgG4, CD8m, or FcγIIIa-hing. In an embodiment, a cysteine in the hinge region may be replaced with a serine. Other examples of hinge regions are well known in the art.

The chimeric receptor described herein may be one or more of SEQ ID NOs: 1, 3, 4, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and 25 to 34, a fragment of any of these, and/or at least 90% sequence identity to one or more of SEQ ID NOs: 1, 3, 4, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and 25 to 34 or fragments thereof. It may contain polypeptides with Examples of chimeric receptors include, but are not limited to, SEQ ID NOs: 35-54 or homologues or fragments thereof. In another embodiment, the polypeptide comprises an amino acid sequence selected from the group consisting of polypeptides having at least 90% sequence identity to one or more of SEQ ID NOs: 35-54.

Embodiments include a nucleic acid sequence comprising a nucleic acid sequence encoding a chimeric receptor as described above. Examples of such nucleic acids include one or more of SEQ ID NOs: 2, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24, fragments of any of these, and/or SEQ ID NOs: 2, 6, 8 , 10, 12, 14, 16, 18, 20, 22, and 24, or a nucleic acid having at least 90% sequence identity to one or more fragments thereof. Additional examples include nucleic acids encoding one or more of SEQ ID NOs: 24-54 and fragments of any of these.

In an embodiment, a chimeric receptor can be glycosylated, pegylated, and/or otherwise post-translationally modified. Additionally, the nucleic acid sequence may be part of a vector. By way of example, the vector may be a plasmid, phage, cosmid, artificial chromosome, viral vector, AAV vector, adenoviral vector, or lentiviral vector. In certain embodiments, a nucleic acid encoding a chimeric receptor may be operably linked to a promoter and/or other regulatory sequences (eg, enhancers, silencers, insulators, locus control regions, cis-acting elements, etc.).

A further embodiment includes a cell comprising a chimeric receptor or a nucleic acid encoding a chimeric receptor. Non-limiting examples of such cells include bone marrow cells, bone marrow progenitor cells, monocytes, neutrophils, basophils, eosinophils, megakaryocytes, T cells, B cells, natural killer cells, leukocytes, lymphocytes, dendritic cells, and macrophages.

Embodiments include contacting a macrophage comprising a chimeric receptor with a ligand for an extracellular domain of the chimeric receptor; and a method of polarizing macrophages by binding the ligand to the extracellular domain of the chimeric receptor. Binding of the ligand to the extracellular domain of the chimeric receptor activates the cytoplasmic portion, and activation of the cytoplasmic portion polarizes the macrophage.

Nucleotide, polynucleotide, or nucleic acid sequence refers to both double-stranded or single-stranded DNA or RNA and transcription products of DNA or RNA in monomeric and dimeric (so-called tandem) forms according to the present invention. It will be understood.

Aspects of the present invention start with separation methods such as, for example, ion exchange chromatography, by exclusion based on molecular size, or by fractionation techniques based on affinity or, alternatively, solubility in different solvents, or Starting from genetic engineering methods such as amplification, cloning, and subcloning, it relates to a nucleotide sequence capable of being isolated, purified, or partially purified, the sequence being capable of being carried by a vector.

A nucleotide sequence fragment will be understood to designate any nucleotide fragment, by way of non-limiting example, at least 8, 12, 20, 25, 50, 75, 100, 200, 300, 400, 500, It may contain a length of 1000 or more contiguous nucleotides.

It will be understood that a specific fragment of a nucleotide sequence designates any nucleotide fragment having one or more fewer nucleotides or bases after alignment and comparison with the corresponding wild-type sequence.

As used herein, a homologous nucleotide sequence is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, or 99.7% or more of a nucleotide sequence having a percent identity with a base of the nucleotide sequence It is understood that this percentage is purely statistical and it is possible to randomly distribute the differences between two nucleotide sequences over their length.

A specific homologous nucleotide sequence in the meaning of the present invention is understood to mean a homologous sequence having one or more sequences of a specific fragment as defined above. A “specific” homologous sequence may include, for example, a sequence corresponding to a sequence of a genomic sequence or a fragment thereof representative of a variant of a genomic sequence. Thus, these specific homologous sequences may correspond to mutations linked to mutations in the sequence, in particular to truncations, substitutions, deletions, and/or additions of one or more nucleotides. Homologous sequences may likewise correspond to variations linked to degeneracy of the genetic code.

The term “degree or percentage of sequence homology” as defined herein refers to “the degree or percentage of sequence identity between two sequences after optimal alignment”.

Two nucleotide sequences are said to be "identical" if the sequences of amino acids or nucleotide residues in the two sequences are identical when aligned for maximal correspondence as described below. Sequence comparisons between two (or more) peptides or polynucleotides typically compare the sequences of two optimally aligned sequences across segments or "comparison windows" to identify and compare local regions of sequence similarity. done by doing Optimal alignment of sequences for comparison is described in Smith and Waterman, Ad. App. Math 2: 482 (1981) by the homology algorithm of Neddleman and Wunsch, J. Mol. Biol. 48: 443 (1970)] by the homology alignment algorithm [Pearson and Lipman, Proc. Natl. Acad. Sci . (USA) 85: 2444 (1988)], a computerized implementation of these algorithms (GAP, BESTFIT, FASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wisconsin). , and TFASTA), or by visual inspection.

"Percentage of sequence identity" (or degree of identity) is determined by comparing two optimally aligned sequences over a window of comparison, wherein the portion of the peptide or polynucleotide sequence within the window of comparison is for optimal alignment of the two sequences. It may contain additions or deletions (i.e., gaps) compared to a reference sequence (which does not contain additions or deletions). Determine the number of positions where the same amino acid residue or nucleic acid base occurs in both sequences to yield the number of matching positions, divide the number of matching positions by the total number of positions within the comparison window, and multiply the result by 100 to obtain the sequence number. The percentage is calculated by calculating the percentage of identity.

The definition of sequence identity given above is the definition to be used by one skilled in the art. The definition itself does not require the aid of any algorithm, an algorithm only helping to achieve optimal alignment of sequences rather than calculating sequence identity.

From the definitions given above, we arrive at the conclusion that there is only one well-defined value for sequence identity between two compared sequences, and that this value corresponds to the value obtained for the best or optimal alignment.

BLAST N, software conventionally used by the present inventors and commonly used by those skilled in the art to compare and determine identity between two sequences, and available at the website worldwideweb.ncbi.nlm.nih.gov/gorf/bl2.html or BLAST P "BLAST 2 sequences", the gap cost, which depends on the sequence length to be compared, is chosen directly by the software (ie 11.2 for lengths greater than 85 for the substitution matrix BLOSUM-62).

As used herein, a complementary nucleotide sequence of a sequence is understood to mean any DNA whose nucleotides are complementary to those of the sequence and whose orientation is reversed (antisense sequence).

Hybridization under stringent conditions with nucleotide sequences as used herein refers to hybridization under conditions of temperature and ionic strength selected in such a way that they allow maintenance of hybridization between two fragments of complementary DNA. I understand.

As an example, the high stringency conditions of the hybridization step with the purpose of defining the nucleotide fragments described above are advantageously as follows.

Hybridization is performed at a preferential temperature of 65° C. in the presence of SSC buffer, 1×SSC corresponding to 0.15 M NaCl and 0.05 M Na citrate. For example, the washing step can be as follows: 2 x SSC at ambient temperature, then 2 x SSC, 0.5% SDS wash at 65 °C twice; 65° C. for 10 minutes each; 2 × 0.5 × SSC, 0.5% SDS.

Medium stringency conditions, e.g., using a temperature of 42°C in the presence of 2×SSC buffer, or lower stringency conditions, e.g., a temperature of 37°C in the presence of 2×SSC buffer, each two sequences Overall, less significant complementarity is required for hybridization between

Following the teachings of Sambrook et al., 1989, the stringent hybridization conditions described above for polynucleotides having a size of approximately 350 bases will be adjusted by one skilled in the art for oligonucleotides of larger or smaller size.

Among the nucleotide sequences described herein are those that can be used as primers or probes in methods that make it possible to obtain homologous sequences, and these methods, such as polymerase chain reaction (PCR), nucleic acid cloning, and sequencing, are well known to those skilled in the art. It is known.

Among the nucleotide sequences are those that can be used as primers or probes in methods that make it possible to determine the presence of a specific nucleic acid, one of its fragments, or one of its variants as defined below. In an embodiment, the nucleotide sequence will comprise fragments of SEQ ID NOs: 2, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 encoding the transmembrane domain, the cytosol domain, or portions thereof. can Additional fragments may include linkers, such as nucleotides encoding one or more of SEQ ID NOs: 26-34, hinges, or nucleotide sequences encoding fragments thereof. Additional fragments may include fragments of nucleotide sequences encoding one or more of SEQ ID NOs: 35-54.

Fragments of nucleotide sequences can be obtained by specific amplification, eg PCR, or after digestion of nucleotide sequences with appropriate restriction enzymes, these methods being described in particular in the work of Sambrook et al., 1989. has been In addition, such fragments can be obtained by standard gene synthesis techniques available from companies such as GenScript®. Such representative fragments can likewise be obtained by chemical synthesis according to methods well known to those skilled in the art.

Modified nucleotide sequences are obtained by mutagenesis according to techniques well known to those skilled in the art and are modifications to the wild-type sequence, for example, in particular regulatory sequences of polypeptide expression which result in alteration of the expression rate of the polypeptide or regulation of the replication cycle and/or It will be understood to mean any nucleotide sequence containing a mutation of the promoter sequence.

A modified nucleotide sequence will likewise be understood to mean any nucleotide sequence encoding a modified polypeptide as defined below.

A nucleotide sequence encoding a chimeric receptor is disclosed comprising a nucleotide sequence selected from SEQ ID NOs: 2, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 or one of fragments thereof. Such fragments may encode specific domains, such as transmembrane domains or cytosol domains or portions thereof. Additional nucleotide sequences encoding the chimeric receptor may include nucleotide sequences encoding linkers, hinges, or fragments thereof, such as nucleotides encoding one or more of SEQ ID NOs: 26-34. The nucleotide sequence encoding the chimeric receptor may further comprise a nucleotide sequence encoding one or more of SEQ ID NOs: 35-54 or fragments thereof.

Embodiments likewise a) a nucleotide sequence encoding one or more of the nucleotide sequences of SEQ ID NOs: 2, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24, or one or more of SEQ ID NOs: 25 to 54 , or one of their fragments; b) the nucleotide sequence of a specific fragment of the sequence as defined in a); c) a homologous nucleotide sequence having at least 80% identity to a sequence as defined in a) or b); d) a complementary nucleotide sequence or sequence of RNA corresponding to a sequence as defined in a), b), or c); and e) a nucleotide sequence selected from a nucleotide sequence modified by a sequence as defined in a), b), c), or d).

Among the nucleotide sequences are the nucleotide sequences of SEQ ID NOs: 2, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24, nucleotide sequences encoding one or more of SEQ ID NOs: 25 to 54, or fragments thereof; A nucleotide sequence encoding at least one of SEQ ID NOs: 2, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24, at least one of SEQ ID NOs: 25 to 54, or fragments thereof and 80 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, There is any nucleotide sequence that has a homology of greater than 97%, 98%, 99%, 99.5%, 99.6%, or 99.7% identity. Homologous sequences can include, for example, sequences corresponding to wild-type sequences. In the same way, these specific homologous sequences may correspond to mutations linked to mutations in the wild-type sequence, in particular to truncations, substitutions, deletions, and/or additions of one or more nucleotides. As will be apparent to those skilled in the art, such homologs are readily generated and identified using standard techniques and publicly available computer programs such as BLAST. As such, each of the homologues noted above is described herein and should be considered fully described.

Embodiments include chimeric receptors encoded by the nucleotide sequences described herein, or fragments thereof, the sequences of which are represented by the fragments. An amino acid sequence corresponding to a polypeptide that can be encoded according to one of the three possible reading frames of one or more of the sequences of SEQ ID NOs: 35-54.

Embodiments likewise include a polypeptide selected from one or more of the amino acid sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and 25 to 54, or fragments thereof It relates to a chimeric receptor characterized in that it comprises one.

Among the polypeptides are polypeptides of the amino acid sequences of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and 25 to 54, or fragments thereof or SEQ ID NO: 1, depending on the embodiment. , 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and one or more of the sequences 25 to 54 or fragments thereof and 80%, 81%, 82%, 83%, 84 %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, There are any other polypeptides that have a homology of 99.6%, or greater than 99.7% identity. As will be apparent to those skilled in the art, such homologs are readily generated and identified using standard techniques and publicly available computer programs such as BLAST. As such, each of the homologues noted above is described herein and should be considered fully described.

Embodiments also include a) specific fragments of at least 5 amino acids of a polypeptide of amino acid sequence; b) a polypeptide homologous to a polypeptide as defined in a); c) a specific biologically active fragment of a polypeptide as defined in a) or b); and d) a polypeptide selected from polypeptides modified by a polypeptide as defined in a), b), or c).

As used herein, the terms polypeptide, peptide, and protein are interchangeable.

In an embodiment, a chimeric receptor can be glycosylated, pegylated, and/or otherwise post-translationally modified. In a further embodiment, glycosylation, pegylation, and/or other post-translational modifications may occur in vivo or in vitro and/or may be performed using chemical techniques. In a further embodiment, any glycosylation, pegylation, and/or other post-translational modifications may be N-linked or O-linked.

In an embodiment, either of the chimeric receptors may be enzymatically or functionally active such that when the extracellular domain is bound by a ligand, a signal is transduced to polarize the macrophage.

As used herein, a “polarized macrophage” is a macrophage that correlates with an M1 or M2 macrophage phenotype. M1 polarized macrophages secrete IL-12 and IL-23. Determination of M1 polarized macrophages can be made by measuring the expression of IL-12 and/or IL-23 using standard cytokine assays and comparing that expression to expression by newly differentiated, unpolarized macrophages. there is. Alternatively, the determination can be made by determining whether the cells are CD14+, CD80+, CD206+, and CDCD163−. M2 polarized macrophages secrete IL-10. Determination of M2 polarized macrophages can be performed by measuring expression of IL-10 using a standard cytokine assay and comparing that expression to expression by newly differentiated, unpolarized macrophages. Alternatively, the determination can be made by determining whether the cells are CD14+, CD80-, CD206+, and CDCD163+.

An aspect of the present invention relates to chimeric receptors obtained by genetic recombination, or alternatively by chemical synthesis, and thus they may contain unnatural amino acids as described below.

A “polypeptide fragment” according to an embodiment is understood to designate a polypeptide containing at least 5 contiguous amino acids, preferably 10 contiguous amino acids or 15 contiguous amino acids.

In this specification, a specific polypeptide fragment is understood to designate a contiguous polypeptide fragment encoded by a specific fragment nucleotide sequence.

A “homologous polypeptide” will be understood to designate a polypeptide that has certain modifications relative to a native polypeptide, particularly deletion, addition, or substitution, truncation, extension, chimeric fusion, and/or mutation of one or more amino acids. Among the homologous polypeptides, those whose amino acid sequences have at least 80% or 90% homology to the amino acid sequences of the polypeptides described herein are preferred.

A “specific homologous polypeptide” will be understood to designate a homologous polypeptide as defined above and having a specific fragment of a polypeptide described herein.

In case of substitution, one or more consecutive or non-contiguous amino acids are replaced by an “equivalent” amino acid. The expression "equivalent" amino acid herein is intended to designate any amino acid which may be substituted by one of the amino acids of the base structure which does not essentially alter the biological activity of the corresponding peptide, which will be defined by will be. As will be apparent to those skilled in the art, such substitutions are readily generated and confirmed using standard molecular biology techniques and publicly available computer programs such as BLAST. As such, each substitution noted above is described herein and should be considered fully described.

These equivalent amino acids can be determined based on their structural homology with the amino acids they replace, or the results of comparative tests of biological activity between different polypeptides that can be performed.

By way of non-limiting example, the possibility of substitutions that can be made without causing extensive modification of the biological activity of the corresponding modified polypeptide, e.g., replacement of leucine by valine or isoleucine, replacement of aspartic acid by glutamic acid , replacement of glutamine by asparagine, replacement of arginine by lysine, etc. will be mentioned, and reverse substitutions can of course be envisioned under the same conditions.

In a further embodiment, substitutions are limited to substitutions at non-conserved amino acids among other proteins with similar identified enzymatic activities. For example, one skilled in the art can align proteins of the same function in similar organisms and determine which amino acids are generally conserved among proteins of that function. An example of a program that can be used to create such an alignment is wordlwideweb.charite.de/bioinf/strap/, with a database provided by NCBI.

Thus, according to one embodiment, substitutions or mutations may be made at positions that are normally conserved among proteins of that function. In a further embodiment, the nucleic acid sequences are mutated or substituted such that the amino acids they encode do not change (degenerate substitutions and/or mutations) and/or any resulting amino acid substitutions or mutations are generally conserved among functional proteins. It can be mutated or substituted to occur in position.

A specific homologous polypeptide likewise corresponds to a polypeptide encoded by a specific homologous nucleotide sequence as defined above, and thus corresponds to variants that may be mutated or present in the wild-type sequence, in particular truncation, substitution of one or more amino acid residues. , deletions, and/or additions are included within this definition.

As used herein, a "specific biologically active fragment of a polypeptide" will be understood to designate a specific polypeptide fragment, as defined above, which has one or more of the characteristics of a polypeptide described in particular. In certain embodiments, the peptide may behave as a chimeric antigen receptor that polarizes macrophages when activated.

As used herein, a "modified polypeptide" of a polypeptide is understood to designate a polypeptide obtained by chemical synthesis or genetic recombination, as described below, having one or more modifications relative to the wild-type sequence. These modifications may or may not relate to amino acids that are at the origin of the specificity and/or activity, or at the origin of the structural conformation, localization, and membrane insertion ability of the polypeptides described herein. Thus, it would be possible to generate equivalent, increased or reduced activity polypeptides, and equivalent, narrower or broader specificity polypeptides. Among modified polypeptides, it is necessary to mention polypeptides in which up to 5 or more amino acids can be modified, truncated at the N-terminal or C-terminal end, even deleted or added.

Methods that make it possible to demonstrate control on eukaryotic or prokaryotic cells are well known to those skilled in the art. It is likewise well understood and described below that it will be possible to use nucleotide sequences encoding modified polypeptides for regulation, eg via vectors.

Modified polypeptides described above can be obtained by using combinatorial chemistry, wherein portions of the polypeptide are systematically varied before testing them on models, cell cultures, or microorganisms, e.g., to determine which is the most active or desired. It is possible to select compounds with the properties that

Chemical synthesis likewise has the advantage of being able to use non-natural amino acids or non-peptide linkages.

Thus, it may be of interest to use non-natural amino acids, eg in the D form, or amino acid analogs, particularly eg sulfur-containing forms, to improve the duration of life of polypeptides.

Finally, it will be possible to incorporate the structure of the polypeptide, its specific or modified homologous form, into a polypeptide type or other chemical structure. Thus, it may be of interest to provide compounds that are not recognized by proteases at the N-terminal and C-terminal ends.

Nucleotide sequences encoding the polypeptides are likewise disclosed herein.

Embodiments likewise relate to nucleotide sequences that can be used as primers or probes, characterized in that the sequences are selected from the nucleotide sequences described herein.

Likewise various embodiments include chimeric receptors encoded by nucleotide sequences obtainable by purification from natural polypeptides, genetic recombination, or chemical synthesis by procedures well known to those skilled in the art and particularly described below. It is well understood that it relates to specific polypeptides. In the same way, labeled or unlabeled monoclonal or polyclonal antibodies directed against a specific polypeptide encoded by a nucleotide sequence are also encompassed by the present invention.

Embodiments further relate to the use of the nucleotide sequences as primers or probes for detection and/or amplification of nucleic acid sequences.

Thus, nucleotide sequences according to embodiments are in particular PCR techniques (polymerase chain reaction) (Erlich, 1989); Innis et al., 1990; Rolfs et al., 1991; and White et al., 1997]) to amplify nucleotide sequences.

These oligodeoxyribonucleotides or oligoribonucleotide primers are advantageously at least 8 nucleotides in length, preferably at least 12 nucleotides and even more preferably at least 20 nucleotides in length.

As an alternative to PCR, other amplification techniques of target nucleic acids can advantageously be used.

The nucleotide sequences described herein, particularly the primers, can likewise be used in other procedures of amplification of target nucleic acids, such as: the TAS technology described by Kwoh et al., 1989 (transcription-based amplification system); the 3SR technique (self-maintaining sequence replication) described by Guatelli et al., 1990; the NASBA technique (nucleic acid sequence based amplification) described by Kievitis et al., 1991; SDA technology (strand displacement amplification) (Walker et al., 1992); TMA technology (transcription-mediated amplification).

Polynucleotides comprising chimeric receptors can also serve as probes and be used in the following techniques of amplification or modification of nucleic acids: described by Landegren et al., 1988 and Barany et al., 1991. LCR technology using thermostable ligases, improved by (ligase chain reaction); the RCR technique (repair chain reaction) described by Segev, 1992; the CPR technique described by Duck et al., 1990 (cyclic probe reaction); Described by Miele et al., 1983 and notably Chu et al., 1986, Lizardi et al., 1988, followed by Burg et al. as well as Stone et al. , 1996], an amplification technique using Q-beta replicase.

If the target polynucleotide to be detected is probably RNA, eg mRNA, before using an amplification reaction with the help of one or more primers, or before using a detection procedure with the help of one or more probes, from the RNA contained in the biological sample It will be possible to use enzymes of the reverse transcriptase type to obtain cDNA. Thus, the resulting cDNA will serve as a target for primer(s) or probe(s) used in an amplification or detection procedure.

The detection probe will be selected in such a way that it hybridizes with the target sequence or an amplicon generated from the target sequence. As a sequence, such a probe will advantageously have a sequence of at least 12 nucleotides, in particular at least 20 nucleotides, preferably at least 100 nucleotides.

Embodiments also include nucleotide sequences that can be used as probes or primers, characterized in that they are labeled with a radioactive compound or a non-radioactive compound.

Unlabeled nucleotide sequences can be used directly as probes or primers, but to obtain probes that can be used in many applications, sequences are usually radioactive isotopes ( ³² P, ³⁵ S, ³ H, ¹²⁵ I) or non-radioactive Labeled with molecules (biotin, acetylaminofluorene, digoxigenin, 5-bromodeoxyuridine, fluorescein).

Examples of non-radioactive labeling of nucleotide sequences are described, for example, in French Patent No. 78.10975 or Urdea et al. or Sanchez-Pescador et al., 1988.

In the latter case, it would also be possible to use one of the labeling methods described in patents FR-2 422 956 and FR-2 518 755.

Hybridization techniques can be performed in a variety of ways (Matthews et al., 1988). The most common method consists of immobilizing a nucleic acid extract of a cell on a support (eg nitrocellulose, nylon, polystyrene), and incubating the immobilized target nucleic acid with a probe under well-defined conditions. After hybridization, excess probe is removed, and the resulting hybrid molecule is detected by an appropriate method (measurement of radioactivity, fluorescence, or enzymatic activity linked to the probe).

Various embodiments likewise include the nucleotide sequences or polypeptide sequences described herein, characterized in that they are covalently or non-covalently immobilized on a support.

According to another advantageous mode of using a nucleotide sequence, the latter can be used immobilized on a support and thus can act to capture, by specific hybridization, a target nucleic acid obtained from a biological sample to be tested. If necessary, the solid support is separated from the sample, and then the hybridization complex formed between the capture probe and the target nucleic acid is detected with the aid of a second probe labeled with a readily detectable element, a so-called detection probe.

Another aspect is a vector for cloning and/or expression of a sequence, characterized in that it contains a nucleotide sequence described herein.

Vectors are likewise provided which are characterized in that they contain elements enabling integration, expression, and/or secretion of the nucleotide sequence in the determined host cell.

The vector may then contain a promoter, signals for initiation and termination of translation, as well as appropriate transcriptional regulatory regions. It can be stably maintained within the host cell and optionally have specific signals that direct the secretion of the translated protein. These different elements can be selected as a function of the host cell used. For this purpose, the nucleotide sequences described herein can be inserted into an autonomously replicating vector in a host of choice, or into an integrated vector of a host of choice.

Such vectors will be prepared according to methods currently used by those skilled in the art, and the resulting clones can be subjected to standard methods such as, for example, calcium phosphate precipitation, lipofection, electroporation, and heat shock. can be introduced into a suitable host by

Vectors are thus, for example, of plasmid or viral origin. Examples of vectors for expression of the polypeptides described herein include plasmids, phages, cosmids, artificial chromosomes, viral vectors, AAV vectors, baculovirus vectors, adenovirus vectors, lentiviral vectors, retroviral vectors, chimeric viral vectors, and chimeric adenoviridae, such as AD5/F35.

These vectors are useful for transforming host cells for cloning or expressing the nucleotide sequences described herein.

Embodiments likewise include host cells transformed with the vector.

These cells can be obtained by introducing into a host cell a nucleotide sequence inserted into a vector as defined above, followed by culturing the cell under conditions allowing replication and/or expression of the transfected nucleotide sequence.

Host cells include, for example, bacterial cells (Olins and Lee, 1993), but likewise yeast cells (Buckholz, 1993), as well as plant cells, such as Arabidopsis sp ., and animal cells. , in particular mammalian cells (Edwards and Aruffo, 1993), eg HEK 293 cells, HEK 293T cells, Chinese Hamster Ovary (CHO) cells, bone marrow cells, bone marrow progenitor cells, monocytes, neutrophils, basophils, eosinophils , megakaryocytes, T cells, B cells, natural killer cells, leukocytes, lymphocytes, dendritic cells, and macrophages; [Luckow, 1993]), prokaryotic or eukaryotic systems.

Embodiments likewise relate to organisms comprising one of the transformed cells.

Obtaining a transgenic organism expressing one or more of the nucleic acids or portions of the nucleic acids can be performed, for example, in rats, mice, or rabbits according to methods well known to those skilled in the art, such as by viral or non-viral transfection. Transgenic organisms expressing more than one gene can be obtained by transfection of multiple copies of a gene, either of a ubiquitous nature or under the control of a strong promoter that is selective for one type of tissue. It will likewise be possible to obtain transgenic organisms by homologous recombination in embryonic cell lines, transfer of these cell lines into embryos, selection of affected chimeras at the germline level, and growth of the chimeras.

Transformed cells as well as transformed organisms can be used in procedures for the production of recombinant polypeptides.

It is currently possible to produce recombinant polypeptides in relatively large quantities either using cells transformed by expression vectors or by genetic engineering using transformed organisms.

A procedure for the preparation of a polypeptide, such as a chimeric receptor, in recombinant form, characterized by the use of a vector and/or a cell transformed by the vector and/or a transformed organism comprising one of the transformed cells, is itself an invention. included in

As used herein, “transformation” and “transformed” refer to the introduction of a nucleic acid into a cell, whether prokaryotic or eukaryotic. Additionally, as used herein, "transformation" and "transformed" need not refer to growth control or growth deregulation.

During the procedure for making a polypeptide, such as a chimeric receptor, in recombinant form, one of the vector, and/or the cell transformed by the vector, and/or the transformed cell, containing a nucleotide sequence, such as those encoding the chimeric receptor, is used. There are manufacturing procedures that use transgenic organisms that include.

Thus, variants, as used herein, can consist of producing a recombinant polypeptide fused to a "carrier" protein (chimeric protein). An advantage of this system is that it will allow stabilization of the recombinant product and/or reduction of proteolysis, increase of solubility during in vitro renaturation and/or simplification of purification when the fusion partner has affinity for a specific ligand. that it can

More specifically, embodiments relate to a procedure for producing a polypeptide comprising the following steps: a) culturing the transformed cell under conditions allowing expression of the recombinant polypeptide of the nucleotide sequence; b) recovering the recombinant polypeptide, if necessary.

When a procedure for producing a polypeptide, such as a chimeric receptor, uses a transgenic organism, the recombinant polypeptide can then be extracted from the organism or left in situ.

Embodiments also relate to polypeptides obtainable by procedures such as those previously described.

Embodiments also include procedures for making synthetic polypeptides, characterized by using the sequence of amino acids of the polypeptide.

The present invention likewise relates to synthetic polypeptides such as chimeric receptors obtained by procedures.

Polypeptides such as chimeric receptors can likewise be prepared by techniques conventional in the field of peptide synthesis. This synthesis can be carried out in a homogeneous solution or solid phase.

For example, the synthesis technique in homogeneous solution described by Houben-Weyl, 1974 can be used.

This synthetic method comprises the successive condensation of two consecutive amino acids in the required order, or fragments and amino acids previously formed and already containing some amino acids in the appropriate order, or alternatively, several previously prepared in this way. consisting of condensing the fragments, according to well-known methods in peptide synthesis, in particular after activation of the carboxyl function, to form one amine function and another carboxyl or vice versa, which should normally participate in the formation of a peptide bond. It is understood that it will be necessary to pre-protect all reactive functions of these amino acids or fragments, except in some cases.

The technique described by Merrifield can also be used.

To prepare a peptide chain according to the Merrifield procedure, a highly porous polymeric resin is used, onto which the first C-terminal amino acid of the chain is anchored. This amino acid is fixed on the resin through its carboxyl group and its amine function is protected. Thus, on each previously deprotected amino group of the portion of the peptide chain already formed and attached to the resin, the amino acid to form the peptide chain is fixed in turn. When the entirety of the desired peptide chain has been formed, the protecting groups of the different amino acids forming the peptide chain are removed and the peptide is desorbed from the resin with the aid of an acid.

These hybrid molecules are in part, possibly immunogenic, in particular diphtheria toxin, tetanus toxin, surface antigen of hepatitis B virus (patent FR 79 21811), VP1 antigen of poliomyelitis virus, or any other virus or bacterium Polypeptide carrier molecules associated with epitopes of toxins or antigens or fragments thereof.

Polypeptides comprising chimeric receptors, antibodies described below, and nucleotide sequences encoding any of the foregoing may advantageously be used in procedures for polarization of macrophages.

In an embodiment, a nucleic acid sequence encoding a chimeric receptor is provided to a cell. The cells can then express the encoded chimeric receptor. The expressed chimeric receptor may be on the surface of a cell or in the cytoplasm. In a specific embodiment, the cell expressing the chimeric receptor is a macrophage. A macrophage-expressed chimeric receptor can bind a ligand, and binding of the ligand can activate the chimeric receptor to induce polarization of the macrophage, as previously described.

In an embodiment, cells provided with a nucleic acid sequence encoding a chimeric receptor can be isolated from a subject. After the cells have been provided with the nucleic acid, the cells can be returned to the subject from which they were obtained, for example by injection or blood transfusion. In other embodiments, cells provided with nucleic acids may be provided by a donor. After the nucleic acid is provided to the donor cells, the cells can then be provided to an individual other than the donor. Examples of donor cells include, but are not limited to, cells from cell lines and primary cells from a subject.

In another embodiment, chimeric receptors can be introduced directly into cells. Any method of introducing a protein into a cell may be used, including but not limited to microinjection, electroporation, membrane fusion, and the use of protein transduction domains. After the cells are provided with the chimeric receptor, the cells can be returned to the subject from which they were obtained, for example by injection or blood transfusion. In another embodiment, the cells provided with the chimeric receptor are provided by a donor. After the nucleic acid is provided to the donor cells, the cells can then be provided to an individual other than the donor. Examples of donor cells include, but are not limited to, cells from cell lines and primary cells from a subject.

Embodiments likewise relate to polypeptides, such as chimeric receptors, labeled with the aid of a suitable label, such as an enzymatic, fluorescent, or radioactive type.

Polypeptides make it possible to prepare monoclonal or polyclonal antibodies, which are characterized in that they specifically recognize the polypeptide. It would advantageously be possible to make monoclonal antibodies from hybridomas according to the techniques described by Kohler and Milstein, 1975. For example, immunization of an animal, particularly a mouse, with a polypeptide or DNA in conjunction with an adjuvant of an immune response, and subsequent specific determination of the polypeptide that served as an antigen contained in the serum of the immunized animal on a previously immobilized affinity column. It will be possible to prepare polyclonal antibodies by purifying the antibodies. Polyclonal antibodies can also be prepared by purification of the antibody, or polypeptide or fragment thereof, contained in the serum of an animal immunologically challenged with a chimeric receptor, on an affinity column on which the polypeptide has previously been immobilized.

Also included are IgA, IgD, IgE, IgG, IgM, Fab fragments, F(ab') ₂ fragments, monovalent antibodies, scFv fragments, scRv-Fc fragments, IgNARs, hcIgG, VhH antibodies, nanobodies, and alphabodies. Antibodies can be used to produce other types of binding molecules, but not limited thereto.

Likewise, embodiments may include monoclonal or polyclonal antibodies or fragments thereof, or chimeric antibodies, or fragments thereof, characterized in that they are capable of specifically recognizing a polypeptide or polypeptide described herein and/or a ligand of a chimeric receptor. It is about.

It would likewise be possible to label antibodies in the same manner as previously described for nuclear probes, such as labeling of the enzymatic, fluorescent, or radioactive type. It will also be possible to include such antibodies and/or fragments thereof as part of a chimeric receptor. As a non-limiting example, such an antibody or fragment thereof may constitute part of the extracellular domain of a chimeric receptor.

Embodiments further relate to a procedure for detection and/or identification of a chimeric receptor in a sample, characterized in that it comprises the following steps: a) contacting the sample with a monoclonal or polyclonal (possibly biological under conditions that allow for an immunological reaction between the antibody and the chimeric receptor present in the sample); b) verification of possibly formed antigen-antibody complexes.

Embodiments are described in further detail in the following illustrative examples. Although the examples may represent only selected embodiments, it should be understood that the following examples are illustrative and not restrictive.

Example

Example 1: Isolation of ScFv fragments for specific ligands

cDNA was purified from monoclonal antibody hybridoma cells (CB1) expressing an antibody specific for human TK1. Using the isolated cDNA, the heavy and light chains of the CB1 variable region were amplified via polymerase chain reaction (PCR). Sequences from heavy and light chains were verified using NCBI Blast. The CB1 heavy and light chains were fused together via site overlap extension (SOE) PCR to form a single chain fragment variable (scFv) using a G4S linker. The G4S linker was codon-optimized for yeast and human using the codon optimization tool provided by IDT (https://www.idtdna.com/CodonOpt) to maximize protein expression. The CB1 scFv was excised using restriction enzymes and inserted into the pMP71 CAR vector.

TK-1 and HPRT-specific human scFv fragments were isolated from yeast antibody libraries. TK-1 and HPRT proteins were isolated, His-tagged and purified. TK-1 and HPRT proteins were labeled with anti-His biotinylated antibodies and added to the library to select TK-1 and HPRT-specific antibody clones. TK-1 and HPRT antibody clones were alternately stained with streptavidin or anti-biotin microbeads and concentrated using magnetic columns. Two additional rounds of sorting and selection were performed to isolate TK-1 and HPRT specific antibodies. For final selection, potential TK-1 and HPRT antibody clones and their respective proteins were sorted by fluorescence-activated cell sorting (FACS) by alternating labeling with fluorescence-conjugated anti-HA or anti-c-myc antibodies TK-1 and HPRT specific antibodies were isolated. High affinity clones were selected for chimeric receptor construction. Other human or humanized antibodies from other animals could be selected or altered to be TK-1 or HPRT specific by using phage display or other recombination methods.

The selected scFv clones were then combined with human IgG1 constant domains to generate antibodies for use in applications such as Western blot or ELISA to confirm the binding specificity of the scFv. The antibody construct was inserted into the pPNL9 yeast secretion vector, and YVH10 yeast were transformed with the construct and induced to produce antibodies. Other expression systems such as E. coli or mammalian systems could also be used to secrete the antibody.

Isolation and characterization of protein-specific antibody fragments.

Referring to Figure 26, 105 yeasts were incubated with 2.5 ug of the protein of interest labeled with a fluorescently tagged APC. The higher left (red) peak represents the yeast population that did not bind the protein of interest (negative control). The lower left (blue) peak on the left illustrates yeast not expressing its surface protein, while the higher (blue) peak on the right indicates binding of the expressed antibody fragment to the protein of interest.

Structural Consensus among Antibodies Defines the Antigen 5 Binding Site, PLoS Comput. Biol . 8(2): e1002388. doi:10.1371/journal.pcbi.1002388, Kunik V, Ashkenazi S, Ofran Y (2012)]. Paratome: An online tool for systematic identification of antigen binding regions in antibodies based on sequence or structure, Nucleic Acids Res . 2012 Jul; 40 (Web Server issue): W521-4. doi: 10.1093/nar/gks480. Epub 2012 Jun 6].

Example 2: Generation of Chimeric Receptors

Construction of Chimeric Receptor Vectors:

The first step in the process is the design of the nucleotide sequence for the synthetic chimeric receptor gene and the selection of an appropriate lentiviral vector. All vector designs were run on Genious software version 9.1.6. Sequences are retrieved from Uniprot and the Human Protein Reference Data base and also NCBI.

Vectors are synthesized by a combination of recombinant DNA technology and gene synthesis.

Sequences for short chain variable fragments are generated with humanized antibody yeast display libraries or phage display libraries. TK1, HPRT, ROR1 , MUC-16, EGFRvIII, mesothelin, HER2, CEA, BCMA , GPC3, FAP, EphA2, NKG2D ligand, GD2, CD19, CD20, CD30, CD33, CD123, CD133, CD138, and CD171 respectively A nucleic acid encoding a ScFv specific for All possible combinations of nucleic acids encoding the chimeric receptor have at least one each of a), b), c), d), and e), wherein a), b), c), d) and e) are equal to:

a) TK1, HPRT, ROR1 , MUC-16, EGFRvIII, mesothelin, HER2, CEA, BCMA , GPC3, FAP, EphA2, NKG2D ligand, GD2, CD19, CD20, CD30, CD33, CD123, CD133, CD138, and CD171 ScFv specific for;

b) GS linker or no GS linker;

c) a hinge region selected from LRR 5 amino acid short hinge, LRR long hinge, IgG4 short hinge, IgG 119 amino acid middle hinge, and IgG4 long hinge, CD8 hinge, CD8 hinge with cysteine converted to serine, and no hinge;

d) a transmembrane domain selected from the transmembrane domains of MYD88, TLR3, TLR4, TLR7, TLR8, TLR9, MAL, IRAK1, FCGR2A, FCGR3A, and FCER1G; and

e) a cytosol domain selected from the cytosol domains of MYD88, TLR3, TLR4, TLR7, TLR8, TLR9, MAL, IRAK1, FCGR2A, FCGR3A, and FCER1G.

A combination of recombinant DNA techniques and gene synthesis synthesizes the aforementioned nucleic acid encoding the chimeric receptor.

Lentivirus-mediated gene transfer is an integrated gene transfer method that genetically modifies macrophages to provide nucleic acids encoding chimeric receptors. A third generation lentiviral system from Addgene is used to package our lentiviral vectors. pHIV-dTomato (#21374) and pUltra-chilli (#48687) are gene transfer plasmids. pCMV-VSV-G (#8454), pMDLg/pRRE (#12251), pRSV-Rev (#12253), and pHCMV-AmphoEnv (#15799) are packaging plasmids. Lentivirus-mediated gene delivery of human lymphocytes, which achieves efficiencies of up to 50% transduction, has previously been standardized. HEK293T cells are transfected with the calcium phosphate method (SIGMA CAPHOS). Approximately 10 μg of each packaging plasmid and 20 μg of the vector encoding the chimeric receptor are used per transfection. After 48-36 hours, viral particles are harvested and sterile filtered. Virus titers infecting HT1080 and U937 cells are determined.

The assay is performed by flow cytometry, which detects red fluorescent protein. After viral titer, human monocytes are transduced using retronectin plates (Clonetech, T100B) and spin infection method.

Prior to lentiviral transduction, monocytes are isolated from whole PBMNCs by negative selection and magnetic sorting using Human Monocyte Isolation Kit II (MACS130-091-153). After monocyte isolation, cells are split into two Nunclon 6-well plates (Thermo, 145380), and 1.5X10 6 cells are seeded in each well for each vector. One plate is immediately transduced while the second plate is used for ex vivo differentiation of monocytes into M1 macrophages. M1-macrophage production medium DFX (Promocell, C-28055) was used to generate M1 macrophages. After 7 days, macrophages are transduced and activated on day 9 with LPS (500X) (Affimetryx, 00-4976-03) and IFN-γ (Promokine, C-60724). Transduction efficiency is analyzed by flow cytometry. Transduced cells are isolated by cell sorting using a FACS Aria cell sorter. After cell sorting, transduced monocytes are cultured ex vivo for 2 days before differentiation, whereas differentiated macrophages can persist for 1 month.

Example 3: Polarization of macrophages via chimeric receptors

Transduced macrophages prepared in Example 2 were treated with TK1, HPRT, ROR1 , MUC-16, EGFRvIII, mesothelin, HER2, CEA, BCMA , GPC3, FAP, EphA2, NKG2D conjugated ligand, GD2, CD19, CD20, Testing for polarization towards the M1 phenotype by exposure to CD30, CD33, CD123, CD133, CD138, and CD171 separately and monitoring secretion of IL-12 and IL-23 or measuring RNA production using standard cytokine assays did Macrophages harboring chimeric receptors are polarized to an M1 phenotype when exposed to ligands specific for particular chimeric receptors, as determined by increased secretion of IL-12 and/or IL-23. Ligands other than specific ligands for specific chimeric receptors do not show increases in IL-12 and/or IL-21.

Example 4: Generation and transduction of monocyte-derived macrophages

After 7 days of differentiation, monocyte-derived macrophages underwent phenotypic changes. These changes were compared between transduced and non-transduced cells. As can be seen in Figure 27, the transduced cells have a more aggressive phenotype similar to M1 or classically activated macrophages. 27 shows images of non-transduced monocyte-derived macrophages and transduced monocyte-derived macrophages at day 8 of differentiation. Interferon gamma and LPS were not added at this time point. It can be observed that the phenotype of macrophages transduced with the chimeric receptor is different from non-transduced macrophages. Transduced cells exhibited a traditionally activated phenotype or M1-like phenotype indicating macrophage activation. The altered phenotype may be the combined effect of the transduction process and the expression of new synthetic receptors.

28 provides confirmation of insertion and expression of constructs encoding chimeric receptors as confirmed by expression of dTomato 48-72 hours post-transduction. This demonstrates successful transduction of human monocyte-derived macrophages.

Example 5: Transduction Efficiency

After day 10 of differentiation, transduction efficiency was assessed and macrophages expressing the chimeric receptor were cell sorted. Lentiviral transduction is difficult in macrophages. However, almost 30% macrophage transduction was achieved using the HIV-1 based system with the EF1-α promoter. Transduction of cells in the early stages of macrophage differentiation showed different transduction efficiencies. Monocytes or macrophages in the early stages of differentiation are easier to transduce. Adenoviral transduction by the chimeric adenovirus AD5/F35 has emerged as another alternative for macrophage transduction. 29 shows the results of transduced macrophages cell sorted using the FACSAria system. Approximately 30% of macrophage transduction was achieved using the lentiviral approach. The leftmost plot represents a control where only 0.58% of the cells fluoresce to show expression of dTomato. The right two plots show a transduction efficiency of 27.1% after transduction.

Example 6: Immunophenotyping of transduced macrophages

The activation state of the transduced cells was confirmed by immunophenotyping of the macrophages transduced with the vector for expression of the chimeric receptor. It has been reported that modification of the extracellular domain of TLR-4 can induce constant activation of its signaling domain (Gay et al., 2014). Constitutive activation of TLR-4 signaling could lead to macrophage activation or M1 phenotype. It is not known whether the constructs used, based on TLR-4, can trigger constitutive activation of signaling through the TIR domain taken from TLR-4. However, after the transduction process, changes in phenotype and expression of cell surface markers in macrophages were observed. This is likely due to a combination of lentiviral transduction and expression of the chimeric receptor protein. Expression of CD14, CD80, D206 and low expression of CD163 were indicative of macrophage polarization towards the M1 phenotype. Expression of these cell surface markers was observed in transduced cells. 30 shows six scatter plots of fluorescence activated cell sorting demonstrating retention of the dye (Alexa 647) and expression of CD80, CD163, CD206, and CD14 in macrophages transduced with the chimeric receptor.

31 shows a histogram of relative expression levels of M1 cell surface markers in macrophages transduced with a vector for expressing the chimeric receptor.

Example 7: In vitro toxicity of TK1 targeting chimeric receptor transduced macrophages against NCI-H460 cells.

The oncolytic activity of TK1-targeting chimeric receptor-transduced macrophages was tested against NCI-H460-GFP cells. The E:T ratio used was 1:10. Analysis was performed by confocal microscopy. Detection of fluorescence was performed every 5 minutes for a period of 12 hours. It was observed over time that TK1 targeting chimeric receptor transduced macrophages migrate towards and attack H460-GFP cells. After synapsis, specific cell death is induced in the target cell. As demonstrated by the images in FIG. 32 , TK1-targeting chimeric receptor-transduced macrophages are able to detect, attack, and induce apoptosis in lung cancer cell lines expressing TK1. NCI-H460 cells were transformed to express GFP. The oncolytic activity of TK1-targeting chimeric receptor-transduced macrophages was detected by confocal microscopy as loss of fluorescence in the target cells.

The invention may be further modified within the spirit and scope of the invention. Accordingly, this application is intended to cover any variations, uses, or adaptations of this invention using its general principles. Additionally, this application is intended to cover such departures from this invention as come within known or customary practice in the art to which this invention pertains, which are within the scope of the appended claims and their legal equivalents.

table of references

1. Hanahan, D., & Weinberg, R. a. (2011). Hallmarks of cancer: the next generation. Cell , 144 (5), 646-74. http://doi.org/10.1016/j.cell.2011.02.013

2. American Cancer Society. (2015). Cancer Facts & Figures 2015 .

3. Hoyert, DL, & Xu, J. (2012). National Vital Statistics Reports Deaths: Preliminary Data for 2011 (Vol. 61).

4. Kurahara, H., Shinchi, H., Mataki, Y., Maemura, K., Noma, H., Kubo, F., . Takao, S. (2011). Significance of M2-polarized tumor-associated macrophage in pancreatic cancer. The Journal of Surgical Research , 167 (2), e211-9. http://doi.org/10.1016/j.jss.2009.05.026

5. Steidl, C., Lee, T., & Shah, S. (2010a). Tumor-associated macrophages and survival in classic Hodgkin's lymphoma. The New England Journal of Medicine , 875-885. Retrieved from http://www.nejm.org/doi/full/10.1056/NEJMoa0905680

, N., & Vizoso, F. J. (2012). Inflammation and cancer. World Journal of Gastrointestinal Surgery, 4(3), 62-72. http://doi.org/10.4240/wjgs.v4.i3.626.

, N., & Vizoso, F.J. (2012). Inflammation and cancer. World Journal of Gastrointestinal Surgery , 4 (3), 62-72. http://doi.org/10.4240/wjgs.v4.i3.62

7. Kelly, PM, Davison, RS, Bliss, E., & McGee, JO (1988). Macrophages in human breast disease: a quantitative immunohistochemical study. British Journal of Cancer , 57 (2), 174-7. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2246436&tool=pmcentrez&rendertype=abstract

8. Lewis, C., & Leek, R. (1995). Cytokine regulation of angiogenesis in breast cancer: the role of tumor-associated macrophages. Journal of Leukocytes … , 57 (May), 747-751. Retrieved from http://www.jleukbio.org/content/57/5/747.short

9. Mantovani, A., Biswas, SK, Galdiero, MR, Sica, A., & Locati, M. (2013). Macrophage plasticity and polarization in tissue repair and remodelling. The Journal of Pathology , 229 (2), 176-85. http://doi.org/10.1002/path.4133

10. Porta, C., Rimoldi, M., Raes, G., Brys, L., Ghezzi, P., Di Liberto, D., . . . Sica, A. (2009). Tolerance and M2 (alternative) macrophage polarization are related processes orchestrated by p50 nuclear factor kappaB. Proceedings of the National Academy of Sciences of the United States of America , 106 (35), 14978-83. http://doi.org/10.1073/pnas.0809784106

11. Sica, A., & Mantovani, A. (2012). Macrophage plasticity and polarization: in vivo veritas. The Journal of Clinical Investigation , 122 (3), 787-796. http://doi.org/10.1172/JCI59643DS1

12. Anderson, CF, & Mosser, DM (2002). A novel phenotype for an activated macrophage: the type 2 activated macrophage. Journal of Leukocyte Biology , 72 (1), 101-6. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/12101268

, W., Van Ginderachter, J. a, Meerschaut, S., … Raes, G. (2006). Identification of a common gene signature for type II cytokine-associated myeloid cells elicited in vivo in different pathologic conditions. Blood, 108(2), 575-83. http://doi.org/10.1182/blood-2005-04-148513. Ghassabeh, G. H., De Baetselier, P., Brys, L.,

, W., Van Ginderachter, J. a, Meerschaut, S., ... Raes, G. (2006). Identification of a common gene signature for type II cytokine-associated myeloid cells elicited in vivo in different pathologic conditions. Blood , 108 (2), 575-83. http://doi.org/10.1182/blood-2005-04-1485

factor 4 regulates macrophage polarization. The Journal of Clinical Investigation, 121(7). http://doi.org/10.1172/JCI45444DS114. Liao, X., Sharma, N., & Kapadia, F. (2011).

factor 4 regulates macrophage polarization. The Journal of Clinical Investigation , 121 (7). http://doi.org/10.1172/JCI45444DS1

15. Davis, MJ, Tsang, TM, Qiu, Y., Dayrit, JK, Freij, JB, Huffnagle, GB, & Olszewski, MA (2013). Macrophage M1/M2 polarization dynamically adapts to changes in cytokine microenvironments in Cryptococcus neoformans infection. mBio , 4 (3), e00264-13. http://doi.org/10.1128/mBio.00264-13

16. Mantovani, A., Sozzani, S., Locati, M., Allavena, P., & Sica, A. (2002). Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends in Immunology , 23 (11), 549-55. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/12401408

, J., Oberg, A., Oldenborg, P.-A., & Palmqvist, R. (2012). The distribution of macrophages with a m1 or m2 phenotype in relation to prognosis and the molecular characteristics of colorectal cancer. PloS One, 7(10), e47045. http://doi.org/10.1371/journal.pone.004704517. Edin, S., Wikberg, M. L., Dahlin, A. M.;

, J., Oberg, A., Oldenborg, P.-A., & Palmqvist, R. (2012). The distribution of macrophages with a m1 or m2 phenotype in relation to prognosis and the molecular characteristics of colorectal cancer. PloS One , 7 (10), e47045. http://doi.org/10.1371/journal.pone.0047045

18. Forssell, J., Oberg, A., Henriksson, M L, Stenling, R., Jung, A., & Palmqvist, R. (2007). High macrophage infiltration along the tumor front correlates with improved survival in colon cancer. Clinical Cancer Research , 13 (5), 1472-9. http://doi.org/10.1158/1078-0432.CCR-06-2073

19. Guiducci, C., Vicari, AP, Sangaletti, S., Trinchieri, G., & Colombo, MP (2005). Redirecting in vivo elicited tumor infiltrating macrophages and dendritic cells towards tumor rejection. Cancer Research , 65 (8), 3437-46. http://doi.org/10.1158/0008-5472.CAN-04-4262

20. Baccala, R., Hoebe, K., Kono, DH, Beutler, B., & Theofilopoulos, A. N. (2007). TLR-dependent and TLR-independent pathways of type I interferon induction in systemic autoimmunity. Nature Medicine , 13 (5), 543-51. http://doi.org/10.1038/nm1590

21. Banerjee, S., Xie, N., Cui, H., Tan, Z., Yang, S., Icyuz, M., . Liu, G. (2013). MicroRNA let-7c regulates macrophage polarization. Journal of Immunology (Baltimore , Md.: 1950) , 190 (12), 6542-9. http://doi.org/10.4049/jimmunol.1202496

22. Murray, PJ, Allen, JE, Biswas, SK, Fisher, EA, Gilroy, DW, Goerdt, S., . Wynn, T. A. (2014). Macrophage Activation and Polarization: Nomenclature and Experimental Guidelines. Immunity , 41 (1), 14-20. http://doi.org/10.1016/j.immuni.2014.06.008

, M.-H., Fan, Y.-H., Cao, Y.-L., Zhang, Z.-R., & Yang, S.-M. (2012). Macrophages in tumor microenvironments and the progression of tumors. Clinical & Developmental Immunology, 2012, 948098. http://doi.org/10.1155/2012/94809823. Hao, N. -B.,

, M.-H., Fan, Y.-H., Cao, Y.-L., Zhang, Z.-R., & Yang, S.-M. (2012). Macrophages in tumor microenvironments and the progression of tumors. Clinical & Developmental Immunology , 2012 , 948098. http://doi.org/10.1155/2012/948098

24. Sinha, P., Clements, VK, & Ostrand-Rosenberg, S. (2005). Reduction of myeloid-derived suppressor cells and induction of M1 macrophages facilitate the rejection of established metastatic disease. Journal of Immunology , 174 (2), 636-45. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/15634881

25. Bingle, L., Brown, NJ, & Lewis, CE (2002). The role of tumour-associated macrophages in tumour progression: implications for new anticancer therapies. The Journal of Pathology , 196 (3), 254-65. http://doi.org/10.1002/path.1027

26. Herbeuval, J.-P., Lambert, C., Sabido, O., Cottier, M., Fournel, P., Dy, M., & Genin, C. (2003). Macrophages from cancer patients: analysis of TRAIL, TRAIL receptors, and colon tumor. Journal of the National Cancer Institute , 95 (8), 611-21. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/12697854

27. Ma, J., Liu, L., Che, G., Yu, N., Dai, F., & You, Z. (2010). The M1 form of tumor-associated macrophages in non-small cell lung cancer is positively associated with survival time. BMC Cancer , 10 , 112. http://doi.org/10.1186/1471-2407-10-112

28. Ohri, CM, Shikotra, A., Green, RH, Waller, D. a, & Bradding, P. (2009). Macrophages within NSCLC tumor islets are predominantly of a cytotoxic M1 phenotype associated with extended survival. The European Respiratory Journal , 33 (1), 118-26. http://doi.org/10.1183/09031936.00065708

29. Urban, JL, Shepard, HM, Rothstein, JL, Sugarman, BJ, & Schreiber, H. (1986). Tumor necrosis factor: a potent effector molecule for tumor cell killing by activated macrophages. Proceedings of the National Academy of Sciences of the United States of America , 83 (14), 5233-7. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=323925&tool=pmcentrez&rendertype=abstract

30. Wong, S.-C., Puaux, A.-L., Chittezhath, M., Shalova, I., Kajiji, TS, Wang, X., . Biswas, SK (2010). Macrophage polarization to a unique phenotype driven by B cells. European Journal of Immunology , 40 (8), 2296-307. http://doi.org/10.1002/eji.200940288

31. Hardison, SE, Herrera, G., Young, M L, Hole, CR, Wozniak, KL, & Wormley, FL (2012). Protective immunity against pulmonary cryptococcosis is associated with STAT1-mediated classical macrophage activation. Journal of Immunology (Baltimore , Md.: 1950) , 189 (8), 4060-8. http://doi.org/10.4049/jimmunol.1103455

32. Wang, Y.-C., He, F., Feng, F., Liu, X.-W., Dong, G.-Y., Qin, H.-Y., . Han, H. (2010). Notch signaling determines the M1 versus M2 polarization of macrophages in antitumor immune responses. Cancer Research , 70 (12), 4840-9. http://doi.org/10.1158/0008-5472.CAN-10-0269

33. Cai, X., Yin, Y., Li, N., Zhu, D., Zhang, J., Zhang, C.-Y., & Zen, K. (2012). Re-polarization of tumor-associated macrophages to pro-inflammatory M1 macrophages by microRNA-155. Journal of Molecular Cell Biology , 4 (5), 341-3. http://doi.org/10.1093/jmcb/mjs044

, J., … Schober, A. (2013). The microRNA-342-5p fosters inflammatory macrophage activation through an Akt1- and microRNA-155-dependent pathway during atherosclerosis. Circulation, 127(15), 1609-19. http://doi.org/10.1161/CIRCULATIONAHA.112.00073634. Wei, Y., Nazari-Jahantigh, M., Chan, L., Zhu, M., Heyll, K.,

, J., … Schober, A. (2013). The microRNA-342-5p fosters inflammatory macrophage activation through an Akt1- and microRNA-155-dependent pathway during atherosclerosis. Circulation , 127 (15), 1609-19. http://doi.org/10.1161/CIRCULATIONAHA.112.000736

35. Squadrito, M. L., Etzrodt, M., De Palma, M., & Pittet, M. J. (2013). MicroRNA-mediated control of macrophages and its implications for cancer. Trends in Immunology , 34 (7), 350-9. http://doi.org/10.1016/j.it.2013.02.003

36. Biswas, SK, Gangi, L., Paul, S., Schioppa, T., Saccani, A., Sironi, M., . Sica, A. (2006). A distinct and unique transcriptional program expressed by tumor-associated macrophages (defective NF-kappaB and enhanced IRF-3/STAT1 activation). Blood , 107 (5), 2112-22. http://doi.org/10.1182/blood-2005-01-0428

37. Steidl, C., Lee, T., & Shah, S. (2010b). Tumor-associated macrophages and survival in classic Hodgkin's lymphoma. The New England Journal of Medicine , 362 (10), 875-885. Retrieved from http://www.nejm.org/doi/full/10.1056/NEJMoa0905680

38. Lin, EY, Li, J.-F., Gnatovskiy, L., Deng, Y., Zhu, L., Grzesik, D. a, . Pollard, JW (2006). Macrophages regulate the angiogenic switch in a mouse model of breast cancer. Cancer Research , 66 (23), 11238-46. http://doi.org/10.1158/0008-5472.CAN-06-1278

, A., … Balkwill, F. R. (2006). Ovarian cancer cells polarize macrophages toward a tumor-associated phenotype. The Journal of Immunology, 176(8), 5023-32. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/1658559939. Hagemann, T., Wilson, J., Burke, F., Kulbe, H., Li, N.F.;

, A., … Balkwill, F. R. (2006). Ovarian cancer cells polarize macrophages toward a tumor-associated phenotype. The Journal of Immunology , 176 (8), 5023-32. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/16585599

40. Hagemann, T., Lawrence, T., McNeish, I., Charles, K. a, Kulbe, H., Thompson, RG, . Balkwill, F. R. (2008). "Re-educating" tumor-associated macrophages by targeting NF-kappaB. The Journal of Experimental Medicine , 205 (6), 1261-8. http://doi.org/10.1084/jem.20080108

41. Mandal, P., Pratt, BT, Barnes, M., McMullen, MR, & Nagy, LE (2011). Molecular mechanism for adiponectin-dependent M2 macrophage polarization: link between the metabolic and innate immune activity of full-length adiponectin. The Journal of Biological Chemistry , 286 (15), 13460-9. http://doi.org/10.1074/jbc.M110.204644

42. Mantovani, A., Allavena, P., Sica, A., & Balkwill, F. (2008). Cancer-related inflammation. Nature , 454 (7203), 436-44. http://doi.org/10.1038/nature07205

43. Cortez-Retamozo, V., Etzrodt, M., Newton, A., Rauch, PJ, Chudnovskiy, A., Berger, C., . Pittet, M.J. (2012). Origins of tumor-associated macrophages and neutrophils. Proceedings of the National Academy of Sciences of the United States of America , 109 (7), 2491-6. http://doi.org/10.1073/pnas.1113744109

44. Hercus, TR, Thomas, D., Guthridge, MA, Ekert, PG, King-Scott, J., Parker, MW, & Lopez, AF (2009). The granulocyte-macrophage colony-stimulating factor receptor: linking its structure to cell signaling and its role in disease. Blood , 114 (7), 1289-98. http://doi.org/10.1182/blood-2008-12-164004

45. Smith, HO, Stephens, ND, Qualls, CR, Fligelman, T., Wang, T., Lin, C.-Y., . Pollard, JW (2013). The clinical significance of inflammatory cytokines in primary cell culture in endometrial carcinoma. Molecular Oncology , 7 (1), 41-54. http://doi.org/10.1016/j.molonc.2012.07.002

46. West, RB, Rubin, BP, Miller, MA, Subramanian, S., Kaygusuz, G., Montgomery, K., . van de Rijn, M. (2006). A landscape effect in tenosynovial giant-cell tumor from activation of CSF1 expression by a translocation in a minority of tumor cells. Proceedings of the National Academy of Sciences of the United States of America , 103 (3), 690-5. http://doi.org/10.1073/pnas.0507321103

47. Lin, EY, & Pollard, JW (2007). Tumor-associated macrophages press the angiogenic switch in breast cancer. Cancer Research , 67 (11), 5064-6. http://doi.org/10.1158/0008-5472.CAN-07-0912

48. Dalton, HJ, Armaiz-Pena, GN, Gonzalez-Villasana, V., Lopez-Berestein, G., Bar-Eli, M., & Sood, AK (2014). Monocyte subpopulations in angiogenesis. Cancer Research , 74 (5), 1287-93. http://doi.org/10.1158/0008-5472.CAN-13-2825

49. Saccani, A., Schioppa, T., Porta, C., Biswas, SK, Nebuloni, M., Vago, L., . Sica, A. (2006). p50 nuclear factor-kappaB overexpression in tumor-associated macrophages inhibits M1 inflammatory responses and antitumor resistance. Cancer Research , 66 (23), 11432-40. http://doi.org/10.1158/0008-5472.CAN-06-1867

50. Gazzaniga, S., Bravo, AI, Guglielmotti, A., van Rooijen, N., Maschi, F., Vecchi, A., . Wainstok, R. (2007). Targeting tumor-associated macrophages and inhibition of MCP-1 reduce angiogenesis and tumor growth in a human melanoma xenograft. The Journal of Investigative Dermatology , 127 (8), 2031-41. http://doi.org/10.1038/sj.jid.5700827

51. Luo, Y., Zhou, H., & Krueger, J. (2006). Targeting tumor-associated macrophages as a novel strategy against breast cancer. Journal of Clinical Investigation , 116 (8), 2132-2141. http://doi.org/10.1172/JCI27648.2132

, a H. M., Ballmer-Hofer, K., & Schwendener, R. a. (2006). Clodronate-liposome-mediated depletion of tumour-associated macrophages: a new and highly effective antiangiogenic therapy approach. British Journal of Cancer, 95(3), 272-81. http://doi.org/10.1038/sj.bjc.660324052. Zeisberger, SM, Odermatt, B., Marty, C.,

, a HM, Ballmer-Hofer, K., & Schwendener, R. a. (2006). Clodronate-liposome-mediated depletion of tumor-associated macrophages: a new and highly effective antiangiogenic therapy approach. British Journal of Cancer , 95 (3), 272-81. http://doi.org/10.1038/sj.bjc.6603240

53. Bettencourt-Dias, M., Giet, R., Sinka, R., Mazumdar, a, Lock, WG, Balloux, F., . Glover, DM (2004). Genome-wide survey of protein kinases required for cell cycle progression. Nature , 432 (7020), 980-7. http://doi.org/10.1038/nature03160

54. Geschwind, JH, Vali, M., & Wahl, R. (2006). Effects of 3 bromopyruvate (hexokinase 2 inhibitor) on glucose uptake in lewis rats using 2-(F-18) fluoro-2-deoxy-d-glucose. In 2006 Gastrointestinal Cancers Symposium (pp. 12-14).

55. Wolf, A., Agnihotri, S., Micallef, J., Mukherjee, J., Sabha, N., Cairns, R., . Guha, A. (2011). Hexokinase 2 is a key mediator of aerobic glycolysis and promotes tumor growth in human glioblastoma multiforme. The Journal of Experimental Medicine , 208 (2), 313-26. http://doi.org/10.1084/jem.20101470

56. Blagih, J., & Jones, RG (2012). Polarizing macrophages through reprogramming of glucose metabolism. Cell Metabolism , 15 (6), 793-5. http://doi.org/10.1016/j.cmet.2012.05.008

57. Haschemi, A., Kosma, P., Gille, L., Evans, CR, Burant, CF, Starkl, P., . Wagner, O. (2012). The sedoheptulose kinase CARKL directs macrophage polarization through control of glucose metabolism. Cell Metabolism , 15 (6), 813-26. http://doi.org/10.1016/j.cmet.2012.04.023

58. Arranz, A., Doxaki, C., Vergadi, E., Martinez de la Torre, Y., Vaporidi, K., Lagoudaki, ED, . Tsatsanis, C. (2012). Akt1 and Akt2 protein kinases differentially contribute to macrophage polarization. Proceedings of the National Academy of Sciences of the United States of America , 109 (24), 9517-22. http://doi.org/10.1073/pnas.1119038109

59. Jones, RG, & Thompson, CB (2007). Revving the engine: signal transduction fuels T cell activation. Immunity , 27 (2), 173-8. http://doi.org/10.1016/j.immuni.2007.07.008

60. Shu, CJ, Guo, S., Kim, YJ, Shelly, SM, Nijagal, A., Ray, P., … Witte, ON (2005). Visualization of a primary anti-tumor immune response by positron emission tomography. Proceedings of the National Academy of Sciences of the United States of America , 102 (48), 17412-7. http://doi.org/10.1073/pnas.0508698102

61. Van Ginderachter, JA, Movahedi, K., Hassanzadeh Ghassabeh, G., Meerschaut, S., Beschin, A., Raes, G., & De Baetselier, P. (2006). Classical and alternative activation of mononuclear phagocytes: Picking the best of both worlds for tumor promotion. Immunobiology , 211 (6), 487-501. Retrieved from http://www.sciencedirect.com/science/article/pii/S0171298506000829

62. Mills, CD, Shearer, J., Evans, R., & Caldwell, MD (1992). Macrophage arginine metabolism and the inhibition or stimulation of cancer. Journal of Immunology (Baltimore , Md.: 1950) , 149 (8), 2709-14. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/1401910

63. Ji, Y., Sun, S., Xu, A., Bhargava, P., Yang, L., Lam, KSL, … Qi, L. (2012). Activation of natural killer T cells promotes M2 Macrophage polarization in adipose tissue and improves systemic glucose tolerance via interleukin-4 (IL-4)/STAT6 protein signaling axis in obesity. The Journal of Biological Chemistry , 287 (17), 13561-71. http://doi.org/10.1074/jbc.M112.350066

64. Andreesen, R., Scheibenbogen, C., & Brugger, W. (1990). Adoptive transfer of tumor cytotoxic macrophages generated in vitro from circulating blood monocytes: a new approach to cancer immunotherapy. Cancer Research , 7450-7456. Retrieved from http://cancerres.aacrjournals.org/content/50/23/7450.short

65. Korbelik, M., Naraparaju, VR, & Yamamoto, N. (1997). Macrophage-directed immunotherapy as adjuvant to photodynamic therapy of cancer. British Journal of Cancer , 75 (2), 202-7. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2063270&tool=pmcentrez&rendertype=abstract

66. Ellem, KAO, Rourke, MGEO, Johnson, GR, Parry, G., Misko, IS, Schmidt, CW, . Mulligan, RC (1997). A case report: immune responses and clinical course of the first human use of granulocyte/macrophage-colony-stimulating-factor-transduced autologous melanoma. Cancer Immunology , Immunotherapy , 10-20. Retrieved from http://www.springerlink.com/index/JQ4EB21E4C7ADMT7.pdf

, H. (2000). immunotherapy with subcutaneous granulocyte macrophage colony-stimulating factor, low-dose interleukin 2, and interferon

in progressive metastatic melanoma. Clinical Cancer Research. Retrieved from http://clincancerres.aacrjournals.org/content/6/4/1267.short67. Gast, G. de, &

, H. (2000). immunotherapy with subcutaneous granulocyte macrophage colony-stimulating factor, low-dose interleukin 2, and interferon

in progressive metastatic melanoma. Clinical Cancer Research . Retrieved from http://clincancerres.aacrjournals.org/content/6/4/1267.short

68. Hill, H., Jr, TC, & Sabel, M. (2002). Immunotherapy with Interleukin 12 and Granulocyte-Macrophage Colony-stimulating Factor-encapsulated Microspheres Coinduction of Innate and Adaptive Antitumor. Cancer Research . Retrieved from http://cancerres.aacrjournals.org/content/62/24/7254.short

69. Lokshin, A., Mayotte, J., & Levitt, M. (1995). Mechanism of Interferon Beta-Induced Squamous Differentiation and Programmed Cell Death in Human Non-Small-Cell Lung Cancer Cell Lines. Journal of the National Cancer Institute , 87 , 206-212. Retrieved from http://jnci.oxfordjournals.org/content/87/3/206.short

. Journal of the National Cancer Institute, (type II), 1185-1190. Retrieved from http://jnci.oxfordjournals.org/content/84/15/118570. Johns, T., & Mackay, I. (1992). Antiproliferative potencies of interferons on melanoma cell lines and xenografts: higher efficacy of interferon

. Journal of the National Cancer Institute , (type II), 1185-1190. Retrieved from http://jnci.oxfordjournals.org/content/84/15/1185

71. Qin, X.-Q., Runkel, L., Deck, C., DeDios, C., & Barsoum, J. (1997). Interferon-beta induces S phase accumulation selectively in human transformed cells. Journal of Interferon & Cytokine Research , 17 (6), 355-367. http://doi.org/10.1089/jir.1997.17.355

72. Zhang, F., Lu, W., & Dong, Z. (2002). Tumor-infiltrating macrophages are involved in suppressing growth and metastasis of human prostate cancer cells by INF-β gene therapy in nude mice. Clinical Cancer Research , 2942-2951. Retrieved from http://clincancerres.aacrjournals.org/content/8/9/2942.short

73. Simpson, KD, Templeton, DJ, & Cross, JV (2012). Macrophage Migration Inhibitory Factor Promotes Tumor Growth and Metastasis by Inducing Myeloid-Derived Suppressor Cells in the Tumor Microenvironment. The Journal of Immunology . http://doi.org/10.4049/jimmunol.1201161

74. Sanford, DE, Belt, BA, Panni, RZ, Mayer, A., Deshpande, AD, Carpenter, D., … Linehan, DC (2013). Inflammatory monocyte mobilization decreases patient survival in pancreatic cancer: a role for targeting the CCL2/CCR2 axis. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research , 19 (13), 3404-15. http://doi.org/10.1158/1078-0432.CCR-13-0525

75. Schmall, A., Al-Tamari, H. M., Herold, S., Kampschulte, M., Weigert, A., Wietelmann, A., . Savai, R. (2014). Macrophage and Cancer Cell Crosstalk via CCR2 and CX3CR1 is a Fundamental Mechanism Driving Lung Cancer. American Journal of Respiratory and Critical Care Medicine . http://doi.org/10.1164/rccm.201406-1137OC

76. Kimura, YN, Watari, K., Fotovati, A., Hosoi, F., Yasumoto, K., Izumi, H., … Ono, M. (2007). Inflammatory stimuli from macrophages and cancer cells synergistically promote tumor growth and angiogenesis. Cancer Science , 98 (12), 2009-18. http://doi.org/10.1111/j.1349-7006.2007.00633.x

77. Chen, H., Li, P., Yin, Y., Cai, X., Huang, Z., Chen, J., … Zhang, J. (2010). The promotion of type 1 T helper cell responses to cationic polymers in vivo via toll-like receptor-4 mediated IL-12 secretion. Biomaterials , 31 (32), 8172-80. http://doi.org/10.1016/j.biomaterials.2010.07.056

78. Rogers, T.L., & Holen, I. (2011). Tumor macrophages as potential targets of bisphosphonates. Journal of Translational Medicine , 9 (1), 177. http://doi.org/10.1186/1479-5876-9-177

79. Junankar, S., Shay, G., Jurczyluk, J., Ali, N., Down, J., Pocock, N., . Rogers, M.J. (2015). Real-time intravital imaging establishes tumor-associated macrophages as the extraskeletal target of bisphosphonate action in cancer. Cancer Discovery , 5 (1), 35-42. http://doi.org/10.1158/2159-8290.CD-14-0621

80. Huang, Z., Yang, Y., Jiang, Y., Shao, J., Sun, X., Chen, J., . Zhang, J. (2013). Anti-tumor immune responses of tumor-associated macrophages via toll-like receptor 4 triggered by cationic polymers. Biomaterials , 34 (3), 746-55. http://doi.org/10.1016/j.biomaterials.2012.09.062

81. Q. He, T. Fornander, H. Johansson et al., "Thymidine kinase 1 in serum predicts increased risk of distant or loco-regional recurrence following surgery in patients with early breast cancer," Anticancer Research, vol. 26, no. 6, p. 4753-4759, 2006.

82. K. L. O'Neill, M. Hoper, and G. W. Odling-Smee, "Can thymidine kinase levels in breast tumors predict disease recurrence?" Journal of the National Cancer Institute, vol. 84, no. 23, p. 1825-1828, 1992.

<110> O'Neill, Kim <120> TRANSGENIC MACROPHAGES, CHIMERIC ANTIGEN RECEPTORS, AND ASSOCIATED METHODS <130> 3787-P13797 <160> 54 <170> PatentIn version 3.5 <210> 1 <211> 904 <212> PRT <213> Homo sapiens <400> 1 Met Arg Gln Thr Leu Pro Cys Ile Tyr Phe Trp Gly Gly Leu Leu Pro 1 5 10 15 Phe Gly Met Leu Cys Ala Ser Ser Thr Thr Lys Cys Thr Val Ser His 20 25 30 Glu Val Ala Asp Cys Ser His Leu Lys Leu Thr Gln Val Pro Asp Asp 35 40 45 Leu Pro Thr Asn Ile Thr Val Leu Asn Leu Thr His Asn Gln Leu Arg 50 55 60 Arg Leu Pro Ala Ala Asn Phe Thr Arg Tyr Ser Gln Leu Thr Ser Leu 65 70 75 80 Asp Val Gly Phe Asn Thr Ile Ser Lys Leu Glu Pro Glu Leu Cys Gln 85 90 95 Lys Leu Pro Met Leu Lys Val Leu Asn Leu Gln His Asn Glu Leu Ser 100 105 110 Gln Leu Ser Asp Lys Thr Phe Ala Phe Cys Thr Asn Leu Thr Glu Leu 115 120 125 His Leu Met Ser Asn Ser Ile Gln Lys Ile Lys Asn Asn Pro Phe Val 130 135 140 Lys Gln Lys Asn Leu Ile Thr Leu Asp Leu Ser His Asn Gly Leu Ser 145 150 155 160 Ser Thr Lys Leu Gly Thr Gln Val Gln Leu Glu Asn Leu Gln Glu Leu 165 170 175 Leu Leu Ser Asn Asn Lys Ile Gln Ala Leu Lys Ser Glu Glu Leu Asp 180 185 190 Ile Phe Ala Asn Ser Ser Leu Lys Lys Leu Glu Leu Ser Ser Asn Gln 195 200 205 Ile Lys Glu Phe Ser Pro Gly Cys Phe His Ala Ile Gly Arg Leu Phe 210 215 220 Gly Leu Phe Leu Asn Asn Val Gln Leu Gly Pro Ser Leu Thr Glu Lys 225 230 235 240 Leu Cys Leu Glu Leu Ala Asn Thr Ser Ile Arg Asn Leu Ser Leu Ser 245 250 255 Asn Ser Gln Leu Ser Thr Thr Ser Asn Thr Thr Phe Leu Gly Leu Lys 260 265 270 Trp Thr Asn Leu Thr Met Leu Asp Leu Ser Tyr Asn Asn Leu Asn Val 275 280 285 Val Gly Asn Asp Ser Phe Ala Trp Leu Pro Gln Leu Glu Tyr Phe Phe 290 295 300 Leu Glu Tyr Asn Asn Ile Gln His Leu Phe Ser His Ser Leu His Gly 305 310 315 320 Leu Phe Asn Val Arg Tyr Leu Asn Leu Lys Arg Ser Phe Thr Lys Gln 325 330 335 Ser Ile Ser Leu Ala Ser Leu Pro Lys Ile Asp Asp Phe Ser Phe Gln 340 345 350 Trp Leu Lys Cys Leu Glu His Leu Asn Met Glu Asp Asn Asp Ile Pro 355 360 365 Gly Ile Lys Ser Asn Met Phe Thr Gly Leu Ile Asn Leu Lys Tyr Leu 370 375 380 Ser Leu Ser Asn Ser Phe Thr Ser Leu Arg Thr Leu Thr Asn Glu Thr 385 390 395 400 Phe Val Ser Leu Ala His Ser Pro Leu His Ile Leu Asn Leu Thr Lys 405 410 415 Asn Lys Ile Ser Lys Ile Glu Ser Asp Ala Phe Ser Trp Leu Gly His 420 425 430 Leu Glu Val Leu Asp Leu Gly Leu Asn Glu Ile Gly Gln Glu Leu Thr 435 440 445 Gly Gln Glu Trp Arg Gly Leu Glu Asn Ile Phe Glu Ile Tyr Leu Ser 450 455 460 Tyr Asn Lys Tyr Leu Gln Leu Thr Arg Asn Ser Phe Ala Leu Val Pro 465 470 475 480 Ser Leu Gln Arg Leu Met Leu Arg Arg Val Ala Leu Lys Asn Val Asp 485 490 495 Ser Ser Pro Ser Pro Phe Gln Pro Leu Arg Asn Leu Thr Ile Leu Asp 500 505 510 Leu Ser Asn Asn Asn Ile Ala Asn Ile Asn Asp Asp Met Leu Glu Gly 515 520 525 Leu Glu Lys Leu Glu Ile Leu Asp Leu Gln His Asn Asn Leu Ala Arg 530 535 540 Leu Trp Lys His Ala Asn Pro Gly Gly Pro Ile Tyr Phe Leu Lys Gly 545 550 555 560 Leu Ser His Leu His Ile Leu Asn Leu Glu Ser Asn Gly Phe Asp Glu 565 570 575 Ile Pro Val Glu Val Phe Lys Asp Leu Phe Glu Leu Lys Ile Ile Asp 580 585 590 Leu Gly Leu Asn Asn Leu Asn Thr Leu Pro Ala Ser Val Phe Asn Asn 595 600 605 Gln Val Ser Leu Lys Ser Leu Asn Leu Gln Lys Asn Leu Ile Thr Ser 610 615 620 Val Glu Lys Lys Val Phe Gly Pro Ala Phe Arg Asn Leu Thr Glu Leu 625 630 635 640 Asp Met Arg Phe Asn Pro Phe Asp Cys Thr Cys Glu Ser Ile Ala Trp 645 650 655 Phe Val Asn Trp Ile Asn Glu Thr His Thr Asn Ile Pro Glu Leu Ser 660 665 670 Ser His Tyr Leu Cys Asn Thr Pro Pro His Tyr His Gly Phe Pro Val 675 680 685 Arg Leu Phe Asp Thr Ser Ser Cys Lys Asp Ser Ala Pro Phe Glu Leu 690 695 700 Phe Phe Met Ile Asn Thr Ser Ile Leu Leu Ile Phe Ile Phe Ile Val 705 710 715 720 Leu Leu Ile His Phe Glu Gly Trp Arg Ile Ser Phe Tyr Trp Asn Val 725 730 735 Ser Val His Arg Val Leu Gly Phe Lys Glu Ile Asp Arg Gln Thr Glu 740 745 750 Gln Phe Glu Tyr Ala Ala Tyr Ile Ile His Ala Tyr Lys Asp Lys Asp 755 760 765 Trp Val Trp Glu His Phe Ser Ser Met Glu Lys Glu Asp Gln Ser Leu 770 775 780 Lys Phe Cys Leu Glu Glu Arg Asp Phe Glu Ala Gly Val Phe Glu Leu 785 790 795 800 Glu Ala Ile Val Asn Ser Ile Lys Arg Ser Arg Lys Ile Ile Phe Val 805 810 815 Ile Thr His His Leu Leu Lys Asp Pro Leu Cys Lys Arg Phe Lys Val 820 825 830 His His Ala Val Gln Gln Ala Ile Glu Gln Asn Leu Asp Ser Ile Ile 835 840 845 Leu Val Phe Leu Glu Glu Ile Pro Asp Tyr Lys Leu Asn His Ala Leu 850 855 860 Cys Leu Arg Arg Gly Met Phe Lys Ser His Cys Ile Leu Asn Trp Pro 865 870 875 880 Val Gln Lys Glu Arg Ile Gly Ala Phe Arg His Lys Leu Gln Val Ala 885 890 895 Leu Gly Ser Lys Asn Ser Val His 900 <210> 2 <211> 2715 <212> DNA <213> Homo sapiens <400> 2 atgagacaga ctttgccttg tatctacttt tggggggggcc ttttgccctt tgggatgctg 60 tgtgcatcct ccaccaccaa gtgcactgtt agccatgaag ttgctgactg cagccacctg 120 aagttgactc aggtacccga tgatctaccc acaaacataa cagtgttgaa ccttacccat 180 aatcaactca gaagattacc agccgccaac ttcacaaggt atagccagct aactagcttg 240 gatgtaggat ttaacaccat ctcaaaactg gagccagaat tgtgccagaa acttcccatg 300 ttaaaagttt tgaacctcca gcacaatgag ctatctcaac tttctgataa aacctttgcc 360 ttctgcacga atttgactga actccatctc atgtccaact caatccagaa aattaaaaat 420 aatccctttg tcaagcagaa gaatttaatc acattagatc tgtctcataa tggcttgtca 480 tctacaaaat taggaactca ggttcagctg gaaaatctcc aagagcttct attatcaaac 540 aataaaattc aagcgctaaa aagtgaagaa ctggatatct ttgccaattc atctttaaaa 600 aaattagagt tgtcatcgaa tcaaattaaa gagttttctc cagggtgttt tcacgcaatt 660 ggaagattat ttggcctctt tctgaacaat gtccagctgg gtcccagcct tacagagaag 720 ctatgtttgg aattagcaaa cacaagcatt cggaatctgt ctctgagtaa cagccagctg 780 tccaccacca gcaatacaac tttcttggga ctaaagtgga caaatctcac tatgctcgat 840 ctttcctaca acaacttaaa tgtggttggt aacgattcct ttgcttggct tccacaacta 900 gaatatttct tcctagagta taataatata cagcatttgt tttctcactc tttgcacggg 960 cttttcaatg tgaggtacct gaatttgaaa cggtctttta ctaaacaaag tatttccctt 1020 gcctcactcc ccaagattga tgatttttct tttcagtggc taaaatgttt ggagcacctt 1080 aacatggaag ataatgatat tccaggcata aaaagcaata tgttcacagg attgataaac 1140 ctgaaatact taagtctatc caactccttt acaagtttgc gaactttgac aaatgaaaca 1200 tttgtatcac ttgctcattc tcccttacac atactcaacc taaccaagaa taaaatctca 1260 aaaatagaga gtgatgcttt ctcttggttg ggccacctag aagtacttga cctgggcctt 1320 aatgaaattg ggcaagaact cacaggccag gaatggagag gtctagaaaa tattttcgaa 1380 atctatcttt cctacaacaa gtacctgcag ctgactagga actcctttgc cttggtccca 1440 agccttcaac gactgatgct ccgaagggtg gcccttaaaa atgtggatag ctctccttca 1500 ccattccagc ctcttcgtaa cttgaccatt ctggatctaa gcaacaacaa catagccaac 1560 ataaatgatg acatgttgga gggtcttgag aaactagaaa ttctcgattt gcagcataac 1620 aacttagcac ggctctgggaa acacgcaaac cctggtggtc ccatttattt cctaaagggt 1680 ctgtctcacc tccacatcct taacttggag tccaacggct ttgacgagat cccagttgag 1740 gtcttcaagg atttatttga actaaagatc atcgatttag gattgaataa tttaaacaca 1800 cttccagcat ctgtctttaa taatcaggtg tctctaaagt cattgaacct tcagaagaat 1860 ctcataacat ccgttgagaa gaaggttttc gggccagctt tcaggaacct gactgagtta 1920 gatatgcgct ttaatccctt tgattgcacg tgtgaaagta ttgcctggtt tgttaattgg 1980 attaacgaga cccataccaa catccctgag ctgtcaagcc actacctttg caacactcca 2040 cctcactatc atgggttccc agtgagactt tttgatacat catcttgcaa agacagtgcc 2100 ccctttgaac tctttttcat gatcaatacc agtatcctgt tgatttttat ctttattgta 2160 cttctcatcc actttgaggg ctggaggata tctttttat ggaatgtttc agtacatcga 2220 gttcttggtt tcaaagaaat agacagacag acagaacagt ttgaatatgc agcatatata 2280 attcatgcct ataaagataa ggattgggtc tgggaacatt tctcttcaat ggaaaaggaa 2340 gaccaatctc tcaaattttg tctggaagaa agggactttg aggcgggtgt ttttgaacta 2400 gaagcaattg ttaacagcat caaaagaagc agaaaaatta tttttgttat aacacaccat 2460 ctattaaaag acccattatg caaaagattc aaggtacatc atgcagttca acaagctatt 2520 gaacaaaatc tggattccat tatattggtt ttccttgagg agattccaga ttataaactg 2580 aaccatgcac tctgtttgcg aagaggaatg tttaaatctc actgcatctt gaactggcca 2640 gttcagaaag aacggatagg tgcctttcgt cataaattgc aagtagcact tggatccaaa 2700 aactctgtac attaa 2715 <210> 3 <211> 839 <212> PRT <213> Homo sapiens <400> 3 Met Met Ser Ala Ser Arg Leu Ala Gly Thr Leu Ile Pro Ala Met Ala 1 5 10 15 Phe Leu Ser Cys Val Arg Pro Glu Ser Trp Glu Pro Cys Val Glu Val 20 25 30 Val Pro Asn Ile Thr Tyr Gln Cys Met Glu Leu Asn Phe Tyr Lys Ile 35 40 45 Pro Asp Asn Leu Pro Phe Ser Thr Lys Asn Leu Asp Leu Ser Phe Asn 50 55 60 Pro Leu Arg His Leu Gly Ser Tyr Ser Phe Phe Ser Phe Pro Glu Leu 65 70 75 80 Gln Val Leu Asp Leu Ser Arg Cys Glu Ile Gln Thr Ile Glu Asp Gly 85 90 95 Ala Tyr Gln Ser Leu Ser His Leu Ser Thr Leu Ile Leu Thr Gly Asn 100 105 110 Pro Ile Gln Ser Leu Ala Leu Gly Ala Phe Ser Gly Leu Ser Ser Leu 115 120 125 Gln Lys Leu Val Ala Val Glu Thr Asn Leu Ala Ser Leu Glu Asn Phe 130 135 140 Pro Ile Gly His Leu Lys Thr Leu Lys Glu Leu Asn Val Ala His Asn 145 150 155 160 Leu Ile Gln Ser Phe Lys Leu Pro Glu Tyr Phe Ser Asn Leu Thr Asn 165 170 175 Leu Glu His Leu Asp Leu Ser Ser Asn Lys Ile Gln Ser Ile Tyr Cys 180 185 190 Thr Asp Leu Arg Val Leu His Gln Met Pro Leu Leu Asn Leu Ser Leu 195 200 205 Asp Leu Ser Leu Asn Pro Met Asn Phe Ile Gln Pro Gly Ala Phe Lys 210 215 220 Glu Ile Arg Leu His Lys Leu Thr Leu Arg Asn Asn Phe Asp Ser Leu 225 230 235 240 Asn Val Met Lys Thr Cys Ile Gln Gly Leu Ala Gly Leu Glu Val His 245 250 255 Arg Leu Val Leu Gly Glu Phe Arg Asn Glu Gly Asn Leu Glu Lys Phe 260 265 270 Asp Lys Ser Ala Leu Glu Gly Leu Cys Asn Leu Thr Ile Glu Glu Phe 275 280 285 Arg Leu Ala Tyr Leu Asp Tyr Tyr Leu Asp Asp Ile Ile Asp Leu Phe 290 295 300 Asn Cys Leu Thr Asn Val Ser Ser Phe Ser Leu Val Ser Val Thr Ile 305 310 315 320 Glu Arg Val Lys Asp Phe Ser Tyr Asn Phe Gly Trp Gln His Leu Glu 325 330 335 Leu Val Asn Cys Lys Phe Gly Gln Phe Pro Thr Leu Lys Leu Lys Ser 340 345 350 Leu Lys Arg Leu Thr Phe Thr Ser Asn Lys Gly Gly Asn Ala Phe Ser 355 360 365 Glu Val Asp Leu Pro Ser Leu Glu Phe Leu Asp Leu Ser Arg Asn Gly 370 375 380 Leu Ser Phe Lys Gly Cys Cys Ser Gln Ser Asp Phe Gly Thr Thr Ser 385 390 395 400 Leu Lys Tyr Leu Asp Leu Ser Phe Asn Gly Val Ile Thr Met Ser Ser 405 410 415 Asn Phe Leu Gly Leu Glu Gln Leu Glu His Leu Asp Phe Gln His Ser 420 425 430 Asn Leu Lys Gln Met Ser Glu Phe Ser Val Phe Leu Ser Leu Arg Asn 435 440 445 Leu Ile Tyr Leu Asp Ile Ser His Thr His Thr Arg Val Ala Phe Asn 450 455 460 Gly Ile Phe Asn Gly Leu Ser Ser Leu Glu Val Leu Lys Met Ala Gly 465 470 475 480 Asn Ser Phe Gln Glu Asn Phe Leu Pro Asp Ile Phe Thr Glu Leu Arg 485 490 495 Asn Leu Thr Phe Leu Asp Leu Ser Gln Cys Gln Leu Glu Gln Leu Ser 500 505 510 Pro Thr Ala Phe Asn Ser Leu Ser Ser Leu Gln Val Leu Asn Met Ser 515 520 525 His Asn Asn Phe Phe Ser Leu Asp Thr Phe Pro Tyr Lys Cys Leu Asn 530 535 540 Ser Leu Gln Val Leu Asp Tyr Ser Leu Asn His Ile Met Thr Ser Lys 545 550 555 560 Lys Gln Glu Leu Gln His Phe Pro Ser Ser Leu Ala Phe Leu Asn Leu 565 570 575 Thr Gln Asn Asp Phe Ala Cys Thr Cys Glu His Gln Ser Phe Leu Gln 580 585 590 Trp Ile Lys Asp Gln Arg Gln Leu Leu Val Glu Val Glu Arg Met Glu 595 600 605 Cys Ala Thr Pro Ser Asp Lys Gln Gly Met Pro Val Leu Ser Leu Asn 610 615 620 Ile Thr Cys Gln Met Asn Lys Thr Ile Ile Gly Val Ser Val Leu Ser 625 630 635 640 Val Leu Val Val Ser Val Val Val Ala Val Leu Val Tyr Lys Phe Tyr Phe 645 650 655 His Leu Met Leu Leu Ala Gly Cys Ile Lys Tyr Gly Arg Gly Glu Asn 660 665 670 Ile Tyr Asp Ala Phe Val Ile Tyr Ser Ser Gln Asp Glu Asp Trp Val 675 680 685 Arg Asn Glu Leu Val Lys Asn Leu Glu Glu Gly Val Pro Pro Phe Gln 690 695 700 Leu Cys Leu His Tyr Arg Asp Phe Ile Pro Gly Val Ala Ile Ala Ala 705 710 715 720 Asn Ile Ile His Glu Gly Phe His Lys Ser Arg Lys Val Ile Val Val 725 730 735 Val Ser Gln His Phe Ile Gln Ser Arg Trp Cys Ile Phe Glu Tyr Glu 740 745 750 Ile Ala Gln Thr Trp Gln Phe Leu Ser Ser Arg Ala Gly Ile Ile Phe 755 760 765 Ile Val Leu Gln Lys Val Glu Lys Thr Leu Leu Arg Gln Gln Val Glu 770 775 780 Leu Tyr Arg Leu Leu Ser Arg Asn Thr Tyr Leu Glu Trp Glu Asp Ser 785 790 795 800 Val Leu Gly Arg His Ile Phe Trp Arg Arg Leu Arg Lys Ala Leu Leu 805 810 815 Asp Gly Lys Ser Trp Asn Pro Glu Gly Thr Val Gly Thr Gly Cys Asn 820 825 830 Trp Gln Glu Ala Thr Ser Ile 835 <210> 4 <211> 23 <212> PRT <213> Homo sapiens <400> 4 Ile Gly Val Ser Val Leu Ser Val Leu Val Val Ser Val Val Ala Val 1 5 10 15 Leu Val Tyr Lys Phe Tyr Phe 20 <210> 5 <211> 183 <212> PRT <213> Homo sapiens <400> 5 His Leu Met Leu Leu Ala Gly Cys Ile Lys Tyr Gly Arg Gly Glu Asn 1 5 10 15 Ile Tyr Asp Ala Phe Val Ile Tyr Ser Ser Gln Asp Glu Asp Trp Val 20 25 30 Arg Asn Glu Leu Val Lys Asn Leu Glu Glu Gly Val Pro Pro Phe Gln 35 40 45 Leu Cys Leu His Tyr Arg Asp Phe Ile Pro Gly Val Ala Ile Ala Ala 50 55 60 Asn Ile Ile His Glu Gly Phe His Lys Ser Arg Lys Val Ile Val Val 65 70 75 80 Val Ser Gln His Phe Ile Gln Ser Arg Trp Cys Ile Phe Glu Tyr Glu 85 90 95 Ile Ala Gln Thr Trp Gln Phe Leu Ser Ser Arg Ala Gly Ile Ile Phe 100 105 110 Ile Val Leu Gln Lys Val Glu Lys Thr Leu Leu Arg Gln Gln Val Glu 115 120 125 Leu Tyr Arg Leu Leu Ser Arg Asn Thr Tyr Leu Glu Trp Glu Asp Ser 130 135 140 Val Leu Gly Arg His Ile Phe Trp Arg Arg Leu Arg Lys Ala Leu Leu 145 150 155 160 Asp Gly Lys Ser Trp Asn Pro Glu Gly Thr Val Gly Thr Gly Cys Asn 165 170 175 Trp Gln Glu Ala Thr Ser Ile 180 <210> 6 <211> 2520 <212> DNA <213> Homo sapiens <400> 6 atgatgtctg cctcgcgcct ggctgggact ctgatcccag ccatggcctt cctctcctgc 60 gtgagaccag aaagctggga gccctgcgtg gaggtggttc ctaatattac ttatcaatgc 120 atggagctga atttctacaa aatccccgac aacctcccct tctcaaccaa gaacctggac 180 ctgagcttta atcccctgag gcatttaggc agctatagct tcttcagttt cccagaactg 240 caggtgctgg atttatccag gtgtgaaatc cagacaattg aagatggggc atatcagagc 300 ctaagccacc tctctacctt aatattgaca ggaaacccca tccagagttt agccctggga 360 gccttttctg gactatcaag tttacagaag ctggtggctg tggagacaaa tctagcatct 420 ctagagaact tccccattgg acatctcaaa actttgaaag aacttaatgt ggctcacaat 480 cttatccaat ctttcaaatt acctgagtat ttttctaatc tgaccaatct agagcacttg 540 gacctttcca gcaacaagat tcaaagtatt tattgcacag acttgcgggt tctacatcaa 600 atgcccctac tcaatctctc tttagacctg tccctgaacc ctatgaactt tatccaacca 660 ggtgcattta aagaaattag gcttcataag ctgactttaa gaaataattt tgatagttta 720 aatgtaatga aaacttgtat tcaaggtctg gctggtttag aagtccatcg tttggttctg 780 ggagaattta gaaatgaagg aaacttggaa aagtttgaca aatctgctct agagggcctg 840 tgcaatttga ccattgaaga attccgatta gcatacttag actactacct cgatgatatt 900 attgacttat ttaattgttt gacaaatgtt tcttcatttt ccctggtgag tgtgactatt 960 gaaagggtaa aagacttttc ttataatttc ggatggcaac atttagaatt agttaactgt 1020 aaatttggac agtttcccac attgaaactc aaatctctca aaaggcttac tttcacttcc 1080 aacaaaggtg ggaatgcttt ttcagaagtt gatctaccaa gccttgagtt tctagatctc 1140 agtagaaatg gcttgagttt caaaggttgc tgttctcaaa gtgattttgg gacaaccagc 1200 ctaaagtatt tagatctgag cttcaatggt gttattacca tgagttcaaa cttcttgggc 1260 ttagaacaac tagaacatct ggatttccag cattccaatt tgaaacaaat gagtgagttt 1320 tcagtattcc tatcactcag aaacctcatt taccttgaca tttctcatac tcacaccaga 1380 gttgctttca atggcatctt caatggcttg tccagtctcg aagtcttgaa aatggctggc 1440 aattctttcc aggaaaactt ccttccagat atcttcacag agctgagaaa cttgaccttc 1500 ctggacctct ctcagtgtca actggagcag ttgtctccaa cagcatttaa ctcactctcc 1560 agtcttcagg tactaaatat gagccacaac aacttctttt cattggatac gtttccttat 1620 aagtgtctga actccctcca ggttcttgat tacagtctca atcacataat gacttccaaa 1680 aaacaggaac tacagcattt tccaagtagt ctagctttct taaatcttac tcagaatgac 1740 tttgcttgta cttgtgaaca ccagagtttc ctgcaatgga tcaaggacca gaggcagctc 1800 ttggtggaag ttgaacgaat ggaatgtgca acaccttcag ataagcaggg catgcctggg 1860 ctgagtttga atatcacctg tcagatgaat aagaccatca ttggtgtgtc ggtcctcagt 1920 gtgcttgtag tatctgttgt agcagttctg gtctataagt tctattttca cctgatgctt 1980 cttgctggct gcataaagta tggtagaggt gaaaacatct atgatgcctt tgttatctac 2040 tcaagccagg atgaggactg ggtaaggaat gagctagtaa agaatttaga agaagggggtg 2100 cctccatttc agctctgcct tcactacaga gactttattc ccggtgtggc cattgctgcc 2160 aacatcatcc atgaaggttt ccataaaagc cgaaaggtga ttgttgtggt gtcccagcac 2220 ttcatccaga gccgctggtg tatctttgaa tatgagattg ctcagacctg gcagtttctg 2280 agcagtcgtg ctggtatcat cttcattgtc ctgcagaagg tggagaagac cctgctcagg 2340 cagcaggtgg agctgtaccg ccttctcagc aggaacactt acctggagtg ggaggacagt 2400 gtcctggggc ggcacatctt ctggagacga ctcagaaaag ccctgctgga tggtaaatca 2460 tggaatccag aaggaacagt gggtacagga tgcaattggc aggaagcaac atctatctga 2520 2520 <210> 7 <211> 1049 <212> PRT <213> Homo sapiens <400> 7 Met Val Phe Pro Met Trp Thr Leu Lys Arg Gln Ile Leu Ile Leu Phe 1 5 10 15 Asn Ile Ile Leu Ile Ser Lys Leu Leu Gly Ala Arg Trp Phe Pro Lys 20 25 30 Thr Leu Pro Cys Asp Val Thr Leu Asp Val Pro Lys Asn His Val Ile 35 40 45 Val Asp Cys Thr Asp Lys His Leu Thr Glu Ile Pro Gly Gly Ile Pro 50 55 60 Thr Asn Thr Thr Asn Leu Thr Leu Thr Ile Asn His Ile Pro Asp Ile 65 70 75 80 Ser Pro Ala Ser Phe His Arg Leu Asp His Leu Val Glu Ile Asp Phe 85 90 95 Arg Cys Asn Cys Val Pro Ile Pro Leu Gly Ser Lys Asn Asn Met Cys 100 105 110 Ile Lys Arg Leu Gln Ile Lys Pro Arg Ser Phe Ser Gly Leu Thr Tyr 115 120 125 Leu Lys Ser Leu Tyr Leu Asp Gly Asn Gln Leu Leu Glu Ile Pro Gln 130 135 140 Gly Leu Pro Pro Ser Leu Gln Leu Leu Ser Leu Glu Ala Asn Asn Ile 145 150 155 160 Phe Ser Ile Arg Lys Glu Asn Leu Thr Glu Leu Ala Asn Ile Glu Ile 165 170 175 Leu Tyr Leu Gly Gln Asn Cys Tyr Tyr Arg Asn Pro Cys Tyr Val Ser 180 185 190 Tyr Ser Ile Glu Lys Asp Ala Phe Leu Asn Leu Thr Lys Leu Lys Val 195 200 205 Leu Ser Leu Lys Asp Asn Asn Val Thr Ala Val Pro Thr Val Leu Pro 210 215 220 Ser Thr Leu Thr Glu Leu Tyr Leu Tyr Asn Asn Met Ile Ala Lys Ile 225 230 235 240 Gln Glu Asp Asp Phe Asn Asn Leu Asn Gln Leu Gln Ile Leu Asp Leu 245 250 255 Ser Gly Asn Cys Pro Arg Cys Tyr Asn Ala Pro Phe Pro Cys Ala Pro 260 265 270 Cys Lys Asn Asn Ser Pro Leu Gln Ile Pro Val Asn Ala Phe Asp Ala 275 280 285 Leu Thr Glu Leu Lys Val Leu Arg Leu His Ser Asn Ser Leu Gln His 290 295 300 Val Pro Pro Arg Trp Phe Lys Asn Ile Asn Lys Leu Gln Glu Leu Asp 305 310 315 320 Leu Ser Gln Asn Phe Leu Ala Lys Glu Ile Gly Asp Ala Lys Phe Leu 325 330 335 His Phe Leu Pro Ser Leu Ile Gln Leu Asp Leu Ser Phe Asn Phe Glu 340 345 350 Leu Gln Val Tyr Arg Ala Ser Met Asn Leu Ser Gln Ala Phe Ser Ser 355 360 365 Leu Lys Ser Leu Lys Ile Leu Arg Ile Arg Gly Tyr Val Phe Lys Glu 370 375 380 Leu Lys Ser Phe Asn Leu Ser Pro Leu His Asn Leu Gln Asn Leu Glu 385 390 395 400 Val Leu Asp Leu Gly Thr Asn Phe Ile Lys Ile Ala Asn Leu Ser Met 405 410 415 Phe Lys Gln Phe Lys Arg Leu Lys Val Ile Asp Leu Ser Val Asn Lys 420 425 430 Ile Ser Pro Ser Gly Asp Ser Ser Glu Val Gly Phe Cys Ser Asn Ala 435 440 445 Arg Thr Ser Val Glu Ser Tyr Glu Pro Gln Val Leu Glu Gln Leu His 450 455 460 Tyr Phe Arg Tyr Asp Lys Tyr Ala Arg Ser Cys Arg Phe Lys Asn Lys 465 470 475 480 Glu Ala Ser Phe Met Ser Val Asn Glu Ser Cys Tyr Lys Tyr Gly Gln 485 490 495 Thr Leu Asp Leu Ser Lys Asn Ser Ile Phe Phe Val Lys Ser Ser Asp 500 505 510 Phe Gln His Leu Ser Phe Leu Lys Cys Leu Asn Leu Ser Gly Asn Leu 515 520 525 Ile Ser Gln Thr Leu Asn Gly Ser Glu Phe Gln Pro Leu Ala Glu Leu 530 535 540 Arg Tyr Leu Asp Phe Ser Asn Asn Arg Leu Asp Leu Leu His Ser Thr 545 550 555 560 Ala Phe Glu Glu Leu His Lys Leu Glu Val Leu Asp Ile Ser Ser Asn 565 570 575 Ser His Tyr Phe Gln Ser Glu Gly Ile Thr His Met Leu Asn Phe Thr 580 585 590 Lys Asn Leu Lys Val Leu Gln Lys Leu Met Met Asn Asp Asn Asp Ile 595 600 605 Ser Ser Ser Thr Ser Arg Thr Met Glu Ser Glu Ser Leu Arg Thr Leu 610 615 620 Glu Phe Arg Gly Asn His Leu Asp Val Leu Trp Arg Glu Gly Asp Asn 625 630 635 640 Arg Tyr Leu Gln Leu Phe Lys Asn Leu Leu Lys Leu Glu Glu Leu Asp 645 650 655 Ile Ser Lys Asn Ser Leu Ser Phe Leu Pro Ser Gly Val Phe Asp Gly 660 665 670 Met Pro Pro Asn Leu Lys Asn Leu Ser Leu Ala Lys Asn Gly Leu Lys 675 680 685 Ser Phe Ser Trp Lys Lys Leu Gln Cys Leu Lys Asn Leu Glu Thr Leu 690 695 700 Asp Leu Ser His Asn Gln Leu Thr Thr Val Pro Glu Arg Leu Ser Asn 705 710 715 720 Cys Ser Arg Ser Leu Lys Asn Leu Ile Leu Lys Asn Asn Gln Ile Arg 725 730 735 Ser Leu Thr Lys Tyr Phe Leu Gln Asp Ala Phe Gln Leu Arg Tyr Leu 740 745 750 Asp Leu Ser Ser Asn Lys Ile Gln Met Ile Gln Lys Thr Ser Phe Pro 755 760 765 Glu Asn Val Leu Asn Asn Leu Lys Met Leu Leu Leu His His Asn Arg 770 775 780 Phe Leu Cys Thr Cys Asp Ala Val Trp Phe Val Trp Trp Val Asn His 785 790 795 800 Thr Glu Val Thr Ile Pro Tyr Leu Ala Thr Asp Val Thr Cys Val Gly 805 810 815 Pro Gly Ala His Lys Gly Gln Ser Val Ile Ser Leu Asp Leu Tyr Thr 820 825 830 Cys Glu Leu Asp Leu Thr Asn Leu Ile Leu Phe Ser Leu Ser Ile Ser 835 840 845 Val Ser Leu Phe Leu Met Val Met Met Thr Ala Ser His Leu Tyr Phe 850 855 860 Trp Asp Val Trp Tyr Ile Tyr His Phe Cys Lys Ala Lys Ile Lys Gly 865 870 875 880 Tyr Gln Arg Leu Ile Ser Pro Asp Cys Cys Tyr Asp Ala Phe Ile Val 885 890 895 Tyr Asp Thr Lys Asp Pro Ala Val Thr Glu Trp Val Leu Ala Glu Leu 900 905 910 Val Ala Lys Leu Glu Asp Pro Arg Glu Lys His Phe Asn Leu Cys Leu 915 920 925 Glu Glu Arg Asp Trp Leu Pro Gly Gln Pro Val Leu Glu Asn Leu Ser 930 935 940 Gln Ser Ile Gln Leu Ser Lys Lys Thr Val Phe Val Met Thr Asp Lys 945 950 955 960 Tyr Ala Lys Thr Glu Asn Phe Lys Ile Ala Phe Tyr Leu Ser His Gln 965 970 975 Arg Leu Met Asp Glu Lys Val Asp Val Ile Ile Leu Ile Phe Leu Glu 980 985 990 Lys Pro Phe Gln Lys Ser Lys Phe Leu Gln Leu Arg Lys Arg Leu Cys 995 1000 1005 Gly Ser Ser Val Leu Glu Trp Pro Thr Asn Pro Gln Ala His Pro Tyr 1010 1015 1020 Phe Trp Gln Cys Leu Lys Asn Ala Leu Ala Thr Asp Asn His Val Ala 1025 1030 1035 1040 Tyr Ser Gln Val Phe Lys Glu Thr Val 1045 <210> 8 <211> 3150 <212> DNA <213> Homo sapiens <400> 8 atggtgtttc caatgtggac actgaagaga caaattctta tcctttttaa cataatccta 60 atttccaaac tccttggggc tagatggttt cctaaaactc tgccctgtga tgtcactctg 120 gatgttccaa agaaccatgt gatcgtggac tgcacagaca agcatttgac agaaattcct 180 ggaggtattc ccacgaacac cacgaacctc accctcacca ttaaccacat accagacatc 240 tccccagcgt cctttcacag actggaccat ctggtagaga tcgatttcag atgcaactgt 300 gtacctattc cactggggtc aaaaaacaac atgtgcatca agaggctgca gattaaaccc 360 agaagcttta gtggactcac ttatttaaaa tccctttacc tggatggaaa ccagctacta 420 gagataccgc agggcctccc gcctagctta cagcttctca gccttgaggc caacaacatc 480 ttttccatca gaaaagagaa tctaacagaa ctggccaaca tagaaatact ctacctgggc 540 caaaactgtt attatcgaaa tccttgttat gtttcatatt caatagagaa agatgccttc 600 ctaaacttga caaagttaaa agtgctctcc ctgaaagata acaatgtcac agccgtccct 660 actgttttgc catctacttt aacagaacta tatctctaca acaacatgat tgcaaaaatc 720 caagaagatg attttaataa cctcaaccaa ttacaaattc ttgacctaag tggaaattgc 780 cctcgttgtt ataatgcccc atttccttgt gcgccgtgta aaaataattc tcccctacag 840 atccctgtaa atgcttttga tgcgctgaca gaattaaaag ttttacgtct acacagtaac 900 tctcttcagc atgtgccccc aagatggttt aagaaca acaaactcca ggaactggat 960 ctgtcccaaa acttcttggc caaagaaatt ggggatgcta aatttctgca ttttctcccc 1020 agcctcatcc aattggatct gtctttcaat tttgaacttc aggtctatcg tgcatctatg 1080 aatctatcac aagcattttc ttcactgaaa agcctgaaaa ttctgcggat cagaggatat 1140 gtctttaaag agttgaaaag ctttaacctc tcgccattac ataatcttca aaatcttgaa 1200 gttcttgatc ttggcactaa ctttataaaa attgctaacc tcagcatgtt taaacaattt 1260 aaaagactga aagtcataga tctttcagtg aataaaatat caccttcagg agattcaagt 1320 gaagttggct tctgctcaaa tgccagaact tctgtagaaa gttatgaacc ccaggtcctg 1380 gaacaattac attatttcag atatgataag tatgcaagga gttgcagatt caaaaacaaa 1440 gaggcttctt tcatgtctgt taatgaaagc tgctacaagt atgggcagac cttggatcta 1500 agtaaaaata gtatattttt tgtcaagtcc tctgattttc agcatctttc tttcctcaaa 1560 tgcctgaatc tgtcaggaaa tctcattagc caaactctta atggcagtga attccaacct 1620 ttagcagagc tgagatattt ggacttctcc aacaaccggc ttgatttact ccattcaaca 1680 gcatttgaag agcttcacaa actggaagtt ctggatataa gcagtaatag ccattatttt 1740 caatcagaag gaattactca tatgctaaac tttaccaaga acctaaaggt tctgcagaaa 1800 ctgatgatga acgacaatga catctcttcc tccaccagca ggaccatgga gagtgagtct 1860 cttagaactc tggaattcag aggaaatcac ttagatgttt tatggagaga aggtgataac 1920 agatacttac aattattcaa gaatctgcta aaattagagg aattagacat ctctaaaaat 1980 2040 tctttggcca aaaatgggct caaatctttc agttggaaga aactccagtg tctaaagaac 2100 ctggaaactt tggacctcag ccacaaccaa ctgaccactg tccctgagag attatccaac 2160 tgttccagaa gcctcaagaa tctgattctt aagaataatc aaatcaggag tctgacgaag 2220 tattttctac aagatgcctt ccagttgcga tatctggatc tcagctcaaa taaaatccag 2280 atgatccaaa agaccagctt cccagaaaat gtcctcaaca atctgaagat gttgcttttg 2340 catcataatc ggtttctgtg cacctgtgat gctgtgtggt ttgtctggtg ggttaaccat 2400 acggaggtga ctattcctta cctggccaca gatgtgactt gtgtggggcc aggagcacac 2460 aagggccaaa gtgtgatctc cctggatctg tacacctgtg agttagatct gactaacctg 2520 attctgttct cactttccat atctgtatct ctctttctca tggtgatgat gacagcaagt 2580 cacctctatt tctgggatgt gtggtatatt taccatttct gtaaggccaa gataaagggg 2640 tatcagcgtc taatatcacc agactgttgc tatgatgctt ttattgtgta tgacactaaa 2700 gacccagctg tgaccgagtg ggttttggct gagctggtgg ccaaactgga agacccaaga 2760 gagaaacatt ttaatttatg tctcgaggaa agggactggt taccagggca gccagttctg 2820 gaaaaccttt cccagagcat acagcttagc aaaaagacag tgtttgtgat gacagacaag 2880 tatgcaaaga ctgaaaattt taagatagca ttttacttgt cccatcagag gctcatggat 2940 gaaaaagttg atgtgattat cttgatattt cttgagaagc cctttcagaa gtccaagttc 3000 ctccagctcc ggaaaaggct ctgtgggagt tctgtccttg agtggccaac aaacccgcaa 3060 gctcacccat acttctggca gtgtctaaag aacgccctgg ccacagacaa tcatgtggcc 3120 tatagtcagg tgttcaagga aacggtctag 3150 <210> 9 <211> 1041 <212> PRT <213> Homo sapiens <400> 9 Met Glu Asn Met Phe Leu Gln Ser Ser Met Leu Thr Cys Ile Phe Leu 1 5 10 15 Leu Ile Ser Gly Ser Cys Glu Leu Cys Ala Glu Glu Asn Phe Ser Arg 20 25 30 Ser Tyr Pro Cys Asp Glu Lys Lys Gln Asn Asp Ser Val Ile Ala Glu 35 40 45 Cys Ser Asn Arg Arg Leu Gln Glu Val Pro Gln Thr Val Gly Lys Tyr 50 55 60 Val Thr Glu Leu Asp Leu Ser Asp Asn Phe Ile Thr His Ile Thr Asn 65 70 75 80 Glu Ser Phe Gln Gly Leu Gln Asn Leu Thr Lys Ile Asn Leu Asn His 85 90 95 Asn Pro Asn Val Gln His Gln Asn Gly Asn Pro Gly Ile Gln Ser Asn 100 105 110 Gly Leu Asn Ile Thr Asp Gly Ala Phe Leu Asn Leu Lys Asn Leu Arg 115 120 125 Glu Leu Leu Leu Glu Asp Asn Gln Leu Pro Gln Ile Pro Ser Gly Leu 130 135 140 Pro Glu Ser Leu Thr Glu Leu Ser Leu Ile Gln Asn Asn Ile Tyr Asn 145 150 155 160 Ile Thr Lys Glu Gly Ile Ser Arg Leu Ile Asn Leu Lys Asn Leu Tyr 165 170 175 Leu Ala Trp Asn Cys Tyr Phe Asn Lys Val Cys Glu Lys Thr Asn Ile 180 185 190 Glu Asp Gly Val Phe Glu Thr Leu Thr Asn Leu Glu Leu Leu Ser Leu 195 200 205 Ser Phe Asn Ser Leu Ser His Val Pro Pro Lys Leu Pro Ser Ser Leu 210 215 220 Arg Lys Leu Phe Leu Ser Asn Thr Gln Ile Lys Tyr Ile Ser Glu Glu 225 230 235 240 Asp Phe Lys Gly Leu Ile Asn Leu Thr Leu Leu Asp Leu Ser Gly Asn 245 250 255 Cys Pro Arg Cys Phe Asn Ala Pro Phe Pro Cys Val Pro Cys Asp Gly 260 265 270 Gly Ala Ser Ile Asn Ile Asp Arg Phe Ala Phe Gln Asn Leu Thr Gln 275 280 285 Leu Arg Tyr Leu Asn Leu Ser Ser Thr Ser Leu Arg Lys Ile Asn Ala 290 295 300 Ala Trp Phe Lys Asn Met Pro His Leu Lys Val Leu Asp Leu Glu Phe 305 310 315 320 Asn Tyr Leu Val Gly Glu Ile Ala Ser Gly Ala Phe Leu Thr Met Leu 325 330 335 Pro Arg Leu Glu Ile Leu Asp Leu Ser Phe Asn Tyr Ile Lys Gly Ser 340 345 350 Tyr Pro Gln His Ile Asn Ile Ser Arg Asn Phe Ser Lys Leu Leu Ser 355 360 365 Leu Arg Ala Leu His Leu Arg Gly Tyr Val Phe Gln Glu Leu Arg Glu 370 375 380 Asp Asp Phe Gln Pro Leu Met Gln Leu Pro Asn Leu Ser Thr Ile Asn 385 390 395 400 Leu Gly Ile Asn Phe Ile Lys Gln Ile Asp Phe Lys Leu Phe Gln Asn 405 410 415 Phe Ser Asn Leu Glu Ile Ile Tyr Leu Ser Glu Asn Arg Ile Ser Pro 420 425 430 Leu Val Lys Asp Thr Arg Gln Ser Tyr Ala Asn Ser Ser Ser Phe Gln 435 440 445 Arg His Ile Arg Lys Arg Arg Ser Thr Asp Phe Glu Phe Asp Pro His 450 455 460 Ser Asn Phe Tyr His Phe Thr Arg Pro Leu Ile Lys Pro Gln Cys Ala 465 470 475 480 Ala Tyr Gly Lys Ala Leu Asp Leu Ser Leu Asn Ser Ile Phe Phe Ile 485 490 495 Gly Pro Asn Gln Phe Glu Asn Leu Pro Asp Ile Ala Cys Leu Asn Leu 500 505 510 Ser Ala Asn Ser Asn Ala Gln Val Leu Ser Gly Thr Glu Phe Ser Ala 515 520 525 Ile Pro His Val Lys Tyr Leu Asp Leu Thr Asn Asn Arg Leu Asp Phe 530 535 540 Asp Asn Ala Ser Ala Leu Thr Glu Leu Ser Asp Leu Glu Val Leu Asp 545 550 555 560 Leu Ser Tyr Asn Ser His Tyr Phe Arg Ile Ala Gly Val Thr His His 565 570 575 Leu Glu Phe Ile Gln Asn Phe Thr Asn Leu Lys Val Leu Asn Leu Ser 580 585 590 His Asn Asn Ile Tyr Thr Leu Thr Asp Lys Tyr Asn Leu Glu Ser Lys 595 600 605 Ser Leu Val Glu Leu Val Phe Ser Gly Asn Arg Leu Asp Ile Leu Trp 610 615 620 Asn Asp Asp Asp Asn Arg Tyr Ile Ser Ile Phe Lys Gly Leu Lys Asn 625 630 635 640 Leu Thr Arg Leu Asp Leu Ser Leu Asn Arg Leu Lys His Ile Pro Asn 645 650 655 Glu Ala Phe Leu Asn Leu Pro Ala Ser Leu Thr Glu Leu His Ile Asn 660 665 670 Asp Asn Met Leu Lys Phe Phe Asn Trp Thr Leu Leu Gln Gln Phe Pro 675 680 685 Arg Leu Glu Leu Leu Asp Leu Arg Gly Asn Lys Leu Leu Phe Leu Thr 690 695 700 Asp Ser Leu Ser Asp Phe Thr Ser Ser Leu Arg Thr Leu Leu Leu Ser 705 710 715 720 His Asn Arg Ile Ser His Leu Pro Ser Gly Phe Leu Ser Glu Val Ser 725 730 735 Ser Leu Lys His Leu Asp Leu Ser Ser Asn Leu Leu Lys Thr Ile Asn 740 745 750 Lys Ser Ala Leu Glu Thr Lys Thr Thr Thr Lys Leu Ser Met Leu Glu 755 760 765 Leu His Gly Asn Pro Phe Glu Cys Thr Cys Asp Ile Gly Asp Phe Arg 770 775 780 Arg Trp Met Asp Glu His Leu Asn Val Lys Ile Pro Arg Leu Val Asp 785 790 795 800 Val Ile Cys Ala Ser Pro Gly Asp Gln Arg Gly Lys Ser Ile Val Ser 805 810 815 Leu Glu Leu Thr Thr Cys Val Ser Asp Val Thr Ala Val Ile Leu Phe 820 825 830 Phe Phe Thr Phe Phe Ile Thr Thr Met Val Met Leu Ala Ala Leu Ala 835 840 845 His His Leu Phe Tyr Trp Asp Val Trp Phe Ile Tyr Asn Val Cys Leu 850 855 860 Ala Lys Val Lys Gly Tyr Arg Ser Leu Ser Thr Ser Gln Thr Phe Tyr 865 870 875 880 Asp Ala Tyr Ile Ser Tyr Asp Thr Lys Asp Ala Ser Val Thr Asp Trp 885 890 895 Val Ile Asn Glu Leu Arg Tyr His Leu Glu Glu Ser Arg Asp Lys Asn 900 905 910 Val Leu Leu Cys Leu Glu Glu Arg Asp Trp Asp Pro Gly Leu Ala Ile 915 920 925 Ile Asp Asn Leu Met Gln Ser Ile Asn Gln Ser Lys Lys Thr Val Phe 930 935 940 Val Leu Thr Lys Lys Tyr Ala Lys Ser Trp Asn Phe Lys Thr Ala Phe 945 950 955 960 Tyr Leu Ala Leu Gln Arg Leu Met Asp Glu Asn Met Asp Val Ile Ile 965 970 975 Phe Ile Leu Leu Glu Pro Val Leu Gln His Ser Gln Tyr Leu Arg Leu 980 985 990 Arg Gln Arg Ile Cys Lys Ser Ser Ile Leu Gln Trp Pro Asp Asn Pro 995 1000 1005 Lys Ala Glu Gly Leu Phe Trp Gln Thr Leu Arg Asn Val Val Leu Thr 1010 1015 1020 Glu Asn Asp Ser Arg Tyr Asn Asn Met Tyr Val Asp Ser Ile Lys Gln 1025 1030 1035 1040 Tyr <210> 10 <211> 3126 <212> DNA <213> Homo sapiens <400> 10 atggaaaaca tgttccttca gtcgtcaatg ctgacctgca ttttcctgct aatatctggt 60 tcctgtgagt tatgcgccga agaaaatttt tctagaagct atccttgtga tgagaaaaag 120 caaaatgact cagttatgc agagtgcagc aatcgtcgac tacaggaagt tccccaaacg 180 gtgggcaaat atgtgacaga actagacctg tctgataatt tcatcacaca cataacgaat 240 gaatcatttc aagggctgca aaatctcact aaaataaatc taaaccacaa ccccaatgta 300 cagcaccaga acggaaatcc cggtatacaa tcaaatggct tgaatatcac agacggggca 360 ttcctcaacc taaaaaacct aagggagtta ctgcttgaag acaaccagtt accccaaata 420 ccctctggtt tgccagagtc tttgacagaa cttagtctaa ttcaaaacaa tatatacaac 480 ataactaaag agggcatttc aagacttata aacttgaaaa atctctattt ggcctggaac 540 tgctatttta acaaagtttg cgagaaaact aacatagaag atggagtatt tgaaacgctg 600 acaaatttgg agttgctatc actatctttc aattctcttt cacacgtgcc acccaaactg 660 ccaagctccc tacgcaaact ttttctgagc aacacccaga tcaaatacat tagtgaagaa 720 gatttcaagg gattgataaa tttaacatta ctagatttaa gcgggaactg tccgaggtgc 780 ttcaatgccc catttccatg cgtgccttgt gatggtggtg cttcaattaa tatagatcgt 840 tttgcttttc aaaacttgac ccaacttcga tacctaaacc tctctagcac ttccctcagg 900 aagattaatg ctgcctggtt taaaaatatg cctcatctga aggtgctgga tcttgaattc 960 aactatttag tgggaaat agcctctggg gcatttttaa cgatgctgcc ccgcttagaa 1020 atacttgact tgtcttttaa ctatataaag gggagttatc cacagcatat taatatttcc 1080 agaaacttct ctaaactttt gtctctacgg gcattgcatt taagaggtta tgtgttccag 1140 gaactcagag aagatgattt ccagcccctg atgcagcttc caaacttatc gactatcaac 1200 ttgggtatta attttattaa gcaaatcgat ttcaaacttt tccaaaattt ctccaatctg 1260 gaaattattt acttgtcaga aaacagaata tcaccgttgg taaaagatac ccggcagagt 1320 tatgcaaata gttcctcttt tcaacgtcat atccggaaac gacgctcaac agattttgag 1380 tttgacccac attcgaactt ttatcatttc acccgtcctt taataaagcc acaatgtgct 1440 gcttatgggaa aagccttaga tttaagcctc aacagtattt tcttcattgg gccaaaccaa 1500 tttgaaaatc ttcctgacat tgcctgttta aatctgtctg caaatagcaa tgctcaagtg 1560 ttaagtgggaa ctgaattttc agccattcct catgtcaaat atttggattt gacaaacaat 1620 agactagact ttgataatgc tagtgctctt actgaattgt ccgacttgga agttctagat 1680 ctcagctata attcacacta tttcagaata gcaggcgtaa cacatcatct agaatttatt 1740 caaaatttca caaatctaaa agttttaaac ttgagccaca acaacattta tactttaaca 1800 gataagtata acctggaaag caagtccctg gtagaattag ttttcagtgg caatcgcctt 1860 gacattttgt ggaatgatga tgacaacagg tatatctcca ttttcaaagg tctcaagaat 1920 ctgacacgtc tggatttatc ccttaatagg ctgaagcaca tcccaaatga agcattcctt 1980 aatttgccag cgagtctcac tgaactacat ataaatgata atatgttaaa gttttttaac 2040 tggacattac tccagcagtt tcctcgtctc gagttgcttg acttacgtgg aaacaaacta 2100 ctctttttaa ctgatagcct atctgacttt acatcttccc ttcggacact gctgctgagt 2160 cataacagga tttcccacct accctctggc tttctttctg aagtcagtag tctgaagcac 2220 ctcgatttaa gttccaatct gctaaaaaca atcaacaaat ccgcacttga aactaagacc 2280 accaccaaat tatctatgtt ggaactacac ggaaacccct ttgaatgcac ctgtgacatt 2340 ggagattcc gaagatggat ggatgaacat ctgaatgtca aaattcccag actggtagat 2400 gtcatttgtg ccagtcctgg ggatcaaaga gggaagagta ttgtgagtct ggagctaaca 2460 acttgtgttt cagatgtcac tgcagtgata ttatttttct tcacgttctt tatcaccacc 2520 atggttatgt tggctgccct ggctcaccat ttgttttact gggatgtttg gtttatatat 2580 aatgtgtgtt tagctaaggt aaaaggctac aggtctcttt ccacatccca aactttctat 2640 gatgcttaca tttcttatga caccaaagat gcctctgtta ctgactgggt gataaatgag 2700 ctgcgctacc accttgaaga gagccgagac aaaaacgttc tcctttgtct agaggagagg 2760 gattgggacc cgggattggc catcatcgac aacctcatgc agagcatcaa ccaaagcaag 2820 aaaacagtat ttgttttaac caaaaaatat gcaaaaagct ggaactttaa aacagctttt 2880 tacttggctt tgcagaggct aatggatgag aacatggatg tgattatatt tatcctgctg 2940 gagccagtgt tacagcattc tcagtatttg aggctacggc agcggatctg taagagctcc 3000 atcctccagt ggcctgacaa cccgaaggca gaaggcttgt tttggcaaac tctgagaaat 3060 gtggtcttga ctgaaaatga ttcacggtat aacaatatgt atgtcgattc cattaagcaa 3120 tactaa 3126 <210> 11 <211> 1032 <212> PRT <213> Homo sapiens <400> 11 Met Gly Phe Cys Arg Ser Ala Leu His Pro Leu Ser Leu Leu Val Gln 1 5 10 15 Ala Ile Met Leu Ala Met Thr Leu Ala Leu Gly Thr Leu Pro Ala Phe 20 25 30 Leu Pro Cys Glu Leu Gln Pro His Gly Leu Val Asn Cys Asn Trp Leu 35 40 45 Phe Leu Lys Ser Val Pro His Phe Ser Met Ala Ala Pro Arg Gly Asn 50 55 60 Val Thr Ser Leu Ser Leu Ser Ser Asn Arg Ile His His Leu His Asp 65 70 75 80 Ser Asp Phe Ala His Leu Pro Ser Leu Arg His Leu Asn Leu Lys Trp 85 90 95 Asn Cys Pro Pro Val Gly Leu Ser Pro Met His Phe Pro Cys His Met 100 105 110 Thr Ile Glu Pro Ser Thr Phe Leu Ala Val Pro Thr Leu Glu Glu Leu 115 120 125 Asn Leu Ser Tyr Asn Asn Ile Met Thr Val Pro Ala Leu Pro Lys Ser 130 135 140 Leu Ile Ser Leu Ser Leu Ser His Thr Asn Ile Leu Met Leu Asp Ser 145 150 155 160 Ala Ser Leu Ala Gly Leu His Ala Leu Arg Phe Leu Phe Met Asp Gly 165 170 175 Asn Cys Tyr Tyr Lys Asn Pro Cys Arg Gln Ala Leu Glu Val Ala Pro 180 185 190 Gly Ala Leu Leu Gly Leu Gly Asn Leu Thr His Leu Ser Leu Lys Tyr 195 200 205 Asn Asn Leu Thr Val Val Pro Arg Asn Leu Pro Ser Ser Leu Glu Tyr 210 215 220 Leu Leu Leu Ser Tyr Asn Arg Ile Val Lys Leu Ala Pro Glu Asp Leu 225 230 235 240 Ala Asn Leu Thr Ala Leu Arg Val Leu Asp Val Gly Gly Asn Cys Arg 245 250 255 Arg Cys Asp His Ala Pro Asn Pro Cys Met Glu Cys Pro Arg His Phe 260 265 270 Pro Gln Leu His Pro Asp Thr Phe Ser His Leu Ser Arg Leu Glu Gly 275 280 285 Leu Val Leu Lys Asp Ser Ser Leu Ser Trp Leu Asn Ala Ser Trp Phe 290 295 300 Arg Gly Leu Gly Asn Leu Arg Val Leu Asp Leu Ser Glu Asn Phe Leu 305 310 315 320 Tyr Lys Cys Ile Thr Lys Thr Lys Ala Phe Gln Gly Leu Thr Gln Leu 325 330 335 Arg Lys Leu Asn Leu Ser Phe Asn Tyr Gln Lys Arg Val Ser Phe Ala 340 345 350 His Leu Ser Leu Ala Pro Ser Phe Gly Ser Leu Val Ala Leu Lys Glu 355 360 365 Leu Asp Met His Gly Ile Phe Phe Arg Ser Leu Asp Glu Thr Thr Leu 370 375 380 Arg Pro Leu Ala Arg Leu Pro Met Leu Gln Thr Leu Arg Leu Gln Met 385 390 395 400 Asn Phe Ile Asn Gln Ala Gln Leu Gly Ile Phe Arg Ala Phe Pro Gly 405 410 415 Leu Arg Tyr Val Asp Leu Ser Asp Asn Arg Ile Ser Gly Ala Ser Glu 420 425 430 Leu Thr Ala Thr Met Gly Glu Ala Asp Gly Gly Glu Lys Val Trp Leu 435 440 445 Gln Pro Gly Asp Leu Ala Pro Ala Pro Val Asp Thr Pro Ser Ser Glu 450 455 460 Asp Phe Arg Pro Asn Cys Ser Thr Leu Asn Phe Thr Leu Asp Leu Ser 465 470 475 480 Arg Asn Asn Leu Val Thr Val Gln Pro Glu Met Phe Ala Gln Leu Ser 485 490 495 His Leu Gln Cys Leu Arg Leu Ser His Asn Cys Ile Ser Gln Ala Val 500 505 510 Asn Gly Ser Gln Phe Leu Pro Leu Thr Gly Leu Gln Val Leu Asp Leu 515 520 525 Ser His Asn Lys Leu Asp Leu Tyr His Glu His Ser Phe Thr Glu Leu 530 535 540 Pro Arg Leu Glu Ala Leu Asp Leu Ser Tyr Asn Ser Gln Pro Phe Gly 545 550 555 560 Met Gln Gly Val Gly His Asn Phe Ser Phe Val Ala His Leu Arg Thr 565 570 575 Leu Arg His Leu Ser Leu Ala His Asn Asn Ile His Ser Gln Val Ser 580 585 590 Gln Gln Leu Cys Ser Thr Ser Leu Arg Ala Leu Asp Phe Ser Gly Asn 595 600 605 Ala Leu Gly His Met Trp Ala Glu Gly Asp Leu Tyr Leu His Phe Phe 610 615 620 Gln Gly Leu Ser Gly Leu Ile Trp Leu Asp Leu Ser Gln Asn Arg Leu 625 630 635 640 His Thr Leu Leu Pro Gln Thr Leu Arg Asn Leu Pro Lys Ser Leu Gln 645 650 655 Val Leu Arg Leu Arg Asp Asn Tyr Leu Ala Phe Phe Lys Trp Trp Ser 660 665 670 Leu His Phe Leu Pro Lys Leu Glu Val Leu Asp Leu Ala Gly Asn Gln 675 680 685 Leu Lys Ala Leu Thr Asn Gly Ser Leu Pro Ala Gly Thr Arg Leu Arg 690 695 700 Arg Leu Asp Val Ser Cys Asn Ser Ile Ser Phe Val Ala Pro Gly Phe 705 710 715 720 Phe Ser Lys Ala Lys Glu Leu Arg Glu Leu Asn Leu Ser Ala Asn Ala 725 730 735 Leu Lys Thr Val Asp His Ser Trp Phe Gly Pro Leu Ala Ser Ala Leu 740 745 750 Gln Ile Leu Asp Val Ser Ala Asn Pro Leu His Cys Ala Cys Gly Ala 755 760 765 Ala Phe Met Asp Phe Leu Leu Glu Val Gln Ala Ala Val Pro Gly Leu 770 775 780 Pro Ser Arg Val Lys Cys Gly Ser Pro Gly Gln Leu Gln Gly Leu Ser 785 790 795 800 Ile Phe Ala Gln Asp Leu Arg Leu Cys Leu Asp Glu Ala Leu Ser Trp 805 810 815 Asp Cys Phe Ala Leu Ser Leu Leu Ala Val Ala Leu Gly Leu Gly Val 820 825 830 Pro Met Leu His His Leu Cys Gly Trp Asp Leu Trp Tyr Cys Phe His 835 840 845 Leu Cys Leu Ala Trp Leu Pro Trp Arg Gly Arg Gln Ser Gly Arg Asp 850 855 860 Glu Asp Ala Leu Pro Tyr Asp Ala Phe Val Val Phe Asp Lys Thr Gln 865 870 875 880 Ser Ala Val Ala Asp Trp Val Tyr Asn Glu Leu Arg Gly Gln Leu Glu 885 890 895 Glu Cys Arg Gly Arg Trp Ala Leu Arg Leu Cys Leu Glu Glu Arg Asp 900 905 910 Trp Leu Pro Gly Lys Thr Leu Phe Glu Asn Leu Trp Ala Ser Val Tyr 915 920 925 Gly Ser Arg Lys Thr Leu Phe Val Leu Ala His Thr Asp Arg Val Ser 930 935 940 Gly Leu Leu Arg Ala Ser Phe Leu Leu Ala Gln Gln Arg Leu Leu Glu 945 950 955 960 Asp Arg Lys Asp Val Val Val Leu Val Ile Leu Ser Pro Asp Gly Arg 965 970 975 Arg Ser Arg Tyr Val Arg Leu Arg Gln Arg Leu Cys Arg Gln Ser Val 980 985 990 Leu Leu Trp Pro His Gln Pro Ser Gly Gln Arg Ser Phe Trp Ala Gln 995 1000 1005 Leu Gly Met Ala Leu Thr Arg Asp Asn His His Phe Tyr Asn Arg Asn 1010 1015 1020 Phe Cys Gln Gly Pro Thr Ala Glu 1025 1030 <210> 12 <211> 3099 <212> DNA <213> Homo sapiens <400> 12 atgggtttct gccgcagcgc cctgcacccg ctgtctctcc tggtgcaggc catcatgctg 60 gccatgaccc tggccctggg taccttgcct gccttcctac cctgtgagct ccagccccac 120 ggcctggtga actgcaactg gctgttcctg aagtctgtgc cccacttctc catggcagca 180 ccccgtggca atgtcaccag cctttccttg tcctccaacc gcatccacca cctccatgat 240 tctgactttg cccacctgcc cagcctgcgg catctcaacc tcaagtggaa ctgcccgccg 300 gttggcctca gcccccatgca cttcccctgc cacatgacca tcgagcccag caccttcttg 360 gctgtgccca ccctggaaga gctaaacctg agctacaaca acatcatgac tgtgcctgcg 420 ctgcccaaat ccctcatatc cctgtccctc agccatacca acatcctgat gctagactct 480 gccagcctcg ccggcctgca tgccctgcgc ttcctattca tggacggcaa ctgttattac 540 aagaacccct gcaggcaggc actggaggtg gccccgggtg ccctccttgg cctgggcaac 600 ctcacccacc tgtcactcaa gtacaacaac ctcactgtgg tgccccgcaa cctgccttcc 660 agcctggagt atctgctgtt gtcctacaac cgcatcgtca aactggcgcc tgaggacctg 720 gccaatctga ccgccctgcg tgtgctcgat gtgggcggaa attgccgccg ctgcgaccac 780 gctcccaacc cctgcatgga gtgccctcgt cacttccccc agctacatcc cgataccttc 840 agccacctga gccgtcttga aggcctggtg ttgaaggaca gttctctctc ctggctgaat 900 gccagttggt tccgtgggct gggaaacctc cgagtgctgg acctgagtga gaacttcctc 960 tacaaatgca tcactaaaac caaggccttc cagggcctaa cacagctgcg caagcttaac 1020 ctgtccttca attaccaaaa gagggtgtcc tttgcccacc tgtctctggc cccttccttc 1080 ggggagcctgg tcgccctgaa ggagctggac atgcacggca tcttcttccg ctcactcgat 1140 gagaccacgc tccggccact ggcccgcctg cccatgctcc agactctgcg tctgcagatg 1200 aacttcatca accaggccca gctcggcatc ttcagggcct tccctggcct gcgctacgtg 1260 gacctgtcgg acaaccgcat cagcggagct tcggagctga cagccaccat gggggaggca 1320 gatggagggg agaaggtctg gctgcagcct ggggaccttg ctccggcccc agtggacact 1380 cccagctctg aagacttcag gcccaactgc agcaccctca acttcacctt ggatctgtca 1440 cggaacaacc tggtgaccgt gcagccggag atgtttgccc agctctcgca cctgcagtgc 1500 ctgcgcctga gccacaactg catctcgcag gcagtcaatg gctcccagtt cctgccgctg 1560 accggtctgc aggtgctaga cctgtcccac aataagctgg acctctacca cgagcactca 1620 ttcacggagc taccgcgact ggaggccctg gacctcagct acaacagcca gccctttggc 1680 atgcagggcg tgggccacaa cttcagcttc gtggctcacc tgcgcaccct gcgccacctc 1740 agcctggccc acaacaat ccacagccaa gtgtcccagc agctctgcag tacgtcgctg 1800 cgggccctgg acttcagcgg caatgcactg ggccatatgt gggccgaggg agacctctat 1860 ctgcacttct tccaaggcct gagcggtttg atctggctgg acttgtccca gaaccgcctg 1920 cacaccctcc tgccccaaac cctgcgcaac ctccccaaga gcctacaggt gctgcgtctc 1980 cgtgacaatt acctggcctt ctttaagtgg tggagcctcc acttcctgcc caaactggaa 2040 gtcctcgacc tggcaggaaa ccagctgaag gccctgacca atggcagcct gcctgctggc 2100 acccggctcc ggaggctgga tgtcagctgc aacagcatca gcttcgtggc ccccggcttc 2160 ttttccaagg ccaaggagct gcgagagctc aaccttagcg ccaacgccct caagacagtg 2220 gaccactcct ggtttgggcc cctggcgagt gccctgcaaa tactagatgt aagcgccaac 2280 cctctgcact gcgcctgtgg ggcggccttt atggacttcc tgctggaggt gcaggctgcc 2340 gtgcccggtc tgcccagccg ggtgaagtgt ggcagtccgg gccagctcca gggcctcagc 2400 atctttgcac aggacctgcg cctctgcctg gatgaggccc tctcctggga ctgtttcgcc 2460 ctctcgctgc tggctgtggc tctgggcctg ggtgtgccca tgctgcatca cctctgtggc 2520 tgggacctct ggtactgctt ccacctgtgc ctggcctggc ttccctggcg ggggcggcaa 2580 agtgggcgag atgaggatgc cctgccctac gatgccttcg tggtcttcga caaaacgcag 2640 agcgcagtgg cagactgggt gtacaacgag cttcgggggc agctggagga gtgccgtggg 2700 cgctgggcac tccgcctgtg cctggaggaa cgcgactggc tgcctggcaa aaccctcttt 2760 gagaacctgt gggcctcggt ctatggcagc cgcaagacgc tgtttgtgct ggcccacacg 2820 gaccgggtca gtggtctctt gcgcgccagc ttcctgctgg cccagcagcg cctgctggag 2880 gaccgcaagg acgtcgtggt gctggtgatc ctgagccctg acggccgccg ctcccgctac 2940 gtgcggctgc gccagcgcct ctgccgccag agtgtcctcc tctggcccca ccagcccagt 3000 ggtcagcgca gcttctgggc ccagctggggc atggccctga ccagggacaa ccaccacttc 3060 tataaccgga acttctgcca gggacccacg gccgaatag 3099 <210> 13 <211> 317 <212> PRT <213> Homo sapiens <400> 13 Met Thr Met Glu Thr Gln Met Ser Gln Asn Val Cys Pro Arg Asn Leu 1 5 10 15 Trp Leu Leu Gln Pro Leu Thr Val Leu Leu Leu Leu Ala Ser Ala Asp 20 25 30 Ser Gln Ala Ala Ala Pro Pro Lys Ala Val Leu Lys Leu Glu Pro Pro 35 40 45 Trp Ile Asn Val Leu Gln Glu Asp Ser Val Thr Leu Thr Cys Gln Gly 50 55 60 Ala Arg Ser Pro Glu Ser Asp Ser Ile Gln Trp Phe His Asn Gly Asn 65 70 75 80 Leu Ile Pro Thr His Thr Gln Pro Ser Tyr Arg Phe Lys Ala Asn Asn 85 90 95 Asn Asp Ser Gly Glu Tyr Thr Cys Gln Thr Gly Gln Thr Ser Leu Ser 100 105 110 Asp Pro Val His Leu Thr Val Leu Ser Glu Trp Leu Val Leu Gln Thr 115 120 125 Pro His Leu Glu Phe Gln Glu Gly Glu Thr Ile Met Leu Arg Cys His 130 135 140 Ser Trp Lys Asp Lys Pro Leu Val Lys Val Thr Phe Phe Gln Asn Gly 145 150 155 160 Lys Ser Gln Lys Phe Ser His Leu Asp Pro Thr Phe Ser Ile Pro Gln 165 170 175 Ala Asn His Ser His Ser Gly Asp Tyr His Cys Thr Gly Asn Ile Gly 180 185 190 Tyr Thr Leu Phe Ser Ser Lys Pro Val Thr Ile Thr Val Gln Val Pro 195 200 205 Ser Met Gly Ser Ser Ser Pro Met Gly Ile Ile Val Ala Val Val Ile 210 215 220 Ala Thr Ala Val Ala Ala Ile Val Ala Ala Val Val Ala Leu Ile Tyr 225 230 235 240 Cys Arg Lys Lys Arg Ile Ser Ala Asn Ser Thr Asp Pro Val Lys Ala 245 250 255 Ala Gln Phe Glu Pro Pro Gly Arg Gln Met Ile Ala Ile Arg Lys Arg 260 265 270 Gln Leu Glu Glu Thr Asn Asn Asp Tyr Glu Thr Ala Asp Gly Gly Tyr 275 280 285 Met Thr Leu Asn Pro Arg Ala Pro Thr Asp Asp Asp Lys Asn Ile Tyr 290 295 300 Leu Thr Leu Pro Pro Asn Asp His Val Asn Ser Asn Asn 305 310 315 <210> 14 <211> 954 <212> DNA <213> Homo sapiens <400> 14 atgactatgg agacccaaat gtctcagaat gtatgtccca gaaacctggg gctgcttcaa 60 ccattgacag ttttgctgct gctggcttct gcagacagtc aagctgcagc tcccccaaag 120 gctgtgctga aacttgagcc cccgtggatc aacgtgctcc aggaggactc tgtgactctg 180 acatgccagg gggctcgcag ccctgagagc gactccattc agtggttcca caatgggaat 240 ctcattccca cccacacgca gcccagctac aggttcaagg ccaacaacaa tgacagcggg 300 gagtacacgt gccagactgg ccagaccagc ctcagcgacc ctgtgcatct gactgtgctt 360 tccgaatggc tggtgctcca gacccctcac ctggagttcc aggagggaga aaccatcatg 420 ctgaggtgcc acagctggaa ggacaagcct ctggtcaagg tcacattctt ccagaatgga 480 aaatcccaga aattctccca tttggatccc accttctcca tcccacaagc aaaccacagt 540 cacagtggtg attaccactg cacaggaaac ataggctaca cgctgttctc atccaagcct 600 gtgaccatca ctgtccaagt gcccagcatg ggcagctctt caccaatggg gatcattgtg 660 gctgtggtca ttgcgactgc tgtagcagcc attgttgctg ctgtagtggc cttgatctac 720 tgcaggaaaa agcggatttc agccaattcc actgatcctg tgaaggctgc ccaatttgag 780 ccacctggac gtcaaatgat tgccatcaga aagagacaac ttgaagaaac caacaatgac 840 tatgaaacag ctgacggcgg ctacatgact ctgaacccca gggcacctac tgacgatgat 900 aaaaacatct acctgactct tcctcccaac gaccatgtca acagtaataa ctaa 954 <210> 15 <211> 254 <212> PRT <213> Homo sapiens <400> 15 Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala 1 5 10 15 Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25 30 Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40 45 Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60 Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr 65 70 75 80 Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95 Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110 Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125 His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140 Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro 145 150 155 160 Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Phe 165 170 175 Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 180 185 190 Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln 195 200 205 Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly 210 215 220 Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp 225 230 235 240 Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys 245 250 <210> 16 <211> 765 <212> DNA <213> Homo sapiens <400> 16 atgtggcagc tgctcctccc aactgctctg ctacttctag tttcagctgg catgcggact 60 gaagatctcc caaaggctgt ggtgttcctg gagcctcaat ggtacagggt gctcgagaag 120 gacagtgtga ctctgaagtg ccagggagcc tactcccctg aggacaattc cacacagtgg 180 tttcacaatg agagcctcat ctcaagccag gcctcgagct acttcattga cgctgccaca 240 gtcgacgaca gtggagagta caggtgccag acaaacctct ccaccctcag tgacccggtg 300 cagctagaag tccatatcgg ctggctgttg ctccaggccc ctcggtgggt gttcaaggag 360 gaagacccta ttcacctgag gtgtcacagc tggaagaaca ctgctctgca taaggtcaca 420 tatttacaga atggcaaagg caggaagtat tttcatcata attctgactt ctacattcca 480 aaagccacac tcaaagacag cggctcctac ttctgcaggg ggctttttgg gagtaaaaat 540 gtgtcttcag agactgtgaa catcaccatc actcaaggtt tggcagtgtc aaccatctca 600 tcattctttc cacctgggta ccaagtctct ttctgcttgg tgatggtact cctttttgca 660 gtggacacag gactatattt ctctgtgaag acaaacattc gaagctcaac aagagactgg 720 aaggaccata aatttaaatg gagaaaggac cctcaagaca aatga 765 <210> 17 <211> 86 <212> PRT <213> Homo sapiens <400> 17 Met Ile Pro Ala Val Val Leu Leu Leu Leu Leu Leu Leu Val Glu Gln Ala 1 5 10 15 Ala Ala Leu Gly Glu Pro Gln Leu Cys Tyr Ile Leu Asp Ala Ile Leu 20 25 30 Phe Leu Tyr Gly Ile Val Leu Thr Leu Leu Tyr Cys Arg Leu Lys Ile 35 40 45 Gln Val Arg Lys Ala Ala Ile Thr Ser Tyr Glu Lys Ser Asp Gly Val 50 55 60 Tyr Thr Gly Leu Ser Thr Arg Asn Gln Glu Thr Tyr Glu Thr Leu Lys 65 70 75 80 His Glu Lys Pro Pro Gln 85 <210> 18 <211> 261 <212> DNA <213> Homo sapiens <400> 18 atgattccag cagtggtctt gctcttactc cttttggttg aacaagcagc ggccctggga 60 gagcctcagc tctgctatat cctggatgcc atcctgtttc tgtatggaat tgtcctcacc 120 ctcctctact gtcgactgaa gatccaagtg cgaaaggcag ctataaccag ctatgagaaa 180 tcagatggtg tttacacggg cctgagcacc aggaaccagg agacttacga gactctgaag 240 catgagaaac caccacagta g 261 <210> 19 <211> 296 <212> PRT <213> Homo sapiens <400> 19 Met Ala Ala Gly Gly Pro Gly Ala Gly Ser Ala Ala Pro Val Ser Ser 1 5 10 15 Thr Ser Ser Leu Pro Leu Ala Ala Leu Asn Met Arg Val Arg Arg Arg 20 25 30 Leu Ser Leu Phe Leu Asn Val Arg Thr Gln Val Ala Ala Asp Trp Thr 35 40 45 Ala Leu Ala Glu Glu Met Asp Phe Glu Tyr Leu Glu Ile Arg Gln Leu 50 55 60 Glu Thr Gln Ala Asp Pro Thr Gly Arg Leu Leu Asp Ala Trp Gln Gly 65 70 75 80 Arg Pro Gly Ala Ser Val Gly Arg Leu Leu Glu Leu Leu Thr Lys Leu 85 90 95 Gly Arg Asp Asp Val Leu Leu Glu Leu Gly Pro Ser Ile Glu Glu Asp 100 105 110 Cys Gln Lys Tyr Ile Leu Lys Gln Gln Gln Glu Glu Ala Glu Lys Pro 115 120 125 Leu Gln Val Ala Ala Val Asp Ser Ser Val Pro Arg Thr Ala Glu Leu 130 135 140 Ala Gly Ile Thr Thr Leu Asp Asp Pro Leu Gly His Met Pro Glu Arg 145 150 155 160 Phe Asp Ala Phe Ile Cys Tyr Cys Pro Ser Asp Ile Gln Phe Val Gln 165 170 175 Glu Met Ile Arg Gln Leu Glu Gln Thr Asn Tyr Arg Leu Lys Leu Cys 180 185 190 Val Ser Asp Arg Asp Val Leu Pro Gly Thr Cys Val Trp Ser Ile Ala 195 200 205 Ser Glu Leu Ile Glu Lys Arg Cys Arg Arg Met Val Val Val Val Ser 210 215 220 Asp Asp Tyr Leu Gln Ser Lys Glu Cys Asp Phe Gln Thr Lys Phe Ala 225 230 235 240 Leu Ser Leu Ser Pro Gly Ala His Gln Lys Arg Leu Ile Pro Ile Lys 245 250 255 Tyr Lys Ala Met Lys Lys Glu Phe Pro Ser Ile Leu Arg Phe Ile Thr 260 265 270 Val Cys Asp Tyr Thr Asn Pro Cys Thr Lys Ser Trp Phe Trp Thr Arg 275 280 285 Leu Ala Lys Ala Leu Ser Leu Pro 290 295 <210> 20 <211> 954 <212> DNA <213> Homo sapiens <400> 20 atgcgacccg accgcgctga ggctccagga ccgcccgcca tggctgcagg aggtcccggc 60 gcggggtctg cggccccggt ctcctccaca tcctcccttc ccctggctgc tctcaacatg 120 cgagtgcggc gccgcctgtc tctgttcttg aacgtgcgga cacaggtggc ggccgactgg 180 accgcgctgg cggaggagat ggactttgag tacttggaga tccggcaact ggagacacaa 240 gcggacccca ctggcaggct gctggacgcc tggcagggac gccctggcgc ctctgtaggc 300 cgactgctcg agctgcttac caagctgggc cgcgacgacg tgctgctgga gctgggaccc 360 agcattgagg aggattgcca aaagtatatc ttgaagcagc agcaggagga ggctgagaag 420 cctttacagg tggccgctgt agacagcagt gtcccacgga cagcagagct ggcgggcatc 480 accacacttg atgaccccct ggggcatatg cctgagcgtt tcgatgcctt catctgctat 540 tgccccagcg acatccagtt tgtgcaggag atgatccggc aactggaaca gacaaactat 600 cgactgaagt tgtgtgtgtc tgaccgcgat gtcctgcctg gcacctgtgt ctggtctatt 660 gctagtgagc tcatcgaaaa gaggttggct agaaggccac ggggtgggtg ccgccggatg 720 gtggtggttg tctctgatga ttacctgcag agcaaggaat gtgacttcca gaccaaattt 780 gcactcagcc tctctccagg tgcccatcag aagcgactga tcccccatcaa gtacaaggca 840 atgaagaaag agttccccag catcctgagg ttcatcactg tctgcgacta caccaacccc 900 tgcaccaaat cttggttctg gactcgcctt gccaaggcct tgtccctgcc ctga 954 <210> 21 <211> 153 <212> PRT <213> Homo sapiens <400> 21 Met Ala Pro Ala Ala Ala Thr Gly Gly Ser Thr Leu Pro Ser Gly Phe 1 5 10 15 Ser Val Phe Thr Thr Leu Pro Asp Leu Leu Phe Ile Phe Glu Phe Ile 20 25 30 Phe Gly Gly Leu Val Trp Ile Leu Val Ala Ser Ser Leu Val Pro Trp 35 40 45 Pro Leu Val Gln Gly Trp Val Met Phe Val Ser Val Phe Cys Phe Val 50 55 60 Ala Thr Thr Thr Leu Ile Ile Leu Tyr Ile Ile Gly Ala His Gly Gly 65 70 75 80 Glu Thr Ser Trp Val Thr Leu Asp Ala Ala Tyr His Cys Thr Ala Ala 85 90 95 Leu Phe Tyr Leu Ser Ala Ser Val Leu Glu Ala Leu Ala Thr Ile Thr 100 105 110 Met Gln Asp Gly Phe Thr Tyr Arg His Tyr His Glu Asn Ile Ala Ala 115 120 125 Val Val Phe Ser Tyr Ile Ala Thr Leu Leu Tyr Val Val His Ala Val 130 135 140 Phe Ser Leu Ile Arg Trp Lys Ser Ser 145 150 <210> 22 <211> 462 <212> DNA <213> Homo sapiens <400> 22 atggcccccg cagcggcgac ggggggcagc accctgccca gtggcttctc ggtcttcacc 60 accttgcccg acttgctctt catctttgag tttatcttcg ggggcctggt gtggatcctg 120 gtggcctcct ccctggtgcc ctggcccctg gtccagggct gggtgatgtt cgtgtctgtg 180 ttctgcttcg tggccaccac caccttgatc atcctgtaca taattggagc ccacggtgga 240 gagacttcct gggtcacctt ggacgcagcc taccactgca ccgctgccct cttttacctc 300 agcgcctcag tcctggaggc cctggccacc atcacgatgc aagacggctt cacctacagg 360 cactaccatg aaaacattgc tgccgtggtg ttctcctaca tagccactct gctctacgtg 420 gtccatgcgg tgttctcttt aatcagatgg aagtcttcat aa 462 <210> 23 <211> 712 <212> PRT <213> Homo sapiens <400> 23 Met Ala Gly Gly Pro Gly Pro Gly Glu Pro Ala Ala Pro Gly Ala Gln 1 5 10 15 His Phe Leu Tyr Glu Val Pro Pro Trp Val Met Cys Arg Phe Tyr Lys 20 25 30 Val Met Asp Ala Leu Glu Pro Ala Asp Trp Cys Gln Phe Ala Ala Leu 35 40 45 Ile Val Arg Asp Gln Thr Glu Leu Arg Leu Cys Glu Arg Ser Gly Gln 50 55 60 Arg Thr Ala Ser Val Leu Trp Pro Trp Ile Asn Arg Asn Ala Arg Val 65 70 75 80 Ala Asp Leu Val His Ile Leu Thr His Leu Gln Leu Leu Arg Ala Arg 85 90 95 Asp Ile Ile Thr Ala Trp His Pro Pro Ala Pro Leu Pro Ser Pro Gly 100 105 110 Thr Thr Ala Pro Arg Pro Ser Ser Ile Pro Ala Pro Ala Glu Ala Glu 115 120 125 Ala Trp Ser Pro Arg Lys Leu Pro Ser Ser Ala Ser Thr Phe Leu Ser 130 135 140 Pro Ala Phe Pro Gly Ser Gln Thr His Ser Gly Pro Glu Leu Gly Leu 145 150 155 160 Val Pro Ser Pro Ala Ser Leu Trp Pro Pro Pro Pro Ser Pro Ala Pro 165 170 175 Ser Ser Thr Lys Pro Gly Pro Glu Ser Ser Val Ser Leu Leu Gln Gly 180 185 190 Ala Arg Pro Phe Pro Phe Cys Trp Pro Leu Cys Glu Ile Ser Arg Gly 195 200 205 Thr His Asn Phe Ser Glu Glu Leu Lys Ile Gly Glu Gly Gly Phe Gly 210 215 220 Cys Val Tyr Arg Ala Val Met Arg Asn Thr Val Tyr Ala Val Lys Arg 225 230 235 240 Leu Lys Glu Asn Ala Asp Leu Glu Trp Thr Ala Val Lys Gln Ser Phe 245 250 255 Leu Thr Glu Val Glu Gln Leu Ser Arg Phe Arg His Pro Asn Ile Val 260 265 270 Asp Phe Ala Gly Tyr Cys Ala Gln Asn Gly Phe Tyr Cys Leu Val Tyr 275 280 285 Gly Phe Leu Pro Asn Gly Ser Leu Glu Asp Arg Leu His Cys Gln Thr 290 295 300 Gln Ala Cys Pro Pro Leu Ser Trp Pro Gln Arg Leu Asp Ile Leu Leu 305 310 315 320 Gly Thr Ala Arg Ala Ile Gln Phe Leu His Gln Asp Ser Pro Ser Leu 325 330 335 Ile His Gly Asp Ile Lys Ser Ser Asn Val Leu Leu Asp Glu Arg Leu 340 345 350 Thr Pro Lys Leu Gly Asp Phe Gly Leu Ala Arg Phe Ser Arg Phe Ala 355 360 365 Gly Ser Ser Pro Ser Gln Ser Ser Met Val Ala Arg Thr Gln Thr Val 370 375 380 Arg Gly Thr Leu Ala Tyr Leu Pro Glu Glu Tyr Ile Lys Thr Gly Arg 385 390 395 400 Leu Ala Val Asp Thr Asp Thr Phe Ser Phe Gly Val Val Val Leu Glu 405 410 415 Thr Leu Ala Gly Gln Arg Ala Val Lys Thr His Gly Ala Arg Thr Lys 420 425 430 Tyr Leu Lys Asp Leu Val Glu Glu Glu Ala Glu Glu Ala Gly Val Ala 435 440 445 Leu Arg Ser Thr Gln Ser Thr Leu Gln Ala Gly Leu Ala Ala Asp Ala 450 455 460 Trp Ala Ala Pro Ile Ala Met Gln Ile Tyr Lys Lys His Leu Asp Pro 465 470 475 480 Arg Pro Gly Pro Cys Pro Pro Glu Leu Gly Leu Gly Leu Gly Gln Leu 485 490 495 Ala Cys Cys Cys Leu His Arg Arg Ala Lys Arg Arg Pro Pro Met Thr 500 505 510 Gln Val Tyr Glu Arg Leu Glu Lys Leu Gln Ala Val Val Ala Gly Val 515 520 525 Pro Gly His Ser Glu Ala Ala Ser Cys Ile Pro Pro Ser Pro Gln Glu 530 535 540 Asn Ser Tyr Val Ser Ser Thr Gly Arg Ala His Ser Gly Ala Ala Pro 545 550 555 560 Trp Gln Pro Leu Ala Ala Pro Ser Gly Ala Ser Ala Gln Ala Ala Glu 565 570 575 Gln Leu Gln Arg Gly Pro Asn Gln Pro Val Glu Ser Asp Glu Ser Leu 580 585 590 Gly Gly Leu Ser Ala Ala Leu Arg Ser Trp His Leu Thr Pro Ser Cys 595 600 605 Pro Leu Asp Pro Ala Pro Leu Arg Glu Ala Gly Cys Pro Gln Gly Asp 610 615 620 Thr Ala Gly Glu Ser Ser Trp Gly Ser Gly Pro Gly Ser Arg Pro Thr 625 630 635 640 Ala Val Glu Gly Leu Ala Leu Gly Ser Ser Ala Ser Ser Ser Ser Ser Glu 645 650 655 Pro Pro Gln Ile Ile Ile Asn Pro Ala Arg Gln Lys Met Val Gln Lys 660 665 670 Leu Ala Leu Tyr Glu Asp Gly Ala Leu Asp Ser Leu Gln Leu Leu Ser 675 680 685 Ser Ser Ser Leu Pro Gly Leu Gly Leu Glu Gln Asp Arg Gln Gly Pro 690 695 700 Glu Glu Ser Asp Glu Phe Gln Ser 705 710 <210> 24 <211> 2139 <212> DNA <213> Homo sapiens <400> 24 atggccgggg ggccgggccc gggggagccc gcagccccccg gcgcccagca cttcttgtac 60 gaggtgccgc cctgggtcat gtgccgcttc tacaaagtga tggacgccct ggagcccgcc 120 gactggtgcc agttcgccgc cctgatcgtg cgcgaccaga ccgagctgcg gctgtgcgag 180 cgctccgggc agcgcacggc cagcgtcctg tggccctgga tcaaccgcaa cgcccgtgtg 240 gccgacctcg tgcacatcct cacgcacctg cagctgctcc gtgcgcggga catcatcaca 300 gcctggcacc ctcccgcccc gcttccgtcc ccaggcacca ctgccccgag gcccagcagc 360 atccctgcac ccgccgaggc cgaggcctgg agcccccgga agttgccatc ctcagcctcc 420 accttcctct ccccagcttt tccaggctcc cagacccatt cagggcctga gctcggcctg 480 gtcccaagcc ctgcttccct gtggcctcca ccgccatctc cagccccttc ttctaccaag 540 ccaggcccag agagctcagt gtccctcctg cagggagccc gcccctttcc gttttgctgg 600 cccctctgg agatttcccg gggcacccac aacttctcgg aggagctcaa gatcggggag 660 ggtggctttg ggtgcgtgta ccgggcggtg atgaggaaca cggtgtatgc tgtgaagagg 720 ctgaaggaga acgctgacct ggaggtggact gcagtgaagc agagcttcct gaccgaggtg 780 gagcagctgt ccaggtttcg tcacccaaac attgtggact ttgctggcta ctgtgctcag 840 aacggcttct actgcctggt gtacggcttc ctgcccaacg gctccctgga ggaccgtctc 900 cactgccaga cccaggcctg cccacctctc tcctggcctc agcgactgga catccttctg 960 ggtacagccc gggcaattca gtttctacat caggacagcc ccagcctcat ccatggagac 1020 atcaagagtt ccaacgtcct tctggatgag aggctgacac ccaagctggg agactttggc 1080 ctggcccggt tcagccgctt tgccgggtcc agccccagcc agagcagcat ggtggcccgg 1140 acacagacag tgcggggcac cctggcctac ctgcccgagg agtacatcaa gacgggaagg 1200 ctggctgtgg acacggacac cttcagcttt ggggtggtag tgctagagac cttggctggt 1260 cagagggctg tgaagacgca cggtgccagg accaagtatc tgaaagacct ggtggaagag 1320 gaggctgagg aggctggagt ggctttgaga agcacccaga gcacactgca agcaggtctg 1380 gctgcagatg cctgggctgc tcccatcgcc atgcagatct acaagaagca cctggacccc 1440 aggcccgggc cctgcccacc tgagctgggc ctgggcctgg gccagctggc ctgctgctgc 1500 ctgcaccgcc gggccaaaag gaggcctcct atgacccagg tgtacgagag gctagagaag 1560 ctgcaggcag tggtggcggg ggtgcccggg cattcggagg ccgccagctg catcccccct 1620 tccccgcagg agaactccta cgtgtccagc actggcagag cccacagtgg ggctgctcca 1680 tggcagcccc tggcagcgcc atcaggagcc agtgcccagg cagcagagca gctgcagaga 1740 ggccccaacc agcccgtgga gagtgacgag agcctaggcg gcctctctgc tgccctgcgc 1800 tcctggcact tgactccaag ctgccctctg gacccagcac ccctcaggga ggccggctgt 1860 cctcaggggg acacggcagg agaatcgagc tgggggagtg gcccaggatc ccggcccaca 1920 gccgtggaag gactggccct tggcagctct gcatcatcgt cgtcagagcc accgcagatt 1980 atcatcaacc ctgcccgaca gaagatggtc cagaagctgg ccctgtacga ggatggggcc 2040 ctggacagcc tgcagctgct gtcgtccagc tccctcccag gcttgggcct ggaacaggac 2100 aggcaggggc ccgaagaaag tgatgaattt cagagctga 2139 <210> 25 <211> 257 <212> PRT <213> artificial sequence <220> <223> ScFv fragments <400> 25 Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser 1 5 10 15 Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser 20 25 30 Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys 35 40 45 Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg 50 55 60 Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile Ser Arg 65 70 75 80 Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser 85 90 95 Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Ser Gly 100 105 110 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Ala 115 120 125 Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser Gly Ala 130 135 140 Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr Ala Ser 145 150 155 160 Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln Arg Pro 165 170 175 Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn Gly Asn 180 185 190 Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr Thr Asp 195 200 205 Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr Ser Glu 210 215 220 Asp Thr Ala Val Tyr Cys Ala Lys Val Gly Tyr Gly His Trp Tyr 225 230 235 240 Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Val Asp 245 250 255 Leu <210> 26 <211> 5 <212> PRT <213> artificial sequence <220> <223> short hinge linker <400> 26 Met Asn Lys Thr Ile 1 5 <210> 27 <211> 55 <212> PRT <213> artificial sequence <220> <223> long hinge linker <400> 27 Asn Asp Phe Ala Cys Thr Cys Glu His Gln Ser Phe Leu Gln Trp Ile 1 5 10 15 Lys Asp Gln Arg Gln Leu Leu Val Glu Val Glu Arg Met Glu Cys Ala 20 25 30 Thr Pro Ser Asp Lys Gln Gly Met Pro Val Leu Ser Leu Asn Ile Thr 35 40 45 Cys Gln Met Asn Lys Thr Ile 50 55 <210> 28 <211> 327 <212> PRT <213> Homo sapiens <400> 28 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 29 <211> 12 <212> PRT <213> Homo sapiens <400> 29 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 1 5 10 <210> 30 <211> 119 <212> PRT <213> Homo sapiens <400> 30 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gln Pro Arg 1 5 10 15 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 20 25 30 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 35 40 45 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 50 55 60 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 65 70 75 80 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 85 90 95 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 100 105 110 Leu Ser Leu Ser Leu Gly Lys 115 <210> 31 <211> 228 <212> PRT <213> Homo sapiens <400> 31 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Pro Val 1 5 10 15 Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 20 25 30 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 35 40 45 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 50 55 60 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser Thr 65 70 75 80 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 85 90 95 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 100 105 110 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 115 120 125 Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 130 135 140 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 145 150 155 160 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165 170 175 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 180 185 190 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 195 200 205 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 210 215 220 Ser Leu Gly Lys 225 <210> 32 <211> 235 <212> PRT <213> Homo sapiens <400> 32 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Ser Gln Phe Arg Val Ser Pro Leu Asp Arg Thr 20 25 30 Trp Asn Leu Gly Glu Thr Val Glu Leu Lys Cys Gln Val Leu Leu Ser 35 40 45 Asn Pro Thr Ser Gly Cys Ser Trp Leu Phe Gln Pro Arg Gly Ala Ala 50 55 60 Ala Ser Pro Thr Phe Leu Leu Tyr Leu Ser Gln Asn Lys Pro Lys Ala 65 70 75 80 Ala Glu Gly Leu Asp Thr Gln Arg Phe Ser Gly Lys Arg Leu Gly Asp 85 90 95 Thr Phe Val Leu Thr Leu Ser Asp Phe Arg Arg Glu Asn Glu Gly Tyr 100 105 110 Tyr Phe Cys Ser Ala Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe 115 120 125 Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg 130 135 140 Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg 145 150 155 160 Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly 165 170 175 Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr 180 185 190 Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His 195 200 205 Arg Asn Arg Arg Arg Val Cys Lys Cys Pro Arg Pro Val Val Lys Ser 210 215 220 Gly Asp Lys Pro Ser Leu Ser Ala Arg Tyr Val 225 230 235 <210> 33 <211> 68 <212> PRT <213> artificial sequence <220> <223> Mutated CD8 hinge <400> 33 Ser Ala Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe Val Pro Val 1 5 10 15 Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr 20 25 30 Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala 35 40 45 Ser Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe 50 55 60 Ala Ser Asp Ile 65 <210> 34 <211> 15 <212> PRT <213> artificial sequence <220> <223> gs linker <400> 34 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 35 <211> 496 <212> PRT <213> artificial sequence <220> <223> Chimeric antigen receptor <220> <221> SIGNAL <222> (1)..(18) <220> <221> MISC_FEATURE <222> (19)..(275) <223> TK1-ScFv <220> <221> MISC_FEATURE <222> (276)..(290) <223> GS-Linker <220> <221> MISC_FEATURE <222> (291)..(313) <223> TLR4-Transmembrane domain <220> <221> MISC_FEATURE <222> (314).. (496) <223> TLR4-cytosolic domain <400> 35 Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile 20 25 30 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 35 40 45 Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser 50 55 60 Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile 85 90 95 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser 145 150 155 160 Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr 165 170 175 Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln 180 185 190 Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn 195 200 205 Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr 210 215 220 Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr 225 230 235 240 Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His 245 250 255 Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 260 265 270 Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 275 280 285 Gly Ser Ile Gly Val Ser Val Leu Ser Val Leu Val Val Ser Val Val 290 295 300 Ala Val Leu Val Tyr Lys Phe Tyr Phe His Leu Met Leu Leu Ala Gly 305 310 315 320 Cys Ile Lys Tyr Gly Arg Gly Glu Asn Ile Tyr Asp Ala Phe Val Ile 325 330 335 Tyr Ser Ser Gln Asp Glu Asp Trp Val Arg Asn Glu Leu Val Lys Asn 340 345 350 Leu Glu Glu Gly Val Pro Phe Gln Leu Cys Leu His Tyr Arg Asp 355 360 365 Phe Ile Pro Gly Val Ala Ile Ala Ala Asn Ile Ile His Glu Gly Phe 370 375 380 His Lys Ser Arg Lys Val Ile Val Val Val Ser Gln His Phe Ile Gln 385 390 395 400 Ser Arg Trp Cys Ile Phe Glu Tyr Glu Ile Ala Gln Thr Trp Gln Phe 405 410 415 Leu Ser Ser Arg Ala Gly Ile Ile Phe Ile Val Leu Gln Lys Val Glu 420 425 430 Lys Thr Leu Leu Arg Gln Gln Val Glu Leu Tyr Arg Leu Leu Ser Arg 435 440 445 Asn Thr Tyr Leu Glu Trp Glu Asp Ser Val Leu Gly Arg His Ile Phe 450 455 460 Trp Arg Arg Leu Arg Lys Ala Leu Leu Asp Gly Lys Ser Trp Asn Pro 465 470 475 480 Glu Gly Thr Val Gly Thr Gly Cys Asn Trp Gln Glu Ala Thr Ser Ile 485 490 495 <210> 36 <211> 500 <212> PRT <213> artificial sequence <220> <223> Chimeric antigen receptor <220> <221> SIGNAL <222> (1)..(18) <220> <221> MISC_FEATURE <222> (19)..(275) <223> TK1-ScFv <220> <221> MISC_FEATURE <222> (276)..(290) <223> GS-Linker <220> <221> MISC_FEATURE <222> (291)..(295) <223> LRR Short Hinge <220> <221> MISC_FEATURE <222> (296)..(318) <223> TLR4 transmembrane domain <220> <221> MISC_FEATURE <222> (319).. (500) <223> TLR4 cytosolic domain <400> 36 Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile 20 25 30 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 35 40 45 Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser 50 55 60 Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile 85 90 95 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser 145 150 155 160 Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr 165 170 175 Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln 180 185 190 Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn 195 200 205 Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr 210 215 220 Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr 225 230 235 240 Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His 245 250 255 Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 260 265 270 Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 275 280 285 Gly Ser Met Asn Lys Thr Ile Ile Gly Val Ser Val Leu Ser Val Leu 290 295 300 Val Val Ser Val Val Ala Val Leu Val Tyr Lys Phe Tyr Phe His Leu 305 310 315 320 Met Leu Leu Ala Gly Cys Ile Lys Tyr Gly Arg Gly Glu Asn Ile Tyr 325 330 335 Asp Ala Phe Val Ile Tyr Ser Ser Gln Asp Glu Asp Trp Val Arg Asn 340 345 350 Glu Leu Val Lys Asn Leu Glu Glu Gly Val Pro Pro Phe Gln Leu Cys 355 360 365 Leu His Tyr Arg Asp Phe Ile Pro Gly Val Ala Ile Ala Ala Asn Ile 370 375 380 Ile His Glu Gly Phe His Lys Ser Arg Lys Val Ile Val Val Val Ser 385 390 395 400 Gln His Phe Ile Gln Ser Arg Trp Cys Ile Phe Glu Tyr Glu Ile Ala 405 410 415 Gln Thr Trp Gln Phe Leu Ser Ser Arg Ala Gly Ile Ile Phe Ile Val 420 425 430 Leu Gln Lys Val Glu Lys Thr Leu Leu Arg Gln Gln Val Glu Leu Tyr 435 440 445 Arg Leu Leu Ser Arg Asn Thr Tyr Leu Glu Trp Glu Asp Ser Val Leu 450 455 460 Gly Arg His Ile Phe Trp Arg Arg Leu Arg Lys Ala Leu Leu Asp Gly 465 470 475 480 Lys Ser Trp Asn Pro Glu Gly Thr Val Gly Thr Gly Cys Asn Trp Gln 485 490 495 Glu Ala Thr Ser 500 <210> 37 <211> 551 <212> PRT <213> artificial sequence <220> <223> Chimeric antigen receptor <220> <221> SIGNAL <222> (1)..(18) <223> Singal peptide <220> <221> MISC_FEATURE <222> (19)..(275) <223> TK1-ScFv <220> <221> MISC_FEATURE <222> (269)..(551) <223> TLR4 cytosolic domain <220> <221> MISC_FEATURE <222> (276)..(290) <223> GS-Linker <220> <221> MISC_FEATURE <222> (291)..(345) <223> LRR long hinge <220> <221> MISC_FEATURE <222> (346).. (368) <223> TLR4 transmembrane domain <400> 37 Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile 20 25 30 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 35 40 45 Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser 50 55 60 Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile 85 90 95 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser 145 150 155 160 Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr 165 170 175 Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln 180 185 190 Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn 195 200 205 Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr 210 215 220 Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr 225 230 235 240 Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His 245 250 255 Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 260 265 270 Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 275 280 285 Gly Ser Asn Asp Phe Ala Cys Thr Cys Glu His Gln Ser Phe Leu Gln 290 295 300 Trp Ile Lys Asp Gln Arg Gln Leu Leu Val Glu Val Glu Arg Met Glu 305 310 315 320 Cys Ala Thr Pro Ser Asp Lys Gln Gly Met Pro Val Leu Ser Leu Asn 325 330 335 Ile Thr Cys Gln Met Asn Lys Thr Ile Ile Gly Val Ser Val Leu Ser 340 345 350 Val Leu Val Val Ser Val Val Val Ala Val Leu Val Tyr Lys Phe Tyr Phe 355 360 365 His Leu Met Leu Leu Ala Gly Cys Ile Lys Tyr Gly Arg Gly Glu Asn 370 375 380 Ile Tyr Asp Ala Phe Val Ile Tyr Ser Ser Gln Asp Glu Asp Trp Val 385 390 395 400 Arg Asn Glu Leu Val Lys Asn Leu Glu Glu Gly Val Pro Pro Phe Gln 405 410 415 Leu Cys Leu His Tyr Arg Asp Phe Ile Pro Gly Val Ala Ile Ala Ala 420 425 430 Asn Ile Ile His Glu Gly Phe His Lys Ser Arg Lys Val Ile Val Val 435 440 445 Val Ser Gln His Phe Ile Gln Ser Arg Trp Cys Ile Phe Glu Tyr Glu 450 455 460 Ile Ala Gln Thr Trp Gln Phe Leu Ser Ser Arg Ala Gly Ile Ile Phe 465 470 475 480 Ile Val Leu Gln Lys Val Glu Lys Thr Leu Leu Arg Gln Gln Val Glu 485 490 495 Leu Tyr Arg Leu Leu Ser Arg Asn Thr Tyr Leu Glu Trp Glu Asp Ser 500 505 510 Val Leu Gly Arg His Ile Phe Trp Arg Arg Leu Arg Lys Ala Leu Leu 515 520 525 Asp Gly Lys Ser Trp Asn Pro Glu Gly Thr Val Gly Thr Gly Cys Asn 530 535 540 Trp Gln Glu Ala Thr Ser Ile 545 550 <210> 38 <211> 508 <212> PRT <213> artificial sequence <220> <223> Chimeric antigen receptor <220> <221> SIGNAL <222> (1)..(18) <220> <221> MISC_FEATURE <222> (19)..(275) <223> TK1 ScFv <220> <221> MISC_FEATURE <222> (276)..(290) <223> gs linker <220> <221> MISC_FEATURE <222> (291).. (302) <223> IgG4 short hinge <220> <221> MISC_FEATURE <222> (303).. (325) <223> TLR4 transmembrane domain <220> <221> MISC_FEATURE <222> (326).. (508) <223> TLR4 cytosolic domain <400> 38 Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile 20 25 30 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 35 40 45 Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser 50 55 60 Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile 85 90 95 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser 145 150 155 160 Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr 165 170 175 Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln 180 185 190 Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn 195 200 205 Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr 210 215 220 Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr 225 230 235 240 Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His 245 250 255 Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 260 265 270 Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 275 280 285 Gly Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ile Gly 290 295 300 Val Ser Val Leu Ser Val Leu Val Val Ser Val Val Ala Val Leu Val 305 310 315 320 Tyr Lys Phe Tyr Phe His Leu Met Leu Leu Ala Gly Cys Ile Lys Tyr 325 330 335 Gly Arg Gly Glu Asn Ile Tyr Asp Ala Phe Val Ile Tyr Ser Ser Gln 340 345 350 Asp Glu Asp Trp Val Arg Asn Glu Leu Val Lys Asn Leu Glu Glu Glu Gly 355 360 365 Val Pro Pro Phe Gln Leu Cys Leu His Tyr Arg Asp Phe Ile Pro Gly 370 375 380 Val Ala Ile Ala Ala Asn Ile Ile His Glu Gly Phe His Lys Ser Arg 385 390 395 400 Lys Val Ile Val Val Val Ser Gln His Phe Ile Gln Ser Arg Trp Cys 405 410 415 Ile Phe Glu Tyr Glu Ile Ala Gln Thr Trp Gln Phe Leu Ser Ser Arg 420 425 430 Ala Gly Ile Ile Phe Ile Val Leu Gln Lys Val Glu Lys Thr Leu Leu 435 440 445 Arg Gln Gln Val Glu Leu Tyr Arg Leu Leu Ser Arg Asn Thr Tyr Leu 450 455 460 Glu Trp Glu Asp Ser Val Leu Gly Arg His Ile Phe Trp Arg Arg Leu 465 470 475 480 Arg Lys Ala Leu Leu Asp Gly Lys Ser Trp Asn Pro Glu Gly Thr Val 485 490 495 Gly Thr Gly Cys Asn Trp Gln Glu Ala Thr Ser Ile 500 505 <210> 39 <211> 615 <212> PRT <213> artificial sequence <220> <223> Chimeric antigen receptor <220> <221> SIGNAL <222> (1)..(18) <223> signal peptide <220> <221> MISC_FEATURE <222> (19)..(275) <223> TK1 ScFv <220> <221> MISC_FEATURE <222> (276)..(290) <223> gs linker <220> <221> MISC_FEATURE <222> (291)..(409) <223> IgG4 medium hinge <220> <221> MISC_FEATURE <222> (410).. (432) <223> TLR4 transmembrane domain <220> <221> MISC_FEATURE <222> (433)..(615) <223> TLR4 cytosolic domain <400> 39 Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile 20 25 30 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 35 40 45 Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser 50 55 60 Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile 85 90 95 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser 145 150 155 160 Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr 165 170 175 Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln 180 185 190 Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn 195 200 205 Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr 210 215 220 Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr 225 230 235 240 Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His 245 250 255 Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 260 265 270 Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 275 280 285 Gly Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gln 290 295 300 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 305 310 315 320 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 325 330 335 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 340 345 350 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 355 360 365 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 370 375 380 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 385 390 395 400 Lys Ser Leu Ser Leu Ser Leu Gly Lys Ile Gly Val Ser Val Leu Ser 405 410 415 Val Leu Val Val Ser Val Val Val Ala Val Leu Val Tyr Lys Phe Tyr Phe 420 425 430 His Leu Met Leu Leu Ala Gly Cys Ile Lys Tyr Gly Arg Gly Glu Asn 435 440 445 Ile Tyr Asp Ala Phe Val Ile Tyr Ser Ser Gln Asp Glu Asp Trp Val 450 455 460 Arg Asn Glu Leu Val Lys Asn Leu Glu Glu Gly Val Pro Pro Phe Gln 465 470 475 480 Leu Cys Leu His Tyr Arg Asp Phe Ile Pro Gly Val Ala Ile Ala Ala 485 490 495 Asn Ile Ile His Glu Gly Phe His Lys Ser Arg Lys Val Ile Val Val 500 505 510 Val Ser Gln His Phe Ile Gln Ser Arg Trp Cys Ile Phe Glu Tyr Glu 515 520 525 Ile Ala Gln Thr Trp Gln Phe Leu Ser Ser Arg Ala Gly Ile Ile Phe 530 535 540 Ile Val Leu Gln Lys Val Glu Lys Thr Leu Leu Arg Gln Gln Val Glu 545 550 555 560 Leu Tyr Arg Leu Leu Ser Arg Asn Thr Tyr Leu Glu Trp Glu Asp Ser 565 570 575 Val Leu Gly Arg His Ile Phe Trp Arg Arg Leu Arg Lys Ala Leu Leu 580 585 590 Asp Gly Lys Ser Trp Asn Pro Glu Gly Thr Val Gly Thr Gly Cys Asn 595 600 605 Trp Gln Glu Ala Thr Ser Ile 610 615 <210> 40 <211> 724 <212> PRT <213> artificial sequence <220> <223> Chimeric antigen receptor <220> <221> SIGNAL <222> (1)..(18) <220> <221> MISC_FEATURE <222> (19)..(275) <223> TK1 ScFv <220> <221> MISC_FEATURE <222> (276)..(290) <223> gs linker <220> <221> MISC_FEATURE <222> (291)..(518) <223> IgG4 long hinge <220> <221> MISC_FEATURE <222> (519).. (541) <223> TLR4 transmembrane domain <220> <221> MISC_FEATURE <222> (542)..(724) <223> TLR4 cytosolic domain <400> 40 Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile 20 25 30 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 35 40 45 Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser 50 55 60 Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile 85 90 95 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser 145 150 155 160 Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr 165 170 175 Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln 180 185 190 Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn 195 200 205 Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr 210 215 220 Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr 225 230 235 240 Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His 245 250 255 Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 260 265 270 Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 275 280 285 Gly Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 290 295 300 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 305 310 315 320 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 325 330 335 Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 340 345 350 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln 355 360 365 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 370 375 380 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 385 390 395 400 Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 405 410 415 Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn 420 425 430 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 435 440 445 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 450 455 460 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg 465 470 475 480 Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys 485 490 495 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 500 505 510 Ser Leu Ser Leu Gly Lys Ile Gly Val Ser Val Leu Ser Val Leu Val 515 520 525 Val Ser Val Val Ala Val Leu Val Tyr Lys Phe Tyr Phe His Leu Met 530 535 540 Leu Leu Ala Gly Cys Ile Lys Tyr Gly Arg Gly Glu Asn Ile Tyr Asp 545 550 555 560 Ala Phe Val Ile Tyr Ser Ser Gln Asp Glu Asp Trp Val Arg Asn Glu 565 570 575 Leu Val Lys Asn Leu Glu Glu Gly Val Pro Pro Phe Gln Leu Cys Leu 580 585 590 His Tyr Arg Asp Phe Ile Pro Gly Val Ala Ile Ala Ala Asn Ile Ile 595 600 605 His Glu Gly Phe His Lys Ser Arg Lys Val Ile Val Val Val Ser Gln 610 615 620 His Phe Ile Gln Ser Arg Trp Cys Ile Phe Glu Tyr Glu Ile Ala Gln 625 630 635 640 Thr Trp Gln Phe Leu Ser Ser Arg Ala Gly Ile Ile Phe Ile Val Leu 645 650 655 Gln Lys Val Glu Lys Thr Leu Leu Arg Gln Gln Val Glu Leu Tyr Arg 660 665 670 Leu Leu Ser Arg Asn Thr Tyr Leu Glu Trp Glu Asp Ser Val Leu Gly 675 680 685 Arg His Ile Phe Trp Arg Arg Leu Arg Lys Ala Leu Leu Asp Gly Lys 690 695 700 Ser Trp Asn Pro Glu Gly Thr Val Gly Thr Gly Cys Asn Trp Gln Glu 705 710 715 720 Ala Thr Ser Ile <210> 41 <211> 564 <212> PRT <213> artificial sequence <220> <223> Chimeric antigen receptor <220> <221> SIGNAL <222> (1)..(18) <223> signal peptide <220> <221> MISC_FEATURE <222> (19)..(275) <220> <221> MISC_FEATURE <222> (276)..(290) <223> gs linker <220> <221> MISC_FEATURE <222> (291)..(358) <223> Mutated CD8 hinge <220> <221> MISC_FEATURE <222> (359)..(381) <223> TLR4 transmembrane domain <220> <221> MISC_FEATURE <222> (382)..(564) <223> TLR4 cytosolic domain <400> 41 Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile 20 25 30 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 35 40 45 Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser 50 55 60 Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile 85 90 95 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser 145 150 155 160 Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr 165 170 175 Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln 180 185 190 Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn 195 200 205 Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr 210 215 220 Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr 225 230 235 240 Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His 245 250 255 Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 260 265 270 Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 275 280 285 Gly Ser Ser Ala Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe Val 290 295 300 Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro 305 310 315 320 Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro 325 330 335 Glu Ala Ser Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 340 345 350 Asp Phe Ala Ser Asp Ile Ile Gly Val Ser Val Leu Ser Val Leu Val 355 360 365 Val Ser Val Val Ala Val Leu Val Tyr Lys Phe Tyr Phe His Leu Met 370 375 380 Leu Leu Ala Gly Cys Ile Lys Tyr Gly Arg Gly Glu Asn Ile Tyr Asp 385 390 395 400 Ala Phe Val Ile Tyr Ser Ser Gln Asp Glu Asp Trp Val Arg Asn Glu 405 410 415 Leu Val Lys Asn Leu Glu Glu Gly Val Pro Pro Phe Gln Leu Cys Leu 420 425 430 His Tyr Arg Asp Phe Ile Pro Gly Val Ala Ile Ala Ala Asn Ile Ile 435 440 445 His Glu Gly Phe His Lys Ser Arg Lys Val Ile Val Val Val Ser Gln 450 455 460 His Phe Ile Gln Ser Arg Trp Cys Ile Phe Glu Tyr Glu Ile Ala Gln 465 470 475 480 Thr Trp Gln Phe Leu Ser Ser Arg Ala Gly Ile Ile Phe Ile Val Leu 485 490 495 Gln Lys Val Glu Lys Thr Leu Leu Arg Gln Gln Val Glu Leu Tyr Arg 500 505 510 Leu Leu Ser Arg Asn Thr Tyr Leu Glu Trp Glu Asp Ser Val Leu Gly 515 520 525 Arg His Ile Phe Trp Arg Arg Leu Arg Lys Ala Leu Leu Asp Gly Lys 530 535 540 Ser Trp Asn Pro Glu Gly Thr Val Gly Thr Gly Cys Asn Trp Gln Glu 545 550 555 560 Ala Thr Ser Ile <210> 42 <211> 564 <212> PRT <213> artificial sequence <220> <223> Chimeric antigen receptor <220> <221> SIGNAL <222> (1)..(18) <223> Sginal peptide <220> <221> MISC_FEATURE <222> (19)..(275) <223> TK1 ScFv <220> <221> MISC_FEATURE <222> (276)..(290) <223> gs linker <220> <221> MISC_FEATURE <222> (291)..(358) <223> gs linker <220> <221> MISC_FEATURE <222> (359)..(381) <223> TLR4 transmembrane domain <220> <221> MISC_FEATURE <222> (382)..(564) <223> TLR4 cytosolic domain <400> 42 Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile 20 25 30 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 35 40 45 Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser 50 55 60 Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile 85 90 95 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser 145 150 155 160 Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr 165 170 175 Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln 180 185 190 Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn 195 200 205 Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr 210 215 220 Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr 225 230 235 240 Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His 245 250 255 Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 260 265 270 Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 275 280 285 Gly Ser Ser Ala Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe Val 290 295 300 Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro 305 310 315 320 Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro 325 330 335 Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 340 345 350 Asp Phe Ala Cys Asp Ile Ile Gly Val Ser Val Leu Ser Val Leu Val 355 360 365 Val Ser Val Val Ala Val Leu Val Tyr Lys Phe Tyr Phe His Leu Met 370 375 380 Leu Leu Ala Gly Cys Ile Lys Tyr Gly Arg Gly Glu Asn Ile Tyr Asp 385 390 395 400 Ala Phe Val Ile Tyr Ser Ser Gln Asp Glu Asp Trp Val Arg Asn Glu 405 410 415 Leu Val Lys Asn Leu Glu Glu Gly Val Pro Pro Phe Gln Leu Cys Leu 420 425 430 His Tyr Arg Asp Phe Ile Pro Gly Val Ala Ile Ala Ala Asn Ile Ile 435 440 445 His Glu Gly Phe His Lys Ser Arg Lys Val Ile Val Val Val Ser Gln 450 455 460 His Phe Ile Gln Ser Arg Trp Cys Ile Phe Glu Tyr Glu Ile Ala Gln 465 470 475 480 Thr Trp Gln Phe Leu Ser Ser Arg Ala Gly Ile Ile Phe Ile Val Leu 485 490 495 Gln Lys Val Glu Lys Thr Leu Leu Arg Gln Gln Val Glu Leu Tyr Arg 500 505 510 Leu Leu Ser Arg Asn Thr Tyr Leu Glu Trp Glu Asp Ser Val Leu Gly 515 520 525 Arg His Ile Phe Trp Arg Arg Leu Arg Lys Ala Leu Leu Asp Gly Lys 530 535 540 Ser Trp Asn Pro Glu Gly Thr Val Gly Thr Gly Cys Asn Trp Gln Glu 545 550 555 560 Ala Thr Ser Ile <210> 43 <211> 378 <212> PRT <213> artificial sequence <220> <223> Chimeric antigen receptor <220> <221> SIGNAL <222> (1)..(18) <223> signal peptide <220> <221> MISC_FEATURE <222> (19)..(275) <223> TK1 ScFv <220> <221> MISC_FEATURE <222> (276)..(290) <223> gs linker <220> <221> MISC_FEATURE <222> (291)..(311) <223> FCGR3A transmembrane domain <220> <221> MISC_FEATURE <222> (312)..(336) <223> FCGR3A cytosolic domain <220> <221> MISC_FEATURE <222> (337)..(378) <223> FCER1G cytosolic domain <400> 43 Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile 20 25 30 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 35 40 45 Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser 50 55 60 Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile 85 90 95 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser 145 150 155 160 Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr 165 170 175 Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln 180 185 190 Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn 195 200 205 Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr 210 215 220 Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr 225 230 235 240 Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His 245 250 255 Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 260 265 270 Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 275 280 285 Gly Ser Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp 290 295 300 Thr Gly Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg 305 310 315 320 Asp Trp Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys 325 330 335 Arg Leu Lys Ile Gln Val Arg Lys Ala Ala Ile Thr Ser Tyr Glu Lys 340 345 350 Ser Asp Gly Val Tyr Thr Gly Leu Ser Thr Arg Asn Gln Glu Thr Tyr 355 360 365 Glu Thr Leu Lys His Glu Lys Pro Pro Gln 370 375 <210> 44 <211> 446 <212> PRT <213> artificial sequence <220> <223> Chimeric antigen receptor <220> <221> SIGNAL <222> (1)..(18) <223> signal peptide <220> <221> MISC_FEATURE <222> (19)..(275) <220> <221> MISC_FEATURE <222> (276)..(290) <223> gs linker <220> <221> MISC_FEATURE <222> (291)..(358) <223> Modified CD8a linker <220> <221> MISC_FEATURE <222> (359)..(379) <223> FCGR3A transmembrane domain <220> <221> MISC_FEATURE <222> (380).. (404) <223> FCGR3A cytosolic domain <220> <221> MISC_FEATURE <222> (405).. (446) <223> FCER1G cytosolic domain <400> 44 Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile 20 25 30 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 35 40 45 Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser 50 55 60 Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile 85 90 95 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser 145 150 155 160 Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr 165 170 175 Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln 180 185 190 Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn 195 200 205 Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr 210 215 220 Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr 225 230 235 240 Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His 245 250 255 Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 260 265 270 Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 275 280 285 Gly Ser Ser Ala Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe Val 290 295 300 Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro 305 310 315 320 Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro 325 330 335 Glu Ala Ser Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 340 345 350 Asp Phe Ala Ser Asp Ile Val Ser Phe Cys Leu Val Met Val Leu Leu 355 360 365 Phe Ala Val Asp Thr Gly Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg 370 375 380 Ser Ser Thr Arg Asp Trp Lys Asp His Lys Phe Lys Trp Arg Lys Asp 385 390 395 400 Pro Gln Asp Lys Arg Leu Lys Ile Gln Val Arg Lys Ala Ala Ile Thr 405 410 415 Ser Tyr Glu Lys Ser Asp Gly Val Tyr Thr Gly Leu Ser Thr Arg Asn 420 425 430 Gln Glu Thr Tyr Glu Thr Leu Lys His Glu Lys Pro Pro Gln 435 440 445 <210> 45 <211> 446 <212> PRT <213> artificial sequence <220> <223> Chimeric antigen receptor <220> <221> SIGNAL <222> (1)..(18) <223> signal peptide <220> <221> MISC_FEATURE <222> (19)..(275) <223> TK1 ScFv <220> <221> MISC_FEATURE <222> (276)..(290) <223> gs linker <220> <221> MISC_FEATURE <222> (291)..(358) <223> Portion of CD8 hinge <220> <221> MISC_FEATURE <222> (359)..(379) <223> FCGR3A transmembrane domain <220> <221> MISC_FEATURE <222> (380).. (404) <223> FCGR3A cytosolic domain <220> <221> MISC_FEATURE <222> (405).. (446) <223> FCER1G cytosolic domain <400> 45 Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile 20 25 30 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 35 40 45 Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser 50 55 60 Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile 85 90 95 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser 145 150 155 160 Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr 165 170 175 Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln 180 185 190 Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn 195 200 205 Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr 210 215 220 Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr 225 230 235 240 Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His 245 250 255 Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 260 265 270 Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 275 280 285 Gly Ser Ser Ala Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe Val 290 295 300 Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro 305 310 315 320 Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro 325 330 335 Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 340 345 350 Asp Phe Ala Cys Asp Ile Val Ser Phe Cys Leu Val Met Val Leu Leu 355 360 365 Phe Ala Val Asp Thr Gly Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg 370 375 380 Ser Ser Thr Arg Asp Trp Lys Asp His Lys Phe Lys Trp Arg Lys Asp 385 390 395 400 Pro Gln Asp Lys Arg Leu Lys Ile Gln Val Arg Lys Ala Ala Ile Thr 405 410 415 Ser Tyr Glu Lys Ser Asp Gly Val Tyr Thr Gly Leu Ser Thr Arg Asn 420 425 430 Gln Glu Thr Tyr Glu Thr Leu Lys His Glu Lys Pro Pro Gln 435 440 445 <210> 46 <211> 391 <212> PRT <213> artificial sequence <220> <223> Chimeric antigen receptor <220> <221> SIGNAL <222> (1)..(18) <223> signal peptide <220> <221> MISC_FEATURE <222> (19)..(275) <223> TK1 ScFv <220> <221> MISC_FEATURE <222> (276)..(290) <223> gs linker <220> <221> MISC_FEATURE <222> (291)..(303) <223> IgG4 short hinge <220> <221> MISC_FEATURE <222> (304).. (324) <223> FCGR3A transmembrane domain <220> <221> MISC_FEATURE <222> (325).. (349) <223> FCGR3A cytosolic domain <220> <221> MISC_FEATURE <222> (350).. (391) <223> FCER1G cytosolic domain <400> 46 Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile 20 25 30 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 35 40 45 Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser 50 55 60 Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile 85 90 95 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser 145 150 155 160 Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr 165 170 175 Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln 180 185 190 Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn 195 200 205 Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr 210 215 220 Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr 225 230 235 240 Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His 245 250 255 Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 260 265 270 Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 275 280 285 Gly Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ile Val 290 295 300 Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly Leu 305 310 315 320 Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp Lys 325 330 335 Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys Arg Leu Lys 340 345 350 Ile Gln Val Arg Lys Ala Ala Ile Thr Ser Tyr Glu Lys Ser Asp Gly 355 360 365 Val Tyr Thr Gly Leu Ser Thr Arg Asn Gln Glu Thr Tyr Glu Thr Leu 370 375 380 Lys His Glu Lys Pro Pro Gln 385 390 <210> 47 <211> 497 <212> PRT <213> artificial sequence <220> <223> Chimeric antigen receptor <220> <221> SIGNAL <222> (1)..(18) <223> signal peptide <220> <221> MISC_FEATURE <222> (19)..(275) <223> TK1 ScFv <220> <221> MISC_FEATURE <222> (276)..(290) <223> gs linker <220> <221> MISC_FEATURE <222> (291)..(409) <223> IgG4 119 aa hinge <220> <221> MISC_FEATURE <222> (410).. (430) <223> FCGR3A transmembrane domain <220> <221> MISC_FEATURE <222> (431).. (455) <223> FCGR3A cytosolic domain <220> <221> MISC_FEATURE <222> (456)..(497) <223> FCER1G cytosolic domain <400> 47 Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile 20 25 30 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 35 40 45 Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser 50 55 60 Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile 85 90 95 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser 145 150 155 160 Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr 165 170 175 Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln 180 185 190 Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn 195 200 205 Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr 210 215 220 Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr 225 230 235 240 Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His 245 250 255 Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 260 265 270 Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 275 280 285 Gly Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gln 290 295 300 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 305 310 315 320 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 325 330 335 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 340 345 350 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 355 360 365 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 370 375 380 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 385 390 395 400 Lys Ser Leu Ser Leu Ser Leu Gly Lys Val Ser Phe Cys Leu Val Met 405 410 415 Val Leu Leu Phe Ala Val Asp Thr Gly Leu Tyr Phe Ser Val Lys Thr 420 425 430 Asn Ile Arg Ser Ser Thr Arg Asp Trp Lys Asp His Lys Phe Lys Trp 435 440 445 Arg Lys Asp Pro Gln Asp Lys Arg Leu Lys Ile Gln Val Arg Lys Ala 450 455 460 Ala Ile Thr Ser Tyr Glu Lys Ser Asp Gly Val Tyr Thr Gly Leu Ser 465 470 475 480 Thr Arg Asn Gln Glu Thr Tyr Glu Thr Leu Lys His Glu Lys Pro Pro 485 490 495 Gln <210> 48 <211> 607 <212> PRT <213> artificial sequence <220> <223> Chimeric angtigen receptor <220> <221> SIGNAL <222> (1)..(18) <223> signal peptide <220> <221> MISC_FEATURE <222> (19)..(275) <223> TK1 ScFv <220> <221> MISC_FEATURE <222> (276)..(290) <223> gs linker <220> <221> MISC_FEATURE <222> (291)..(519) <223> IgG4 long hinge <220> <221> MISC_FEATURE <222> (520).. (540) <223> FCGR3A transmembrane domain <220> <221> MISC_FEATURE <222> (541).. (565) <223> FCGR3A cytosolic domain <220> <221> MISC_FEATURE <222> (566).. (607) <223> FCER1G cytosolic domain <400> 48 Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile 20 25 30 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 35 40 45 Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser 50 55 60 Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile 85 90 95 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser 145 150 155 160 Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr 165 170 175 Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln 180 185 190 Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn 195 200 205 Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr 210 215 220 Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr 225 230 235 240 Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His 245 250 255 Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 260 265 270 Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 275 280 285 Gly Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 290 295 300 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 305 310 315 320 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 325 330 335 Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 340 345 350 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln 355 360 365 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 370 375 380 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 385 390 395 400 Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 405 410 415 Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn 420 425 430 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 435 440 445 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 450 455 460 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg 465 470 475 480 Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys 485 490 495 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 500 505 510 Ser Leu Ser Leu Gly Lys Ile Val Ser Phe Cys Leu Val Met Val Leu 515 520 525 Leu Phe Ala Val Asp Thr Gly Leu Tyr Phe Ser Val Lys Thr Asn Ile 530 535 540 Arg Ser Ser Thr Arg Asp Trp Lys Asp His Lys Phe Lys Trp Arg Lys 545 550 555 560 Asp Pro Gln Asp Lys Arg Leu Lys Ile Gln Val Arg Lys Ala Ala Ile 565 570 575 Thr Ser Tyr Glu Lys Ser Asp Gly Val Tyr Thr Gly Leu Ser Thr Arg 580 585 590 Asn Gln Glu Thr Tyr Glu Thr Leu Lys His Glu Lys Pro Pro Gln 595 600 605 <210> 49 <211> 390 <212> PRT <213> artificial sequence <220> <223> Chimeric antigen receptor <220> <221> SIGNAL <222> (1)..(18) <223> signal peptide <220> <221> MISC_FEATURE <222> (19)..(275) <223> TK1 ScFv <220> <221> MISC_FEATURE <222> (276)..(290) <223> gs linker <220> <221> MISC_FEATURE <222> (291)..(312) <223> FCGR2A transmembrane domain <220> <221> MISC_FEATURE <222> (313).. (390) <223> FCGR2A cytosolic domain <400> 49 Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile 20 25 30 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 35 40 45 Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser 50 55 60 Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile 85 90 95 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser 145 150 155 160 Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr 165 170 175 Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln 180 185 190 Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn 195 200 205 Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr 210 215 220 Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr 225 230 235 240 Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His 245 250 255 Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 260 265 270 Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 275 280 285 Gly Ser Ile Ile Val Ala Val Val Ile Ala Thr Ala Val Ala Ala Ile 290 295 300 Val Ala Ala Val Val Ala Leu Ile Tyr Cys Arg Lys Lys Arg Ile Ser 305 310 315 320 Ala Asn Ser Thr Asp Pro Val Lys Ala Ala Gln Phe Glu Pro Pro Gly 325 330 335 Arg Gln Met Ile Ala Ile Arg Lys Arg Gln Leu Glu Glu Thr Asn Asn 340 345 350 Asp Tyr Glu Thr Ala Asp Gly Gly Tyr Met Thr Leu Asn Pro Arg Ala 355 360 365 Pro Thr Asp Asp Asp Lys Asn Ile Tyr Leu Thr Leu Pro Pro Asn Asp 370 375 380 His Val Asn Ser Asn Asn 385 390 <210> 50 <211> 458 <212> PRT <213> artificial sequence <220> <223> Chimeric antigen receptor <220> <221> SIGNAL <222> (1)..(18) <223> signal peptide <220> <221> MISC_FEATURE <222> (19)..(275) <223> TK1 ScFv <220> <221> MISC_FEATURE <222> (276)..(290) <223> gs linker <220> <221> MISC_FEATURE <222> (291)..(358) <223> Modified CD8a hinge <220> <221> MISC_FEATURE <222> (359).. (380) <223> FCGR2A transmembrane domain <220> <221> MISC_FEATURE <222> (381)..(458) <223> FCGR2A cytosolic domain <400> 50 Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile 20 25 30 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 35 40 45 Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser 50 55 60 Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile 85 90 95 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser 145 150 155 160 Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr 165 170 175 Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln 180 185 190 Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn 195 200 205 Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr 210 215 220 Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr 225 230 235 240 Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His 245 250 255 Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 260 265 270 Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 275 280 285 Gly Ser Ser Ala Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe Val 290 295 300 Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro 305 310 315 320 Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro 325 330 335 Glu Ala Ser Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 340 345 350 Asp Phe Ala Ser Asp Ile Ile Ile Val Ala Val Val Ile Ala Thr Ala 355 360 365 Val Ala Ala Ile Val Ala Ala Val Val Ala Leu Ile Tyr Cys Arg Lys 370 375 380 Lys Arg Ile Ser Ala Asn Ser Thr Asp Pro Val Lys Ala Ala Gln Phe 385 390 395 400 Glu Pro Pro Gly Arg Gln Met Ile Ala Ile Arg Lys Arg Gln Leu Glu 405 410 415 Glu Thr Asn Asn Asp Tyr Glu Thr Ala Asp Gly Gly Tyr Met Thr Leu 420 425 430 Asn Pro Arg Ala Pro Thr Asp Asp Asp Lys Asn Ile Tyr Leu Thr Leu 435 440 445 Pro Pro Asn Asp His Val Asn Ser Asn Asn 450 455 <210> 51 <211> 458 <212> PRT <213> artificial sequence <220> <223> Chimeric antigen receptor <220> <221> SIGNAL <222> (1)..(18) <223> signal peptide <220> <221> MISC_FEATURE <222> (19)..(275) <223> TK1 ScFv <220> <221> MISC_FEATURE <222> (276)..(290) <223> gs linker <220> <221> MISC_FEATURE <222> (291)..(358) <223> Portion of CD8a linker <220> <221> MISC_FEATURE <222> (359).. (380) <223> FCGR2A transmembrane domain <220> <221> MISC_FEATURE <222> (381)..(458) <223> FCGR2A cytosolic domain <400> 51 Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile 20 25 30 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 35 40 45 Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser 50 55 60 Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile 85 90 95 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser 145 150 155 160 Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr 165 170 175 Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln 180 185 190 Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn 195 200 205 Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr 210 215 220 Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr 225 230 235 240 Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His 245 250 255 Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 260 265 270 Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 275 280 285 Gly Ser Ser Ala Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe Val 290 295 300 Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro 305 310 315 320 Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro 325 330 335 Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 340 345 350 Asp Phe Ala Cys Asp Ile Ile Ile Val Ala Val Val Ile Ala Thr Ala 355 360 365 Val Ala Ala Ile Val Ala Ala Val Val Ala Leu Ile Tyr Cys Arg Lys 370 375 380 Lys Arg Ile Ser Ala Asn Ser Thr Asp Pro Val Lys Ala Ala Gln Phe 385 390 395 400 Glu Pro Pro Gly Arg Gln Met Ile Ala Ile Arg Lys Arg Gln Leu Glu 405 410 415 Glu Thr Asn Asn Asp Tyr Glu Thr Ala Asp Gly Gly Tyr Met Thr Leu 420 425 430 Asn Pro Arg Ala Pro Thr Asp Asp Asp Lys Asn Ile Tyr Leu Thr Leu 435 440 445 Pro Pro Asn Asp His Val Asn Ser Asn Asn 450 455 <210> 52 <211> 403 <212> PRT <213> artificial sequence <220> <223> Chimeric antigen receptor <220> <221> SIGNAL <222> (1)..(18) <223> signal peptide <220> <221> MISC_FEATURE <222> (19)..(275) <223> TK1 ScFv <220> <221> MISC_FEATURE <222> (276)..(290) <223> gs linker <220> <221> MISC_FEATURE <222> (291)..(303) <223> IgG4 short hinge <220> <221> MISC_FEATURE <222> (304).. (325) <223> FCGR2A transmembrane domain <220> <221> MISC_FEATURE <222> (326).. (403) <223> FCGR2A cytosolic domain <400> 52 Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile 20 25 30 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 35 40 45 Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser 50 55 60 Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile 85 90 95 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser 145 150 155 160 Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr 165 170 175 Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln 180 185 190 Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn 195 200 205 Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr 210 215 220 Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr 225 230 235 240 Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His 245 250 255 Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 260 265 270 Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 275 280 285 Gly Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ile Ile 290 295 300 Ile Val Ala Val Val Ile Ala Thr Ala Val Ala Ala Ile Val Ala Ala 305 310 315 320 Val Val Ala Leu Ile Tyr Cys Arg Lys Lys Arg Ile Ser Ala Asn Ser 325 330 335 Thr Asp Pro Val Lys Ala Ala Gln Phe Glu Pro Pro Gly Arg Gln Met 340 345 350 Ile Ala Ile Arg Lys Arg Gln Leu Glu Glu Thr Asn Asn Asp Tyr Glu 355 360 365 Thr Ala Asp Gly Gly Tyr Met Thr Leu Asn Pro Arg Ala Pro Thr Asp 370 375 380 Asp Asp Lys Asn Ile Tyr Leu Thr Leu Pro Pro Asn Asp His Val Asn 385 390 395 400 Ser Asn Asn <210> 53 <211> 509 <212> PRT <213> artificial sequence <220> <223> Chimeric antigen receptor <220> <221> SIGNAL <222> (1)..(18) <223> signal peptide <220> <221> MISC_FEATURE <222> (19)..(275) <223> TK1 ScFv <220> <221> MISC_FEATURE <222> (276)..(290) <223> gs linker <220> <221> MISC_FEATURE <222> (291)..(409) <223> IgG4 119 aa hinge <220> <221> MISC_FEATURE <222> (410).. (431) <223> FCGR2A transmembrane domain <220> <221> MISC_FEATURE <222> (432).. (509) <223> FCGR2A cytosolic domain <400> 53 Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile 20 25 30 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 35 40 45 Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser 50 55 60 Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile 85 90 95 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser 145 150 155 160 Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr 165 170 175 Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln 180 185 190 Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn 195 200 205 Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr 210 215 220 Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr 225 230 235 240 Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His 245 250 255 Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 260 265 270 Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 275 280 285 Gly Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gln 290 295 300 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 305 310 315 320 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 325 330 335 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 340 345 350 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 355 360 365 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 370 375 380 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 385 390 395 400 Lys Ser Leu Ser Leu Ser Leu Gly Lys Ile Ile Val Ala Val Val Ile 405 410 415 Ala Thr Ala Val Ala Ala Ile Val Ala Ala Val Val Ala Leu Ile Tyr 420 425 430 Cys Arg Lys Lys Arg Ile Ser Ala Asn Ser Thr Asp Pro Val Lys Ala 435 440 445 Ala Gln Phe Glu Pro Pro Gly Arg Gln Met Ile Ala Ile Arg Lys Arg 450 455 460 Gln Leu Glu Glu Thr Asn Asn Asp Tyr Glu Thr Ala Asp Gly Gly Tyr 465 470 475 480 Met Thr Leu Asn Pro Arg Ala Pro Thr Asp Asp Asp Lys Asn Ile Tyr 485 490 495 Leu Thr Leu Pro Pro Asn Asp His Val Asn Ser Asn Asn 500 505 <210> 54 <211> 619 <212> PRT <213> artificial sequence <220> <223> Chimeric antigen receptor <220> <221> SIGNAL <222> (1)..(18) <223> signal peptide <220> <221> MISC_FEATURE <222> (19)..(275) <223> TK! ScFvs <220> <221> MISC_FEATURE <222> (276)..(290) <223> gs linker <220> <221> MISC_FEATURE <222> (291)..(519) <223> IgG4 long hinge <220> <221> MISC_FEATURE <222> (520).. (541) <223> FCGR2A transmembrane domain <220> <221> MISC_FEATURE <222> (542)..(619) <223> FCGR2A cytosolic domain <400> 54 Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile 20 25 30 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 35 40 45 Ser Ser Val Ser Tyr Met His Phe Tyr Gln Gln Lys Pro Gly Ser Ser 50 55 60 Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile 85 90 95 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 Ser Ser Asn Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Met Ala Val Val Thr Gly Val Asn Ser Glu Val Gln Leu Gln Gln Ser 145 150 155 160 Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Thr 165 170 175 Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln 180 185 190 Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn 195 200 205 Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr 210 215 220 Thr Asp Thr Ser Phe Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr 225 230 235 240 Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Gly Tyr Gly His 245 250 255 Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 260 265 270 Val Asp Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 275 280 285 Gly Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 290 295 300 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 305 310 315 320 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 325 330 335 Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 340 345 350 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln 355 360 365 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 370 375 380 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 385 390 395 400 Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 405 410 415 Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn 420 425 430 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 435 440 445 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 450 455 460 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg 465 470 475 480 Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys 485 490 495 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 500 505 510 Ser Leu Ser Leu Gly Lys Ile Ile Ile Val Ala Val Val Ile Ala Thr 515 520 525 Ala Val Ala Ala Ile Val Ala Ala Val Val Ala Leu Ile Tyr Cys Arg 530 535 540 Lys Lys Arg Ile Ser Ala Asn Ser Thr Asp Pro Val Lys Ala Ala Gln 545 550 555 560 Phe Glu Pro Pro Gly Arg Gln Met Ile Ala Ile Arg Lys Arg Gln Leu 565 570 575 Glu Glu Thr Asn Asn Asp Tyr Glu Thr Ala Asp Gly Gly Tyr Met Thr 580 585 590 Leu Asn Pro Arg Ala Pro Thr Asp Asp Asp Lys Asn Ile Tyr Leu Thr 595 600 605 Leu Pro Pro Asn Asp His Val Asn Ser Asn Asn 610 615

Claims (21)

cytoplasmic domain;
transmembrane domain; and
contains an extracellular domain;
wherein the cytoplasmic domain comprises the cytoplasmic portion of a receptor that, when activated, polarizes macrophages to M2 macrophages;
wherein the wild-type protein comprising a cytoplasmic portion does not comprise an extracellular domain;
A chimeric receptor wherein the extracellular domain is an antibody or a fragment thereof. The chimeric receptor of claim 1 , wherein binding of the ligand to the extracellular domain activates the cytoplasmic portion. delete delete delete delete delete The chimeric receptor according to claim 1, wherein the antibody or fragment thereof is a ScFv fragment. The chimeric receptor according to claim 1, further comprising a linker between the transmembrane domain and the extracellular domain. 10. The chimeric receptor according to claim 9, wherein the linker is a GS linker. The chimeric receptor according to claim 1, further comprising a hinge region between the transmembrane domain and the extracellular domain. 10. The chimeric receptor according to claim 9, further comprising a hinge region between the transmembrane domain and the linker. A nucleic acid comprising a polynucleotide encoding the chimeric receptor of claim 1. 14. The nucleic acid of claim 13, further comprising a promoter operably linked to the polynucleotide. A vector comprising the nucleic acid of claim 13. 16. The vector according to claim 15, which is a lentiviral vector. A cell comprising the chimeric receptor of claim 1. 18. The cell according to claim 17, which is a monocyte or macrophage. A cell comprising the nucleic acid of claim 13 . 20. The cell according to claim 19, which is a monocyte or macrophage. contacting a macrophage containing the chimeric receptor of claim 1 with a ligand for the extracellular domain of the chimeric receptor in vitro; and
binding the ligand to the extracellular domain of the chimeric receptor;
wherein binding of the ligand to the extracellular domain of the chimeric receptor activates the cytoplasmic portion;
wherein the activation of the cytoplasmic portion polarizes the macrophage.

Images