JP2022189893A - Transgenic macrophages, chimeric antigen receptors and related methods - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide chimeric antigen receptors, nucleic acids encoding chimeric antigen receptors, macrophages having the chimeric antigen receptors and/or the coding nucleic acids, as well as related methods.

SOLUTION: A chimeric receptor is disclosed. The chimeric receptor comprises a cytoplasmic domain, a transmembrane domain and an extracellular domain. In embodiments, the cytoplasmic domain comprises a cytoplasmic moiety of the receptor that polarizes the macrophage on activation. In further embodiments, a wild type protein comprising the cytoplasmic moiety dose not contain the extracellular domain of the chimeric antigen receptor. In embodiments, binding of a ligand to the extracellular domain of the chimeric receptor activates the intracellular moiety of the chimeric receptor. The activation of the intracellular moiety of the chimeric receptor can polarize the macrophage into an M1 or M2 macrophage.

SELECTED DRAWING: None

COPYRIGHT: (C)2023,JPO&INPIT

Description

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

Cancer is characterized by uncontrolled cell proliferation and death, genomic instability and mutation, tumor-promoting inflammation, induction of angiogenesis, immune system evasion, deregulation of metabolic pathways, tumor cell replication, and metastatic tissue invasion. It consists of a group of associated diseases [1]. 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 expected to be diagnosed each year, more than 580,000 Americans are expected to die (approximately 1600 cancer deaths per day), It accounts for nearly one in four all deaths [2, 3].

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

Macrophages are cells derived from the myeloid system and belong to the innate immune system. They are derived from blood monocytes that migrate into tissues. One of their major functions is to phagocytize microorganisms and remove cellular debris. They also play an important role in both the initiation and resolution of inflammation [9,10]. Moreover, macrophages can display different responses, ranging from pro-inflammatory to anti-inflammatory, depending on the type of stimulation 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), IFNγ, IL-1β, TNFα, and Toll-like receptor engagement. M1 macrophages constitute a powerful arm of the immune system deployed to fight infections. These can be either direct (pathogen-type recognizing receptors) or indirect (Fc receptors, complement receptors) recognition of pathogens. They also have the ability to generate reactive oxygen species (ROS) as a means to help kill pathogens. In addition, M1 macrophages secrete pro-inflammatory cytokines and chemokines that attract other types of immune cells and coordinate/orchestrate 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 surrogate 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 that allow recruitment of regulatory T cells to sites of inflammation. M2 macrophages do not constitute a homogeneous population and are often further subdivided into M2a, M2b and M2c categories. A common factor for all three subpopulations is high production of IL-10 accompanied by low production of IL-12. One of these distinguishing properties is the production of the enzyme arginine 1, which depletes L-arginine, thereby suppressing T cell responses and depleting its substrate iNOS.

The in vivo molecular mechanisms of macrophage polarization are not well characterized [10, 14] due to the various signals that macrophages experience in the cellular microenvironment. In recent years, progress has 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, it has been shown that macrophage polarization in vivo is plastic and macrophages can be polarized back and forth to either phenotype with the help of cytokines [15, 16]. Interferon gamma (IFNγ) and IL-4 are two cytokines that can polarize macrophages into M1 and M2 phenotypes, respectively [15].

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

Polarization of macrophages The tumor microenvironment can affect the polarization of macrophages. The polarization process can be diverse 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 is known to involve transcriptional regulation. For example, macrophages exposed to LPS or IFNγ polarize towards the M1 phenotype, whereas macrophages exposed to IL-4 or IL-13 polarize towards the M2 phenotype. LPS or IFNγ interact with Toll-like receptor 4 (TLR4) on the surface of macrophages that induces the Trif and MyD88 pathways, induces activation of the transcription factors IRF3, AP-1, and NFκB, thus inducing inflammation. It can activate TNF genes, interferon genes, CXCL10, NOS2, IL-12, etc., which are required for sexual M1 macrophage response [20]. Similarly, IL-4 and IL-13 bind IL-4R and regulate the expression of genes associated with the anti-inflammatory response (M2 response) such as CCL17, ARG1, IRF4, IL-10, SOCS3, Jak /Stat6 pathway.

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

Polarization of macrophages was a complex process, and macrophage behavior elicited different responses in response to microenvironmental stimuli. Macrophage polarization is therefore better represented by successive states of activation where the M1 and M2 phenotypes are the extremes of the spectrum. In recent years, there has been much debate about the definition/description of macrophage activation and macrophage polarization. A recent paper published by Murray et al. describes a set of criteria to be considered for the consensus definition/description of macrophage activation, polarization, activators and markers. This publication was greatly needed for the definition and characterization of activated/polarized macrophages [22].

M1 Phenotype M1 pro-inflammatory or classically activated macrophages are active and highly phagocytic and generate large amounts of reactive oxygen and nitrogen species, thereby promoting Th1 responses [11]. M1 macrophages secrete high levels of IL-12 and IL-23, two important inflammatory cytokines. IL-12 induces activation and clonal expansion of Th17 cells, which secrete large amounts of IL-17, which contributes to inflammation [23]. These characteristics enable M1 macrophages to control metastasis, suppress tumor growth, and control microbial infection [24]. Moreover, infiltration and recruitment of M1 macrophages to tumor sites correlates with better prognosis and higher overall survival in patients with solid tumors [17, 18, 25-28].

Polarization of macrophages to the M1 phenotype is regulated in vitro by inflammatory signals such as IFNγ, TNFα, IL-1β, and LPS, as well as transcription factors and miRNAs [29, 30]. Classically activated macrophages initiate induction of the STAT1 transcription factor, which targets CXCL9, CXCL10 (also known as IP-10), IFN regulator 1, and suppressor of cytokine signaling 1 [31 ]. The cytokine signaling 1 protein functions downstream of cytokine receptors and participates in a negative feedback loop to attenuate cytokine signaling. In the tumor microenvironment, Notch signaling plays a key role in the polarization of M1 macrophages, thereby allowing the transcription factor RBP-J to regulate canonical activation. Macrophages deficient in Notch signaling express the M2 phenotype regardless of other exogenous inducers [32]. One key miRNA, miRNA-155, is upregulated during the transition of macrophages from M2 to M1, and M1 macrophages overexpressing miRNA-155 are generally more active and contribute to tumor shrinkage. Related [33]. Furthermore, miRNA-342-5p has been found to promote higher inflammatory responses in macrophages by targeting Akt1 in mice. This miRNA further promotes upregulation of Nos2 and IL-6, both of which act as inflammatory signals in macrophages [34]. Other miRNAs such as miRNA-125 and miRNA-378 have further 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, as their presence usually indicates a favorable prognosis. Following 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 signals such as the tumor microenvironment or tissue-resident cells can polarize M1 macrophages to M2 macrophages. In vivo studies of mouse macrophages show that macrophages are plastic in cytokine and surface marker expression, and that repolarization of macrophages to the M1 phenotype in the presence of cancer may help the immune system to reject tumors. have shown that it is possible [19].

M2 Phenotype M2 macrophages are anti-inflammatory and aid in the processes of angiogenesis and tissue repair. They express scavenger receptors and produce large amounts of IL-10 and other anti-inflammatory cytokines [33,36]. Expression of IL-10 by M2 macrophages promotes Th2 responses. As a result, Th2 cells upregulate the production of IL-3 and IL-4. IL-3 stimulates proliferation of all cells in the myeloid lineage (granulocytes, monocytes, and dendritic cells) and other cytokines such as erythropoietin (EPO), granulocyte-macrophage colony-stimulating factor (GM-CSF), and in combination with IL-6. IL-4 is an important cytokine in the healing process as it contributes to the production of extracellular matrix [23]. M2 macrophages exhibit a function that may aid tumor progression by allowing blood vessels to nourish malignant cells, thus promoting proliferation. The presence of macrophages (considered M2) in the majority of solid tumors is negatively correlated with successful treatment and longer survival [37]. Furthermore, the presence of M2 macrophages has been linked to metastatic potential in breast cancer. Lin and colleagues found that early recruitment of macrophages to mammary tumor sites in mice increased angiogenesis and malignant tumor incidence [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 M2 responses [41].

Tumor-associated macrophages (TAM)
Cells exposed to the tumor microenvironment behave differently. For example, tumor-associated macrophages found in the periphery of solid tumors help promote tumor growth and metastasis and are thought to have an M2-like phenotype [42]. Tumor-associated macrophages can be either tissue-resident macrophages or bone marrow-derived recruited macrophages (macrophages that differentiate from monocytes to macrophages and migrate to tissues). A study by Cortez-Retamozo found that a large number of TAM precursors in the spleen translocated into the tumor stroma, further suggesting this organ as a reservoir for TAMs [43]. TAM precursors found in the spleen were found to initiate translocation through the CCR2 chemokine receptor [43]. Recent studies have found CSF-1 as a major factor in attracting macrophages to the tumor periphery, with CSF-1 production by cancer cells predicting reduced survival and an overall poor prognosis. [44-46]. Other cytokines such as TNF-α and IL-6 have also been linked to accumulation/recruitment of macrophages to the peritumoral area [45].

Macrophages recruited around the tumor border are thought to be regulated by an "angiogenic switch" that is activated within the tumor. The angiogenic switch is defined as the process by which a tumor develops a dense network of blood vessels that potentially allow the tumor to metastasize and is required for malignant metastasis. It was observed that a complete angiogenic switch required the presence of macrophages in a breast cancer mouse model. The angiogenic switch is further delayed when the maturation, migration and accumulation of macrophages around the tumor is delayed, the angiogenic switch does not occur in the absence of macrophages, and the presence of macrophages is required for malignant tumor progression. [47]. In addition, tumor stromal cells produce chemokines such as CSF1, CCL2, CCL3, CCL5, and placental growth factor that will recruit macrophages to the peritumoral area. These chemokines provide an environment in which macrophages activate the angiogenic switch, and 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 elimination. Macrophages are attracted to a hypoxic tumor environment and will respond by producing hypoxia-inducible factor-1α (HIF-1α), which regulates the transcription of genes associated with angiogenesis, and HIF-2α. Important to note. During the angiogenic switch, macrophages can also secrete VEGF (stimulated by the NF-κB pathway) that may promote vascular maturation and vascular permeability [48].

Tumor-associated macrophages maintain their M2-like phenotype by receiving polarizing signals from malignant cells such as IL-1R, and MyD88 mediated through the IkB kinase β and NF-kB signaling cascades. It is considered possible. Inhibition of NF-kB in TAMs promotes canonical activation [40]. In addition, another study suggested that the p50 NF-kB subunit was involved in the suppression of M1 macrophages, and reduced inflammation promoted tumor growth. p50 NF-κB knockout mice generated by Saccani et al. suggested that p50 NF-κB knockout restored M1 activity and reduced tumor survival [49].

Because tumor masses contain large numbers of M2-like macrophages, TAMs can be used as targets for cancer therapy. Reducing the number of TAMs or polarizing them towards the M1 phenotype can help destroy cancer cells and reduce tumor growth [50-52]. Luo and colleagues used vaccines against the cysteine proteases upregulated in TAMs, which are considered potential tumor targets, and the stress protein legumain [52]. Genes controlling angiogenesis were down-regulated and tumor growth was halted when a vaccine against legumain was administered to mice [52].

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

M1 and M2 macrophages demonstrate distinct metabolic patterns that reflect their distinct behavior [56]. The M1 phenotype increases glycolysis and tilts glucose metabolism towards the oxidative pentose phosphate pathway, which reduces oxygen consumption, resulting in large amounts of radical oxygen and nitrogen species, as well as TNF-α, Inflammatory cytokines such as IL-12, IL-6 are produced [56, 57]. Reducing flux to the pentose phosphate pathway while increasing the redox potential of the whole cell, the M2 phenotype increases fatty acid uptake and oxidation, resulting in scavenger receptors and IL-10 and TGF-β upregulates immunomodulatory cytokines such as [56].

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

L-arginine metabolism shows an important discrete shift in cytokine expression in macrophages, illustrating a distinct metabolic pathway that alters TAM-tumor cell interactions [61]. Classically activated (M1) macrophages support inducible nitric oxide synthase (iNOS). The iNOS pathway produces cytotoxic nitric oxide (NO), resulting in anti-tumor behavior. Alternatively, activated (M2) macrophages have been shown to support the arginase pathway and produce ureum and l-ornithine that contribute to progressive tumor cell proliferation [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 signature [56, 57]. When CARKL is knocked down by RNAi, macrophages tend to adopt an M1-like metabolic pathway (metabolism skewed toward glycolysis and reduced oxygen consumption). When CARKL is overexpressed, macrophages adopt an M2-like metabolism (decreased glycolytic flux and higher oxygen consumption) [56]. When macrophages adopt an M1-like metabolic state through LPS/TLR4 engagement, CARKL levels are reduced and genes regulated by the NFκB pathway are activated (TNF-α, IL-12, and IL-6). , NADH:NAD+ and GSH:GSSSG complexes increase the redox potential of the cell. During the M2-like metabolic state, macrophages upregulate genes regulated by CARKL and STAT6/IL-4 (IL-10 and TGF-β).

Obesity can further affect macrophage polarization. Obesity is associated with conditions of chronic inflammation, an environment that drives the IL4/STAT6 pathway to activate NKT cells that drive macrophages toward the M2 response. During late-stage dietary obesity, macrophages migrate to adipose tissue and immune cells alter the level of T _H 1 or T _H 2 cytokine expression in adipose tissue, predisposing to an M2 phenotype and possibly increased insulin sensitivity. cause [63].

M1 phenotyping by targeting metabolic pathways in TAMS may provide an alternative means of reducing tumor growth and metastasis.

Macrophage Immunotherapy Approaches to Cancer The role of cancer immunotherapy is to stimulate the immune system to recognize, reject, and destroy cancer cells. Cancer immunotherapy with monocytes/macrophages aims to polarize macrophages towards a pro-inflammatory response (M1), thus enabling macrophages and other immune cells to destroy tumors. Many cytokines and bacterial compounds can accomplish this in vitro, but typically the side effects are very 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 each year [64, 65]. Early immunotherapy has established a good foundation for better cancer therapy and increased survival in patients treated with immunotherapy [66].

Several approaches to cancer immunotherapy include the use of cytokines or chemokines to recruit activated macrophages and other immune cells to the tumor site, thereby allowing tumor site recognition and target destruction. [67, 68]. IFN-α and IFN-β have been shown to inhibit tumor progression by inducing cell differentiation and apoptosis [69]. Furthermore, IFN treatment is anti-proliferative and can increase the duration of the S phase in the cell cycle [70,71]. Zhang and colleagues have conducted studies in nude mice using IFN-β gene therapy to target human prostate cancer cells. These results indicate that adenovirus-delivered IFN-β gene therapy accompanies macrophages and helps suppress proliferation and metastasis [72].

Macrophage inhibitory factor (MIF) is another cytokine that can be used in cancer immunotherapy. MIF is commonly found in solid tumors and is associated with a poor prognosis. MIF inhibits active macrophage function and drives macrophages toward the M2 phenotype, which can support tumor growth and progression. Simpson, Templeton, and Cross (2012) found that MIF induced the differentiation of macrophage precursor myeloid lineage cells into a suppressive population of myeloid lineage cells expressing the M2 phenotype [73]. By targeting MIF, we were able to deplete this suppressive population of macrophages and inhibit their proliferation, thus controlling tumor growth and metastasis [73].

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

Other studies have used liposomes to deliver drugs to deplete M2 macrophages from tumors and halt angiogenesis. Cancer cells that express high levels of IL-1β grow faster and induce more angiogenesis in vivo. Kimura and colleagues have shown that macrophages exposed to IL-1β-expressing tumor cells develop high levels of angiogenic agents such as vascular endothelial growth factor A (VEG-A), IL-8, and monocyte chemoattractant protein-1. It was found to produce factors and chemokines and promote tumor growth and angiogenesis [76]. When clodronate liposomes were used to deplete macrophages, they found fewer IL-1β-producing tumor cells. Furthermore, it was found that inhibition of NF-κB and AP-1 transcription factors in cancer cells reduced tumor growth and angiogenesis. These findings may suggest that macrophages surrounding the tumor site may be involved in promoting tumor growth and angiogenesis [76].

Compounds such as methionine enkephalin (MENK) have antitumor properties in vivo and in vitro. MENK polarizes M2 macrophages to M1 macrophages by downregulating CD206 and Arginase-1 (M2 markers) while upregulating CD64, MHC-II, and nitric oxide production (M1 markers) have the ability. In addition, MENK can 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 prevent skeletal complications such as bone resorption and to treat patients with metastatic breast cancer [78]. While bisphosphonates remain in the body for a short period of time, bisphosphonates can target cells of the same family as osteoclasts, macrophages, due to their high affinity for hydroxyapatite. Once the bisphosphonate is bound to bone, the bone matrix internalizes the bisphosphonate by endocytosis. Once in the cytoplasm, bisphosphonates can inhibit protein prenylation, an event that prevents integrin signaling and endosomal trafficking, thereby causing the cell to undergo apoptosis [69]. Until recently, it was unclear whether bisphosphonates could target tumor-associated macrophages, but recent studies by Junankar et al. show that macrophages take up nitrogen-containing bisphosphonate compounds by phagocytosis and phagocytosis, an event that affects peritumoral epithelial cells. [79]. Using bisphosphonates to force TAMs into apoptosis could reduce angiogenesis and metastasis.

Additional approaches to cancer immunotherapy involve the use of biomaterials capable of eliciting an immune response. Cationic polymers are used in immunotherapy because they react when dissolved in water. Chen et al. used cationic polymers including PEI, polylysine, cationic dextran, and cationic gelatin to generate strong Th1 immune responses [77]. They were also able to induce proliferation of CD4+ cells and secretion of IL-12 typical of M1 macrophages [77]. Huang and colleagues used biomaterials to induce TAMs to elicit anti-tumor responses by targeting TLR4 [80]. This study found that TAMs were able to polarize to the M1 phenotype and express IL-12. These cationic molecules were found to have direct tumoricidal activity, demonstrating tumor reduction in mice [80].

TLR4
Toll-like receptor 4 is a human protein encoded by the TLR4 gene. TLR4 detects lipopolysaccharide (LPS) on Gram-negative bacteria and thus plays a fundamental role in recognizing danger and activating 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 involved in the activation of M1 macrophages upon detecting the presence of LPS. This is the functional portion of the receptor that binds to MOTO-CAR (ie, chimeric receptor) to induce activation of monocytes/macrophages upon binding of the CAR to its target protein.

The adapter proteins MyD88 and TIRAP are involved in the activation of some and possibly all pathways through direct interaction with the TLR4 Toll/interleukin-1 receptor (IL-1R) (TIR) domain. contribute. However, there may be additional adapters required for activation of specific subsets 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 largely studied in the context of overexpression in tumors. TK1 was first generalized due to its expression in the serum of cancer patients (sTK), and thus its diagnostic and prognostic potential has been extensively studied. For example, several studies have demonstrated that sTK1 in many different cancer patients is elevated in a staged fashion with higher levels of TK1, indicative of more advanced tumors [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 showed a significant decrease in sTK1 levels when patients responded to therapy, whereas sTK1 levels continue to rise in patients who do not appear to respond to their therapy. . It is also known that sTK1 levels begin to rise before relapse, and it is also widely known that sTK1 levels in some cases can predict relapse "1-6 months before the onset of clinical symptoms". Several other studies confirm the abundant potential of TK1 as a diagnostic and prognostic indicator for cancer [82].

Although the diagnostic and prognostic potential of TK1 is well established, the therapeutic potential of TK1 in comparison remains unclear. While it is true that HSV-TK has been used in gene therapy and that PET imaging utilizes TK1 to identify proliferating cancer cells, any researcher addressing the potential of TK1 immunotherapy is unlikely. For research, almost none. This is probably mainly because TK1 is a known cytosolic protein. It has recently been discovered that TK1 is expressed not only on cancer cells, but also on the surface membrane of multiple tumor types and is therefore a very viable target for tumor immunotherapy.

Described herein are chimeric receptors. A chimeric receptor comprises a cytoplasmic domain, a transmembrane domain and an extracellular domain. In embodiments, the cytoplasmic domain comprises the cytoplasmic portion of the receptor that polarizes macrophages when activated. In a further embodiment, the wild-type protein, including this cytoplasmic portion, does not include the extracellular domain of the chimeric receptor (see, eg, Figure 21). In embodiments, binding of the ligand to the extracellular domain of the chimeric receptor activates the intracellular portion of the chimeric receptor (see, eg, Figure 22). Activation of the intracellular portion of the chimeric receptor can polarize macrophages into M1 or M2 macrophages (see, eg, FIGS. 23 and 24(A) and 25).

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

Further embodiments include a cell comprising a chimeric receptor or nucleic acid encoding a chimeric receptor.

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

These and other aspects of the disclosure will become apparent to those skilled in the art in light of the teachings contained herein.

A block diagram of the alignment of the constituent molecules in the chimeric receptor TKs1-MOTO1 is shown. Shown is the sequence of TK1-MOTO1 (SEQ ID NO:35). 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 291-313 are the TLR4 transmembrane domain, and amino acids 314- 496 is the TLR4 cytosolic domain. A block diagram of the alignment of the constituent molecules in the chimeric receptor TK1-MOTO2 is shown. Shown is 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, amino acids 296-318. is the TLR4 transmembrane domain and amino acids 319-500 is the TLR4 cytosolic domain. A block diagram of the alignment of the constituent molecules in the chimeric receptor TK1-MOTO3 is shown. Shown is 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, amino acids 346-368. is the TLR4 transmembrane domain and amino acids 269-501 is the TLR4 cytosolic domain. A block diagram of the alignment of the constituent molecules in the chimeric receptor TK1-MOTO4 is shown. Shown is 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, amino acids 303-325. is the TLR4 transmembrane domain and amino acids 326-508 are the TLR4 cytosolic domain. A block diagram of the alignment of the constituent molecules in the chimeric receptor TK1-MOTO5 is shown. Shown is the sequence of TK1-MOTO5 (SEQ ID NO:39). 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 291-409 are the IgG 119 amino acid-mediated hinge, amino acids 410- 432 is the TLR4 transmembrane domain and amino acids 433-615 are the TLR4 cytosolic domain. A block diagram of the alignment of the constituent molecules in the chimeric receptor TK1-MOTO6 is shown. Shown is 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, amino acids 519-541. is the TLR4 transmembrane domain and amino acids 542-724 is the TLR4 cytosolic domain. A block diagram of the alignment of the constituent molecules in the chimeric receptor TK1-MOTO7 is shown. Shown is 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, and amino acids 291-358 are the hinge of CD8 mutated with C339S and C356S. Amino acids 359-381 are the TLR4 transmembrane domain, and amino acids 382-564 are the TLR4 cytosolic domain. A block diagram of the alignment of the constituent molecules in the chimeric receptor TK1-MOTO8 is shown. Shown is 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, amino acids 359- 381 is the TLR4 transmembrane domain and amino acids 382-564 are the TLR4 cytosolic domain. A block diagram of the alignment of the constituent molecules in the chimeric receptor TK1-MO-FCGRA-CAR-1 is shown. Shown is the sequence of TK1-MO-FCGRA-CAR-1 (SEQ ID NO:43). 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 291-311 are the FCGR3A transmembrane domain, amino acids 312-336. is the FCGR3A cytosolic domain and amino acids 337-378 are the FCER1G cytosolic domain. A block diagram of the alignment of the constituent molecules in the chimeric receptor TK1-MO-FCGRA-CAR-2 is shown. Shown is 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, and amino acids 291-358 are the hinge of CD8 mutated with C339S and C356S. Amino acids 359-379 are the FCGR3A transmembrane domain, amino acids 380-404 are the FCGR3A cytosolic domain, and amino acids 405-446 are the FCER1G cytosolic domain. A block diagram of the alignment of the constituent molecules in the chimeric receptor TK1-MO-FCGRA-CAR-3 is shown. Shown is 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, amino acids 359- 379 is the FCGR3A transmembrane domain, amino acids 380-404 are the FCGR3A cytosolic domain, and amino acids 405-446 are the FCER1G cytosolic domain. A block diagram of the alignment of the constituent molecules in the chimeric receptor TK1-MO-FCGRA-CAR-4 is shown. Shown is the sequence of TK1-MO-FCGRA-CAR-4 (SEQ ID NO:46). 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, amino acids 304-324. is the FCGR3A transmembrane domain, amino acids 325-349 are the FCGR3A cytosolic domain, and amino acids 350-391 are the FCER1G cytosolic domain. A block diagram of the alignment of the constituent molecules in the chimeric receptor TK1-MO-FCGRA-CAR-5 is shown. Shown is the sequence of TK1-MO-FCGRA-CAR-5 (SEQ ID NO:47). 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 291-409 are the 119 amino acid hinge of IgG4, amino acids 410- 430 is the FCGR3A transmembrane domain, amino acids 431-455 are the FCGR3A cytosolic domain, and amino acids 456-497 are the FCER1G cytosolic domain. A block diagram of the alignment of the constituent molecules in the chimeric receptor TK1-MO-FCGRA-CAR-6 is shown. Shown is the sequence of TK1-MO-FCGRA-CAR-6 (SEQ ID NO:48). 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, amino acids 520-540. is the FCGR3A transmembrane domain, amino acids 541-565 are the FCGR3A cytosolic domain, and amino acids 566-607 are the FCER1G cytosolic domain. A block diagram of the alignment of the constituent molecules in the chimeric receptor TK1-MO-FCG2A-CAR-1 is shown. Shown is the sequence of TK1-MO-FCG2A-CAR-1 (SEQ ID NO:49). 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 291-312 are the FCGR2A transmembrane domain, amino acids 313-390. is the FCGR2A cytosolic domain. A block diagram of the alignment of the constituent molecules in the chimeric receptor TK1-MO-FCG2A-CAR-2 is shown. Shown is 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, and amino acids 291-358 are the hinge of CD8 mutated with C339S and C356S. Amino acids 359-380 are the FCGR2A transmembrane domain and amino acids 381-458 are the FCGR2A cytosolic domain. A block diagram of the alignment of the constituent molecules in the chimeric receptor TK1-MO-FCG2A-CAR-3 is shown. Shown is the sequence of TK1-MO-FCG2A-CAR-3 (SEQ ID NO:51). 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 hinge of CD8, amino acid 359. ~380 is the FCGR2A transmembrane domain and amino acids 381-458 are the FCGR2A cytosolic domain. A block diagram of the alignment of the constituent molecules in the chimeric receptor TK1-MO-FCG2A-CAR-4 is shown. Shown is 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, amino acids 304-325. is the FCGR2A transmembrane domain and amino acids 326-403 are the FCGR2A cytosolic domain. A block diagram of the alignment of the constituent molecules in the chimeric receptor TK1-MO-FCG2A-CAR-5 is shown. Shown is the sequence of TK1-MO-FCG2A-CAR-5 (SEQ ID NO:53). 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 291-409 are the 119 amino acid hinge of IgG4, amino acids 410- 431 is the FCGR2A transmembrane domain and amino acids 432-509 are the FCGR2A cytosolic domain. A block diagram of the alignment of the constituent molecules in the chimeric receptor TK1-MO-FCG2A-CAR-6 is shown. Shown is 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, amino acids 520-541. is the FCGR2A transmembrane domain and amino acids 542-619 are the FCGR2A cytosolic domain. Schematic representation of a chimeric receptor. Schematic representation of macrophages expressing chimeric receptors. As shown, the chimeric receptor comprises a Toll-like receptor cytosolic domain, a transmembrane domain, and a ligand-specific ScFv. Arrows indicate signaling that polarizes macrophages upon ScFv binding to ligand. Schematic diagram showing different macrophage receptors that can be utilized to construct chimeric receptors. Schematic showing the Fc gamma receptor III signaling cascade leading to cell activation. Schematic showing the Fc gamma receptor III signaling cascade leading to inhibition of calcium flux and proliferation. Schematic showing the Fc gamma receptor III signaling cascade leading to apoptosis. Schematic representation of the Toll-like receptor signaling cascade. A graph is presented showing flow cytometry confirming that the expressed antibody fragment binds the ligand of interest. Two images are presented showing phenotypic changes in macrophages after transduction with chimeric receptors. Two images are presented confirming the expression of chimeric receptors in monocytes. Three scatterplots of fluorescence-activated cell sorting demonstrating expression of dTomato are shown. The leftmost plot shows a control in which only 0.58% of cells show fluorescence that would indicate expression of dTomato. The right two plots show a transduction efficiency of 27.1 percent after transduction. Six scatterplots of fluorescence-activated cell sorting demonstrating retention of the dye (Alexa 647) and expression of CD80, CD163, CD206, and CD14 in macrophages transduced with chimeric receptors are presented. Histograms showing relative expression levels of CD80, CD163, CD206, and CD14 in macrophages transduced with chimeric receptors are presented. Six images of transduced macrophages expressing chimeric receptors that induce detection, attack, and cell death in a lung cancer cell line (NCI-H460) are presented.

Described herein are chimeric receptors. A chimeric receptor comprises a cytoplasmic domain, a transmembrane domain and an extracellular domain. In embodiments, the cytoplasmic domain comprises the cytoplasmic portion of the receptor that polarizes macrophages when activated. In a further embodiment, the wild-type protein, including this cytoplasmic portion, does not include the extracellular domain of the chimeric receptor. In embodiments, 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 into M1 or M2 macrophages.

In certain embodiments, the cytoplasmic portion of the chimeric receptor comprises toll-like receptor, myeloid lineage primary response protein (MYD88) (SEQ ID NO: 19), toll-like receptor 3 (TLR3) (SEQ ID NO: 1), 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: 9) 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), low affinity immunoglobulin gamma Fc region receptor II-a (FCGR2A) (SEQ ID NO: 13), high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) (SEQ ID NO: 19) and sequences having at least 90% sequence identity with the cytoplasmic domain of any one of the foregoing. 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 embodiments, the cytosolic moiety can be any polypeptide that, upon activation, will result in polarization of macrophages.

In further embodiments, examples of ligands that bind 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), carcinoembryonic antigen (CEA), B cell maturation antigen (BCMA), glypican 3 ( GPC3), fibroblast activation protein (FAP), erythropoietin-producing hepatocellular carcinoma A2 (EphA2), natural killer group 2D (NKG2D) ligand, disialoganglioside 2 (GD2), CD19, CD20, CD30, CD33, CD123, Can be, but are not limited to, CD133, CD138, and CD171. In certain embodiments, the ligand is not TK1 or HPRT.

Antibodies that can be engineered to produce chimeric receptor extracellular domains are well known in the art and commercially available. Examples of commercially available antibodies include 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-CD30 [SP266] includes (ab199432) (Abcam), anti-CD123 (ab53698) (Abcam), anti-CD133 (BioLegend), anti-CD123 (1A3H4) ab181789 (Abcam), and anti-CD171 (L1.1) (Invitrogen antibodies) but not limited to these. Techniques for generating antibody fragments, such as ScFv, from known antibodies are routine in the art. Furthermore, it is also routine 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 comprise 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, IgNAR, hcIgG, VhH antibodies, nanobodies. , and alpha bodies. In further embodiments, the extracellular domain may comprise any amino acid sequence that allows specific binding of a ligand, including but not limited to dimerization domains, receptors, binding pockets, and the like.

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

The chimeric receptors described herein include one or more of SEQ ID NOs: 1, 3, 4, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and 25-34; fragments of any of them and/or at least one of SEQ ID NOS: 1, 3, 4, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and 25-34 Polypeptides having at least 90% sequence identity, or fragments thereof, may be included. 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 with at least one of SEQ ID NOs:35-54.

Embodiments include nucleic acid sequences that include nucleic acid sequences that encode the chimeric receptors 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 thereof, and/or Nucleic acids having at least 90% sequence identity with at least one of SEQ ID NOs: 2, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24, or fragments thereof, may be included. Further examples include nucleic acids encoding one or more of SEQ ID NOS:24-54, and fragments of any thereof.

In embodiments, the chimeric receptor may be glycosylated, pegylated, and/or otherwise post-translationally modified. Additionally, the nucleic acid sequence can be part of a vector. By way of example, vectors can be plasmids, phages, cosmids, artificial chromosomes, viral vectors, AAV vectors, adenoviral vectors, or lentiviral vectors. In certain embodiments, a nucleic acid encoding a chimeric receptor can be operably linked to promoters and/or other regulatory sequences (e.g., enhancers, silencers, insulators, locus control regions, cis-acting regions, etc.). .

Further embodiments include a cell comprising a chimeric receptor or nucleic acid encoding a chimeric receptor. Non-limiting examples of such cells include myeloid cells, myeloid precursor cells, monocytes, neutrophils, basophils, eosinophils, megakaryocytes, T cells, B cells, natural killer cells, leukocytes, Included are lymphocytes, dendritic cells, and macrophages.

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

In accordance with this disclosure, nucleotide, polynucleotide, or nucleic acid sequences are understood to mean both double- or single-stranded DNA or RNA in monomeric and dimeric form, and transcripts of DNA or RNA. will be done.

Aspects of the present disclosure begin with separation methods such as, for example, ion exchange chromatography, and isolate, purify, and isolate by preparative techniques based on molecular size exclusion, affinity, or alternatively solubility in different solvents. Or it relates to a nucleotide sequence that has been allowed to be partially purified or initiated from genetic engineering methods such as amplification, cloning, subcloning, etc., and that the sequence can be carried by a vector.

A nucleotide sequence fragment may be understood as denoting any nucleotide fragment, including, as non-limiting examples, at least 8, 12, 20, 25, 50, 75, 100, 200, 300, of the sequence from which it originates. Lengths of 400, 500, 1000 or more contiguous nucleotides can be included.

A specific fragment of a nucleotide sequence will be understood to refer to any nucleotide fragment that has at least one less nucleotide or base after alignment and comparison with the corresponding wild-type sequence.

A homologous nucleotide sequence, as used herein, refers to a nucleotide sequence that has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, Percent identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, or 99.7% and this proportion is purely statistical, allowing the differences between the two nucleotide sequences to be distributed randomly and over their entire length.

A specific homologous nucleotide sequence in the context of the present disclosure is understood as meaning a homologous sequence having at least one sequence of the specific fragment as defined above. A "specific" homologous sequence can include, for example, a sequence corresponding to a sequence of a fragment thereof representing a genomic sequence or a variant of a genomic sequence. Thus, these particular homologous sequences can accommodate changes associated with mutations in the sequences, and in particular can accommodate truncations, substitutions, deletions and/or additions of at least one nucleotide. Similarly, homologous sequences can correspond to changes associated with the degeneracy of the genetic code.

The term "degree or percentage of sequence homology" refers to the "degree or percentage of sequence identity between two sequences after optimal alignment" as defined in this application.

As described below, two nucleotide sequences are said to be "identical" if the sequences of amino acids or nucleotide residues in the two sequences are aligned for maximum correspondence. Typically, sequence comparison between two (or more) peptides or polynucleotides involves comparing the sequences of the two sequences that are optimally aligned over a segment or "comparison window" to localize sequence similarity. It is performed by identifying and comparing regions. Optimal alignment of sequences for comparison is determined by the local homology algorithm of Smith and Waterman, Ad. App. Math 2:482 (1981), the homology alignment algorithm of Neddleman and Wunsch, J. Am. Mol. Biol. 48:443 (1970), Pearson and Lipman's Search for Similar Methods, Proc. Natl. Acad. Sci. (U.S.A.) 85:2444 (1988), Electronic implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr. ., Madison, Wis.), or by visual inspection.

A "percentage of sequence identity" (or degree of identity) is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the peptide or polynucleotide sequence in the comparison window is may contain additions or deletions (ie, gaps) relative to the reference sequence (not containing additions or deletions) for optimal alignment of . The percentage is obtained by determining the number of positions where the same amino acid residue or nucleobase occurs in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions within the comparison window, Multiply the result by 100 to determine percent sequence identity.

The above definition of sequence identity is the definition used by those skilled in the art. The definition itself does not require the aid of any algorithm, and algorithms serve only to achieve optimal alignment of sequences, not to calculate sequence identity.

From the above definitions it follows that there is only one well-defined value for sequence identity between two compared sequences, corresponding to the value obtained in the best or optimal alignment.

In BLAST N or BLAST P "BLAST2 Sequences", www.worldwideweb.com. ncbi. nlm. nih. gov/golf/bl2. html website and is commonly used by the inventors and generally by those skilled in the art to compare and determine the identity between two sequences, depending on the length of the sequences being compared. The gap cost to do is chosen directly by the software (ie 11.2 for a permutation matrix BLOSUM-62 of length >85).

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

Hybridization under conditions of stringency with nucleotide sequences as used herein involves temperature and temperature selected in such a way as to allow maintenance of hybridization between two pieces of complementary DNA. It is understood to mean hybridization under conditions of ionic strength.

By way of illustration, the highly stringent conditions of the hybridization step aimed at defining the nucleotide fragments described above are advantageous in the following.

Hybridization is performed at a favored temperature of 65° C. in the presence of 1×SSC corresponding to SSC buffer, 0.15 M NaCl and 0.05 M Na citrate. Washing steps are, for example, 2×SSC, ambient temperature, followed by two washes in 2×SSC, 0.5% SDS at 65° C.; 2×0.5×SSC, 0.5% SDS; can be minutes.

For example, medium stringency conditions using a temperature of 42° C. in the presence of 2×SSC buffer, or low stringency conditions of a temperature of 37° C. in the presence of 2×SSC buffer, respectively An overall low degree of significant complementarity is required for hybridization between sequences.

The stringent hybridization conditions described above for polynucleotides having a size of approximately 350 bases are described in Sambrook et al. , 1989, larger or smaller size oligonucleotides will be adapted by those skilled in the art.

Some of the nucleotide sequences described herein can be used as primers or probes in methods that allow obtaining homologous sequences, such as polymerase chain reaction (PCR), nucleic acid cloning, and These methods, such as sequencing, are well known to those skilled in the art.

Within the nucleotide sequence are primers or probes in a method that allows to determine the presence of a particular nucleic acid, one of its fragments, or one of its variants as defined below. There is something that can be used as In embodiments, the nucleotide sequences are fragments of SEQ ID NOs: 2, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 that encode transmembrane domains, cytosolic domains, or portions thereof. can contain. Additional fragments can include nucleotide sequences encoding linkers, hinges, or fragments thereof, such as nucleotides encoding one or more of SEQ ID NOs:26-34. Additional fragments may include fragments of the nucleotide sequences encoding one or more of SEQ ID NOs:35-54.

Fragments of nucleotide sequences can be obtained, for example, by specific amplification, such as PCR, or after digestion of the nucleotide sequences with appropriate restriction enzymes, certain of these methods being described in Sambrook et al. , 1989. Further, such fragments may be obtained using standard gene synthesis techniques available from commercial 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 may be modified relative to the wild-type sequence obtained by mutagenesis according to techniques well known to those skilled in the art, such as, among other things, the regulation of the expression rate of the polypeptide or the regulation of the replication cycle of the polypeptide expression. It will be understood to mean any nucleotide sequence that contains mutations in the regulatory and/or promoter sequences.

A modified nucleotide sequence is likewise understood to mean any nucleotide sequence that encodes a modified polypeptide as defined below.

Nucleotide sequences encoding chimeric receptors are disclosed, the nucleotide sequences from SEQ ID NOs: 2, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24, or one of fragments thereof. Contains a selected nucleotide sequence. Such fragments may encode specific domains such as the transmembrane domain, or the cytosolic domain, or portions thereof. Nucleotide sequences encoding additional chimeric receptors can include nucleotide sequences encoding linkers, hinges, or fragments thereof, such as nucleotides encoding one or more of SEQ ID NOs:26-34. A nucleotide sequence encoding a chimeric receptor can further be a nucleotide sequence encoding one or more of SEQ ID NOs:35-54, or fragments thereof.

Similarly, embodiments provide: or one of its fragments, b) the nucleotide sequence of a particular fragment of the sequence as defined in a), c) as defined in a) or b) d) a homologous nucleotide sequence having at least 80% identity to the sequence in question, d) a complementary nucleotide sequence or sequence of RNA corresponding to a sequence as defined in a), b) or c), and e) a) A nucleotide sequence, characterized in that it comprises a nucleotide sequence selected from: a nucleotide sequence modified by a sequence as defined in b), c), or d).

Among the nucleotide sequences encoding the nucleotide sequences of SEQ ID NOs: 2, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24, and at least one of SEQ ID NOs: 25-54 or fragments thereof, and at least 80%, 81%, 82% with at least one of the sequences of SEQ ID NOs: 2, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% %, 99.5%, 99.6%, or 99.7% identity homology and any nucleotide sequence encoding at least one of SEQ ID NOS: 25-54, or there is a fragment. A homologous sequence can include, for example, a sequence corresponding to a wild-type sequence. Likewise, these particular homologous sequences can correspond to changes associated with mutations in the wild-type sequence, particularly truncations, substitutions, deletions and/or additions of at least one nucleotide. can. As will be apparent to those skilled in the art, such homologues are readily created and identified using standard techniques and publicly available computer programs such as BLAST. Accordingly, each homologue referenced above should be evaluated as disclosed and fully described herein.

Embodiments include chimeric receptors encoded by the nucleotide sequences described herein, or fragments thereof where the sequences are represented by fragments. Amino acid sequences corresponding to polypeptides can be encoded according to one of three readable frames of at least one of the sequences of SEQ ID NOs:35-54.

Similarly, embodiments include at least one of the amino acid sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and 25-54, or fragments thereof. Chimeric receptors characterized in that they comprise a polypeptide selected from one of

Among the polypeptides, according to embodiments, are polypeptides of amino acid sequences among SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and 25-54, or fragment or at least 80%, 81%, 82% with at least one of the sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and 25-54 , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% There are other polypeptides, or fragments thereof, with 99.5%, 99.6%, or 99.7% identity homology. As will be apparent to those skilled in the art, such homologues are readily created and identified using standard techniques and publicly available computer programs such as BLAST. Accordingly, each homologue referenced above should be evaluated as disclosed and fully described herein.

Embodiments also include a) a specific fragment 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 polypeptide defined in a) or b). and d) a polypeptide selected from a polypeptide modified by a polypeptide as defined in a), b) or c). It relates to a polypeptide characterized by

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

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

In embodiments, any one of the chimeric receptors may be enzymatically or functionally active, and when the extracellular domain is bound by a ligand, a signal is transduced to polarize the macrophage.

As used herein, "polarized macrophages" are macrophages that correlate with the M1 or M2 macrophage phenotype. M1 polarized macrophages secrete IL-12 and IL-23. Determination of M1-polarized macrophages is by measuring IL-12 and/or IL-23 expression using standard cytokine assays and comparing that expression to that by newly differentiated, non-polarized macrophages. can be performed by Alternatively, this determination can be performed 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 IL-10 expression using standard cytokine assays and comparing that expression to that by newly differentiated, non-polarized macrophages. Alternatively, this determination can be performed by determining whether the cells are CD14+, CD80-, CD206+, and CDCD163+.

Aspects of the present disclosure relate to chimeric receptors that are either genetically engineered or chemically synthesized, and thus may contain unnatural amino acids, as explained below.

According to an embodiment a "polypeptide fragment" is understood to denote a polypeptide comprising at least 5 contiguous amino acids, preferably 10 contiguous amino acids or 15 contiguous amino acids.

A specified polypeptide fragment is understood herein to refer to a contiguous polypeptide fragment encoded by a specified fragment of a nucleotide sequence.

A "homologous polypeptide" is intended to indicate a polypeptide having specific modifications with respect to the native polypeptide, such as deletion, addition or substitution of at least one amino acid, truncation, extension, chimeric fusion and/or mutation. will be understood. Among homologous polypeptides, it is preferred that the amino acid sequence has at least 80% or 90% homology with the amino acid sequence of the polypeptides described herein.

A "specific homologous polypeptide" refers to a homologous polypeptide as defined above and will be understood as having specific fragments of the polypeptides described herein.

In the case of substitutions, one or more consecutive or non-consecutive amino acids are replaced with "equivalent" amino acids. As used herein, the term "equivalent" amino acid refers to any amino acid that can be substituted for one of the basic structural amino acids without essentially altering the biological activity of the corresponding peptide. For purposes of illustration, it is defined as follows: As will be apparent to those skilled in the art, such substitutions are readily created and identified using standard molecular biology techniques and publicly available computer programs such as BLAST. Accordingly, each permutation referred to above should be evaluated as disclosed and fully described herein.

These equivalent amino acids can be determined according to their structural homology with the amino acids they replace or according to the results of comparative tests of biological activity between different polypeptides that are feasible.

As non-limiting examples, mention is made of possible substitutions that can be made without causing extensive modification of the biological activity of the corresponding modified polypeptides, e.g. replacement of leucine by valine or isoleucine, Under the same conditions of natural reverse substitution, exchange of aspartic acid, exchange of glutamine by asparagine, exchange of arginine by lysine, etc. are envisioned.

In a further embodiment, substitutions are limited to substitutions at amino acids that are not conserved among other proteins with similarly identified enzymatic activity. 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 generate such alignments is available at wordwideweb.com in conjunction with the database provided by NCBI. charite. de/bioinf/strap/.

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

Likewise, certain homologous polypeptides correspond to polypeptides encoded by certain homologous nucleotide sequences as defined above, thus in this definition, in particular truncation, substitution of at least one amino acid residue , deletions, and/or additions that may be present in the wild-type sequence corresponding to mutant or variant forms and corresponding polypeptides.

As used herein, a "particular biologically active fragment of a polypeptide", as defined above, has at least one of the characteristics of a polypeptide described herein, in particular It will be understood as referring to specific polypeptide fragments. Peptides in certain embodiments are capable of behaving as chimeric antigen receptors that polarize macrophages when activated.

As used herein, a "modified polypeptide" of a polypeptide is a polypeptide obtained by genetic recombination or chemical synthesis, as will be described below, having at least one modification with respect to the wild-type sequence. is understood to indicate Such modifications may be responsible for the amino acids that are responsible for the specificity and/or activity of the polypeptides described herein, or for the structural compatibility, localization, and membrane insertion capabilities, or I can't. Thus, it will be possible to generate polypeptides of equal, increased or decreased activity and equal, more restricted or broader specificity. Among the modified polypeptides, reference should be made to polypeptides in which up to 5 or more amino acids have been modified and can be truncated at the N- or C-terminus, or even deleted or added.

Methods that allow demonstration of eukaryotic or prokaryotic regulation are well known to those skilled in the art. It will be appreciated that it would also be possible to use the nucleotide sequences encoding the modified polypeptides for regulation, eg, via a vector as described below.

Such modified polypeptides may be produced by systematically altering portions of the polypeptides to, for example, select compounds that are most active or have desired properties, prior to testing them in models, cell cultures or microorganisms. It can be obtained by using combinatorial chemistry, which is selectable.

Similarly, chemical synthesis has the advantage of being able to use non-natural amino acids or non-peptide bonds.

Thus, it may be important to use, for example, D-forms of unnatural amino acids, or, for example, amino acid analogues, especially sulfur-containing forms, to improve the longevity of polypeptides.

Finally, the structure of a polypeptide, specific or modified homologous forms thereof, could be incorporated into polypeptide-type or other chemical structures. Therefore, it may be important to provide N- and C-terminal compounds that are not recognized by proteases.

Nucleotide sequences encoding the polypeptides are also disclosed herein.

Similarly, embodiments relate to nucleotide sequences usable as primers or probes, characterized in that the sequences are selected from the nucleotide sequences described herein.

Similarly, various embodiments are directed to nucleotides that can be obtained by purification, genetic recombination, or chemical synthesis from naturally occurring polypeptides by procedures well known to those of skill in the art, particularly as described below. It is well understood that this relates to specific polypeptides containing chimeric receptors encoded by the sequences. Similarly, labeled or unlabeled mono- or polyclonal antibodies to the specific polypeptides encoded by the nucleotide sequences are also encompassed by this disclosure.

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

Thus, according to embodiments the nucleotide sequences can be used to amplify nucleotide sequences, in particular by means of PCR technology (polymerase chain reaction) (Erlich, 1989, Innis et al., 1990, Rolfs et al., 1991, and White et al., 1997).

These oligodeoxyribonucleotide or oligoribonucleotide primers advantageously have a length of at least 8 nucleotides, preferably at least 12 nucleotides, even more preferably at least 20 nucleotides.

Other amplification techniques for target nucleic acids can be used to advantage as an alternative to PCR.

The nucleotide sequences, particularly primers, described herein may be used in other procedures for amplification of target nucleic acids, such as the TAS technique (transcription-based amplification system) described by Kwoh et al., 1989, Guatelli et al., 1990. 3SR technology (Self-Sustained Sequence Replication) described by Kievitis et al. in 1991, NASBA technology (nucleic acid sequence-based amplification), SDA technology (strand displacement amplification) (Walker et al. , 1992), which can also be utilized in TMA technology (transcription-mediated amplification).

Polynucleotides containing chimeric receptors were described by Landegren et al., 1988, utilizing a thermostable ligase, LCR technology (ligase chain reaction) improved by Barany et al., 1991, Segev, 1992. RCR technique (repair chain reaction), CPR technique (cycling probe reaction) described by Duck et al. 1990, Miele et al. It can further be utilized in techniques for amplification or modification of nucleic acids that serve as probes, such as the Q-beta replicase-based amplification technique improved by Burg et al. and Stone et al., 1996.

before utilizing an amplification reaction with the aid of at least one primer, or before utilizing a detection procedure with the aid of at least one probe, where the target polynucleotide to be detected is likely RNA, e.g., mRNA. , it would be possible to use the reverse transcriptase class of enzymes to obtain cDNA from RNA contained within a biological sample. The resulting cDNA will thus serve as a target for primer(s) or probe(s) utilized in amplification or detection procedures.

Detection probes are selected in such a manner as to hybridize with the target sequence or amplicons generated from the target sequence. As a sequence such probes advantageously have a sequence of at least 12 nucleotides, in particular of at least 20 nucleotides and preferably of at least 100 nucleotides.

Embodiments further include nucleotide sequences usable as probes or primers, characterized in that they are labeled with radioactive or non-radioactive compounds.

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

Examples of non-radioactive labeling of nucleotide sequences are described, for example, in French Patent No. 78,10975, or by Urdea et al. in 1988 or by Sanchez Pescador et al.

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 involves immobilizing a nucleic acid extract of cells on a support (nitrocellulose, nylon, polystyrene, etc.) and incubating the immobilized target nucleic acid with a probe under well-defined conditions. consists of After hybridization, excess probe is removed and the hybrid molecules formed are detected by a suitable method (measurement of radioactivity, fluorescence or enzymatic activity linked to the probe).

Similarly, various embodiments include the nucleotide sequences or polypeptide sequences described herein characterized by being immobilized on a support either covalently or non-covalently.

According to another advantageous mode of employing nucleotide sequences, the latter can be used immobilized on a support and thus serve by specific hybridization to capture target nucleic acids obtained from the biological sample to be tested. be able to. Optionally, the solid support is separated from the sample and the hybridization complex formed between the capture probe and the target nucleic acid is then isolated from a second probe labeled with a readily detectable element, the so-called Detected using a detection probe.

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

Vectors are also provided, characterized in that they contain elements that allow the integration, expression and/or secretion of the nucleotide sequences in the determined host cell.

The vector may then include a promoter, translation initiation and termination signals, and appropriate regions for the regulation of transcription. It can be stably maintained in a host cell, and can optionally have specific signals specifying secretion of the translated protein. These different elements can be selected as a function of the host cell used. To this end, the nucleotide sequences described herein can be inserted into an autonomously replicating vector within the chosen host or into an integrative vector of the chosen host.

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

Vectors are, for example, according to vectors 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, baculoviral vectors, adenoviral vectors, lentiviral vectors, retroviral vectors, Chimeric viral vectors, and chimeric adenoviruses such as AD5/F35.

These vectors are useful for transforming host cells to clone or express the nucleotide sequences described herein.

Similarly, embodiments include host cells transformed by the vectors.

These cells are prepared by introducing a nucleotide sequence inserted into a vector as defined above into a host cell and then culturing the cell under conditions that allow replication and/or expression of the transduced nucleotide sequence. can be obtained by

Host cells can be prokaryotic or eukaryotic systems, such as bacterial cells (Olins and Lee, 1993), but also yeast cells (Buckholz, 1993), and plant cells such as Arabidopsis spp., and animal cells, especially mammals. Cell cultures (Edwards and Aruffo, 1993) such as HEK293 cells, HEK 293T cells, Chinese Hamster Ovary (CHO) cells, myeloid lineage cells, myeloid progenitor cells, monocytes, neutrophils, basophils, eosinophils, megakaryocytes, T cells, B cells, natural killer cells, leukocytes, lymphocytes, dendritic cells, and macrophages, but also insect cells amenable to baculovirus-based procedures; For example, it can be selected from sf9 insect cells (Luckow, 1993).

Likewise, embodiments relate to organisms containing one of the transformed cells.

Obtaining transgenic organisms expressing one or more nucleic acids or parts of nucleic acids, for example in rats, mice or rabbits, according to methods well known to those skilled in the art, such as viral or non-viral gene transfer. can be performed. It may be possible to obtain transgenic organisms expressing more than one gene under the control of a ubiquitous strong promoter or by transgenesis of multiple copies of genes selective for one tissue. Similarly, it is possible to obtain transgenic organisms by homologous recombination in embryonic cell lines, introduction of these cell lines into embryos, selection of affected chimeras at the germline level, and growth of chimeras. be.

Transformed cells and transgenic organisms are available in procedures for the preparation of recombinant polypeptides.

Genetic engineering techniques, using cells transformed with expression vectors, or using transgenic organisms, now make it possible to produce relatively large amounts of recombinant polypeptides.

Recombinant chimeric receptors, etc., characterized by using a vector and/or a cell transformed with the vector and/or a transgenic organism containing one of the transformed cells. Procedures for preparing the polypeptides are themselves included in this disclosure.

As used herein, "transformation" and "transformed" refer to the introduction of nucleic acid into a cell, whether prokaryotic or eukaryotic. Furthermore, "transformed" and "transformed" as used herein need not relate to growth regulation or growth regulation.

Among the procedures for the preparation of polypeptides such as recombinant chimeric receptors, the preparation procedures include vectors and/or cells transformed with vectors and/or those encoding chimeric receptors. A transgenic organism containing one of the transformed cells containing the nucleotide sequence of is utilized.

A variant as used herein may consist of producing a recombinant polypeptide fused to a "carrier" protein (a chimeric protein). Advantages of this system include stabilization and/or reduction of proteolytic degradation of the recombinant product, increased solubility during the process of renaturation in vitro, and/or ease of purification when the fusion partner has an affinity for a particular ligand. It is possible to make simplification possible.

More particularly, embodiments provide for a) culturing of transformed cells under conditions that allow expression of the recombinant polypeptide of the nucleotide sequence, and b) recovery of the recombinant polypeptide, if necessary. It relates to procedures for the preparation of polypeptides, including steps.

When utilizing transgenic organisms in procedures for the preparation of polypeptides such as chimeric receptors, the recombinant polypeptide can be extracted from the organism or left intact.

Embodiments also relate to polypeptides obtainable by a procedure as described above.

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

Similarly, the present disclosure relates to synthetic polypeptides, such as chimeric receptors, obtained by procedures.

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

For example, one can resort to the technique of synthesis in homogeneous solution described by Houben-Weyl in 1974.

This method of synthesis consists of successively condensing consecutive amino acids two by two in the required order, or condensing previously formed amino acids and fragments already containing some amino acids in the preferred order, or alternatively , consisting of several fragments, previously prepared in this way, according to methods well known in the synthesis of peptides, especially after activation of the carboxyl function, usually have to be involved in the formation of peptide bonds. It is understood that all reactive functional groups carried by these amino acids or fragments must be pre-protected, except , an amine function on one side and a carboxyl group on the other, or vice versa. .

Additionally, one may rely on the technique described by Merrifield.

To prepare peptide chains according to the Merrifield procedure, one can resort to a highly porous polymeric resin on which the first C-terminal amino acid of the chain is immobilized. The amino acid is immobilized on the resin through its carboxyl group and its amine function is protected. Therefore, the amino acids that are to form a peptide chain are immobilized one after another on the amino groups attached to the resin, with the already formed peptide chain portion being deprotected in advance each time. Once the entire desired peptide chain is formed, the protecting groups of the different amino acids forming the peptide chain are removed and the peptide is cleaved from the resin with the aid of acid.

These hybrid molecules are in part possibly immunogenic moieties, in particular diphtheria toxin, tetanus toxin, surface antigen of hepatitis B virus (Patent FR 79 21811), VP1 antigen of poliovirus, or any other virus or It may be formed from a polypeptide carrier molecule or fragment thereof associated with an epitope of a bacterial toxin or antigen.

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

In embodiments, a nucleic acid sequence encoding a chimeric receptor is provided to the cell. The cells can then express the encoded chimeric receptor. The expressed chimeric receptor can be on the surface of the cell or within the cytoplasm. In certain embodiments, the chimeric receptor-expressing cell is a macrophage. The macrophage-expressed chimeric receptor can bind the ligand, and binding with the ligand can activate the chimeric receptor to induce polarization of the macrophage as described above.

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

In other embodiments, chimeric receptors can be introduced directly into cells. Any method of introducing proteins into cells can be used including, but not limited to, microinjection, electroporation, membrane fusion, and the use of protein transduction domains. After the cell has been provided with the chimeric receptor, the cell can be returned to the subject from which it was obtained, eg, by injection or infusion. In other embodiments, the chimeric receptor provided cells are provided by a donor. After the nucleic acid has been provided to the donor cell, the cell can then be provided to an individual other than the donor. Examples of donor cells include, but are not limited to, primary cells from a subject and cells from cell lines.

Likewise, embodiments relate to polypeptides, such as chimeric receptors, labeled with the aid of a suitable label, such as a fluorescent or radioactive enzyme.

The polypeptide allows monoclonal or polyclonal antibodies to be prepared which are characterized by specific recognition of the polypeptide. It may be advantageously possible to prepare monoclonal antibodies from hybridomas according to the technique described by Kohler and Milstein in 1975. For example, an animal, particularly a mouse, is immunized with a polypeptide or DNA associated with an adjuvant of the immune response, and then in the serum of the immunized animal on an affinity column pre-immobilized with a polypeptide serving as an antigen. It may be possible to prepare polyclonal antibodies by purifying the specific antibodies contained in . Polyclonal antibodies are prepared by purification of antibodies contained within the serum of animals immunologically challenged with the chimeric receptor, or a polypeptide or fragment thereof, on an affinity column to which the polypeptide has been pre-immobilized. can do more.

Furthermore, antibodies can be used to generate IgA, IgD, IgE, IgG, IgM, Fab fragments, F(ab') ₂ fragments, monovalent antibodies, scFv fragments, scRv-Fc fragments, IgNAR, hcIgG, VhH antibodies, nanobodies, Other forms of binding molecules can be prepared including, but not limited to, alphabodies and alphabodies.

Similarly, embodiments include mono- or polyclonal antibodies or fragments thereof, or chimeric antibodies, characterized in that they are capable of specifically recognizing the polypeptides described herein, or the ligands of the polypeptides and/or chimeric receptors. or related to fragments thereof.

Similarly, antibodies could be labeled in the same manner as described above for nucleic acid probes, such as fluorescent or radioactive enzymatic labeling. It would also be possible to include such antibodies and/or fragments thereof as part of a chimeric receptor. As a non-limiting example, such antibodies or fragments thereof may constitute part of the extracellular domain of a chimeric receptor.

Embodiments include: a) mono- or polyclonal contact of the sample with the sample (under conditions that allow an immunological reaction between the antibody and chimeric receptors that may be present in the biological sample); A further object is a procedure for the detection and/or identification of chimeric receptors in a sample, characterized in that it comprises the step of demonstrating potential antigen-antibody complexes.

Embodiments are described in further detail in the following illustrative examples. While the examples may represent only selected embodiments, it is to be understood that the following examples are illustrative, not limiting.

EXAMPLES Example 1 Isolation of ScFv Fragments Against Specific Ligands cDNA was purified from monoclonal antibody hybridoma cells (CB1), which express antibodies specific to human TK1. The isolated cDNA was used to amplify the heavy and light chains of the CB1 variable region via polymerase chain reaction (PCR). Sequences from the heavy and light chains were confirmed using NCBI Blast. The CB1 heavy and light chains were fused together via site overlap extension (SOE) PCR to form single chain fragment variable (scFv) using G4S linkers. 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 cut with restriction enzymes and inserted into the pMP71 CAR vector.

TK-1 and HPRT-specific human scFv fragments were isolated from a yeast antibody library. 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 enriched using a magnetic column. 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 immunolabeled with fluorescently labeled anti-HA or anti-c-myc antibodies to isolate TK-1 and HPRT specific antibodies. were sorted by fluorescence-activated cell sorting (FACS) by alternating labeling with . High affinity clones were selected for chimeric receptor construction. Other human antibodies or humanized antibodies from other animals can be selected or modified specifically for TK-1 or HPRT by using phage display or other recombinant methods.

Selected scFv clones were then combined with human IgG1 constant domains to generate antibodies for use in applications such as Western blots or ELISAs to confirm binding specificity of the scFv. The antibody construct was inserted into the pPNL9 yeast secretion vector and YVH10 yeast was transformed with the construct and induced to produce antibody. Antibodies can also be secreted using other expression systems such as E. coli or mammalian systems.

Isolation and characterization of protein-specific antibody fragments.
Referring to Figure 26, 105 yeast were incubated with 2.5ug of protein of interest labeled with the fluorescent tag APC. The taller peak on the left (red) represents the yeast population not bound to the protein of interest (negative control). The lower left peak on the left (blue) shows yeast that do not express that surface protein, while the high peak on the right (blue) shows binding of the expressed antibody fragment to the protein of interest.

Structural identities between antibodies are shown in the antigen 5 binding site, PLoS ComputBiol. 8(2): e1002388. doi: 10.1371/journal. pcbi. 1002388, Kunik V, Ashkenazi S, Ofran Y (2012). Paratope: An online tool for systematically identifying antigen-binding regions in antibodies based on sequence or structure is published in Nucleic Acids Res. 2012 Jul;40 (Web Server issue): W521-4. doi: 10.1093/nar/gks480. Epub 2012 Jun 6.

Example 2: Construction of Chimeric Receptors Construction of Chimeric Receptor Vectors:
The first step in the process is the design of the nucleotide sequence of the synthetic chimeric receptor gene and the selection of a suitable lentiviral vector. All vector designs are performed with advanced software version 9.1.6. Sequences are also obtained from the Uniprot and Human Protein Reference Databases and NCBI.

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

Single chain variable fragment sequences are generated using humanized antibody yeast display libraries or phage display libraries. TK1, HPRT, ROR1, MUC-16, EGFRvIII, mesothelin, HER2, CEA, BCMA, GPC3, FAP, EphA2, NKG2D ligands, GD2, CD19, CD20, CD30, CD33, CD123, CD133, CD138, and CD171, respectively Nucleic acid encoding a ScFv specific for. all possible combinations of nucleic acids encoding chimeric receptors having at least one of each of a), b), c), d) and e), wherein a), b), c ), d), and e) are
a) to TK1, HPRT, ROR1, MUC-16, EGFRvIII, mesothelin, HER2, CEA, BCMA, GPC3, FAP, EphA2, NKG2D ligands, GD2, CD19, CD20, CD30, CD33, CD123, CD133, CD138, and CD171 ScFv specific for
b) GS linker or no GS linker,
c) selected from LRR 5 amino acid short hinge, LRR long hinge, IgG4 short hinge, IgG 119 amino acid mediated hinge and IgG4 long hinge, CD8 hinge, CD8 hinge with cysteine converted to serine hinge region as well as no hinge,
d) a transmembrane domain selected from the transmembrane domains of MYD88, TLR3, TLR4, TLR7, TLR8, TLR9, MAL, IRAK1, FCGR2A, FCGR3A and FCER1G e) Myd88, TLR3, TLR4, TLR7, TLR8, TLR9, MAL , IRAK1, FCGR2A, FCGR3A, and FCER1G.

Nucleic acids encoding the aforementioned chimeric receptors are synthesized by a combination of recombinant DNA techniques and gene synthesis.

Macrophages are genetically modified by the method of integrated gene delivery via lentiviral-mediated gene transfer to provide nucleic acids encoding chimeric receptors. The third generation lentiviral system from Addgene is used to package the lentiviral vectors. pHIV-dTomato (#21374) and pUltra-chilli (#48687) are gene transfer plasmids. pCMV-VSV-G (#8454), pMDLg/pRRE (#12251), pRSV-Rev (#12253), pHCMV-AmphoEnv (#15799) are packaging plasmids. Lentiviral-mediated gene transfer of human lymphocytes has previously been standardized to obtain transduction efficiencies of up to 50%. HEK293T cells are transfected using the calcium phosphate method (SIGMA CAPHOS). Approximately 10 μg of each packaging plasmid and 20 ug of chimeric receptor-encoding vector are used per transfection. After 48-36 hours, virus particles are harvested and sterile filtered. A titer of virus is determined to infect HT1080 and U937 cells.

Analysis is performed by flow cytometry, which detects red fluorescent protein. Human monocytes after viral titration are transduced using RetroNectin plates (Clonetech, T100B) and the spin infection method.

Prior to lentiviral transduction, monocytes are isolated from whole PBMNCs by negative selection and magnetic sorting using the Monocyte Isolation Kit II, human (MACS 130-091-153). After monocyte isolation, the cells are split into two nunclon 6-well plates (Thermo, 145380) and each well is seeded with 1.5×10 6 cells for each vector. One plate is transduced immediately, while the second plate is used for ex vivo differentiation of monocytes to M1 macrophages. M1 macrophages are generated using M1-Macrophage Generation Medium DFX (Promocell, C-28055) medium. Macrophages are transduced after 7 days and activated on day 9 with LPS (500X) (Affimetryx, 00-4976-03) and IFNγ (Promokine, C-60724). Transduction efficiency is analyzed by flow cytometry. Transduced cells are separated by cell sorting using a FACS Aria cell sorter. After cell sorting, transformed monocytes can be cultured in vitro for several days prior to differentiation, while differentiated macrophages can persist for a month.

Example 3 Polarization of Macrophages Via Chimeric Receptors Separately exposed to EphA2, NKG2D conjugated ligands, GD2, CD19, CD20, CD30, CD33, CD123, CD133, CD138, and CD171, monitoring IL-12 and IL-23 secretion using standard cytokine assays or tested for polarization to the M1 phenotype by measuring RNA production. Chimeric receptor-bearing macrophages develop an M1 phenotype as determined by exposure to ligands specific for a particular chimeric receptor and increased secretion of IL-12 and/or IL-23. polarized. Ligands other than the specific ligand for the particular chimeric receptor do not show an increase in IL-12 and/or IL-21.

Example 4: Production and transduction of monocyte-derived macrophages Monocyte-derived macrophages after 7 days of differentiation had undergone phenotypic changes. These changes were compared between transduced and non-transduced cells. As can be observed in Figure 27, the transduced cells have a more active phenotype similar to M1 or classically activated macrophages. FIG. 27 shows images of non-transduced and transduced monocyte-derived macrophages at day 8 of differentiation. Interferon gamma and LPS were not added at this time. It can be observed that the phenotype of macrophages transduced with chimeric receptors is different from non-transduced macrophages. Transduced cells displayed a classically activated or M1-like phenotype indicative of macrophage activation. The altered phenotype may be the combined effect of the transduction process and the expression of new synthetic receptors.

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

Example 5: Transduction Efficiency Transduction efficiency was assessed after 10 days of differentiation and macrophages expressing chimeric receptors were cell sorted. Lentiviral transduction is difficult in macrophages. However, nearly 30% macrophage transduction was achieved using an HIV-1 based system with an EF1-α promoter. Transduction of cells at early stages of macrophage differentiation displayed different transduction efficiencies. Monocytes or macrophages at early stages of differentiation are easier to transduce. Adenoviral transduction using the chimeric adenovirus AD5/F35 has emerged as another option for macrophage transduction. Figure 29 shows the results of cell sorting of transduced macrophages using the FACSAria system. Approximately 30% of macrophage transduction was achieved using the lentiviral approach. The leftmost plot shows a control in which only 0.58% of cells show fluorescence that would indicate expression of dTomato. The right two plots show a transduction efficiency of 27.1 percent after transduction.

Example 6 Immunophenotyping of Transduced Macrophages Immunophenotyping of macrophages transduced with vectors for expression of chimeric receptors was performed to determine the activation state of the transduced cells. . 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). Constant activation of TLR-4 signaling can lead to macrophage activation or the M1 phenotype. It is not known whether constructs used based on TLR-4 can trigger constant activation of signaling through TIR domains derived from TLR-4. However, after the transduction process, phenotypic changes were observed, with altered expression of cell surface markers within macrophages. This is likely due to the combination of lentiviral transduction and expression of the chimeric receptor protein. Low expression of CD14, CD80, D206 and CD163 was indicative of macrophage polarization towards the M1 phenotype. Expression of these cell surface markers was observed in transduced cells. FIG. 30 presents six scatterplots of fluorescence-activated cell sorting demonstrating dye (Alexa 647) retention and expression of CD80, CD163, CD206, and CD14 in macrophages transduced with chimeric receptors. do.

FIG. 31 shows histograms of relative expression levels of M1 cell surface markers in macrophages transduced with vectors to express chimeric receptors.

Example 7: In vitro toxicity of TK1 targeting chimeric receptor-transduced macrophages against NCI-H460 cells.
The tumor activity of TK1 targeting macrophages transduced with chimeric receptors was tested against NCI-H460-GFP cells. The E:T ratio used was 1:10. Analysis was performed using confocal microscopy. Fluorescence detection was performed every 5 minutes for 12 hours. During the time-lapse, TK1 targeting macrophages transduced with chimeric receptors was observed to migrate toward H460-GFP cells and attack them. Post-synapsis, target-cell-specific cell death is induced. As demonstrated by the images in FIG. 32, macrophages transduced with TK1-targeted chimeric receptors can be detected, attacked, and induce cell death in TK1-expressing lung cancer cell lines. NCI-H460 cells were modified to express GFP. The tumoricidal activity of TK1 targeting macrophages transduced with chimeric receptors was detected using confocal microscopy as loss of fluorescence in target cells.

This disclosure may be further modified within the spirit and scope of this disclosure. This application is therefore intended to cover any variations, uses, or adaptations of the disclosure using its general principles. Further, the present application submits this disclosure to the extent that this disclosure pertains, falls within the scope of the appended claims and their legal equivalents, and falls within the scope of known or customary practice in the art. is intended to cover such deviations from

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Claims (21)

a chimeric receptor,
a cytoplasmic domain;
a transmembrane domain;
an extracellular domain, and
said cytoplasmic domain comprises a cytoplasmic portion of a receptor that polarizes macrophages when activated;
A chimeric receptor wherein the wild-type protein comprising said cytoplasmic portion does not comprise said extracellular domain. 2. The chimeric receptor of claim 1, wherein binding of ligand to said extracellular domain activates said cytoplasmic portion. 2. The chimeric receptor of Claim 1, wherein said cytoplasmic portion is activated, which polarizes macrophages to M1 macrophages. 2. The chimeric receptor of Claim 1, wherein said cytoplasmic portion is activated, which polarizes macrophages into M2 macrophages. The cytoplasmic portion comprises toll-like receptor, myeloid differentiation primary response protein (MYD88), toll-like receptor 3 (TLR3), toll-like receptor 4 (TLR4), toll-like receptor 7 (TLR7), toll-like receptor body 8 (TLR8), toll-like receptor 9 (TLR9), myelin and lymphocyte protein (MAL), interleukin-1 receptor-associated kinase 1 (IRAK1), low-affinity immunoglobulin gamma Fc region receptor III-A (FCGR3A), low-affinity immunoglobulin gamma Fc region receptor II-a (FCGR2A), and a cytoplasmic domain from high-affinity immunoglobulin epsilon receptor subunit gamma (FCER1G). receptor. the ligand is 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), carcinoembryonic antigen (CEA), B cell maturation antigen (BCMA), glypican 3 (GPC3), fibroblast activation protein (FAP), selected from the group consisting of erythropoietin-producing hepatocellular carcinoma A2 (EphA2), natural killer group 2D (NKG2D) ligands, disialoganglioside 2 (GD2), CD19, CD20, CD30, CD33, CD123, CD133, CD138, and CD171 3. The chimeric receptor of claim 2. the extracellular domain is thymidine kinase (TK1), hypoxanthine-guanine phosphoribosyltransferase (HPRT), receptor tyrosine kinase-like orphan receptor 1 (ROR1), mucin-16 (MUC-16), epidermal growth factor receptor body vIII (EGFRvIII), mesothelin, human epidermal growth factor receptor 2 (HER2), carcinoembryonic antigen (CEA), B cell maturation antigen (BCMA), glypican 3 (GPC3), fibroblast activation protein (FAP) ), erythropoietin-producing hepatocellular carcinoma A2 (EphA2), natural killer group 2D (NKG2D) ligands, disialoganglioside 2 (GD2), CD19, CD20, CD30, CD33, CD123, CD133, CD138, and CD171. 2. The chimeric receptor of claim 1, which is an antibody or fragment thereof specific for a ligand to be administered. 8. The chimeric receptor of claim 7, wherein said antibody or fragment thereof is a ScFv fragment. 2. The chimeric receptor of claim 1, wherein said chimeric receptor further comprises a linker between said transmembrane domain and said extracellular domain. 10. The chimeric receptor of claim 9, wherein said linker is a GS linker. 2. The chimeric receptor of claim 1, wherein said chimeric receptor further comprises a hinge region between said transmembrane domain and said extracellular domain. 10. The chimeric receptor of claim 9, wherein said chimeric receptor further comprises a hinge region between said transmembrane domain and said 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 said polynucleotide. A vector comprising the nucleic acid of claim 13 . 16. The vector of claim 15, wherein said vector is a lentiviral vector. A cell comprising the chimeric receptor of claim 1 . 16. The cells of claim 15, wherein said cells are monocytes or macrophages. A cell comprising the nucleic acid of claim 13 . 20. The cells of claim 19, wherein said cells are monocytes or macrophages. A method of polarizing macrophages, comprising:
contacting a macrophage comprising the chimeric receptor of claim 1 with a ligand of said extracellular domain of said chimeric receptor;
binding the ligand to the extracellular domain of the chimeric receptor;
said binding of said ligand to said extracellular domain of said chimeric receptor activates said cytoplasmic portion;
The method, wherein activation of said cytoplasmic portion polarizes said macrophages.

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