Tandem receptor CAR

Abstract

The present invention describes a bispecific tandem receptor CAR, called RfuCAR, that contains an scFv that recognizes and ligates surface molecules on neoplastic cells (CD33, CD123, or other tumor targets) and IL-1 receptor type II (IL-1R2). This selected IL-1R2 as the ideal receptor for the RfuCAR structure, allowing it to capture IL-1β with high affinity and specificity. These properties indicate that it is a good candidate for reducing the neurotoxicity and CRS effects of CAR-T therapy. Furthermore, this invention addresses methods for modulating the tumor microenvironment in other cancer types, including, but not limited to, acute myeloid leukemia, acute lymphoblastic leukemia, pancreatic, lung, and ovarian cancer.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, more precisely to the fields of immunology, cell, biology, and molecular biology, and describes the bispecificity of a tandem receptor CAR called RfuCAR. This includes tumor cells (CD33, CD123, or other tumor targets, not limited to but similar to CD19, mesothelin, and BCMA, etc.), surface molecules recognized above, and ScFv for ligation. Interleukin-1 receptor type 2 (IL-1R2), and methods for modulating the tumor microenvironment, such as in the case of acute myeloid leukemia, or other cancer types, not limited to but similar to acute lymphoblastic leukemia, pancreas, lung, ovarian cancer, etc.

Background Art

[0002] Interleukin-1 (IL-1α and IL-1β) is a prototype multifunctional cytokine different from other site cytokines, and can affect or not affect almost all cell types simultaneously with other site cytokines and small mediator molecules (DINARELLO, 1996). This site cytokine has a multifaceted speech effect as an immune and inflammatory mediator. (DINARELLO, 1996; APTE et al., 2002)

[0003] The main members of the IL-1 family are IL-1α, IL-1β, interleukin-1 receptor antagonist (IL-1ra), interleukin-1 receptor type I (IL-1RI), interleukin-1 receptor type II (IL-1RII), and interleukin-1 receptor accessory protein (IL-1RAcP) (DINARELLO, 1996, DUNN et al., 2001). Other site cytokines such as IL-18, IL-1F5, IL-1F6, IL-1F7, IL-1F9 are also included in the IL-1 family (DUNN et al., 2001; DINARELLO, et al., 2010).

[0004] The IL-1 family are site kine that regulate inflammatory responses in response to pathogen-related tissue damage and / or danger-related molecular patterns; therefore, they are major mediators of innate immune responses (Weber, et al., 2010).

[0005] IL-1 signal transduction and expression are tightly regulated events involving gene expression regulation, synthesis, secretion, and control of surface receptors, soluble receptors, and receptor antagonists (DINARELLO, 1996). This site kine has numerous effects, including fever, increased acute liver response, increased metastasis, angiogenesis, increased antibody and lymphokine production, cartilage destruction, fibroblast proliferation, smooth muscle and mesenteric cell proliferation, and increased HIV-1 gene expression (AURON, 1998).

[0006] IL-1 mediates the increase of endothelial adhesion molecules that promote the migration of neutrophils to tissues and also affects the metastatic niche (Vidal-VANACLOCHA et al. 1996). This site kine is also involved in angiogenesis and vascular endothelial growth factor (VEGF) production (COXON, et al., 2002; VORONOV, et al. 2003; SONG et al. 2003). In patients with acute myeloid leukemia (AML), levels of all members of the IL-1 family are elevated, and normal progenitor cell clonal genicity and disease progression are significantly suppressed (Carey, et al. 2017). Despite high sequence similarity between IL-1α and IL-1β (DUNN et al., 2001), there are significant differences in their biological effects. IL-1α suppresses tumor development by inducing antitumor immunity, while IL-1β promotes host tumor angiogenesis and immunosuppression, among other aggressive effects (Song, et al. 2003). This protumorogenic effect of IL-1β has been observed in various cancer models, including melanoma, breast cancer, and prostate cancer, as well as in tumor cell and macrophage cultures (VORONOV, et al. 2003).

[0007] Deregulation or overactivation of the IL-1 receptor is a potential cause of dangerous and harmful local or systemic inflammatory responses, as well as autoimmune or allergic reactions (BONECCHI et al. 2016). More recently, the roles of IL-6 and IL-1 in CRS and the neurotoxicity induced by CAR-T therapy have been demonstrated, showing that IL-1 is a better target for controlling both of these adverse events (NORELLI, et al. 2018; GIAVRIDIS, et al. 2018; TARASEVICIUTE, et al. 2018). Blocking IL-1 expression in myeloma patients reduces IL-6 production and extends progression-free survival (LUST, et al. 2009). IL-1 receptor antagonists (IL1-RAs) are used to treat a variety of diseases, including rheumatoid arthritis, asthma, septic shock, graft-versus-host disease, Alzheimer's disease, arteriosclerosis, multiple myeloma, and adult T-cell leukemia (HALLEGUA, 2002). There is sufficient evidence to support IL-1 occlusion as an excellent approach for treating metastatic patients (DINARELLO, et al. 1991).

[0008] IL-1 receptor IL-1 has two major receptors, type I and type II, located on the long arm of chromosome 2 and encoded as 552 amino acids, 80 kDa, and 336 amino acids, 68 kDa (DINARELLO, 1996; DINARELLO, et al. 1991). These two receptors are members of the immunoglobulin superfamily, each consisting of three IgG-like regions, and share significant homology to each other (DINARELLO, 1996). Type I receptors are found primarily in T cells, endothelial cells, keratinocytes, hepatocytes, and fibroblasts, while type II receptors are found in neutrophils, B cells, and myeloid cells. However, some cells may be able to express both types (DINARELLO, et al. 1991).

[0009] IL-1R1 and IL-1R2 have different affinities to the three major ligands of the IL-1 family (IL-1α, IL-1β, and IL-1Ra). IL-1R1 binds to IL-1α with high affinity, while IL-1R2 binds to IL-1β with high affinity. Furthermore, IL-1R2 binds IL-1Ra 100 less efficiently than IL-1R1 (MANTOVANI, et al. 1998; COLOTTA, et al. 1993). IL-1R2 shares 28% amino acid homology with the extracellular portion of IL-1R1, but lacks a TIR region and has a cytoplasmic tail of only 29 amino acids (BONECCHI, et al. 2016; MCMAHAN, et al. 1991). IL-1R2 cannot transduce the decoy receptor singnal (AURON, 1998; Thomas, et al. 2014). IL-1 acts on bone marrow monocytes via IL-1R1 and IL-1R2, inhibiting the site-caine activity that acts as a trap for IL-1 agonists (COLOTTA, et al. 1993). Type II soluble IL-1R blocks the interaction between mature IL-1β and the type 1 IL-1 receptor by inhibiting IL-1β in two steps and preventing propeptide processing (SYMONS, et al. 1995; BOURKE, et al. 1995).

[0010] IL-1R2 levels increase in the presence of glucocorticoid hormones (such as dexamethasone), prostaglandins, aspirin, Th2 site kines, IL-10, and IL-27, contributing to immunosuppressive and anti-inflammatory activities (BONECCHI et al. 2016, RE. et al. 1994). The anti-inflammatory effects of IL-1R2 have been demonstrated in several diseases, including chronic dermatitis (RAUSCHMAYR et al. 1997), arthritis (BESSIS, et al. 2000; DAWSON, et al. 1999; ATTUR, et al. 2000), endometriosis (KHOUFACHE, et al. 2012; BELLEHUMEUR, et al. 2005; GUAY, et al. 2007), heart transplantation (SIMEONI, et al. 2007), and autoimmune myocarditis induced by Th17 cells (CHANG et al. 2013).

[0011] Spacer and Linker CAR-mediated T cell recognition is defined by the antibody region and is independent of MHC expression. This recognition extends to any target the antibody can target (CHMIELEWSKI, et al. 2013). This interaction is strongly influenced by the structure and density of the target molecule on the tumor, and the location of the epitope, indicating that the arrangement between scFv and the T cell membrane is important and needs to provide flexibility (HUDECEK, et al. 2015). The length of the spacer may vary depending on the target molecule, and this logical reasoning can also be applied to the region between the two recognition sites of Bee-specific CARs (HUDECEK, et al. 2015; GRADA, et al. 2013; HEGDE, et al. 2016). Furthermore, the properties of the spacer are important factors that influence the capabilities of CARs in modulating transgenic cell phenotype, activation state, mobility, and tumor recognition (WATANABE, et al. 2016; NORELLI, et al. 2016).

[0012] Many spacer combinations have been tested based on the literature, and their suitability is directly related to the disease being treated and the chosen target (CHMIELEWSKI et al. 2013). GRADA et al. showed that in CD19 and Her2 tandem CARs, Her2-scFv must be positioned at a multimembrane location and CD19-scFv at a distal location to allow for more relaxed co-binding (GRADA, et al. 2013). CARs targeting carcinoembryonic antigen (CEA) exhibit the same behavior as CD19-ScFv, showing higher T cell activation when targeting epitopes close to the cell membrane (HOMBACH, et al. 2000). This has also been reported for CARs targeting other epitopes (GUEST, et al. 2005, James, et al. 2008).

[0013] These findings suggest that a dynamic separation model targeting distal membrane epitopes increases CAR ligand clusters, leading to stronger input from phosphatase molecules in synapses and suppressing TCR signaling transduction compared to proximal epitopes (DAVIS, et al. 2006). However, this model does not rule out the need for accessible and flexible epitopes. This indicates that the optimal target epitope and binding affinity for optimal CAR T cell activation have so far been evaluated empirically in each case (CHMIELEWSKI et al. 2013).

[0014] Specific requirements for spacers or non-antigen-binding components of CARs within the extracellular domain must be carefully selected and demonstrated in vitro and in vivo (HUDECEK, et al. 2015). Short spacer sequences derived from CD8α can cross the scFv into the intracellular signal transduction region (PORTER, et al. 2011; KALOS, et al. 2011) of the IgG1 hinge and Fc (HUDECEK, et al. 2015; SAVOLDO, et al. 2011) or without a spacer (REN-HEIDENREICH, et al. 2000; Moritz, et al. 1995). Patel (Patel, et al. 1999) reported that CARs targeting HIVenv containing CD7 or IgG1-derived spacers showed optimal cytolysis compared to CARs containing CD8, CD4, or IgG1-derived spacer truncation. He emphasized that spacer selection is disease-dependent and must be target-specific.

[0015] Previous studies have shown that even when spacer regions provide flexibility to the extracellular region, increasing its distance from membranes and other antigen-binding regions, they may still inhibit T cell activation, indicating that improved binding does not necessarily lead to increased CAR signaling (HOMBACH, et al. 2000; Hawkins 2014). Controversial data in the literature vary strongly among different target CARs, highlighting the need for in vitro and in vivo putative structure testing.

[0016] Linker, Spacer, Hinge Gly - Ser Linker GRADA (GRADA, et al. 2013) constructed a tandem CAR for CD19 and HER2. The spacer / linker between the two recognition motifs was a series of glucose and serine amino acids. Tandem repeats occur in at least 14% of proteins with fewer than 2,000 residues and do not form standard secondary structures such as α-helices or β-sheets (MATSUSHIMA, et al. 2008). The high flexibility and non-cleavability of the Gly-Ser tandem repeat indicates that the movement of the CAR subunit is almost free (MATSUSHIMA, et al. 2008). The tandem repeat appears to enable structural flexibility that allows for interactions with a variety of ligands, including metal ions and other proteins (MATSUSHIMA, et al. 2008).

[0017] IgG1 IgG1 is the most abundant immunoglobulin class and is widely applied, for example, as a spacer in CAR structures (VIDARSSON, et al 2014). The spacer consists of the entire IgG1 FC region (CH2CH3), the FC region and hinge, or the hinge alone. The hinge is 15 amino acids between the CH1 and CH2 regions (VIDARSSON, et al 2014). Guest et al showed that the IgG1 Fc region space is unnecessary for optimal function of CD19 CARs (GUEST et al. 2005) and can abolish the efficacy of CD19 CAR-T cells in mice (ALMASBAK, et al 2015). Also, Hombach (HOMBACH, et al 2000) showed that the extracellular IgG1 region impairs antigen-dependent cell activation in anti-CD30 models. On the other hand, several authors have proposed efficient CAR structures using the IgG1 Fc region. For example, CAR-PSCA and CAR-MUC1 (ATHAPAN et al. 2014), NGFR-spaced CD44v6, NGFR-spaced CD19, and NGFR-spaced CEA (CASUCCI, et al. 2015, CASUCCI, et al. 2018), and CAR-PSA (WATANABE et al. 2016). Moritz (Moritz, et al. 1995) tested various structures using hinge regions between functional CAR regions. One concern with IgG1 Fc adaptation is that this region is responsible for complement cascade activation, which is enhanced when the antibody Fab region is deleted (WANG, et al. 2016). Despite the estimated effect of the FC chain on immune activation, experimental results using IgG1 suggest it may be a good spacer candidate, although its effect may only be detectable in experimental assays.

[0018] IgG4 The IgG4 class is less abundant than IgG1 and has a structure very similar in hinge region size (12aa) (VIDARSSON, et al 2014). IgG4 antibodies are often formed after repeated or prolonged exposure to the antigen in a non-infectious environment (VIDARSSON, et al 2014). IgG4 is functionally monovalent and does not appear to be very suitable for undesirable crosslinking (AALBERSE, et al 2002). Due to these properties, the IgG4 Fc and hinge are already applied to the CAR structure. Generally, IgG4 sequences consist of a hinge and Fc CH2CH3 region (Qin, et al 2017) applied or sequences from other IgGs. Replacing the first six amino acids of the CH2 region of IgG4 (APEFLG) with the corresponding five amino acids of IgG2 (APPVA) inactivates Fc receptor binding, which is necessary for tumor recognition in vivo (HUDECEK, et al 2015). The need for alteration of the IgG4 CH2 region has been reported by other authors, and mutations of the CH2 region by altering two sites (L235E; N297Q) can reduce Fc receptor binding without mediating antigen-specific lysis (JONNALAGADDA et al. 2015) because the deletion is incorporated. Deletion and mutation of the IgG4 CH2 region eliminated cytotoxicity and significantly reduced complement activation by IgG4 (Montano, et al 2002, DORAI, et al 1992).

[0019] Other spacers The transmembrane and hinge regions of CD4, CD8, and CD28 are widely applied to CAR structures (NORELLI, et al 2016). In CD19 CARs, the hinge and transmembrane regions of CD8α or CD28 have similar functions in mice, although CD8α appears to have lower levels of inflammatory site kine production and activation-induced cell death (REN-HEIDENREICH, et al 2000, ALABANZA, et al 2017). These motifs are typically applied as spacers between the scFv and T cell membrane of single-specific CARs.

[0020] Acute myeloid leukemia CAR spacer trial Based on the above information, we select the IgG4 Fc region and hinge (hinge only, hinge-CH2CH3, and CH2CH3 only) to be applied as a spacer between the scFv receptor and the IL1-R2 receptor in the RfuCAR structure, including variations. As previously mentioned, this spacer is unresponsive and does not appear to affect the normal function of the CAR (JONNALAGADDA, et al 2015, Montano, et al 2002, DORAI, et al 1992, Jena, et al 2010). It is also important to associate the spacer with the target disease of RfuCAR. Therefore, for other tumor targets, this option may be modified by other spacers that are more appropriate. The disease to be treated first is acute myeloid leukemia (AML) with anti-CD33 and anti-CD123 CARs. In this regard, the use of IgG4 Fc and hinge has also been tested with other structures. Many authors have discussed CD33 (KENDERIAN, et al 2015), CD123 (MARDIROS, et al 2015; THOKALA, et al 2016), and other targets (LABORDA, et al 2017) in relation to CAR-Ts against AML (KENDERIAN, et al 2017) (CD33 and CD123 Novartis patents).

[0021] Conventional technology The use of recombinant chimeric T cell receptor antigens expressing CD33 and / or CD123 antigens in cancer treatment has attracted considerable attention in the scientific community in recent years. For example:

[0022] US 2013 / 280220 A1 refers to a computational modeling tool that guides the design and construction of novel single CAR molecules (TanCARs) that can individually recognize each target molecule and mediate bifacial-specific activation and T cell targeting. Accordingly, paragraph 4 of this document states: The present invention is directed to a method and configuration relating to cell therapy. In particular, the cell therapy is for cancer, including solid tumors. However, the inventors use glycine, serine, or both as linkers. The structure of the present invention, in addition to having different targets, uses the IgG4 FC region as a spacer between the scFv receptor and the IL1-R2 receptor and the CD8 hinge, and as a spacer between the scFv and the T cell membrane in the RfuCAR structure.

[0023] Document No. WO 2014 / 186469 A2 relates to a method and composition of immunotherapy using modified T cells composed of clinical-grade antigen chimeric (CAR) receptors that can be directly administered for cancer treatment. Such modifications allow this invention to recycle effector function within the tumor microenvironment. However, the inventors use a transpososystem to transduce T cells and utilize lentiviral vectors. Furthermore, instead of constructing CARs using two different receptors simultaneously for different targets, they fused mutant IL15 and CD19 receptors within the structure.

[0024] Document No. WO 2014 / 055442 A2 relates to the composition and methods of treatment for human cancer. The methodology in this document describes a method for inducing cells into the tumor microenvironment. This method involves a chimeric antigen receptor (CAR) composed of an antigen-binding domain, a transmembrane domain, a costimulatory signal transduction domain, and a zeta-CD3 signal transduction domain on which the antigen-binding domain binds to stromal cell antigens. The inventors of this patent aim to influence the tumor microenvironment, but attempt to achieve this in a different way than the RfuCAR of the current invention. They want to reduce IL-1 present in the microenvironment, but the current invention consists of an antigen-binding domain that binds to stromal cell antigens such as FAP (fibroblast-activating protein).

[0025] Document WO 2017 / 222593 A1 relates to the composition and methods associated with chimeric antigen receptors. More precisely, it relates to genetically modified cells in which the chimeric antigen receptor is directed to at least two targets (e.g., CD33 and CD123). However, such documents show an alternative method for assembling CARs. In the same CAR structure, two antigen recognition sites are inserted, and furthermore, a fusion protein is placed in the structure to function as an enhancer. Our CAR presents two different receptors linked to each other. A switch-off mechanism is realized via a suicide gene, and RfuCAR has an on / off switch, allowing for fine-tuning of the response.

[0026] U.S. Patent No. 9,815,901 B2 is related to the treatment of diseases associated with CD123 expression. For this reason, this document proposes a method of administering genetically modified cells expressing a CD123 binding region and is related to a chimeric antigen receptor specific to CD123. Such a document is different from the current invention in that it does not suggest the structure of the receptor, but only mentions the recognition of the CD123 antigen for the treatment of diseases associated with the expression of such antigens. Furthermore, this construct uses Glycine / Serine as a linker. Also, a CAR targeting the antigen recognition sites of CD123 and CD19 was constructed. The activation of this CAR occurs when two antigens are bound by the receptor. To control toxicity, the authors have proposed a dimerization switch. In this switch, the signal transduction generated by the recognition of CART is transmitted only when there is a molecule that dimerizes two CART moieties. In the current invention, the regulation of the immune microenvironment through the isolation of IL-1 from the tumor microenvironment and the possibility of toxicity management by administering peptides directed to the described receptor are proposed.

[0027] Document No. WO 2017 / 173256 A1 is related to compositions and methods composed of genetically modified immune cells expressing an antigen (CAR) receptor or a T cell receptor (CAR-T) aimed at killing cancer cells. This document mentions immunotherapy with CAR-T, but it is not specific to the recognition of CD33 and CD123 antigens and does not aim at constructing a chimeric receptor using genetically modified cells as in the current invention. Furthermore, the CAR structure composed in this document is composed of a single antigen site and a truncated hinge region. The goals used in such a document are different from the current invention.

[0028] Document WO 2015 / 164594 A1 is related to chimeric antigen receptors directed against cells expressing antigens. In this document, treatments modifying T cells are referred to, but these do not necessarily pertain to the recognition of CD123 or CD33 antigens. Furthermore, such a document is related to CART cells targeting the EGFR antigen. Furthermore, a second transgene, which is a sequence expressing an IL15 / IL15Ra fusion protein, is inserted. Its structure was made for the work through the target density.

[0029] The document titled "Cancer Therapy by Engineering Chimeric Antigen Receptor-T Cells" analyzes the treatment using chimeric antigen receptor (CAR) cells that have been successful in treating B-cell malignancies and emphasizes its great potential in anti-tumor therapy. CAR-T cells can be designed to specifically kill malignant cells or modify the tumor microenvironment. To do so, they release soluble factors that regulate the functions of stromal cells and immune cells, providing a powerful tool to target multiple components of the tumor ecosystem. Such a document is a review of the literature on CART cell technology. It explains the concepts of the tumor ecosystem, cancer immune phenotypes, and the T-cell exhaustion mechanism in immune evasion. It also explains the functional challenges of CART cell technology. The immune regulation mechanism proposed in the current invention is cited at all times.

[0030] The document titled "Perspectives on Chimeric Antigen Receptor T-Cell Immunotherapy for Solid Tumors" is related to a specific chimeric antigen receptor CAR and may cause robust activation of T cells leading to the death of target tumor cells. This document explains the recent approaches and technological innovations for the genetic reconstruction of CAR T cells to combat the suppressive effects found in the tumor microenvironment. Furthermore, it is mentioned that the treatment of solid tumors is difficult and the tumor microenvironment may contribute to this problem. Therefore, this publication supports the current invention.

[0031] The document, "T cells expressing a CD123-specific chimeric antigen receptor exhibit specific cytolytic effector function and antitumor activity against human acute myeloid leukemia," relates to a CD123-specific chimeric antigen receptor expressed on T cells, exhibiting specific cytolytic effector function and antitumor activity against acute myeloid leukemia. As mentioned above, the receptor proposed in this document is specific to the recognition of the CD123 antigen. However, such documents are experimental articles and are designed to use two characteristic epitopes for CD123 in the structure. The CART design in this article is similar to ours, which uses our IgG4 Fc receptor hinge, CD28 as a co-stimulator and CD3 zeta as a signal transduction exchanger, but our CART design also explores the use of a second co-stimulatory molecule, 4-1BB. Another major difference in our CART is the immune microenvironment regulatory mechanism we propose. The CART design for the document does not take into account mechanisms related to optional functions.

[0032] A document titled "Cell Switching: A Novel Modular Platform for Retargeting T Cells to AML Blasts" describes a proposed treatment for AML where CD33 and CD123 are expressed either individually or in combination in patients with acute myeloid leukemia (AML), and subsequently, chimeric T cell antigen receptors expressing both CD33 and CD123 are used. This document is very similar to the current invention, as it suggests the use of recombinant T cells that recognize CD123 or CD33 antigens in the treatment of acute myeloid leukemia. Such papers have shown modular CAR structures with dual-targeting modules for tumor antigens that can be switched on / off by the appropriate structure. The present invention is constructed using two very different receptors, one for tumor antigens and one for IL-IRA.

[0033] As described above, the proposed structure (RfuCAR) in the current application is concluded to be different from other structures. This is because it can modulate the tumor microenvironment and is composed of a mechanism that can be switched off by the administration of both receptor epitope peptides. IL-1R2, which has not yet been used for this purpose, is used as the receptor to constitute a regulatory safety switch other than the tumor receptor target. If a toxic CAR-T effect is detected in the patient, the action of RfuCAR cells can be temporarily switched off and modulated by the administration of various peptides linked to IL-1R2, IL-1-IL-1R2, and / or scFv epitopes. These peptides can link two RfuCAR cells to tumor cells to inhibit ligation, or inhibit or modulate RfuCAR activity and / or bind to IL-1. The RfuCAR cells can proliferate in the patient and kill tumor cells once the peptide administration is discontinued. Therefore, the structure and mechanism of this invention represent a novel approach that controls the tumor microenvironment not only through cellular signaling and transduction pathways, but also through molecules secreted by macrophages and other cells.

[0034] Outline of the invention This invention aims to propose a dual capability of a tandem receptor CAR named RfuCAR. This involves scFv, which recognizes and ligands surface molecules on tumor cells (CD33, CD123, or other tumor targets such as CD19, mesothelin, BCMA, etc.) and IL-1 receptor type 2 (IL-1R2), and a method for modulating the tumor microenvironment.

[0035] RfuCAR The structure and mechanism represent a novel approach to controlling the tumor microenvironment not only through cellular signaling and transduction pathways, but also through molecules secreted by macrophages and other cells. In silico and in vitro studies are being conducted to demonstrate the benefits of Celluris RfuCAR applications. [Brief explanation of the drawing]

[0036] [Figure 1] Figure 1 shows a schematic diagram of the RfuCAR structure. Here, A is the vector scheme and B is the Rfu scheme. [Figure 2] Figure 2 shows a scheme illustrating the switching and tuning of RfuCAR. Combinations of peptides can either turn off or modulate RfuCAR action. Three peptides—one targeting IL-1R and a tumor target, another targeting IL-1R-IL-1 and a tumor target, and both—can turn off RfuCAR. Administering only one peptide modulates RfuCAR action. [Figure 3] Figure 3 shows a schematic diagram of the estimated anti-CD33 RfuCAR. [Figure 4] Figure 4 shows the predicted structures (RaptorX) of the anti-CD33 scFv region, the IL1-R2 extracellular region, and the IgG4 Hine-CH2CH3 region. [Figure 5] Figure 5 shows predictive three-dimensional models of tested sequences of the extracellular region of anti-CD33 RfuCAR. The numbers in the boxes correspond to models 1 through 6. Two software programs, IntFold and RaptorX, are shown, each displaying the model best suited to its structure. [Figure 6] Figure 6 shows all the optimal quality plots for each model of the CD33-RfuCAR model (1-6). [Figure 7]Figure 7 shows the disordered plots of the anti-CD33 RfuCAR prediction models. Each plot represents the failure prediction for each model 1 through 6. [Figure 8] Figure 8 shows the estimated binding sites for RfuCAR Model 2. [Figure 9] Figure 9 shows a schematic diagram of the estimated anti-CD123RfuCAR. [Figure 10] Figure 10 shows the predicted structures (RaptorX) of the anti-CD123cFv region, the IL1-R2 extracellular region, and the IgG4 Hinge-CH2CH3 region. [Figure 11] Figure 11 shows predictive three-dimensional models of tested sequences of the extracellular region of anti-CD123 RfuCAR. The numbers in the boxes correspond to model numbers 7 through 12. Two software programs, IntFold and RaptorX, are shown, each displaying the model best suited to the respective structure. [Figure 12] Figure 12 shows all the optimal quality plots for each model of the CD123-RfuCAR model (7-12). [Figure 13] Figure 13 shows the disordered plots of the prediction models for anti-CD123 RfuCAR. Each plot represents the disordered prediction for models 7 through 12. [Figure 14] Figure 14 shows the estimated binding sites for the RfuCAR Model 8. [Modes for carrying out the invention]

[0037] Detailed description of the invention This invention describes the bispecificity of a tandem receptor CAR, named RfuCAR. This involves scFv, which recognizes and ligands surface molecules on tumor cells (CD33, CD123, CD19-like but not limited thereto, mesothelin, BCMA, and other tumor targets) and IL-1 receptor type 2 (IL-1R2), and a method for modulating the tumor microenvironment. Furthermore, such a mechanism can be switched off by administering both receptor epitope peptides, which constitute a regulatory safety switch (Figure 1A and B).

[0038] The scFv motif ligands tumor cells, leading to apoptosis identical to that of third-generation CARs. Secreted IL-1β binds to and traps IL-1R on RfuCAR, inhibiting its binding to IL-1R1 and IL-1 signaling transduction. This inhibition reduces IL-1 pathway activation, leading to modulation of tumor cell proliferation. Regulating IL-1 content in the microbial environment is thought to modulate other site-kine activation in the tumor environment, preventing the excessive secretion of site-kines observed in CRS and neurotoxic events.

[0039] If a toxic CAR-T effect is detected in a patient, the action of RfuCAR cells can be temporarily switched off and modified by administering various peptides linked to the IL-1R, IL-1-IL-1R, and scFv epitopes (Figure 2). These peptides can link two RfuCARs to tumor cells that inhibit ligation and / or conjugate IL-1 that inhibits or modulates RfuCAR activity. Once the peptide administration is discontinued, the RfuCAR cells can proliferate in the patient and kill tumor cells.

[0040] RfuCAR Estimation Structure The extracellular putative structure of RfuCAR was tested using two IntFold online structure prediction functions (MCGUFFIN et al. 2010; MCGUFFIN, et al. 2018; MCGUFFIN, et al. 2015; BUENAVISTA, et al. 2012; Roche, et al. 2012) and RaptorX (KA LLBERG, et al. 2012; MA, et al. 2012; Peng, et al. 2011; Peng, et al. 2011; MA, et al. 2013). These functions predict the putative structure by comparing it to the submitted primary structure with secondary and tertiary structural characteristics in the PDB database and provide a similarity score. They also provide disordered regions and putative binding sites.

[0041] The following points should be considered when interpreting the data: · RaptorX - Score: The alignment score is between 0 and the (region) sequence length, with 0 indicating the worst. In practice, the score may slightly exceed the sequence length due to estimation errors. - uSeqID and SeqID: These represent the number of identical residues in the alignment. SeqID is uSeqID normalized by the protein (or region) sequence length and multiplied by 100. A higher uSeqID (SeqID) indicates better results. A SeqID greater than 30% and a protein (or region) with more than 200 residues usually indicates that the predictive model has the correct fold. - uGDT and GDT: uGDT is, This is an unnormalized GDT (Global Distance Test) score defined as 1 * N(1) + 0.75 * N(2) + 0.5 * N(4) + 0.25 * N(8). N(x) is the number of residues (in Å) where a modeling error less than x is predicted. GDT is calculated by dividing uGDT by the length of the protein (or region) and multiplying by 100. uGDT (GDT) measures the absolute quality of the model. For proteins with more than 100 residues, uGDT > 50 is a good indicator. For proteins with 100 or fewer residues, GDT > 50 is a good indicator. If the model has a good uGDT (>50) but the GDT (<50) is incorrect, it indicates that only a small portion of the model may be correct. - P-value: The P-value assesses the relative quality of a model because a predictive model may perform worse than the best of a randomly generated set of models for this protein (or region). A smaller P-value indicates better model quality. For alpha proteins, a P-value less than 10^-3 is a good indicator. For Manly beta proteins, a P-value less than 10^-4 is a good indicator.

[0042] IntFold The results table is ranked according to the decrease in the global model quality score. The global model quality score ranges from 0 to 1. Generally, a score below 0.2 indicates a possible mismodeling of the domain, while a score above 0.4 generally indicates a complete and reliable model that closely resembles the native structure. Each model is also assigned a confidence level, color-coded according to its p-value.

[0043] [Table 1]

[0044] - The confidence score should be considered in conjunction with the quality of the local model (score per residue) and the range of the target protein by template / multi-template. The score per residue indicates the predicted distance (in Angstroms) between the CA atom of the residue in the model and the CA atom of the equivalent residue in the native structure. - A 3D cartoon view of a model color-coded by residual error according to the RasMol temperature coloring scheme. - Failure Prediction - This image shows a plot of failure probability (on the y-axis) for each numbered amino acid (on the x-axis) in the sequence. The threshold for disorder / order probability is shown as a dashed line on the plot. Residues above the threshold are mostly considered to be out of order or less, but this threshold is used only to guide the user. - Region Boundary Prediction - The image displays a predicted top-level 3D model color-coded to indicate the predicted region. - Color changes indicate potential region boundaries. - Binding Site Prediction - The image displays a predicted top-level 3D model with annotations indicating residues at the estimated binding site. The cartoon view of the model is shown in blue, and the binding site is shown as a blue bar with labeled residues. A list of binding residues is provided, along with the most likely (numerous) ligands, the ligand closest to the center of the predicted binding pocket, a list of potentially interacting ligands, and the number of ligands identified in the associated template structure.

[0045] RfuCAR anti-CD33 A tested model of RfuCAR anti-CD33 is shown in Figure 3, with the sequence used represented by SEQ. ID. No. 1 to 6. The color scheme is the same in the schematic view and the sequence.

[0046] In the tested sequences, the following three-dimensional predicted structures were generated for each region only (Figure 4) and for the estimated structure (Figure 5).

[0047] Clearly, the structure of Model 2 (represented by SEQ; ID. No. 2) maintains the correct structure of the components it contains. This is supported by the technical data reported by each software, summarized in Table 1 (regions only) and Table 2 (anti-CD33 RfuCAR models). Model 2 achieved the highest quality score (0.4465) in IntFold compared to the other models. While this score is not high, it is sufficient to indicate good structural prediction. Furthermore, analysis of the quality plots for all models (Figure 6) shows that the mismatch between the primary sequence of the model and the matched template is minor in Model 2, indicating more accurate prediction.

[0048] [Table 2]

[0049] [Table 3]

[0050] [Table 4]

[0051] Model 2 yields the best results for the structure predicted by both software. The matching template in the database also resembles the expected function of RfuCAR. Model 2 matches the structure of the IL-1 receptor complex and the scFv motif (Table 3 reports all matched templates), suggesting that this model likely maintains the structures of the anti-CD33 and IL-1R2 receptors. The only spacer applied in this model is the IgG4 hinge, but given the proportion of disorder and regions (Table 2, Figure 7), appropriate mobility of the motif seems possible. The most disordered regions, regions lacking normal secondary structure, and more flexible regions are located in the IgG4 hinge region and in the linker between the two chains of the scFv (anti-CD33) region. Other models have more disordered regions, resulting in lower accuracy in the predictive model or showing proteins with tertiary structures different from the prediction.

[0052] [Table 5]

[0053] [Table 6]

[0054] The predicted binding sites and locations for Model 2 are sites 50, 227, 228, 229, 272, and 360. The most likely ligands (type) for each site are ILE and FUL. The central ligands (TypeID) for each site are SER657 and FUL641. All ligands (type-frequency) within the cluster are GLY-1, TRP-1, GLU-2, ILE-3, PRO-3, SEL-2, THR-2, TYR-3, ASP-1, LEU-1, ASN-1, FUL-1, and FUC-1. The likely central ligands for each site are ILE650 and FUL641. The predicted binding sites are shown in Figure 8.

[0055] RfuCAR anti-CD123 A tested model of RfuCAR anti-CD123 is shown in Figure 9, with the sequence used represented by SEQ. ID. No. 7-12. The color scheme is the same in the schematic view and the sequence.

[0056] In the tested sequences, the following three-dimensional predictive structures were generated for each region only (Figure 10) and for the predictive structure (Figure 11).

[0057] Outwardly, the structure of Model 8 (ID. No. 8, represented by SEQ) maintains the proper structure of its constituent components. This is supported by technical data reported by each software collected in Table 1 (regions only) and Table 4 (Anti-CD123 RfuCAR model). This model has the second-best quality score in IntFold analysis (0.4404). Model 8's quality score is lower than Model 11's, and the quality plots have very similar distributions (Figure 12). Nevertheless, Model 8 has already proven to retain the CD8 hinge near the cell membrane constructed for anti-CD33 and anti-CD123 CA-TS. Model 11 lacks the CD8 hinge region.

[0058] Model 8 is also one of the best results from the RaptorX software. The matched template in the database is more similar to the expected function of RfuCAR. Model 8 matches the structure of the IL-1 receptor complex and the scFv motif (Table 3 reports all matched templates), suggesting that this model likely maintains the structures of the anti-CD123 and IL-1R2 receptors. The only spacer applied in this model is the IgG4 hinge, but given the disordered regions and rates (Table 11, Figure 13), it appears to allow for motif mobility. The most disordered regions, regions lacking normal secondary structure, and more flexible regions are located in the IgG4 hinge region and in the linker between the two chains of the scFv (anti-CD123) region. Models 7, 9, 10, and 12 have many highly disordered regions, indicating regions where predictive folding is not good.

[0059] [Table 7]

[0060] [Table 8]

[0061] The predicted binding sites and locations for Model 8 are 33 and 235. The most likely ligand (type) for each site is TYR. Each site + center point ligand (TypeID): PRO656. All ligands within the cluster (type - frequency): GLY-3, TRP-2, GLU-2, ILE-3, PRO-4, SER-3, ARG-1k, THR-2, TYR-5, ASP-1, LEU-1, ASN-4, ALA-2, CYS-1. The likely center point ligand for each site is TYR672. The predicted binding sites are shown in Figure 14.

[0062] Therefore, according to the reported predicted structures, the optimal choice for a spacer that is likely to be maintained in vivo is to make the structure of the RfuCAR region for anti-CD33 RfuCAR and Model 8 (SEQ ID. No. 8) anti-CD123 RfuCAR the same as Model 2 (SEQ ID. No. 2). Both contain an antitumor scFv and include an IL1-R2 receptor with an IgG4 hinge as a spacer between the receptor and the CD8 hinge, and a CD8 hinge as a spacer from the cell membrane.

[0063] Although the invention is well described, it is evident to those skilled in the art that various changes and modifications may be made to enhance the design without such changes falling outside the scope of the invention.

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Claims

1. A tandem receptor CAR characterized by comprising an extracellular domain composed of the sequence of SEQ ID No. 2, a CD28 transmembrane domain, a 4-1BB domain, and a CD3ζ domain.

2. The tandem receptor CAR according to Claim 1, characterized in that it has presumed binding sites at positions 50, 227, 228, 229, 272, and 360.

Images