Immune cells expressing reverse universal chimeric antigen receptors for targeting different multiple antigens, preparation methods thereof, and use thereof in treating cancer, infection, and autoimmune disorders

Abstract

The present invention relates to an immune cell-based anti-cancer therapeutic agent and a method for treating cancer using the same.

Description

[0001] The present invention relates to immune cell-based therapeutics and methods of using the same to treat cancer, infection, and autoimmune disorders.

[0002] Chimeric antigen receptor (CAR) is an artificial receptor consisting of a binding moiety that provides antigen specificity and one or more signal transduction chains derived from immune receptors (Cartellieri et al., J. Biomed. Biotechnol. doi: 10.1155 / 2010 / 956304 (2010)). The two main CAR domains are connected by a connecting peptide chain comprising a transmembrane domain, which anchors CAR in the plasma membrane. Immune cells, especially T and NK lymphocytes, can be genetically modified to express CAR inserted into their plasma membranes. If such CAR-modified immune cells encounter appropriate targets expressing CAR binding moieties or other cells or tissue structures decorated with appropriate targets of CAR binding moieties, then after the CAR binding moiety binds to the target antigen, the CAR-modified immune cells are cross-linked with the target. Cross-linking results in the induction of signal pathways by the CAR signal transduction chain, which will change the biological properties of the immune cells implanted by CAR. For example, CAR triggering in effector CD4+ and CD8+ T cells will activate typical effector functions, such as the secretion of lytic compounds and cytokines, which will ultimately lead to the killing of the corresponding target cells. Currently, adoptive transfer of immune cells engineered with chimeric antigen receptors (CARs) is considered a very promising therapeutic option for the treatment of otherwise incurable malignant, infectious or autoimmune diseases. To date, two CAR-T cell therapeutics have received marketing authorization for the treatment of B-cell-derived malignancies, demonstrating the clinical feasibility of this approach.

[0003] However, conventional CAR technology is accompanied by many key issues, which need to be resolved before this treatment can be widely used in clinical treatment. First, several safety issues must be addressed. So far, it has been difficult to control the immune response of T cells engineered with conventional CAR after infusion into the patient. In particular, unexpected target gene expression on healthy tissues may stimulate a rapid and stringent immune response of engineered T cells against healthy cells, which may lead to serious side effects (Lamers et al. J. Clin. Oncol. 24e20-e22 (2006), Morgan et al. Mol. Ther. 18: p. 843-851 (2010)). Accidental cross-reactions of CAR binding domains with antigens expressed on healthy tissues may also induce organ damage. Although this phenomenon has not been reported for CAR-T cells until now because many trials are still in the early stages, it has been observed in clinical trials using T cells genetically engineered to express recombinant T cell receptors (Linette et al., Blood 2013, Morgan et al. J. Immunother. 2013). In addition, since CAR-T cells are a new class of self-amplifying cell drugs, the infused T cells may undergo dramatic expansion in the presence of heavy tumor burden, leading to tumor lysis syndrome and cytokine release syndrome (Brudno and Kochenderfer, Blood 2016; Maude et al. Cancer J. 2014). Another disadvantage of conventional CAR technology is the limitation of engineered T cells to retarget a single antigen. This single treatment approach implies the risk of tumor escape variants that lose the target antigen during treatment. In clinical trials, the emergence of tumor escape variants has been observed after several months under conventional CAR T cell therapy (Grupp et al. N. Engl. J. Med. 368: 1509–1518 (2013)). In summary, these obstacles limit the application of CAR-T cells to very few indications. In fact, to date, examples of clinical effectiveness have been limited to CD19+ CAR-T cells.

[0004] Mardiros et al. disclose T cells expressing CD123-specific chimeric antigen receptors for human acute myeloid leukemia (AML) (Mardiros et al. Blood 122:3138-3148 (2014)). Interleukin 3 receptor alpha chain (CD123) is described as a potential immunotherapy target because it is overexpressed in AML compared to normal hematopoietic stem cells.

[0005] Lee et al. disclosed a chimeric antigen receptor (ACAR) based on "proliferation-inducing ligand" (APRIL) for dual targeting of B cell maturation antigen (BCMA) and transmembrane activator and calcium regulator and cyclophilin ligand (TACI) in multiple myeloma (Lee et al. Blood 131:746-758 (2018)). For use in CAR constructs, a truncated form of APRIL (residues 116 to 250) was used.

[0006] WO 2016154621 A1 discloses a switchable chimeric receptor comprising a non-antibody extracellular domain that interacts with a chimeric receptor binding partner presented on a switch. The switch comprises a chimeric receptor binding partner (preferably a protein or peptide, more preferably an antibody or antibody fragment) and a targeting moiety.

[0007] The modular "universal" CAR-T (UniCAR) approach can overcome these limitations by separating the antigen recognition and activation domains of the CAR into two separate operational units. T cells are engineered to express a CAR with a universal binding domain that recognizes a tag (Cartellieri et al. Blood cancer J. 2016; Rodgers et al. 2016 PNAS). Antigen specificity is provided by a soluble adapter protein consisting of the antigen binding domain fused to the tag recognized by the universal CAR.

[0008] WO 2012082841 A2 discloses T cells expressing universal anti-tag chimeric antigen receptors and methods for treating cell-related disorders such as cancer.

[0009] In addition, WO 2013044225 A1 discloses a universal immune receptor expressed by T cells, which is used to target a variety of antigens.

[0010] Both approaches describe the use of modified T cells expressing universal anti-tag immune receptors. These T cells can be redirected to disease-associated cell surface antigens by additionally applying modules that bind to these surface antigens and carry the corresponding tags. A problem with these approaches is that redirecting genetically modified T cells using exogenous tags is likely to be immunogenic, which could put patients at risk and negatively impact the effectiveness of treatment.

[0011] WO 2017112784 A1 describes the use of SpyCatcher and SpyTag for universal immune receptors of T cells, in particular, a nucleic acid sequence encoding a universal immune receptor, wherein the universal immune receptor comprises a SpyCatcher or SpyTag extracellular binding domain bound to an extracellular hinge region, a transmembrane domain, and a T cell receptor intracellular signaling domain. It also describes vectors and cells comprising the nucleic acid sequence, as well as an isolated universal immune receptor comprising SpyCatcher or SpyTag.

[0012] WO 2016030414 A1 provides genetically modified immune cells that allow for redirection of a variety of disorders in a safe and effective manner using endogenous nucleoprotein-based tags.

[0013] Mitwasi et al. disclose a novel targeting module (TM) for retargeting UniCAR T cells to disialoganglioside GD2-positive tumor cells (Mitwasi et al. Oncotarget 8: 108584-108603 (2017)). GD2-specific TMs were constructed by fusing the UniCAR epitope E5B9 to respective anti-GD2 scFvs, with different orientations (VL-VH or VH-VL) and spacer peptides tested in vitro and in vivo. UniCAR comprises the anti-La5B9 scFv, a hinge domain, a transmembrane domain, and a signaling domain.

[0014] However, the binding portion of these UniCAR T cells is still a single-chain variable fragment (scFv), and therefore an active binding domain. Such an active binding domain has several disadvantages. First, if the target tag is present on healthy tissue, it still has the risk of on-target / off-tumor activity. Second, there is a theoretical risk of cross-reactivity of the binding domain with non-target cell surface proteins, leading to off-tumor activation and potentially causing severe tissue damage. Finally, the coding sequence of scFv is quite long, which is disadvantageous for making genetically engineered cells.

[0015] Therefore, one object of the present invention is to provide a genetically modified immune cell that allows for redirection against a variety of diseases in a safe and effective manner using a small cell surface binding moiety, which will minimize the risk of cross-reactivity of CAR-modified immune cells and significantly shorten the coding sequence, thereby allowing genetic modification of immune cells with several CAR constructs. Another object of the present invention is to provide a method for treating a variety of cell-related disorders, wherein the length and intensity of treatment can be adjusted in a simple manner according to clinical needs.

[0016] The present invention provides a reverse universal modular chimeric antigen receptor (RevCAR) system that allows retargeting of RevCAR-implanted immune cells against multiple antigens. The system uses a gene therapy platform to generate immune cells capable of recognizing multiple antigens, which have broad and valuable clinical significance for the use of immune cell-based therapies, especially T cell- and NK cell-based therapies.

[0017] In a first aspect, the present invention provides an isolated nucleic acid sequence encoding a reverse universal chimeric antigen receptor, wherein the receptor comprises three domains, wherein the first domain is a peptide epitope tag that serves as a cell surface binding domain, the second domain is a connecting peptide chain comprising an extracellular hinge and a transmembrane domain, and the third domain is a signal transduction domain, wherein the peptide epitope tag that serves as a cell surface binding domain is a linear or conformational epitope.

[0018] The peptide epitope tag according to the present invention non-covalently binds to a tag binding domain, which may be an antibody or a ligand.Preferably, the peptide epitope tag comprises 10 to 20 amino acids.

[0019] Particularly suitable cell surface peptide epitope tags are human peptide sequences, particularly peptide sequences derived from human nuclear proteins (i.e., La protein), and most particularly peptide sequences that are known not to be targets of autoantibodies in autoimmune patients (i.e., the 5B9 epitope of La protein), thereby making the tag less likely to be immunogenic in the context of the reverse universal chimeric receptor.

[0020] Preferably, the nucleic acid according to the present invention is a nucleic acid encoding a reverse universal chimeric antigen receptor according to sequences SEQ. ID 24 to 27.

[0021] More preferably, the nucleic acid according to the present invention is the sequence SEQ. ID 1, 8, 10 or 12.

[0022] The optional fourth domain is a short peptide linker in the extracellular portion of RevCAR, which forms a linear epitope for a monoclonal antibody (mab) specifically binding to the fourth domain. The additional domain is not required for the function of the RevCAR system, but additional clinical benefits can be added to the present invention. Preferably, the present invention provides an isolated nucleic acid sequence encoding a reverse universal chimeric antigen receptor according to the present invention, wherein the nucleic acid sequence encodes an artificial chimeric fusion protein, and wherein the nucleic acid sequence is provided as cDNA.

[0023] On the other hand, the present invention provides an adapter module (AdMo) consisting of a binding portion having specificity for a certain human cell surface protein or protein complex and a binding portion having specificity for a peptide epitope tag that is a cell surface binding domain of the RevCAR according to the present invention.

[0024] In another aspect, the present invention provides a nucleic acid encoding an adapter module according to the present invention. Preferably, the present invention provides an isolated nucleic acid sequence encoding an adapter module according to the present invention, wherein the isolated nucleic acid is provided as cDNA.

[0025] In another aspect, the present invention provides a cell comprising a nucleic acid encoding a reverse universal chimeric antigen receptor according to the present invention, the reverse universal chimeric antigen receptor comprising three domains, wherein the first domain is a peptide epitope tag serving as a cell surface binding domain, the second domain is a connecting peptide chain comprising an extracellular hinge and a transmembrane domain, and the third domain is a signal transduction domain.

[0026] In another aspect, the invention provides a cell comprising nucleic acids encoding two or more reverse universal chimeric antigen receptors according to the invention, each reverse universal chimeric antigen receptor comprising three domains, wherein the first domain is a peptide epitope tag that serves as a cell surface binding domain, the second domain is a connecting peptide chain comprising an extracellular hinge and a transmembrane domain, and the third domain is a signal transduction domain.

[0027] In another aspect, the present invention provides a vector comprising a nucleic acid encoding a reverse universal chimeric antigen receptor according to the present invention, wherein the reverse universal chimeric antigen receptor comprises three domains, wherein the first domain is a peptide epitope tag serving as a cell surface binding domain, the second domain is a connecting peptide chain comprising an extracellular hinge and a transmembrane domain, and the third domain is a signal transduction domain.

[0028] In another aspect, the present invention provides a kit comprising a vector according to the present invention comprising a nucleic acid sequence encoding a reverse universal chimeric antigen receptor according to the present invention and an adapter module according to the present invention and / or a vector encoding an isolated nucleic acid sequence encoding an adapter module according to the present invention.

[0029] Furthermore, the present invention includes pharmaceutical compositions comprising cells and adaptor modules according to the present invention in combination with a pharmaceutically acceptable diluent or carrier.Preferably, the pharmaceutical composition is in a form suitable for intravenous administration.

[0030] Preferably, the composition comprises a cell comprising a nucleic acid encoding a reverse universal chimeric antigen receptor according to the invention, and an adapter module according to the invention.

[0031] The pharmaceutical compositions according to the present invention include various administration forms. The pharmaceutical compositions are preferably administered parenterally, particularly preferably intravenously. In one embodiment of the present invention, the parenteral pharmaceutical composition is in an administration form suitable for injection. Therefore, a particularly preferred composition is a solution, emulsion, or suspension of cells and adapter modules in a pharmaceutically acceptable diluent or carrier.

[0032] As carriers, water, buffered water, 0.9% saline solutions, glycine solutions and similar solvents are preferably used. The solutions are sterile. The pharmaceutical compositions are sterilized by conventional, well-known techniques. The composition preferably contains pharmaceutically acceptable excipients, for example, those required to provide approximate physiological conditions and / or increase the stability of the adapter module, such as reagents and buffers for adjusting the pH value, preferably selected from sodium acetate, sodium chloride, sodium citrate, potassium phosphate, potassium chloride, calcium chloride, sodium lactate and histidine. In these formulations, the concentration of the adapter module according to the present invention is variable depending on the application; they are preferably less than 0.01% by weight, preferably at least 0.1% by weight, further preferably 1 to 5% by weight, and they are selected primarily based on the fluid volume, viscosity, etc. or according to the corresponding mode of administration.

[0033] The pharmaceutical composition must be sterile and stable under the conditions of manufacture and storage.The composition can be formulated as a solution, microemulsion, dispersion, in liposomes or in other ordered structures suitable for the purpose and known to the skilled artisan.

[0034] The cells and adapter modules according to the present invention are preferably introduced into a composition suitable for parenteral administration. Preferably, the pharmaceutical composition is an injectable buffered solution comprising 1 ng / mL to 500 mg / ml of AdMo, particularly preferably 5 ng / mL to 250 mg / ml of the adapter module (particularly together with 1 to 500 mmol / l (mM), particularly preferably 5 to 20 mM of a buffer). The injectable solution may be in liquid form. The buffer may preferably be histidine (preferably 1 to 50 mM, particularly preferably 5 to 20 mM) with a pH of 5.0 to 7.0 (particularly preferably pH 6.0).

[0035] Other suitable buffers include, but are not limited to, sodium succinate, sodium citrate, sodium phosphate, or potassium phosphate. Preferably, 0 to 300 mM, particularly preferably 150 mM, of sodium chloride is used for liquid administration forms. In liquid administration forms, a stabilizer is preferably used, preferably 0.0001% (w / v) to 1% (w / v), particularly preferably 0.001% (w / v) to 0.1% (w / v) of polysorbate-80.

[0036] Typical dosage rates delivered per patient per day are in the range of 1 ng to 1000 mg, preferably 3 ng to 3 mg, with the dose being administered one or more times daily or weekly or continuously over a period of up to several weeks.

[0037] In another aspect, the present invention provides the use of a cell according to the present invention comprising a nucleic acid encoding a universal chimeric antigen receptor according to the present invention and an adapter module according to the present invention for stimulating a universal chimeric antigen receptor-mediated immune response in a mammal. Preferably, the present invention provides the use of a cell according to the present invention comprising a nucleic acid encoding a reverse universal chimeric antigen receptor according to the present invention and an adapter module according to the present invention for use as a medicament, more preferably as a medicament for treating cancer or an autoimmune disease. Autoimmune diseases are caused by an abnormal immune response (autoimmunity) of the body to substances and tissues normally present in the body.

[0038] The invention further comprises the use of cells and adaptor modules according to the invention for the preparation of a medicament for therapeutic and / or diagnostic use in the context of cancer or autoimmune diseases.

[0039] The invention also includes methods of treating humans suffering from cancer, infectious, inflammatory or autoimmune diseases by administering cells and adaptor modules according to the invention.

[0040] For therapeutic applications, a sterile pharmaceutical composition comprising a pharmacologically effective amount of cells and adaptor modules according to the present invention is administered to a patient to treat the above-mentioned diseases.

[0041] The invention will be explained in more detail with the aid of the following figures and embodiments, without restricting the invention to them.

[0042] Figure 1 A schematic diagram of a reverse universal chimeric antigen receptor (RevCAR) is depicted. LP, a leader peptide for translation in the endoplasmic reticulum and transport to the cell membrane; PE, a peptide epitope that serves as the passive binding domain of RevCAR; PL; an optional fourth domain for recognition and / or purification; ECD, an extracellular domain that serves as a hinge domain; TM, a transmembrane domain; ICD 1 and ICD2, intracellular signaling domains, which can be composed of one domain or two or more domains.

[0043] Figure 2A schematic diagram of a reverse universal chimeric antigen receptor (RevCAR) platform for retargeting antigen-specific immune cells is shown. In an example, the immune cell is a T cell (T). AdMo, an adapter molecule, is composed of two single-chain variable fragments in this example; PE, a peptide epitope as a passive binding domain of RevCAR; RevCAR, a reverse universal chimeric antigen receptor; TCR, an endogenous T cell receptor.

[0044] Figure 3 Depicted is a schematic diagram of two adapter modules, both of which specifically bind to CD123 (an antigen commonly expressed on leukemias). The adapter molecules are constructed in such a way that they can recruit RevCAR with a La5B9 epitope tag (AdMo CD123-La5B9) or with a La7B6 tag (AdMo CD123-La7B6).

[0045] Figure 4 Shown are the affinities of two CD123-specific adaptor modules for the target antigen CD123 (as determined by binding assay on CD123-expressing OCI-AML3 blasts) (A) and for their RevCAR tags, which are either La5B9 or La7B6 tags (B).

[0046] Figure 5 Shown are the specific lysis of CD123-positive AML blasts by human primary T cells genetically engineered to express RevCAR with La5B9 tags (A) or La7B6 tags (B). In the presence of the corresponding CD123-specific adapter modules recognizing La5B9 tags (AdMo CD123-La5B9) or La7B6 tags (AdMo CD123-La7B6), specific lysis was induced in a concentration-dependent manner.

[0047] Figure 6 A schematic diagram of the lentiviral vector pLVX-EF1α-IRES-ZsGreen1 is shown.

[0048] Figure 7 A schematic diagram of the lentiviral packaging plasmid psPAX2 is shown.

[0049] Figure 8 A schematic diagram of the envelope plasmid pMD2.G is shown.

[0050] Figure 9 The number of RevCAR constructs 1-4 per cell on the cell surface of lentiviral-transduced T cells was summarized. The number of molecules was quantified using QIFIKIT (Agilent, Santa Clara, CA, USA) and monoclonal anti-La antibodies 5B9 or 7B6, respectively.

[0051] Effector cells

[0052] The effector cells used in the method of the present invention can be autologous, isogenic or allogeneic, and their selection depends on the disease to be treated and the means available for doing so. Suitable effector cell groups that can be used in the method include any immune cells with cell lysis, phagocytosis or immunosuppressive activity, such as T cells, including regulatory T cells, NK cells and macrophages. In one aspect, effector cells are from a certain HLA background and are used for autologous or allogeneic systems. Effector cells can be separated from any source, including separation of cells within the tumor explant of the treated subject or the treated subject. In one embodiment, effector cells can be produced by in vitro differentiation from pluripotency or multipotent stem cells or progenitor cells before or after genetic manipulation to express RevCAR to the corresponding cells. Hereinafter, the term "effector cell" refers to any type of above-mentioned immune cells that have been genetically altered to express RevCAR on its cell surface.

[0053] Reverse universal chimeric antigen receptor (RevCAR)

[0054] The RevCAR expressed by the effector cells used in the method of the present invention allows modular, highly flexible and tightly controllable re-targeting of immune cells expressing RevCAR in an antigen-specific manner. The only requirement of the RevCAR used in the method is that (i) RevCAR has binding specificity to a specific tag binding portion through its cell surface peptide epitope tag, and the specific tag binding portion can be conjugated to an adapter module, which in turn binds to a cell surface protein or extracellular structure of the target cell, and (ii) immune cells can be engineered to express RevCAR.

[0055] RevCAR contains three domains ( Figure 1 ). The first domain is a cell surface peptide epitope tag. The cell surface peptide epitope tag is generally present at the amino terminus of the polypeptide comprising RevCAR. Positioning the cell surface peptide epitope tag at the amino terminus allows the cell surface peptide epitope tag to approach the adapter module bound to the target cell without hindrance. The cell surface peptide epitope tag is generally a linear peptide epitope.

[0056] In a preferred embodiment, the cell surface peptide epitope tag is a short linear peptide epitope derived from a human protein.

[0057] In a more preferred embodiment, the cell surface peptide epitope tag is a short linear peptide epitope derived from human nucleoprotein.

[0058] Preferably, the nucleic acid encoding the peptide epitope tag is a nucleic acid encoding a peptide epitope tag according to sequence SEQ. ID 28 or 29, more preferably the nucleic acid encoding the peptide epitope tag is a nucleic acid according to sequence SEQ. ID 3 or 11.

[0059] In the most preferred embodiment, the cell surface peptide epitope tag is a short linear peptide epitope derived from the human nuclear La protein. Preferably, the tag is selected from the human La epitope E5B9 or E7B6. The use of E5B9 and E7B6 La epitopes in the RevCAR system is advantageous because under normal physiological conditions, anti-E5B9 and anti-E7B6 scFv do not interact with the native La protein bound to the cell surface, and in autoimmune patients who are often reported to have autoantibodies to the La protein, no autoantibodies to these epitopes are observed.

[0060] The second domain of RevCAR is extracellular hinge and transmembrane (TM) domain.Hinge domain allows RevCAR to protrude from the effector cell surface for the best combination with its corresponding scFv.TM domain anchors RevCAR in the cell membrane of effector cells.Exemplary hinge and TM domain include but are not limited to the hinge and transmembrane region of the part of human CD28 molecule, CD8a chain, NK cell receptor such as natural killer group 2D (NKG2D), DAP12 or antibody constant region and various hinge and TM domain combinations.

[0061] In other embodiments, the hinge and transmembrane region are selected from the hinge and transmembrane region of a portion of a human CD28 molecule, a CD8a chain, an NK cell receptor (preferably natural killer group NKG2D), DAP12, an Fc receptor or an antibody constant region, and combinations of different hinge and transmembrane domains thereof, wherein the hinge region is part of the extracellular region.

[0062] In the presence of, the tertiary domain is a signal transduction domain. When effector cells are cross-linked with cells or extracellular structures, the domain transmits cell signals to the effector cells carrying RevCAR. The cross-linking between effector and target cell is mediated and depends on the following existence: (i) adapter module, which is incorporated into its specific binding portion on target cell or target cell extracellular structure and carries RevCAR binding portion and (ii) RevCAR (its presentation can be recognized by adapter module and combined peptide epitope tags) expressed on the surface of effector cells. Effector cell activation includes the induction of cytokines or chemokines and the cell dissolution, engulfment or inhibition of active activation of effector cells. Exemplary effector cell signal transduction domains include but are not limited to the cytoplasmic regions of CD28, CD137 (41BB), CD134 (OX40), DAP10 and CD27, which act to enhance T cell survival and proliferation; Inhibitory receptors such as programmed cell death 1 (PD-1) and cytotoxic T lymphocyte antigen 4 (CTLA-4) and CD3 chains (e.g., CD3zeta), DAP12 and Fc receptors, which induce T and NK cell activation. RevCAR may include one or more than one signal transduction domains, such as two, three, four or more immune cell activation or costimulatory domains.

[0063] In other embodiments, the signal transduction domain is selected from the group consisting of CD28, CD137 (41BB), CD134 (OX40), DAP10 and the cytoplasmic region of CD27, programmed cell death 1 (PD-1), cytotoxic T lymphocyte antigen 4 (CTLA-4) and the CD3 chain, DAP12 and the cytoplasmic region of the T cell activation-inducing Fc receptor, wherein the signal transduction domain and the cytoplasmic region are signaling domains.

[0064] In another embodiment, RevCAR comprises a fourth domain which is a short peptide linker ( Figure 1). In terms of its functionality, the fourth domain is required to form a linear epitope that allows specific monoclonal antibodies to be bound with reasonable affinity. One or more linear epitopes may be included in the fourth domain, and they may be located as a linker in the tag binding domain, between the tag binding domain and the extracellular linker, or may be a component of the extracellular hinge domain. With the aid of an optional fourth domain, immune cells implanted in RevCAR can be specifically stimulated so that, compared with unimplanted immune cells, immune cells implanted in RevCAR preferentially proliferate and last longer in vitro or in vivo. The fourth domain can also be used to purify immune cells implanted in RevCAR from a mixed cell population. It can also be used to suppress immune responses mediated by immune cells implanted in RevCAR and eliminate immune cells implanted in RevCAR in vivo.

[0065] In order to allow expression on the cell surface of effector cells, signal peptide (sometimes also referred to as signal sequence, targeting signal or leader peptide) is placed in front of the peptide epitope used as extracellular binding domain, at the 5' end of the RevCAR nucleic acid sequence encoding its N-terminal. Signal peptide targets protein to secretory pathway after co-translation or translation. For this purpose, the signal peptide of protein from various species can be utilized, but preferentially using the leader peptide of the heavy chain or light chain of protein such as CD28, CD8 α, IL-2 or antibody from human source to avoid immunogenic reaction.

[0066] adapter module

[0067] The adapter module is composed of a binding portion with specificity for a certain human cell surface protein or protein complex and a binding portion that is bound to the cell surface peptide epitope of the RevCAR receptor according to the present invention. Before, simultaneously with, or after the administration of the effector cells expressing RevCAR, the adapter module is administered to the subject. Alternatively, before infusion into the receptor, the effector cells expressing RevCAR can be modified with an adapter module. The potential binding portion of the adapter module includes but is not limited to an antibody or fragment thereof that is bound to a surface antigen, and the surface antigen is, for example: CD2, CD3, CD4, CD8, CD10, CD19, CD20, CD22, CD25, CD23, CD30, CD33, CD38, CD44, CD52, CD90, CD99, CD123, CD223, CD269 (B cell maturation antigen, BCMA), CD274, CD276, CD279 and CD366, epidermal growth factor receptor members of the ErbB1, ErbB2, ErbB3, ErbB4 and their mutants), epidermal growth factor receptor (EGFR), members of the tumor necrosis factor receptor superfamily, members of the ephrin receptor family (EphA1-10, EphB1-6), so-called prostate-specific antigens (e.g., prostate stem cell antigen PSCA, prostate-specific membrane antigen PSMA), embryonic antigens (e.g., carcinoembryonic antigen CEA, fetal acetylcholine receptor), members of the vascular endothelial growth factor family (VEGFR 1-3), epithelial cell adhesion molecule EpCAM, alpha-fetoprotein AFP, members of the mucin family (e.g., MUC1, MUC16), follicle-stimulating hormone receptor (FSHR), human high molecular weight melanoma-associated antigen (HMW-MAA), folate binding protein FBP, α-folate receptor, ligands of NKG2D receptor, cytokine receptors [e.g., IL-8Rα (CXCR1), IL-8Rβ (CXCR2), IL-11Rα, IL-11Rβ, IL-13Rα1 and 2, CXCR4], members of the epithelial glycoprotein family Members (e.g., EGP-2, EGP-4), disialogangliosides (e.g., GD2, GD3), members of the carbonic anhydrase family (e.g., CAIX), members of the carbohydrate antigen family (e.g., Ley), Notch ligands (e.g., Delta-like 1 and 4), melanoma-associated chondroitin sulfate proteoglycans (MCSP), glycoprotein A33, and tumor-specific glycans [e.g., serine or threonine-linked N-acetylgalactosamine (Tn) or derivatives such as sialyl-Tn], including mutants of the proteins and protein families. In addition, the binding portion of the adapter module includes, but is not limited to, antibodies or fragments thereof that bind to cytoplasmic or nuclear antigens such as La / SSB antigens, members of the Rho family of GTPases, members of the high-mobility group protein, etc.Similarly, the binding portion of the adapter module can be composed of the α and β or γ and δ chains of the T cell receptor (TCR) or fragments thereof. Such TCR-derived binding portions recognize and bind to peptides presented by human leukocyte antigen (HLA) class I and class II protein complexes. Examples include, but are not limited to, TCRs that are specific for peptides derived from proteins such as the EGFR family, survivin, the Sry-like high mobility group box (SOX) protein family, melanoma-associated antigens (e.g., the autoimmune cancer / testis antigen NY-ESO-1, members of the melanoma antigen family A (MAGEA), antigens preferentially expressed in melanoma (PRAME), and antigens associated with leukemia (e.g., Wilms tumor gene 1 WT1). The binding portion of the adapter module can also comprise a protein or protein complex Ligand (which is further referred to as receptor) or fragment thereof. Such ligands may bind to, but are not limited to, cytokine receptors (e.g., IL-13 receptor), ligands of NKG2D receptors, ligands of EGFR family members, checkpoint molecules such as PD-1, CTLA-4, lymphocyte activation gene 3 (LAG-3) or T cell immunoglobulin and mucin domain-containing-3 (TIM-3) ligands, or autoreactive TCRs. In addition, the target binding portion may also be a chemically synthesized peptide derivative fused to the tag binding portion by a chemical reaction (i.e., click chemistry).In a preferred embodiment, the peptide having binding specificity for a membrane receptor has binding specificity for a membrane receptor selected from the group consisting of CD2, CD3, CD4, CD2, CD3, CD4, CD8, CD10, CD19, CD20, CD22, CD23, CD25, CD30, CD33, CD38, CD44, CD52, CD90, CD99, CD123, CD223, CD269, CD274, CD276, CD279 and CD366, interleukin receptors, particularly preferably IL-8Rα (CXCR1), IL-8Rβ (CXCR2), IL-11Rα, I IL-11Rβ, IL-13Rα1 and 2, CXCR4; c-Met, transforming growth factor β receptor, erbB1, erbB2, erbB3, erbB4 and mutants thereof, members of the tumor necrosis factor receptor superfamily, ephrin receptors, particularly preferably EphA1-10, EphA5 or EphB1-6; prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), carcinoembryonic antigen (CEA), fetal acetylcholine receptor, carcinoembryonic antigen, tumor-specific glycans [e.g., serine- or threonine-linked N-acetylgalactosamine (Tn) or derivatives such as sialyl-Tn]; VEGFR 1, VEGFR 2 or VEGFR 3, neuropilin-1, epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), alpha-fetoprotein (AFP), mucin, particularly preferably MUC1, MUC16 or MUC18; follicle-stimulating hormone receptor (FSHR), human high molecular weight melanoma-associated antigen (HMW-MAA), folate binding protein (FBP), folate receptor, NKG2D, major histocompatibility complex (MHC) class I molecule, particularly preferably MHC class I chain-related gene A (MICA) or B (MICB), UL16 binding protein (ULPB) 1, ULPB 2, ULPB 3, a member of the ribonucleic acid export 1 (Rae-1) family or histocompatibility 60 (H-60); a chaperone protein and a heat shock protein, particularly preferably heat shock protein (HSP) 90 or 78 kDa glucose-regulated protein (GRP78); EGP-2 or EGP-4, disialoganglioside 2 (GD2) or GD3, carbonic anhydrase 9 (CAIX), Lewis Y (LeY), C-type lectin-like molecule 1 (CLL-1), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor, apoptosis antigen 1 (APO-1, Fas, CD95), a member of the keratin family or an integrin, particularly preferably avβ3 or avβ5, aminopeptidase A, aminopeptidase N or neural / glial antigen 2 (NG2).

[0068] The binding portion of the adapter module can comprise a single antigen specificity (monospecific), two, three, or more antigen specificities (bispecific and multispecific). Examples of bispecific and multispecific antigen specificities include, but are not limited to, adapter modules that bind to: PSCA and PSMA antigens, CD19 and CD20 antigens, CD19, CD20, and CD22 antigens, CD33 and CD123 antigens, CD33 and CD99, CD33 and TIM-3, ErbB-1 and ErbB-2, PSCA and ErbB-2, and other combinations. Preferred examples of bispecific and multispecific antigen specificities include adapter modules that bind to: PSCA and PSMA, CD19 and CD20, CD19 and CD22, CD19, CD20 and CD22, CD19 and CD123, CD33 and CD123, CD33 and CD99, CD33 and TIM-3, ErbB-1 and ErbB-2, PSCA and ErbB-2, IL-13Rα2 and ErbB-2, CD38 and CD269. The binding portion of the adapter module can also contain monovalent binding as well as bivalent and multivalent binding sites. Examples of bivalent and multivalent targeting strategies include, but are not limited to, adapter modules that incorporate two scFvs that recognize different epitopes of PSCA, CEA, CD19, and CD33, and ligand-scFv combinations that recognize different epitopes of the ErbB1 receptor.

[0069] The adapter module can also carry other ligands that do not participate in the binding of the target antigen, further referred to as payloads. Such payloads can include, but are not limited to, co-stimulatory ligands or cytokines fused to the N or C terminus of the adapter module, particularly the extracellular domains of CD28, CD137 (41BB), CD134 (OX40), and CD27, as well as IL-2, IL-7, IL-12, IL-15, IL-17, and IL-21, all of which stimulate different types of immune cells. Other payloads can be radionuclides or chemical compounds that induce cell death in target cells and neighboring cells.

[0070] In an embodiment of the present invention, the cell surface peptide epitope tag of the RevCAR receptor is a determined tag. The identity of the tag is limited only by the identity of the binding domain of the corresponding adapter module. The tag can be derived from any structure, and antibodies or other binding domains for the structure are available. Antibodies specific for the tag can be obtained from any animal species, although preferably obtained from mammals, such as humans, monkeys, mice, rats, rabbits, guinea pigs, horses, cattle, sheep, goats, pigs, dogs or cats. Preferably, the antibody is a human or humanized antibody. There is no restriction on the specific categories of antibodies that can be used (including IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE antibodies). Antibody fragments include single-chain variable fragments (scFv), single-chain antibodies, F (ab') 2 fragments, Fab fragments and fragments produced by Fab expression libraries, the only limitation being that the antibody fragment retains the ability to bind to the selected tag. The antibody may also be a polyclonal, monoclonal or chimeric antibody, for example, in which the antigen binding region (e.g., F(ab')2 or hypervariable region) of a non-human antibody is transferred into the framework of a human antibody by recombinant DNA technology to produce a substantially human molecule. Antigen binding fragments, such as scFv, can be prepared therefrom. Antibodies to a selected tag can be produced by immunizing various hosts (including but not limited to goats, rabbits, rats, mice, humans) with a specific protein or any portion, fragment, or oligopeptide that retains the immunogenic properties of the protein.

[0071] In some embodiments, the present invention relates to adjuvants for the treatment of immunologic diseases. Adjuvants include, but are not limited to, detoxification heat-labile toxins, Freund's, mineral gels such as aluminum hydroxide and surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol from Escherichia coli. BCG (BCG (Bacillus Calmette-Guerin)) and Corynebacterium parvum (Corynebacterium parvum) are also potentially useful adjuvants. Antibodies and fragments thereof can be prepared using any technology that provides the generation of antibody molecules, for example, by cultivating continuous cell lines for monoclonal antibody production. Such techniques include, but are not limited to, the hybridoma technique originally described by Koehler and Milstein (Nature 256:495-497 (1975)), the human B cell hybridoma technique (Kosbor et al., Immunol Today 4:72 (1983); Cote et al., Proc Natl. Acad. Sci 80:2026-2030 (1983)), and the EBV hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss Inc, New York NY, pp 77-96 (1985)). It is also possible to use techniques developed for producing "chimeric antibodies," i.e., splicing mouse antibody genes to human antibody genes to obtain molecules with appropriate antigenic specificity and biological activity (Morrison et al., Proc Natl. Acad. Sci 81: 6851-6855 (1984); Neuberger et al., Nature 312: 604-608 (1984); Takeda et al., Nature 314: 452-454 (1985)). Alternatively, the techniques described for the production of single-chain antibodies can be adjusted to be suitable for producing tag-specific single-chain antibodies.

[0072] In one aspect, the tag binding domain is a single-chain variable fragment (scFv). ScFv comprises the variable regions of the heavy chain (VH) and light chain (VL) of an antibody, typically connected by a short peptide of 5 to about 25 amino acids. A linker can connect the N-terminus of VH to the C-terminus of VL, or vice versa.

[0073] In a preferred embodiment, the cell surface peptide epitope tag (tag) of the RevCAR receptor is a short linear epitope from human nuclear La protein. The tag binding domain of the adapter module can constitute an antibody or an antibody-derived antigen binding fragment, for example, a single chain variable fragment (scFv) that binds to the corresponding La epitope.

[0074] Preferably, the tag is selected from the human La epitope E5B9 or E7B6.

[0075] A method for stimulating a reverse universal chimeric antigen receptor-mediated immune response in a mammal, the method comprising:

[0076] - administering to a mammal an effective amount of effector cells that have been genetically modified to express one or more reverse universal chimeric antigen receptors, wherein the reverse universal chimeric antigen receptor comprises three domains, wherein the first domain is a peptide epitope tag, the second domain is an extracellular hinge and transmembrane domain, and the third domain is a signal transduction domain, wherein the tag is recognized by a binding domain suitable for generating a soluble adaptor module ( Figure 2 ).

[0077] - Administration of one or more adapter modules consisting of a binding moiety specific for a particular human cell surface protein or protein complex and a binding domain, wherein the latter binding domain recognizes the cell surface peptide epitope tag of RevCAR ( Figure 2 ).

[0078] wherein the adapter module is administered to the subject prior to, concurrently with, or after administration of effector cells expressing the reverse universal chimeric antigen receptor.

[0079] In a preferred embodiment, the effector cells and adaptor modules are administered to a human.

[0080] Generation of RevCAR effector cells

[0081] In one embodiment of the present invention, immune cells can be genetically engineered to express RevCAR by a variety of methods. Typically, the polynucleotide vector encoding RevCAR and all the necessary elements to ensure its expression in genetically engineered immune cells are transferred into the cell. The transfer of the vector can be, but is not limited to, electroporation or transfection of nucleic acids or by means of a viral vector system (such as, but not limited to, adenovirus, adeno-associated virus, retrovirus, foamy virus or lentiviral gene transfer).

[0082] In another embodiment, lentiviral gene transfer can be applied for stable expression of RevCAR in immune cells by first constructing a lentiviral vector encoding the selected RevCAR. Exemplary lentiviral vectors include but are not limited to Figure 6 The vector shown is pLVX-EF1alpha RevCAR 28 / ζ, wherein the lentiviral part of the vector is derived from human immunodeficiency virus (HIV).

[0083] For the described applications, the MSC / IRES / ZxGreenI portion was replaced with the RevCAR construct. Figure 6 The abbreviations used are as follows:

[0084] 5'LTR: 5' long terminal repeat, PBS: primer binding site, Ψ: packaging signal, RRE: Rev response element, cPPT / CTS: (central polypurine tract / central termination sequence, PEF1α: human elongation factor 1α promoter, MCS: multiple cloning site, IRES: internal ribosome entry site, ZsGreen1: human codon-optimized, WPRE: woodchuck hepatitis virus posttranscriptional regulatory element, 3'LTR: 3' long terminal repeat, pUC: origin of replication, Ampr: ampicillin resistance gene; β-lactamase.

[0085] Lentiviral particles are typically produced by transiently transfecting human embryonic kidney (HEK) 293T (ACC 635) cells with a lentiviral vector plasmid encoding RevCAR and using a Figure 7 The group-specific antigen (gag) and polymerase (pol) encoding plasmids (e.g., psPAX2, Addgene plasmid 12260) as shown in Figure 8 The packaging plasmids are generated by co-transfection of the indicated envelope encoding plasmids (e.g. pMD2.G, Addgene plasmid 12259). After transfection, the packaging plasmids express the HIV-1 Gag and Pol proteins. Figure 7 The abbreviations used in the text are as follows: CMVenh: CMV enhancer and promoter, SD: splice donor, SA: splice acceptor, Gag: group-specific antigen, Pro: precursor protein encoding protease protein, Pol: protein encoding reverse transcriptase and integrase, RRE: rev response element, Amp: ampicillin. Plasmid MD2.G ( Figure 8 ) encodes the glycoprotein of vesicular stomatitis virus (VSV-G). VSV-G protein is used in lentiviral vectors to transduce a wide range of mammalian cells. Figure 8 The abbreviations used in the present invention are as follows: CMV: CMV enhancer and promoter, β-globin intron: β-globin intron, β-globin pA: β-globin polyadenosine tail.

[0086] Various envelopes from different viral species can be used for this purpose. Lentiviral vectors can be successfully pseudotyped using, but not limited to, the envelope glycoprotein (Env) of amphotropic murine leukemia virus (MLV) or the G protein of vesicular stomatitis virus (VSV-G), modified envelopes of prototype foamy viruses (PFV), or chimeric envelope glycoprotein variants derived from gibberish ape leukemia virus (GaLV) and MLV. The supernatant of transfected HEK293T cells can be harvested 24 to 96 hours after transfection, and viral particles can (but are not necessarily) concentrated from the supernatant by ultracentrifugation or other methods. For lentiviral transduction of immune cells, various established protocols can be applied. In one aspect, peripheral blood mononuclear cells (PBMCs) or isolated T cells can be activated with monoclonal antibodies specific for the CD3 complex (e.g., clones OKT3 or UCHT1, which can be in solution or coated on plastic cell culture dishes or magnetic beads). The activation of PBMC or isolated T cells can be further enhanced by stimulating the co-stimulatory pathway with monoclonal antibodies or ligands specific for (but not limited to) CD27, CD28, CD134 or CD137 (alone or in various combinations) and providing exogenous recombinant cytokines (such as but not limited to interleukin (IL) -2, IL-7, IL-12, IL-15 and IL-21). 24h to 96h after the initial administration of activating CD3 antibodies and / or recombinant cytokines in a single dose or multiple doses, concentrated or unconcentrated viral particles are added to PBMC or T cell cultures. The stable transduction of T cells can be determined by flow cytometry after staining with an anti-tag antibody for the surface expression of RevCAR or using a monoclonal antibody for the fourth domain of RevCAR on the third day after the final administration of the viral supernatant. RevCAR-transduced T cells can be cultured and activated with anti-CD3 monoclonal antibodies under the supply of recombinant cytokines to propagate in vitro.

[0087] In another embodiment, immune cells can be genetically engineered using gene editing technology (e.g., transcription activator-like effector nuclease (TALEN) or clustered regularly interspaced short palindromic repeats (CRISPR / Cas)) to stably integrate the coding sequence of RevCAR into the host cell genome and promote RevCAR surface expression.

[0088] If RevCAR carries an optional fourth domain (a peptide sequence that forms a linear epitope of a monoclonal antibody), immune cells genetically modified to express RevCAR can be specifically propagated in vitro by coating a monoclonal antibody or its antibody fragment that binds to a RevCAR tag or an optional fourth RevCAR domain on the surface of a culture dish or beads of any kind, which is added to the cell culture at a determined ratio (but not limited to) of 1 bead to 1-4 effector cells implanted with RevCAR. The binding of the surface-coated monoclonal antibody to the RevCAR tag or the fourth domain induces cross-linking of RevCAR expressed on the cell surface and the formation of immune synapses, resulting in the activation of signaling pathways specifically triggered by the signaling domain of RevCAR. Depending on the induced signaling pathway, this may result in enhanced proliferation and sustained resistance to cell death induced by activation of immune cells carrying RevCAR, and thus lead to the enrichment of immune cells genetically modified by RevCAR in a mixed population.

[0089] The RevCAR tag or the optional fourth domain can be further used to enrich and purify immune cells expressing RevCAR from a mixed population. Enrichment and purification can be carried out with the help of a monoclonal antibody or its antibody fragment bound to the RevCAR tag or the fourth RevCAR domain to mark cells expressing RevCAR for cell sorting or to transiently connect immune cells expressing RevCAR to small particles, which can be used for cell separation. In one aspect, immune cells implanted with RevCAR are incubated with monoclonal antibodies that recognize the RevCAR tag or the fourth domain. Next, magnetic beads are added that are conjugated to antibodies or their fragments that are specific for species and isotype-specific heavy and light chains of monoclonal antibodies that bind to the optional fourth domain. Thus, immune cells expressing RevCAR are connected to magnetic beads and can be captured in a magnetic field and separated from other immune cells.

[0090] In another embodiment of the present invention, the RevCAR tag or the optional fourth domain can be used to detect UniCAR surface expression ( Figure 9 ). Figure 9 Depicts that RevCAR surface expression can be detected by using a monoclonal antibody against the RevCAR tag followed by staining with an anti-species secondary antibody conjugated with a fluorescent dye.

[0091] RevCAR-expressing immune cell populations can be formulated for administration to a subject using techniques known to those skilled in the art.

[0092] The preparation comprising the immune cell group expressing RevCAR can include one or more pharmaceutically acceptable excipients. The excipients included in the preparation will have different purposes, depending on, for example, the properties of the label constituting RevCAR, the immune cell group used and the mode of administration. Examples of commonly used excipients include, but are not limited to, saline, buffered saline, dextrose, water for injection, glycerol, ethanol and combinations thereof, stabilizers, solubilizers and surfactants, buffers and preservatives, tonicity agents, fillers and lubricants. Preparations comprising immune cell groups expressing RevCAR will typically be prepared and cultured in the absence of any non-human components such as animal serum (e.g., bovine serum albumin).

[0093] The preparation may include a population of immune cells expressing RevCAR or more than one, for example, two, three, four, five, six or more populations. The different populations of immune cells implanted with RevCAR can vary based on the identity of the tag domain, the identity of the signal transduction domain, the identity of the subpopulation, the pattern of generation and cultivation, or a combination thereof. For example, the preparation may include a population of T and NK cells expressing RevCAR that recognizes and binds to one or more than one (e.g., two, three, four, five, six or more) different adapter modules.

[0094] The preparation of one or more groups of RevCAR immune cells can be administered to the subject using patterns and techniques known to technicians. Exemplary patterns include but are not limited to intravenous injection. Other patterns include but are not limited to intratumoral, intradermal, subcutaneous (sc, sq, sub-Q, Hypo), intramuscular (im), intraperitoneal (ip), intraarterial, intramedullary, intracardiac, intraarticular (joint), intrasynovial (joint fluid area), intracranial, intraspinal and intrathecal (cerebrospinal fluid). Any known device that can be used for parenteral injection or infusion of preparations can be used to achieve this administration. Injection can be carried out in the form of batch injection or continuous flow injection.

[0095] The preparation containing one or more immune cell populations expressing RevCAR administered to the subject contains many RevCAR-expressing immune cells that are effective in treating and / or preventing a specific indication or disease. Therefore, when practicing the method of the present invention, a therapeutically effective RevCAR-expressing immune cell population is administered to the subject. The number of RevCAR-expressing immune cells administered to the subject will vary over a wide range, depending on the site, source, identity, extent and severity of the disease, the age and condition of the individual to be treated, etc. Typically, administration of a RevCAR-expressing immune cell population containing about 1x10 4 to about 1x10 10 In most cases, the preparation will contain approximately 1x10 5 to about 1x10 9RevCAR-expressing immune cells, approximately 5×10 5 to about 5×10 8 RevCAR-expressing immune cells, or approximately 1×10 6 to about 1x10 9 The doctor will ultimately determine the appropriate dose to use. In the event of an adverse event, the RevCAR-implanted immune cells can be removed from the individual by administering a monoclonal antibody against the RevCAR tag or the fourth domain (if present).

[0096] Adaptor module generation

[0097] The adapter module comprises two domains, a binding portion specific to a specific human cell surface protein or protein complex and a tag binding domain for a cell surface peptide epitope tag of RevCAR. The adapter module can be manufactured by techniques known to technicians. These techniques include, but are not limited to, recombinant expression in prokaryotic or eukaryotic cells, artificial synthesis of polypeptide chains, or chemical synthesis.

[0098] On the one hand, the adapter module can be expressed in Chinese hamster ovary (CHO, ACC-110) cells, which are suitable for synthesizing a large amount of recombinant proteins in their biologically active form. The nucleic acid sequence encoding the adapter module can be transferred to CHO cells by established genetic engineering techniques such as, but not limited to, naked nucleic acid transfection, electroporation, or viral gene transfer. High-yield single-cell clones can be selected from parental lines using, for example, a dihydrofolate reductase (DHFR) selection system. In this system, DHFR-deficient CHO cell mutants (e.g., CHO subline DXB11 or DG44) are genetically modified by co-transfecting a functional copy of the DHFR gene in addition to the nucleic acid sequence encoding the adapter module. Clonal selection is then performed by growing in a culture medium that does not contain glycine, hypoxanthine, and thymidine. High-yield clones can be further selected by culturing cells in high levels of methotrexate (MTX), a folic acid analog that blocks DHFR activity. Since genetically modified cells must cope with a decrease in DHFR activity (which cannot be rescued by the presence of only a single copy of DHFR), under these conditions, clones with amplified copies of the DHFR gene are preferred. The genetic linkage between DHFR and the gene of interest ensures that the transgene is also co-amplified, thereby increasing the chance of obtaining high-yield cell clones. The selected cell clones are grown under good production conditions, preferably in the absence of any animal serum. The adapter module can be separated from the cell culture supernatant by a preparative protein purification method established, which includes preliminary steps such as precipitation or ultracentrifugation and various purification techniques, such as but not limited to size exclusion, affinity or ion exchange chromatography. On the one hand, the nucleic acid sequence of the adapter module carries a coding sequence of six to ten continuous histidine amino acids that form a polyhistidine tag. Polyhistidines are strongly bound to divalent metal ions (such as nickel and cobalt). The cell culture supernatant can be moved through a column containing fixed nickel ions, which binds the polyhistidine tag, and all untagged proteins pass through the column. The adapter module can be eluted with imidazole (which competes with the polyhistidine tag for binding to the column) or by lowering the pH (which reduces the affinity of the tag for the resin). In one aspect, the adapter module is suitable for affinity chromatography based on protein L (e.g., Capto L). In another aspect, the adapter molecule can carry a variable region of the heavy chain family 3, which enables purification by a column resin containing a specific domain of Staphylococcal protein A (e.g., MabSelect).

[0099] Adaptor module application

[0100] One adapter module or more than one, eg, two, three, four, or more, adapter modules can be formulated for administration to a subject using techniques known to the skilled artisan.

[0101] Formulations comprising one or more adapter modules may include one or more pharmaceutically acceptable excipients. Depending on, for example, the nature of the adapter module and the mode of administration, the excipients included in the formulation will have different purposes. Examples of commonly used excipients include, but are not limited to, saline, buffered saline, dextrose, water for injection, glycerol, ethanol, and combinations thereof, stabilizers, solubilizers and surfactants, buffers and preservatives, tonicity agents, fillers, and lubricants. Formulations comprising adapter modules will typically be prepared and cultured in the absence of any non-human components, such as animal serum (e.g., bovine serum albumin).

[0102] The formulation can include one adapter module or more than one, for example, two, three, four, five, six or more adapter modules. The adapter modules can vary based on the identity of the binding moiety, the identity of the tag, the mode of generation, or a combination thereof. For example, a formulation can include adapter modules that recognize and bind to one or more than one, for example, two, three, four, five, six or more different human cell surface proteins, protein complexes or extracellular matrix structures.

[0103] A preparation comprising one or more immune cell populations expressing RevCAR can be incubated in vitro with a preparation comprising one or more adapter modules to modify the RevCAR-expressing immune cells with the adapter modules before administration to the subject. Alternatively, the preparation comprising one or more adapter modules can be administered directly to the subject, or a combination of the two strategies can be selected. The route and dosage will vary over a wide range, depending on the site, source, identity, extent and severity of the disease, the age and condition of the individual to be treated, etc. The doctor will ultimately determine the appropriate route of application and dosage to be used.

[0104] The formulation comprising the adapter module is administered to a subject in an amount effective to treat and / or prevent a specific indication or disease. 2 Typical dosage rates for daily delivery are 1 ng to 1000 mg, preferably 5 ng to 1 mg, with dosages administered daily or once or more weekly or continuously over a period of several weeks. However, the amount of adapter module in the formulation administered to a subject will vary widely, depending on the site, source, identity, extent, and severity of the cancer, the age and condition of the individual being treated, etc. A physician will ultimately determine the appropriate dosage to use.

[0105] The present invention relates to a method of treating a subject having cancer, an infection, or an autoimmune disorder, comprising administering to a subject in need of treatment one or more preparations of an adapter module, wherein the adapter module binds to cancer cells, and administering one or more therapeutically effective populations of RevCAR-expressing immune cells, wherein the RevCAR-expressing immune cells bind to the adapter module and induce cell death.

[0106] The term "cancer" is intended to be broadly construed and encompasses all aspects of abnormal cell growth and / or cell division. Examples include: carcinomas, including but not limited to adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, anaplastic carcinoma, large cell carcinoma, small cell carcinoma, and cancers of the skin, breast, prostate, bladder, vagina, cervix, uterus, liver, kidney, pancreas, spleen, lung, trachea, bronchus, colon, small intestine, stomach, esophagus, and gallbladder; sarcomas, including but not limited to chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant nerve sheath tumor, osteosarcoma, soft tissue sarcoma, and cancers of bone, cartilage, fat, muscle, blood vessels, and hematopoietic tissue; lymphomas and leukemias, including but not limited to mature B-cell neoplasms such as chronic lymphocytic leukemia / Small lymphocytic lymphomas, B-cell prolymphocytic leukemias, lymphomas and plasma cell neoplasms, including multiple myeloma, mature T-cell and natural killer (NK) cell neoplasms, such as T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, aggressive NK-cell leukemia and adult T-cell leukemia / lymphoma, Hodgkin lymphoma and lymphoproliferative disorders associated with immunodeficiency; germ cell tumors, including but not limited to testicular cancer and ovarian cancer; blastomas, including but not limited to hepatoblastoma, medulloblastoma, Wilms' tumor, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma and retinoblastoma. The term also encompasses benign tumors.

[0107] As used herein, the terms "treat," "treat," and "therapy" have their ordinary and customary meanings and include one or more of: blocking, alleviating, or reducing the severity and / or frequency of symptoms of cancer in a subject, and / or inhibiting the growth, division, spread, or proliferation of cancer cells or the progression of cancer (e.g., the appearance of new tumors) in a subject. Treating means blocking, alleviating, reducing, or inhibiting by about 1% to about 100% compared to a subject who has not practiced the methods of the invention. Preferably, the blocking, alleviating, reducing, or inhibiting is about 100%, 99%, 98%, 97%, 96%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% compared to a subject who has not practiced the methods of the invention.

[0108] The frequency of administration of both the formulation comprising a population of RevCAR-expressing immune cells and the formulation of the adapter module will vary depending on factors including the disease being treated, the elements comprising the RevCAR-expressing immune cells and the adapter module, and the mode of administration. Each formulation can be independently administered 4, 3, 2, or once daily, every other day, every three days, every four days, every five days, every six days, once a week, every eight days, every nine days, every ten days, every two weeks, every month, and every two months.

[0109] The duration of treatment will depend on the condition being treated and will be best determined by the attending physician. However, the duration of treatment is expected to last for days, weeks or months.

[0110] The present invention provides flexibility in treatment methods, and therefore one or more preparations of the adapter module and one or more immune cell populations expressing RevCAR can be administered to the subject in any order. Thus, one or more preparations of the adapter module can be administered to the subject before, after, or simultaneously with one or more immune cell populations expressing RevCAR. Alternatively, when more than one preparation of the adapter module and / or more than one population of immune cells expressing RevCAR are administered to the subject, they can be staggered. For example, the first preparation of the adapter module can be administered, followed by a first population of immune cells expressing RevCAR, followed by a second preparation of a tagged protein, and then a second population of immune cells expressing RevCAR.

[0111] The present invention also includes such a method, wherein the population of RevCAR-expressing immune cells is coated with an adapter module before the RevCAR-expressing immune cells are administered to a subject.

[0112] In each embodiment of the invention, the subject being treated is a human or a non-human animal, such as a non-human primate, bird, horse, cow, goat, sheep, companion animal, such as a dog, cat or rodent, or other mammal.

[0113] In one embodiment, RevCAR genetically engineered T cells can be specifically redirected to tumor cells expressing CD123, a surface marker frequently detected in >80% of acute myeloid leukemia (AML) and nearly 100% of acute lymphoblastic leukemia (ALL). Figure 3 Two CD123-specific adapter modules were designed, cDNAs were synthesized and cloned into lentiviral vectors, and stable CHO production cell lines were generated by lentiviral gene transfer. The adapter modules were purified from cell supernatants using standard immobilized metal affinity chromatography (IMAC) via a C-terminal His-tag. One adapter module variant had an active scFv binding domain that recognized the La 5B9 epitope; alternatively, the other adapter module contained an scFv that recognized the La 7B6 epitope. Figure 3 The two modules bound to AML cells expressing CD123 with comparable affinities of 15 pM and 11 pM, respectively ( Figure 4 A). Binding to the RevCAR tag can also be confirmed by binding experiments on target cells expressing RevCAR ( Figure 4B) The affinities of the two adapter modules for their specific RevCAR tags are comparable, with a K of 0.05 for the La5B9 RevCAR tag. D The K of La7B6 RevCAR tag is 12 pM. D 14 pM ( Figure 4 B).

[0114] In order to redirect immune cells with CD123-specific adapter modules, two RevCARs with La5B9 or La7B6 epitopes as RevCAR tags were constructed. Human natural T cells were genetically engineered to express either of the two RevCARs by lentiviral gene transfer. After incubation with AML blasts expressing CD123, primary human RevCAR T cells mediated specific lysis of AML blasts in a concentration-dependent manner in the presence of the corresponding adapter molecules ( Figure 5 ).

[0115] In another embodiment, a nucleic acid encoding a reverse universal chimeric antigen receptor (referred to as RevCAR1) is provided according to SEQ.ID 1. The nucleic acid sequence encodes a human IL-2m leader peptide according to SEQ.ID 2, a human La5B9 epitope according to SEQ.ID 3, a human CD28 portion according to SEQ.ID 4 to 6 (including a human CD28 extracellular portion with a mutant binding motif according to SEQ.ID.ID 4, a CD28 transmembrane domain according to SEQ.ID 5, and a human CD28 intracellular portion including a mutant internalization motif according to SEQ.ID 6), and a human CD3 zeta intracellular domain according to SEQ.ID 7.

[0116] The protein expression product of the nucleic acid according to SEQ.ID 1 can be obtained in SEQ.ID 24.

[0117] The nucleic acid sequence of the human La 5B9 epitope according to SEQ. ID 3 encodes the protein domain according to SEQ. ID 28.

[0118] In another embodiment, a nucleic acid sequence encoding a reverse universal chimeric antigen receptor (referred to as RevCAR2) is provided according to SEQ.ID 8. The nucleic acid sequence encodes a human IL-2m leader peptide according to SEQ.ID 2, a human La 5B9 epitope according to SEQ.ID 3, an extracellular hinge and transmembrane region according to SEQ.ID 4 and 5, a human CD137 intracellular signaling domain according to SEQ.ID.ID 9, and a human CD3 zeta intracellular domain according to SEQ.ID 7.

[0119] The product of protein expression according to the isolated nucleic acid sequence of SEQ.ID 8 can be obtained in SEQ.ID 25.

[0120] In another embodiment, a nucleic acid sequence encoding a reverse universal chimeric antigen receptor (referred to as RevCAR3) is provided according to SEQ.ID 10. The nucleic acid sequence encodes a human IL-2m leader peptide according to SEQ.ID 2, a human La 7B6 epitope according to SEQ.ID 11, a human CD28 portion according to SEQ.ID 4 to 6 (including a human CD28 extracellular portion comprising a binding motif with a mutation according to SEQ.ID.ID 4, a CD28 transmembrane domain according to SEQ.ID 5, and a human CD28 intracellular portion comprising an internalization motif with a mutation according to SEQ.ID 6), and a human CD3 zeta intracellular domain according to SEQ.ID 7.

[0121] The product of protein expression according to the isolated nucleic acid sequence of SEQ.ID 10 can be obtained in SEQ.ID 26.

[0122] The nucleic acid sequence of the human La 7B6 epitope according to SEQ. ID 11 encodes the protein domain according to SEQ. ID 29.

[0123] In another embodiment, a nucleic acid sequence encoding a reverse universal chimeric antigen receptor (referred to as RevCAR4) is provided according to SEQ.ID 12. The nucleic acid sequence encodes the human IL-2m leader peptide according to SEQ.ID 2, the human La 7B6 epitope according to SEQ.ID 11, the extracellular hinge and transmembrane regions according to SEQ.ID 4 and 5, the human CD137 intracellular signaling domain according to SEQ.ID.ID 9, and the human CD3 zeta intracellular domain according to SEQ.ID 7.

[0124] The product of protein expression according to the isolated nucleic acid sequence of SEQ.ID 12 can be obtained in SEQ.ID 27.

[0125] In other embodiments of the invention, nucleic acids encoding adapter modules are provided, wherein the tag binding portion of the adapter module comprises an antibody or fragment thereof that binds to the 5B9 or 7B6 epitope of the La / SSB antigen, preferably according to SEQ. ID 14 and 15 or 18 and 19.

[0126] In a further embodiment of the invention, a nucleic acid encoding an adapter module is provided, wherein the tag binding portion of the adapter module comprises an antibody or fragment thereof that binds PSMA or CD123, preferably according to SEQ. ID 20 and 21 or 22 and 23.

[0127] In another embodiment of the present invention, an adapter module having a binding moiety against prostate-specific membrane antigen (PSMA) is provided. The nucleic acid encoding the adapter module comprises a sequence encoding an IgG kappa leader peptide according to SEQ. ID 13, a humanized heavy chain of an anti-La 5B9 scFv according to SEQ. ID 14, a humanized light chain of an anti-La 5B9 scFv according to SEQ. ID 15, a humanized heavy chain of an anti-PSMA scFv according to SEQ. ID 20, a humanized light chain of an anti-PSMA scFv according to SEQ. ID 21, a myc tag according to SEQ. ID 16, and a his tag according to SEQ. ID 17.

[0128] In another embodiment of the present invention, an adapter module having a binding moiety against prostate-specific membrane antigen (PSMA) is provided. The nucleic acid encoding the adapter module comprises a sequence encoding an IgG kappa leader peptide according to SEQ. ID 13, a humanized heavy chain of an anti-La 7B6 scFv according to SEQ. ID 18, a humanized light chain of an anti-La 7B6 scFv according to SEQ. ID 19, a humanized heavy chain of an anti-PSMA scFv according to SEQ. ID 20, a humanized light chain of an anti-PSMA scFv according to SEQ. ID 21, a myc tag according to SEQ. ID 16, and a his tag according to SEQ. ID 17.

[0129] In another embodiment of the present invention, an adapter module having a binding portion for the leukemia antigen CD123 is provided. The nucleic acid encoding the adapter module comprises a sequence encoding an IgGκ leader peptide according to SEQ.ID 13, a humanized heavy chain of an anti-La 5B9 scFv according to SEQ.ID 14, a humanized light chain of an anti-La 5B9 scFv according to SEQ.ID 15, a humanized heavy chain of an anti-CD123 scFv according to SEQ.ID 22, a humanized light chain of an anti-CD123 scFv according to SEQ.ID 23, a myc tag according to SEQ.ID 16, and a his tag according to SEQ.ID 17.

[0130] In another embodiment of the present invention, an adapter module having a binding portion against the leukemia antigen CD 123 is provided. The nucleic acid encoding the adapter module comprises a sequence encoding an IgG kappa leader peptide according to SEQ. ID 13, a humanized heavy chain of an anti-La 7B6 scFv according to SEQ. ID 18, a humanized light chain of an anti-La 7B6 scFv according to SEQ. ID 19, a humanized heavy chain of an anti-CD123 scFv according to SEQ. ID 22, a humanized light chain of an anti-CD123 scFv according to SEQ. ID 23, a myc tag according to SEQ. ID 16, and a his tag according to SEQ. ID 17.

[0131] The nucleic acid sequence of the humanized anti-La 5B9 variable region heavy chain according to SEQ. ID 14 encodes the protein according to SEQ. ID 30, while the humanized anti-La 5B9 variable region light chain according to SEQ. ID 15 encodes the protein according to SEQ. ID 31.

[0132] The nucleic acid sequence of the myc tag according to SEQ. ID 16 encodes the protein according to SEQ. ID 32, whereas the his tag according to SEQ. ID 17 encodes the protein according to SEQ. ID 33.

[0133] The nucleic acid sequence of the humanized anti-7B6 variable region heavy chain according to SEQ.ID 18 encodes the protein according to SEQ.ID 34, while the humanized anti-7B6 variable region light chain according to SEQ.ID 19 encodes the protein according to SEQ.ID 35.

[0134] The nucleic acid sequence of the humanized anti-PSMA variable region heavy chain according to SEQ. ID 20 encodes the protein according to SEQ. ID 36, while the humanized anti-PSMA variable region light chain according to SEQ. ID 21 encodes the protein according to SEQ. ID 37.

[0135] The nucleic acid sequence of the humanized anti-CD123 variable region heavy chain according to SEQ.ID 22 encodes the protein according to SEQ.ID 38, while the humanized anti-CD123 variable region light chain according to SEQ.ID 23 encodes the protein according to SEQ.ID 39. Sequence Listing <110> GEMoaB Monoclonals GmbH <120> Immune cells expressing reverse universal chimeric antigen receptors for targeting different multiple antigens, preparation methods thereof, and use thereof in treating cancer, infection, and autoimmune disorders <130> 01281P0017EPWO <150> 18177502.4 <151> 2018-06-13 <160> 39 <170> PatentIn version 3.5 <210> 1 <211> 1101 <212> DNA <213> Artificial sequence <220> <223> Human <400> 1 atgcgccgca tgcagctgct gcttctgatc gctctgagcc tggctcttgt gaccaactct 60 gaattcaaac ccctacctga agtgactgat gagtatgctg ccgctgggcc cggaggaggc 120 ggcagcaaga tcctggtcaa acagtcccct atgctggtcg cttacgacaa cgccgttaat 180 ctgagttgca aatatagtta caacctgttt agccgggaat ttcgcgcatc tctccacaag 240 ggactggatt ctgcggttga ggtttgtgtg gtctatggca attatagcca gcaactgcaa 300 gtgtacagca aaacaggctt taactgcgac gggaaactcg ggaacgaatc agtgaccttc 360 tatctgcaga acctgtacgt taaccaaaca gatatttact tctgcaagat agaggtgatg 420 gctccaccgc cagcactgga taacgagaag tccaatggaa ccatcattca cgtcaagggg 480 aagcatctgt gtccttcccc gttgttccct gggccgagca aacccttttg ggtgcttgtg 540 gtagttggcg gggtattggc ctgctattcc cttctcgtaa ctgtggcctt catcatcttc 600 tgggtcagat ctaagaggtc taggggcggg catagcgact acatgaacat gacacccagg 660 cggcctggcc ccactcgcaa acactaccag ccatacgcac caccaagaga ctttgccgca 720 tatcggagtg gtggcggcgg gtcaggaggt ggagctagcg gtggaggagg ttccttctct 780 aggtcagctg atgctcccgc ctatcagcaa ggtcagaacc agctctacaa tgagctgaat 840 ctgggacgtc gggaggagta cgacgtgctg gataaacgaa gaggacgcga tcccgagatg 900 ggtgggaagc ctaggcgcaa gaatccccag gaaggcctct acaatgaact gcagaaagac 960 aagatggccg aagcctacag cgagattggc atgaaagggg agcgacggag aggaaaggga 1020 catgacgggt tgtatcaggg tctttccact gcgacaaagg atacctatgg ggctctgcac 1080 atgcaagcac tgccacctag a 1101[[ID=१९]] <210> 2 <211> 60 <212> DNA <213> Human <400> 2 atgcgccgca tgcagctgct gcttctgatc gctctgagcc tggctcttgt gaccaactct 60 <210> ३ <211> ३० <212> DNA <213> human <400> 3 aaacccctac ctgaagtgac tgatgagtat 30 <210> 4 <211> 399 <212> DNA <213> human <400> 4 aagatcctgg tcaaacagtc ccctatgctg gtcgcttacg acaacgccgt taatctgagt 60 tgcaaatata gttacaacct gtttagccgg gaatttcgcg catctctcca caagggactg 120 gattctgcgg ttgaggtttg tgtggtctat ggcaattata gccagcaact gcaagtgtac 180 agcaaaacag gctttaactg cgacgggaaa ctcgggaacg aatcagtgac cttctatctg 240 cagaacctgt acgttaacca aacagatatt tacttctgca agatagaggt gatggctcca 300 ccgccagcac tggataacga gaagtccaat ggaaccatca ttcacgtcaa ggggaagcat 360 ctgtgtcctt ccccgttgtt ccctgggccg agcaaaccc 399 <210> 5 <211> 81 <212> DNA <213> human [[ID=4~1]]<400> 5 ttttgggtgc ttgtggtagt tggcggggta ttggcctgct attcccttct cgtaactgtg 60 gccttcatca tcttctgggt c 81 <210> 6 <211> 123 <212> DNA <213> human <400> 6 agatctaaga ggtctagggg cgggcatagc gactacatga acatgacacc caggcggcct 60 ggccccactc gcaaacacta ccagccatac gcaccaccaa gagactttgc cgcatatcgg 120 agt 123 <210> 7 <211> 327 <212> DNA <213> human <400> 7 ttctctaggt cagctgatgc tcccgcctat cagcaaggtc agaaccagct ctacaatgag 60 ctgaatctgg gacgtcggga ggagtacgac gtgctggata aacgaagagg acgcgatccc 120 gagatgggtg ggaagcctag gcgcaagaat ccccaggaag gcctctacaa tgaactgcag 180 aaagacaaga tggccgaagc ctacagcgag attggcatga aaggggagcg acggagagga 240 aagggacatg acgggttgta tcagggtctt tccactgcga caaaggatac ctatggggct 300 ctgcacatgc aagcactgcc acctaga 327 <210> 8 <211> 1137 <212> DNA <213> Artificial Sequence <220> <223> human <400> 8 atgcgccgca tgcagctgct gcttctgatc gctctgagcc tggctcttgt gaccaactct 60 gaattcaaac ccctacctga agtgactgat gagtatgctg ccgctgggcc cggaggaggc 120 ggcagcaaga tcctggtcaa acagtcccct atgctggtcg cttacgacaa cgccgttaat 180 ctgagttgca aatatagtta caacctgttt agccgggaat ttcgcgcatc tctccacaag 240 ggactggatt ctgcggttga ggtttgtgtg gtctatggca attatagcca gcaactgcaa 300 gtgtacagca aaacaggctt taactgcgac gggaaactcg ggaacgaatc agtgaccttc 360 tatctgcaga acctgtacgt taaccaaaca gatatttact tctgcaagat agaggtgatg 420 gctccaccgc cagcactgga taacgagaag tccaatggaa ccatcattca cgtcaagggg 480 aagcatctgt gtccttcccc gttgttccct gggccgagca aacccttttg ggtgcttgtg 540 gtagttggcg gggtattggc ctgctattcc cttctcgtaa ctgtggcctt catcatcttc 600 tgggtcagat ctaagaggtc taggggcggg ggcgggtcaa aacgcggacg gaagaaactg 660 ctgtacatct tcaagcagcc cttcatgcgc cccgtgcaga caacacagga agaggacggt 720 tgcagctgcc gatttcccga agaggaggag ggaggctgtg aattgggtgg cggcgggtca 780 ggaggtggag ctagcggtgg aggaggttcc ttctctaggt cagctgatgc tcccgcctat 840 cagcaaggtc agaaccagct ctacaatgag ctgaatctgg gacgtcggga ggagtacgac 900 gtgctggata aacgaagagg acgcgatccc gagatgggtg ggaagcctag gcgcaagaat 960 ccccaggaag gcctctacaa tgaactgcag aaagacaaga tggccgaagc ctacagcgag 1020 attggcatga aaggggagcg acggagagga aagggacatg acgggttgta tcagggtctt 1080 tccactgcga caaaggatac ctatggggct ctgcacatgc aagcactgcc acctaga 1137 <210> 9<00​​​​​​​​​​​​​​​​​​​​​​​​​​​ atgcgccgca tgcagctgct gcttctgatc gctctgagcc tggctcttgt gaccaactct 60 gaattcgaga aagaagcact gaagaaaata atagaagacc aacaagaatc cctaaacaaa 120 gctgccgctg ggcccggagg aggcggcagc aagatcctgg tcaaacagtc ccctatgctg 180 gtcgcttacg acaacgccgt taatctgagt tgcaaatata gttacaacct gtttagccgg 240 gaatttcgcg catctctcca caagggactg gattctgcgg ttgaggtttg tgtggtctat 300 ggcaattata gccagcaact gcaagtgtac agcaaaacag gctttaactg cgacgggaaa 360 ctcgggaacg aatcagtgac cttctatctg cagaacctgt acgttaacca aacagatatt 420 tacttctgca agatagaggt gatggctcca ccgccagcac tggataacga gaagtccaat 480 ggaaccatca ttcacgtcaa ggggaagcat ctgtgtcctt ccccgttgtt ccctgggccg 540 agcaaaccct tttgggtgct tgtggtagtt ggcggggtat tggcctgcta ttcccttc 600 gtaactgtgg ccttcatcat cttctgggtc agatctaaga ggtctagggg cgggcatagc 660 gactacatga acatgacacc caggcggcct ggccccactc gcaaacacta ccagccatac 720 gcaccaccaa gagactttgc cgcatatcgg agtggtggcg gcgggtcagg aggtggagct 780 agcggtggag gaggttcctt ctctaggtca gctgatgctc ccgcctatca gcaaggtcag 840 aaccagctct acaatgagct gaatctggga cgtcgggagg agtacgacgt gctggataaa 900 cgaagaggac gcgatcccga gatgggtggg aagcctaggc gcaagaatcc ccaggaaggc 960 ctctacaatg aactgcagaa agacaagatg gccgaagcct acagcgagat tggcatgaaa 1020 ggggagcgac ggagaggaaa gggacatgac gggttgtatc agggtctttc cactgcgaca 1080 aaggatacct atggggctct gcacatgcaa gcactgccac ctaga 1125 <210> 11 <211> 54 <212> DNA <213> Human <400> 11 gagaaagaag cactgaagaa aataatagaa gaccaacaag aatccctaaa caaa 54 <210> 12 <211> 1161 <212> DNA <213> Artificial Sequence <220> <223> Human <400> 12 atgcgccgca tgcagctgct gcttctgatc gctctgagcc tggctcttgt gaccaactct 60 gaattcgaga aagaagcact gaagaaaata atagaagacc aacaagaatc cctaaacaaa 120 gctgccgctg ggcccggagg aggcggcagc aagatcctgg tcaaacagtc ccctatgctg 180 gtcgcttacg acaacgccgt taatctgagt tgcaaatata gttacaacct gtttagccgg 240 gaatttcgcg catctctcca caagggactg gattctgcgg ttgaggtttg tgtggtctat 300 ggcaattata gccagcaact gcaagtgtac agcaaaacag gctttaactg cgacgggaaa 360 ctcgggaacg aatcagtgac cttctatctg cagaacctgt acgttaacca aacagatatt 420 tacttctgca agatagaggt gatggctcca ccgccagcac tggataacga gaagtccaat 480 ggaaccatca ttcacgtcaa ggggaagcat ctgtgtcctt ccccgttgtt ccctgggccg 540 agcaaaccct tttgggtgct tgtggtagtt ggcggggtat tggcctgcta ttcccttc 600 gtaactgtgg ccttcatcat cttctgggtc agatctaaga ggtctagggg cgggggcggg 660 tcaaaacgcg gacggaagaa actgctgtac atcttcaagc agcccttcat gcgccccgtg 720 cagacaacac aggaagagga cggttgcagc tgccgatttc ccgaagagga ggaggggc 780 tgtgaattgg gtggcggcgg gtcaggaggt ggagctagcg gtggaggagg ttccttctct 840 aggtcagctg atgctcccgc ctatcagcaa ggtcagaacc agctctacaa tgagctgaat 900 ctgggacgtc gggaggagta cgacgtgctg gataaacgaa gaggacgcga tcccgagatg 960 ggtgggaagc ctaggcgcaa gaatccccag gaaggcctct acaatgaact gcagaaagac 1020 aagatggccg aagcctacag cgagattggc atgaaagggg agcgacggag aggaaaggga 1080 catgacgggt tgtatcaggg tctttccact gcgacaaagg atacctatgg ggctctgcac 1140 atgcaagcac tgccacctag a 1161 <210> 13 <211> 63 <212> DNA <213> human <400> 13 atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60 gac 63 <210> 14 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> Mouse-Human Chimeric <400> 14 caggtgcagc tggtgcagag cggagccgag gtgaagaagc ctggagcctc tgtgaaggtg 60 agctgcaagg cttctggcta caccttcacc cactactaca tctactgggt gagacaggct 120 cccggacagg gcctggagtg gatgggaggc gtgaacccca gcaacggagg cacccacttc 180 aacgagaagt tcaagtctcg cgtgaccatg acccgcgaca ccagcatctc taccgcttac 240 atggagctga gccgcctgcg ctctgatgat accgctgtgt actactgcgc tcgcagcgag 300 tacgattacg gactgggctt cgcctactgg ggccagggaa ccctggtgac cgtgagctct 360 <210> 15 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> Mouse-Human Chimeric[[ID=^21]] <400> 15 gatatcgtga tgacccagtc tcctgatagc ctggctgtga gcctgggcga gagagctacc 60 atcaactgca agagcagcca gagcctgctg aactctcgca cccctaagaa ctaccttgct 120 tggtaccagc agaagcctgg acagccccct aagctgctga tctactgggc ttctacccgc 180 aagagcggcg tgcccgacag attctctggc agcggaagcg gcaccgattt caccctgacc 240 atcagcagcc tgcaggctga ggacgtggcc gtgtactact gcaagcagtc ttacaacctg 300 ctgaccttcg gaggcggaac caaggtggag atcaag 336 <210> 16 <211> 30 <212> DNA <213> human <400> 16 gaacaaaaac tcatctcaga agaggatctg 30 <210> 17 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Hexahistidine <400> 17 catcatcatc atcatcat 18 <210> 18 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> Mouse-Human Chimeric <400> 18 gacattgtta tgacccagag cccggactct ctcgctgtta gtcttggtga gcgagcgact 60 attaactgcc ggagcagtca gagtttgttg gactctcgga cgaaaaagaa ctacctggca 120 tggtaccagc agaagccggg ccaaccacct aaattactga tatattgggc gtcgactcgt 180 gagtcagggg taccggacag gttttctgga agcggatcag gaacagactt cactttgacg 240 atctcttcgc ttcaagccga ggacgttgcg gtttattatt gtaagcaaag ctataatctg 300 ccgacatttg gtggcggcac caaggttgaa attaag 336 <210> 19 <211> 354 <212> DNA <213> Artificial sequence <220> <223> Mouse - human chimeric <400> 19 gaggtgcaac tggtcgaaag tggcggtggt ttagttcagc ctggtggaag tctacggctt 60 agctgcgcag catccggttt cacctttagc gacttttgga tgaactgggt tcggcaggct 120 ccgggcaaag gactggagtg ggttgggcaa atccgcaaca aaccgaataa ctacgaaact 180 tattactcag atagcctgaa gggtcgattc accatcagca gggatgattc aaagtcaatc 240 acttacctac agatgaactc attaagagcg gaggatactg cggtgtatta ctgtacacta 300 ggtaactcct ggttcgcgta ttggggacag ggcacccttg taaccgtctc cagc 354 <21l> 20 <211> 345 <212> DNA <213> Artificial sequence <220> <223> Human - mouse chimeric <400> 20 gaggtgcagc tgcagcagtc aggacctgaa ctggtgaagc ctgggacttc agtgaggata 60 tcctgcaaga cttctggata cacattcact gaatatacca tacactgggt gaagcagagc 120 catggaaaga gccttgagtg gattggaaac atcaatccta acaatggtgg taccacctac 180 aatcagaagt tcgaggacaa ggccacattg actgtagaca agtcctccag tacagcctac 240 atggagctcc gcagcctaac atctgaggat tctgcagtct attattgtgc agctggttgg 300 aactttgact actggggcca agggaccacg gtcaccgtct cctca 345 <210> 21 <211> 321 <212> DNA <213> Artificial sequence <220> <223> Human - mouse chimeric <400> 21 gacattgtga tgacccagtc tcacaaattc atgtccacat cagtaggaga cagggtcagc 60 atcatctgta aggccagtca agatgtgggt actgctgtag actggtatca acagaaacca 120 ggacaatctc ctaaactact gatttattgg gcatccactc ggcacactgg agtccctgat 180 cgcttcacag gcagtggatc tgggacagac ttcactctca ccattactaa tgttcagtct 240 gaagacttgg cagattattt ctgtcagcaa tataacagct atcccctcac gttcggtgct 300 gggaccatgc tggacctgaa a 321 <210> 22 <211> 357 <212> DNA <213> Artificial sequence<000<220> <223> Human-mouse chimeric <400> 22 gaagtgcagc tgcagcagtc tggccccgag ctggtcaaac caggcgccag cgtgaagatg 60 agctgcaagg ccagcggcta caccttcacc gactactaca tgaagtgggt caagcagagc 120 cacggcaaga gcctggaatg gatcggcgac atcatcccca gcaacggcgc caccttctac 180 aaccagaagt tcaagggcaa ggccaccctg accgtggaca gaagcagcag caccgcctac 240 atgcacctga acagcctgac cagcgaggac agcgccgtgt actactgcac cagaagccat 300 ctgctgcggg ccagttggtt cgcttattgg ggccagggca ccctggtcac agtgtct 357 <210> 23 <211> 339 <212> DNA <213> Artificial sequence <220> <223> Human-mouse chimeric <400> 23 gacttcgtga tgacccagag ccctagcagc ctgaccgtga cagccggcga gaaagtgacc 60 atgagctgca agagcagcca gagcctgctg aactccggca accagaagaa ctacctgacc 120 tggtatctgc agaagcccgg acagcccccc aagctgctga tctactgggc cagcaccaga 180 gaaagcggcg tgcccgatag attcacaggc agcggcagcg gcaccgactt caccctgaca 240 atcagcagcg tgcaggccga ggacctggcc gtgtactatt gccagaacga ctacagctac 300 ccctacacct tcggaggcgg gaccaagctg gaaatcaag 339 <210> 24 <211> 367 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens <400> 24 Met Arg Arg Met Gln Leu Leu Leu Leu Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser Glu Phe Lys Pro Leu Pro Glu Val Thr Asp Glu Tyr 20 25 30 Ala Ala Ala Gly Pro Gly Gly Gly Gly Ser Lys Ile Leu Val Lys Gln 35 40 45 Ser Pro Met Leu Val Ala Tyr Asp Asn Ala Val Asn Leu Ser Cys Lys 50 55 60 Tyr Ser Tyr Asn Leu Phe Ser Arg Glu Phe Arg Ala Ser Leu His Lys 65 70 75 80 Gly Leu Asp Ser Ala Val Glu Val Cys Val Val Tyr Gly Asn Tyr Ser 85 90 95 Gln Gln Leu Gln Val Tyr Ser Lys Thr Gly Phe Asn Cys Asp Gly Lys 100 105 110 Leu Gly Asn Glu Ser Val Thr Phe Tyr Leu Gln Asn Leu Tyr Val Asn 115 120 125 Gln Thr Asp Ile Tyr Phe Cys Lys Ile Glu Val Met Ala Pro Pro Pro 130 135 140 Ala Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile His Val Lys Gly 145 150 155 160 Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe 165 170 175 Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu 180 185 190 Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg 195 200 205 Gly Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro 210 215 220 Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala 225 230 235 240 Tyr Arg Ser Gly Gly Gly Gly Ser Gly Gly Gly Ala Ser Gly Gly Gly 245 250 255 Gly Ser Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln 260 265 270 Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp 275 280 285 Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro 290 295 300 Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp 305 310 315 320 Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg 325 330 335 Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr 340 345 350 Lys Asp Thr Tyr Gly Ala Leu His Met Gln Ala Leu Pro Pro Arg 355 360 365 <210> 25 <211> 379 <212> PRT <213> Artificial Sequence <220> <223> Human <400> 25 Met Arg Arg Met Gln Leu Leu Leu Leu Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser Glu Phe Lys Pro Leu Pro Glu Val Thr Asp Glu Tyr 20 25 30 Ala Ala Ala Gly Pro Gly Gly Gly Gly Ser Lys Ile Leu Val Lys Gln 35 40 45 Ser Pro Met Leu Val Ala Tyr Asp Asn Ala Val Asn Leu Ser Cys Lys 50 55 60 Tyr Ser Tyr Asn Leu Phe Ser Arg Glu Phe Arg Ala Ser Leu His Lys 65 70 75 80 Gly Leu Asp Ser Ala Val Glu Val Cys Val Val Tyr Gly Asn Tyr Ser 85 90 95 Gln Gln Leu Gln Val Tyr Ser Lys Thr Gly Phe Asn Cys Asp Gly Lys 100 105 110 Leu Gly Asn Glu Ser Val Thr Phe Tyr Leu Gln Asn Leu Tyr Val Asn 115 120 125 Gln Thr Asp Ile Tyr Phe Cys Lys Ile Glu Val Met Ala Pro Pro Pro 130 135 140 Ala Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile His Val Lys Gly 145 150 155 160 Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe 165 170 175 Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu 180 185 190 Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg 195 200 205 Gly Gly Gly Gly Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe 210 215 220 Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly 225 230 235 240 Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Gly 245 250 255 Gly Gly Gly Ser Gly Gly Gly Ala Ser Gly Gly Gly Gly Ser Phe Ser 260 265 270 Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr 275 280 285 Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys 290 295 300 Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn 305 310 315 320 Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu 325 330 335 Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly 340 345 350 His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr 355 360 365 Gly Ala Leu His Met Gln Ala Leu Pro Pro Arg 370 375 <210> 26 <211> 375 <212> PRT <213> Artificial Sequence <220> <223> Human <400> 26 Met Arg Arg Met Gln Leu Leu Leu Leu Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser Glu Phe Glu Lys Glu Ala Leu Lys Lys Ile Ile Glu 20 25 30 Asp Gln Gln Glu Ser Leu Asn Lys Ala Ala Ala Gly Pro Gly Gly Gly 35 40 45 Gly Ser Lys Ile Leu Val Lys Gln Ser Pro Met Leu Val Ala Tyr Asp 50 55 60 Asn Ala Val Asn Leu Ser Cys Lys Tyr Ser Tyr Asn Leu Phe Ser Arg 65 70 75 80 Glu Phe Arg Ala Ser Leu His Lys Gly Leu Asp Ser Ala Val Glu Val 85 90 95 Cys Val Val Tyr Gly Asn Tyr Ser Gln Gln Leu Gln Val Tyr Ser Lys 100 105 110 Thr Gly Phe Asn Cys Asp Gly Lys Leu Gly Asn Glu Ser Val Thr Phe 115 120 125 Tyr Leu Gln Asn Leu Tyr Val Asn Gln Thr Asp Ile Tyr Phe Cys Lys 130 135 140 Ile Glu Val Met Ala Pro Pro Pro Ala Leu Asp Asn Glu Lys Ser Asn 145 150 155 160 Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu 165 170 175 Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly 180 185 190 Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe 195 200 205 Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn 210 215 220 Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr 225 230 235 240 Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Gly Gly Gly Gly Ser 245 250 255 Gly Gly Gly Ala Ser Gly Gly Gly Gly Ser Phe Ser Arg Ser Ala Asp 260 265 270 Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 275 280 285 Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 290 295 300 Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly 305 310 315 320 Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu 325 330 335 Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu 340 345 350 Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Gly Ala Leu His 355 360 365 Met Gln Ala Leu Pro Pro Arg 370 375 <210> 27 <211> 387 <212> PRT <213> Artificial Sequence <220> <223> Human <400> 27 Met Arg Arg Met Gln Leu Leu Leu Leu Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser Glu Phe Glu Lys Glu Ala Leu Lys Lys Ile Ile Glu 20 25 30 Asp Gln Gln Glu Ser Leu Asn Lys Ala Ala Ala Gly Pro Gly Gly Gly 35 40 45 Gly Ser Lys Ile Leu Val Lys Gln Ser Pro Met Leu Val Ala Tyr Asp 50 55 60 Asn Ala Val Asn Leu Ser Cys Lys Tyr Ser Tyr Asn Leu Phe Ser Arg 65 70 75 80 Glu Phe Arg Ala Ser Leu His Lys Gly Leu Asp Ser Ala Val Glu Val 85 90 95 Cys Val Val Tyr Gly Asn Tyr Ser Gln Gln Leu Gln Val Tyr Ser Lys 100 105 110 Thr Gly Phe Asn Cys Asp Gly Lys Leu Gly Asn Glu Ser Val Thr Phe 115 120 125 Tyr Leu Gln Asn Leu Tyr Val Asn Gln Thr Asp Ile Tyr Phe Cys Lys 130 135 140 Ile Glu Val Met Ala Pro Pro Pro Ala Leu Asp Asn Glu Lys Ser Asn 145 150 155 160 Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu 165 170 175 Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly 180 185 190 Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe 195 200 205 Trp Val Arg Ser Lys Arg Ser Arg Gly Gly Gly Gly Ser Lys Arg Gly 210 215 220 Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val 225 230 235 240 Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu 245 250 255 Glu Glu Gly Gly Cys Glu Leu Gly Gly Gly Gly Ser Gly Gly Gly Ala 260 265 270 Ser Gly Gly Gly Gly Ser Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr 275 280 285 Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg 290 295 300 Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met 305 310 315 320 Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu 325 330 335 Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys 340 345 350 Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu 355 360 365 Ser Thr Ala Thr Lys Asp Thr Tyr Gly Ala Leu His Met Gln Ala Leu 370 375 380 Pro Pro Arg 385 <210> 28 <211> 10 <212> PRT <213> human <400> 28 Lys Pro Leu Pro Glu Val Thr Asp Glu Tyr 1 5 10 <210> 29 <211> 17 <212> PRT <213> human <400> 29 Glu Lys Glu Ala Leu Lys Lys Ile Ile Glu Asp Gln Gln Glu Ser Leu 1 5 10 15 Asn <210> 30 <211> 120 <212> PRT <213> artificial sequence <220> <223> mouse - human chimeric <400> 30 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr His Tyr 20 25 30 Tyr Ile Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Val Asn Pro Ser Asn Gly Gly Thr His Phe Asn Glu Lys Phe 50 55 60 Lys Ser Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Glu Tyr Asp Tyr Gly Leu Gly Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 31 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Mouse-Human Chimeric <400> 31 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Arg Thr Pro Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Lys Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Asn Leu Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 32 <211> 10 <212> PRT <213> human <400> 32 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 1 5 10 <210> 33 <211> 6 <212> PRT <213> human <400> 33 His His His His His His 1 5 <210> 34 <211> 112 <212> PRT <213> synthetic sequence <220> <223> Mouse-human chimera <400> 34 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Arg Thr Lys Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Asn Leu Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 35 <211> 118 <212> PRT <213> Artificial sequence <220> <223> Mouse-human chimera <400> 35 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Phe 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Gln Ile Arg Asn Lys Pro Asn Asn Tyr Glu Thr Tyr Tyr Ser Asp 50 55 60 Ser Leu Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile 65 70 75 80 Thr Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Leu Gly Asn Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 36 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Human - Mouse Chimeric <400> 36 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Thr 1 5 10 15 [[ID=四十七]]Ser Val Arg Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr[[ID=四十八]] [[ID=四十九]]20 25 30[[ID=五十]] Thr Ile His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe 50 55 60 Glu Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser 115 <210> 37 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Human-Mouse Chimeric <400> 37 Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Ile Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25 30 Val Asp Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn Val Gln Ser 65 70 75 80 Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Met Leu Asp Leu Lys 100 105 <210> 38 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Human - Mouse Chimeric <400> 38 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Met Lys Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Asp Ile Ile Pro Ser Asn Gly Ala Thr Phe Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Arg Ser Ser Ser Thr Ala Tyr 65 70 75 80 甲硫氨酸-组氨酸-亮氨酸-天冬酰胺-丝氨酸-亮氨酸-苏氨酸-丝氨酸-谷氨酸-天冬氨酸-丝氨酸-丙氨酸-缬氨酸-酪氨酸-酪氨酸-半胱氨酸 85 90 95 苏氨酸-精氨酸-丝氨酸-组氨酸-亮氨酸-亮氨酸-精氨酸-丙氨酸-丝氨酸-色氨酸-苯丙氨酸-丙氨酸-酪氨酸-色氨酸-甘氨酸-谷氨酰胺 100 105 110 甘氨酸-苏氨酸-亮氨酸-缬氨酸-苏氨酸-缬氨酸-丝氨酸 115 <210> 39 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Human-Mouse Chimeric <400> 39 天冬氨酸-苯丙氨酸-缬氨酸-甲硫氨酸-苏氨酸-谷氨酰胺-丝氨酸-脯氨酸-丝氨酸-丝氨酸-亮氨酸-苏氨酸-缬氨酸-苏氨酸-丙氨酸-甘氨酸 1 5 10 15 谷氨酸-赖氨酸-缬氨酸-苏氨酸-甲硫氨酸-丝氨酸-半胱氨酸-赖氨酸-丝氨酸-丝氨酸-谷氨酰胺-丝氨酸-亮氨酸-亮氨酸-天冬酰胺-丝氨酸 20 25 30 甘氨酸-天冬酰胺-谷氨酰胺-赖氨酸-天冬酰胺-酪氨酸-亮氨酸-苏氨酸-色氨酸-酪氨酸-亮氨酸-谷氨酰胺-赖氨酸-脯氨酸-甘氨酸-谷氨酰胺 35 40 45 脯氨酸-脯氨酸-赖氨酸-亮氨酸-亮氨酸-异亮氨酸-酪氨酸-色氨酸-丙氨酸-丝氨酸-苏氨酸-精氨酸-谷氨酸-丝氨酸-甘氨酸-缬氨酸 50 55 60 脯氨酸-天冬氨酸-精氨酸-苯丙氨酸-苏氨酸-甘氨酸-丝氨酸-甘氨酸-丝氨酸-甘氨酸-天冬氨酸-苯丙氨酸-苏氨酸-亮氨酸-苏氨酸 65 70 75 80 异亮氨酸-丝氨酸-丝氨酸-缬氨酸-谷氨酰胺-丙氨酸-谷氨酸-天冬氨酸-亮氨酸-丙氨酸-缬氨酸-酪氨酸-酪氨酸-半胱氨酸-谷氨酰胺-天冬酰胺 85 90 95 Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys

Claims

1. A combination comprising: (a) a reverse universal chimeric antigen receptor or a nucleic acid encoding the reverse universal chimeric antigen receptor or a cell or vector comprising a nucleic acid encoding the reverse universal chimeric antigen receptor, wherein the receptor comprises three domains, wherein - the first domain is a peptide epitope tag, wherein the peptide epitope tag is a short linear epitope from human nucleoprotein according to the sequence shown in SEQ ID 28 or 29, - the second domain is the extracellular hinge and transmembrane domain, and - the third domain is a signal transduction domain; and (b) an adapter module or a nucleic acid encoding the adapter module, wherein the adapter module consists of a binding portion specific for a human cell surface protein or protein complex and a tag binding domain, wherein the tag binding domain is an antibody or fragment thereof or a ligand that binds to the peptide epitope tag of the reverse universal chimeric antigen receptor.

2. The combination according to claim 1, wherein the nucleic acid encoding the peptide epitope tag is the nucleic acid according to SEQ. ID 3 and 11.

3. The combination according to claim 1 , wherein the hinge and transmembrane domain are selected from the hinge and transmembrane domain of part of the human CD28 molecule, CD8a chain, NK cell receptor, DAP12, Fc receptor or antibody constant region, and combinations of different hinge and transmembrane domains thereof, wherein the hinge domain is part of the extracellular domain.

4. The combination according to any one of claims 1 to 3, wherein the signal transduction domain is selected from the group consisting of: CD28, CD137 (41BB), CD134 (OX40), DAP10 and the cytoplasmic region of CD27, programmed cell death 1 (PD-1), cytotoxic T lymphocyte antigen 4 (CTLA-4), and the cytoplasmic region of the CD3 chain, DAP12 and T cell activation-inducing Fc receptor, wherein the signal transduction domain and the cytoplasmic region are signaling domains.

5. The combination according to any one of claims 1 to 3, wherein the reverse universal chimeric antigen receptor is represented by one of the sequences SEQ. ID 24 to 27.

6. A combination according to any one of claims 1 to 3, wherein the receptor comprises a fourth domain which is a short peptide linker in the extracellular part of the receptor.

7. The combination according to any one of claims 1 to 3, wherein the nucleic acid encoding the reverse universal chimeric antigen receptor is represented by one of the sequences SEQ. ID 1, 8, 10 or 12.

8. The combination of claim 1 , wherein the binding portion of the adapter module comprises an antibody or fragment thereof that binds to a surface antigen of: CD2, CD3, CD4, CD8, CD10, CD19, CD20, CD22, CD23, CD25, CD30, CD33, CD38, CD44, CD52, CD90, CD99, CD123, CD181, CD182, CD184, CD223, CD269 (BCMA), CD274, CD276, CD279 and CD366, an interleukin receptor, CXCR4, a member of the epidermal growth factor receptor family, a member of the tumor necrosis factor receptor superfamily, an ephrin receptor, prostate specific antigen, prostate stem cell antigen (PSCA), and prostate Specific membrane antigen (PSMA), embryonic antigen carcinoembryonic antigen (CEA) and fetal acetylcholine receptor, members of the vascular endothelial growth factor family, epithelial cell adhesion molecule EpCAM, alpha-fetoprotein AFP, members of the mucin family, follicle-stimulating hormone receptor (FSHR), human high molecular weight melanoma-associated antigen (HMW-MAA), folate binding protein FBP, α-folate receptor, ligand of NKG2D receptor, cytokine receptor, members of the epithelial glycoprotein family, disialoganglioside, members of the carbonic anhydrase family, members of the carbohydrate antigen family, Notch ligand, melanoma-associated chondroitin sulfate proteoglycan (MCSP), glycoprotein A33 and tumor-specific glycans, and the binding portion includes antibodies or fragments thereof that bind to cytoplasmic or nuclear antigens.

9. The combination according to claim 8, wherein the cytoplasmic or nuclear antigen is a member of the Rho family of GTPases or a member of the high mobility group protein.

10. The combination according to claim 1 , wherein the binding portion of the adapter module consists of the α and β chains of the T cell receptor (TCR), or of the γ and δ chains of the TCR, or of fragments of the α and β chains, or of fragments of the γ and δ chains, wherein the TCR-derived binding portion recognizes and binds to peptides presented by human leukocyte antigen (HLA) class I and class II protein complexes.

11. The combination according to claim 10, wherein the TCR is specific for peptides derived from proteins including the EGFR family, survivin, the SRY-like high-mobility group box (SOX) protein family, melanoma-associated antigens and leukemia-associated antigens.

12. The combination according to claim 1, wherein the binding portion of the adapter module comprises a ligand of a protein or protein complex or a fragment thereof, wherein the ligand binds a cytokine receptor, a ligand of an NKG2D receptor, a ligand of a member of the EGFR family, a ligand of a checkpoint molecule, or an autoreactive TCR.

13. The combination according to claim 12, wherein the checkpoint molecule is PD-1, CTLA-4, lymphocyte activation gene 3 (LAG-3) or TIM-3.

14. The combination according to claim 1, wherein the binding portion of the adapter module comprises a chemically synthesized peptide derivative fused to the tag binding portion by a chemical reaction.

15. The combination according to claim 14, wherein the chemical reaction is click chemistry.

16. A combination according to any one of claims 1 and 8 to 15, wherein the binding portion of the adapter moiety comprises bispecific and multispecific antigen specificities, including binding to both PSCA and PSMA, binding to both CD19 and CD20, binding to both CD19 and CD22, binding to all three of CD19, CD20 and CD22, binding to both CD19 and CD123, binding to both CD33 and CD123, binding to both CD33 and CD99, binding to both CD33 and TIM-3, binding to both ErbB-1 and ErbB-2, binding to both PSCA and ErbB-2, binding to both IL-13Rα2 and ErbB-2, binding to both CD38 and CD269.

17. The combination of claim 1 , wherein in the nucleic acid encoding the adapter module, the tag-binding portion of the adapter module comprises an antibody or fragment thereof that binds to the 5B9 or 7B6 epitope of the La / SSB antigen, wherein the antibody or fragment thereof is according to SEQ. ID 14 and 15, or SEQ. ID 18 and 19.

18. The combination according to claim 1, wherein in the nucleic acid encoding the adapter module, the target binding portion of the adapter module comprises an antibody or fragment thereof that binds PSMA or CD123.

19. The combination according to claim 18, wherein in the nucleic acid encoding the adapter module, the antibody or fragment thereof that binds to PSMA or CD123 is according to SEQ. ID 20 and 21, or SEQ. ID 22 and 23.

20. The combination according to claim 1, wherein the cells comprising the nucleic acid encoding the reverse universal chimeric antigen receptor are selected from the group of immune cells comprising T cells, natural killer cells, cytotoxic T lymphocytes and regulatory T cells.

21. A kit comprising: - a vector according to claim 1 comprising a nucleic acid encoding a reverse universal chimeric antigen receptor, and - An adapter module in a combination according to any one of claims 1 to 16 and / or a vector encoding a nucleic acid as defined in any one of claims 17 to 19.

22. A formulation for administration to a subject, comprising an immune cell comprising a nucleic acid encoding the reverse universal chimeric antigen receptor as defined in claim 20 and an adapter module according to the combination of any one of claims 1 to 16.

23. Use of a cell as defined in claim 20 and an adapter module in combination with any one of claims 1 to 16 in the preparation of a medicament for treating cancer, infection or autoimmune disorder.

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