HK1236225B - Universal chimeric antigen receptor expressing immune cells for targeting of diverse multiple antigens and method of manufacturing the same and use of the same for treatment of cancer, infections and autoimmune disorders - Google Patents

Description

Universal chimeric antigen receptor-expressing immune cells targeting diverse multiple antigens, methods for their manufacture, and their use in the treatment of cancer, infection, and autoimmune diseases

Technical Field

The present invention relates to immune cell-based therapeutics and methods of utilizing the same in the treatment of cancer, infection, and autoimmune diseases.

Background Art

Chimeric antigen receptors (CARs) are artificial receptors composed of a binding moiety that provides antigen specificity and one or more signaling 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 containing a transmembrane domain, which anchors the CAR in the cell plasma membrane. Immune cells, particularly T and NK lymphocytes, can be genetically modified to express CARs inserted into their plasma membranes. If such CAR-modified immune cells encounter other cells or tissue structures that express or are stained with the appropriate target of the CAR binding moiety, when the CAR binding moiety binds to the target antigen, the CAR-modified immune cell cross-links with the target. Cross-linking causes the induction of signaling pathways via the CAR signaling chain, which will change the biological properties of the CAR-implanted immune cells. For example, CAR triggering in effector CD4+ and CD8+ T cells will activate typical effector cell functions, such as secretion of lytic compounds and cytokines, which will ultimately lead to killing of the corresponding target cells. Adoptive transfer of immune cells engineered with chimeric antigen receptors (CARs) is currently considered a highly promising treatment option for malignant, infectious, or autoimmune diseases that are otherwise incurable. The first clinical trial demonstrated that this treatment strategy is both safe and feasible (Lamers et al. J. Clin. Oncol. 24e20-e22 (2006), Kershaw et al. Clin. Cancer Res. 12 6106–6115 (2006)). In ongoing trials, the majority of patients with advanced B-cell-derived tumors showed complete or at least partial responses to treatment with autologous T cells equipped with a CD19-specific CAR, which lasted for several months (Brentjens et al. Blood. 118 4817–4828 (2011), Sci. Transl. Med. 20(5) doi: 10.1126/scitranslmed.3005930 (2013)., Kalos et al. 2011 Sci. Transl. Med. 3(95) doi: 10.1126/scitranslmed.3002842, Grupp et al. N. Engl. J. Med. 368: 1509–1518 (2013)).

However, conventional CAR technology is accompanied by many major problems that need to be solved before this treatment can be widely used in clinical treatment. First of all, several safety issues must be resolved. So far, the immune response of T cells engineered with conventional CARs after being injected into patients has been difficult to control. In particular, unexpected target gene expression in healthy tissues may provoke a rapid and intense immune response of engineered T cells against healthy cells, which can lead to serious side effects (Lamers et al. J. Clin. Oncol. 24e20-e22 (2006), Morgan et al. Mol. Ther. 18: 843-851 (2010). Another disadvantage of conventional CAR technology is the limitation of engineered T cells to re-target a single antigen. Such a single treatment approach means the risk of generating tumor escape variants that lose the target antigen during treatment. In clinical trials, tumor escape variants have been observed to appear after a few months under conventional CART cell therapy (Grupp et al. N. Engl. J. Med. 368: 1509–1518 (2013)).

WO 2012082841 A2 discloses universal tag-resistant chimeric antigen receptor-expressing T cells and methods for treating cell-related disorders, such as cancer.

In addition, WO 2013044225 A1 discloses universal immune receptors expressed by T cells that target diverse and multiple antigens.

These two approaches describe the use of modified T cells expressing universal anti-tag immune receptors. By additionally applying modules that bind to these surface antigens and carry the corresponding tags, these T cells can be redirected to disease-associated cell surface antigens. A problem with these approaches is that redirecting genetically modified T cells using exogenous tags can be immunogenic, placing patients at risk and negatively impacting treatment efficacy.

Therefore, the object of the present invention is to provide genetically modified immune cells that utilize endogenous nuclear protein-based tags to allow for redirection against a variety of disorders in a safe and effective manner. Another object of the present invention is to provide treatment methods for a variety of cell-related disorders, wherein the length and intensity of treatment can be adjusted in a simple manner.

Summary of the Invention

The present invention provides a universal, modular, anti-tag chimeric antigen receptor (UniCAR) system that allows UniCAR-implanted immune cells to be retargeted to multiple antigens. The system utilizes a gene therapy platform to generate immune cells that can recognize a variety of antigens and have broad and valuable clinical significance for use in immune cell-based therapies, particularly T and NK cell-based therapies.

In the first aspect, the present invention provides an isolated nucleic acid sequence encoding a universal chimeric antigen receptor, wherein the receptor comprises three domains, wherein the first domain is a tag 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 tag binding domain binds to a tag derived from any human nuclear protein. In particular, a suitable tag is a peptide sequence from a nuclear antigen that cannot be approached and bound by the corresponding tag binding domain in the environment of the natural protein under physiological conditions. In addition, the peptide sequence should not be a target for autoantibodies in autoimmune patients, thereby making it unlikely that the tag is immunogenic in the environment of the universal chimeric receptor. The optional fourth domain is a short peptide linker in the extracellular portion of UniCAR that forms a linear epitope to which a monoclonal antibody (mab) specifically binds to the fourth domain. This additional domain is not required for the functionality of the UniCAR system, but can add additional clinical benefits to the present invention. Preferably, the present invention provides an isolated nucleic acid sequence encoding a universal chimeric antigen receptor of the present invention, wherein the nucleic acid sequence encodes an artificial chimeric fusion protein and wherein the nucleic acid sequence is provided as cDNA.

In another aspect, the present invention provides a targeting module consisting of a binding moiety specific for a certain human cell surface protein or protein complex, and a tag, wherein the tag is derived from any human nuclear protein.

In another aspect, the present invention provides a nucleic acid encoding a target module of the present invention. Preferably, the present invention provides an isolated nucleic acid sequence encoding a target module of the present invention, wherein the isolated nucleic acid is provided as cDNA.

In another aspect, the present invention provides a cell comprising a nucleic acid encoding a universal chimeric antigen receptor of the present invention, wherein the receptor comprises three domains, wherein the first domain is a tag binding domain, the second domain is a connecting peptide chain including an extracellular hinge and a transmembrane domain, and the third domain is a signal transduction domain, and wherein the tag binding domain binds to a tag derived from any human nuclear protein.

In another aspect, the present invention provides a vector comprising a nucleic acid encoding a universal chimeric antigen receptor of the present invention, wherein the universal chimeric antigen receptor comprises three domains, wherein the first domain is a tag 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, and wherein the tag binding domain binds to a tag derived from any human nuclear protein.

In another aspect, the present invention provides a kit comprising a vector comprising a nucleic acid sequence encoding a universal chimeric antigen receptor of the present invention and a target module of the present invention and/or a vector encoding an isolated nucleic acid sequence encoding a target module of the present invention.

The present invention also includes a pharmaceutical composition comprising the cells and target modules of the present invention in combination with a pharmaceutically acceptable diluent or carrier. Preferably, the pharmaceutical composition is in a form suitable for intravenous administration.

Preferably, the composition comprises a cell comprising a nucleic acid encoding a universal chimeric antigen receptor of the present invention and a targeting moiety of the present invention.

The pharmaceutical compositions of the present invention encompass 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. Particularly preferred compositions are solutions, emulsions, or suspensions of the cells and target module in a pharmaceutically acceptable diluent or carrier.

As carriers, water, buffered water, 0.4% saline solution, 0.3% glycine, and similar solvents are preferably used. These solutions are sterile. The pharmaceutical compositions are sterilized by conventional, well-known techniques. These compositions preferably contain pharmaceutically acceptable excipients, such as those required to provide near-physiological conditions and/or enhance the stability of the target moiety, such as pH-regulating agents and buffers, agents for regulating toxicity, and the like, preferably selected from sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. The concentration of the target moiety of the present invention in these formulations varies depending on the application; it is preferably less than 0.01% by weight, preferably at least 0.1% by weight, and more preferably between 1 and 5% by weight, and is primarily selected based on the volume, viscosity, and other factors, or in accordance with the respective mode of administration.

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 this purpose and known to the skilled artisan.

The cells and target modules of the present invention are preferably incorporated into a composition suitable for parenteral administration. Preferably, the pharmaceutical composition is an injectable buffered solution containing between 0.001 and 500 mg/ml of the antibody, particularly preferably between 0.001 and 250 mg/ml of the target module, in particular together with 1 to 500 mmol/l (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 10 mM) at a pH of 5.0 to 7.0 (particularly preferably pH 6.0).

Other suitable buffers include, but are not limited to, sodium succinate, sodium citrate, sodium phosphate or potassium phosphate. Preferably, the liquid dosage form uses 0 to 300 mM, particularly preferably 150 mM sodium chloride. In the liquid dosage form, a stabilizer is preferably used, particularly preferably between 1 and 50 mM L-methionine (preferably between 5 and 10 mM).

Typical dosage rates delivered per m2 per day are between 1 μg and 1000 mg, preferably 10 μg to 1 mg, with the dosage being administered one or more times daily or weekly or continuously over a period of several weeks.

In another aspect, the present invention provides the use of a cell of the present invention comprising a nucleic acid encoding a universal chimeric antigen receptor of the present invention and a targeting module of 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 of the present invention comprising a nucleic acid encoding a universal chimeric antigen receptor of the present invention and a targeting module of the present invention as a medicament, more preferably as a medicament for treating cancer or an autoimmune disease. Autoimmune diseases result from an abnormal immune response (autoimmunity) to substances and tissues normally present in the body.

The present invention also encompasses the use of the cells and target modules of the present invention for the preparation of a medicament for therapeutic and/or diagnostic applications in the context of cancer or autoimmune diseases.

The invention also includes methods of treating humans suffering from cancer or autoimmune disease by administering the cells and targeting moieties of the invention.

For therapeutic applications, a sterile pharmaceutical composition containing a pharmacologically effective amount of the cells of the invention and a targeting module is administered to a patient to treat the aforementioned condition.

The invention will be explained in more detail with the aid of the following figures and exemplary embodiments, without however restricting the invention to these.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 depicts a schematic diagram of the universal chimeric antigen receptor (UniCAR),

FIG2 shows a schematic diagram of the universal chimeric antigen receptor (UniCAR) platform for retargeting antigen-specific immune cells.

FIG3 shows a schematic diagram of the lentiviral vector pLVX-EF1α-IRES-ZsGreen1.

Figure 4 shows a schematic diagram of the lentiviral packaging plasmid psPAX2,

Figure 5 shows a schematic diagram of the envelope plasmid pMD2.G,

FIG6 depicts a graph showing UniCAR surface expression detected using a monoclonal antibody against an optional fourth domain,

FIG7 shows a graph showing the effector function of UniCAR engineered T cells against tumor cells expressing prostate stem cell antigen and prostate membrane antigen.

FIG8 shows a graph of concentration-response curves for different target modules.

FIG9 shows a graph showing the effector function of UniCAR engineered T cells against acute myeloid leukemia,

FIG10 depicts a graph showing T cell redirection following implantation of a UniCAR targeting two antigens simultaneously, and

Figure 11 depicts a graph showing the in vivo pharmacokinetics of the bispecific αCD123-CD33 targeting moiety.

DETAILED DESCRIPTION

Effector cells

The effector cells used in the method of the present invention can be autologous, homologous or allogeneic, and the selection depends on the disease to be treated and the means that can be used to do so. Suitable effector cell colonies that can be used in the method include any immune cells with cytolysis, cytophagy or immunosuppression activity, such as T cells, including regulatory T cells, NK cells and macrophages. In one aspect, the effector cells are from a certain HLA background and are utilized in an autologous or allogeneic system. The effector cells can be isolated from any source, including separation of cells within the tumor of a treated subject or a tumor of a treated subject. In the following, the term "effector cell" refers to any type of the aforementioned immune cells that are genetically altered and express UniCAR on the cell surface.

Universal Chimeric Antigen Receptor (UniCAR)

The UniCARs expressed by effector cells used in the methods of the present invention allow modular, highly flexible, and tightly controllable retargeting of UniCAR-expressing immune cells in an antigen-specific manner. The only requirements for the UniCARs used in the methods are that (i) the UniCARs have binding specificity for a specific tag that can be engaged by a target module that in turn binds to a cell surface protein or extracellular structure, and (ii) immune cells can be engineered to express the UniCARs.

The UniCAR comprises three domains ( FIG. 1 ). The first domain is a tag binding domain. This domain is typically present at the amino terminus of the polypeptide comprising the UniCAR. The tag binding domain is positioned at the amino terminus to allow unimpeded access of the tag binding domain to the tagged target module bound to the target cell. The tag binding domain is typically, but not limited to, an antibody or an antigen-binding fragment thereof. The identity of the antibody or fragment is limited only by the identity of the tag of the tagged target module. The tag can be derived from any human nuclear protein for which antibodies or other binding domains are already available. The antibody can be obtained from any animal species, but is preferably obtained from a mammal such as a human, ape, mouse, rat, rabbit, guinea pig, horse, cow, sheep, goat, pig, dog or cat. Preferably, the antibody is a human or humanized antibody. There is no limitation on the specific antibody class that can be used, including IgG1, IgG2, IgG3, IgG4, IgM, IgAl, 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 fragments retain the ability to bind to the selected tag. The antibodies may also be polyclonal, monoclonal or chimeric antibodies, for example, non-human antigen binding regions (e.g., F(ab')2 or hypervariable regions) are transferred into the framework of human antibodies by recombinant DNA technology to produce essentially human molecules. Antigen binding fragments, such as scFv, can be prepared therefrom. Antibodies against selected tags can be produced by immunizing various hosts, including, but not limited to, goats, rabbits, rats, mice, and humans, with a particular protein or any portion, fragment, or oligopeptide that retains the immunogenic properties of the protein by injection.

Depending on the host species, various adjuvants can be used to increase the immune response. Such adjuvants include, but are not limited to, detoxified heat-labile toxins from Escherichia coli (E. coli), Freund's adjuvant, mineral gels such as aluminum hydroxide, and surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum are also potential adjuvants. Antibodies and fragments thereof can be prepared using any technology that provides for the production of antibody molecules, such as monoclonal antibody production by continuous cell lines in culture. 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 N.Y., pp. 77-96 (1985)). Techniques developed for the production of "chimeric antibodies," i.e., splicing mouse and human antibody genes to obtain molecules with appropriate antigenic specificity and biological activity, can also be used (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 also be adapted to produce tag-specific single-chain antibodies.

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, the variable regions typically connected via a short peptide of ten to about 25 amino acids. The linker can connect the N-terminus of VH to the C-terminus of VL, or vice versa.

As noted above, the binding specificity of the tag-binding domain will depend on the identity of the tag that is conjugated to the protein used to bind the target structure.

In a preferred embodiment, the tag is a short linear epitope from the human nuclear La protein (E5B9), and the tag binding domain can constitute an antibody or an antibody-derived antigen-binding fragment, such as a single-chain variable fragment (scFv) that binds to the corresponding La epitope (5B9). Since the anti-La epitope scFv does not interact with the natural La protein bound to the cell surface under normal physiological conditions, it is advantageous to use the 5B9 anti-La epitope in the UniCAR system. Therefore, it is impossible for UniCAR-expressing immune cells, such as T or NK cells, to interact undesirably with the 5B9 epitope on natural La. This results in minimizing the risk of uncontrolled on-target off-site toxicities caused by UniCAR-expressing immune cells, such as the release of toxic levels of cytokines, variously referred to as cytokine storms or cytokine release syndrome (CRS). The reactivity of the corresponding mab with denatured La protein in Western blotting, but no reactivity with natural La protein in immunoprecipitation experiments (Figure 3) was confirmed. Furthermore, although patients with Sjögren's syndrome and systemic lupus erythematosus produce autoantibodies against various La epitopes, no autoantibodies against E5B9 have been identified, suggesting that this epitope is not immunogenic (Yiannaki et al., Clin Exp Immunol., 112(1):152-8 (1998)).

The second domain of the UniCAR is an extracellular hinge and transmembrane (TM) domain. The hinge domain allows the UniCAR to extend from the surface of the effector cell to optimally bind to its specific tag. The TM domain anchors the UniCAR to the cell membrane of the effector cell. Exemplary hinge and TM domains include, but are not limited to, hinges and transmembrane regions of portions of human CD28 molecules, CD8a chains, NK cell receptors such as natural killer cell class 2D (NKG2D), or antibody constant regions, and combinations of various hinge and TM domains.

The third domain, when present, is a signal transduction domain. This domain transmits cell signals to the effector cells when the effector cells carrying UniCAR cross-link with cells or extracellular structures. The cross-link between effector cells and target cells is mediated and depends on the presence of (i) a target module that is combined with a special binding portion on a target cell or target cell extracellular structure and carries a label, and (ii) the UniCAR expressed on the surface of the effector cells can identify and bind to the label included in the target module. Effector cell activation includes inducing cytokines or chemokines and activating the cytolysis, phagocytosis or suppression activity of the effector cells. Exemplary effector cell signal transduction domains include, but are not limited to, CD28, CD137 (41BB), CD134 (OX40), DAP10 and CD27 cytoplasmic regions, which are used to improve 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., CD3 ξ), DAP12 and Fc receptor cytoplasmic regions, which induce T and NK cell activation. The UniCAR may include one or more than one signal transduction domains, such as two, three, four or more immune cell activation or costimulatory domains.

In another embodiment, the UniCAR comprises a fourth domain, which is a short peptide linker in the extracellular portion of the UniCAR (Figure 1). Its functionality is required, that is, this fourth domain forms a linear epitope that allows specific monoclonal antibodies with moderate affinity to bind. One or more linear epitopes can be included in the fourth domain and they can be located within the tag binding domain as a linker, between the tag binding domain and the extracellular linker or the component of the extracellular hinge domain. With the help of the optional fourth domain, the immune cells implanted by UniCAR can be specifically stimulated so that the immune cells implanted by UniCAR are preferentially proliferated and persist longer in vitro or in vivo compared to non-implanted immune cells. The fourth domain can also be used to purify the immune cells implanted by UniCAR from a mixed cell population. It can also be used to weaken the immune response mediated by the immune cells implanted by UniCAR and eliminate the immune cells implanted by UniCAR in vivo.

In order to allow expression on the cell surface of effector cells, a signal peptide (sometimes also referred to as a signal sequence, a guide signal, or a leader peptide) is placed in front of the tag binding domain at the N-terminus of the UniCAR nucleic acid sequence. The signal peptide directs the protein to the secretory pathway during or after translation. For this purpose, signal peptides from proteins of various species can be utilized, but in order to avoid immunogenic reactions, leader peptides from heavy or light chains of proteins such as CD28, CD8α, IL-2, or antibodies of human origin are preferably used.

Target module

The target module is composed of a binding moiety specific for a human cell surface protein or protein complex and a tag. The target module is administered to the subject before, simultaneously with, or after administration of the UniCAR-expressing effector cells. Alternatively, the UniCAR-expressing effector cells can be coated with the target module and then infused into the recipient. The binding portion of the target moiety includes, but is not limited to, antibodies or fragments thereof that bind to the following antigens: surface antigens such as CD2, CD3, CD4, CD8, CD10, CD19, CD20, CD22, CD23, CD33, CD38, CD44, CD52, CD99, CD123, CD274 and TIM-3, members of the epidermal growth factor receptor family (erb1, erb2, erb3, erb4 and mutants thereof), 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, ligand of the NKG2D receptor, members of the epithelial glycoprotein family (e.g., EGP-2, EGP-4), disialogangliosides (e.g., GD2, GD3), members of the carbonic anhydrase family (e.g., CAIX), and members of the carbohydrate antigen family (e.g., Ley), including mutants of the named proteins and protein families. In addition, the binding portion of the target 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 target module can be composed of the α and β or γ and δ chains of the T cell receptor (TCR) or fragments thereof. This TCR-derived binding portion recognizes and binds to peptides presented by class I and II human leukocyte antigen (HLA) protein complexes. Examples include, but are not limited to, 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., autoimmune cancer/testis antigen NY-ESO-1, members of the melanoma antigen family AMAGEA, antigens preferentially expressed in melanoma PRAME), and leukemia-associated antigens (e.g., Wilms tumor gene 1 WT1). The binding portion of the target module can also include ligands of proteins and protein complexes, also known as receptors. Such ligands can bind to, but are not limited to, cytokine receptors (e.g., IL-13 receptor), ligands of NKG2D receptors, ligands of the EGFR family members, or autoreactive TCRs.

The binding portion of a targeting 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, targeting 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, erb-1 and -2, PSCA and erb-2, and other combinations. The binding portion of a targeting module can also comprise monovalent binding, as well as bivalent and multivalent binding sites. Examples of bivalent and multivalent targeting strategies include, but are not limited to, targeting modules that incorporate two scFvs that recognize different epitopes of PSCA, CD19, and CD33, and ligand-scFv combinations that recognize different epitopes of the erb1 receptor.

The target module can also carry additional ligands that do not participate in the target antigen binding, otherwise known as payloads. Such payloads may include, but are not limited to, co-stimulatory ligands or cytokines fused to the N or C terminus of the target 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 11-21, all of which stimulate different types of immune cells. Other payloads may be radionuclides or compounds that induce cell death in target cells and adjacent cells.

method

A method of stimulating a universal chimeric antigen receptor-mediated immune response in a mammal, the method comprising:

- administering to a mammal an effective amount of effector cells that are genetically modified to express a universal chimeric antigen receptor, wherein the universal chimeric antigen receptor comprises three domains, wherein the first domain is a tag binding domain, the second domain is an extracellular hinge and transmembrane domain, and the third domain is a signal transduction domain, wherein the tag binding domain binds to a tag derived from any human nuclear protein, and

- administering a targeting module consisting of a binding moiety specific for a certain human cell surface protein or protein complex and a tag, wherein the tag is derived from any human nuclear protein,

The targeting module is administered to the subject before, simultaneously with, or after administration of the universal chimeric antigen receptor-expressing effector cells.

In a preferred embodiment, the effector cells and targeting moieties are administered to humans.

UniCAR effector cell manufacturing

In embodiments of the present invention, immune cells can be genetically engineered to express UniCAR by various methods. Generally, a polynucleotide vector encoding the UniCAR and all necessary elements to ensure its expression in the genetically engineered immune cells is transferred to the cells. The transfer of the vector can be, but is not limited to, performed by electroporation or transfection of nucleic acids or with the aid of viral gene transfer using viral vector systems such as, but not limited to, adenovirus, adeno-associated virus, retrovirus, foamy virus, or lentivirus.

In another embodiment, lentiviral gene transfer can be applied to stably express UniCAR in immune cells by first constructing a lentiviral vector encoding the selected UniCAR. Exemplary lentiviral vectors include, but are not limited to, the vector pLVX-EF1αUniCAR 28/ζ (Clontech, Takara Bio Group) shown in Figure 3, wherein the lentiviral portion of the vector is derived from human immunodeficiency virus (HIV).

For the described applications, the MSC/IRES/ZxGreenI portion was replaced by the UniCAR construct. With respect to Figure 3, the following abbreviations are used:

5′LTR: 5′ long terminal repeat, PBS: primer binding site, Ψ: packaging signal, RRE: Rev-responsive 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: optimized human codons, WPRE: woodchuck hepatitis virus posttranscriptional regulatory element, 3′LTR: 3′ long terminal repeat, pUC: origin of replication, Ampr: ampicillin resistance gene; β-lactamase.

Lentiviral particles are typically produced by transiently transfecting human embryonic kidney (HEK) 293T (ACC 635) cells with a UniCAR-encoding lentiviral vector plasmid and co-transfecting with a group-specific antigen (gag) and polymerase (pol) encoding plasmid (e.g., psPAX2, Addgene plasmid 12260) as depicted in FIG4 , plus an envelope encoding plasmid (e.g., pMD2.G, Addgene plasmid 12259) as depicted in FIG5 . Following transfection, the packaging plasmid expresses the HIV-1 Gag and Pol proteins. The abbreviations used in FIG4 are as follows: CMVenh: CMV enhancer and promoter, SD: splice donor, SA: splice acceptor, Gag: group-specific antigen, Pro: precursor protein encoding the protease protein, Pol: protein encoding the reverse transcriptase and integrase, RRE: rev response element, Amp: ampicillin.

The plasmid MD2.G ( Figure 5 ) encodes the glycoprotein of vesicular stomatitis virus (VSV-G). The VSV-G protein is used in lentiviral vectors to transduce a wide range of mammalian cells. The abbreviations used in Figure 5 are as follows: CMV: CMV enhancer and promoter, beta-globin intron: beta-globin intron, beta-globin pA: beta-globin polyadenylation tail.

Various envelopes from different viral species can be used for this purpose. Lentiviral vectors can be successfully, but are not limited to, pseudotyped with 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. Supernatant from transfected HEK293T cells can be harvested 24h to 96h after transfection, and viral particles can, but do not necessarily have to, be concentrated from the supernatant by ultracentrifugation or other methods. Regarding lentiviral transduction of immune cells, various established protocols can be applied. In one aspect, peripheral blood mononuclear cells (PBMC) or isolated T cells can be activated with a mab specific for the CD3 complex, such as clone OKT3 or UCHT, which is administered 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 and supplying exogenous recombinant cytokines such as, but not limited to, CD27, CD28, CD134 or CD137 specific mabs or ligands alone or in various combinations and supplying exogenous recombinant cytokines such as, but not limited to, interleukin (IL) -2, IL-7, IL-12, IL-15 and IL-21. PBMC or T cell cultures are initially administered with a single or multiple dose of activating CD3 antibodies and/or recombinant cytokines 24h to 96h, and concentrated or non-concentrated viral particles are added. From 3 days after the last administration of viral supernatant, after staining the surface-expressed UniCAR or mab directly against the fourth domain of the UniCAR with a target module containing a tag, stable T cell transduction can be determined by flow cytometry. UniCAR-transduced T cells can be propagated in vitro by culturing them under the supply of recombinant cytokines and activating anti-CD3 mabs.

In the case where the UniCAR comprises the optional fourth domain, a peptide sequence that forms a linear epitope for a mAb, immune cells genetically modified to express the UniCAR can be specifically propagated in vitro by coating the surface of a culture dish or beads of any type with a mAb or antibody fragment that binds to the fourth UniCAR domain. The beads are then added to the cell culture at a defined ratio of, but not limited to, 1 bead to 1-4 UniCAR-engrafted effector cells. Binding of the surface-coated mAb to the UniCAR peptide domain induces cross-linking of UniCARs expressed on the cell surface and formation of immune synapses, which leads to activation of signaling pathways specifically triggered by the UniCAR signaling domain. Depending on the signaling pathway induced, this can result in increased proliferation of the UniCAR-bearing immune cells and sustained resistance to activation-induced cell death, thereby enriching for UniCAR-genetically modified immune cells in a mixed population.

The optional fourth domain, i.e., the peptide sequence that forms the linear epitope of the mab, can also be used to enrich and purify UniCAR-expressing immune cells from a mixed population. Enrichment and purification can be performed with the aid of a mab or its antibody fragment that binds to the fourth UniCAR domain to mark UniCAR-expressing cells for cell sorting or to transiently link the UniCAR-expressing immune cells to small particles that can be used for cell separation. In one aspect, UniCAR-implanted immune cells are incubated with a mab that recognizes the fourth domain. Next, magnetic beads are added that are conjugated to antibodies or fragments thereof that are directed against the species and isotype-specific heavy and light chains of the mab that binds to the optional fourth domain. Thus, the UniCAR-expressing immune cells are linked to the magnetic beads and can be captured and separated from other immune cells in a magnetic field.

In another embodiment of the present invention, the optional fourth domain can be used to detect UniCAR surface expression, as shown in Figure 6. Figure 6 depicts that UniCAR surface expression can be detected by using a monoclonal antibody directed against the optional fourth domain and then staining with a fluorescent dye-conjugated anti-species secondary antibody. The optional fourth domain can also be used to purify UniCAR-engrafted T cells to a high purity, as depicted in Figure 6).

UniCAR immune cell delivery

Populations of UniCAR-expressing immune cells can be formulated for administration to a subject using techniques known to the skilled artisan.

The formulation comprising the UniCAR-expressing immune cell population may include a pharmaceutically acceptable excipient. The excipients included in the formulation will have different purposes depending on, for example, the properties of the UniCAR-containing tag binding domain, the immune cell population used, and the mode of administration. Examples of commonly used excipients include, but are not limited to: physiological saline, buffered saline, glucose, water for injection (water-for-infection), glycerol, ethanol, and combinations thereof, stabilizers, solubilizers and surfactants, buffers and preservatives, tonicity agents, volume extenders, and lubricants. The formulation comprising the UniCAR-expressing immune cell population will typically be prepared and cultured without any non-human components such as animal serum (e.g., bovine serum albumin).

The preparation can include one population or more than one, for example, two, three, four, five, six or more populations of UniCAR-expressing immune cells. Different UniCAR-engrafted immune cell populations can vary based on the identity of the tag binding domain, the identity of the signal transduction domain, the identity of the subpopulation, the method of generation and culture, or a combination thereof. For example, the preparation can contain UniCAR-expressing T and NK cell populations that recognize and bind one or more than one, for example, two, three, four, five, six or more different tag proteins.

The preparation comprising the UniCAR immune cell population can be administered to a subject using methods and techniques known to technicians. Exemplary methods include, but are not limited to, intravenous injection. Other methods include, but are not limited to, intratumoral, intradermal, subcutaneous (s.c, s.q., sub-Q, Hypo), intramuscular (i.m.), intraperitoneal (i.p.), intraarterial, intramedullary, intracardiac, intraarticular, intrasynovial (synovial fluid area), intracranial, intraspinal, and intrathecal (spinal fluid). Any known device for parenteral injection or infusion of the preparation can be used to achieve such administration. Injection can be performed by push or continuous infusion.

The formulation comprising the UniCAR-expressing immune cell population administered to the subject comprises a plurality of UniCAR-expressing immune cells effective for treating and/or preventing a particular indication or disease. Thus, when practicing the methods of the present invention, a therapeutically effective population of UniCAR-expressing immune cells is administered to the subject. The number of UniCAR-expressing immune cells administered to the subject will vary within broad limits depending on the location, origin, identity, extent, and severity of the disease, the age and condition of the individual to be treated, and the like. Generally, a formulation comprising between about 1 x 10 ⁴ and about 1 x 10 ¹⁰ UniCAR-expressing immune cells is administered. In most cases, the formulation will comprise between about 1 x 10 ⁵ and about 1 x 10 ⁹ UniCAR-expressing immune cells, from about 5 x 10 ⁵ to about 5 x 10 ⁸ UniCAR-expressing immune cells, or from about 1 x 10 ⁶ to about 1 x 10 ⁹ UniCAR-expressing immune cells. The physician will ultimately determine the appropriate dosage for use. In the event of an adverse event, UniCAR-engrafted immune cells can be depleted from the individual by administering a mab directed against the peptide domain (fourth domain) of the UniCAR that forms the linear epitope for the corresponding antibody.

Target module generation

The target module comprises two domains: a binding moiety specific for a human cell surface protein or protein complex and a tag that the tag-binding domain of the UniCAR directly targets. The target module can be manufactured using techniques known to those skilled in the art. These techniques include, but are not limited to, recombinant expression in prokaryotic or eukaryotic cells or artificial synthesis of polypeptide chains.

In one aspect, the target module can be expressed in Chinese hamster ovary (CHO, ACC-110) cells, which are suitable for synthesizing large amounts of biologically active forms of recombinant proteins. The nucleic acid sequence encoding the target 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. Highly productive single-cell clones can be selected from parent lines utilizing, for example, a dihydrofolate reductase (DHFR) selection system. In this system, DHFR-deficient CHO cell mutants (e.g., CHO sublines 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 target module. Clonal selection is then performed by growing in a culture medium lacking glycine, hypoxanthine, and thymidine. Highly productive clones can be further selected by culturing the cells in high levels of methotrexate (MTX), a folic acid analog that blocks DHFR activity. Because genetically modified cells must cope with reduced DHFR activity, which cannot be rescued by the presence of only a single DHFR copy, clones with amplified copies of the DHFR gene are advantageous under these conditions. The genetic linkage between DHFR and the target gene ensures that the transgene is also co-amplified, thereby increasing the chance of obtaining highly productive cell clones. The selected cell clones are grown under good manufacturing conditions, preferably in the absence of any animal serum. The target module can be isolated from the cell culture supernatant by established preparative protein purification methods, which include preliminary steps such as precipitation or ultracentrifugation and various purification techniques such as, but not limited to, size exclusion or ion exchange chromatography. In one aspect, the nucleic acid sequence of the target module carries a coding sequence of six to eight consecutive histidine amino acids that form a polyhistidine tag. The polyhistidine strongly binds to divalent metal ions such as nickel and cobalt. The cell culture supernatant can be passed through a column containing immobilized nickel ions that bind to the polyhistidine tag, while the untagged protein passes through the column. The target module can be eluted with imidazole, which competes with the polyhistidine tag for binding to the column, or eluted by lowering the pH to reduce the affinity of the tag for the resin.

Targeted module drug delivery

One targeting moiety or more than one, such as two, three, four or more targeting moieties, can be formulated for administration to a subject using techniques known to the skilled artisan.

The formulation containing one or more than one target moiety may include a pharmaceutically acceptable excipient. The excipients included in the formulation will have different purposes depending on, for example, the nature of the target moiety and the mode of administration. Examples of commonly used excipients include, but are not limited to: physiological saline, buffered saline, dextrose, water for injection, glycerol, ethanol, and combinations thereof, stabilizers, solubilizers and surfactants, buffers and preservatives, tonicity agents, volume extenders, and lubricants. The formulation containing the target moiety will typically be prepared and cultured without any non-human components, such as animal serum (e.g., bovine serum albumin).

The formulation can include one targeting module or more than one, for example, two, three, four, five, six or more targeting modules. The targeting module 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, the formulation can include a targeting module that recognizes and binds 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.

A preparation comprising a population of UniCAR-expressing immune cells can be incubated ex vivo with a preparation comprising one or more target modules to coat the UniCAR-expressing immune cells with the target modules prior to administration to a subject. Alternatively, the preparation comprising one or more target modules can be administered directly to a subject, or a combination of these two strategies can be selected. The route and dosage will vary within wide limits depending on the location, origin, identity, extent, and severity of the disease, the age and condition of the individual to be treated, and the like. The physician will ultimately determine the appropriate route of application and dosage to use.

The formulation containing the targeting moiety is administered to a subject in an amount effective to treat and/or prevent the specific indication or disease. Typical daily dosage rates per m² are between 1 μg and 1000 mg, preferably 10 μg to 1 mg, with the dosage administered one or more times daily or weekly, or continuously, over a period of several weeks. However, the amount of the targeting moiety in the formulation administered to a subject will vary within wide limits depending on the location, origin, identity, extent, and severity of the cancer, the age and condition of the individual being treated, and other factors. The physician will ultimately determine the appropriate dosage to use.

The present invention relates to methods for treating a subject suffering from cancer, infection, or autoimmune disease, the methods comprising administering to a subject in need of treatment one or more preparations of a target moiety, wherein the target moiety binds to cancer cells, and administering one or more therapeutically effective populations of UniCAR-expressing immune cells, wherein the UniCAR-expressing immune cells bind to the target moiety and induce cell death.

The term "cancer" is intended to be broadly interpreted and includes 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, bronchi, colon, small intestine, stomach, esophagus, gall bladder; sarcomas, including but not limited to chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, 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 lymphoma; The term "tumor" includes but is not limited to hepatoblastoma, medulloblastoma, Wilms' tumor, neuroblastoma, pancreatic blastoma, leuropulmonary blastoma, and retinoblastoma. The term also includes benign tumors.

As used herein, the term "treating" has its ordinary and customary meaning and includes one or more of the following: 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 in a subject (e.g., the appearance of new tumors). Treating refers to blocking, alleviating, reducing or inhibiting by about 1% to about 100% relative to a subject who has not practiced the methods of the present 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% relative to a subject who has not practiced the methods of the present invention.

The frequency of administration of both the formulation comprising the UniCAR-expressing immune cell population and the formulation comprising the target module will vary depending on factors including the disease being treated, the elements comprising the UniCAR-expressing immune cells and the target module, and the mode of administration. Each formulation can be administered independently 4, 3, 2 times 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, two weeks, monthly, and bimonthly.

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

The present invention provides flexibility in treatment methods, so that the target module and the preparation of the UniCAR-expressing immune cell population can be administered to the subject in any order. Thus, the preparation of the target module can be administered to the subject before, after, or simultaneously with the UniCAR-expressing immune cell population. Alternatively, in the case where more than one target module and/or more than one preparation of the UniCAR-expressing immune cell population is administered to the subject, the administration can be staggered. For example, a first preparation of the target module can be administered, followed by a first population of UniCAR-expressing immune cells, followed by a second preparation of the tag protein, and then a second population of UniCAR-expressing immune cells.

The present invention also includes methods of coating a population of UniCAR-expressing immune cells with a targeting moiety prior to administering the UniCAR-expressing immune cells to a subject.

In various embodiments of the invention, the subject being treated is a human or non-human animal, e.g., a non-human primate, bird, horse, cow, goat, sheep, companion animal such as a dog, cat or rodent, or other mammal.

In one embodiment, UniCAR genetically engineered T cells can be specifically redirected against tumor cells expressing PSCA and/or PSMA, which are frequently detected in biopsies, such as from prostate, bladder, pancreatic, and breast tumors ( FIG. 7 ). Human T cells were mock-transduced (white bars) or transduced with lentiviral vectors encoding UniCARs containing dual CD28/CD3ζ signaling domains (black bars), lacking any signaling domains (hatched bars), or expressing only an EGFP-tagged protein (striped bars). Specific lysis of prostate tumor cells (PC3) genetically engineered to express prostate stem cell antigen (PC3-PSCA) or prostate membrane antigen (PC3-PSMA) in the presence of engineered human T cells and a target module was analyzed in a 51Cr release assay. T cells were incubated with 51Cr-loaded PC3 target cells at an effector cell to target ratio (e:t) of 5:1 or 1:1. Target modules (TMs) specific for PSCA (αPSCA™) or PSMA (αPSMA™) were added at a concentration of 15 nMol. After 20 hours of incubation, target cell lysis (chromium release) was measured. The graph shows the mean and SD values from experiments with three individual T cell donors ( FIG7A ). Similarly, the killing ability of UniCAR-engineered T cells against LNCap-4-2B cancer stem cells expressing both PSCA and PSMA antigens was demonstrated by 51Cr release analysis at the indicated e:t ratios in the presence of PSCA- or PSMA™ (1 nMol). The mean and SD values from experiments with three individual T cell donors are shown ( FIG7B ). The experiments in FIG7B were repeated at a lower e:t ratio of 1:2, with the addition of 1 nMol of PSCA- or PSMA-specific TMs or a combination of these two TMs at a total concentration of 1 nMol. Specific lysis, as measured by 51Cr release, was measured after 24 and 48 hours. Shown are the mean and SD values for experiments with three individual healthy T cell donors ( FIG7C ). This experiment demonstrates that the combination of two different TMs improves the killing capacity of UniCAR-redirected T cells against tumor cells compared to a single antigen retargeting strategy, even when the total amount of both TMs in the dual-targeted sample is equal to the total amount of each TM in the single antigen retargeting control sample ( FIG7C ). Furthermore, human T cells armed with the UniCAR secrete inflammatory and proliferative cytokines when cross-linked with antigen-expressing tumor cells in the presence of the corresponding TM ( FIG7D ). Human T cells were incubated with PC3 cells expressing PSCA or PSMA antigens in the presence or absence of TMs specific for PSCA or PSMA. After 24 h of culture, cell-free supernatants were harvested and subsequently analyzed for T cell-specific cytokine release using commercially available ELISA kits. Statistical analysis for FIG7C and D was performed using a nonparametric one-way ANOVA (Kruskal-Wallis test) and a post hoc Dunn's multiple comparison test (**p < 0.01).

In another embodiment, FIG8 shows the concentration response curve of human T cells genetically modified with UniCAR in the presence of target modules that bind to various antigens on the surface of tumor cells from different sources. Human T cells were transduced with a lentiviral vector encoding UniCAR containing dual CD28/CD3ξ signaling domains. UniCAR-implanted T cells were incubated with 51Cr-loaded PC3 target cells genetically engineered to express PSCA or PSMA antigens at an e:t ratio of 5:1. Target modules (TMs) specific for PSCA (αPSCA™) or PSMA (αPSMA™) were added at increasing concentrations ( FIG7A ). The half-maximal effective dose (EC50) of these two TMs was measured to be close to 12 pMol. Additional experiments as in FIG7A were performed, but the e:t ratio was selected to be 1:1 and the acute myeloid leukemia cell line MOLM-13 was used as the target tumor cell. TM specific for the CD33 antigen (αCD33 TM) or the CD123 antigen (αCD123 TM) was added at increasing concentrations and the EC50 values for αCD33 TM were measured to be 137 pMol and 45 pMol for αCD123 TM. These experiments demonstrated that UniCAR-implanted T cells were effective at very low concentrations of TM, which were 10 to 100 times lower than the EC50 values determined for monoclonal antibody drugs approved for cancer treatment (e.g., Herter et al. Mol Cancer Ther 12(10):2031-2042 (2013)).

In another embodiment, FIG9 demonstrates that UniCAR engineered T cells can effectively kill acute myeloid leukemia (AML) blasts. Human T cells were pseudo-transduced (wt, diamond) or transduced with a lentiviral vector encoding a U-CAR (CAR 28/ζ, hollow and filled circles) containing a dual CD28/CD3ζ signaling domain or lacking any signaling domain (CAR stop, upward triangle) or expressing only EGFP marker protein (vc, downward triangle). T cells were incubated with 2*10 ⁴ Alexa eFluor 674-labeled target cells from three AML cell lines (MOLM-13, MV4-11, OCI-AML3) at an e: t ratio of 1: 1 for 24 h ( FIG9A ). The number of live, propidium iodide (PI)-negative, but Alexa eFluor 674-positive target cells was determined by flow cytometry using an analyzer. The number of live leukemia target cells was normalized to a control sample with target cells but no T cells. This experiment demonstrated that UniCAR-implanted T cells effectively lysed AML blasts regardless of antigen density after cross-linking with the corresponding TM, with CD33 being high on MOLM-13, medium on MV4-1,1, and low on OCI-AML3, while the order of CD123 antigen density was opposite on the three cell lines. In addition, after TM-mediated cross-linking, the T cells implanted by UniCAR were able to eliminate AML blasts even at low e:t ratios as found in typical patient samples (Figure 9B). The experimental setup was similar to Figure 9A, but a low e:t ratio of 1:5 was selected. The number of live, PI-negative T cells and target cells was determined by flow cytometry using an analyzer at the specified time points. The number of live leukemia target cells is normalized relative to the control sample with target cells but lacking any T cells (Fig. 9 B). After activation via CAR-mediated signal transduction, the T cells implanted by UniCAR begin to proliferate, as shown in Figure 9 C. The experimental setting is as described for Figure 9 B, and the number of T cells is determined by flow cytometry using an analyzer. T cell expansion is calculated by the ratio of the number of T cells present in the sample after 144h (d6) to the number of inoculations at the beginning of the experiment (d0). The statistical analysis of Figures 8 A and B is performed using non-parametric one-way ANOVA (Kruskal-Wallis test) and Dunn's multiple comparison test afterwards. The statistical results of significance are pointed out for Figure 9 A, and for Figure 9 B, the results are given in the table below the figure (ns=non-significant, *p<0.05, **p<0.01, ***p<0.001).

In another embodiment, Figure 10 shows the redirection of T cells implanted with UniCAR for two antigens at the same time. Due to its modular nature, the UniCAR technology allows the immune cells (e.g., T cells) implanted with UniCAR to utilize two separate TMs (TM 1+TM 2, Figure 10A left), or bispecific TMs (TM1-2, Figure 10A right) arranged as a combination of bispecific single chain tandem constructs, simultaneously or sequentially redirected for two antigens (10A). The use of bispecific TMs targeting two antigens can be more effective than the combination of two single antigen-specific TMs, as demonstrated by the concentration-response curves of CD33- and CD123-specific retargeting of UniCAR T cells against AML cell lines (Figure 10B). Human T cells were transduced with a lentiviral vector encoding a UniCAR containing a dual CD28/CD3ζ signaling domain. UniCAR-implanted T cells were incubated with Cr51-labeled MOLM-13 (average value from experiments with 4 different healthy human donors, triangles) and OCI-AML3 (average value from experiments with 2 different healthy human donors, open circles) at an e:t ratio of 1:1 for 24 hours. Target modules (TM) specific for CD33 (αCD33™), CD123 (αCD123™), or bispecific CD33-CD123™ (αCD123-CD33™) were added at increasing concentrations. EC50 values were determined to be: αCD33+αCD123™: EC50 MOLM-13 = 70.2 pMol, EC50 OCI-AML3 = 80.2 pMol, αCD33-αCD123™: EC50 MOLM-13 = 2.9 pMol, EC50 OCI-AML3 = 11.7 pMol. Secondly, it was demonstrated that UniCAR-engrafted T cells from both healthy human donors and AML patients could be successfully redirected against AML cell lines as well as AML blasts isolated from patients in blast crisis (Figures 10C, D, E, F). Human T cells were mock-transduced (open circles in Figures 10C, D, open bars in Figures 10E, F) or transduced with lentiviral vectors encoding UniCARs containing dual CD28/CD3ζ signaling domains (triangles in Figures 10C, D, black bars in Figures 10E, F) or lacking any signaling domains (diamonds in Figures 10C, D, gray bars in Figures 10E, F). In the presence of a combination of αCD33TM and αCD123TM or the dual-targeting αCD123-CD33TM, but not in the absence of the TM, UniCAR-engrafted T cells mediated highly effective AML cell elimination, again demonstrating that cross-linking via the TM is essential for antigen-specific redirection of UniCAR-engrafted T cells (Figures 10C, D, E). T cells are incubated with 2* ¹⁰ Alexa eFluor 674 labeled target cells from 3 AML cell lines (MOLM-13, MV4-11, OCI-AML3) in the presence (+) or absence (-) of 100 pMol TM for 144 h with an e:t ratio of 1:5. TM is updated after 48 h. The quantity of target cells that are alive, PI negative but Alexa eFluor 674 positive utilizes an analyzer to measure by flow cytometry, and with target cells but without any T cell control samples (10C, open circles) compared. In Figure 10 D, T cell amplification is calculated by the ratio of the quantity of T cells and the test start (d0) inoculation in the sample after 144 h (d6). Shown are the results from the test with 6 donors, with meansigma methods and sd indicated. These results strongly demonstrate that the activation of UniCAR signaling after TM-mediated cross-linking not only causes target cell killing, but also the T cells implanted by UniCAR receive proliferation stimulation by the CD28/CD3ζUniCAR signaling chain of the combination and begin to divide. Next, genetically modified T cells from healthy donors were incubated with 5*10 ⁴ Alexa eFluor 674 labeled leukemia cells from AML patients with a 1: 1 e: t ratio (Figure 10 E) in the presence (+) or absence (-) 0.5nMol TM. After 24h (left figure) and 48h (right figure), the number of live, Alexa eFluor 674 positive target cells was determined by flow cytometry using an analyzer. The number of live leukemia target cells was normalized relative to a control sample with target cells but without any T cells. The results from 5 paired experiments in Figure 10 E demonstrate that UniCAR engineering T cells are able to lyse the patient's AML blasts over time. It can also be demonstrated that, by adding AML antigen-specific TM, UniCAR genetically modified T cells from AML patients are able to attack and lyse AML cells. Modified T cells were incubated with 2*10 ⁴ Alexa eFluor 674-labeled target cells from three AML cell lines (MOLM-13, MV4-11, OCI-AML3) in the presence (+) or absence (-) of a total of 0.5 nMol TM at an e: 1 ratio. The number of live, PI-negative but Alexa eFluor 674-positive target cells was determined by flow cytometry using an analyzer.

In another embodiment, Figure 11 depicts a graph showing the in vivo pharmacokinetics of the bispecific αCD123-CD33 target module. NSG mice (NOD/SCID IL2Rγ-/-) were injected intravenously (i.v., Figure 11A) or intraperitoneally (i.p., Figure 11B) with 250 μg/g body weight of αCD123-CD33 TM and serum samples were taken at the specified time points. Capture ELISA was used to determine the concentration of TM in the sample. The results show the mean and standard deviation (n=3). The half-life of i.v. injection was determined using an exponential single-phase decay model (GraphPad Prism software).

In another embodiment, an isolated nucleic acid sequence encoding a universal chimeric antigen receptor is provided as SEQ.ID 1. The encoding isolated nucleic acid sequence is as follows: human IL-2m leader peptide of SEQ.ID 2, humanized anti-La 5B9 antibody variable region heavy chain of SEQ.ID 3, humanized anti-La 5B9 antibody variable region light chain of SEQ.ID 4, La 7B6 epitope of SEQ.ID 5, human CD28 of SEQ.IDs 6 to 8, including the extracellular portion of human CD28 with a mutant binding motif of SEQ.ID 6, the CD28 transmembrane domain of SEQ.ID 7, the intracellular portion of human CD28 including a mutant internalization motif of SEQ.ID 8, and the human CD3ζ intracellular domain of SEQ.ID 9.

The protein expression product of the isolated nucleic acid sequence of SEQ.ID 1 can be obtained in SEQ.ID 27. The nucleic acid sequence of the humanized anti-La5B9 antibody variable region heavy chain of SEQ.ID 3 encodes the protein of SEQ.ID 33, while the humanized anti-La5B9 antibody variable region light chain of SEQ.ID 4 encodes the protein of SEQ.ID 34.

The nucleic acid sequence of the human La 7B6 epitope of SEQ.ID 5 encodes the protein domain of SEQ.ID 35.

In another embodiment of the present invention, an isolated nucleic acid sequence encoding a targeting moiety having a binding portion for the prostate-specific antigen PSCA is provided in SEQ. ID 10. The encoding isolated nucleic acid sequence is as follows: the leader peptide IgGκ of SEQ. ID 11, the humanized light chain of the anti-PSCA scFv of SEQ. ID 12, the humanized heavy chain of the anti-PSCA scFv of SEQ. ID 13, the La5B9 epitope of SEQ. ID 14, the myc tag of SEQ. ID 15, and the his tag of SEQ. ID 16.

The protein expression product of the nucleic acid of SEQ.ID 10 can be obtained from SEQ.ID 28. The nucleic acid sequence of the humanized light chain of the anti-PSCA scFv of SEQ.ID 12 encodes the protein domain of SEQ.ID 36, while the humanized heavy chain of the anti-PSCA scFv of SEQ.ID 13 encodes the protein domain of SEQ.ID 37. The La5B9 epitope of SEQ.ID 14 encodes the protein of SEQ.ID 44.

In another embodiment of the present invention, an isolated nucleic acid sequence encoding a targeting moiety having a binding moiety for prostate-specific antigen (PSMA) is provided in SEQ. ID 17. The encoding isolated nucleic acid sequence is as follows: leader peptide IgGκ of SEQ. ID 11, humanized heavy chain of anti-PSMA scFv of SEQ. ID 18, humanized light chain of anti-PSMA scFv of SEQ. ID 19, La5B9 epitope of SEQ. ID 14, myc tag of SEQ. ID 15, and his tag of SEQ. ID 16.

The protein expression product of the nucleic acid of SEQ.ID 17 can be obtained from SEQ.ID 29. The nucleic acid sequence of the humanized heavy chain of the anti-PSMA scFv of SEQ.ID 18 encodes the protein domain of SEQ.ID 38, while the humanized light chain of the anti-PSMA scFv of SEQ.ID 19 encodes the protein domain of SEQ.ID 39. The La5B9 epitope of SEQ.ID 14 encodes the protein of SEQ.ID 44.

In another embodiment of the present invention, an isolated nucleic acid sequence encoding a targeting moiety having a binding portion of an anti-CD33 antigen antibody is provided in SEQ.ID 20. The encoding isolated nucleic acid sequence is as follows: leader peptide IgGκ of SEQ.ID 11, humanized light chain of anti-CD33 scFv of SEQ.ID 21, humanized heavy chain of anti-CD33 scFv of SEQ.ID 22, La5B9 epitope of SEQ.ID 14, myc tag of SEQ.ID 15, and his tag of SEQ.ID 16.

The protein expression product of the nucleic acid of SEQ.ID 20 can be obtained from SEQ.ID 30. The nucleic acid sequence of the humanized light chain of the anti-CD33 scFv of SEQ.ID 21 encodes the protein domain of SEQ.ID 40, while the humanized heavy chain of the anti-CD33 scFv of SEQ.ID 22 encodes the protein domain of SEQ.ID 41. The La5B9 epitope of SEQ.ID 14 encodes the protein of SEQ.ID 44.

In another embodiment of the present invention, an isolated nucleic acid sequence encoding a target moiety having a binding portion of an anti-CD123 antigen antibody is provided in SEQ.ID 23. The encoding isolated nucleic acid sequence is as follows: the leader peptide IgGκ of SEQ.ID 11, the humanized heavy chain of the anti-CD123 scFv of SEQ.ID 24, the humanized light chain of the anti-CD123 scFv of SEQ.ID 25, the La 5B9 epitope of SEQ.ID 14, the myc tag of SEQ.ID 15, and the his tag of SEQ.ID 16.

The protein expression product of the nucleic acid of SEQ.ID 23 can be obtained from SEQ.ID 31. The nucleic acid sequence of the humanized heavy chain of the anti-CD123 scFv of SEQ.ID 24 encodes the protein domain of SEQ.ID 42, while the humanized light chain of the anti-CD123 scFv of SEQ.ID 25 encodes the protein domain of SEQ.ID 43. The La5B9 epitope of SEQ.ID 14 encodes the protein of SEQ.ID 44.

In another embodiment of the present invention, an isolated nucleic acid sequence encoding a target moiety having a binding portion of an anti-CD123 anti-CD33 antigen antibody is provided in SEQ.ID 26. The encoding isolated nucleic acid sequence is as follows: leader peptide IgGκ of SEQ.ID 11, humanized heavy chain of anti-CD123 scFv of SEQ.ID 24, humanized light chain of anti-CD123 scFv of SEQ.ID 25, La 5B9 epitope of SEQ.ID 14, humanized heavy chain of anti-CD33 scFv of SEQ.ID 22, humanized light chain of anti-CD33 scFv of SEQ.ID 21, myc tag of SEQ.ID 15, and his tag of SEQ.ID 16.

The protein expression product of the nucleic acid of SEQ.ID 26 can be obtained from SEQ.ID 32. The nucleic acid sequence of the humanized heavy chain of the anti-CD123 scFv of SEQ.ID 24 encodes the protein domain of SEQ.ID 42, while the humanized light chain of the anti-CD123 scFv of SEQ.ID 25 encodes the protein domain of SEQ.ID 43. The La5B9 epitope of SEQ.ID 14 encodes the protein of SEQ.ID 44. The humanized heavy chain of the anti-CD33 scFv of SEQ.ID 22 encodes the protein domain of SEQ.ID 41, while the nucleic acid sequence of the humanized light chain of the anti-CD33 scFv of SEQ.ID 21 encodes the protein domain of SEQ.ID 40.

Sequence Listing

<110> Gemo Abo Monoclonal Co., Ltd.

<120> Universal chimeric antigen receptor-expressing immune cells targeting diverse antigens, methods for their manufacture, and their use in the treatment of cancer, infection, and autoimmune diseases

<130> 01281P0002EPWO

<150> EP 14 182 945.7

<151> 2014-08-29

<160> 44

<170> PatentIn version 3.5

<210> 1

<211> 2685

<212> DNA

<213> Artificial Sequence

<220>

<223> Chimeric DNA Sequence mouse/human

<400> 1

atgcgccgca tgcagctgct gcttctgatc gctctgagcc tggctcttgt gaccaactct 60

gaattccagg tgcagctggt gcagagcgga gccgaggtga agaagcctgg agcctctgtg 120

aaggtgagct gcaaggcttc tggctacacc ttcacccact actacatcta ctgggtgaga 180

caggctcccg gacagggcct ggagtggatg ggaggcgtga accccagcaa cggaggcacc 240

cacttcaacg agaagttcaa gtctcgcgtg accatgaccc gcgacaccag catctctacc 300

gcttacatgg agctgagccg cctgcgctct gatgataccg ctgtgtacta ctgcgctcgc 360

agcgagtacg attacggact gggcttcgcc tactggggcc agggaaccct ggtgaccgtg 420

agctctggag gcggaggcag cggaggcggc ggatctggag gcggaggaag cgatatcgtg 480

atgacccagt ctcctgatag cctggctgtg agcctgggcg agagagctac catcaactgc 540

aagagcagcc agagcctgct gaactctcgc acccctaaga actaccttgc ttggtaccag 600

cagaagcctg gacagccccc taagctgctg atctactggg cttctacccg caagagcggc 660

gtgcccgaca gattctctgg cagcggaagc ggcaccgatt tcaccctgac catcagcagc 720

ctgcaggctg aggacgtggc cgtgtactac tgcaagcagt cttacaacct gctgaccttc 780

ggaggcggaa ccaaggtgga gatcaaggct gccgctctgg agaaggaggc cctgaagaag 840

atcatcgagg atcagcagga ggctctgaac aagtgggctg ccgctgggcc cggaggaggc 900

ggcagcaaga tcctggtcaa acagtcccct atgctggtcg cttacgacaa cgccgttaat 960

ctgagttgca aatatagtta caacctgttt agccgggaat ttcgcgcatc tctccacaag 1020

ggactggatt ctgcggttga ggtttgtgtg gtctatggca attatagcca gcaactgcaa 1080

gtgtacagca aaacaggctt taactgcgac gggaaactcg ggaacgaatc agtgaccttc 1140

tatctgcaga acctgtacgt taaccaaaca gatatttact tctgcaagat agaggtgatg 1200

gctccaccgc cagcactgga taacgagaag tccaatggaa ccatcattca cgtcaagggg 1260

aagcatctgt gtccttcccc gttgttccct gggccgagca aacccttttg ggtgcttgtg 1320

gtagttggcg gggtattggc ctgctattcc cttctcgtaa ctgtggcctt catcatcttc 1380

tgggtcagat ctaagaggtc taggggcggg catagcgact acatgaacat gacacccagg 1440

cggcctggcc ccactcgcaa acactaccag ccatacgcac caccaagaga ctttgccgca 1500

tatcggagtg gtggcggcgg gtcaggaggt ggagctagcg gtggaggagg ttccttctct 1560

aggtcagctg atgctcccgc ctatcagcaa ggtcagaacc agctctacaa tgagctgaat 1620

ctgggacgtc gggagggagta cgacgtgctg gataaacgaa gaggacgcga tcccgagatg 1680

ggtgggaagc ctaggcgcaa gaatccccag gaaggcctct acaatgaact gcagaaagac 1740

aagatggccg aagcctacag cgagattggc atgaaagggg agcgacggag aggaaaggga 1800

catgacgggt tgtatcaggg tctttccact gcgacaaagg atacctatgg ggctctgcac 1860

atgcaagcac tgccacctag aggatccggc tcgagcggtg agggcagagg aagtcttcta 1920

acatgcggtg acgtggagga gaatcccggc ccaccggtcg ccaccatggt gagcaagggc 1980

gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc 2040

cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg 2100

aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg 2160

acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc 2220

aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc 2280

aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag 2340

ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac 2400

tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac 2460

ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag 2520

aacacccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag 2580

tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg 2640

accgccgccg ggatcactct cggcatggac gagctgtaca agtaa 2685

<210> 2

<211> 60

<212> DNA

<213> Homo sapiens

<400> 2

atgcgccgca tgcagctgct gcttctgatc gctctgagcc tggctcttgt gaccaactct 60

<210> 3

<211> 360

<212> DNA

<213> mouse

<400> 3

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> 4

<211> 336

<212> DNA

<213> mouse

<400> 4

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> 5

<211> 60

<212> DNA

<213> Homo sapiens

<400> 5

ctggagaagg aggccctgaa gaagatcatc gaggatcagc aggaggctct gaacaagtgg 60

<210> 6

<211> 399

<212> DNA

<213> Homo sapiens

<400> 6

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> 7

<211> 81

<212> DNA

<213> Homo sapiens

<400> 7

ttttgggtgc ttgtggtagt tggcggggta ttggcctgct attcccttct cgtaactgtg 60

gccttcatca tcttctgggt c 81

<210> 8

<211> 123

<212> DNA

<213> Homo sapiens

<400> 8

agatctaaga ggtctagggg cgggcatagc gactacatga acatgacacc caggcggcct 60

ggccccactc gcaaacacta ccagccatac gcaccaccaa gagactttgc cgcatatcgg 120

agt 123

<210> 9

<211> 327

<212> DNA

<213> Homo sapiens

<400> 9

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> 10

<211> 981

<212> DNA

<213> Artificial Sequence

<220>

<223> Chimeric DNA sequence mouse/human

<400> 10

atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60

gacgcggccc agccggccgg atccgatatc cagatgactc aaagtcctag ttccctgtct 120

gcatcagtgg gagaccgggt gaccattaca tgcggtacat cccaagacat caataattat 180

ctcaactggt atcagcagaa gccaggcaaa gttcctaagt tattaatcta ctacacatcc 240

aggctgcatt ccggggtgcc ctcccgcttt tcgggctccg ggtcgggaac cgactttacc 300

ctaaccatat cttccctgca gcctgaagac gttgcaacgt actattgtca gcagtcaaag 360

acattaccat ggacatttgg tggtgggacg caactcactg tacttggtgg aggtggcagt 420

ggtggaggag ggagcggagc aagtgccgct ggaggcggag gttcaggcgg tggtggaagc 480

caggtgcagc tagtggagtc cggtggcggc ctcgttaagc cgggcggatc gctgcgcctt 540

tcatgtgccg catcaggatt cacattctcc agttatactcta tgtcatggat tcggcaggca 600

cctggcaagg gattggaatg ggtctcgtac attaatgatt caggtggaag tacattctat 660

ccggacacgg ttaaaggtag atttaccatc agccgtgata acgcgaagaa tagcttgtac 720

ttacagatga atagcctgcg tgcagaggat actgctgtat attattgcgc tcgacgtatg 780

tattatggca atagtcactg gcactttgac gtctggggcc agggcacgac agttactgtc 840

tcttcgggag gaggaggatc cgcggccgct aaacccctac ctgaagtgac tgatgagtat 900

gctcgaggag ggcccgaaca aaaactcatc tcagaagagg atctgaatag cgccgtcgac 960

catcatcatc atcatcattg a 981

<210> 11

<211> 63

<212> DNA

<213> Homo sapiens

<400> 11

atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60

gac 63

<210> 12

<211> 321

<212> DNA

<213> mouse

<400> 12

gatatccaga tgactcaaag tcctagttcc ctgtctgcat cagtgggaga ccgggtgacc 60

attacatgcg gtacatccca agacatcaat aattatctca actggtatca gcagaagcca 120

ggcaaagttc ctaagttatt aatctactac acatccaggc tgcattccgg ggtgccctcc 180

cgcttttcgg gctccgggtc gggaaccgac tttaccctaa ccatatcttc cctgcagcct 240

gaagacgttg caacgtacta ttgtcagcag tcaaagacat taccatggac atttggtggt 300

gggacgcaac tcactgtact t 321

<210> 13

<211> 366

<212> DNA

<213> mouse

<400> 13

caggtgcagc tagtggagtc cggtggcggc ctcgttaagc cgggcggatc gctgcgcctt 60

tcatgtgccg catcaggatt cacattctcc agttatactcta tgtcatggat tcggcaggca 120

cctggcaagg gattggaatg ggtctcgtac attaatgatt caggtggaag tacattctat 180

ccggacacgg ttaaaggtag atttaccatc agccgtgata acgcgaagaa tagcttgtac 240

ttacagatga atagcctgcg tgcagaggat actgctgtat attattgcgc tcgacgtatg 300

tattatggca atagtcactg gcactttgac gtctggggcc agggcacgac agttatactgtc 360

tcttcg 366

<210> 14

<211> 30

<212> DNA

<213> Homo sapiens

<400> 14

aaacccctac ctgaagtgac tgatgagtat 30

<210> 15

<211> 30

<212> DNA

<213> Homo sapiens

<400> 15

gaacaaaaac tcatctcaga agaggatctg 30

<210> 16

<211> 18

<212> DNA

<213> Homo sapiens

<400> 16

catcatcatc atcatcat 18

<210> 17

<211> 909

<212> DNA

<213> Artificial Sequence

<220>

<223> chimeric DNA construct human/mouse

<400> 17

atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60

gacgcggccc agccggccga ggtgcagctg cagcagtcag gacctgaact ggtgaagcct 120

gggacttcag tgaggatatc ctgcaagact tctggataca cattcactga atataccata 180

cactgggtga agcagagcca tggaaagagc cttgagtgga ttggaaacat caatcctaac 240

aatggtggta ccacctacaa tcagaagttc gaggacaagg ccacattgac tgtagacaag 300

tcctccagta cagcctacat ggagctccgc agcctaacat ctgaggattc tgcagtctat 360

tattgtgcag ctggttggaa ctttgactac tggggccaag ggaccacggt caccgtctcc 420

tcaggtggag gtggatcagg tggaggtgga tctggtggag gtggatctga cattgtgatg 480

acccagtctc acaaattcat gtccacatca gtaggagaca gggtcagcat catctgtaag 540

gccagtcaag atgtgggtac tgctgtagac tggtatcaac agaaaccagg acaatctcct 600

aaactactga tttattgggc atccactcgg cacactggag tccctgatcg cttcacaggc 660

agtggatctg ggacagactt cactctcacc attactaatg ttcagtctga agacttggca 720

gattatttct gtcagcaata taacagctat cccctcacgt tcggtgctgg gaccatgctg 780

gacctgaaag cggccgctaa acccctacct gaagtgactg atgagtatgc tcgaggaggg 840

cccgaacaaa aactcatctc agaagaggat ctgaatagcg ccgtcgacca tcatcatcat 900

catcattga 909

<210> 18

<211> 345

<212> DNA

<213> mouse

<400> 18

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> 19

<211> 321

<212> DNA

<213> mouse

<400> 19

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> 20

<211> 972

<212> DNA

<213> Artificial Sequence

<220>

<223> chimeric DNA sequence mouse/human

<400> 20

atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60

gacgcggccc agccggccgg atccgatata gttttaaccc aatcccctgc tagtctggcc 120

gtatccccag gccagagggc tactataacc tgcactgcaa gctcatctgt caactacatc 180

cattggtacc agcagaaacc tggacaaccg ccgaaacttc tgatatacga caccagcaag 240

gtcgcgtccg gggtgcctgc tcgattcagc ggcagcggat caggtactga cttcactttg 300

actatcaatc cagtggaagc gaacgatact gcgaactact actgccagca atggaggtcg 360

taccccttga catttggcca aggtactaaa ctagagataa aaggtggagg tggcagtggt 420

ggaggaggga gcggagcaag tggcgccgga ggcggaggtt caggcggtgg tggaagccag 480

gtacaactgg tccaatctgg agccgaagtc aagaaaccag gagcttctgt gaaagtcagt 540

tgcaaggcgt ctgggtatac attcacagat tacgtagtac actgggttag gcaagctcct 600

ggtcaagggc ttgaatggat gggatatatt aatccgtaca acgacggaac aaaataac 660

gagaagttta agggtagagt aactatgacc agggacacaa gcatcagtac agcgtatatg 720

gaactgagtc gtctccggtc tgatgacacc gctgtctatt attgtgcaag agattaccgt 780

tacgaggttt acggcatgga ctattggggc caaggcactc tcgttaccgt gtcaagcgga 840

ggaggagat ccgcggccgc taaaccccta cctgaagtga ctgatgagta tgctcgagga 900

gggcccgaac aaaaactcat ctcagaagag gatctgaata gcgccgtcga ccatcatcat 960

catcatcatt ga 972

<210> 21

<211> 318

<212> DNA

<213> mouse

<400> 21

gatatagttt taacccaatc ccctgctagt ctggccgtat ccccaggcca gagggctact 60

ataacctgca ctgcaagctc atctgtcaac tacatccatt ggtaccagca gaaacctgga 120

caaccgccga aacttctgat atacgacacc agcaaggtcg cgtccggggt gcctgctcga 180

ttcagcggca gcggatcagg tactgacttc actttgacta tcaatccagt ggaagcgaac 240

gatactgcga actactactg ccagcaatgg aggtcgtacc ccttgacatt tggccaaggt 300

actaaactag agataaaa 318

<210> 22

<211> 360

<212> DNA

<213> mouse

<400> 22

caggtacaac tggtccaatc tggagccgaa gtcaagaaac caggagcttc tgtgaaagtc 60

agttgcaagg cgtctgggta tacattcaca gattacgtag tacactgggt taggcaagct 120

cctggtcaag ggcttgaatg gatgggatat attaatccgt acaacgacgg aacaaaatat 180

aacgagaagt ttaagggtag agtaactatg accagggaca caagcatcag tacagcgtat 240

atggaactga gtcgtctccg gtctgatgac accgctgtct attattgtgc aagagattac 300

cgttacgagg tttacggcat ggactattgg ggccaaggca ctctcgttac cgtgtcaagc 360

<210> 23

<211> 963

<212> DNA

<213> Artificial Sequence

<220>

<223> chimeric DNA sequence mouse/human

<400> 23

atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60

gacgcggccc agccggccga agtgcagctg cagcagtctg gccccgagct ggtcaaacca 120

ggcgccagcg tgaagatgag ctgcaaggcc agcggctaca ccttcaccga ctactacatg 180

aagtgggtca agcagagcca cggcaagagc ctggaatgga tcggcgacat catccccagc 240

aacggcgcca ccttctacaa ccagaagttc aagggcaagg ccaccctgac cgtggacaga 300

agcagcagca ccgcctacat gcacctgaac agcctgacca gcgaggacag cgccgtgtac 360

tactgcacca gaagccatct gctgcgggcc agttggttcg cttattgggg ccagggcacc 420

ctggtcacag tgtctgccgc ctctggagga ggaggtagtg gcggaggtgg gtccggtggc 480

ggtggctctg acttcgtgat gacccagagc cctagcagcc tgaccgtgac agccggcgag 540

aaagtgacca tgagctgcaa gagcagccag agcctgctga actccggcaa ccagaagaac 600

tacctgacct ggtatctgca gaagccccgga cagccccccca agctgctgat ctactgggcc 660

agcaccagag aaagcggcgt gcccgataga ttcacaggca gcggcagcgg caccgacttc 720

accctgacaa tcagcagcgt gcaggccgag gacctggccg tgtactattg ccagaacgac 780

tacagctacc cctacacctt cggaggcggg accaagctgg aaatcaaggg aggaggagga 840

tccgcggccg ctaaacccct acctgaagtg actgatgagt atgctcgagg agggcccgaa 900

caaaaactca tctcagaaga ggatctgaat agcgccgtcg accatcatca tcatcatcat 960

tga 963

<210> 24

<211> 357

<212> DNA

<213> mouse

<400> 24

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> 25

<211> 339

<212> DNA

<213> mouse

<400> 25

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> 26

<211> 1671

<212> DNA

<213> Artificial Sequence

<220>

<223> chimeric DNA sequence mouse/human

<400> 26

atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60

gacgcggccc agccggccga agtgcagctg cagcagtctg gccccgagct ggtcaaacca 120

ggcgccagcg tgaagatgag ctgcaaggcc agcggctaca ccttcaccga ctactacatg 180

aagtgggtca agcagagcca cggcaagagc ctggaatgga tcggcgacat catccccagc 240

aacggcgcca ccttctacaa ccagaagttc aagggcaagg ccaccctgac cgtggacaga 300

agcagcagca ccgcctacat gcacctgaac agcctgacca gcgaggacag cgccgtgtac 360

tactgcacca gaagccatct gctgcgggcc agttggttcg cttattgggg ccagggcacc 420

ctggtcacag tgtctgccgc ctctggagga ggaggtagtg gcggaggtgg gtccggtggc 480

ggtggctctg acttcgtgat gacccagagc cctagcagcc tgaccgtgac agccggcgag 540

aaagtgacca tgagctgcaa gagcagccag agcctgctga actccggcaa ccagaagaac 600

tacctgacct ggtatctgca gaagccccgga cagccccccca agctgctgat ctactgggcc 660

agcaccagag aaagcggcgt gcccgataga ttcacaggca gcggcagcgg caccgacttc 720

accctgacaa tcagcagcgt gcaggccgag gacctggccg tgtactattg ccagaacgac 780

tacagctacc cctacacctt cggaggcggg accaagctgg aaatcaaggc ggccgctaaa 840

cccctacctg aagtgactga tgagtatgct cgaggacagg tacaactggt ccaatctgga 900

gccgaagtca agaaaccagg agcttctgtg aaagtcagtt gcaaggcgtc tgggtataca 960

ttcacagatt acgtagtaca ctgggttagg caagctcctg gtcaagggct tgaatggatg 1020

ggatatatta atccgtacaa cgacggaaca aaatataacg agaagtttaa gggtagagta 1080

actatgacca gggacacaag catcagtaca gcgtatatgg aactgagtcg tctccggtct 1140

gatgacaccg ctgtctatta ttgtgcaaga gattaccgtt acgaggttta cggcatggac 1200

tattggggcc aaggcactct cgttaccgtg tcaagcggcg gcggcggatc cggcggtggc 1260

ggttccggag gaggcggatc cgatatagtt ttaacccaat cccctgctag tctggccgta 1320

tccccaggcc agagggctac tataacctgc actgcaagct catctgtcaa ctacatccat 1380

tggtaccagc agaaacctgg acaaccgccg aaacttctga tatacgacac cagcaaggtc 1440

gcgtccgggg tgcctgctcg attcagcggc agcggatcag gtactgactt cactttgact 1500

atcaatccag tggaagcgaa cgatactgcg aactactact gccagcaatg gaggtcgtac 1560

cccttgacat ttggccaagg tactaaacta gagataaaag ggcccgaaca aaaactcatc 1620

tcagaagagg atctgaatag cgccgtcgac catcatcatc atcatcattg a 1671

<210> 27

<211> 894

<212> PRT

<213> Artificial Sequence

<220>

<223> chimeric fusion protein mouse/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 Gln Val Gln Leu Val Gln Ser Gly Ala Glu

20 25 30

Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly

35 40 45

Tyr Thr Phe Thr His Tyr Tyr Ile Tyr Trp Val Arg Gln Ala Pro Gly

50 55 60

Gln Gly Leu Glu Trp Met Gly Gly Val Asn Pro Ser Asn Gly Gly Thr

65 70 75 80

His Phe Asn Glu Lys Phe Lys Ser Arg Val Thr Met Thr Arg Asp Thr

85 90 95

Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp

100 105 110

Thr Ala Val Tyr Tyr Cys Ala Arg Ser Glu Tyr Asp Tyr Gly Leu Gly

115 120 125

Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val

145 150 155 160

Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala

165 170 175

Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Arg Thr Pro

180 185 190

Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys

195 200 205

Leu Leu Ile Tyr Trp Ala Ser Thr Arg Lys Ser Gly Val Pro Asp Arg

210 215 220

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

225 230 235 240

Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Lys Gln Ser Tyr Asn

245 250 255

Leu Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ala Ala Ala

260 265 270

Leu Glu Lys Glu Ala Leu Lys Lys Ile Ile Glu Asp Gln Gln Glu Ala

275 280 285

Leu Asn Lys Trp Ala Ala Ala Gly Pro Gly Gly Gly Gly Ser Lys Ile

290 295 300

Leu Val Lys Gln Ser Pro Met Leu Val Ala Tyr Asp Asn Ala Val Asn

305 310 315 320

Leu Ser Cys Lys Tyr Ser Tyr Asn Leu Phe Ser Arg Glu Phe Arg Ala

325 330 335

Ser Leu His Lys Gly Leu Asp Ser Ala Val Glu Val Cys Val Val Tyr

340 345 350

Gly Asn Tyr Ser Gln Gln Leu Gln Val Tyr Ser Lys Thr Gly Phe Asn

355 360 365

Cys Asp Gly Lys Leu Gly Asn Glu Ser Val Thr Phe Tyr Leu Gln Asn

370 375 380

Leu Tyr Val Asn Gln Thr Asp Ile Tyr Phe Cys Lys Ile Glu Val Met

385 390 395 400

Ala Pro Pro Pro Ala Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile

405 410 415

His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro

420 425 430

Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys

435 440 445

Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser

450 455 460

Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg

465 470 475 480

Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg

485 490 495

Asp Phe Ala Ala Tyr Arg Ser Gly Gly Gly Gly Ser Gly Gly Gly Ala

500 505 510

Ser Gly Gly Gly Gly Ser Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

515 520 525

Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg

530 535 540

Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met

545 550 555 560

Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu

565 570 575

Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys

580 585 590

Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu

595 600 605

Ser Thr Ala Thr Lys Asp Thr Tyr Gly Ala Leu His Met Gln Ala Leu

610 615 620

Pro Pro Arg Gly Ser Gly Ser Ser Ser Gly Glu Gly Arg Gly Ser Leu Leu

625 630 635 640

Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Pro Val Ala Thr Met

645 650 655

Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val

660 665 670

Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu

675 680 685

Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys

690 695 700

Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu

705 710 715 720

Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln

725 730 735

His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg

740 745 750

Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val

755 760 765

Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile

770 775 780

Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn

785 790 795 800

Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly

805 810 815

Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val

820 825 830

Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro

835 840 845

Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser

850 855 860

Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val

865 870 875 880

Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys

885 890

<210> 28

<211> 326

<212> PRT

<213> Artificial Sequence

<220>

<223> chimeric fusion protein mouse/human

<400> 28

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Ala Ala Gln Pro Ala Gly Ser Asp Ile Gln Met

20 25 30

Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr

35 40 45

Ile Thr Cys Gly Thr Ser Gln Asp Ile Asn Asn Tyr Leu Asn Trp Tyr

50 55 60

Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile Tyr Tyr Thr Ser

65 70 75 80

Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

85 90 95

Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala

100 105 110

Thr Tyr Tyr Cys Gln Gln Ser Lys Thr Leu Pro Trp Thr Phe Gly Gly

115 120 125

Gly Thr Gln Leu Thr Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

Ser Gly Ala Ser Ala Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

165 170 175

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

180 185 190

Ser Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

195 200 205

Ser Tyr Ile Asn Asp Ser Gly Gly Ser Thr Phe Tyr Pro Asp Thr Val

210 215 220

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

225 230 235 240

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

245 250 255

Ala Arg Arg Met Tyr Tyr Gly Asn Ser His Trp His Phe Asp Val Trp

260 265 270

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Ala

275 280 285

Ala Ala Lys Pro Leu Pro Glu Val Thr Asp Glu Tyr Ala Arg Gly Gly

290 295 300

Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Ser Ala Val Asp

305 310 315 320

His His His His His His

325

<210> 29

<211> 302

<212> PRT

<213> Artificial Sequence

<220>

<223> chimeric fusion protein mouse/human

<400> 29

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Ala Ala Gln Pro Ala Glu Val Gln Leu Gln Gln

20 25 30

Ser Gly Pro Glu Leu Val Lys Pro Gly Thr Ser Val Arg Ile Ser Cys

35 40 45

Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr Thr Ile His Trp Val Lys

50 55 60

Gln Ser His Gly Lys Ser Leu Glu Trp Ile Gly Asn Ile Asn Pro Asn

65 70 75 80

Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe Glu Asp Lys Ala Thr Leu

85 90 95

Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu

100 105 110

Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Ala Gly Trp Asn Phe

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met

145 150 155 160

Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser

165 170 175

Ile Ile Cys Lys Ala Ser Gln Asp Val Gly Thr Ala Val Asp Trp Tyr

180 185 190

Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser

195 200 205

Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly

210 215 220

Thr Asp Phe Thr Leu Thr Ile Thr Asn Val Gln Ser Glu Asp Leu Ala

225 230 235 240

Asp Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Leu Thr Phe Gly Ala

245 250 255

Gly Thr Met Leu Asp Leu Lys Ala Ala Ala Lys Pro Leu Pro Glu Val

260 265 270

Thr Asp Glu Tyr Ala Arg Gly Gly Pro Glu Gln Lys Leu Ile Ser Glu

275 280 285

Glu Asp Leu Asn Ser Ala Val Asp His His His His His

290 295 300

<210> 30

<211> 323

<212> PRT

<213> Artificial Sequence

<220>

<223> chimeric construct

<400> 30

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Ala Ala Gln Pro Ala Gly Ser Asp Ile Val Leu

20 25 30

Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly Gln Arg Ala Thr

35 40 45

Ile Thr Cys Thr Ala Ser Ser Ser Val Asn Tyr Ile His Trp Tyr Gln

50 55 60

Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Asp Thr Ser Lys

65 70 75 80

Val Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr

85 90 95

Asp Phe Thr Leu Thr Ile Asn Pro Val Glu Ala Asn Asp Thr Ala Asn

100 105 110

Tyr Tyr Cys Gln Gln Trp Arg Ser Tyr Pro Leu Thr Phe Gly Gln Gly

115 120 125

Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Gly Ala Ser Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

145 150 155 160

Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser

165 170 175

Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Val

180 185 190

Val His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly

195 200 205

Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe Lys

210 215 220

Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr Met

225 230 235 240

Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala

245 250 255

Arg Asp Tyr Arg Tyr Glu Val Tyr Gly Met Asp Tyr Trp Gly Gln Gly

260 265 270

Thr Leu Val Thr Val Ser Ser Ser Gly Gly Gly Gly Ser Ala Ala Ala Lys

275 280 285

Pro Leu Pro Glu Val Thr Asp Glu Tyr Ala Arg Gly Gly Pro Glu Gln

290 295 300

Lys Leu Ile Ser Glu Glu Asp Leu Asn Ser Ala Val Asp His His

305 310 315 320

His His His

<210> 31

<211> 320

<212> PRT

<213> Artificial Sequence

<220>

<223> chimeric fusion protein mouse/human

<400> 31

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Ala Ala Gln Pro Ala Glu Val Gln Leu Gln Gln

20 25 30

Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys

35 40 45

Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Tyr Met Lys Trp Val Lys

50 55 60

Gln Ser His Gly Lys Ser Leu Glu Trp Ile Gly Asp Ile Ile Pro Ser

65 70 75 80

Asn Gly Ala Thr Phe Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu

85 90 95

Thr Val Asp Arg Ser Ser Ser Thr Ala Tyr Met His Leu Asn Ser Leu

100 105 110

Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Thr Arg Ser His Leu Leu

115 120 125

Arg Ala Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

130 135 140

Ser Ala Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

145 150 155 160

Gly Gly Ser Asp Phe Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val

165 170 175

Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu

180 185 190

Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Leu Gln Lys

195 200 205

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu

210 215 220

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

225 230 235 240

Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr

245 250 255

Cys Gln Asn Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys

260 265 270

Leu Glu Ile Lys Gly Gly Gly Gly Ser Ala Ala Ala Lys Pro Leu Pro

275 280 285

Glu Val Thr Asp Glu Tyr Ala Arg Gly Gly Pro Glu Gln Lys Leu Ile

290 295 300

Ser Glu Glu Asp Leu Asn Ser Ala Val Asp His His His His His

305 310 315 320

<210> 32

<211> 556

<212> PRT

<213> Artificial Sequence

<220>

<223> chimeric fusion protein mouse/human

<400> 32

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Ala Ala Gln Pro Ala Glu Val Gln Leu Gln Gln

20 25 30

Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys

35 40 45

Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Tyr Met Lys Trp Val Lys

50 55 60

Gln Ser His Gly Lys Ser Leu Glu Trp Ile Gly Asp Ile Ile Pro Ser

65 70 75 80

Asn Gly Ala Thr Phe Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu

85 90 95

Thr Val Asp Arg Ser Ser Ser Thr Ala Tyr Met His Leu Asn Ser Leu

100 105 110

Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Thr Arg Ser His Leu Leu

115 120 125

Arg Ala Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

130 135 140

Ser Ala Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

145 150 155 160

Gly Gly Ser Asp Phe Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val

165 170 175

Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu

180 185 190

Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Leu Gln Lys

195 200 205

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu

210 215 220

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

225 230 235 240

Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr

245 250 255

Cys Gln Asn Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys

260 265 270

Leu Glu Ile Lys Ala Ala Ala Lys Pro Leu Pro Glu Val Thr Asp Glu

275 280 285

Tyr Ala Arg Gly Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys

290 295 300

Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr

305 310 315 320

Phe Thr Asp Tyr Val Val His Trp Val Arg Gln Ala Pro Gly Gln Gly

325 330 335

Leu Glu Trp Met Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr

340 345 350

Asn Glu Lys Phe Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile

355 360 365

Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala

370 375 380

Val Tyr Tyr Cys Ala Arg Asp Tyr Arg Tyr Glu Val Tyr Gly Met Asp

385 390 395 400

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly

405 410 415

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Leu Thr

420 425 430

Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly Gln Arg Ala Thr Ile

435 440 445

Thr Cys Thr Ala Ser Ser Ser Ser Val Asn Tyr Ile His Trp Tyr Gln Gln

450 455 460

Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Asp Thr Ser Lys Val

465 470 475 480

Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

485 490 495

Phe Thr Leu Thr Ile Asn Pro Val Glu Ala Asn Asp Thr Ala Asn Tyr

500 505 510

Tyr Cys Gln Gln Trp Arg Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr

515 520 525

Lys Leu Glu Ile Lys Gly Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp

530 535 540

Leu Asn Ser Ala Val Asp His His His His His

545 550 555

<210> 33

<211> 120

<212> PRT

<213> Mouse

<400> 33

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> 34

<211> 112

<212> PRT

<213> mouse

<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 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> 35

<211> 20

<212> PRT

<213> Homo sapiens

<400> 35

Leu Glu Lys Glu Ala Leu Lys Lys Ile Ile Glu Asp Gln Gln Glu Ala

1 5 10 15

Leu Asn Lys Trp

20

<210> 36

<211> 107

<212> PRT

<213> mouse

<400> 36

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gly Thr Ser Gln Asp Ile Asn Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Lys Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Gln Leu Thr Val Leu

100 105

<210> 37

<211> 122

<212> PRT

<213> mouse

<400> 37

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ser Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Asn Asp Ser Gly Gly Ser Thr Phe Tyr Pro Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Met Tyr Tyr Gly Asn Ser His Trp His Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 38

<211> 115

<212> PRT

<213> mouse

<400> 38

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Thr

1 5 10 15

Ser Val Arg Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr

20 25 30

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> 39

<211> 107

<212> PRT

<213> mouse

<400> 39

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> 40

<211> 106

<212> PRT

<213> mouse

<400> 40

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Thr Ala Ser Ser Ser Val Asn Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr

35 40 45

Asp Thr Ser Lys Val Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Pro Val Glu Ala Asn

65 70 75 80

Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Trp Arg Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 41

<211> 120

<212> PRT

<213> mouse

<400> 41

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 Asp Tyr

20 25 30

Val Val His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly 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 Asp Tyr Arg Tyr Glu Val Tyr Gly Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 42

<211> 119

<212> PRT

<213> mouse

<400> 42

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

Met His Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Ser His Leu Leu Arg Ala Ser Trp Phe Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser

115

<210> 43

<211> 113

<212> PRT

<213> mouse

<400> 43

Asp Phe Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Leu 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 Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 44

<211> 10

<212> PRT

<213> Homo sapiens

<400> 44

Lys Pro Leu Pro Glu Val Thr Asp Glu Tyr

1 5 10

Claims (13)

1. A nucleic acid encoding a universal chimeric antigen receptor, wherein the receptor comprises three domains, wherein - The first domain is a tag-binding domain, wherein the tag-binding domain is an antibody or its antigen-binding fragment capable of specifically binding to the amino acid sequence of SEQ.ID44. - The second domain is the extracellular hinge and transmembrane domain, and - The third structural domain is the signal transduction structural domain. The antibody or its antigen-binding fragment comprises the amino acid sequences of SEQ.ID 33 and SEQ.ID 34. 2. The nucleic acid according to claim 1, wherein the hinge and transmembrane region are selected from the hinge and transmembrane region of a portion of the constant region of a human CD28 molecule, a CD8a chain, an NK cell receptor, or an antibody, and combinations thereof of different hinge and transmembrane domains. 3. The nucleic acid according to claim 1, wherein the signal transduction domain is selected from the signal transduction domains of CD28, CD137 (41BB), CD134 (OX40), DAP10, CD27, programmed cell death-1 (PD-1), cytotoxic T-lymphocyte antigen 4 (CTLA-4), CD3 chain, DAP12 and T cell activation-inducible Fc receptor. 4. The nucleic acid of claim 1, wherein the receptor comprises a fourth domain, the fourth domain being a short peptide linker between the tag-binding domain and the extracellular hinge and transmembrane domain. 5. The nucleic acid according to claim 1, which is composed of the nucleotide sequence of SEQ ID 1. 6. A vector comprising the nucleic acid according to any one of claims 1 to 5. 7. A cell comprising the nucleic acid according to any one of claims 1 to 5. 8. The cell according to claim 7, wherein the cell is selected from T cells, natural killer cells, cytotoxic T lymphocytes, and regulatory T cells. 9. A reagent kit comprising: - The carrier according to claim 6, and - Target modules consisting of amino acid sequences of SEQ.ID 28, 29, 30, 31 or 32 and/or vectors containing nucleic acids having sequences selected from SEQ.ID 10, 17, 20, 23 and 26. 10. A formulation comprising cells according to claim 7 or 8, or comprising cells according to claim 7 or 8 and a target module consisting of an amino acid sequence of SEQ ID 28, 29, 30, 31 or 32. 11. Use of the cell according to claim 7 or 8 in the preparation of a medicament for stimulating a universal chimeric antigen receptor-mediated immune response in mammals. 12. A polypeptide comprising an amino acid sequence encoded by a nucleic acid according to any one of claims 1 to 5. 13. A cell comprising the polypeptide according to claim 12.