KR20180054600A - The macrophage chimeric antigen receptor (MOTO-CAR) - Google Patents

Abstract

Modified macrophage immune cells are provided for the treatment of cancer and other diseases. In particular, the macrophages express the chimeric antigen receptor (CAR). The short chain variable fragment (scFv) may be derived against thymidine kinase 1 (TK1) or hypoxanthine guanine phosphoribosyltransferase (HPRT). The signal transduction domain may be derived from a toll-like receptor (TLR).

Description

The macrophage chimeric antigen receptor (MOTO-CAR)

Cancer refers to a group of diseases involving uncontrolled cell growth and death, genomic instability and mutation, tumor-promoting inflammation, induction of angiogenesis, immune system avoidance, deregulation of the metabolic pathway, immortal cell replication, and metastatic tissue invasion [One]. Cancer is the second leading cause of death in the United States after heart disease [2]. More than 1,600,000 new cases of cancer are estimated to be diagnosed each year, with more than 580,000 Americans expected to die (about 1,600 cancer deaths per day), accounting for almost one out of every four US deaths [2,3].

The immune system plays an important role in the development and progression of cancer. Mononuclear cells that differentiate into macrophages develop different responses according to various stimuli and exhibit different functions depending on the microenvironment surrounding them. Macrophages may be pro-inflammatory (M1) or anti-inflammatory (M2). Surveys have shown that macrophage infiltration into the tumor site can account for more than 50% of the mass, thus inducing angiogenesis to assist metastasis and produce a poor prognosis. The remaining macrophages that migrate to tumor sites that promote angiogenesis and metastasis are called tumor associated macrophages (TAM) and are thought to express the anti-inflammatory M2 phenotype.

Macrophages are derived from the myeloid lineage and belong to the congenital immune system. They are derived from blood mononuclear cells that migrate into the tissues. One of their main functions is to infect microorganisms and remove cell debris. They also play an important role in both initiation and dissipation of inflammation [9, 10]. In addition, macrophages can exhibit different responses in the range of antiinflammation from proinflammation, depending on the type of stimulation they receive from the surrounding microenvironment [11]. Extreme macrophage response: Two major macrophage phenotypes have been proposed that are associated with M1 and M2.

M1 proinflammatory macrophages are activated upon contact with certain molecules such as lipopolysaccharide (LPS), IFN-y, IL-1 [beta], TNF- [alpha], and Toll-like receptor engagement. M1 Macrophages constitute a powerful segment of the immune system that is deployed to fight infections. They can recognize pathogens directly (pathogen pattern recognition receptors) or indirect (Fc receptors, complement receptors). They are also armed with their ability to generate reactive oxygen species (ROS) as a means to aid pathogen killings. In addition, M1 macrophages secrete pro-inflammatory cytokines and chemokines that attract other types of immune cells and integrate / regulate the immune response. M1 activation is induced by IFN-g, TNFa, GM-CSF, LPS and other toll-like receptor (TLR) ligands.

In contrast, M2 antiinflammatory macrophages, also known as alternatively activated macrophages, are activated by anti-inflammatory molecules such as IL-4, IL-13, and IL-10 [12,13]. M2 macrophages exhibit immunoregulatory, tissue repair, and angiogenic properties that can mobilize regulatory T cells into inflammatory sites. M2 Macrophages do not constitute a homogeneous population but are often subdivided into the M2a, M2b and M2c categories. The common denominator of all three subpopulations is high IL-10 production with low production of IL-12. One of their characteristics is the production of the enzyme arginine-1, which down-regulates L-arginine and inhibits T cell responses and deprives its substrate iNOS.

In vivo molecular mechanisms of macrophage polarization have been characterized inadequately due to the various signals experienced by macrophages in the cell microenvironment [10,14]. Advances have been made in the identification of in vivo macrophage polarization in physiological conditions such as onset, pregnancy, and pathological conditions such as allergies, chronic inflammation and cancer. However, it is not known that in vitro macrophage polarization is plastic, and that macrophages can be polarized back and forth with both phenotypes with the help of cytokines [15, 16]. Interferon gamma (IFN-γ) and IL-4 are two cytokines that can polarize macrophages into M1 and M2 phenotypes, respectively [15].

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

Macrophage  Polarization

Tumor microenvironment may affect macrophage polarization. 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 they are known to be involved in transcriptional regulation. For example, macrophages exposed to LPS or IFN-y will be polarized against the M1 phenotype whereas macrophages exposed to IL-4 or IL-13 will be polarized against the M2 phenotype. LPS or IFN-? Can interact with toll-like receptor 4 (TLR4) on the surface of macrophages to induce Trif and MyD88 pathways and induce the activation of transcription factors IRF3, AP-1, and NFkB to produce proinflammatory M1 macrophages TNF gene, interferon, CXCL10, NOS2, IL-12, etc. which are essential for the reaction can be activated [20]. Similarly, IL-4 and IL-13 bind to IL-4R and bind to Jak / Stat6, which regulates the expression of genes associated with the anti-inflammatory response (M2 response), CCL17, ARG1, IRF4, IL-10, SOCS3, Activate the path.

Additional mechanisms of macrophage polarization include microRNA (miRNA) microarray management. miRNAs are small noncoding RNAs of 22 nucleotides in length that regulate gene expression after transcription, since they affect the rate of mRNA degradation. Some miRNAs, particularly miRNA-155, miRNA-125, miRNA-378 (M1 polarization), and miRNA let-7c, miRNA-9, miRNA-21, miRNA-146, miRNA147, miRNA- It has been shown to be highly expressed in macrophages [21].

Macrophage polarization is a complex process in which macrophages act in response to microenvironmental stimuli to induce different responses. Thus, macrophage polarization is better expressed as a continuum of activation states of the extreme spectra of the M1 and M2 phenotypes. Recently, there has been considerable controversy regarding the definition / explanation of macrophage activation and macropa polarization. In a recent paper published by Murray et al., They describe a set of standards considered for common definition / description of macrophage activation, polarization, activation factors, and markers. This publication required much more definition and characterization of activated / polarized macrophages [22].

M1  Phenotype

M1 proinflammatory macrophages or classically activated macrophages are aggressive, highly bacterial, and produce large amounts of reactive oxygen species and nitrogen species, promoting Th1 responses [11]. M1 macrophages secrete high levels of two important inflammatory cytokines, IL-12 and IL-23. IL-12 induces activation and clonal expansion of Th17 cells, which secrete large amounts of IL-17 that cause inflammation [23]. These features allow M1 macrophages to control metastasis, inhibit tumor growth, and control microbial infection [24]. In addition, invasion and mobilization of M1 macrophages into the tumor site is associated with better prognosis and higher overall survival in patients with solid tumors [17, 18, 25-28].

Polarization of macrophages as M1 phenotypes is controlled in vitro by transcription factors and miRNAs, including inflammatory signals such as IFN-y, TNF-a, IL-lß and LPS [29,30]. The classically activated macrophages initiate the induction of STAT1 transcription factors targeting CXCL9, CXCL10 (also known as IP-10), IFN regulatory factor-1, and inhibitor-1 of cytokine signaling [31]. The cytokine signaling-1 protein functions downstream of the cytokine receptor and participates in the negative feedback loop to attenuate cytokine signaling. In tumor microenvironment, Notch signaling plays an important role in the polarization of M1 macrophages as the transcription factor RBP-J can regulate classical activation.

Macrophages lacking Notch signaling express the M2 phenotype irrespective of other external inducers [32]. One crucial miRNA, miRNA-155, is up-regulated when macrophages transition from M2 to M1, and M1 macrophages over-expressing miRNA-155 are generally more aggressive and associated with tumor reduction [33]. In addition, miRNA-342-5p has been shown to target Akt1 in mice, resulting in a greater inflammatory response in macrophages. These miRNAs also promote upregulation of Nos2 and IL-6, both of which act as inflammatory signals to macrophages [34]. Other miRNAs such as miRNA-125 and miRNA-378 have also been found to be involved in the classical activation pathway (M1) of macrophages [35].

The classically activated macrophages are believed to play an important role in the recognition and destruction of cancer cells because their presence generally implies a good prognosis. After recognition, malignant cells can be destroyed by M1 macrophages through several mechanisms including contact-dependent phagocytosis and cytotoxicity (ie, they release cytokines such as TNF-α) [24]. However, environmental signals such as tumor microenvironment or tissue-resident cells can be polarized from M1 macrophages to M2 macrophages. In vivo studies of macrophages and animal macrophages show that macrophages are plastic in their cytokine and surface marker expression and re-polarization of macrophages into the M1 phenotype in the presence of cancer can help the immune system reject the tumor [19].

M2 phenotype

M2 Macrophages are anti-inflammatory and aid in the process of angiogenesis and tissue repair. They express scavenger receptors and produce large amounts of IL-10 and other anti-inflammatory cytokines [33, 36]. M2 expression of IL-10 by macrophages promotes Th2 response. Th2 cells consequently upregulate the production of IL-3 and IL-4. IL-3 is a potent inhibitor of the myeloid lineage (granulocyte, monocyte, and dendritic cells), along with other cytokines such as erythropoietin (EPO), granulocyte macrophage colony stimulating factor (GM- Stimulates the proliferation of all the cells of the. IL-4 is an important cytokine in the healing process because it contributes to the production of extracellular matrix [23]. M2 Macrophages are able to supply blood vessels to malignant cells and thus function to support tumor progression by promoting their growth. The presence of macrophages (considered as M2) in most solid tumors is negatively correlated with treatment success and longer survival rates [37]. In addition, the presence of M2 macrophages was associated with the metastatic potential of breast cancer. Lin and colleagues have shown that the early mobilization of macrophages into breast tumor areas in mice increases the incidence of malignant tumors and angiogenesis [38]. The tumor microenvironment is thought to help macrophages maintain the M2 phenotype [23, 39]. Anti-inflammatory signals such as adiponectin and IL-10 present in the tumor microenvironment may enhance the M2 response [41].

Tumor-associated macrophages (TAM)

Cells exposed to tumor microcysts behave differently. For example, tumor-associated macrophages present in the periphery of solid tumors are thought to help promote tumor growth and metastasis, and have an M2-like phenotype [42]. Tumor-associated macrophages may be tissue resident macrophages or mobilized macrophages derived from bone marrow (macrophages that have differentiated into macrophages and migrated to tissues). Experiments by Cortez-Retamozo have shown that a high number of TAM precursors in the spleen migrate to the tumor matrix, suggesting that this organ also has TAM storage [43]. TAM precursors present in the spleen were found to initiate migration through their CCR2 chemokine receptors [43]. Recent experiments have found CSF-1 as a major factor that attracts macrophages to the periphery of the tumor, and predicts a low survival rate of CSF-1 production by cancer cells, which in turn is a bad prognosis overall. [44-46]. Other cytokines such as TNF-a and IL-6 have also been associated with the accumulation / mobilization of macrophages into the tumor periphery [45].

Macrophages mobilized around the periphery of the tumor are thought to be regulated by "angiogenic switches" activated in the tumor. Angiogenesis switch is defined as the process by which a tumor can potentially make a tumor metastatic and develop a dense network of blood vessels essential for malignant metastasis. In a breast cancer mouse model, the presence of macrophages was observed to be necessary for a complete angiogenic switch. In the case of delayed maturation, migration, and accumulation of macrophages around the tumor, the angiogenic switch was also delayed, suggesting that the angiogenic switch is not generated in the absence of macrophages and the presence of macrophages is necessary for malignant progression [47]. In addition, tumor stromal cells produce chemokines such as CSF1, CCL2, CCL3, CCL5, and placental growth factors that mobilize macrophages around the tumor. These chemokines will provide an environment for macrophages to activate angiogenic switches, where they will produce IL-10, TGF-beta, ARG-1 and low levels of IL-12, TNF-a and IL-6 . The expression of these cytokines suggests that macrophages control immune avoidance. It is noted that macrophages will react by generating hypoxia-inducible factor-1α (HIF-1α) and HIF-2α, which are attracted to the hypoxic tumor environment and regulate the transcription of genes involved in angiogenesis It is important. During angiogenesis switches, macrophages can also secrete VEGF (stimulated by the NF-κB pathway), which also promotes vascular maturation and vascular permeability [48].

Tumor-associated macrophages are thought to be able to accommodate their M2-like phenotype by accommodating the polarization signals from malignant cells, such as IL-1R and MyD88, mediated through the IkB kinase beta and the NF- kappa B signaling cascade. Inhibition of NF-kB in TAM promotes classical activation [40]. In addition, another experiment suggested that the p50 NF-kB subunit was involved in the inhibition of M1 macrophages and that a decrease in inflammation promoted tumor growth. Saccani et al. Suggested that p50 NF-κB knockout mice restored M1 invasion by p50 NF-kB knockout and reduced tumor survival [49].

Since the mass contains a large number of M2-like macrophages, TAM can be used as a target for cancer therapy. Decreasing the number of TAM or their polarization with the M1 phenotype may help to destroy cancer cells and impair tumor growth [50-52]. Luo and his colleagues used a vaccine against regmain, a stress protein and cysteine protease up-regulated in TAM, which is considered a potential tumor target [52]. When vaccines against the regumain were given to mice, genes controlling angiogenesis were down-regulated and tumor growth was discontinued [52].

Metabolism and activation pathway

Metabolism changes present in tumor cells are controlled by the same gene mutations that cause cancer [53]. As a result of these metabolic alterations, cancer cells can modify the polarization of macrophages and produce signals that can promote tumor growth [54, 55].

M1 and M2 macrophages have been shown to have distinct metabolic patterns that reflect their different behavior [56]. The M1 phenotype increases the action and bends glucose metabolism toward the oxidative pentose phosphate pathway thereby reducing oxygen consumption resulting in the production of inflammatory cytokines including TNF-a, IL-12, And IL-6 [56, 57]. The M2 phenotype increases fatty acid uptake and oxidation, leading to a decrease in flow towards the pentose phosphate pathway while an increase in the total cellular redox potential, resulting in the formation of scavenger receptors and immunoregulatory cytokines such as IL-10 and TGF- β is upregulated [56].

Many metabolic pathways play an important role in macrophage polarization. Protein kinases such as Akt1 and Akt2 alter the macrophage polarization by allowing cells to survive, proliferate, and utilize intermediate metabolism [58]. Other protein kinases can specify macrophage polarization through glucose metabolism by increasing their action and reducing oxygen consumption [57, 59]. Shu and colleagues first visualized macrophage metabolism and immune responses in vivo using PET scans and glucose analogs [60].

L-arginine metabolism also represents a distinct metabolic pathway that represents a distinct shift in cytokine expression in macrophages and alters TAM-tumor cell interactions [61]. The classically activated (M1) macrophages prefer inducible nitric oxide synthase (iNOS). The iNOS pathway produces cytotoxic nitric oxide (NO) and, consequently, antitumor behavior. Alternatively, activated (M2) macrophages prefer the arginase pathway to produce I-ornithine and urea that cause progressive tumor cell growth [61, 62].

Direct manipulation of the metabolic pathway may alter the macropargic polarization. Carbohydrate kinase-like protein (CARKL) proteins, which play a role in glucose metabolism, have been used to alter macrophage cytokine characteristics [56, 57]. When CARKL is knocked down by RNAi, macrophages tend to adopt the M1 analogous metabolic pathway (metabolism leans towards the action and oxygen consumption is reduced). When CARKL is overexpressed, macrophages adopt M2-like metabolism (corresponding action flow is reduced and more oxygen is consumed) [56]. When macrophages adopt the M1-like metabolism status via LPS / TLR4 involvement, CARKL levels are reduced, NKKB-regulated genes are activated (TNF-a, IL-12, and IL-6) Dislocations are increased by increasing concentrations of NADH: NAD + and GSH: GSSSG complexes. During the M2-like metabolic state, macrophages upregulate the genes and CARKL regulated by STAT6 / IL-4 (IL-10 and TGF-β).

Macrophage Immunotherapy Approach to Cancer

The role of cancer immunotherapy is to stimulate the immune system to recognize, reject, and destroy cancer cells. Cancer immunotherapy by monocyte / macrophage has the goal of polarizing macrophages to the proinflammatory response (M1) so that macrophages and other immune cells can destroy the tumor. Many cytokine and bacterial compounds can be obtained in vitro, but typically the side effects are too severe in vivo. The key is to find compounds with minimal or easily managed patient side effects. Immunotherapy using monocytes / macrophages has been used for decades and a new approach has been developed annually [64, 65]. Early immunotherapy established a good basis for better cancer therapy and increased the survival rate of patients treated with immunotherapy [66].

Some approaches to cancer immunotherapy include the use of cytokines or chemokines to mobilize activated macrophages and other immune cells to the tumor site to enable recognition and targeted destruction of tumor sites [67, 68]. IFN-α and IFN-β have been shown to inhibit tumor progression by inducing cell differentiation and apoptosis [69]. In addition, IFN treatment is antiproliferative and can increase the S phase of the cell cycle [70, 71]. Zhang and colleagues conducted experiments in nude mice using IFN-beta gene therapy targeting human prostate cancer cells. They suggested that adenovirus-mediated IFN-β gene therapy may contribute to macrophage and inhibit growth and metastasis [72].

Macrophage inhibitory factor (MIF) is another cytokine that can be used in cancer immunotherapy. MIF is generally present in solid tumors and represents a bad prognosis. MIF promotes macrophages as an M2 phenotype that can inhibit aggressive macrophage function and aid tumor growth and progression. Simpson, Templeton and Cross (2012) found that MIF induces the differentiation of bone marrow cells and macrophage precursors as an inhibitory population of bone marrow cells expressing the M2 phenotype [73]. By targeting MIF, they were able to reduce these inhibitory populations of macrophages, inhibit their growth and thereby control tumor growth and metastasis [73].

The type 2 chemokine receptor, CCR2, is central to the mobilization of mononuclear cells into inflammatory sites and has been identified as a target to prevent mobilization, angiogenesis, and metastasis of macrophages to tumor sites. Sanford and colleagues (2013) tested a novel CCR2 inhibitor (PF-04136309) in a pancreatic mouse model to demonstrate that CCR2 inhibitors reduce mononuclear / macrophage mobilization to tumor sites, reduce tumor growth and metastasis, And increased anti-tumor immunity [74]. Other recent experiments by Schmall et al. Have confirmed that macrophages co-cultured with 10 different human lung cancers upregulated CCR2 expression. They also showed that tumor growth and metastasis were reduced in CCR2 antagonist treated lung mouse models [75].

Other experiments used liposomes to deliver drugs to diminish M2 macrophages and to stop angiogenesis from tumors. Cancer cells expressing high levels of IL-1 [beta] grow faster in vivo and further induce angiogenesis. Kimura and colleagues found that macrophages exposed to IL-1β-expressing tumor cells express high levels of angiogenic factors and chemokines such as vascular endothelial growth factor A (VEG-A), IL-8, monocyte chemoattractant protein 1 To promote tumor growth and angiogenesis [76]. When they used the creatinide liposomes to reduce macrophages, they found a small number of IL-1β-producing tumor cells. They also inhibited NK-κB and AP-1 transcription factors in cancer cells and found that tumor growth and angiogenesis were reduced. These findings suggest that macrophages around the tumor site may be involved in promoting tumor growth and angiogenesis [76].

The compounds such as methionine enkephalin (MENK) have antitumor properties in vivo and in vitro. MENK down-regulates CD206 and arginase-1 (M2 marker) while the production of CD64, MHC-II, and nitric oxide (M1 marker) is upregulated to polarize M2 macrophages to M1 macrophages. MENK can also upregulate TNF-a and down-regulate IL-10 [77].

Recent experiments have focused on bisphosphonates as potential inhibitors of M2 macrophages. Bisphosphonates are commonly used to treat patients with metastatic breast cancer to prevent skeletal complications such as bone resorption [78]. While bisphosphonates remain in the body for a short period of time, bisphosphonates can target osteoclasts, which are cells of the same family as macrophages, due to their high affinity for hydroxyapatite. When a bisphosphonate binds to a bone, the bone matrix internalizes the bisphosphonate by intracellular entry. When present in the cytoplasm, bisphosphonates can prevent integrin signaling and endosome trafficking, thereby inhibiting protein prenylation, an event that causes cells to become apoptotic [69]. Until recently, it has not been known whether bisphosphonates can target tumor-associated macrophages. However, recent experiments with Junankar et al. Have shown that by immunocytochemical and phagocytic action, events that do not occur in epithelial cells around the tumor Showed that macrophages absorb nitrogen-containing bisphosphonate compounds [79]. Using bisphosphonates to direct TAM to apoptosis may reduce angiogenesis and metastasis.

An additional approach to cancer immunotherapy involves the use of biomaterials that can trigger an immune response. Cationic polymers are used in immunotherapy because of their reactivity when dissolved in water. Chen et al. Used cationic polymers including PEI, polylysine, cationic dextran and cationic gelatin to generate a strong Th1 immune response [77]. They were also able to induce IL-12 secretion and CD4 + cell proliferation typical of M1 macrophages [77]. Huang and colleagues also used biomaterials to trigger TAM to target TLR4 and generate antitumoral responses [80]. This experiment found that TAM could polarize to the M1 phenotype and express IL-12. They have shown that these cationic molecules have direct tumor-destructive activity and have demonstrated tumor reduction in mice [80].

CAR T cell immunotherapy

Artificial T cell receptors (also known as chimeric T cell receptors, chimeric immunoreceptors, chimeric antigen receptors (CARs)) are engineered receptors that have been engineered with random specificity on immunoactive effector cells. Typically, these receptors are used to combine the specificity of monoclonal antibodies on T cells with the delivery of their coding sequences facilitated by retroviral vectors.

Recently, therapies have been developed that use these engineered T cells to target and destroy cells containing cancer-specific or cancer-associated biomarkers. Once an appropriate target is established, the extracellular domain of the T cell receptor (TCR) is replaced with a single-chain variable fragment (scFv) derived from the antibody against the target. Such scFvs contain antibody variable regions that determine binding. As a result, upon contact with the target, the scFv will bind to and initiate signaling cascades that will activate T cells. These engineered immune cells are called chimeric antigen receptors (CARs) due to their combinatorial properties and represent a novel new therapy for cancer treatment. However, CAR has limited availability of appropriate targets.

T cells were transfected with chimeric antigen receptors (CARs) with extracellular antibody fragments directed against tumor epitopes fused to intracellular T cell signaling domains to give them new specificities for non-MHC restricted epitopes [3]. The chimeric antigen receptor (CAR) is a recombinant receptor that provides both surface antigen binding and T cell activation functions. Numerous CARs targeting cell surface tumor antigen aggregates have been reported for the past decade. Their biological function has changed dramatically after the introduction of a three-part receptor containing a coplanar domain, called second-generation CAR. They have recently been shown to have clinical benefit in patients treated with CD19-targeted autologous T cells. CAR can be combined with a coplanar ligand, a chimeric confocal receptor, or a cytokine to further enhance T cell titer, specificity and safety. CAR is a new class of drugs with an exciting potential for cancer immunotherapy.

Although T cells can induce potent antitumoral responses, T cells that most efficiently respond to peptide-MHC epitopes on the tumor surface are often very similar or identical to magnetic epitopes, often undergoing clonal resistance or deletion do. T-cell therapy has involved genetic modification of T cells by the introduction of TCR against tumor-associated T cell epitopes. Although this strategy has been identified as promising, there still remains a pairing error of the endogenous and introduced TCRs, including the various challenges surrounding T cell epitopes in general. It has been proposed to allow T cells to respond to traditional antibody epitopes, exploiting the forces of T cells in the fight against tumors.

BiTE (bispecific T-cell engager)

Another strategy for targeting T cells with the correct antibody epitope utilizes a long-studied type of molecule called a "bispecific antibody" that links an antibody that recognizes the CD3 subunit with an anti-cancer antibody. These are recently called BiTE (bispecific T cell inducer). The short-chain variable fragment (scFv) that binds to the tumor epitope is linked to a second scFv that binds to the constant part of the T cell receptor complex to activate and target the effector T cells against the tumor epitope, regardless of the TCR mediated specificity of the T cell ≪ / RTI > The evidence shows that these reagents are significantly more potent than antibodies against tumor cells alone. BiTE targets more than ten tumor-associated epitopes, including blinda tomomab (in the case of B cell leukemia) against CD19 and MT-110 (in the case of various adenocarcinomas and cancer stem cells) against EpCAM , Both of which are currently undergoing clinical trials. A high response rate for recurrence-free survival and minimal residual disease was found in patients with refractory acute lymphoblastic leukemia (ALL) who received blini or tomomab in clinical trials.

Thymidine Kinase ( Thymidine Kinase  1: TK1)

Human thymidine kinase 1 (TK1) is a well-known nucleotide bypass pathway enzyme that has been extensively studied for its overexpression in tumors. Because TK1 was initially introduced by its expression in the serum of cancer patients (sTK), its diagnostic and prognostic potential has been extensively studied. For example, some experiments have demonstrated that sTKl in many different cancers is elevated in a stepwise fashion with higher levels of TK1 suggesting more advanced tumors [81].

Other experiments examined the prognostic potential of TK1. One such experiment has demonstrated that TK1 levels can be used to predict relapse in primary breast tumors. Other exciting TK1 prognostic tests have shown a significant reduction in sTK1 levels when patients respond to treatment, while sTK1 levels continue to rise in patients who do not appear to respond to their treatment. It is also known that the level of sTK1 begins to rise before relapse and in some cases the level of sTK1 noted that it is predictable to recur at 1-6 months before the onset of clinical symptoms. Several other experiments have identified the abundant potential of TK1 as a diagnostic and prognostic indicator of cancer [82].

The diagnostic and prognostic potential of TK1 has been well established, but the therapeutic potential of TK1 remains unclear. Although it is true that HSV-TK is used in gene therapy and TK1 is used to identify cancer cells that are proliferating by PET imaging, few studies have demonstrated the possibility of TK1 immunotherapy. Perhaps this is mainly because TK1 is a known cytoplasmic protein. It has recently been shown that TK1 is expressed on the surface membrane of most tumor types as well as cancer cells and is thus a very clear target of tumor immunotherapy.

The diagnostic and prognostic potential of TK1 has been demonstrated using traditional TK activity radioactivity assays for both hematologic malignant tumors and solid tumors. TK1 has been extensively tested in association with cancer diagnostic biomarkers that have been found to be upregulated in tissues and serum of both solid tumors and hematological malignancies.

TK1 levels in serum have also been found to have diagnostic potential in other cancers such as bladder, cervical carcinoma, stomach, non-small cell lung, and kidney and rectal cancer. In summary, high levels of TK1 serum correlate with tumor aggressiveness and may be indicative of early events of carcinogenesis. However, the mechanism by which TK1 enters the serum and its function in serum have not been investigated in general. Perhaps, its function in serum is related to the regulation of the immune system. Further analysis is needed to understand this connectivity and its significance.

In its most basic structure, human TK1 (hTK1) as a monomer has a molecular weight of 25.5 kDa and is 234 amino acids in length. TK1 adopts a variety of oligomeric forms but is most commonly identified as a dimer or tetramer of approximately 53 kDa and 100 kDa respectively. In 1993, Munch-Petersen reported that the TK1 dimer is a low-efficiency form of the enzyme with high Km (15 μM). On the other hand, the TK1 tetramer is a high-efficiency form of low Km (0.7 μM) and has been reported to have a 30-fold increased efficiency in catalyzing the phospholyl transfer reaction compared with the dimer. The crystallization of TK1 means that the tetrameric form is composed of the dimer of the dimer. As such, there are two different monomer-monomer interfaces marked as strong and weak. The weak interface is mainly stabilized indirectly by ATP donor molecules, while the strong interface is directly stabilized through many polar interactions. Each monomer has an alpha / beta-domain that most closely resembles a DNA binding protein containing RecA.

Thymidine kinase 1 (TK1) is a nucleotide bypass pathway enzyme primarily responsible for converting deoxy thymidine to deoxy thymidine monophosphate and is highly upregulated during cell replication. During DNA synthesis, the nucleotides are synthesized either through a bypass path where they are regenerated from intracellular and extracellular sources, or are newly synthesized.

TK1 is one of the two major bypass pathway kinases responsible for maintaining the cell nucleotide pool. TK1 is primarily responsible for the phosphorylation of deoxy thymidine (dT). Its product, dTMP, is then phosphorylated and introduced into DNA as deoxytimidine triphosphate (dTTP). As predicted, dTTP is the rate limiting step of this process, which helps control this process by inhibiting TK1. Under normal growth conditions, TK1 is regulated by the cell cycle. TK1 levels are very low or almost undetectable during the G1 phase and begin to increase during the late G1 phase. The level of TK1 is at least 10 times higher than the level during the G1 phase, the highest during the S phase with a concentration of almost 200 nM. Interestingly, Sherley et al. Reported that under normal conditions, TK1 mRNA increased only three-fold or less, compared with a 15-fold increase in protein activity levels during the cell cycle. They also determined that the [35S] incorporation rate during the S phase was 12 times more efficient than during the G1 phase. The rapid increase in TK1 levels during the S phase was not an increase in transcription but an increase in TK1 translation efficiency. This finding is particularly interesting from an experimental standpoint such as Chou et al. That the 5'-untranslated region (5'UTR) can render translation of TK1 mRNA cap-independent. Munsch-Peterson et al. Have demonstrated that this rapid increase in TK1 is also the result of the conversion of the active dimer to the active tetramer TK1 form. Some experiments have confirmed that TK1 levels are increased as a result of DNA damage, especially after irradiation or chemotherapy.

In 2010, Chen et al. Further characterize the association between TK1 and DNA by showing that p53 - / - tumor cells increase TK1 levels in response to DNA damage, whereas p53 wild-type tumor cells do not. This association between TK1 and p53 has been confirmed in other experiments that demonstrate that normal p53 function is required to maintain cell cycle-dependent regulation of TK1 and that there is a compensatory increase in TK1 upon p53 loss. A final analysis of this association revealed that the increase in TK1 levels following p21 DNA damage was p21-dependent. In fact, Huang et al. (2001) confirmed that the C-terminal domain of p21 interacts with TK1 and overexpression of TK1 prevents p21-dependent growth inhibition. These results challenged the traditional role of TK1 in tumor cells. For example, Chen et al. Found that TK1 knockdown did not affect tumor cell growth, even when dTTP levels were significantly reduced (p <0.01). Their results support the conclusion that TK1's primary role in tumor cells is DNA repair rather than providing sufficient levels of dTTP for replication and growth. However, these conclusions support that the biochemical role of TK1 is still unclear. In normal cells, TK1 is responsible for maintaining the dTTP nucleotide pool in a cell cycle dependent manner. In addition, TK1 plays a valuable role in DNA repair and survival of tumor cells after DNA damage. The biological significance of TK1 is less understood and somewhat catchy. Normal TK1 function is essential for proper development and function of the kidneys and salivary glands, although these mechanisms are not understood. TK1 also appears to be essential for normal functioning of the immune system and can play a role in its deregulation. Another unexplained feature of TK1 is its role in the circulatory system of cancer patients.

Hypoxanthine  Guanine Phospholibosyltransferase ( Hypoxanthine  Guanine Phosphoribosyltransferase: HPRT).

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) or HPRT is a key enzyme for the large-scale production of guanine and inosine bases. HPRT functions by transferring phospholipids from PRPP to hypoxanthine or guanine base to form IMP and GMP, respectively. Due to its role in DNA maintenance, HGPRT is known as a housekeeping gene and is often used as a standard for quantitative analysis because of its constant expression in all eukaryotic cells.

Hypoxanthin-guanine phosphoribosyltransferase (HGPRT) is an enzyme encoded in humans by the HPRT1 locus. This enzyme makes it possible for cells to recycle the building block type of DNA and its chemical cousin RNA, purine. Purine manufacturing uses more energy and takes more time than recycling purines, so recycling makes these molecules more efficient. Purine recycling ensures that cells have an abundant supply of building blocks for the production of DNA and RNA. The purine recycling process is also known as the purine bypass route.

Hyposantoin phospholyl transferase 1 (HGPRT)

Hypoxanthin guanine phospholibosyltransferase (HPRT or HGPRT), also critical for cell division and the generation of nucleotides used for successful DNA replication, is an important enzyme for the large-scale production of guanine and inosine in the purine bypass pathway. Bypass pathway enzymes act as recyclers, using the components of the old nucleotide to bypass the consumption of energy required for nucleotide synthesis. This generation method dominates most cell cycles in humans since 90% of the free purines are recycled. As a key enzyme in this process, HPRT is essential for cell survival and propagation. However, its role in cancer cell proliferation remains largely unknown. Through initial work to assess these relationships, preliminary data suggest that cancer cells can regulate HPRT and exist exclusively on proteins on the cell surface.

Hypoxanthin guanine phosphoribosyltransferase (HGPRT) is a bypass pathway enzyme involved in the purine synthesis of guanine and inosine (Caskey & Kruh, 1979). HGPRT is a transferase that cleaves ribosmonophosphate from PRPP and covalently attaches it to a guanine base to form GMP. When ribose monophosphate is released from PRPP, it releases pyrophosphate (PPi) as a by-product. As GMP is generated, additional enzymes will introduce more phosphate groups to form functional GTP. This same process is also consistent with inosine nucleotide synthesis since HGPRT will transfer ribosmonophosphate from PRPP to hypoxanthine base to form IMP. These enzymes transfer phospholipids from PRPP to hypoxanthine or guanine bases (Stout & Caskey, 1985; Wilson, Tarrt, & Kelley, 1983). The HGPRT enzyme consists of 10 beta-strands with 37 to 189 residues forming the center of the enzyme and 6 alpha helices (Eads, Scapin, Xu, Grubmeyer, & Sacchettini, 1994). Depending on the pH of the surrounding tissue, this protein may be present as a dimer or tetramer with the same or small subunit (Eads et al., 1994; Keough, Brereton, De Jersey, & Guddat, 2005; Zhang et al. 2016). The molecular weight of each protein subunit is 48.8783 kDa and the molecule has an instability index of 21.69, which is classified as stable. The homo-tetramer contains four subunits labeled A, A ', B, and B' (Eads et al., 1994).

Figure 8 shows the HGPRT biochemical pathway. Homogeneous tetrameric structures of human HGPRT have beta-sheet, beta-strand, alpha helical, and betaturn. This protein has only 27% alpha helices and 27% beta sheets and the remaining 46% of the enzymes are betatons and arbitrary coils. The structure has a subunit labeled A, A 'and B, B'. Each subunit is relatively identical and is translated from the same mRNA message.

The enzyme has several regions with different functions in substrate recognition and reactivity. The carboxy terminus of the central beta sheet is primarily involved in substrate recognition. The core region of the protein contains five beta strands surrounded by four alpha helices and twisted parallel beta sheets. The 65-74 residues form the most elastic part of the protein, creating a loop in which they bind to the pyrophosphate. The residue of the enzyme that binds to the PRPP substrate is 129 to 140 and is located at the bottom of the active site. In order for the enzyme activity of the active site to be successful, the metal ion Mg ²⁺ is required (Eads et al., 1994; Zhang et al., 2016).

The gene coding for HGPRT is called HPRT. These 47,827 bp genes are present in the long segment of the X chromosome and are relatively large, and it is believed that only a small fraction of the transcribed DNA is actually translated. This gene contains nine exons encoding 217 amino acid proteins, which represent only 1.3% of the original genomic message (Fuscoe, Fenwick, Ledbetter, & Caskey, 1983; Stout & Caskey, 1985; Wilson et al. , 1983). Because the final protein product is involved in cell maintenance, the control sequence upstream of the HPRT gene contains the characteristics of the mammalian housekeeping gene and the absence of the 5 'transcription sequence, including the TATA and CAAT boxes, and exceptionally the 5' Thus, there are GC-rich sequences with many GC hexanucleotide motifs (Kim et al., 1986). As a housekeeping gene, HPRT is present in all somatic tissues in a low order (Melton, Mcewan, Reid, & Mckie, 1986). In most human cells, the HPRT mRNA transcript contains only 0.005 to 0.01% of the total mRNA (Caskey, 1981). The only exception is central nervous tissue, where there is an ideally elevated level of HPRT expression ranging from 0.02 to 0.04% of total mRNA, which is a four-fold increase compared to other somatic tissues (Caskey, 1981; Zoref-shani, Frishberg , &Amp; Bromberg, 2000). This elevated expression is not fully understood because the cells of the central nervous system (CNS) are not stimulated to divide and the mechanism for nucleotide synthesis is less required. In addition, the human genome contains a non-functional HPRT homology region of autosomal DNA of chromosomes 5, 11, and 13 (Fuscoe et al., 1983). These DNA sequences are not known to be transcribed, and most likely are probably stomach genes, but their exact origin and expression are not fully understood (Nyhan & Diego, 2012).

Due to the proliferative capacity of cancer cells and the large demand for nucleotide production, HPRT is expected to upregulate these environments (Linehan & Goedegebuure, 2005). Through preliminary experiments to determine whether HPRT is upregulated in cancerous situations, we have found that there is a strong association between HPRT and the plasma membrane of cancer cells. This association has been observed in a variety of cancer types and cell lines in a number of different assays. Confocal imaging and flow cytometry analyzes were obtained for a number of different cancer cell lines and HPRT was shown to be consistently expressed on the surface of all cancer types tested. This same expression is not observed in the by-pass enzymes DCK and APRT, meaning that HPRT has a role in a cancerous environment that is not shared by all by-pass enzymes. The reason for this surface expression is unknown and can only be guessed as to why it is externally present in cancer. This unique surface expression can indicate the secondary role of HGPRT over its predominant role as a purine synthetase and it is possible to provide additional information on the unique ecosystem of the tumor microenvironment.

Important factors for the success of this system for cancer therapy include modifications of macrophages with chimeric antigen receptors (MOTO-CAR), tumor antigens are associated with cancer cells and associated with normal cells It does not.

Macrophage

One aspect is the use of macrophages modified against cancer antigens. Using CAR technology, macrophages have antigen receptors against cancer antigens.

As mentioned above, CAR technology has been used to develop T cells with antigen receptors against cancer antigens. These antigens are often substances that do not activate the immune response in the normal state because they are the same as or similar to the human product. For this reason, T-cells have been modified to have such receptors. Therapies involving such T cells with a chimeric antigen receptor (CAR), in which antigen receptors are induced against tumor epitopes, have been tested. T cells can induce potent antitumor responses, as mentioned in the background section above, and these therapies are promising, but have presented problems.

For example, normal T cells that most efficiently respond to peptide-MHC epitopes on the tumor surface have often experienced clonal resistance or deletion, since many of these epitopes are very similar or identical to magnetic epitopes. T-cell therapy included genetic modification of T cells in vitro by the introduction of TCR against tumor-associated T cell epitopes. While this strategy has been shown to be promising, potential pairing errors of endogenous TCR and introduced TCR remain, including the various challenges generally associated with T cell epitopes. It is proposed that T cells can respond to traditional antibody epitopes, exploiting the forces of T cells in fighting against tumors.

T-cells can live for long periods, infinitely exist in the body, and can also experience antigens against cancer antigens. This means that antigen-specific T cells against tumor antigen markers can be present after treatment and removal of the cancer. This can be a problem because tumor antigens are generally human-produced (primarily requiring CAR) and may be present in small amounts for other bodily functions. The persistent presence of modified CAR T cells and the potential innate presence of target antigens can lead to deleterious and unwanted activation of T cells. This can endanger important processes in the body or induce a cytokine storm, resulting in the destruction of the cytokine production / activation feedback loop for T cells resulting in uncontrolled and expanded activation of immune cells, resulting in an excessive immune response do. Cytokine storms can cause significant damage and potentially death.

Modifying the macrophage cells to produce macrophage CAR cells (MOTO-CAR) against cancer antigens solves the problem in the subject system. Macrophages may last several weeks after infection, but unlike CAR T cells, they do not appear to possess memory. Thus, leaving a CAR will reduce the potential harm from response to low concentrations of cancer antigen-free solutions. In addition, macrophages do not participate in the cytokine storm phenomenon, eliminating the problem of T cell CAR.

Antigens Associated with Cancer

An aspect of the therapy is that certain cancers and tumor antigens are associated with cancer and tumors, and not with noncancerous tissues. For example, it has been demonstrated that TK1 and HGPRT are expressed on many, possibly all, cancerous types of surfaces, with little or no expression on the surface of normal cells. They provide antigenic markers that can detect and target cancer cells and kill cancerous cells without harming non-cancerous cells.

One aspect is the combination of modified macrophage specific CAR technology and monocyte / macrophage to combat cancer by combining human / humanized antibodies against human thymidine kinase 1 (: TK1) and hypoxanthine-guanine phosphoribosyltransferase (HPRT) . It can also be used against other common tumor targets such as CD19, CD20, epithelial growth factor (EGFR), receptor tyrosine kinase-like oropharyngeal receptor 1 (ROR1) and other novel tumor targets to produce potentially activated macrophages for many different tumors RTI ID = 0.0 &gt; humanized &lt; / RTI &gt;

We believe that there are additional potential antigen markers that can be used by this therapy targeting cancer cells than normal cells. These may include, for example, bypass pathway enzymes that contribute to metastasis, substrates that aid in angiogenesis. Any normal antigen that is not present on the surface of normal cells can be expressed on the surface of cancer cells as any mutated normal human protein that can differ significantly from the normal protein to distinguish it from CAR or MOTO CAR. Some fetal antigens that can be expressed exclusively on cancer cells can be used as targets if mutant proteins produced as a result of tumorigenesis are also significantly different from unmodified proteins so that they can be distinguished by antibodies.

Both TK1 and HPRT were upregulated in many types of cancer and were found on the surface of many cancer cells. Neither is found on normal cells and is therefore a major target of immunotherapy. A preliminary finding implies that HGPRT is present on the surface at the same rate as TK1, in other words, HGPRT is also high when TK1 is high, and HGPRT is also low when TK1 is low. While not intending to be bound by theory, they can be a complex together.

The technique may be used for the treatment of diseases such as cancer, without limitation, from humanized or nonhuman mammalian (e.g. mouse) monoclonal antibodies to HGPRT or TK1, which can be used as appropriate genetic manipulation to manipulate macrophages ultimately derived from the patient 0.0 &gt; scFv &lt; / RTI &gt; and produced CAR or BiTE. The fact that antigenic substances (e.g., TK1, HGPRT) are present on the surface of cancer cells and not on any normal cell surface is a major part of the discovery, and this knowledge has been shown to induce macrophages to specifically induce tumor cells Because it can be used to.

The uniqueness of this technique is due in part to the fact that antibodies specifically raised against human cancer antigens associated with cancer cells, but not normal cells, can be used to target tumors. For example, antigens expressed in this manner on the surface of cancer cells, such as TK1 and HGPRT, can be used to target CAR, MOTO CAR, and BiTE into tumors.

Antibodies specific for human HGPRT are known, for example, as "anti-HPRT antibody (ab10479)" at http://www.abcam.com/hprt-antibody-ab10479.html.

Antibodies specific for human TK are known, for example, as disclosed in U.S. Patent Nos. 9267948, 7837998, 7311906, and 5698409.

One aspect is the use of macrophages or monocytes or other immune cells containing a MOTO-CAR vector designed for specific tumor-associated antigens (scFV fused to an activation region in a toole-like receptor cell), and the use of monocytes Or the use of Macrophage MOTO-CAR technology. Techniques can be used for any specific antigen using a vector to cause an immune response using monocytes or macrophages.

One aspect is the use of macrophages or monocytes or other immune cells containing MOTO-CAR vectors designed for specific tumor-associated antigens such as TK1 and HPRT (scFV fused to an activation region in a toll-like receptor cell).

One aspect is a method for treating a tumor when the specific tumor antigen is specifically HPRT. TK1 has a high level in patient-derived serum with aggressive tumors and can bind to MOTO-CAR and activate CAR before it reaches the tumor site. HPRT has been shown to have low serum levels and appears to be more abundantly distributed in cancer cell membranes, not normal cells.

One aspect is a method for polarizing macrophages to the M1 phenotype in a cancerous environment. MOTO-CAR is designed to activate macrophages to be converted to M1 aggressive death macrophages as opposed to M2, which is attached to TK1 or HPRT on the surface of cancer cells and is associated with tumors and protects them from immune destruction.

One aspect is the use of a macrophage specific promoter for activation of macrophage CAR. Since MOTO-CAR can bind to soluble TK1 in serum, it can be activated without being near the tumor. A possible solution to this is to isolate patient-derived monocytes and infect them with MOTO-CAR constructs that are under the control of a macrophage-specific promoter. Monocytes become macrophages only when they move from blood to tissue. Placing MOTO-CAR under the control of a macrophage-specific promoter will allow MOTO-CAR to be expressed only in tissues and avoid activation in serum.

Another aspect is the use of a cytoplasmic macrophage activating molecule / signal transduction cascade such as a toll-like receptor. MOTO-CAR can be activated using a toll-like receptor cytoplasmic domain. There are other activating signaling molecules that may have similar functions. And different activating molecules are contemplated. The molecule used should not be a toll-like receptor in which there are other signaling pathways available for this technique.

Other aspects include the use of scFV derived from human / humanized monoclonal. The use of mouse or human derived scFv is contemplated. For example, human antibody acquisition against TK1 and HPRT using, for example, MOTO-CAR with scFv (specific for TK1) derived from mice and humans, or a yeast library producing human monoclonal antibodies is contemplated.

Another aspect is the use of this technology for target diseases such as cancer and further development for use in other diseases (i. E. Infectious diseases and autoimmune diseases). MOTO-CAR technology does not have to be limited to attack cancer, and there may be other diseases where this technique may be effective.

Another aspect is the use of co-polar molecules to enhance macrophage activation. (MD2, CD14) Other molecules involved in macrophage activation may be used as part of the MOTO-CAR construct. Most immune cells require stimulation from other molecules before they are fully activated. In some applications, co-activation of moiety molecules may be required for MOTO-CAR to be fully activated. These molecules may include (without limitation) MD-2 and CD14.

Another aspect is the use of a bispecific macrophage engager (BIME) for use in immunotherapy. In addition to MOTO-CAR, you can use the bi-specific Macrophage Ingerizer (BIME). BIME uses macrophage activation and new tumor antigens. A scFv or a macrophage activator protein protein linked by an amino acid spacer to an scFv against a tumor antigen. As an example, there are three different BIME examples. The first is a molecule composed of a molecule of IFN-y linked by an amino acid spacer to any scFv for TK1, HPRT or any other tumor antigen. The second is designed as a combination of scFv for the CSF-1 receptor and scFv for the tumor antigen. The third involves a bispecific antibody to the hydrophobic pocket of the MD2 protein, which causes close activation of two TLR4 proteins that trigger signaling cascades by the physical encounters of two TLR4s in the TIR domain in the cell fluid. MOTO-CARS and BIMES are part of a new generation of cancer immunotherapy technology, both of which are available in the treatment of many different cancer types.

Figure 1 is a schematic diagram illustrating a macrophage chimeric antigen receptor.
FIG. 2 is a schematic diagram showing a macrophage toll-like receptor CAR (MOTO CAR). The intracellular and transmembrane domains of tol-like receptors, FC-gamma III receptors, IL-1 or IFN-gamma receptors are fused to appropriate hinge and scFv for tumor antigens to activate macrophages upon binding to specific tumor antigens .
Figure 3A is a schematic diagram showing different macrophage receptors that can be used to construct macrophages CAR.
Figure 3b is a schematic diagram illustrating signaling of Fc gamma receptor III.
Fig. 4 is a schematic diagram showing bispecific macrophage-induced _IFN-? (BIME _IFN-? ). M2 Tumor stays Macrophages can be polarized and anchored to tumor cells using molecules of IFN-? That are linked by an amino acid spacer to the scFv for tumor antigens.
FIG. 5 is a schematic diagram showing a bispecific macrophage inducer (BIME). FIG. M2 macrophages can be polarized against the M1 phenotype and can be induced against tumor cells. Bispecific antibodies can block the CSF-1 receptor blocking the CSF-1 receptor induced by the M2 profile. At the same time, macrophages can be anchored in scFvs for tumor antigens. The patient can then receive IFN- [gamma] and macrophages can be polarized toward the M1 phenotype for tumor clearance.
6 is a schematic diagram showing the macrophage activation factor _MD2 (BIME _MD2 ). Toll-like receptor 4 dimerization can be triggered using scFv for the hydrophobic pocket of the MD2 protein. Next, BIME can be added to anchor macrophages in tumor cells.
Figure 7 is a schematic diagram illustrating toll-like receptor signaling.
Figure 8 shows the HGPRT biochemical pathway.
Figure 9 is a graph illustrating HGPRT protein surface expression compared to the other APRT and dCK two bypass pathway enzymes. (b) confirmed the presence of HGPRT on the cell surface using flow cytometry.

TK1 and HPRT were exclusively expressed on the surface membrane of tumor cells and led to the development of a monoclonal antibody range for human TK1 and HPRT. The specific binding capacity of these specific monoclonal antibodies can be used in macrophages transfected with modified macrophage-specific antigen receptors to treat cancer patients. Methods for modifying monocytes / macrophages to have receptors for human TK1 (MOTO CAR) can involve the generation of TK1 and HPRT specific human / humanized monoclonal antibodies (FIG. 1). These TK1 and HPRT-specific monoclonal antibodies are derived from the macrophage (MO) signaling domain (FIG. 2) (eg, the Toll-like receptor (TO), FC gamma III, IL-1 or INF- Can be used to generate a chimeric antigen receptor (CAR) by fusion of a short chain variable fragment to the cytoplasmic domain portion (Figure 7) (Figure 3a, b). The premise is that monocytes / macrophages are removed from the patient and transfected in vitro with a macrophage-specific chimeric antigen receptor lentiviral vector. This will allow macrophages to recognize and bind to cells expressing TK1, HPRT, or any other tumor antigen on their surface membrane and stimulate macrophage activation and cancer cell death. Since TK1 is present on the surface of many different tumors and is not present on the surface of normal cells, it can be used to treat many different types of cancer.

MOTO-CAR construction

cDNA is purified from monoclonal antibody hybridoma cells (CB1) having antibodies specific for human TK1 and used to amplify the heavy and light chains of the CB1 variable region through polymerase chain reaction (PCR). Sequences of 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 generate short chain fragment (scFv) using a G4S linker. The G4S linker codon optimized for yeast and humans using the codon optimizer provided by IDT ( https://www.idtdna.com/CodonOpt ) to maximize protein expression. CB1 scFv was digested with restriction enzymes and inserted into pMP71 CAR vector.

TK-1 and HPRT-specific human scFv antibodies were isolated from the enzyme 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 TK-1 and HPRT-specific antibody clones were added to the library to screen. TK-1 and HPRT antibody clones were alternatively stained with streptavidin or anti-biotin microbeads and concentrated using a magnetic column. Two further sorting and sorting were performed to isolate TK-1 and HPRT specific antibodies. For final screening, possible TK-1 and HPRT antibody clones and their individual proteins are alternatively labeled with an anti-HA or anti-c-myc antibody that is fluorescently conjugated to isolate TK-1 and HPRT specific antibodies (FACS) using fluorescence activated cell sorting (FACS). High affinity clones were selected for CAR construction. Humanized antibodies from other human antibodies or other animals can be altered or selected to be TK-1 or HPRT-specific using phage display or other recombinant methods.

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

Isolation and characterization of protein - specific antibody fragments.

Referring to FIG. 9, 10 ⁵ yeast was incubated with 2.5 μg of the protein of interest labeled with the fluorescent tag APC. The higher left (red) peak means a yeast population that is not bound to the protein of interest (our negative control). The lower left (blue) peak on the left illustrates the yeast that does not express their surface protein while the higher (blue) peak on the right refers to the binding of antibody fragments expressed in the protein of interest.

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

discovery

(E.g., murine) monoclonal antibody-derived scFvs for HPRT and TK1 that can use appropriate genetic manipulations to manipulate macrophage lymphocytes ultimately derived from a patient to treat diseases, such as cancer, without limitation, And the use of CAR or BiTE generated with one is an aspect. The fact that HPRT and TK1 are present on the surface of cancer cells but not on the surface of any normal cells is a major part of the discovery because this knowledge can be used to derive lymphocytes specifically into tumor cells.

As an aspect of the present system, using antibodies specifically raised against human HPRT or TK1, CAR and BiTE are targeted against the tumor by believing that HPRT and TK1 are expressed on the surface of human cancer cells and not on the surface of normal cells Based on the discovery that it can be used to Although T cells have been extensively used in CAR therapy as a result of varying T cells, genetically modified macrophages using a scFv derived from a unique antibody attached to the cytosolic domain of a toll-like receptor, such as toll-like receptor 4, Is also proposed. This unique approach overcomes many unique problems associated with current T cell CAR technology. Utilizing the killing power of macrophages induced by specific unique targets on tumor cells enables enhanced response without major disadvantages, such as cytokine storms, memory activation, and off-target targeting problems.

One aspect is to couple the potential of a specific monoclonal antibody to a human tumor antigen to the activated receptor of the patient &apos; s macrophage to ensure a localized M1 response that is specifically induced in the tumor. Such applications include, but are not limited to, humanization or mouse monoclonal antibodies against HPRT, TK1 or other tumor antigens that can be used with appropriate genetic engineering to manipulate macrophages, neutrophils or other immune cells from patients, Lt; RTI ID = 0.0 &gt; biTE &lt; / RTI &gt; The scFv from the humanized mouse monoclonal is engineered to attach to the transmembrane and cytoplasmic domains of TLR4, resulting in the TLR4 macrophage chimeric antigen receptor. The fact that HPRT is present on the surface of cancer cells and does not exist on the surface of any normal cells indicates that this knowledge and these techniques specifically induce (using the HPRT monoclonal portion) and activation (TLR4 (Using the cytoplasmic domain portion), which is a major part of the discovery.

It is clear that macrophages play a significant role in cancer progression and that immunotherapy, including macrophages, should be included in the treatment of this disease. Polarization of macrophages with M1 responses with minimal side effects can be a powerful therapy for solid tumors. Inflammatory signals such as LPS or TNF-a can easily polarize macrophages into the M1 phenotype in vitro. However, in vivo, materials such as LPS and TNF-a exacerbate the systemic inflammatory response involving cells of the innate and acquired immune system. They can cause fever and inflammation in some tissues, including mucosal surfaces and the lungs. These inflammatory signals are also highly cytotoxic (Apostolaki, Armaka, Victoratos, & Kollias, 2010; Kolb & Granger, 1968; Michel & Nagy, 1997). Immunotherapy requires activation of the immune system, but it is difficult to find cytokines, chemokines, compounds, or biological materials that do not produce some side effects. Macrophages belong to the innate immune system and exhibit proinflammatory and anti-inflammatory properties, so they are ideal immunotherapy candidates.

Although the present invention has been described with reference to certain specific embodiments and examples, many variations are possible without departing from the scope and spirit of the invention, and as set forth in the claims, the present invention may be embodied in other forms without departing from the spirit of the invention, Those skilled in the art will understand that all changes and modifications are intended to be included herein.

Table A

Table B of the reference literature

Claims (23)

As a therapy for treating a disease,
The disease is characterized by an antigen associated with a diseased cell that is not associated with normal cells,
Treatment with macrophage cells with a transfected chimeric antigen receptor (CAR) containing a fusion of a single-chain variable fragment (scFv) of a monoclonal antibody to the signaling domain Thereby killing the diseased cell,
Wherein said monoclonal antibody is specific for said antigen. 2. The method according to claim 1, wherein the disease is cancer. The therapy according to claim 1, wherein said antigen is TK1 (Thymidine Kinase 1) and said monoclonal antibody is specific for TK1. The therapy according to claim 1, wherein the antigen is Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and the monoclonal antibody is HGPRT specific. As a therapy for treating a disease,
The disease is characterized by an antigen associated with a diseased cell that is not associated with normal cells,
Macrophasia with a bispecific T-cell engager (BiTE) in which a short chain variable fragment (scFv) derived from a monoclonal antibody is fused to an scFv derived from another antibody that binds to macrophages through the macrophage signal transduction domain And destroying the diseased cell by treatment with the cell,
Wherein said monoclonal antibody is specific for said antigen. A method of treating a tumor, comprising treating the tumor with an immune cell having a MOTO-CAR vector (scFV fused to a toll-like receptor intracellular activation region), wherein the scFV is a monoclonal antibody specific for a tumor- Lt; / RTI &gt; antibody. A method of treating a tumor, comprising treating with a macrophage immunocell with a MOTO-CAR vector (scFV fused to an activation region in a toll-like receptor cell), wherein the scFV is derived from a monoclonal antibody specific for a tumor- Lt; RTI ID = 0.0 &gt; a &lt; / RTI &gt; 13. The method of claim 12, wherein the antigen is present on or in a tumor cell. 14. The method of claim 13, wherein the antigen is present on the surface of the tumor cells. 13. The method according to claim 12, wherein the antigen is TK1 or HPRT (Hypoxanthine Guanine Phosphoribosyltransferase). A method of treating a disease comprising treating with a macrophage immunocell with a MOTO-CAR vector (scFV fused to an activation region in a toll-like receptor cell), wherein the scFV is derived from a monoclonal antibody specific for a tumor- Or a pharmaceutically acceptable salt thereof. As a method of treating a tumor, one of a macrophage, monocyte, leukocyte, lymphocyte, and dendritic cell comprising a transduced chimeric antigen receptor (CARS) comprising a fusion of a short chain variable fragment (scFv) of a monoclonal antibody to the signaling domain Lt; RTI ID = 0.0 &gt; transformed &lt; / RTI &gt; A bispecific macrophage inducer (BIME) comprising a macrophage activator protein or scFv linked by an amino acid spacer to an scFv against a tumor antigen. 13. The method according to claim 12, wherein the modified immune cell is a monocyte, wherein the monocyte further comprises a macrophage-specific promoter which becomes a macrophage after migration from the blood to the tissue. 13. The method of claim 12, wherein the signal transduction domain is a signal transduction domain other than a cytoplasmic domain portion from a toll-like receptor. 13. The method of claim 12, wherein the monoclonal antibody is human or mouse. 13. The method of claim 12, wherein said immune cells are macrophages stimulated by co-stimulatory molecules. 18. The method of claim 17, wherein said co-polar molecule is MD2. A method for modifying a macrophage immune cell to have a receptor against human TK1 or HPRT,
Producing a monoclonal antibody specific for TK1 or HPRT,
Generating a chimeric antigen receptor (CAR) by fusion of a short chain variable fragment (scFv) of said monoclonal antibody to a signaling domain; and
And transducing said CAR into said immune cell. 20. The method of claim 19, wherein the signal transduction domain is a cytoplasmic domain portion from a toll-like receptor. 20. The method of claim 19, wherein the toll-like receptor is TLR4. A modified immune cell selected from one of macrophage, monocyte, leukocyte, lymphocyte, and dendritic cell comprising a transduced chimeric antigen receptor (CARS) comprising a fusion of a short chain variable fragment (scFv) of a monoclonal antibody to the signaling domain . 23. The modified immune cell of claim 22, wherein said monoclonal antibody is specific for TK1 or HPRT.

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