Conditionally active chimeric antigen receptors for modified T cells

Abstract

The present invention relates to a chimeric antigen receptor for binding to a tumor-specific target antigen. The chimeric antigen receptor comprises at least one antigen-specific targeting region evolved from a parent protein or a fragment thereof, and the antigen-specific targeting region has the following characteristics: the activity in a determination under normal physiological conditions is decreased compared to the activity in a determination under abnormal conditions. A method for producing the chimeric antigen receptor is also provided.

Description

[0001] Related application data

[0002] This application is a continuation-in-part of U.S. patent application No. 16 / 053,166 filed on August 2, 2018 (currently under review), which is in turn a divisional application of the now abandoned U.S. patent application No. 15 / 052,487, which is in turn a continuation-in-part of International Application No. PCT / US15 / 47197 filed on August 27, 2015 (designated country: the United States), which claims priority to U.S. Provisional Application No. 62 / 043,067 filed on August 28, 2014 (now expired), all of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to the field of protein evolution. In particular, the present invention relates to a method for generating conditionally active chimeric antigen receptors from a parent or wild-type protein. The conditionally active chimeric antigen receptors are reversibly or irreversibly inactivated under normal physiological conditions of the wild type, but are active under abnormal conditions. Background Art

[0004] There is a large literature describing the potential to evolve various properties of proteins, particularly enzymes. For example, enzymes can be evolved to stabilize them in order to operate under different conditions, such as elevated temperatures. In the case of increased activity at elevated temperatures, much of this increase is due to higher kinetic activity, which is often described by the Q10 law, which is estimated for enzymes as a doubling of their turnover rate for every 10°C increase in temperature.

[0005] In addition, there are examples of natural mutations that render proteins unstable under normal working conditions. Some mutants may be active at lower temperatures, but at reduced levels of activity compared to the parent or wild-type protein. This is also often described as a decrease in activity governed by Q10 or similar rules.

[0006] It is desirable to obtain useful conditionally activated molecules. For example, it is desirable to obtain molecules that are almost inactive under wild-type working conditions, but are active under conditions other than wild-type working conditions, and their activity levels are the same or higher than the activity under wild-type working conditions, or they are activated or inactivated in a specific microenvironment, or they are activated or inactivated after a period of time. In addition to temperature, other conditions that can evolve or optimize proteins include pH, osmotic pressure, weight molar osmotic pressure concentration (osmolality), oxidative stress, and electrolyte concentration. Other target properties that can be optimized during the evolution process include chemical resistance and resistance to proteolysis.

[0007] Many strategies for evolving or engineering molecules have been published. However, engineering or evolving a protein to be inactive or nearly inactive (less than 10% activity, preferably less than 1% activity) under wild-type working conditions, while maintaining activity comparable to or greater than that of its corresponding parent or wild-type protein under conditions other than wild-type working conditions, requires the coexistence of destabilizing mutations with activity-enhancing mutations that do not antagonize the destabilizing effect. It is expected that the destabilizing reduction in the activity of the protein will have a greater effect than predicted by standard rules such as Q10, and thus, by evolving the ability of a protein to be inactive under normal working conditions of the corresponding parent or wild-type protein but to work effectively at low temperatures, for example, an unexpected class of new proteins can be prepared.

[0008] Chimeric antigen receptors (CARs) have been used to treat cancer. US 2013 / 0280220 discloses methods and compositions for providing improved cells encoding chimeric antigen receptors specific for two or more antigens, including tumor antigens. Cells expressing chimeric antigen receptors can be used for cell therapy. Although in a specific embodiment this cell therapy is for cancer (including cancer involving solid tumors), the cell therapy may be applicable to any medical condition.

[0009] The present invention provides engineered conditionally active chimeric antigen receptors that are inactive or less active under normal physiological conditions but active under abnormal physiological conditions.

[0010] In this application, various publications are cited by author and date. In order to more fully describe the state of the art as described in the claims and the dates of these publications known to those skilled in the art, these publications are hereby incorporated by reference in their entirety into this application. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 A schematic diagram of a chimeric antigen receptor according to one embodiment of the present invention is depicted. ASTR is an antigen-specific targeting region, L is a linker, ESD is an extracellular spacer domain, TM is a transmembrane domain, CSD is a co-stimulatory domain, and ISD is an intracellular signaling domain.

[0012] Figure 2 and 3 It was shown that expressing the conditionally active antibodies of Example 1 as bivalent or monovalent antibodies did not significantly alter the selectivity of these antibodies above pH 6.0 and pH 7.4.

[0013] Figure 4 The size exclusion chromatography spectrum shows that the conditionally active antibody of Example 2 does not aggregate.

[0014] Figure 5 Shown are the association and dissociation rates of the conditionally active antibody of Example 2 measured by surface plasmon resonance (SPR) assay.

[0015] FIG. 6A to FIG. 6B The selectivity of the conditionally active antibody measured by SPR assay of Example 2 is shown.

[0016] Fig. 7A It was shown that CAR-T cells had no effect on a CHO cell population that did not express the target antigen X1 of CAR-T cells. The CAR molecule in the CAR-T cells of this example contained an antibody against the target antigen X1, although the antibody was not conditionally active (Comparative Example A).

[0017] Figure 7B The CAR-T cells were shown to reduce the population of CHO-63 cells expressing the target antigen X1 of the CAR-T cells. These CAR-T cells were used to generate Fig. 7A The cells for which the data are shown are the same as those in (Comparative Example A).

[0018] Fig. 8A It shows that CAR-T cells have no effect on the CHO cell population that does not express the target antigen X1 of CAR-T cells. The CAR molecule in the CAR-T cells of Example 3 contains a conditionally active antibody against the target antigen X1.

[0019] Figure 8B The CAR-T cells showed a reduction in the CHO-63 cell population expressing the target antigen X1 of the CAR-T cells tested in Example 3. These CAR-T cells were used to generate Fig. 8A The cells for which the data are shown are the same.

[0020] FIG. 9A to FIG. 9B Cytokine release induced by binding of CAR-T cells to target antigen X1 is shown as described in Example 3.

[0021] Fig.10 Conditionally active antibodies against target antigen X2 are shown.

[0022] Fig.11A The cytotoxic effect induced by CAR-T cells binding to Daudi cells expressing target antigen X2 and the cytotoxic effect induced by CAR-T cells on HEK293 cells not expressing target antigen X2 are shown, as described in Example 4.

[0023] FIG. 12A to FIG. 12B Cytokine release induced by binding of CAR-T cells to target antigen X1 is shown as described in Example 5.

[0024] FIG. 13A to FIG. 13B Cytokine release induced by binding of CAR-T cells to target antigen X2 is shown as described in Example 5.

[0025] Fig.14 Conditionally active antibodies against the target antigen CD22 suitable for constructing CAR-T cells were shown. Summary of the invention

[0026] In one aspect, the present invention provides a chimeric antigen receptor (CAR) for binding to a tumor-specific target antigen. The chimeric antigen receptor includes at least one antigen-specific targeting region, and the at least one antigen-specific targeting region evolves from a parent protein or its domain. The CAR further comprises a transmembrane domain and an intracellular signaling domain. The at least one antigen-specific targeting region has the following characteristics: the activity in the assay under normal physiological conditions is decreased compared to the activity in the assay under abnormal conditions.

[0027] In another aspect, the present invention provides an expression vector comprising a polynucleotide sequence encoding a chimeric antigen receptor of the present invention, wherein the expression vector is selected from a lentiviral vector, a gamma-retroviral vector, a foamy virus vector, an adeno-associated virus vector, an adenoviral vector, a poxvirus vector, a herpesvirus vector, an engineered hybrid virus, and a transposon-mediated vector.

[0028] In another aspect, the present invention provides a genetically engineered cytotoxic cell comprising a polynucleotide sequence encoding a chimeric antigen receptor of the present invention. The cytotoxic cell may be a T cell, and may be selected from a naive T cell, a central memory T cell, and an effector memory T cell.

[0029] In another aspect, the present invention provides a pharmaceutical composition comprising the chimeric antigen receptor, expression vector and / or genetically engineered cytotoxic cell of the present invention; and a pharmaceutically acceptable excipient.

[0030] On the other hand, the present invention provides a method for producing a chimeric antigen receptor, the chimeric antigen receptor comprising at least one antigen-specific targeting region, a transmembrane domain and an intracellular signaling domain. The method includes the step of generating the at least one antigen-specific targeting region from a parent protein or a domain thereof that specifically binds to a tumor-specific target antigen. These steps include: (i) evolving DNA encoding a parent or wild-type protein or a domain thereof using one or more evolution techniques to produce mutant DNA; (ii) expressing the mutant DNA to obtain a mutant polypeptide; (iii) subjecting the mutant polypeptide to assays under normal physiological conditions and assays under abnormal conditions; and (iv) selecting at least one antigen-specific targeting region from the mutant polypeptides expressed in step (ii), wherein the activity of the at least one antigen-specific targeting region in an assay under normal physiological conditions is decreased compared to the activity in an assay under the abnormal conditions.

[0031] definition

[0032] To facilitate understanding of the examples provided herein, some frequently appearing methods and / or terms will be defined herein.

[0033] As used herein, the term "about" in relation to a measured amount refers to the normal variation in the measured amount that can be expected by one skilled in the art making the measurement and performing the measurement with a level of care commensurate with the purpose of the measurement and the precision of the measuring instrument. Unless otherwise indicated, "about" means a variation of + / - 10% of the value provided.

[0034] The term "agent" as used herein refers to a compound, a mixture of compounds, a spatially localized compound series (e.g., a VLSIPS peptide series, a polynucleotide series, and / or a combinatorial small molecule series), a biomacromolecule, a phage peptide display library, a phage antibody (e.g., scFv) display library, a polyribosomal peptide display library, or an extract obtained from a biological material, such as a cell or tissue of bacteria, plants, fungi, or animals (particularly mammals). The potential enzymatic activity of the agent is evaluated in the screening assay described below. The potential activity of the agent as a conditionally active biotherapeutic enzyme is evaluated in the screening assay described below.

[0035] As used herein, the term "amino acid" refers to any organic compound containing an amino group (-NH2) and a carboxyl group (-COOH); preferably as a free group or as part of a peptide bond after condensation. The "twenty α-amino acids that form naturally encoded polypeptides" are understood in the art to refer to: alanine (ala or A), arginine (arg or R), asparagine (asn or N), aspartic acid (asp or D), cysteine ​​(cys or C), glutamic acid (glu or E), glutamine (gin or Q), glycine (gly or G), histidine (his or H), isoleucine (ile or I), leucine (leu or L), lysine (lys or K), methionine (met or M), phenylalanine (phe or F), proline (pro or P), serine (ser or S), threonine (thr or T), tryptophan (tip or W), tyrosine (tyr or Y) and valine (val or V).

[0036] As used herein, the term "amplification" refers to an increase in the number of copies of a polynucleotide.

[0037] As used herein, the term "antibody" refers to intact immunoglobulin molecules, as well as fragments of immunoglobulin molecules, such as Fab, Fab', (Fab')2, Fv and SCA fragments that are capable of binding to an epitope of an antigen. These antibody fragments retain some ability to selectively bind to the antigen (e.g., a polypeptide antigen) from which the antibody is derived, and can be prepared using methods well known in the art (see, e.g., Harlow and Lane, supra), and are further described below. Antibodies can be used to separate prepared quantities of antigen by immunoaffinity chromatography. Various other uses of such antibodies are therapeutic applications for diagnosis and / or staging of disease (e.g., neoplasia) and treatment of diseases, such as neoplasia, autoimmune diseases, AIDS, cardiovascular disease, infections, and the like. Chimeric, human-like, humanized or fully human antibodies are particularly useful for administration to human patients.

[0038] Fab fragments consist of a monovalent antigen-binding fragment of an antibody molecule and can be prepared by digesting an intact antibody molecule with the enzyme papain to produce a fragment consisting of an entire light chain and a portion of a heavy chain.

[0039] The Fab' fragment of an antibody molecule can be obtained by treating the whole antibody molecule with pepsin, followed by reduction to obtain a molecule consisting of a complete light chain and a portion of a heavy chain. Two Fab' fragments are obtained from each antibody molecule treated in this manner.

[0040] The (Fab')2 fragment of an antibody can be obtained by treating an intact antibody molecule with pepsin without subsequent reduction. The (Fab')2 fragment is a dimer of two Fab' fragments held together by two disulfide bonds.

[0041] The Fv fragment is defined as a genetically engineered fragment containing the light chain variable region and the heavy chain variable region expressed as two chains.

[0042] The term "antigen" or "Ag" as used herein is defined as a molecule that triggers an immune response. This immune response can be involved in antibody production or the activation of specific immune-competent cells or both. It will be understood by those skilled in the art that any macromolecule, including almost all proteins or peptides, can be used as an antigen. In addition, antigens can be derived from recombinant or genomic DNA. It will be understood by those skilled in the art that any DNA containing a nucleotide sequence or a partial nucleotide sequence encoding a protein that triggers an immune response thus encodes the term "antigen" used herein. In addition, it will be understood by those skilled in the art that antigens do not need to be encoded only by the full-length nucleotide sequence of a gene. It is obvious that the present invention includes but is not limited to the use of partial nucleotide sequences of more than one gene, and these nucleotide sequences are arranged in various combinations to trigger the desired immune response. In addition, the technician will understand that antigens do not need to be encoded by "genes" at all. Obviously, antigens can be produced, synthesized, or can be derived from biological samples. Such biological samples can include but are not limited to tissue samples, tumor samples, cells or biological fluids.

[0043] As used herein, "antigen loss escape variant" refers to a cell that exhibits reduced or lost expression of a target antigen that is targeted by a CAR of the present invention.

[0044] The term "autoimmune disease" as used herein is defined as a condition caused by an autoimmune response. Autoimmune diseases are the result of inappropriate and excessive responses to autoantigens. Examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia greata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes (type 1), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, colitis, etc.

[0045] As used herein, the term "autologous" refers to any material derived from an individual that is subsequently reintroduced into the same individual. For example, T cells from a patient can be isolated, genetically engineered to express a CAR, and then reintroduced into the patient.

[0046] As used herein, the term "B cell-related disease" includes B cell immunodeficiencies, autoimmune diseases, and / or excessive / uncontrolled cell proliferation associated with B cells (including lymphomas and / or leukemias). Examples of diseases for which the bispecific CARs of the present invention can be used for their therapeutic methods include, but are not limited to, systemic lupus erythematosus (SLE), diabetes, rheumatoid arthritis (RA), reactive arthritis, multiple sclerosis (MS), pemphigus vulgaris, celiac disease, Crohn's disease, inflammatory bowel disease, ulcerative colitis, autoimmune thyroid disease, X-linked agammaglobulinemia, pre-B acute lymphoblastic leukemia, systemic lupus erythematosus, common variable immunodeficiency, chronic lymphocytic leukemia, diseases associated with selective IgA deficiency and / or IgG subclass deficiency, B-lineage lymphoma (Hodgkin's lymphoma and / or non-Hodgkin's lymphoma), immunodeficiency with thymoma, transient hypogammaglobulinemia and / or hyper-IgM syndrome, as well as virally mediated B cell diseases (such as EBV-mediated lymphoproliferative diseases) and chronic infections in which B cells are involved from a pathobiological perspective.

[0047] The term "blood-brain barrier" or "BBB" refers to a physiological barrier between the peripheral circulation and the brain and spinal cord, which is formed by tight junctions in the plasma membrane of brain capillary endothelial cells, creating a dense barrier that limits the transport of molecules, even very small molecules (such as urea (60 Daltons)) to the brain. The blood-brain barrier in the brain, the blood-spinal cord barrier in the spinal cord, and the blood-retinal barrier in the retina are continuous capillary barriers in the central nervous system (CNS), and are collectively referred to herein as "blood-brain barrier" or "BBB". The BBB also includes a blood-cerebrospinal fluid barrier (choroid plexus), in which the barrier includes ependymal cells rather than capillary endothelial cells.

[0048] As used herein, the terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, B-cell lymphoma (Hodgkin's lymphoma and / or non-Hodgkin's lymphoma), brain tumors, breast cancer, colon cancer, lung cancer, hepatocellular carcinoma, stomach cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, thyroid cancer, kidney cancer, carcinoma, melanoma, head and neck cancer, brain cancer, and prostate cancer (including, but not limited to, androgen-dependent prostate cancer and androgen-independent prostate cancer).

[0049] The term "chimeric antigen receptor" or "CAR" used herein refers to an engineered receptor that is specifically transplanted to cytotoxic cells such as T cells, NK cells and macrophages. The CAR of the present invention may include at least one antigen-specific targeting region (ASTR), an extracellular spacer domain (ESD), a transmembrane domain (TM), one or more co-stimulatory domains (CSD) and an intracellular signaling domain (ISD). In one embodiment, ESD and / or CSD are optional. In another embodiment, CAR is a bispecific CAR that is specific for two different antigens or epitopes. After ASTR specifically binds to the target antigen, ISD activates intracellular signaling. For example, ISD can redirect T cell specificity and reactivity to a selected target in a non-MHC restricted manner, thereby developing the antigen binding properties of antibodies. Non-MHC restricted antigen recognition enables T cells expressing CAR to have the ability to recognize antigens independently of antigen processing, thereby bypassing the main mechanism of tumor escape. In addition, when expressed in T cells, CAR is advantageously not dimerized with endogenous T cell receptor (TCR) α and β chains.

[0050] The term "coexpression" as used herein refers to the simultaneous expression of two or more genes. A gene can be a nucleic acid encoding, for example, a single protein or a chimeric protein as a single polypeptide chain. For example, the CAR of the present invention can be coexpressed with a therapeutic control (e.g., a truncated epidermal growth factor (EGFRt)), wherein the CAR is encoded by a first polynucleotide chain and the therapeutic control is encoded by a second polynucleotide chain. In one embodiment, the first and second polynucleotide chains are connected by a nucleic acid sequence encoding a cleavable joint. Alternatively, the CAR and the therapeutic control are encoded by two different polynucleotides that are not connected by a joint but are encoded by, for example, two different vectors.

[0051] The term "homologous" as used herein refers to gene sequences that are evolutionarily and functionally related between species. For example, but not limited to, the human CD4 gene is a homologous gene of the mouse 3d4 gene in the human genome, because the sequences and structures of the two genes show that they have a high degree of homology, and both genes encode a protein that plays a role in transmitting T cell activation signals through MHC class II restricted antigen recognition.

[0052] The term "conditionally active biologic protein" refers to a variant or mutation of a parent or wild-type protein that is more or less active than the parent or wild-type protein under one or more normal physiological conditions. Such conditionally active proteins also exhibit activity in selected areas of the body, and / or exhibit increased or decreased activity under abnormal or permissive physiological conditions. The term "normal physiological conditions" as used herein refers to one of the following: temperature; pH; osmotic pressure; osmolality, oxidative stress; electrolyte concentration; concentrations of small organic molecules such as glucose, lactate, pyruvate, nutritional components, other metabolites, concentrations of other molecules such as oxygen, carbonate, phosphate, and carbon dioxide, and cell type and nutrient availability, which are considered to be within the normal range at the site of administration to the individual or in the tissue or organ at the site of action of the individual.

[0053] In one embodiment, normal physiological conditions are normal physiological pH in the plasma of a mammalian individual, in the range of greater than 7.0 to about 7.8, or about 7.2 to about 7.8, or about 7.2 to about 7.6, or about 7.3 to about 7.6, or about 7.3 to about 7.5. Abnormal conditions are pH in a tumor microenvironment, in the range of about 6.0 to less than 7.0, or about 6.2 to about 6.9, or about 6.0 to about 6.8, or about 6.2 to about 6.8, or about 6.4 to about 6.8, or about 6.4 to about 6.6.

[0054] The term "abnormal conditions" as used herein refers to conditions that deviate from the normal acceptable range of the condition. In one aspect, the conditionally active biological protein is almost inactive under normal physiological conditions, but is active under abnormal conditions, and its level is equal to or better than the parent or wild-type protein from which it is derived. For example, in one aspect, the evolved conditionally active biological protein is almost inactive at body temperature, but is active at lower temperatures. On the other hand, the conditionally active biological protein is reversibly or irreversibly inactivated under normal physiological conditions. On the other hand, the parent or wild-type protein is a therapeutic protein. In another aspect, the conditionally active biological protein is used as a drug or therapeutic agent. In another aspect, the protein is more or less active in highly oxygenated blood, for example, in a lower pH environment found after flowing through the lungs or in the kidneys.

[0055] "Conservative amino acid substitution" refers to the interchangeability of residues with similar side chains. For example, a group of amino acids with aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; a group of amino acids with aliphatic hydroxyl side chains: serine and threonine; a group of amino acids with amide-containing side chains: asparagine and glutamine; a group of amino acids with aromatic side chains: phenylalanine, tyrosine and tryptophan; a group of amino acids with basic side chains: lysine, arginine and histidine; and a group of amino acids with sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine and asparagine-glutamine.

[0056] The term "corresponding to" as used herein refers to a polynucleotide sequence that is homologous to all or part of a reference polynucleotide sequence (i.e., is identical, not strictly evolutionarily related), or a polypeptide sequence that is identical to a reference polypeptide sequence. In contrast, the term "complementary" as used herein refers to a complementary sequence that is homologous to all or part of a reference nucleotide sequence. For example, the nucleoside sequence "TATAC" corresponds to the reference sequence "TATAC" and is complementary to the reference sequence "GTATA".

[0057] The term "costimulatory ligand" as used herein includes molecules on antigen presenting cells (e.g., dendritic cells, B cells, etc.) that specifically bind to cognate costimulatory molecules on T cells, thereby providing signals that mediate T cell responses (including but not limited to proliferation, activation, differentiation, etc.) in addition to the first signal provided by the binding of the TCR / CD3 complex to the MHC molecule loaded with peptides. Costimulatory ligands may include but are not limited to CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible co-stimulatory ligand (ICOS-L), intercellular adhesion molecules (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind to B7-H3. Co-stimulatory ligands also include antibodies that specifically bind to co-stimulatory molecules present on T cells, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0058] The term "costimulatory molecule" as used herein refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand to mediate a costimulatory response of the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class 1 molecules, BTLA, and Toll ligand receptors.

[0059] As used herein, the term "co-stimulatory signal" refers to a signal that, in conjunction with a first signal (eg, TCR / CD3 ligation), results in T cell proliferation and / or up-regulation or down-regulation of key molecules.

[0060] The term "cytotoxic cell" as used herein refers to a cell that can damage or destroy invading microorganisms, tumor cells or other diseased tissue cells. The term is intended to include cell types such as natural killer (NK) cells, activated NK cells, neutrophils, T cells, eosinophils, basophils, B cells, macrophages and lymphokine-activated killer (LAK) cells. Cytotoxic cells bind to target cells through antibodies, receptors, ligands or their fragments / derivatives to form a stable complex, and stimulate cytotoxic cells to destroy target cells.

[0061] Cytotoxic cells may also include other immune cells with tumor lytic ability, including but not limited to natural killer T cells (Heczey et al., "Invariant NKT cells with chimeric antigen receptorprovide a novel platform for safe and effective cancer immunotherapy," Blood, Vol. 124, pp. 2824-2833, 2014) and granulocytes. In addition, cytotoxic cells may include immune cells with phagocytic ability, including but not limited to macrophages and granulocytes; cells with stem cell and / or progenitor cell characteristics, including but not limited to hematopoietic stem / progenitor cells (Zhen et al., "HIV-specific Immunity Derived From Chimeric Antigen Receptor-engineered Stem Cells," Mol Ther., Vol. 23, pp. 1358-1367, 2015), embryonic stem cells (ESC), umbilical cord blood stem cells and induced pluripotent stem cells (iPSC) (Themeli et al., "New cell sources for T cell engineering and adoptive immunotherapy," Cell Stem Cell., Vol. 16, pp. 357-366, 2015). In addition, cytotoxic cells include “synthetic cells” such as iPSC-derived T cells (TiPSC) (Themeli et al., “Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy,” Nat Biotechnol., Vol. 31, pp. 928-933, 2013) or iPSC-derived NK cells.

[0062] The term "degradation effective" amount refers to the amount of enzyme required to process at least 50% of the substrate compared to the substrate not contacted with the enzyme.

[0063] The term "directional ligation" refers to a ligation in which the 5' and 3' ends of a polynucleotide are different, sufficient to determine the preferred direction of ligation. For example, an untreated and undigested PCR product having two blunt ends generally does not have a preferred direction of ligation when ligated into a cloning vector that has been digested to form blunt ends at the multiple cloning site; thus, directional ligation generally does not occur in these cases. In contrast, directional ligation generally occurs when a digested PCR product having a 5' end treated with EcoR I and a 3' end treated with BamHI is ligated into a cloning vector digested with EcoR I and BamHI at the multiple cloning site.

[0064] The term "genetically modified cytotoxic cell targeted disease" as used herein includes the targeting of genetically modified cells of the present invention to any cell involved in any disease in any way, regardless of whether the genetically modified cells are targeted to diseased cells or healthy cells to achieve therapeutically beneficial results. Genetically modified cells include, but are not limited to, genetically modified T cells, NK cells, and macrophages. Genetically modified cells express the CAR of the present invention, which can target any antigen expressed on the surface of the target cell. Examples of targetable antigens include, but are not limited to, antigens expressed on B cells; antigens expressed on cancer, sarcoma, lymphoma, leukemia, germ cell tumors, and germ cell tumors; antigens expressed on various immune cells; and antigens expressed on cells associated with various blood diseases, autoimmune diseases, and / or inflammatory diseases. Other antigens that can be targeted are obvious to those skilled in the art and can be targeted by the CAR of the present invention together with its alternative embodiments.

[0065] As used herein, the terms "genetically modified cells," "redirected cells," "genetically engineered cells," or "modified cells" refer to cells that express a CAR of the present invention.

[0066] The term "DNA shuffling" as used herein refers to recombination between substantially homologous but not identical sequences, and in some embodiments, DNA shuffling may include crossover by non-homologous recombination, such as by cer / lox and / or flp / frt systems, etc. DNA shuffling may be random or non-random.

[0067] The term "drug" or "drug molecule" refers to a therapeutic agent comprising a substance that produces a beneficial effect on the human or animal body when it is administered to the human or animal body. Preferably, the therapeutic agent comprises a substance that can treat, cure or alleviate one or more symptoms, diseases or abnormal conditions in the human or animal body or a substance that can improve the health of the human or animal body.

[0068] An "effective amount" refers to an amount of a conditionally active biological protein or fragment that is effective for treating or preventing a disease in a living organism when administered to the living organism for a period of time, such as within a desired dosing interval to provide a therapeutic effect.

[0069] The term "electrolyte" as used herein means a mineral with an electric charge in blood or other body fluids. For example, in one aspect, normal physiological conditions and abnormal conditions can be conditions of "electrolyte concentration". In one aspect, the electrolyte concentration to be measured is selected from one or more ionized calcium, sodium, potassium, magnesium, chloride, bicarbonate and phosphate concentrations. For example, in one aspect, the normal range of serum calcium is 8.5 to 10.2 mg / dL. In this regard, abnormal serum calcium concentrations can be selected above or below the normal range. In another example, in one aspect, the normal range of serum chloride is 96-106 milliequivalents per liter (mEq / L). In this regard, abnormal serum chloride concentrations can be selected above or below the normal range. In another example, in one aspect, the normal range of serum magnesium is from 1.7-2.2 mg / dL. In this regard, abnormal serum magnesium concentrations can be selected above or below the normal range. In another example, in one aspect, the normal range of serum phosphorus is from 2.4 mg / dL to 4.1 mg / dL. In this regard, abnormal serum phosphorus concentrations can be selected above or below the normal range. In another example, in one aspect, the range of normal serum or blood sodium is from 135mEq / L to 145mEq / L. In this regard, abnormal serum or blood sodium concentration can be selected above or below the normal range. In another example, in one aspect, the range of normal serum or blood potassium is from 3.7mEq / L to 5.2mEq / L. In this regard, abnormal serum or blood potassium concentration can be selected above or below the normal range. In another aspect, the normal serum bicarbonate range is from 20mEq / L to 29mEq / L. In this regard, the concentration of abnormal serum or blood bicarbonate can be selected above or below the normal range. In a different aspect, the bicarbonate level can be used to indicate the normal level (pH) of acidity in the blood. The term "electrolyte concentration" can also be used to define the conditions of specific electrolytes in tissues or body fluids other than blood or plasma. In this case, normal physiological conditions are considered to be the clinical normal range of the tissue or fluid. In this regard, the electrolyte concentration of abnormal tissues or body fluids can be selected above or below the normal range.

[0070] The term "epitope" as used herein refers to an antigenic determinant on an antigen such as an enzyme polypeptide, to which an antibody paratope such as an enzyme-specific antibody binds. Antigenic determinants are usually composed of chemically active surface combinations of molecules such as amino acids or sugar side chains, and may have specific three-dimensional structural characteristics and specific charge characteristics. "Epitope" as used herein refers to that part of an antigen or other macromolecule that is capable of forming a binding interaction, which interacts with the variable region binding body of an antibody. Typically, this binding interaction is manifested as intermolecular contact with one or more amino acid residues of a CDR.

[0071] As used herein, the terms "evolution" or "evolving" refer to the use of one or more mutagenesis methods to generate new polynucleotides encoding new polypeptides, which are themselves an improved biomolecule and / or contribute to the generation of another improved biomolecule. In a specific non-limiting aspect, the present invention relates to evolving conditionally active biological proteins from parent or wild-type proteins. For example, in one aspect, evolution involves methods using non-random polynucleotide chimerization and non-random site-directed mutagenesis disclosed in U.S. Patent Application Publication No. 2009 / 0130718. More specifically, the present invention provides a method for evolving conditionally active biological enzymes that exhibit decreased activity under normal physiological conditions compared to a parent or wild-type enzyme parent molecule, and enhanced activity under one or more abnormal conditions compared to an antigen-specific targeted region of a parent or wild-type enzyme.

[0072] When the terms "fragment", "derivative" and "analog" are used in reference to a reference polypeptide, they include polypeptides that retain at least one biological function or activity that is at least essentially the same as the reference polypeptide. In addition, examples of the terms "fragment", "derivative" or "analog" are "precursor forms" of molecules, such as low-activity proproteins, which can be modified by cleavage to produce mature enzymes with significantly higher activity.

[0073] The term "gene" as used herein refers to a DNA segment involved in producing a polypeptide chain; it includes regions before and after the coding region (leader and trailer) and intervening sequences (introns) between coding segments (exons).

[0074] As used herein, the term "heterologous" refers to a single-stranded nucleic acid sequence that cannot hybridize with another single-stranded nucleic acid sequence or its complementary sequence. Thus, a heterologous region refers to a region or area in a polynucleotide region or polynucleotide sequence that cannot hybridize with another nucleic acid or polynucleotide. Such a region or area is, for example, a mutation region.

[0075] As used herein, the term "homologous" or "homeologous" means that a single-stranded nucleic acid sequence can hybridize with the complementary sequence of a single-stranded nucleic acid sequence. The degree of hybridization may depend on many factors, including the amount of identity between the sequences and the hybridization conditions such as temperature and salt concentration discussed later. Preferably, the region of identity is greater than about 5 bp, and more preferably, the region of identity is greater than about 10 bp.

[0076] The benefits of the invention extend to "industrial applications" (or industrial processes), which term is used to include appropriate applications in commercial industry (or simply industry) as well as non-commercial industrial applications (e.g., biomedical research in non-profit institutions). Relevant applications include those in the fields of diagnostics, medicine, agriculture, manufacturing, and academia.

[0077] As used herein, the term "immune cell" refers to cells of the mammalian immune system, including but not limited to antigen presenting cells, B cells, basophils, cytotoxic T cells, dendritic cells, eosinophils, granulocytes, helper T cells, leukocytes, lymphocytes, macrophages, mast cells, memory cells, monocytes, natural killer cells, neutrophils, phagocytes, plasma cells and T cells.

[0078] As used herein, the term "immune response" refers to immunity including, but not limited to, innate immunity, humoral immunity, cellular immunity, immunity, inflammatory response, acquired (adaptive) immunity, autoimmunity, and / or hyperimmunity.

[0079] As used herein, the term "isolated" means that the material is removed from its original environment (e.g., the natural environment, if it occurs naturally). For example, a naturally occurring nucleotide or enzyme present in a living animal is not isolated, but the same nucleotide or enzyme separated from some or all of the coexisting materials in the natural system is isolated. Such a polynucleotide can be part of a vector, and / or such a polynucleotide or enzyme can be part of a composition, and it is still isolated because such a vector or composition is not part of its natural environment.

[0080] As used herein, the term "isolated nucleic acid" is used to define a nucleic acid, such as a DNA or RNA molecule, that is not closely linked to the 5' and 3' flanking sequences, as would normally be the case in a naturally occurring genome in the organism from which it is derived. Thus, the term describes, for example, a nucleic acid that is integrated into a vector such as a plasmid or viral vector; a nucleic acid that is integrated into a heterologous cell genome (or a homologous cell genome, but at a site different from that at which it occurs naturally); and a nucleic acid that exists as a separate molecule, such as a DNA fragment generated by PCR amplification or restriction enzyme digestion, or an RNA molecule generated by in vitro transcription. The term also describes a recombinant nucleotide that forms part of a hybrid gene encoding other polypeptide sequences, which can be used, for example, to produce a fusion protein.

[0081] The term "lentivirus" as used herein refers to a genus of the Retroviridae family. Slow viruses are unique among retroviruses in that they are able to infect non-dividing cells; they can deliver large amounts of genetic information into the DNA of host cells, so they are one of the most effective ways to deliver gene delivery vectors. HIV, SIV, and FIV are all examples of slow viruses. Vectors derived from slow viruses provide a means of achieving significant levels of gene transfer in vivo.

[0082] As used herein, the term "ligand" refers to a molecule that can be recognized by a specific receptor, such as a random peptide or a variable region sequence. Those skilled in the art will appreciate that a molecule (or macromolecular complex) can be both a receptor and a ligand. In general, a binding partner with a smaller molecular weight is referred to as a ligand, and a binding partner with a larger molecular weight is referred to as a receptor.

[0083] As used herein, the term "ligation" refers to the process of forming a phosphodiester bond between two double-stranded nucleic acid fragments (Sambrook et al., (1982). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., p. 146; Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., p. 146). nd Ed.), Cold Spring Harbor Laboratory Press, 1989). Unless otherwise specified, ligation can be accomplished using known buffers and conditions, using 10 units of T4 DNA ligase ("ligase") to ligate approximately equimolar amounts of DNA fragments per 0.5 microgram.

[0084] The term "joint" or "spacer" as used herein refers to a molecule or a combination of molecules connecting two molecules such as a DNA binding protein and a random peptide, so that the two molecules are in a preferred configuration, for example, so that the random peptide binds to the receptor of the DNA binding protein with minimal steric hindrance. "Joint" (L) or "joint domain" or "joint region" used herein refers to an oligopeptide or polypeptide region of about 1 to 100 amino acids in length, which connects any domain / region of the CAR of the present invention together. The joint can be composed of flexible residues such as glycine and serine, so that adjacent protein domains move freely relative to each other. When it is necessary to ensure that two adjacent domains do not interfere with each other spatially, a longer joint can be used. The joint can be cleavable or non-cleavable. Examples of cleavable joints include 2A joints (eg, T2A), 2A-like joints or their functional equivalents and combinations thereof. In some embodiments, the linker includes a picornavirus 2A-like linker, a CHYSEL sequence of porcine Teschovirus (P2A), Thosea asignavirus (T2A), or a combination, variant, and functional equivalent thereof. Other linkers will be apparent to those skilled in the art and may be used in conjunction with alternative embodiments of the present invention.

[0085] The term "mammalian cell surface display" used herein refers to the technology of expressing and displaying a protein or antibody or a part of an antibody on the surface of a mammalian host cell for screening purposes; for example, specific antigen binding is screened by a combination of magnetic beads and fluorescence-activated cell sorting. On the one hand, mammalian expression vectors are used to express immunoglobulins in secretory form and cell surface binding form simultaneously, as described in DuBridge et al., US 2009 / 0136950. On the other hand, the technology is used to screen viral vectors encoding antibody or antibody fragment libraries, which are displayed on the cell membrane when expressed in cells, as described in Gao et al., US 2007 / 0111260. Full IgG surface display on mammalian cells is known. For example, Akamatsuu et al. have developed a mammalian cell surface display vector suitable for directly separating IgG molecules based on their antigen binding affinity and biological activity. Using an episomal vector derived from Epstein-Barr virus, the antibody library is displayed on the cell surface as a complete IgG molecule, and specific antigen binding is screened by a combination of magnetic beads and fluorescence-activated cell sorting. Plasmids encoding antibodies with the desired binding characteristics are recovered from the sorted cells and converted into a form suitable for producing soluble IgG. See Akamatsuu et al. J. Immunol. Methods, Vol. 327, pp. 40-52, 2007. Ho et al. used human embryonic kidney 293T cells, which are widely used for transient protein expression, for cell surface display of single-chain Fv antibodies for affinity maturation. Cells expressing rare mutant antibodies with higher affinity were enriched 240 times by single-pass cell sorting from a large excess of cells expressing WT antibodies with slightly lower affinity. In addition, after a single selection of the combinatorial library randomized the intrinsic antibody hotspots, highly enriched mutants with enhanced binding affinity to CD22 were obtained. See Ho et al., “Isolation of anti-CD22 Fv with high affinity by Fv display on human cells,” Proc Natl Acad Sci USA, Vol. 103, pp. 9637–9642, 2006.

[0086] B cells with specificity for antigens can also be used. Such B cells can be directly isolated from peripheral blood mononuclear cells (PBMC) of human donors. Recombinant antigen-specific single-chain Fv (scFv) libraries are produced from the B cell library and screened by mammalian cell surface display using the Sindbis virus expression system. The variable regions (VR) of heavy chains (HC) and light chains (LC) are separated from positive clones and recombinant full human antibodies produced as complete IgG or Fab fragments. In this way, several hypermutated high-affinity antibodies, model virus antigens, and antibodies specific to nicotine that bind to Qβ virus-like particles (VLPs) are separated. See Beerli et al., "Isolation of human monoclonal antibodies by mammalian cell display", Proc Natl Acad Sci USA, Vol. 105, pp. 14336-14341, 2008.

[0087] Yeast cell surface display can also be used in the present invention, for example, see Kondo and Ueda, "Yeast cell-surface display-applications of molecular display", Appl. Microbiol. Biotechnol., Vol. 64, pp. 28-40, 2004, which describes a cell surface engineering system using, for example, Saccharomyces cerevisiae. Several representative display systems for expression in Saccharomyces cerevisiae are described in Lee et al, "Microbial cell-surface display," TRENDS in Bitechnol., Vol. 21, pp. 45-52, 2003; and Boder and Wittrup, "Yeast surface display for screening combinatorial polypeptide libraries," Nature Biotechnol., Vol. 15, p. 553, 1997.

[0088] As used herein, the term "production" refers to the production of a protein in sufficient quantities to allow at least Phase I clinical testing for a therapeutic protein or for regulatory approval for a diagnostic protein.

[0089] As used herein, the term "microenvironment" refers to any part or region of a tissue or body that is persistently or temporally different, physiologically or chemically from other areas of the tissue or region of the body.

[0090] As used herein, the term "molecular properties to be evolved" includes molecules containing polynucleotide sequences, molecules containing polypeptide sequences, and molecules containing partial polynucleotide and partial polypeptide sequences. Examples particularly relevant to the molecular properties to be evolved, but in no way limited to the following, include: the activity of the protein under specific conditions, such as those involving temperature; salinity; osmotic pressure; pH; oxidative stress and the concentration of glycerol, dimethyl sulfoxide, detergents and / or any other molecular species contacted in the reaction environment. Other examples particularly relevant to the molecular properties to be evolved, but in no way limited to the following, include stability, such as the remaining molecular properties exhibited after being exposed to a specific environment for a period of time, such as what would be encountered during storage.

[0091] The term "mutation" as used herein refers to a change in sequence in a parent or wild-type nucleic acid sequence or a change in sequence in a peptide sequence. Such a mutation may be a point mutation, such as a conversion or transversion. The mutation may be a deletion, insertion or duplication.

[0092] The term "multi-specific antibody" as used herein is an antibody with binding affinity to at least two different epitopes. Multi-specific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). Engineered antibodies can bind two, three or more (e.g., four) antigens (see, e.g., US 2002 / 0004587A1). A conditionally active antibody can be engineered into a multi-specific antibody, or two antibodies can be engineered to contain a heterodimer that binds two antigens. Multi-specific antibodies can also be multifunctional.

[0093] As used herein, the degenerate "N,N,G / T" nucleoside sequence represents 32 possible triplets, where "N" can be A, C, G, or T.

[0094] As used herein, the term "naturally occurring" is applied to objects that refer to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including a virus) is naturally occurring, which can be isolated from a natural source and has not been intentionally modified by humans in a laboratory. Generally speaking, the term naturally occurring refers to the presence of an object in a non-pathological (non-diseased) individual, which is common in the species.

[0095] As used herein, "normal physiological conditions" or "wild-type operating conditions" refer to those conditions that are within the normal range that would be considered at the site of administration in an individual or at the site of action in an individual: temperature, pH, osmotic pressure, osmolality, oxidative stress, and electrolyte concentrations.

[0096] The term "nucleic acid molecule" as used herein consists of at least one base or one base pair, depending on whether it is single-stranded or double-stranded, respectively. In addition, nucleic acid molecules may belong exclusively or chimerically to any group of molecules containing nucleosides, as examples but not limited to the following groups of nucleic acid molecules: RNA, DNA, genomic nucleic acids, non-genomic nucleic acids, naturally occurring and non-naturally occurring nucleic acids and synthetic nucleic acids. By way of non-limiting example, this includes nucleic acids associated with any organelle such as mitochondria, ribosomal RNA, and nucleic acid molecules composed of one or more components that are naturally occurring together with naturally occurring components.

[0097] In addition, a "nucleic acid molecule" may contain, in part, one or more non-nucleoside-based components, such as, for example, but not limited to, amino acids and carbohydrates. Thus, by way of example, but not limitation, a ribozyme is partially nucleoside-based and partially protein-based and is considered a "nucleic acid molecule."

[0098] As used herein, the term "nucleic acid sequence encoding..." or "DNA coding sequence..." or "nucleotide sequence encoding..." refers to a DNA sequence that is transcribed and translated into an enzyme when placed under the control of an appropriate regulatory sequence (such as a promoter). A "promoter" is a DNA regulatory region that is capable of binding RNA polymerase intracellularly and initiating transcription of a downstream (3' direction) coding sequence. A promoter is a portion of a DNA sequence. At the 3' end of the sequence is a start codon. The promoter sequence includes a minimum number of bases, and this composition is necessary to initiate transcription at a detectable level above background. However, after RNA polymerase binds to the sequence and transcription begins from the start codon (3' end of the promoter), transcription proceeds in the downstream 3' direction. Within the promoter sequence will be found a transcription start site (conveniently determined by the nuclease S1 map) and a protein binding domain (consensus sequence) responsible for binding RNA polymerase.

[0099] The term "oligonucleotide" (or synonymously "oligonucleotide") refers to a single-stranded polydeoxynucleotide or two complementary polydeoxynucleotide chains that can be chemically synthesized. Such synthetic oligonucleotides may or may not have a 5' phosphate. Those oligonucleotides that do not have a 5' phosphate will not be connected to another oligonucleotide in the presence of a kinase without the addition of phosphate and ATP. The synthetic oligonucleotide will be connected to the undephosphorylated fragment.

[0100] As used herein, the term "operably linked" refers to a connection between polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are generally contiguous and, where necessary to connect two protein coding regions, contiguous and in reading frame.

[0101] A coding sequence is "operably linked" to another coding sequence, and RNA polymerase transcribes the two coding sequences into one mRNA, which is then translated into a polypeptide containing amino acids from both coding sequences. A coding sequence need not be contiguous with another coding sequence as long as the expressed sequence is ultimately processed into the desired protein.

[0102] The term "parental polynucleotide combination" used herein is a combination consisting of one or more different nucleotides. Usually the word is used for the offspring polynucleotide combination preferably obtained by mutagenesis of the parental combination, in which case the terms "parent", "starter" and "template" are used interchangeably.

[0103] The term "patient" or "subject" refers to an animal, such as a mammal such as a human, that is the subject of treatment. The individual or patient may be male or female.

[0104] As used herein, the term "physiological conditions" refers to biochemical parameters such as temperature, pH, osmotic pressure, ionic strength, viscosity, etc., which are compatible with living organisms and / or are usually present in living yeast culture cells or mammalian cells. For example, the intracellular conditions of yeast cells grown under typical laboratory culture conditions are physiological conditions. Suitable in vitro reaction conditions for in vitro transcription cocktails are generally physiological conditions. Typical in vitro physiological conditions include 50-200 mM NaCl or KCl, pH 6.5-8.5, 20-45°C, and 0.001-10 mM divalent cations (e.g., Mg ++ , Ca ++); preferably about 150 mM NaCl or KCl, pH 7.2-7.6, 5 mM divalent cations, and often includes 0.01-1.0% nonspecific protein (e.g., bovine serum albumin (BSA)). Nonionic detergents (Tween, NP-40, Triton X-100) are often present, usually about 0.001-2%, typically 0.05-0.2% (v / v). The specific aqueous solution conditions are selected by the physician in a routine manner. For general guidance, the following buffered aqueous solution conditions may be applicable: 10-250 mM NaCl, 5-50 mM Tris HCl, pH 5-8, optionally with the addition of divalent cations and / or metal chelators and / or nonionic detergents and / or membrane components and / or defoamers and / or scintillants. In some embodiments, the buffer may contain at least one of BSA, carbonate, bicarbonate, chloride salts, etc. Normal physiological conditions refer to temperature, pH, osmotic pressure, osmolality, oxidative stress, and electrolyte concentrations at the site of administration or site of action in a patient or subject that are considered to be within the normal range in the patient.

[0105] Standard convention (5' to 3') is used herein to describe the sequence of double-stranded polynucleotides.

[0106] As used herein, the term "population" refers to a collection of polynucleotides, portions of polynucleotides, or polynucleotide or protein components. A "mixed population" refers to a collection of components that belong to the same nucleic acid or protein family (i.e., are related) but have different sequences (i.e., are not homologous) and thus have different biological activities.

[0107] A molecule in a "pre-pro-form" refers to a molecule that has undergone any combination of one or more covalent and non-covalent chemical modifications (e.g., glycosylation, proteolytic cleavage, dimerization or oligomerization, temperature-induced or pH-induced conformational changes, attachment to cofactors, etc.) to obtain a more mature molecular form with different properties (e.g., increased activity) than a reference precursor molecule. When two or more chemical modifications (e.g., two proteolytic cleavages or one proteolytic cleavage and one deglycosylation) can be distinguished to produce a mature molecule, the reference precursor molecule can be referred to as a "pre-pro-form" molecule.

[0108] As used herein, the term "receptor" refers to a molecule that has an affinity for a given ligand. Receptors can be naturally occurring or artificially synthesized molecules. Receptors can be used in their unchanged state or in aggregates with other species. Receptors can be covalently or non-covalently linked to a binding unit directly or through a specific binding substance. Examples of receptors include, but are not limited to, antibodies, including monoclonal antibodies and antisera that react with specific antigenic determinants (such as those located on viruses, cells or other materials), cell membrane receptors, complexes of carbohydrates and glycoproteins, enzymes and hormone receptors.

[0109] As used herein, the term "reductive reassortment" refers to the increase in molecular diversity generated by deletion (and / or insertion) events mediated by repetitive sequences.

[0110] As used herein, the term "restriction site" refers to a recognition sequence necessary for a restriction enzyme to act, including a catalytic cleavage site. It is understood that the cleavage site may or may not be contained in a portion of a restriction site that contains a low ambiguity sequence (i.e., a sequence that contains the major determinant of the frequency of occurrence of a restriction site). When it is mentioned that an enzyme (e.g., a restriction enzyme) "cuts" a polynucleotide, it is understood that the restriction enzyme catalyzes or promotes the cleavage of the polynucleotide.

[0111] The term "single-chain antibody" as used herein refers to a polypeptide comprising a VH domain and a VL domain connected by a polypeptide, the VH domain and the VL domain are usually connected by a spacer peptide, and it may contain other amino acid sequences at the amino terminal and / or carboxyl terminal. For example, a single-chain antibody may include a tether segment for connecting an encoding polynucleotide. As an example, scFv is a single-chain antibody. Single-chain antibodies are usually proteins composed of one or more polypeptide segments having at least 10 consecutive amino acids, which are substantially encoded by genes of the immunoglobulin superfamily (e.g., see The Immunoglobulin Gene Superfamily, AF Williams and A. N. Barclay, in Immunoglobulin Genes, T. Honjo, F W Alt, and THE. Rabbits, eds., (1989) Academic press: San Diego, Calif., pp. 361-368), most often encoded by rodent, non-human primate, bird, pig, cattle, sheep, goat or human heavy chain or light chain gene sequences. Functional single-chain antibodies usually contain a sufficient portion of immunoglobulin superfamily gene products to maintain the property of binding to a specific target molecule (usually a receptor or antigen (epitope)).

[0112] If they bind to each other with a higher affinity than to other nonspecific molecules, the members of a pair of molecules (e.g., antibody-antigen pairs and ligand-receptor pairs) are described as "specifically binding" to each other. For example, an antibody against an antigen binds to the antigen more effectively than a nonspecific protein and can be described as specifically binding to the antigen.

[0113] As used herein, the term "stimulation" refers to inducing a primary response by binding of a stimulatory molecule (e.g., a TCR / CD3 complex) to its cognate ligand, thereby mediating a signal transduction event, such as, but not limited to, signal transduction by a TCR / CD3 complex. Stimulation can mediate changes in the expression of certain molecules, such as downregulation of TGF-β and / or reorganization of cytoskeletal structure, etc.

[0114] As used herein, the term "stimulatory molecule" refers to a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen presenting cell.

[0115] The term "stimulatory ligand" as used herein refers to a ligand that, when present on an antigen presenting cell (e.g., a dendritic cell, a B cell, etc.), can specifically bind to a cognate binding partner on a T cell (referred to herein as a "stimulatory molecule"), thereby mediating a primary response of the T cell, including but not limited to activation, initiation of an immune response, proliferation, etc. Stimulatory ligands are well known in the art and include, among others, MHC class I molecules loaded with peptides, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0116] The term "target cell" as used herein refers to a cell that is involved in a disease and can be targeted by the genetically modified cytotoxic cells of the present invention (including but not limited to genetically modified T cells, NK cells and macrophages). Other target cells are apparent to those skilled in the art and can be used in conjunction with alternative embodiments of the present invention.

[0117] The terms "T cell" and "T lymphocyte" are interchangeable and used synonymously herein. Examples include, but are not limited to, naive T cells, central memory T cells, effector memory T cells, and combinations thereof.

[0118] As used herein, the term "transduction" refers to the use of a viral vector to introduce exogenous nucleic acid into a cell. "Transfection" as used herein refers to the use of recombinant DNA technology to introduce exogenous nucleic acid into a cell. The term "conversion" refers to the introduction of "exogenous" (i.e., external or extracellular) genes, DNA or RNA sequences into a host cell so that the host cell expresses the introduced gene or sequence to produce a desired substance, such as a protein or enzyme encoded by the introduced gene or sequence. The introduced gene or sequence may also be referred to as a "clone" or "exogenous" gene or sequence, and may include other sequences used by regulatory or control sequences, such as start sequences, termination sequences, promoter sequences, signal sequences, secretory sequences or cell genetic mechanisms. A gene or sequence may include a non-functional sequence or a sequence without known function. A host cell that receives and expresses the introduced DNA or RNA has been "converted" and is a "transformant" or "clone". The DNA or RNA introduced into a host cell may come from any source, including cells of the same genus or species as the host cell, or cells of different genus or species.

[0119] The term "treating" includes: (1) preventing or delaying the onset of clinical symptoms of a state, disease or condition in an animal that may be suffering from or susceptible to the state, disease or condition but does not yet experience or show clinical or subclinical symptoms of the state, disease or condition; (2) inhibiting the state, disease or condition (i.e., arresting, reducing or delaying the development of the disease, or maintaining the recurrence of the disease under treatment, or at least one clinical or subclinical symptom); and / or (3) alleviating the condition (i.e., causing regression of the state, disease or condition or causing regression of at least one clinical or subclinical symptom thereof). The benefit to the patient to be treated is statistically significant, or at least noticeable to the patient or physician.

[0120] As used herein, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.

[0121] As used herein, the term "tumor microenvironment" refers to any and all elements of the tumor environment, including elements that create a structural and / or functional environment for malignant processes to survive and / or expand and / or spread.

[0122] The term "variable segment" as used herein refers to a portion of a nascent peptide that includes a random, pseudo-random or determined core sequence. "Variable segment" refers to a portion of a nascent peptide that includes a random, pseudo-random or determined core sequence. The variable segment may include variable or non-variable residue positions, and the degree of residue variability at the variable residue position may be limited: both options are determined by the physician. Typically, the variable segment is about 5 to 20 amino acid residues in length (e.g., 8 to 10), although the variable segment can be longer and may include antibody portions or receptor proteins, such as antibody fragments, nucleic acid binding proteins, receptor proteins, etc.

[0123] As used herein, "vector", "cloning vector" and "expression vector" refer to vectors that can introduce polynucleotide sequences (e.g., foreign genes) into host cells in order to transform the host and promote the expression (e.g., transcription and translation) of the introduced sequences. Vectors include plasmids, bacteriophages, viruses, etc.

[0124] The term "wild type" as used herein refers to a polynucleotide that does not include any mutations. "Wild-type protein", "wild-type protein", "wild-type biological protein" or "wild-type biological protein" refers to a protein that can be separated from nature and has a certain level of activity under natural conditions and contains an amino acid sequence found under natural conditions. The terms "parent molecule" and "target protein" also refer to wild-type protein. "Wild-type protein" preferably has some desired properties (such as higher binding affinity and / or enzymatic activity), which can be obtained by screening for desired properties in a protein library, and desired properties include better stability, improved selectivity and / or solubility at different temperatures and / or pH.

[0125] The term "work", as in "work sample", for example, simply refers to a sample used for work. Likewise, a "work molecule", for example, refers to a molecule used for work.

[0126] Detailed Description

[0127] For the purpose of illustration, the principles of the present invention are described by reference to various exemplary embodiments. Although certain embodiments of the present invention are specifically described herein, it will be readily appreciated by those of ordinary skill in the art that the same principles are equally applicable and can be used for other systems and methods. Before describing the embodiments disclosed in the present invention in detail, it should be understood that the application of the present invention is not limited to the details of any particular embodiment shown. In addition, the terms used herein are for descriptive purposes, not for limiting purposes. In addition, although certain methods are described with reference to the steps presented in a particular order herein, in many cases, these steps can be performed in any order that can be understood by those skilled in the art. Therefore, the new method is not limited to the specific arrangement of the steps disclosed herein.

[0128] It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Furthermore, the terms "a," "an," "one or more," and "at least one" may be used interchangeably herein. The terms "comprising," "including," "having," and "consisting of" may also be used interchangeably.

[0129] The present invention relates to a chimeric antigen receptor (CAR) for binding to a target antigen, comprising at least one antigen-specific targeting region, the at least one antigen-specific targeting region evolving from a parent or wild-type protein or a domain thereof, and having the following properties: activity in an assay under normal physiological conditions is decreased compared to activity in an assay under abnormal conditions; a transmembrane domain; and an intracellular signaling domain. In some embodiments, the chimeric antigen receptor further comprises an extracellular spacer domain or at least one costimulatory domain. The target antigen may be a tumor-specific antigen, which may be Ax1, ROR2, or CD22.

[0130] The CAR of the present invention may have at least one of the following characteristics: (1) their affinity for the target antigen is reversibly or irreversibly reduced compared to the same CAR without the conditional active antigen-specific targeting region under normal physiological conditions, and (2) the affinity is enhanced compared to the same CAR without the conditional active antigen-specific targeting region under abnormal conditions. These CARs can direct cytotoxic cells to diseased sites where abnormal conditions exist, such as tumor microenvironments or synovial fluid. As a result of these properties, because CAR has a low affinity for normal tissues, CAR can preferentially direct cytotoxic cells to diseased sites. Such CARs can significantly reduce side effects and allow the use of higher doses of therapeutic agents to enhance therapeutic efficacy. CAR is particularly valuable for the development of new therapeutic agents that are required to stay in the individual for a short or limited time. Examples of beneficial applications include high-dose systemic treatments and high-concentration local treatments.

[0131] A chimeric antigen receptor may include an antigen-specific targeting region that has reduced binding affinity for a target antigen under normal physiological conditions compared to the antigen-specific targeting region of a parent or wild-type protein or domain thereof.

[0132] A chimeric antigen receptor may include an antigen-specific targeting region that has enhanced activity in an assay under abnormal conditions compared to the antigen-specific targeting region of a parent or wild-type protein or domain thereof, and has reduced binding affinity for a target antigen under normal physiological conditions compared to the antigen-specific targeting region of a parent or wild-type protein or domain thereof.

[0133] In any of the above chimeric antigen receptors, the antigen-specific targeting region has enhanced selectivity in an assay under abnormal conditions compared to the antigen-specific targeting region of the parent or wild-type protein or domain thereof.

[0134] In some embodiments, the ratio of the activity of the antigen-specific targeting region under abnormal conditions to the same activity under normal physiological conditions can be at least about 1.1, or at least about 1.2, or at least about 1.4, or at least about 1.6, or at least about 1.8, or at least about 2, or at least about 2.5, or at least about 3, or at least about 5, or at least about 7, or at least about 8, or at least about 9, or at least about 10, or at least about 15, or at least about 20.

[0135] The CAR molecule consists of a linker connecting two antigen-specific targeting regions ( Figure 1 ). The linker orients the two antigen-specific targeting regions in such a way that the two antigen-specific targeting regions on the CAR-T cells exhibit improved or optimized activity in binding to the target antigen (Jensen et al., "Design and implementation of adoptive therapy with chimeric antigen receptor-modified T cells," Immunol Rev., Vol. 257, pp. 127-144, 2014). Therefore, the linker is preferably capable of adopting a specific conformation that can improve or optimize the binding of the two antigen-specific targeting regions to the target antigen, thereby increasing the effectiveness of the CAR-T cells.

[0136] In some embodiments, the linker can be a Gly-Ser tandem repeat sequence of 18-25 amino acids in length (Grada, "TanCAR: A Novel Bispecific Chimeric Antigen Receptor for Cancer Immunotherapy," Molecular Therapy Nucleic Acids, Vol. 2, e105, 2013). The flexible linker can adopt many different conformations for improving or optimizing the presentation of two antigen-specific targeting regions for binding to the target antigen.

[0137] In some embodiments, the joint can adopt different conformations under normal physiological conditions and abnormal conditions. In particular, the joint has a first conformation under abnormal conditions, which is improved or optimized for presenting two antigen-specific targeting regions for binding to the target antigen, and the same joint has a second conformation under normal physiological conditions, which is less effective for presenting two antigen-specific targeting regions for binding to the target antigen compared to the first conformation of the joint under abnormal conditions. This joint may be referred to as a "conditional joint", which allows two antigen-specific targeting regions to bind to the target antigen with a higher binding activity under abnormal conditions than under normal physiological conditions. Therefore, CAR-T cells comprising such conditional joints are more active under abnormal conditions than the same CAR-T cells under normal physiological conditions.

[0138] Proteins that change conformation at different pH have been described before, for example in the literature by Di Russo et al. (“pH-Dependent conformational changes in proteins and their effect on experimental pK(a)s: the case of Nitrophorin 4,” PLoS Comput Biol., Vol. 8, e1002761, 2012). In addition, proteins with different conformations at different temperatures are described in Caldwell’s “Temperature-induced protein conformational changes in barley root plasma membrane-enriched microsomes,” Plant Physiol., Vol. 84, pp. 924-929, 1989. The conformation of antibodies affected by pH and / or temperature is discussed in Gandhi’s “Effect of pH and temperature on conformational changes of a humanized monoclonal antibody,” a master’s thesis from the University of Rhode Island, USA.

[0139] The conditional joint selected for CAR molecules falls within the scope of the present invention. The conditional joint can adopt a first conformation under abnormal conditions, which is improved or optimized for presenting two antigen-specific targeting regions for binding to the target antigen, and a second conformation is adopted under normal physiological conditions, which is suboptimal for presenting two antigen-specific targeting regions for binding to the target antigen. In some embodiments, the binding activity of the CAR molecule produced by the suboptimal conformation of the joint under normal physiological conditions to the target antigen is less than about 90% of the binding activity of the CAR molecule with the improved or optimized conformation of the joint under abnormal conditions, or about 80%, or about 70%, or about 60%, or about 50%, or about 40%, or about 30%, or about 20%, or about 10%, or about 5%.

[0140] The conditional linker can be generated from a starting linker selected from a 2A linker, a 2A-like linker, a picornavirus 2A-like linker, a 2A peptide (P2A) of porcine teschovirus, and a 2A peptide (T2A) of a beta-tetrasomal virus of a sphenotype moth, and variants and functional equivalents thereof. The starting linker is evolved to produce a mutant protein; the mutant protein is then assayed under normal physiological conditions and assayed under abnormal conditions. A protein having a conditional linker is selected from the mutant proteins, based on the selected protein exhibiting (a) a conditional linker having a first conformation under abnormal conditions, the first conformation being improved or optimized for presenting two antigen-specific targeting regions for binding to a target antigen, and (b) a second conformation of the conditional linker under normal physiological conditions, the second conformation being suboptimal for presenting two antigen-specific targeting regions for binding to a target antigen.

[0141] The CAR molecule also includes an extracellular spacer domain that connects the two antigen-specific targeting regions to a transmembrane domain that in turn connects the co-stimulatory domain and the intracellular signaling domain within the T cell ( Figure 1 ). The extracellular spacer domain is preferably capable of supporting the antigen-specific targeting region to recognize and bind to the target antigen on the target cell (Hudecek et al., "The non-signaling extracellular spacer domain of chimericantigen receptors is decisive for in vivo antitumor activity," Cancer Immunol Res., Vol. 3, pp. 125-135, 2015). In some embodiments, the extracellular spacer domain is a flexible domain, thereby allowing the antigen-specific targeting region to have a structure that optimally recognizes the specific structure and density of the target antigen on cells such as tumor cells (Hudecek et al., "The non-signaling extracellular spacer domain of chimericantigen receptors is decisive for in vivo antitumor activity," Cancer Immunol Res., Vol. 3, pp. 125-135, 2015). The flexibility of the extracellular spacer domain allows the extracellular spacer domain to adopt many different conformations.

[0142] In some embodiments, the extracellular spacer domain is able to adopt different conformations under normal physiological conditions and abnormal conditions. In particular, the extracellular spacer domain has a first conformation under abnormal conditions, which is improved or optimized for presenting two antigen-specific targeting regions for binding to the target antigen, while the same extracellular spacer domain has a second conformation under normal physiological conditions, which is suboptimal for presenting two antigen-specific targeting regions for binding to the target antigen. Such an extracellular spacer domain may be referred to as a "conditional extracellular spacer domain" because it enables the two antigen-specific targeting regions to bind to the target antigen with a higher binding activity under abnormal conditions than under normal physiological conditions. Therefore, in the case of a conditional extracellular spacer domain, CAR-T cells are more active under abnormal conditions than the same CAR-T cells under normal physiological conditions.

[0143] The conditional extracellular spacer domain selected for CAR molecules falls within the scope of the present invention. In some embodiments, the suboptimal conformation of the extracellular spacer domain under normal physiological conditions produces a CAR molecule that has less binding activity to the target antigen than about 90% of the CAR molecule with the best conformation of the same extracellular spacer domain under abnormal conditions, or about 80%, or about 70%, or about 60%, or about 50%, or about 40%, or about 30%, or about 20%, or about 10%, or about 5%.

[0144] It has been found that for the region including the extracellular spacer domain and the transmembrane domain, the anti-ubiquitination form can enhance CAR-T cell signaling and thereby enhance anti-tumor activity (Kunii et al., "Enhanced function of redirected human t cells expressing linker for activation oft cells that is resistant to ubiquitylation," Human Gene Therapy, Vol. 24, pp. 27-37, 2013). In this region, the extracellular spacer domain is outside the CAR-T cell and is therefore exposed to different conditions and may become conditionally anti-ubiquitinated.

[0145] The extracellular spacer domain is conditionally resistant to ubiquitination, which also falls within the scope of the present invention. In particular, the extracellular spacer domain of the CAR molecule has a higher ubiquitination resistance under abnormal conditions than under normal physiological conditions. Therefore, relative to their cytotoxicity under normal physiological conditions, CAR-T cells with conditionally resistant ubiquitination extracellular spacer domains will have enhanced cytotoxicity under abnormal conditions.

[0146] The conditional anti-ubiquitination extracellular spacer domain can be selected to have higher ubiquitination resistance at abnormal pH or abnormal temperature, and lower ubiquitination resistance at normal physiological pH or normal physiological temperature. In one embodiment, the conditional anti-ubiquitination extracellular spacer domain has higher ubiquitination resistance at the pH of the tumor microenvironment, and lower ubiquitination resistance at normal physiological pH, such as at pH 7.2-7.6 in human plasma.

[0147] In order to generate a conditional extracellular spacer domain, a starting protein fragment selected from an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, and a CH3 region of an antibody is evolved to generate a mutant protein. The mutant protein is measured under normal physiological conditions and measured under abnormal conditions. The conditional extracellular spacer domain is selected from a mutant protein, which shows: (a) a conditional extracellular spacer domain having a first conformation under abnormal conditions and a second conformation under normal physiological conditions, the first conformation being used to allow the antigen-specific targeting region to bind to the target antigen with a higher binding activity, the second conformation being used to allow the antigen-specific targeting region to bind to the target antigen with a lower binding activity than under abnormal conditions, or a protein, which (b) has a higher ubiquitination resistance under abnormal conditions than under normal physiological conditions.

[0148] Any of the above-described chimeric antigen receptors can be constructed such that a protein containing the antigen receptor is expressed at an increased level compared to the parent or wild-type protein or domain thereof.

[0149] In an alternative embodiment, the present invention provides a chimeric antigen receptor (CAR) for binding to a target antigen, comprising at least one antigen-specific targeting region, the at least one antigen-specific targeting region evolving from a parent or wild-type protein or its domain, and selectively enhancing compared to the antigen-specific targeting region of a parent or wild-type protein or its domain in an assay under abnormal conditions; a transmembrane domain; and an intracellular signaling domain. In some embodiments, the chimeric antigen receptor further comprises an extracellular spacer domain or at least one costimulatory domain.

[0150] The present invention also relates to a method for evolving a parent or wild-type protein or its domain to produce a conditionally active protein, wherein the conditionally active protein has at least one of the following characteristics: (a) decreased activity compared to the antigen-specific targeting region of the parent or wild-type protein or its domain in a determination under normal physiological conditions, and (b) enhanced activity compared to the antigen-specific targeting region of the parent or wild-type protein or its domain in a determination under abnormal conditions. The conditionally active protein can be engineered into a CAR.

[0151] The chimeric antigen receptor produced by this method may include an antigen-specific targeting region that has reduced binding affinity for a target antigen under normal physiological conditions compared to the antigen-specific targeting region of the parent or wild-type protein or domain thereof.

[0152] The chimeric antigen receptor produced by this method may include an antigen-specific targeting region that has enhanced activity in an assay under abnormal conditions compared to the antigen-specific targeting region of a parent or wild-type protein or domain thereof, and has reduced binding affinity for a target antigen under normal physiological conditions compared to the antigen-specific targeting region of a parent or wild-type protein or domain thereof.

[0153] In any of the above chimeric antigen receptors produced by the methods, the antigen-specific targeting region has enhanced selectivity in an assay under abnormal conditions compared to the antigen-specific targeting region of the parent or wild-type protein or domain thereof.

[0154] Any of the above chimeric antigen receptors produced by the methods described can be constructed such that a protein containing the antigen receptor is expressed at an increased level compared to the parent or wild-type protein or domain thereof.

[0155] In an optional embodiment of the method, the chimeric antigen receptor (CAR) for binding to a target antigen produced by the method comprises at least one antigen-specific targeting region, the at least one antigen-specific targeting region evolving from a parent or wild-type protein or its domain, and selectively enhancing compared to the antigen-specific targeting region of a parent or wild-type protein or its domain in an assay under abnormal conditions; transmembrane domain; and intracellular signaling domain. In some embodiments, the chimeric antigen receptor further comprises an extracellular spacer domain or at least one costimulatory domain.

[0156] Chimeric Antigen Receptor

[0157] Mammals, especially people's immune system, have cytotoxic cells for targeting and destroying diseased tissue and / or pathogens. Using these cytotoxic cells to remove unwanted tissues (i.e., target tissues) such as tumors is a promising treatment method. Other tissues that can be targeted for removal include glandular (e.g., prostate) hyperplasia, warts, and unwanted adipose tissue. However, this relatively new treatment method has only achieved limited success so far. For example, because cancer cells can adapt to new therapies to reduce the effectiveness of the therapy by reducing the expression of surface antigens, the long-term benefits of using T cells to target and destroy tumors are relatively low. Cancer cells can even dedifferentiate to escape the detection of tumor-specific T cells in response to tumor. See Maher, "Immunotherapy of Malignant Disease Using Chimeric Antigen Receptor Engrafted T Cells," ISRN Oncology, Vol. 2012, article ID 278093, 2012.

[0158] Cytotoxic cells expressing chimeric antigen receptors can significantly improve the specificity and sensitivity of these cytotoxic cells. For example, CAR-expressing T cells (CAR-T cells) can use CAR to direct T cells to target tumor cells expressing cell surface antigens that specifically bind to CAR. Such CAR-T cells can deliver cytotoxic agents more selectively to tumor cells. CAR-T cells can directly recognize target molecules and are therefore generally not limited by polymorphic presentation elements such as human leukocyte antigens (HLA). There are three advantages to this CAR targeting strategy. First, since CAR-T cell function is independent of HLA status, the same CAR-based method can in principle be used for all patients with tumors expressing the same target surface antigen. Second, the corruption of antigen processing and presentation mechanisms is a common attribute of tumor cells and can promote immune escape. However, this does not provide protection against CAR-T cells. Third, the system can be used to target a range of macromolecules, including proteins, carbohydrates, and glycolipids.

[0159] The chimeric antigen receptor of the present invention is a chimeric artificial protein comprising at least one antigen-specific targeting region (ASTR), a transmembrane domain (TM) and an intracellular signaling domain (ISD). In some embodiments, CAR may also include an extracellular spacer domain (ESD) and / or a co-stimulatory domain (CSD). See Figure 1 .

[0160] ASTR is the extracellular region of CAR, which is used to bind to specific target antigens, including proteins, carbohydrates and glycolipids. In some embodiments, ASTR comprises an antibody, particularly a single-chain antibody or a fragment thereof. ASTR may comprise a full-length heavy chain, a Fab fragment, a single-chain Fv (scFv) fragment, a bivalent single-chain antibody or a double antibody, each of which is specific to the target antigen.

[0161] ASTRs may also contain another protein functional domain to recognize and bind to a target antigen. Because a target antigen may have other biological functions, such as acting as a receptor or ligand, an ASTR may alternatively contain a functional domain for specific binding to the antigen. Some examples of proteins with functional domains include linked cytokines (which result in recognition of cells with cytokine receptors), affibodies, ligand binding domains from naturally occurring receptors, soluble protein / peptide ligands of receptors, such as on tumor cells. In fact, as will be appreciated by those skilled in the art, almost any molecule that can bind to a given antigen with high affinity can be used in an ASTR.

[0162] In one embodiment, the CAR of the present invention comprises at least two ASTRs targeting at least two different antigens or two epitopes on the same antigen. In an embodiment, the CAR comprises three or more ASTRs targeting at least three or more different antigens or epitopes. When multiple ASTRs are present in the CAR, the ASTRs can be arranged in series and can be separated by a connecting peptide ( Figure 1 ).

[0163] In one embodiment, if V alone H If the domain is sufficient to confer antigen specificity (a "single domain antibody"), then the ASTR comprises a V domain that is specific for the target antigen and has a V H domain, CH1 domain, hinge domain, and CH2 and CH3 (Fc) Ig domains. If V is required to produce fully active ASTR H Domain and V L domains, then it will contain V H The CAR and full-length lambda light chain (IgL) are introduced into the same cytotoxic cell to produce active ASTR. In another embodiment, each ASTR of the CAR comprises at least two single-chain antibody variable fragments (scFv), each specific for a different target antigen. One of the variable domains (V H or V L ) is connected to another variable domain (V L or V H) at the N-terminus without significantly disrupting antigen binding or binding specificity (Chaudhary et al., "A recombinant single-chain immunotoxin composed of anti-Tac variable regions and atruncated diphtheriatoxin," Proc. Natl. Acad. Sci., Vol. 87, p. 9491, 1990; Bedzyk et al., "Immunological and structural characterization ofa high affinity anti-fluorescein single-chain antibody," J. Biol. Chem., Vol. 265, p. 18615, 1990). These scFvs lack the constant region (Fc) present in the heavy and light chains of natural antibodies. scFvs specific for at least two different antigens are arranged in tandem. In one embodiment, the extracellular spacer domain can be connected between the ASTR and the transmembrane domain.

[0164] In another embodiment, the scFv fragment can be fused to all or part of the constant domain of the heavy chain. In another embodiment, the ASTR of the CAR comprises a bivalent single-chain variable fragment (di-scFv, bi-scFv). In a CAR comprising a di-scFV, two scFvs each having specificity for an antigen are linked together to form a variable domain having two V H area and two V LA single peptide chain of the region (Xiong et al., "Development of tumor targeting anti-MUC-1 multimer: effects of di-scFv unpaired cysteine ​​location on PEGylation and tumor binding," Protein Engineering Design and Selection, Vol. 19, pp. 359-367, 2006; Kufer et al., "A revival of bispecific antibodies," Trends in Biotechnology, Vol. 22, pp. 238-244, 2004).

[0165] In another embodiment, the ASTR comprises a diabody. In a diabody, the scFv is produced using a linker peptide that is too short for the two variable regions to fold together, allowing the scFv to dimerize. Shorter linkers (one or two amino acids) lead to the formation of trimers, so-called tribodies. Tetrabodies can also be used in ASTRs.

[0166] When two or more ASTRs are present in a CAR, the ASTRs are covalently linked to each other on a single polypeptide chain via an oligo- or polypeptide linker, an Fc hinge, or a membrane hinge region.

[0167] The antigen targeted by CAR is present on the cell surface or inside the tissue targeted for removal (e.g., tumors, glandular hyperplasia (e.g., prostate) hyperplasia, warts, and unwanted adipose tissue). Although the ASTR of CAR recognizes and binds to surface antigens more efficiently, CAR can also target intracellular antigens. In some embodiments, the target antigen is preferably specific for cancer, inflammatory diseases, neuronal diseases, diabetes, cardiovascular diseases, or infectious diseases. Examples of target antigens include antigens expressed by various immune cells, cancers, sarcomas, lymphomas, leukemias, germ cell tumors, blastomas, and cells associated with various blood diseases, autoimmune diseases, and / or inflammatory diseases.

[0168] Cancer-specific antigens targeted by ASTR include 4-IBB, 5T4, adenocarcinoma antigen, alpha-fetoprotein, Axl, BAFF, B-lymphoma cells, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extradomain B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2 / neu, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgG1, LI-CM, IL-13, IL-6, insulin-like growth factor I receptor, integrin α5β1, integrin αvβ3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-R a, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, ROR1, ROR2, SCH One or more of 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF-β2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2 or vimentin.

[0169] Inflammatory disease-specific antigens targeted by ASTR include AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (basal immunoglobulin), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (chain of IL-2 receptor), CD3, CD4, CD5, IFN-α, IFN-γ, IgE, IgE Fc region, IL-1, IL-12, IL-23, IL-13, IL-17, IL-17A, IL-22, IL-4, IL-5, IL-5, IL-6, IL-6 receptor, integrin α4, integrin α4β7, llama (Lama glama), LFA-1 (CD11a), MEDI-528, myostatin, OX-40, rhuMAb β7, scleroscin, SOST, TGFβ1, TNF-α, or VEGF-A.

[0170] The neuronal disease-specific antigens targeted by the ASTR of the present invention include one or more of amyloid beta or MABT5102A. The diabetes-specific antigens targeted by the ASTR of the present invention include one or more of L-Iβ or CD3. The cardiovascular disease-specific antigens targeted by the ASTR of the present invention include one or more of C5, cardiac myosin, CD41 (integrin α-lib), fibrin II, β chain, ITGB2 (CD18) and sphingosine-1-phosphate.

[0171] The infectious disease-specific antigens targeted by the ASTR of the present invention include one or more of anthrax toxin, CCR5, CD4, aggregation factor A, cytomegalovirus, cytomegalovirus glycoprotein B, endotoxin, Escherichia coli, hepatitis B surface antigen, hepatitis B virus, HIV-1, Hsp90, influenza A hemagglutinin, lipoteichoic acid, Pseudomonas aeruginosa, rabies virus glycoprotein, respiratory syncytial virus and TNF-α.

[0172] Other examples of target antigens include specific or amplified forms of surface proteins found on cancer cells, for example, IL-14 receptor, CD19, CD20 and CD40 of B cell lymphoma, LewisY and CEA antigens of various cancers, Tag72 antigen of breast cancer and colorectal cancer, EGF-R of lung cancer, folate binding protein and HER-2 protein commonly amplified in human breast cancer and ovarian cancer; or viral proteins, such as HIV gp120 and gp41 envelope proteins, envelope proteins from hepatitis B and C viruses, glycoprotein B and other envelope glycoproteins from human cytomegalovirus, and envelope proteins from cancer viruses such as Kaposi's sarcoma-associated herpes virus. Other potential target antigens include CD4, where the ligand is HIV gp120 envelope glycoprotein; and other viral receptors, such as ICAM, which is a receptor for human rhinovirus, and related receptor molecules for poliovirus.

[0173] In another embodiment, CAR can target an antigen engaged with cancer therapy cells (e.g., NK cells and other cells mentioned herein) to activate cancer therapy cells by acting as immune effector cells. An example of this situation is a CAR that targets CD16A antigens to fight NK cells against malignant tumors expressing CD30. Bispecific, tetravalent AFM13 antibodies are examples of antibodies that can deliver this effect. Further details of this type of embodiment can be found in, for example, the following documents: Rothe, A., et al., "A phase 1 study of the bispecific anti-CD30 / CD16A antibody construct AFM13 in patients with relapsed or refractory Hodgkin lymphoma," Blood, June 25, 2015, Vol. 125, No. 26, pp. 4024-4031.

[0174] In one embodiment, the ASTR targets a tumor-specific antigen selected from Ax1, ROR2, and CD22.

[0175] In some embodiments, ASTR is a single-chain antibody targeting cancer antigen Axl, which may have a nucleotide sequence selected from SEQ ID NO: 2-5 or an amino acid sequence selected from SEQ ID NO: 9-12. These single-chain antibodies targeting cancer antigen Axl contain a human IgG Fc region having a nucleotide sequence of SEQ ID NO: 6 or 7 or an amino acid sequence of SEQ ID NO: 13 or 14. The binding activity of these single-chain antibodies targeting cancer antigen Axl to Axl at pH 6.0 is increased compared to the same binding activity to Axl at pH 7.4.

[0176] In another embodiment, ASTR is a single-chain antibody targeting cancer antigen ROR2, which may have a nucleotide sequence of SEQ ID NO: 16 or an amino acid sequence of SEQ ID NO: 15. The single-chain antibody targeting cancer antigen ROR2 has an increased binding activity to ROR2 at pH 6.0 compared to the same binding activity to ROR2 at pH 7.4.

[0177] Single-chain antibodies targeting Ax1 or ROR2 are suitable for connection with transmembrane domains and intracellular signaling domains to generate CAR structures.

[0178] The extracellular spacer domain of CAR is a hydrophilic region between ASTR and transmembrane domain. In some embodiments, the domain is conducive to the proper protein folding of CAR. The extracellular spacer domain is an optional component of CAR. The extracellular spacer domain may include a domain selected from the Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial spacer sequence, or a combination thereof. Examples of extracellular spacer domains include CD8a hinges, artificial spacers composed of polypeptides that can be as small as three glycine (Gly), and CH1 and CH3 domains of IgG (e.g., human IgG4).

[0179] The transmembrane domain of CAR is a region that can cross the plasma membrane of cytotoxic cells. The transmembrane domain is selected from the transmembrane region of a transmembrane protein, such as a type I transmembrane protein, an artificial hydrophobic sequence, or a combination thereof. Examples of transmembrane domains include α-chains, β-chains, or ζ-chains of T cell receptors, CD8, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. The synthetic transmembrane domain may include a triplet of phenylalanine, tryptophan, and valine. Optionally, a short oligopeptide or polypeptide linker (preferably 2 to 10 amino acids in length) may form a connection between the transmembrane domain of CAR and the intracellular signaling domain. Glycine-serine dimers provide a particularly suitable linker between the transmembrane domain and the intracellular signaling domain.

[0180] The CAR of the present invention also includes an intracellular signaling domain. The intracellular signaling domain transduces effector function signals and instructs cytotoxic cells to perform their specialized functions, i.e., damage and / or destroy target cells. Examples of intracellular signaling domains include ζ chains of T cell receptor complexes or any homologs thereof, such as η chains, FcsRly and β chains, MB1 (Iga) chains, B29 (Ig) chains, etc., human CD3ζ chains, CD3 polypeptides (δ, δ and ε), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.) and other molecules involved in T cell transduction, such as CD2, CD5 and CD28. Specifically, the intracellular signaling domain can be the cytoplasmic tail region of human CD3ζ chains, FcγRIII, FcsRI, Fc receptors, cytoplasmic receptors carrying immunoreceptor tyrosine activation motifs (ITAMs) and combinations thereof.

[0181] The intracellular signaling domain used in the CAR may include several types of intracellular signaling domains of various other immune signaling receptors, including but not limited to first, second, and third generation T cell signaling proteins, including CD3 receptors, B7 family co-stimulatory receptors, and tumor necrosis factor receptor (TNFR) superfamily receptors (Park et al., "Are all chimeric antigen receptors created equal?" J Clin Oncol., Vol. 33, pp. 651-653, 2015). In addition, the intracellular signaling domain includes the signaling domain used by NK and NKT cells (Hermanson, et al., "Utilizing chimeric antigen receptors to direct natural killer cell activity," Front Immunol., Vol. 6, p. 195, 2015), such as NKp30 (B7-H6) (Zhang et al., "AnNKp30-based chimeric antigen receptor promotes T cell effector functions and antitumor efficacy in vivo," J Immunol., Vol. 189, pp. 2290-2299, 2012) and DAP12 (Topfer et al., "DAP12-based activating chimeric antigen receptor for NK cell tumor immunotherapy," J Immunol., Vol. 194, pp. 3201-3212, 2015), NKG2D, NKp44, NKp46, DAP10 and CD3z signaling domains. In addition, the intracellular signaling domain also includes the signaling domain of a human immunoglobulin receptor, which contains an immunoreceptor tyrosine-based activation motif (ITAM), such as FcgammaRI, FcgammaRIIA, FcgammaRIIC, FcgammaRIIIA, FcRL5 (Gillis et al., "Contribution of Human FcγRs to Disease with Evidence from Human Polymorphisms and Transgenic Animal Studies," Front Immunol., Vol. 5, p. 254, 2014).

[0182] In some embodiments, the intracellular signaling domain comprises a cytoplasmic signaling domain of TCR ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD5, CD22, CD79a, CD79b or CD66d. It is particularly preferred that the intracellular signaling domain in the CAR comprises a cytoplasmic signaling domain of human CD3 ζ.

[0183] The CAR of the present invention may include a co-stimulatory domain, which has the function of enhancing cell proliferation, cell survival and memory cell formation for cytotoxic cells expressing CAR. The CAR of the present invention may include one or more co-stimulatory domains, which are selected from proteins in the TNFR superfamily, CD28, CD137 (4-IBB), CD134 (OX40), Dap1O, CD27, CD2, CD7, CD5, ICAM-1, LFA-1 (CD11a / CD18), Lck, TNFR-I, PD-1, TNFR-II, Fas, CD30, CD40, ICOSLIGHT, NKG2C, B7-H3 or a combination thereof. If CAR includes more than one co-stimulatory domain, these domains can be arranged in series, optionally separated by a linker. The co-stimulatory domain is an intracellular domain that can be located between the transmembrane domain and the intracellular signaling domain in CAR.

[0184] In some embodiments, two or more components of the CAR of the present invention are separated by one or more joints. For example, in a CAR comprising at least two ASTRs, two ASTRs can be separated by a joint. A joint is an oligopeptide or polypeptide region having a length of about 1 to 100 amino acids. In some embodiments, the length of the joint can be, for example, 5-12 amino acids, 5-15 amino acids, or 5 to 20 amino acids. The joint can be composed of flexible residues such as glycine and serine so that adjacent protein domains can move freely relative to each other. Longer joints, such as joints longer than 100 amino acids, can be used in conjunction with alternative embodiments of the present invention, and such joints can be selected to, for example, ensure that two adjacent domains do not interfere with each other spatially. Examples of joints that can be used in the present invention include, but are not limited to, 2A joints (eg, T2A), 2A-like joints, or their functional equivalents.

[0185] Conditionally active antigen-specific targeting region

[0186] CAR is a chimeric protein produced by fusing all the above-mentioned different domains together to form a fusion protein. CAR is usually produced by an expression vector comprising a polynucleotide sequence encoding different domains of CAR. The ASTR of the present invention that can recognize and bind to antigens on target cells has conditional activity. Specifically, compared with the ASTR of the corresponding parent or wild-type protein, the ASTR of the present invention has less or no activity in binding to the target antigen under normal physiological conditions, and has the activity of binding to the target antigen under abnormal conditions. The present invention provides a method for producing conditionally active ASTRs from a parent or wild-type protein or its binding domain (parent or wild-type ASTR).

[0187] Wild-type proteins suitable for use in whole or in part in at least the binding domain of a target antigen (as an ASTR in the present invention) can be found by generating a protein library and screening the library to obtain proteins with the desired binding affinity to the target antigen. Wild-type proteins can be found by screening cDNA libraries. A cDNA library is a combination of cloned cDNA (complementary DNA) fragments inserted into a collection of host cells, which together constitute part of the transcriptome of an organism. cDNA is produced from fully transcribed mRNA and therefore contains the coding sequence of the expressed protein of the organism. The information in the cDNA library is a powerful and useful tool for discovering proteins with desired properties by screening the library to obtain proteins with the desired binding affinity to the target antigen.

[0188] In some embodiments, the wild-type protein is an antibody, and wild-type antibodies can be found by generating and screening antibody libraries.Antibody libraries can be polyclonal antibody libraries or monoclonal antibody libraries.Polyclonal antibody libraries for target antigens can be produced by injecting the antigen directly into an animal or by administering the antigen to a non-human animal.The antibodies thus obtained represent polyclonal antibody libraries bound to the antigen.For the preparation of monoclonal antibody libraries, any technology providing antibodies produced by continuous cell line culture can be used.Examples include hybridoma technology, tri-source hybridoma technology, human B cell hybridoma technology and EBV hybridoma technology (see, e.g., Cole (1985) in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).The technology for producing single-chain antibodies (see, e.g., U.S. Patent No. 4,946,778) can be suitable for producing single-chain antibody libraries.

[0189] There are also other methods for generating and screening antibody libraries for finding wild-type antibodies. For example, a fully human antibody display library can be utilized. Such a library is an antibody group displayed on the host cell surface. Preferably, the antibody library represents the human antibody repertoire, and they have the ability to combine a wide range of antigens. Because the antibody is displayed on the cell surface, the effective affinity (caused by avidity) of every kind of antibody in the library increases. Different from other popular library types (such as phage display libraries, in which avidity for screening and identification purposes is not required), it is necessary to provide fabulous avidity by cell surface display in the present invention. Cell surface display libraries enable identification of antibodies with low, medium and high binding affinity, and identify non-immunogenic weak epitopes in screening or selection steps.

[0190] Evolved molecules from parent molecules

[0191] A parent or wild-type protein or its binding domain (parent or wild-type ASTR) is subjected to a mutagenesis process to generate a population of mutant polypeptides, which are then screened to identify mutant ASTRs having the following properties: an enhanced binding affinity to the target antigen under abnormal conditions compared to the parent or wild-type ASTR, and optionally, substantially the same or reduced binding affinity to the target antigen under normal physiological conditions compared to the parent or wild-type ASTR.

[0192] Any chemical synthesis method or recombinant mutagenesis method can be used to produce mutant polypeptide groups. Unless otherwise indicated, the practice of the present invention can use the conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology within the technical scope of the art. Such technology is fully explained in the literature. See, e.g., Molecular Cloning A Laboratory Manual, 2nd Ed., ed. Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Vol. I and Vol. II (DN Glover ed., 1985); Oligonucleotide Synthesis (MJ Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (BD Hames & S. J. Higgins eds. 1984); Transcription And Translation (BD Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (RI Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (immobilized cells and enzymes) (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., NY); Gene Transfer Vectors For Mammalian Cells (JH Miller and MPCabs eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vol. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes 1-IV (DM Weir and CC Blackwell, eds., 1986); Manipulating the Mouse Embryo (Mouse Embryo Operation), (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986). .

[0193] The present invention provides a method for producing a nucleic acid mutant encoding a conditionally active mutant polypeptide, the method comprising: modifying the nucleic acid by (i) replacing one or more nucleotides with different nucleotides, wherein the nucleotides include natural or unnatural nucleotides, (ii) deleting one or more nucleotides, (iii) adding one or more nucleotides, or (iv) any combination thereof. In one aspect, the unnatural nucleotides include inosine. In another aspect, the method further comprises detecting the altered enzymatic activity of the polypeptide encoded by the modified nucleic acid to determine that the encoding has a modified nucleic acid with altered enzymatic activity. In one aspect, the step (a) of modification is composed of PCR, error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, cyclic ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, gene site saturation mutagenesis, ligase chain reaction, in vitro mutagenesis, ligase chain reaction, oligonucleotide synthesis, any DNA-generating technology and any combination thereof. In another aspect, the method further comprises repetition of at least one modification step.

[0194] The present invention further provides a method for generating a polynucleotide from two or more nucleic acids, the method comprising: (a) determining the identical parts and the different parts between two or more nucleic acids, wherein at least one nucleic acid comprises a nucleic acid of the present invention; (b) providing a set of oligonucleotides related to at least two sequences in the two or more nucleic acids; and (c) extending the oligonucleotides with a polymerase, thereby generating a polynucleotide.

[0195] Any mutagenesis technique can be used in various embodiments of the present invention. Stochastic or random mutagenesis exemplifies the situation where a parent molecule is mutated (modified or changed) to produce a set of progeny molecules with predetermined mutations. Thus, in an in vitro random mutagenesis reaction, for example, there is no specific predetermined product of the intended yield; the exact nature of the mutations obtained, as well as the products generated, are uncertain and therefore random. Stochastic mutagenesis occurs in methods such as error-prone PCR and random shuffling, in which the mutations obtained are random and predetermined. Variant forms can be generated by error-prone transcription such as error-prone PCR, or using a polymerase lacking error correction function (see Liao (1990) Gene 88: 107-111), a first variant form, or by replicating the first variant form in a mutagenic strain (mutator strain) (mutator host cells will be discussed in further detail below, which are generally well known). Mutagenic strains can include any mutation in any organism with impaired mismatch repair function. These mutations include mutant gene products of mutS, mutT, mutH, mutL, ovrD, dcm, vsr, umuC, umuD, sbcB, recJ, etc. The damage is obtained by adding agents such as small compounds or expressing antisense RNA or other techniques to produce gene mutations, allele replacement, selective inhibition. The damage can be a gene damage, or damage to any homologous gene in an organism.

[0196] Other mutagenesis methods include oligonucleotide directed mutagenesis, error-prone polymerase chain reaction (error-prone PCR) and cassette mutagenesis, in which specific regions of a parental polynucleotide are replaced by synthetic mutagenic oligonucleotides. In these cases, many mutation sites are generated around certain sites in the parental sequence.

[0197] In oligonucleotide directed mutagenesis, a short sequence is replaced by a synthetic mutagenic oligonucleotide. In oligonucleotide directed mutagenesis, a short sequence of a polynucleotide is removed from a polynucleotide using restriction enzyme digestion, and it is replaced by a synthetic polynucleotide in which multiple bases are changed relative to the starting sequence. The polynucleotide sequence can also be changed by chemical mutagenesis. Chemical mutagens include, for example, sodium bisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid. Other agents of analogs of nucleotide precursors include nitrosoguanidine, 5-bromouracil, 2-aminopurine or acridine. In general, these agents are added to the PCR reaction to replace the nucleotide precursors, thereby mutating the sequence. Inserting agents such as proflavin, acriflavine, quinacrine, etc. can also be used. Random mutations of polynucleotide sequences can also be obtained by X-ray or ultraviolet irradiation. In general, the plasmid polynucleotides so mutated are introduced into Escherichia coli and propagated as a pool or library of hybrid plasmids.

[0198] Error-prone PCR uses low-fidelity polymerization conditions to introduce low levels of random point mutations over long sequences. In a mixture of fragments of unknown sequence, error-prone PCR can be used to mutagenize the mixture.

[0199] In cassette mutagenesis, a single template sequence block is usually replaced by a (partially) random sequence. Reidhaar-Olson JF and Sauer RT: Combinatorial cassette mutagenesis as a probe of the informational content of protein sequences; Science 241(4861):53-57,1988.

[0200] Alternatively, any non-stochastic or non-random mutagenesis techniques can be used in various embodiments of the present invention. Examples of non-stochastic mutagenesis are where the parental molecule is mutated (modified or altered) to produce offspring molecules with one or more predetermined mutations. It is understood that the reality is that there is a certain amount of background product in many reactions where molecular processing occurs, and the presence of these background products does not affect the non-stochastic nature of the mutagenesis method with a predetermined product. Site-directed saturation mutagenesis and synthetic ligation recombination are examples of mutagenesis techniques where the exact chemical structure of the target product is predetermined.

[0201] A method for site-directed saturation mutagenesis is disclosed in U.S. Patent Application Publication No. 2009 / 0130718. The method provides a set of degenerate primers corresponding to codons of a template polynucleotide, and performs polymerase extension to generate polynucleotide progeny containing sequences corresponding to the degenerate primers. The polynucleotide progeny can be expressed and screened for directed evolution. Specifically, this is a method for generating a set of progeny polynucleotides, comprising the steps of (a) providing copies of template polynucleotides, each containing a plurality of codons encoding a template polypeptide sequence; and (b) performing the following steps on the codons of each template polynucleotide: (1) providing a set of degenerate primers, wherein each primer contains a degenerate codon corresponding to a codon of the template polynucleotide and at least one adjacent sequence homologous to an adjacent sequence of the template polynucleotide codon; (2) providing conditions for the primers to anneal to the copies of the template polynucleotide; and (3) performing a polymerase extension reaction starting from the primers along the template; thereby generating polynucleotide progeny, each polynucleotide progeny containing a sequence corresponding to a degenerate codon of the annealed primer; thereby generating a set of polynucleotide progeny. Site-saturation mutagenesis involves the directed evolution of nucleic acids and screening of clones containing the evolved nucleic acids for target binding activity.

[0202] Site-directed saturation mutagenesis generally involves the method of 1) preparing molecular progeny (including molecules consisting of polynucleotide sequences, molecules consisting of polypeptide sequences, and molecules consisting of partial polynucleotide sequences and partial polypeptide sequences) from one or more ancestral or parental templates, mutagenized to obtain at least one point mutation, addition, deletion and / or chimera; 2) screening the progeny molecules - preferably using high-throughput methods - to obtain the desired binding affinity for the target antigen; 3) optionally obtaining and / or cataloging structural and / or functional information related to the parental and / or progeny molecules; and 4) optionally repeating any of steps 1) - step 3).

[0203] In site-directed saturation mutagenesis, polynucleotide progeny, referred to as "codon-directed saturation mutagenesis," are generated (e.g., from a parent polynucleotide template), each polynucleotide progeny containing a set of at least up to three consecutive point mutations (i.e., different bases containing new codons) such that each codon (or each degenerate codon family encoding the same amino acid) is represented at each codon position. Corresponding to and encoded by the polynucleotide progeny, a set of polypeptide progeny are also generated, each having at least one individual amino acid point mutation. In a preferred aspect, so-called "amino acid-directed saturation mutagenesis," a mutant polypeptide, each of 19 naturally encoded polypeptides, is generated, forming an α-amino acid substitution at each amino acid position along the polypeptide. This generates (at each amino acid position along the parent polypeptide) a total of 20 different polypeptide progeny, including the original amino acid, or potentially ≥ 21 different polypeptide progeny, if other amino acids are used to replace or add to the 20 naturally encoded amino acids.

[0204] Other mutagenesis techniques may also be used, including recombination and more specifically methods of preparing polynucleotides encoding polypeptides by in vivo shuffling of polynucleotide sequences containing regions of partial homology, recombining the polynucleotides to generate at least one polynucleotide, and screening the polynucleotides to produce polypeptides having useful properties.

[0205] On the other hand, mutagenesis techniques exploit the natural properties of cells to recombine molecules and / or mediate a reduction process that reduces sequence complexity and the extent of repetitive or continuous sequences having regions of homology.

[0206] Various mutagenesis techniques, used alone or in combination, provide methods for preparing hybrid polynucleotides encoding hybrid polypeptides of biological activity. In achieving these and other objectives, according to one aspect of the invention, a method is provided for introducing a polynucleotide into a suitable host cell and growing the host cell under conditions that produce a hybrid polypeptide.

[0207] A chimeric gene is generated by joining two polynucleotide fragments using compatible sticky ends generated by restriction enzymes, wherein each fragment is derived from a separate ancestral (or parental) molecule. Another example is the mutagenesis (i.e., obtaining a codon substitution, addition, or deletion) of a single codon position of a parental polynucleotide to produce a single polynucleotide progeny encoding a single site-mutagenized polypeptide.

[0208] In addition, in vivo site-specific recombination systems have been used to generate genetic hybrids, as well as random methods in vivo recombination and recombination between homologous but truncated genes in plasmids. Mutagenesis by overlap extension and PCR has also been reported.

[0209] Non-stochastic methods have been used to obtain large numbers of point mutations and / or chimerizations, for example, comprehensive or exhaustive methods have been used to generate all molecules in a particular mutation group, with functions attributed to specific structural groups in the template molecule (e.g., specific single amino acid positions or sequences consisting of two or more amino acid positions) for classification and comparison of specific mutation groups.

[0210] Any of these or other evolutionary methods can be used in the present invention to generate new populations (libraries) of mutant polypeptides from a parent or wild-type protein.

[0211] Expression of Evolved Molecules

[0212] The mutant polynucleotides produced from the evolution step can be size-graded on an agarose gel according to a disclosed protocol or can be inserted into an expression vector without this step, and transfected into a suitable host cell to produce a mutant polypeptide (expression). The expression can use conventional molecular biological techniques. Therefore, the expression step can use various known methods.

[0213] For example, in brief, standard molecular biological techniques are then used to digest the mutant polynucleotides produced in the evolution step and connected to an expression vector, such as plasmid DNA. The vector is then transformed into bacteria or other cells using standard methods (protocol). High throughput expression and screening can be performed in a single well of a multi-well plate, such as a 96-well plate. The method is repeated to obtain each mutant polynucleotide.

[0214] The polynucleotides screened and separated are introduced into suitable host cells. Suitable host cells are any cells that can promote recombination and / or reduction recombination. The polynucleotides screened are preferably present in a vector that includes appropriate regulatory sequences. The host cell can be a higher eukaryotic cell such as a mammalian cell, or a lower eukaryotic cell such as a yeast cell, or preferably, the host cell can be a prokaryotic cell such as a bacterial cell. Introducing the construct into the host cell can be calcium phosphate transfection, DEAE-dextran mediated transfection or electroporation (e.g., Ecker and Davis, 1986, Inhibition of gene expression in plant cells by expression of antisense RNA, Proc Natl Acad Sci USA, 83:5372-5376).

[0215] As representative examples, expression vectors that can be used include viral particles, baculovirus, phage, plasmid, phagemid, cosmid, fosmids, bacterial artificial chromosomes, viral DNA (such as vaccinia virus, adenovirus, fowl pox virus, pseudorabies and derivatives of SV40), P1-based artificial chromosomes, yeast plasmids, yeast artificial chromosomes and any other specific target host (such as Bacillus, Aspergillus niger and yeast). Thus, for example, DNA can be included in any expression vector for expressing a polypeptide. Such vectors include chromosomal, non-chromosomal and artificially synthesized DNA sequences. A large number of suitable vectors are well known in the art and are commercially available. The following vectors are provided by way of example; bacteria: pQE vector (Qiagen), pBluescript plasmid, pNH vector, λ-ZAP vector (Stratagene); ptrc99a, pKK223-3, pDR540, pRIT2T (Pharmacia); eukaryotic: pXT1, pSG5 (Stratagene), pSVK3, pBPV, PMSG, pSVLSV40 (Pharmacia). However, any other plasmid or other vector may be used as long as they are replicable and viable in the host. Low copy number or high copy number vectors may be used in the present invention.

[0216] The mutant polynucleotide sequence in the expression vector is operably connected to an appropriate expression control sequence (promoter) to guide the synthesis of RNA. Specially named bacterial promoters include lacI, lacZ, T3, T7, gpt, λPR, PL and trp. Eukaryotic promoters include CMV early (immediate early), HSV thymidine kinase, early and late SV40, LTR from retrovirus and mouse metallothionein-1. The selection of suitable vectors and promoters is the ordinary technical level of this area. The expression vector also contains ribosome binding sites for translation initiation and transcription termination. The vector also includes appropriate sequences for amplification of expression. Using chloramphenicol transferase (CAT) vectors or other vectors with selection markers, the promoter region can be screened out from any target gene. In addition, it is preferred that the expression vector contains one or more selection marker genes to provide phenotypic traits for screening transformed host cells, such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or tetracycline or ampicillin resistance in Escherichia coli.

[0217] Can increase eukaryotic DNA transcription by inserting enhancer sequence into expression vector.Enhancer is the cis-acting sequence of 10-300bp that enhances transcription by promoter.When in the 5' or 3' direction of transcription unit, enhancer can effectively enhance transcription.If be located in intron or in coding sequence itself, enhancer is also effective.Usually use viral enhancer, comprise SV40 enhancer, cytomegalovirus enhancer, polyoma enhancer and adenovirus enhancer.Enhancer sequence from mammal system is also commonly used, for example mouse immunoglobulin heavy chain enhancer.

[0218] Mammalian expression vector systems also typically include a selective marker gene. Examples of suitable markers include dihydrofolate reductase gene (DHFR), thymidine kinase gene (TK) or prokaryotic genes that confer drug resistance. The first two marker genes preferably use mutant cell lines that lack the ability to grow without adding thymidine to the growth medium. The transformed cells can then be identified by the ability of the transformed cells to grow on non-supplemented medium. Examples of prokaryotic drug resistance genes used as markers include genes that confer resistance to G418, mycophenolic acid and hygromycin.

[0219] Depending on the type of cell production host, the expression vector containing the target DNA segment can be transferred into the host cell by known methods. For example, calcium chloride transfection is commonly used for prokaryotic host cells, while calcium phosphate treatment, lipofection or electroporation can be used for eukaryotic host cells. Other methods for transforming mammalian cell production hosts include the use of polybrene, protoplast fusion, liposomes, electroporation and microinjection (see generally Sambrook et al., supra).

[0220] Once the expression vector has been introduced into a suitable host, the host is maintained under conditions suitable for high-level expression of the introduced mutant polynucleotide sequence to produce mutant polypeptides. These expression vectors are usually reproducible in host organisms as episomes or as components of the host chromosomal DNA. Typically, the expression vector will contain a selective marker, such as tetracycline or neomycin, to allow detection of those cells transformed with the desired DNA sequence (see, for example, U.S. Patent number 4,704,362).

[0221] Therefore, in another aspect of the present invention, mutant polynucleotides can be produced by the method of reduction recombination. The method comprises preparing a construct containing a continuous sequence (original coding sequence), inserting them into a suitable vector, and subsequently introducing them into a suitable host cell. The rearrangement of individual molecular identification (molecular identity) is produced by a combinatorial method between the continuous sequences in the construct with homology regions or between quasi-repeated units. The rearrangement method reorganizes and / or reduces the complexity and scale of the repetitive sequence, and produces new molecules. Various treatments can be used to enhance the recombination rate. These may include using ultraviolet light or chemicals that destroy DNA and / or using a host cell that displays a higher level of "genetic instability". Thus, the recombination method may include homologous recombination or the natural properties of the quasi-repetitive sequence that guides self evolution.

[0222] The cells are then propagated and "reductive recombination" is performed. If necessary, the reductive recombination rate can be stimulated by introducing DNA damage. In vivo recombination focuses on "intermolecular" methods, which are generally regarded as "RecA-dependent" phenomena in bacteria. The present invention relies on the recombination of host cells to reorganize and rearrange sequences, or relies on the ability of cells to mediate the reductive process to reduce the complexity of quasi-repeated sequences in cells through deletions. This "reductive recombination" process occurs through an "intramolecular", RecA-independent method. The end result is the recombination of molecules into all possible combinations.

[0223] In one aspect, the host organism or cell comprises a gram-negative bacterium, a gram-positive bacterium or a eukaryotic organism. In another aspect of the invention, the gram-negative bacterium comprises Escherichia coli or Pseudomonas fluorescens. In another aspect of the invention, the gram-positive bacterium comprises Streptomyces diversa, Lactobacillus gasseri, Lactococcus lactis, Lactococcus cremoris or Bacillus subtilis. In another aspect of the invention, the eukaryotic organism comprises Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Kluyveromyces lactis, Hansenula or Aspergillus niger. As representative examples of suitable hosts, mention may be made of: bacterial cells, such as Escherichia coli, Streptomyces, Salmonella typhimurium; fungal cells, such as yeast; insect cells, such as Drosophila S2 and Spodoptera Sf9; animal cells, such as CHO, COS or Bowes melanoma; adenovirus and plant cells. The selection of an appropriate host is deemed to be within the scope of those skilled in the art.

[0224] In addition to eukaryotic microorganisms such as yeast, mammalian tissue cell cultures can also be used to express the mutant polypeptides of the present invention (see Winnacker, "From Genes to Clones," VCH Publishers, NY, NY (1987)). Eukaryotic cells are preferred because many suitable host cell lines capable of secreting complete immunoglobulins have been developed in the art, including CHO cell lines, various COS cell lines, HeLa cells, myeloma cell lines, B cells or hybridomas. The expression vectors of these cells may include expression control sequences, such as replication origins, promoters, enhancers (Queen et al., Immunol. Rev., Vol. 89, p. 49, 1986) and necessary processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites and transcription terminator sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, cytomegalovirus, SV40, adenovirus, bovine papilloma virus, etc.

[0225] In one embodiment, the eukaryotic host cell is selected from CHO, HEK293, IM9, DS-1, THP-1, Hep G2, COS, NIH 3T3, C33a, A549, A375, SK-MEL-28, DU 145, PC-3, HCT 116, MiaPACA-2, ACHN, Jurkat, MM1, Ovcar 3, HT 1080, Panc-1, U266, 769P, BT-474, Caco-2, HCC 1954, MDA-MB-468, LnCAP, NRK-49F and SP2 / 0 cell lines; and mouse spleen cells and rabbit PBMC. In one aspect, the mammalian host cell is selected from CHO or HEK293 cell lines. In a specific aspect, the mammalian host cell is a CHO-S cell line. In another specific aspect, the mammalian system is a HEK293 cell line. In another embodiment, the eukaryotic host is a yeast cell system. In one aspect, the eukaryotic host is selected from Saccharomyces cerevisiae cells or Pichia pastoris cells.

[0226] In another embodiment, mammalian host cells can be created commercially by a contract research or custom manufacturing organization. For example, for recombinant antibodies or other proteins, the GS gene expression system is available from Lonza (Lonza Group GmbH, Basel, Switzerland). TM The technology uses CHOK1SV or NS0 cell production hosts to produce vectors expressing these products. Host cells containing the target polynucleotides can be cultured in conventional nutrient media modified to activate promoters, screen transformants or amplify genes. The culture conditions, such as temperature, pH, etc., are those previously used to screen host cell expression, which are obvious to those skilled in the art.

[0227] As described above, optimization of expression of conditionally active ASTRs can be achieved by optimization of the vector used (vector components, such as promoters, splice sites, 5' and 3' ends and flanking sequences), genetic modification of host cells to reduce gene deletions and rearrangements, evolution of host cell gene activity by methods of evolving related genes in vivo or in vitro, optimization of host glycosylation enzymes by evolution of related genes, and / or selection of cells with enhanced expression capacity by host cell mutagenesis and selection strategies at the chromosomal level.

[0228] Protein expression can be induced by various known methods, and many genetic systems have been disclosed for inducing protein expression. For example, using a suitable system, the addition of an inducer can induce protein expression. The cells are then centrifuged and the supernatant is discarded. The cells are incubated with DNA enzymes, RNA enzymes, and lysozymes to enrich the periplasmic proteins. After centrifugation, the supernatant containing the new protein is transferred to a new multi-well plate and stored before detection.

[0229] Cells are generally collected by centrifugation, destroyed by physical or chemical methods, and the resulting crude extract is used for further purification. Microbial cells used to express proteins can be destroyed by any convenient method, including freeze-thaw cycles, ultrasound, mechanical disruption or the use of cell lysates. Such methods are well known to those skilled in the art. The expressed polypeptide or fragment thereof can be recovered and purified from recombinant cell cultures using methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography. If necessary, a protein renaturation step can be used to complete the configuration of the polypeptide. If necessary, high performance liquid chromatography (HPLC) can be used as the final purification step. Screening for conditionally active ASTRs can be assisted by using convenient high-throughput screening or selection methods. Cell surface display expression and screening techniques (e.g., as defined above) can be used to screen mutant proteins to obtain conditionally active ASTRs.

[0230] Screening to identify reversible or irreversible mutants

[0231] Identification of target molecules is primarily accomplished directly by measuring protein activity under permissive and wild-type conditions. Mutants with the greatest activity ratio (permissive / wild-type) can then be screened and permutations of point mutations generated by combinations of single mutations using standard methods. This combinatorial permutation protein library is then screened for proteins that show the greatest difference in activity between permissive and wild-type conditions.

[0232] The activity of the supernatant can be screened in a variety of ways, such as high-throughput activity assays, such as fluorescence detection, to identify protein mutants that are sensitive to desired features (temperature, pH, etc.). For example, to screen temperature-sensitive mutants, commercially available substrates are used to measure the enzyme activity or antibody activity of each mutant at a lower temperature (such as 25°C) and the temperature at which the original protein functions (such as 37°C). Screening can be performed in a variety of culture media, such as serum and BSA. The reaction can initially be in the form of a multi-well assay, such as a 96-well assay, and validated in a different format, such as a 14 ml tube.

[0233] In one aspect, the method further comprises modifying at least one nucleic acid or polypeptide prior to determining the candidate polypeptide having conditional biological activity. In another aspect, the determining step (c) further comprises: detecting increased expression of the polypeptide in the host cell or host organism. In another aspect, the determining step (c) further comprises: detecting enzyme activity in a pH range of about pH 3 to about pH 12. In another aspect, the determining step (c) further comprises: detecting enzyme activity in a pH range of about pH 5 to about pH 10. In another aspect, the determining step (c) further comprises: detecting enzyme activity in a pH range of about pH 6 to about pH 8. In another aspect, the determining step (c) further comprises: detecting enzyme activity at pH 6.7 and pH 7.5. In another aspect, the determining step (c) further comprises: detecting enzyme activity in a temperature range of about 4°C to about 55°C. In another aspect, the determining step (c) further comprises: detecting enzyme activity in a temperature range of about 15°C to about 47°C. In another aspect, the determining step (c) further comprises: detecting enzyme activity in a temperature range of about 20°C to about 40°C. In another aspect, the determination step (c) further comprises: detecting the enzyme activity at a temperature range of about 25°C to about 37°C. In another aspect, the determination step (c) further comprises: detecting the enzyme activity under conditions of normal osmotic pressure and abnormal (positive or negative) osmotic pressure. In another aspect, the determination step (c) further comprises: detecting the enzyme activity under conditions of normal electrolyte concentration and abnormal (positive or negative) electrolyte concentration. The tested electrolyte concentration is selected from one of calcium, sodium, potassium, magnesium, chloride, bicarbonate and phosphate concentrations. In another aspect, the determination step (c) further comprises: detecting the enzyme activity that forms a stable reaction product.

[0234] In another aspect, the present invention provides purified antibodies that specifically bind to a polypeptide having enzymatic activity of the present invention or a fragment thereof. In one aspect, the present invention provides antibody fragments that specifically bind to a polypeptide having enzymatic activity.

[0235] Antibodies and Antibody-Based Screening Methods

[0236] The present invention provides isolated or recombinant antibodies that specifically bind to enzymes of the present invention. These antibodies can be used to separate, identify or quantify enzymes of the present invention or related polypeptides. These antibodies can be used to separate other polypeptides or other related enzymes within the scope of the present invention. The antibodies can be designed to bind to the active site of the enzyme. Thus, the present invention provides methods for inhibiting enzymes using the antibodies of the present invention.

[0237] The antibodies can be used for immunoprecipitation, staining, immunoaffinity columns, etc. If necessary, the generation of nucleic acid sequences encoding specific antigens can be carried out by separating, amplifying or cloning polypeptides or nucleic acids after immunization and fixing the polypeptides to the array of the present invention. Alternatively, the method of the present invention can be used to modify the structure of antibodies produced by cells to be modified, for example, the affinity of the antibodies can be increased or decreased. Moreover, the ability to prepare or modify antibodies can be the phenotype of cells transformed by the method of the present invention.

[0238] Immunization methods, methods of preparing and isolating antibodies (polyclonal and monoclonal) are known to those skilled in the art and are described in the scientific and patent literature, see, for example, Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley / Greene, NY (1991); Stites (eds.) BASIC AND CLINICAL IMMUNOLOGY (7th ed.) Lange Medical Publications, Los Altos, Calif. ("Stites"); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2nd ed.) Academic Press, New York, NY (1986); Kohler (1975) "Continuous cultures of fused cells secreting antibody of predefined specificity", Nature 256:495; Harlow (1988) Antibodies, A LABORATORY MANUAL, Cold Spring Harbor Publications, New York. In addition to traditional methods in animals, antibodies can also be produced in vitro, for example, using recombinant antibody binding site expression phage display libraries. See, for example, Hoogenboom (1997) "Designing and optimizing library selection strategies for generating high-affinity antibodies", Trends Biotechnol. 15:62-70; and Katz (1997) "Structural and mechanistic determinants of affinity and specificity of ligands discovered or engineered by phage display", Annu. Rev. Biophys. Biomol. Struct. 26:27-45.

[0239] The polypeptide or peptide can be used to prepare antibodies that specifically bind to a polypeptide, such as an enzyme of the present invention. The resulting antibodies can be used in immunoaffinity chromatography to separate or purify a polypeptide or to determine whether a polypeptide is present in a biological sample. In this method, a protein reagent, such as an extract or a biological sample, is contacted with an antibody that can specifically bind to a polypeptide of the present invention.

[0240] In immunoaffinity methods, the antibody is attached to a solid support, such as beads or other columnar matrices. The protein reagent is contacted with the antibody under conditions in which the antibody can specifically bind to a polypeptide of the invention. Non-specifically bound proteins are removed after washing, and the specifically bound polypeptide is eluted.

[0241] The ability of a protein in a biological sample to bind to an antibody can be determined by any method familiar to those skilled in the art. For example, binding can be determined by labeling the antibody with a detectable marker, such as a fluorescent agent, an enzyme label, or a radioisotope. Alternatively, the ability of an antibody to bind to a sample can be determined using a secondary antibody with a detectable marker. Specific tests include ELISA, double antibody sandwich, radioimmunoassay, and Western blotting.

[0242] The polyclonal antibodies generated against the polypeptides of the present invention can be obtained by injecting the polypeptides directly into animals or by administering the polypeptides to non-human animals. The antibodies obtained then bind to the polypeptides themselves. In this manner, even sequences encoding only fragments of the polypeptides can be used to generate antibodies that can bind to the entire native polypeptide. The antibodies can then be used to isolate the polypeptides from cells expressing the polypeptides.

[0243] To prepare monoclonal antibodies, any technique that produces antibodies by continuous cell line culture can be used. Examples include hybridoma technology. For the preparation of monoclonal antibodies, any technique that provides antibodies produced by continuous cell line culture can be used. Examples include hybridoma technology, trioma technology, human B cell hybridoma technology, and Epstein-Barr virus hybridoma technology (see, e.g., Cole (1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

[0244] The techniques described for producing single-chain antibodies (see, e.g., U.S. Pat. No. 4,946,778) can be applied to produce single-chain antibodies that act on the polypeptides of the present invention. Alternatively, transgenic mice can be used to express humanized antibodies that act on these polypeptides or fragments thereof. The antibodies produced that act on the polypeptides of the present invention can be used to screen similar polypeptides (e.g., enzymes) from other organisms and samples. In these techniques, polypeptides from an organism are contacted with antibodies, and those polypeptides that specifically bind to the antibodies are detected. Any of the methods described above can be used to detect antibody binding.

[0245] Screening methods and "online" detection devices

[0246] In the operation of the method of the present invention, a variety of instruments and methods can be used for the polypeptides and nucleic acids of the present invention, for example, to screen polypeptides having enzymatic activity, to screen compounds that serve as potential regulators, such as activators or inhibitors of enzyme activity, to screen antibodies that bind to the polypeptides of the present invention, to screen nucleic acids that hybridize with the nucleic acids of the present invention, to screen cells that express the polypeptides of the present invention, etc.

[0247] Array or "biochip"

[0248] The nucleic acid or polypeptide of the present invention can be fixed to or applied to an array. The array can be used to screen or monitor a library of compositions (e.g., small molecules, antibodies, nucleic acids, etc.) for their ability to bind to or regulate the activity of the nucleic acid or polypeptide of the present invention. For example, in one aspect of the present invention, the parameter monitored is the transcriptional expression of an enzyme gene. One or more or all transcripts of a cell can be determined by hybridization of a sample containing a cell transcript, or a nucleic acid representative of a cell transcript, or its complementary nucleic acid, by hybridization with a nucleic acid fixed on an array or "biochip". By using a nucleic acid "array" on a microchip, some or all cell transcripts can be quantified simultaneously. Alternatively, an array containing genomic nucleic acids can also be used to determine the genotype of a cell strain newly transformed by the method of the present invention. Polypeptide "arrays" can also be used to quantify multiple proteins simultaneously. The present invention can operate using any known "array", also referred to as a "microarray" or "nucleic acid array" or "polypeptide array" or "antibody array" or "biochip" or variants thereof. Typically, an array is a large number of "spots" or "target elements", each target element comprising a defined amount of one or more biomolecules, e.g., oligonucleotides, immobilized on a specific area of ​​a substrate surface for specific binding to a sample molecule, e.g., mRNA transcript.

[0249] In the practice of the methods of the present invention, any known arrays and / or methods of making and using arrays or their variations may be incorporated herein in whole or in part, as described, for example, in U.S. Pat. Nos. 6,277,628, 6,277,489, 6,261,776, 6,258,606, 6,054,270, 6,048,695, 6,045,996, 6,022,963, 6,013,440, 5,965,452, 5,959,098, 5,856,174, 5,830,645, 5,770,456, 5,632,957, 5,556,752, 5,143,854, 5,807,522, 5,800,992, 5,744,305, 5,700,637, 5,556,752, 5,434,049; see also, for example, WO 99 / 51773, WO 99 / 09217, WO 97 / 46313, WO 96 / 17958; see also, for example, Johnston (1998) "Gene chips: Array of hope for understanding gene regulation", Curr. Biol.8: R171-R174; Schummer (1997) "Inexpensive Handheld Device for the Construction of High-Density Nucleic Acid Arrays", Biotechniques 23: 1087-1092; Kern (1997) "Direct hybridization of large-insert genomic clones on high-density gridded cDNA filter arrays", Biotechniques 23: 120-124; Solinas-Toldo (1997) "Matrix-Based Comparative Genomic Hybridization: Biochips to Screen for Genomic Imbalances", Imbalances), Genes, Chromosomes & Cancer 20: 399-407; Bowtell (1999) "Options Available--From Start to Finish--for Obtaining Expression Data by Microarray", Nature Genetics Supp. 21: 25-32. See also published U.S. patent applications Nos. 20010018642, 20010019827, 20010016322, 20010014449, 20010014448, 20010012537, 20010008765.

[0250] Capillary array

[0251] Capillary arrays, such as GIGAMATRIX TMDiversa Corporation, San Diego, Calif can be used in the method of the present invention. The nucleic acid or polypeptide of the present invention can be fixed to or applied on an array, including a capillary array. The array can be used to screen or monitor a library of compositions (e.g., small molecules, antibodies, nucleic acids, etc.) for their ability to bind to or modulate the activity of the nucleic acid or polypeptide of the present invention. Capillary arrays provide another system for capturing and screening samples. For example, a sample screening device can include a large number of capillaries forming an adjacent capillary array, wherein each capillary includes at least one wall that defines a cavity for retaining the sample. The device can further include an interstitial material placed between adjacent capillaries of the array, and one or more reference markers are formed in the interstitial material. Capillaries for screening samples, wherein the capillaries are suitable for being incorporated into a capillary array, can include a first wall, a cavity defined by the cavity for retaining the sample, and a second wall, which is formed by a filter material, and the filtered excitation energy is provided to the cavity to excite the sample. A polypeptide or nucleic acid, such as a ligand, can be introduced into a first component into a portion of at least one capillary of the capillary array. Each capillary of the capillary array may include at least one wall, the cavity defined by which is used to retain the first component. Bubbles may be introduced into the capillary behind the first component. The second component may be introduced into the capillary, wherein the second component is separated from the first component by bubbles. The target sample may be introduced into the capillary of the capillary array as a first liquid labeled with detectable particles, wherein each capillary of the capillary array includes at least one wall, the cavity defined by which is used to retain the first liquid and detectable particles, wherein at least one wall is coated with a binding substance for making detectable particles bonded to at least one wall. The method may further include removing the first liquid from the capillary, wherein the combined detectable particles are retained in the capillary, and introducing the second liquid into the capillary. The capillary array may include many single capillaries containing at least one outer wall defining the cavity. The outer wall of the capillary may be one or more walls fused together. Similarly, the cavity defined by the wall may be cylindrical, square, hexagonal or any other geometric shape, as long as the wall forms a cavity retaining the liquid or sample. The capillaries of the capillary array may be close together to form a planar structure. The capillaries can be joined together by fusion (e.g., where the capillaries are made of glass), bonding, bonding, or sidewall-to-sidewall clamping. Capillary arrays can consist of any number of individual capillaries, for example, ranging from 100 to 4,000,000 capillaries. Capillary arrays can consist of about 100,000 or more individual capillaries joined together to form a microtiter plate.

[0252] Engineered conditionally active antibodies

[0253] Conditionally active antibodies can be engineered to produce multispecific conditionally active antibodies. Multispecific antibodies can be antibodies with multiple epitope specificities, as described in WO 2013 / 170168. Multispecific antibodies include, but are not limited to, heavy chain variable domains (V H ) and the light chain variable domain (V L ) of antibodies, wherein V H V L Unit has multiple epitope specificity; has two or more V L and V H domain antibody, wherein each V H V L units bind different epitopes; antibodies with two or more single variable domains, wherein each single variable domain binds a different epitope; antibodies comprising one or more antibody fragments; and antibodies comprising antibody fragments that have been covalently or non-covalently linked.

[0254] In order to construct multispecific antibodies including bispecific antibodies, antibody fragments with at least one free thiol group are obtained. Antibody fragments can be obtained from full-length conditionally active antibodies. Conditionally active antibodies can be enzymatically digested to produce antibody fragments. Exemplary enzyme digestion methods include, but are not limited to, pepsin, papain, and Lys-C. Exemplary antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, diabodies (Db); tandem diabodies (taDb), linear antibodies (see U.S. Patent No. 5,641,870; Zapata et al., Protein Eng., Vol. 8, pp. 1057-1062, (1995)); one-armed antibodies, single variable domain antibodies, miniantibodies (Olafsen et al. (2004) Protein Eng. Design & Sel., Vol. 17, pp. 315-323), single-chain antibody molecules, fragments produced by Fab expression libraries, anti-idiotype (anti-Id) antibodies, complementary determining regions (CDRs), and epitope binding fragments. Antibody fragments can also be produced using DNA recombinant technology. The DNA encoding the antibody fragment can be cloned into a plasmid expression vector or a phagemid vector and directly expressed in E. coli. Antibody enzyme digestion methods, DNA cloning and recombinant protein expression methods are well known to those skilled in the art.

[0255] Antibody fragments can be purified using conventional techniques and can be reduced to produce free thiol groups. Antibody fragments with free thiol groups can react with cross-linking agents such as bismaleimide. Such cross-linked antibody fragments are purified and then reacted with a second antibody fragment with free thiol groups. The end product of the cross-linking of two antibody fragments is purified. In certain embodiments, each antibody fragment is Fab, and the end product in which two Fabs are connected by bismaleimide is referred to herein as bismaleimide-(sulfur-Fab)2 or bisFab. Such multispecific antibodies and antibody analogs (including bisFab) can be used to rapidly synthesize a large number of antibody fragment combinations or structural variants of natural antibodies or specific antibody / fragment combinations.

[0256] Multispecific antibodies can be synthesized with modified cross-linkers so that other functional moieties can be attached to the multispecific antibodies. Modified cross-linkers allow attachment of any sulfhydryl reactive moiety. In one embodiment, N-succinimidyl-S-acetylthioacetate (SATA) is attached to bismaleimide to form bismaleimido-acetylthioacetate (BMata). After deprotection of the masked thiol groups, any functional group with a sulfhydryl reactive moiety can be attached to the multispecific antibody.

[0257] Exemplary thiol-reactive reagents include multifunctional linker reagents, capture agents (i.e., affinity labeling reagents such as biotin-linker reagents), detection labels (e.g., fluorophore reagents), solid phase immobilization reagents (e.g., SEPHAROSE TM , polystyrene or glass) or a drug-linker intermediate. An example of a thiol-reactive reagent is N-ethylmaleimide (NEM). Such a multispecific antibody or antibody analog with a modified cross-linker can be further reacted with a drug moiety reagent or other label. The reaction of the multispecific antibody or antibody analog with the drug-linker intermediate provides a multispecific antibody-drug conjugate or an antibody analog-drug conjugate, respectively.

[0258] Many other techniques for preparing multispecific antibodies can also be used in the present invention. References describing these techniques include: (1) Milstein and Cuello, Nature, Vol. 305, p. 537 (1983), WO 93 / 08829, and Traunecker et al., EMBO J., Vol. 10, p. 3655 (1991), regarding the recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities; (2) U.S. Pat. No. 5,731,168, regarding knob-to-hole engineering; (3) WO 93 / 08829, regarding the recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities; (4) WO 93 / 08829, regarding the recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities; (5) WO 93 / 08829, regarding the recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities; (6) WO 93 / 08829, regarding the recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities; (7) WO 93 / 08829, regarding the recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities; (8) WO 93 / 08829, regarding the recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities; (9) WO 93 / 08829, regarding the recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities; (10) WO 93 / 08829, regarding the recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities; (11) WO 93 / 08829, regarding the 2009 / 089004A1, regarding engineered electrostatic steering effects for preparing antibody Fc heterodimer molecules; (4) U.S. Pat. No. 4,676,980, and Brennan et al., Science, Vol. 229, p. 81 (1985), regarding cross-linking two or more antibodies or fragments; (5) Kostelny et al., J. Immunol., Vol. 148, pp. 1547-1553 (1992), regarding the use of leucine zippers to produce bispecific antibodies; (6) Hol Linger et al., Proc. Natl. Acad. Sci. USA, Vol. 90, pp. 6444-6448 (1993), regarding the use of "diabody" technology to prepare bispecific antibody fragments; (7) Gruber et al., J. Immunol., Vol. 152, p. 5368 (1994), regarding the use of single-chain Fv (sFv) dimers; (8) Tutt et al., J. Immunol. 147:60 (1991), regarding the preparation of trispecific antibodies; and (9) US 2006 / 0025576A1 and Wu et al., Nature Biotechnology, Vol. 25, pp. 1290-1297 (2007), regarding engineered antibodies with three or more functional antigen binding sites, including "octopus antibodies" or "dual variable domain immunoglobulins" (DVDs).

[0259] The multispecific antibodies of the present invention can also be produced as described in WO / 2011 / 109726.

[0260] In one embodiment, a conditionally active antibody for crossing the blood-brain barrier (BBB) ​​is engineered to prepare a multispecific antibody (e.g., a bispecific antibody). The multispecific antibody comprises a first antigen binding site that binds to BBB-R and a second antigen binding site that binds to a brain antigen. At least the first antigen binding site of BBB-R is conditionally active. Brain antigens are antigens expressed in the brain that can be targeted by antibodies or small molecules. Examples of such antigens include, but are not limited to, beta-secretase 1 (BACE1), amyloid beta (Aβ), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau, apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), Parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophic factor receptor (p75NTR) and caspase 6. In one embodiment, the antigen is BACE1.

[0261] The BBB has an endogenous transport system mediated by BBB receptors (BBB-R), which are specific receptors that allow macromolecules to be transported across the BBB. For example, antibodies that bind to the BBB-R can be transported across the BBB using an endogenous transport system. Such antibodies can be used as carriers for transporting drugs or other agents across the BBB using an endogenous BBB receptor-mediated transport system that crosses the BBB. Such antibodies do not need to have a high affinity for the BBB-R. Antibodies that are not conditionally active antibodies but have a low affinity for the BBB-R have been described as crossing the BBB more efficiently than high-affinity antibodies, as described in US2012 / 0171120.

[0262] Another method for engineering antibodies to enter the brain is to engineer antibodies so that they are delivered to the brain through the central nervous system lymphatic vessels. Therefore, antibodies can be engineered to bind or simulate immune cells, such as T cells or transported to the synovial fluid or cerebrospinal fluid of the central nervous system through lymphatic vessels. The details of the lymphatic vessels of the central nervous system are described in, for example, Louveau, A., et al., "Structural and functional features of central nervous system lymphatic vessels," Nature 523, pp. 337-341, July 16, 2015, and in articles cited in the article and available to the public before the filing date of the present application.

[0263] Unlike traditional antibodies, conditionally active antibodies do not need to have low affinity for BBB-R to cross the BBB and remain in the brain. Conditionally active antibodies can have high affinity for BBB-R on the blood side of the BBB, but have little or no affinity for the brain side of the BBB. Drugs (e.g., drug conjugates) can be coupled to conditionally active antibodies to cross the BBB with the antibodies and be transported to the brain.

[0264] BBB-R is a transmembrane receptor protein expressed on brain endothelial cells, which can cross the blood-brain barrier transport molecule.The example of BBB-R includes transferrin receptor (TfR), insulin receptor, insulin-like growth factor receptor (IGF-R), low-density lipoprotein receptor (including but not limited to low-density lipoprotein receptor-related protein 1 (LRP1) and low-density lipoprotein receptor-related protein 8 (LRP8)) and heparin-binding epidermal growth factor-like growth factor (HB-EGF).Exemplary BBB-R herein is transferrin receptor (TfR).TfR is a transmembrane glycoprotein (molecular weight is about 180,000) composed of two subunits (each apparent molecular weight is about 90,000) connected by a disulfide bond, which participates in iron uptake in vertebrates.

[0265] In some embodiments, the invention provides conditionally active antibodies produced by parent or wild-type antibodies against BBB-R. The conditionally active antibodies bind to the BBB-R on the blood side of the BBB and have a lower affinity for the BBB-R on the brain side of the BBB than the parent or wild-type antibodies. In some other embodiments, the conditionally active antibodies have an affinity for the BBB-R on the blood side of the BBB and have no affinity for the BBB-R on the brain side of the BBB compared to the wild-type or parent antibodies.

[0266] Plasma is a very different body fluid from brain extracellular fluid (ECF). As described by Somjen (“Ions in the Brain: Normal Function, Seizures, and Stroke,” Oxford University Press, 2004, pp. 16 and 33) and Redzic (“Molecular biology of the blood-brain and the blood-cerebrospinal fluid barriers: similarities and differences,” Fluids and Barriers of the CNS, Vol. 8:3, 2011), brain extracellular fluid has significantly less K than plasma. + 、More Mg 2+ and H +The differences in ion concentrations between plasma and brain ECF result in significant differences in osmotic pressure and osmolality between the two fluids. Table 1 shows the millimolar concentrations of common ions in plasma and brain ECF.

[0267] Table 1. Common ions in plasma (arterial plasma) and brain extracellular fluid (CSF)

[0268]

[0269] Brain ECF also contains significantly more lactate than plasma, and significantly less glucose than plasma (Abi-Saab et al., “Striking Differences in Glucose and Lactate Levels Between Brain Extracellular Fluid and Plasma in Conscious Human Subjects: Effects of Hyperglycemia and Hypoglycemia,” Journal of Cerebral Blood Flow & Metabolism, Vol. 22, pp. 271-279, 2002).

[0270] Thus, there are several physiological conditions that differ between the two sides of the BBB, such as pH, various substances (e.g., lactose, glucose, K + Mg 2+ ) concentration, osmotic pressure, and weight molar osmotic pressure concentration. For physiological conditions of pH, human plasma has a higher pH than human brain ECF. For K + Under physiological conditions of concentration, brain ECF has a lower K than human plasma + Concentration. For Mg 2+ Under physiological conditions of concentration, human brain ECF has significantly more Mg than human plasma. 2+ Regarding the physiological conditions of osmotic pressure, human brain ECF has an osmotic pressure different from that of human plasma. In some embodiments, the physiological conditions of brain ECF may be the composition, pH, osmotic pressure, and weight molar osmotic pressure concentration of brain ECF of patients with a specific neurological disorder, which may be different from the physiological conditions of brain ECF of the general population.

[0271] Therefore, the present invention provides a method for evolving a DNA encoding a template antibody for BBB-R to produce a mutant DNA library. The mutant DNA library is then expressed to obtain mutant antibodies. The mutant antibodies are screened to obtain conditionally active antibodies with the following characteristics: binding to BBB-R under at least one plasma physiological condition, and having a lower affinity or no affinity for BBB-R than the template antibody under at least one brain physiological condition in brain ECF. Therefore, the selected mutant antibody has a low or high affinity for BBB-R on the plasma side, and a low affinity or no affinity for BBB-R on the brain ECF side. The selected mutant antibody can be used as a conditionally active antibody for transport across the BBB.

[0272] Such conditionally active antibodies are favored to cross the BBB and remain in brain ECF. Low affinity for the BBB-R on the brain side reduces the rate (or removal) at which conditionally active antibodies are transported across the BBB out of the brain and back into the blood relative to template antibodies.

[0273] In some other embodiments, the present invention provides a method for evolving a DNA encoding a template antibody for BBB-R to produce a mutant DNA library. The mutant DNA library is then expressed to obtain mutant antibodies. The mutant antibodies are screened to obtain conditionally active antibodies with the following characteristics: binding to BBB-R under at least one plasma physiological condition, and having little or no affinity for BBB-R under at least one brain physiological condition. Therefore, the selected mutant antibody has affinity for BBB-R on the plasma side, and has little or no affinity for BBB-R on the brain ECF side. The selected mutant antibody is a conditionally active antibody.

[0274] Such conditionally active antibodies are advantageous in crossing the BBB and being retained in the brain ECF. After binding to the BBB-R on the plasma side, the conditionally active antibodies are transported across the BBB, and having little or no affinity for the BBB-R on the brain ECF side means that the conditionally active antibodies are unlikely to be transported out of the brain.

[0275] The affinity of a conditionally active antibody for the BBB-R can be measured by its half maximal inhibitory concentration (IC50), which is a measure of how much antibody is needed to inhibit 50% of the binding of a known BBB-R ligand to the BBB-R. A common approach is to perform a competitive binding assay, such as a competitive ELISA assay. An exemplary competitive ELISA assay for measuring the IC50 of TfR (a BBB-R) is a method in which increasing concentrations of anti-TfR antibodies are combined with biotinylated TfR ACompetition ELISA assay for competitive binding to TfR. Anti-TfR antibody competition ELISA can be performed overnight at 4°C in Maxisorp plates (Neptune, NJ) coated with 2.5 μg / ml purified mouse TfR extracellular domain in PBS. The plates are washed with PBS / 0.05% Tween 20 and blocked using Superblock blocking buffer (Thermo Scientific, Hudson, NH) in PBS. Titrations of each individual anti-TfR antibody (1:3 serial dilutions) are mixed with biotinylated anti-TfR A (0.5 nM final concentration) and added to the plate for 1 hour at room temperature. The plate was washed with PBS / 0.05% Tween 20, and HRP-streptavidin (SouthernBiotech, Birmingham, MD) was added to the plate and incubated for 1 hour at room temperature. The plate was washed with PBS / 0.05% Tween 20, and biotinylated anti-TfR bound to the plate was detected using TMB substrate (BioFX Laboratories, Owings Mills, MD). A .

[0276] A high IC50 indicates that more conditionally active antibody is required to inhibit binding of a known ligand of the BBB-R, and thus the antibody has a relatively low affinity for that BBB-R. Conversely, a low IC50 indicates that less conditionally active antibody is required to inhibit binding of a known ligand, and thus the antibody has a relatively high affinity for that BBB-R.

[0277] In some embodiments, the IC50 of a conditionally active antibody from BBB-R in plasma can be from about 1 nM to about 100 μM, or from about 5 nM to about 100 μM, or from about 50 nM to about 100 μM, or from about 100 nM to about 100 μM, or from about 5 nM to about 10 μM, or from about 30 nM to about 1 μM, or from about 50 nM to about 1 μM.

[0278] Conditionally active bioprotein for synovial fluid

[0279] In industrialized countries, joint diseases are the main cause of disability and early retirement. Joint diseases often cause damage in joints that are difficult to repair. Synovial fluid is a body fluid found between the cartilage and synovium facing the joint-forming surface in the synovial cavity of the joint (e.g., knee, hip, shoulder) of a human or animal body. Synovial fluid provides nutrition for cartilage and is also used as a lubricant for joints. The cells of cartilage and synovium secrete fluid, which is used as a lubricant between the joint-forming surfaces. Human synovial fluid contains about 85% water. It is derived from the dialysate of blood plasma, which itself is composed of water, dissolved protein, glucose, coagulation factors, mineral ions, hormones, etc. Proteins such as albumin and globulin are present in synovial fluid, and are considered to play an important role in lubricating the joint region. Some other proteins have also been found in human synovial fluid, including glycoproteins, such as alpha-1-acid glycoprotein (AGP), alpha-1-antitrypsin (A1AT) and lubricin.

[0280] Synovial fluid has a very different composition from other parts of the body. Therefore, synovial fluid has physiological conditions different from other parts of the body (such as plasma). For example, synovial fluid has less than about 10 mg / dL of glucose, while the average normal glucose level in human plasma is about 100 mg / dL, fluctuating within a range of 70-100 mg / dL in a day. In addition, the total protein level in synovial fluid is about one-third of the plasma protein level, because macromolecules such as proteins are not easy to enter synovial fluid through the synovial membrane. It has also been found that the pH of human synovial fluid is higher than the pH in human plasma (Jebens et al., "On the viscosity and pH of synovial fluid and the pH of blood," The Journal of Bone and Joint Surgery, Vol. 41B, pp. 388-400, 1959; Farr et al., "Significance of the hydrogen ion concentration in synovial fluid in Rheumatoid Arthritis," Clinical and Experimental Rheumatology, Vol. 3, pp. 99-104, 1985).

[0281] Thus, synovial fluid has several physiological conditions that are different from those in other parts of the body, such as those in plasma. Synovial fluid has a higher pH than other parts of the body, particularly plasma. Synovial fluid has a lower glucose concentration than other parts of the body, such as plasma. Synovial fluid also has a lower protein concentration than other parts of the body, such as plasma.

[0282] Several antibodies have been used to treat joint diseases by introducing antibodies into synovial fluid. For example, it is known that the synovial fluid in injured joints contains many factors that have an impact on the progression of osteoarthritis (see, for example, Fernandes, et al., "The Role of Cytokines in Osteoarthritis Pathophysiology", Biorheology, Vol. 39, pp. 237-246, 2002). It is known that cytokines (such as interleukin-1 (IL-I) and tumor necrosis factor-α (TNF-α)) produced by activated synovial cells upregulate matrix metalloproteinase (MMP) gene expression. The upregulation of MMP leads to the degradation of matrix and non-matrix proteins in the joint. Antibodies that neutralize cytokines can prevent the progression of osteoarthritis.

[0283] Using antibodies as drugs is a promising strategy for treating joint diseases. For example, antibodies (such as antibodies against aggrecan or aggrecanase) have been developed to treat osteoarthritis, which is the most common in joint diseases so far (WO1993 / 022429A1). Antibodies for acetylated high mobility group protein 1 (HMGB1) have been developed for diagnosis or treatment of joint diseases, which are inflammatory, autoimmune, neurodegenerative or malignant diseases / disorders, such as arthritis. The antibody can be used to detect the acetylated form of HMGB1 in synovial fluid (WO 2011 / 157905A1). Another antibody (CD20 antibody) has also been developed for treating the damage of connective tissue and cartilage in joints.

[0284] However, the antigens of these antibodies are usually expressed in other parts of the body that carry important physiological functions. Although antibodies against these antigens are effective in treating joint diseases, they can also significantly interfere with the normal physiological functions of these antigens in other parts of the body. Therefore, patients may experience serious side effects. Therefore, it is desirable to develop therapeutic agents, such as antibodies against cytokines or other antigens, which can preferentially bind to their antigens (proteins or other macromolecules) with higher affinity in synovial fluid, without binding or only weakly binding to the same antigens in other parts of the body to reduce side effects.

[0285] Such conditionally active biological proteins can be conditionally active antibodies. In some embodiments, the present invention also provides conditionally active biological proteins that are proteins other than antibodies. For example, conditionally active immunomodulators can be developed by the present invention to preferentially modulate immune responses in synovial fluid, which can have little or no effect on immune responses in other parts of the body.

[0286] Conditionally active biological proteins can be conditionally active inhibitors of cytokine signaling (SOCS). Many of these SOCS are involved in inhibiting the JAK-STAT signaling pathway. Conditionally active inhibitors of cytokine signaling can preferentially inhibit cytokine signaling in synovial fluid, while not inhibiting or inhibiting to a lesser extent cytokine signaling in other parts of the body.

[0287] In some embodiments, the present invention provides conditionally active biological proteins derived from parent or wild-type biological proteins. The conditionally active biological protein has lower activity than the parent or wild-type biological protein under at least one physiological condition in certain parts of the body (e.g., in plasma), and has higher activity than the parent or wild-type biological protein under at least one physiological condition in synovial fluid. Such conditionally active biological proteins may preferentially act in synovial fluid, but may not or to a lesser extent act on other parts of the body. Therefore, such conditionally active biological proteins may have reduced side effects.

[0288] In some embodiments, the conditionally active biological protein is an antibody to an antigen in or exposed to synovial fluid. Although the antigen is usually a cytokine, such an antigen can be any protein involved in the immune response / inflammation in joint diseases. The conditionally active antibody has a lower affinity for the antigen under at least one physiological condition in other parts of the body (e.g., plasma) than the parent or wild-type antibody for the same antigen, and has a higher affinity for the antigen under at least one physiological condition in synovial fluid than the parent or wild-type antibody. Such a conditionally active antibody may bind weakly or not at all to the antigen in other parts of the body, but binds (e.g., strongly and tightly binds or more strongly binds) to the antigen in synovial fluid.

[0289] Conditionally Active Biopharmaceuticals for Tumors

[0290] Cancer cells in solid tumors can form a tumor microenvironment around them to support the growth and metastasis of cancer cells. The tumor microenvironment is the cellular environment in which the tumor exists, including surrounding blood vessels, immune cells, fibroblasts, other cells, soluble factors, signal transduction molecules, extracellular matrix and mechanical factors (mechanicalcue) that can promote tumor transformation, support tumor growth and invasion, protect tumors from host immunity, cultivate treatment resistance and provide a microenvironment for dormant metastasis to thrive. Tumors and their surrounding microenvironments are closely related and constantly interact. Tumors can affect their microenvironment by releasing extracellular signals, promoting tumor angiogenesis and inducing peripheral immune tolerance, and immune cells in the microenvironment can affect the growth and evolution of cancer cells. See Warts et al. "Tumor Microenvironment Complexity: Emerging Roles in Cancer Therapy," Cancer Res, 72 volumes, pp. 2473-2480, 2012.

[0291] The tumor microenvironment is usually hypoxic. As the tumor mass increases, the internal growth of the tumor is away from the existing blood supply, which makes it difficult to fully supply oxygen to the tumor microenvironment. Compared with the oxygen partial pressure of about 40mmHg in plasma, in more than 50% of locally advanced solid tumors, the oxygen partial pressure in the tumor environment is less than 5mmHg. In contrast, the rest of the body is not hypoxic. The hypoxic environment causes genetic instability, which is associated with cancer progression by downregulating the pathways of nucleotide excision repair and mismatch repair. Hypoxia also causes the upregulation of hypoxia-inducible factor 1α (HIF1-α), which induces angiogenesis and is associated with poor prognosis and the activation of genes associated with metastasis. See Weber et al., "The tumor microenvironment," Surgical Oncology, Vol. 21, pp. 172-177, 2012 and Blagosklonny, "Antiangiogenic therapy and tumor progression," Cancer Cell, Vol. 5, pp. 13-17, 2004.

[0292] In addition, tumor cells tend to rely on energy produced by lactic acid fermentation, which does not require oxygen. Therefore, tumor cells are unlikely to use normal aerobic respiration that requires oxygen. The result of using lactic acid fermentation is that the tumor microenvironment is acidic (pH 6.5-6.9), which is generally neutral or slightly alkaline compared to other parts of the body. For example, the pH of human plasma is about 7.4. See Estrella et al., "Acidity Generated by the Tumor Microenvironment Drives Local Invasion," Cancer Research, Vol. 73, pp. 1524-1535, 2013. Since the nutritional requirements of proliferative cancer cells are relatively high compared to cells located in other parts of the body, the nutrient availability in the tumor microenvironment is also low.

[0293] In addition, the tumor microenvironment also contains many different cell types that are not usually found in other parts of the body. These cell types include endothelial cells and their precursors, pericytes, smooth muscle cells, fibroblasts, cancer-associated fibroblasts, myofibroblasts, neutrophils, eosinophils, basophils, mast cells, T lymphocytes and B lymphocytes, natural killer cells and antigen presenting cells (APC) (such as macrophages and dendritic cells) (Lorusso et al., "The tumor microenvironment and its contribution to tumor evolution toward metastasis," Histochem Cell Biol, Vol. 130, pp. 1091-1103, 2008).

[0294] Thus, the tumor microenvironment has at least several physiological conditions that are different from the physiological conditions in other parts of the body (e.g., the physiological conditions in plasma). The pH (acidity) of the tumor microenvironment is lower than that of other parts of the body, particularly plasma (pH 7.4). The tumor microenvironment has a lower oxygen concentration than other parts of the body (e.g., plasma). In addition, the tumor microenvironment has a lower nutrient availability than other parts of the body (particularly plasma). The tumor microenvironment also has some different cell types that are not common in other parts of the body (particularly plasma).

[0295] Some cancer drugs include antibodies that can penetrate into the tumor microenvironment and act on cancer cells therein. Antibody-based cancer therapy is well established and has become one of the most successful and important strategies for treating patients with hematological malignancies and solid tumors. There are a series of cell surface antigens expressed by human cancer cells that are overexpressed, mutated, or selectively expressed in cancer cells compared to normal tissues. These cell surface antigens are excellent targets for antibody cancer therapy.

[0296] Cancer cell surface antigens that can be targeted by antibodies are divided into several different categories. Hematopoietic differentiation antigens are glycoproteins that are often associated with differentiation cluster (CD) groupings and include CD20, CD30, CD33, and CD52. Cell surface differentiation antigens are different groups of glycoproteins and carbohydrates found on the surface of normal cells and tumor cells. Antigens involved in growth and differentiation signaling are usually growth factors and growth factor receptors. Growth factors that are targets for antibodies in cancer patients include CEA2, epidermal growth factor receptor (EGFR; also known as ERBB1)12, ERBB2 (also known as HER2)13, ERBB3 (REF.18), MET (also known as HGFR)19, insulin-like growth factor 1 receptor (IGF1R)20, ephrin receptor A3 (EPHA3)21, tumor necrosis factor (TNF)-related apoptosis-inducing ligand receptor 1 (TRAILR1; also known as TNFRSF10A), TRAILR2 (also known as TNFRSF10B), and receptor activator of nuclear factor kappa B ligand (RANKL; also known as TNFSF11)22. Antigens involved in angiogenesis are usually proteins or growth factors that support the formation of new microvasculature, including vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR), integrin αVβ3 and integrin α5β1 (REF.10). Tumor stroma and extracellular matrix are indispensable supporting structures for tumors. Matrix antigens and extracellular matrix antigens as therapeutic targets include fibroblast activation protein (FAP) and tenascin. See Scott et al., "Antibody therapy of cancer," Nature Reviews Cancer, Vol. 12, pp. 278-287, 2012.

[0297] In addition to antibodies, other biological proteins are also expected to be used for cancer treatment. Examples include tumor suppressors such as retinoblastoma protein (pRb), p53, pVHL, APC, CD95, ST5, YPEL3, ST7 and ST14. Some proteins that induce apoptosis of cancer cells can also be introduced into tumors to reduce the size of tumors. There are at least two mechanisms that can induce tumor apoptosis: a tumor necrosis factor-induced mechanism and a Fas-Fas ligand-mediated mechanism. At least some proteins involved in either of the two apoptosis mechanisms can be introduced into tumors for treatment.

[0298] Cancer stem cells are cancer cells that are capable of giving rise to all cell types found in a particular cancer sample and are therefore tumor-forming cells. They can give rise to tumors through a stem cell process of self-renewal and differentiation into a variety of cell types. Cancer stem cells are thought to persist as a distinct population within tumors and cause recurrence and metastasis by giving rise to new tumors. The development of specific therapies targeting cancer stem cells could increase survival and improve quality of life in cancer patients, especially for those with metastatic disease.

[0299] These drugs for treating tumors usually interfere with the normal physiological functions of other parts of the body except the tumor. For example, proteins that induce tumor apoptosis can also induce apoptosis in certain other parts of the body, thereby causing side effects. In embodiments using antibodies to treat tumors, the antigens of the antibodies can also be expressed in other parts of the body where they perform normal physiological functions. For example, the monoclonal antibody bevacizumab (targeting vascular endothelial growth factor) prevents tumor blood vessel growth. The antibody can also prevent blood vessel growth or repair in other parts of the body, thereby causing bleeding, poor wound healing, blood clots and kidney damage. More effective tumor treatment is in great need of developing conditionally active biological proteins that mainly or individually target tumors.

[0300] In some embodiments, the present invention provides conditionally active biological proteins produced by parent or wild-type biological proteins, which can be candidates for tumor treatment. Conditionally active biological proteins have lower activity than parent or wild-type biological proteins under at least one physiological condition in a body part other than the tumor microenvironment (e.g., plasma), and have higher activity than parent or wild-type biological proteins under at least one physiological condition in the tumor microenvironment. Such conditionally active biological proteins can preferentially act on cancer cells in the tumor microenvironment to treat tumors, and are therefore less likely to cause side effects. In embodiments where the biological protein is an antibody against an antigen on the surface of a tumor cell and the antigen is exposed to the tumor microenvironment in the tumor cell, the conditionally active antibody has a lower affinity for the antigen than the parent or wild-type antibody in other parts of the body (e.g., non-tumor microenvironment), and has a higher affinity for the antigen in the tumor microenvironment than the parent or wild-type antibody. Such conditionally active antibodies may be weakly bound to or not bound to the antigen in other parts of the body, but may be better bound to the antigen or strongly and tightly bound to the antigen in the tumor microenvironment.

[0301] In some embodiments, the conditionally active antibody is an antibody against an immune checkpoint protein, resulting in inhibition of the immune checkpoint. Such conditionally active antibodies have at least one of the following properties: (1) an enhanced binding affinity for the immune checkpoint protein in a tumor microenvironment compared to a parent or wild-type antibody from which the conditionally active antibody is derived, and (2) a reduced binding affinity for the immune checkpoint protein in a non-tumor microenvironment compared to a parent or wild-type antibody from which the conditionally active antibody is derived.

[0302] Immune checkpoints are used as endogenous inhibitory pathways of the immune system to maintain self-tolerance and regulate the duration and degree of immune responses to antigenic stimulation (i.e., exogenous molecules, cells, and tissues).See Pardoll, Nature Reviews Cancer, Vol. 12, pp. 252-264, 2012. Inhibition of immune checkpoints by inhibiting one or more checkpoint proteins can cause superactivation of the immune system (particularly T cells), thereby inducing the immune system to attack tumors. Checkpoint proteins suitable for the present invention include CTLA4 and its ligands CD80 and CD86, PD1 and its ligands PDL1 and PDL2, T cell immunoglobulins and mucin protein-3 (TIM3) and its ligands GAL9, B and T lymphocyte attenuators (BTLA) and their ligands HVEM (herpes virus entry mediators), receptors such as killer cell immunoglobulin-like receptors (KIR), lymphocyte activation genes-3 (LAG3) and adenosine A2a receptors (A2aR), and ligands B7-H3 and B7-H4. Other suitable immune checkpoint proteins are described in Pardoll, Nature Reviews Cancer, Vol. 12, pp. 252-264, 2012 and Nirschl & Drake, Clin Cancer Res, Vol. 19, pp. 4917-4924, 2013.

[0303] CTLA-4 and PD1 are the two most well-known immune checkpoint proteins. CTLA-4 can downregulate the pathway of T cell activation (Fong et al., Cancer Res. 69 (2): 609-615, 2009; and Weber, Cancer Immunol. Immunother, 58: 823-830, 2009). Blocking CTLA-4 has been shown to enhance T cell activation and proliferation. CTLA-4 inhibitors include anti-CTLA-4 antibodies. Anti-CTLA-4 antibodies bind to CTLA-4 and block the interaction of CTLA-4 with its ligands CD80 or CD86, thereby blocking the downregulation of the immune response triggered by the interaction of CTLA-4 with its ligands.

[0304] It is known that the checkpoint protein PD1 inhibits the activity of T cells in peripheral tissues and limits autoimmunity when an inflammatory response is produced to infection. In response to the stimulation of specific antigen targets or allogeneic cells in mixed lymphocyte reactions, in vitro PD1 blockade can enhance T cell proliferation and cytokine production. It has been shown that the strong correlation between PD1 expression and reduced immune responses is caused by the inhibitory function of PD1, i.e., by inducing immune checkpoints (Pardoll, Nature Reviews Cancer, 12: 252-264, 2012). PD1 blockade can be achieved by a variety of mechanisms, including antibodies that bind PD1 or its ligands PDL1 or PDL2.

[0305] Past studies have found antibodies against several checkpoint proteins (CTLA4, PD1, PD-L1). These antibodies are used to attack tumors by superactivating the immune system (especially T cells) by inhibiting immune checkpoints, thereby effectively treating tumors (superactivating the immune system) (Pardoll, Nature Reviews Cancer, Vol. 12, pp. 252-264, 2012). However, superactivated T cells can also attack host cells and / or tissues, causing collateral damage to the patient's body. Therefore, treatment based on the use of these known antibodies to inhibit immune checkpoints is difficult to control, and the risk to patients is also a serious problem. For example, FDA-approved antibodies against CTLA-4 carry black box warnings due to their high toxicity.

[0306] The present invention solves the problem of collateral damage of hyperactivated T cells by providing conditionally active antibodies for immune checkpoint proteins. These conditionally active antibodies preferentially activate immune checkpoints in tumor microenvironments. At the same time, conditionally active antibodies do not inhibit or less inhibit immune checkpoints in non-tumor microenvironments (e.g., normal body tissues), so that the possibility of collateral damage to the body is reduced in non-tumor microenvironments. This goal is achieved by engineering conditionally active antibodies to be more active in tumor microenvironments than in non-tumor microenvironments.

[0307] In some embodiments, the ratio of the binding activity of a conditionally active antibody against an immune checkpoint protein to an immune checkpoint protein in a tumor microenvironment to the binding activity to the same immune checkpoint protein in a non-tumor microenvironment is at least about 1.1, or at least about 1.2, or at least about 1.4, or at least about 1.6, or at least about 1.8, or at least about 2, or at least about 2.5, or at least about 3, or at least about 5, or at least about 7, or at least about 8, or at least about 9, or at least about 10, or at least about 15, or at least about 20. A typical assay for measuring the binding activity of an antibody is an ELISA assay.

[0308] Highly immunogenic tumors (e.g., malignant melanoma) are most vulnerable to attack by a hyperactivated immune system acquired through immune system manipulation. Therefore, conditionally active antibodies against immune checkpoint proteins are particularly effective for treating such highly immunogenic tumors. However, other types of tumors are also vulnerable to attack by a hyperactivated immune system.

[0309] In some embodiments, conditional active antibodies for immune checkpoint proteins can be used in combination therapy. For example, combination therapy may include conditional active antibodies for tumor cell surface molecules (tumor-specific antigens) and conditional active antibodies for immune checkpoint proteins. In one embodiment, the binding activity of conditional active antibodies to tumor cell surface molecules and the binding activity of conditional active antibodies to immune checkpoint proteins may be present in a single protein, i.e., in bispecific conditional active antibodies disclosed herein. In some other embodiments, combination therapy may include conditional active antibodies for tumor cell surface molecules (tumor-specific antigens) and two or more conditional active antibodies for two or more different immune checkpoint proteins. In one embodiment, all of these binding activities may be present in a single protein, i.e., in multispecific antibodies disclosed herein.

[0310] Because conditionally active antibodies are more active in the tumor microenvironment than the parent or wild-type antibodies from which they are derived against the same tumor cell surface molecules or checkpoint proteins, these combination therapies can provide enhanced efficacy and significantly reduced toxicity. The reduced toxicity of these conditionally active antibodies (particularly antibodies against immune checkpoint proteins) enables the safe use of effective antibodies such as the ADC antibodies described herein and higher doses of antibodies.

[0311] In some embodiments, conditionally active antibodies against checkpoint proteins can be in the form of prodrugs. For example, conditionally active antibodies can be prodrugs without the desired drug activity before being cleaved and converted into drug form. Or because the enzyme that catalyzes such cleavage is preferentially present in the tumor microenvironment or because the conditionally active antibody makes the cleavage site in the tumor microenvironment more accessible than the cleavage site in the non-tumor microenvironment, the prodrug can be preferentially cleaved in the tumor microenvironment.

[0312] Engineering conditionally active bioproteins for stem cell niches including cancer stem cells

[0313] Stem cells exist in an environment called stem cell niche in the body, which constitutes the basic unit of tissue physiology, integrating signals that mediate the response of stem cells to the needs of the organism. However, niches can also induce pathology by applying abnormal functions to stem cells or other targets. The interaction between stem cells and their niches produces a dynamic system required for maintaining tissues and the final design of stem cell therapy (Scadden, "The stem-cell niche as an entity of action," Nature, Vol. 441, pp. 1075-1079, 2006). Common stem cell niches in vertebrates include germline stem cell niches, hematopoietic stem cell niches, hair follicle stem cell niches, intestinal stem cell niches, and cardiovascular stem cell niches.

[0314] The stem cell niche is a specialized environment that is different from other parts of the body (e.g., plasma) (Drummond-Barbosa, "Stem Cells, Their Niches and the Systemic Environment: An Aging Network," Genetics, Vol. 180, pp. 1787-1797, 2008; Fuchs, "Socializing with the Neighbors: Stem Cells and Their Niche," Cell, Vol. 116, pp. 769-778, 2004). The stem cell niche is hypoxic, where oxidative DNA damage is reduced. Direct measurements of oxygen levels have shown that the bone marrow is generally quite hypoxic (~1%-2% O2) compared to plasma (Keith et al., "Hypoxia-Inducible Factors, Stem Cells, and Cancer," Cell, Vol. 129, pp. 465-472, 2007; Mohyeldin et al., "Oxygen in Stem Cell Biology: A Critical Component of the Stem Cell Niche," Cell Stem Cell, Vol. 7, pp. 150-161, 2010). In addition, the stem cell niche requires several other factors to regulate the stem cell characteristics within the niche: factors of the extracellular matrix composition, growth factors, cytokines, and the physicochemical properties of the environment, including pH, ionic strength (e.g., Ca2+), and ion concentration (e.g., Mg2+). 2+ concentration) and metabolites.

[0315] Thus, the stem cell niche has at least several physiological conditions that are different from the physiological conditions in other parts of the body (e.g., the physiological conditions in plasma). The stem cell niche has a lower oxygen concentration (1-2%) than other parts of the body (particularly plasma). Other physiological conditions of the stem cell niche, including pH and ionic strength, may also be different from other parts of the body.

[0316] Stem cell therapy is an interventional strategy that introduces new adult stem cells into damaged tissues in order to treat disease or injury. This strategy depends on the ability of stem cells to self-renew and subsequently produce offspring with varying degrees of differentiation capacity. Stem cell therapy offers great potential for tissue regeneration, and the regenerated tissue can potentially replace diseased and damaged areas in the body with minimal risk of rejection and side effects. Therefore, delivering drugs (biological proteins (such as antibodies) or chemical compounds) to the stem cell niche to affect the renewal and differentiation of stem cells is an important part of stem cell therapy.

[0317] There are several examples of how stem cell niches influence the renewal and / or differentiation of stem cells in mammals. The first is in the skin, where beta-1 integrin is known to be differentially expressed on primary cells and is involved in the constrained localization of stem cell populations through interactions with matrix glycoprotein ligands. Second, in the nervous system, deficiency of tenascin C alters the number and function of neural stem cells in the subventricular zone. Tenascin C appears to modulate the sensitivity of stem cells to fibroblast growth factor 2 (FGF2) and bone morphogenetic protein 4 (BMP4), resulting in an enhanced stem cell bias. Third, another matrix protein, the Arg-Gly-Asp-containing sialoprotein, osteopontin (OPN), has now been shown to contribute to hematopoietic stem cell regulation. OPN interacts with several receptors known to be on hematopoietic stem cells, CD44 and α4 and α5β1 integrins. The production of OPN is significantly altered, especially under conditions of osteoblast activation. Because deficiency of OPN results in supraphysiological stem cell expansion under stimulatory conditions, animals lacking OPN have increased HS cell numbers. Therefore, OPN appears to act as a constraint on the number of hematopoietic stem cells, limiting the number of stem cells under steady-state conditions or under conditions of stimulation. See Scadden, "The stem-cell niche as an entity of action," Nature, Vol. 441, pp. 1075-1079, 2006.

[0318] Xie et al., "Autocrine signaling based selection of combinatorialantibodies that transdifferentiate human stem cells," Proc Natl Acad Sci USA, Vol. 110, pp. 8099-8104, 2013 discloses a method of using antibodies to affect stem cell differentiation. The antibody is an agonist of the granulocyte colony stimulating factor receptor. Unlike natural granulocyte colony stimulating factor, which activates cells to differentiate along a predetermined pathway, isolated agonist antibodies transdifferentiate human myeloid CD34+ bone marrow cells into neural progenitor cells. Melidoni et al. ("Selecting antagonistic antibodies that control differentiation throughinducible expression in embryonic stem cells," Proc Natl Acad Sci USA, Vol. 110, pp. 17802–17807, 2013) also disclose the use of antibodies to interfere with the interaction between FGF4 and its receptor FGFR1β, thereby blocking autocrine FGF4-mediated embryonic stem cell differentiation.

[0319] Understanding the function of ligands / receptors in stem cell differentiation enables the implementation of strategies that use biological proteins to interfere with these ligands / receptors to regulate or even direct stem cell differentiation. Controlling the differentiation ability of human stem cells that have not been genetically modified by administering antibodies to stem cell niches can provide new in vitro or in vivo methods for stem cell-based therapeutics. In some embodiments, the present invention provides conditionally active biological proteins produced by parent or wild-type biological proteins, which can enter stem cell niches including cancer stem cells to regulate stem cell or tumor development. Conditionally active biological proteins have lower activity than parent or wild-type biological proteins under at least one physiological condition of other parts of the body, and have higher activity than parent or wild-type biological proteins under at least one physiological condition of the stem cell niche (e.g., cancer stem cell environment). This conditionally active biological protein is less likely to cause side effects and preferentially acts on the stem cell niche to regulate the renewal and differentiation of stem cells. In some embodiments, the conditionally active biological protein is an antibody. Such conditionally active antibodies may weakly bind or not bind at all to antigens in other parts of the body, but are firmly and tightly bound to antigens in the stem cell niche.

[0320] The conditionally active proteins for synovial fluid, tumor microenvironment and stem cell niche of the present invention are produced by evolving DNA encoding a parent or wild-type biological protein to produce a mutant DNA library. The mutant DNA library is then expressed to obtain mutant proteins. The mutant proteins are screened to obtain conditionally active biological proteins having the following properties: under at least one physiological condition of a first part of the body selected from synovial fluid, tumor microenvironment and stem cell niche, it has a higher activity than the parent or wild-type biological protein, and under at least one physiological condition of a second part of the body different from the first part of the body, it has a lower activity than the parent or wild-type biological protein. The second part of the body can be plasma. This selected mutant biological protein is a conditionally active biological protein that has high activity in the first part of the body and low activity in the second part of the body.

[0321] Such conditionally active biological proteins are advantageous in reducing the side effects of the parent or wild-type protein because the conditionally active biological protein has lower activity in other parts of the body where it is not intended to act. For example, if the conditionally active biological protein is purposefully introduced into the tumor microenvironment, the fact that the conditionally active biological protein has low activity in parts of the body other than the tumor microenvironment means that such conditionally active biological proteins are less likely to interfere with the normal physiological functions of parts of the body other than the tumor microenvironment. At the same time, the conditionally active biological protein has high activity in the tumor microenvironment, which makes the conditionally active biological protein have a higher efficacy in treating tumors.

[0322] Conditionally active biological proteins will allow for the safe use of significantly higher doses of the protein compared to the parent or wild-type biological protein due to reduced side effects. This is particularly beneficial for antibodies against cytokines or growth factors, which may interfere with the normal physiological function of the cytokines or growth factors in other parts of the body. By using conditionally active biological proteins with reduced side effects, higher doses can be used to achieve higher efficacy.

[0323] Conditionally active biological proteins that work in one of the synovial fluid, tumor microenvironment, or stem cell niches also enable the use of new drug targets. The use of traditional biological proteins as therapeutic agents may lead to unacceptable side effects. For example, inhibition of the epidermal growth factor receptor (EGFR) can be very effective in inhibiting tumor growth. However, drugs that inhibit EGFR also inhibit the growth of the skin and gastrointestinal (GI) tract. Side effects make EGFR unsuitable as a tumor drug target. The use of conditionally active antibodies that bind to EGFR with high affinity only in the tumor microenvironment but do not have or have very low affinity in any other part of the body will significantly reduce side effects and simultaneously inhibit tumor growth. In this case, EGFR can become an effective new tumor drug target by using conditionally active antibodies.

[0324] In another example, inhibiting cytokines is generally beneficial for repairing joint damage. However, inhibiting cytokines in other parts of the body may also inhibit the body's immune response, causing immunodeficiency. Therefore, cytokines in synovial fluid are not ideal targets for developing traditional antibody drugs for treating joint damage. However, by using conditionally active antibodies that preferentially bind to cytokines in synovial fluid while not binding or only weakly binding to the same cytokines in other parts of the body, the side effects of immunodeficiency can be significantly reduced. Therefore, cytokines in synovial fluid can become suitable targets for repairing joint damage by using conditionally active antibodies.

[0325] Conditionally active biopharmaceuticals for inflammation-prone organs / tissues

[0326] In some embodiments, the conditionally active biological protein is designed to preferentially act in organs or tissues that are susceptible to inflammation, such as lymph nodes, tonsils, adenoids, and sinuses. Other organs and tissues susceptible to inflammation can be found in anatomy textbooks, such as Gray's Anatomy by Henry Grey, 41st edition, published by Elsevier in 2015.

[0327] Once they become inflamed, these organs and tissues typically exhibit at least one abnormal condition. For example, these inflamed organs and tissues may have a higher osmotic pressure and / or a lower concentration of one or more ions compared to normal physiological conditions in other parts of the body, such as human plasma. In addition, the concentrations of small molecules, lactate, cytokines, and leukocytes in such inflamed organs and tissues may be higher compared to normal physiological conditions in other parts of the body, such as human plasma.

[0328] In some embodiments, conditionally active biological proteins can be produced by the present invention using abnormal conditions selected from one or more abnormal conditions encountered in an inflamed area and normal physiological conditions in human plasma. Thus, the activity of such conditionally active biological proteins in organs / tissues under inflammatory conditions is higher than that of the parent or wild-type biological protein, and the activity in human plasma is lower than that of the parent or wild-type biological protein. Such conditionally active biological proteins can preferentially act on inflamed areas of the body, but have little or no activity in non-inflamed areas of the body.

[0329] Conditionally active viral particles

[0330] Virus particles have long been used as delivery vectors for transporting proteins, nucleic acid molecules, chemical compounds or radioactive isotopes to target cells or tissues. Virus particles commonly used as delivery vectors include retroviruses, adenoviruses, slow viruses, herpes viruses and adeno-associated viruses. Usually in ligand-receptor binding systems, virus particles recognize their target cells by surface proteins, which are used as recognition proteins for specific binding to cell proteins (Lentz, "The recognition event between virus and host cell receptor:atarget for antiviral agents," J. of Gen. Virol., Vol. 71, pp. 751-765, 1990) of target proteins of target cells. For example, virus recognition proteins can be ligands of receptors on target cells. The specificity between ligands and receptors allows virus particles to specifically recognize and deliver their contents to target cells.

[0331] Techniques for developing artificial viral particles from wild-type viruses are well known to those skilled in the art. As delivery vectors, known artificial viral particles include those based on retroviruses (see, e.g., WO 90 / 07936; WO 94 / 03622; WO 93 / 25698; WO 93 / 25234; U.S. Pat. No. 5,219,740; WO 93 / 11230; WO 93 / 10218; U.S. Pat. No. 4,777,127; British Patent No. 2,200,651; EP 0 345 242; and WO 91 / 02805), alphaviruses (e.g., Sindbis virus vectors, Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246), Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR-1264); 1249; ATCC VR-532)) and adeno-associated virus (see, e.g., WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655).

[0332] Usually, artificial virus particles are constructed by inserting an exogenous recognition protein into the virus particle, and the exogenous recognition protein usually replaces the natural recognition protein by recombinant technology. The exogenous recognition protein can be, for example, an antibody, a receptor, a ligand or a collagen binding domain. The present invention provides conditional active recognition proteins, which do not have the activity or activity that is combined with cells under normal physiological conditions, and have the activity or activity that is combined with cells under abnormal conditions. Therefore, the conditional active recognition protein can preferentially bind to the target cells at the diseased tissue and / or the diseased part based on the presence of abnormal conditions at the diseased tissue and / or the diseased part, and avoid or only minimally bind to the cells of the normal tissues with normal physiological conditions. The conditional active recognition protein can be expressed and displayed on the surface of the virus particle.

[0333] In some embodiments, the present invention provides a method for evolving a parent or wild-type recognition protein and screening a conditional active recognition protein. The conditional active recognition protein has a lower activity of binding cells than the parent or wild-type recognition protein under normal physiological conditions, and has a higher activity of binding cells than the parent or wild-type recognition protein under abnormal conditions. This conditional active recognition protein can be inserted into a virus particle by known recombinant techniques to produce a conditional active virus particle.

[0334] In another embodiment, the present invention provides a conditionally active viral particle comprising a conditionally active recognition protein, which allows the conditionally active viral particle to recognize and bind to target cells in diseased tissues or at diseased sites, but not to recognize and bind to normal tissue cells. Such conditionally active viral particles can preferentially deliver the therapeutic agent in the viral particle to diseased tissues or diseased sites, while the conditionally active viral particles deliver less or no therapeutic agent to cells in normal tissues.

[0335] In some embodiments, compared with the pH or temperature in other parts of the body that are healthy or do not suffer from a specific disease or disease state, the target cells in the diseased part are in a region or microenvironment with abnormal pH (e.g., pH 6.5) or abnormal temperature. In this embodiment, at normal physiological pH or temperature, the conditionally active recognition protein is lower in terms of binding to the target protein of the target cell than the activity of the parent or wild-type recognition protein, and is higher in terms of binding to the target protein of the target cell than the activity of the parent or wild-type recognition protein at abnormal pH or temperature. In this way, the recognition protein will preferentially bind to the position that encounters abnormal pH or temperature, thereby delivering the therapeutic agent to the diseased part.

[0336] In one embodiment, the viral particle may comprise a conditionally active antibody of the invention, in particular a variable region of an antibody (e.g., Fab, Fab', Fv). Such a conditionally active antibody may bind to a target protein (as an antigen) of a target cell with a lower affinity than that of a parent or wild-type antibody under normal physiological conditions, which may be encountered at locations with normal tissue, and may bind to a target protein of a target cell with a higher affinity than that of a parent or wild-type antibody under abnormal physiological conditions, which may be encountered at diseased sites or diseased tissues. Conditionally active antibodies may be derived from parent or wild-type antibodies according to the methods of the invention.

[0337] In one embodiment, the target protein on the target cell includes a tyrosine kinase growth factor receptor that is overexpressed on the cell surface in, for example, many tumors. Exemplary tyrosine kinase growth factors are VEGF receptor, FGF receptor, PDGF receptor, IGF receptor, EGF receptor, TGF-α receptor, TGF-β receptor, HB-EGF receptor, ErbB2 receptor, ErbB3 receptor, and ErbB4 receptor.

[0338] Conditionally Active DNA / RNA Modifying Proteins

[0339] DNA / RNA modifying proteins have been discovered as a form of new genome engineering tools, particularly a form called CRISPR, which can allow researchers to perform microsurgery on genes, accurately and easily changing DNA sequences at precise locations on chromosomes (genome editing, Mali et al., "Cas9 as a versatile tool for engineering biology," Nature Methods, Vol. 10, pp. 957-963, 2013). For example, sickle cell anemia is caused by a single base mutation, which can be corrected by using DNA / RNA modifying proteins. The technology can even precisely delete or edit chromosome sites by changing a single base pair (Makarova et al., "Evolution and classification of the CRISPR-Cas systems," Nature Reviews Microbiology, Vol. 9, pp. 467-477, 2011).

[0340] Genome editing with CRISPR can rapidly and simultaneously change multiple genes in cells. Many human diseases including heart disease, diabetes and neurological diseases are affected by multiple gene mutations. This CRISPR-based technology has the potential to reverse disease-causing mutations and treat these diseases or at least reduce the severity of these diseases. Genome editing relies on CRISPR-related (Cas) proteins (enzyme family) for cutting genomic DNA. Typically, Cas proteins are guided to target regions in the genome by small guide RNAs, where the guide RNAs match the target regions. Because Cas proteins have little or no sequence specificity, guide RNAs act as pointers for Cas proteins to achieve precise genome editing. In one embodiment, a Cas protein can be used together with a variety of guide RNAs to simultaneously correct multiple gene mutations.

[0341] There are many Cas proteins. Examples include Cas1, Cas2, Cas3', Cas3", Cas4, Cas5, Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cas10d, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4 (Makarova et al., "Evolution and classification of the CRISPR–Cas systems,” Nature Reviews Microbiology, Vol. 9, pp. 467-477, 2011).

[0342] In order to perform genome editing, Cas proteins must enter target cells. Cells in individuals can have different intracellular pH inside the cells. Some cells in diseased tissues have abnormal intracellular pH. For example, some tumor cells tend to have an alkaline intracellular pH of about 7.12-7.65, while cells in normal tissues have a neutral intracellular pH in the range of 6.99-7.20 (see Cardone et al., "The role of disturbed pH dynamics and the Na(+) / H(+)exchanger in metastasis," Nat. Rev. Cancer, Vol. 5, pp. 786-795, 2005). In chronic hypoxia, cells in diseased tissues have an intracellular pH of about 7.2-7.5, which is also higher than that of normal tissues (Rios et al., "Chronic hypoxia elevates intracellular pH and activates Na+ / H+exchange inpulmonary arterial smooth muscle cells," American Journal of Physiology-Lung Cellular and Molecular Physiology, Vol. 289, pp. L867-L874, 2005). In addition, in ischemic cells, intracellular pH is usually in the range of 6.55-6.65, which is lower than that of normal tissues (Haqberg, "Intracellular pH during ischemia in skeletal muscle: relationship to membrane potential, extracellular pH, tissue lactic acid and ATP," Pflugers Arch., Vol. 404, pp. 342-347, 1985). Further examples of abnormal intracellular pH in diseased tissues are discussed in Han et al., “Fluorescent Indicators for Intracellular pH,” Chem Rev., Vol. 110, pp. 2709-2728, 2010.

[0343] The present invention provides a method for producing a conditionally active Cas protein from a parent or wild-type Cas protein, wherein the conditionally active Cas protein has at least one of the following characteristics: (1) under normal physiological conditions in normal cells, the enzymatic activity is reduced relative to the parent or wild-type Cas protein, and (2) under abnormal conditions in target cells (e.g., a disease cell described above), the enzymatic activity is increased relative to the parent or wild-type Cas protein. In some embodiments, the normal physiological condition is an intracellular pH of approximately neutral, and the abnormal condition is a different intracellular pH above or below neutral. In one embodiment, the abnormal condition is an intracellular pH of 7.2-7.65 or an intracellular pH of 6.5-6.8.

[0344] In some embodiments, the conditionally active viral particles of the present invention can be used to deliver the conditionally active Cas protein to the target cell. The conditionally active viral particles contain the conditionally active Cas protein and at least one guide RNA for guiding the Cas protein to the location of the genomic DNA that the Cas protein is to edit.

[0345] Multispecific antibodies have high selectivity to preferentially target tissues containing all or most of the targets (antigens) that the multispecific antibodies can bind. For example, bispecific antibodies can provide selectivity for target cells by showing greater preference for target cells expressing both antigens recognized by the bispecific antibodies, compared to non-target cells that can only express one of the two antigens. Therefore, due to the dynamics of the system, at equilibrium, there are more bispecific antibodies that bind to target cells than to non-target cells.

[0346] The multispecific antibodies or antigen recognition fragments thereof engineered herein can be used as ASTRs in the chimeric antigen receptors of the present invention.

[0347] Engineered cytotoxic cells

[0348] Once a conditionally active ASTR is identified through the screening step, a chimeric antigen receptor can be assembled by connecting the polynucleotide sequences encoding the various domains to form a single polynucleotide sequence (the CAR gene encoding the conditionally active CAR). The various domains include conditionally active ASTRs, TMs, and ISDs. In some embodiments, other domains can also be introduced into the CAR, including ab ESDs and CSDs ( Figure 1 ). If the conditionally active CAR is a bispecific CAR, the CAR gene can be, for example, in the N-terminal to C-terminal direction in the following configuration: N-terminal signal sequence-ASTR 1-linker-ASTR 2-extracellular spacer domain-transmembrane domain-costimulatory domain-intracellular signaling domain. In one embodiment, such a CAR gene may include two or more costimulatory domains.

[0349] Alternatively, the polynucleotide sequence encoding the conditionally active CAR may have the following configuration in the N-terminal to C-terminal direction: N-terminal signal sequence-ASTR 1-linker-ASTR 2-transmembrane domain-co-stimulatory domain-intracellular signaling domain. In one embodiment, such a CAR may include two or more co-stimulatory domains. If the CAR includes more than two ASTRs, the polynucleotide sequence encoding the CAR may have the following configuration in the N-terminal to C-terminal direction: N-terminal signal sequence-ASTR 1-linker-ASTR 2-linker-(antigen-specific targeting region) n - transmembrane domain - co-stimulatory domain - intracellular signaling domain. Such a CAR may further comprise an extracellular spacer domain. Each ASTR may be separated by a linker. In one embodiment, such a CAR may comprise two or more co-stimulatory domains.

[0350] Conditionally active CAR is introduced into cytotoxic cells by expression vector. Also provided herein is an expression vector comprising a polynucleotide sequence encoding the conditionally active CAR of the present invention. Suitable expression vectors include lentiviral vectors, gamma retroviral vectors, foamy virus vectors, adeno-associated virus (AAV) vectors, adenoviral vectors, engineered hybrid viruses, naked DNA, including but not limited to transposon-mediated vectors, such as Sleeping Beauty, Piggybak, integrase (such as Phi31). Some other suitable expression vectors include herpes simplex virus (HSV) expression vectors and retroviral expression vectors.

[0351] Adenovirus expression vector is based on adenovirus, which has low ability to integrate into genomic DNA but has high efficiency of transfecting host cells. Adenovirus expression vector contains enough to: (a) support the packaging of expression vector and (b) finally express CAR gene in host cell. Adenovirus genome is 36kb linear double-stranded DNA, in which exogenous DNA sequence (such as CAR gene) can be inserted to replace large adenovirus DNA, to prepare the expression vector of the present invention (Grunhaus and Horwitz, "Adenoviruses as cloning vectors," Seminars Virol., Vol. 3, pp. 237-252, 1992).

[0352] Another expression vector is based on adeno-associated virus (AAV), which utilizes an adenovirus coupling system. This AAV expression vector has a high frequency of integration into the host genome. It can even infect non-dividing cells, so it can be used, for example, in tissue culture or in vivo to deliver genes to mammalian cells. The infectivity of AAV vectors has a wide host range. Details about the production and use of AAV vectors are described in U.S. Patents Nos. 5,139,941 and 4,797,368.

[0353] Retroviral expression vectors can be integrated into the host genome, delivering a large amount of exogenous genetic material, infecting a wide range of species and cell types and packaging in special cell lines. Retroviral vectors are constructed by inserting nucleic acids (e.g., nucleic acids encoding CAR) into viral genomes to produce replication-defective viruses at certain positions. Although retroviral vectors can infect a variety of cell types, the integration and stable expression of CAR genes require the division of host cells.

[0354] Slow virus vector is derived from slow virus, and slow virus is a complex retrovirus, in addition to common retroviral genes gag, pol and env, it also contains other genes with regulatory or structural functions (US Patent No. 6,013,516 and No. 5,994,136). Some examples of slow virus include human immunodeficiency virus (HIV-1, HIV-2) and simian immunodeficiency virus (SIV). Slow virus vector is produced by repeatedly weakening HIV virulence genes, for example, genes env, vif, vpr, vpu and nef are deleted so that the vector is biologically safe. Slow virus vector can infect non-dividing cells, and can be used for in vivo and ex vivo gene transfer and expression of CAR genes (US Patent No. 5,994,136).

[0355] The expression vector comprising the conditionally active CAR gene can be introduced into the host cell by any method known to those skilled in the art. If necessary, the expression vector may include a viral sequence for transfection. Alternatively, the expression vector may be introduced by fusion, electroporation, biolistics, transfection, lipofection, etc. The host cell may be grown and amplified in culture before the expression vector is introduced, and then appropriately treated to introduce and integrate the vector. The host cell is then amplified and screened by markers present in the vector. A variety of markers that can be used include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, etc. The terms "cell", "cell line" and "cell culture" used herein may be used interchangeably. In some embodiments, the host cell is a T cell, a NK cell and a NKT cell.

[0356] On the other hand, the present invention also provides genetically engineered cytotoxic cells comprising and stably expressing the conditionally active CAR of the present invention. In one embodiment, the genetically engineered cells include T lymphocytes (T cells), initial T cells (TN), memory T cells (e.g., central memory T cells (TCM), effector memory T cells (TEM), natural killer cells, and macrophages capable of producing treatment-related offspring. In another embodiment, the genetically engineered cells are autologous cells. Examples of suitable T cells include CD4 + / CD8 - 、CD4 - / CD8 + 、CD4 - / CD8 - or CD4 + / CD8 + T cells. T cells can be CD4 + / CD8 - and CD4 - / CD8 + A mixed population of cells or a single clone. When in vitro with cells expressing the target antigen (e.g., CD20 + and / or CD19 + When co-cultured with tumor cells, the CD4 + T cells can also produce IL-2, IFN-γ, TNF-α and other T cell effector cytokines. + T cells can lyse cells expressing the target antigen. In some embodiments, the T cells can be CD45RA + CD62L + Any one or more of initial cells, CD45ROCD62I7 central memory cells, CD62L" effector memory cells or a combination thereof (Berger et al., "Adoptive transfer of virus-specific and tumor-specific T cell immunity," Curr. Opin. Immunol., Vol. 21, pp. 224-232, 2009).

[0357] Genetically engineered cytotoxic cells can be produced by stably transfecting host cells with an expression vector comprising the CAR gene of the present invention.Other methods using expression vector genetically engineered cytotoxic cells include chemical transformation methods (for example, using calcium phosphate, dendritic macromolecules, liposomes and / or cationic polymers), non-chemical transformation methods (such as electroporation, optical conversion, gene electrotransfer and / or hydrodynamic delivery) and / or particle-based methods (for example, using gene guns and / or magnetic transfection piercing infection (impalefection)).Prove that there is a single integrated unrearranged vector and expresses conditional active CAR transfected cells can be amplified in vitro.

[0358] Physical methods for introducing expression vectors into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). Chemical methods for introducing expression vectors into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0359] After the expression vector containing the CAR gene is introduced into the host cell, the CAR gene will be expressed to produce a CAR molecule that can bind to the target antigen. The produced CAR molecule becomes a transmembrane protein because it has a transmembrane domain. The host cell is then converted into a CAR cell, such as a CAR-T cell. The method for producing engineered cytotoxic cells such as CAR-T cells with CAR molecules has been described in, for example, Cartellieri et al., "Chimeric antigen receptor-engineered T cells for immunotherapy of cancer," Journal of Biomedicine and Biotechnology, Vol. 2010, Article ID 956304, 2010; and Ma et al., "Versatile strategy for controlling the specificity and activity of engineered T cells," PNAS, Vol. 113, E450-E458, 2016.

[0360] Whether before or after genetically modifying the cytotoxic cells to express the desired conditionally active CAR, the cells can be activated and expanded using methods described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and US20060121005. For example, the T cells of the present invention can be amplified by contacting a surface to which a reagent stimulating a CD3 / TCR complex-related signal is attached and to which a ligand stimulating a co-stimulatory molecule on the surface of the T cell is attached. Specifically, a T cell population can be stimulated by contacting an anti-CD3 antibody or its antigen-binding fragment or au anti-CD2 antibody fixed on the surface or by contacting a protein kinase C activator (e.g., bryostatin) bound to a calcium ion carrier. In order to co-stimulate the auxiliary molecules on the surface of the T cells, a ligand that binds to the auxiliary molecules is used. For example, T cells can be contacted with anti-CD3 antibodies and anti-CD28 antibodies under conditions suitable for stimulating T cell proliferation. In order to stimulate CD4 + T cells or CD8 + Proliferation of T cells using anti-CD3 antibodies and anti-CD28 antibodies. Examples of anti-CD28 antibodies include 9.3, B-T3 and XR-CD28 (Diaclone, Besancon, France), and these can be used in the present invention, and other methods generally known in the art can also be used in the present invention (Berg et al., Transplant Proc. 30 (8): 3975-3977, 1998; Haanen et al., J. Exp. Med. 190 (9): 13191328, 1999; Garland et al., J. Immunol. Meth. 227 (1-2): 53-63, 1999).

[0361] In various embodiments, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a therapeutically effective amount of a conditionally active CAR of the present invention. The conditionally active CAR in the composition can be any one or more of a polynucleotide encoding a CAR, a protein comprising a conditionally active CAR, or a genetically modified cell expressing a CAR protein. The CAR protein can be in the form of a pharmaceutically acceptable salt. Pharmaceutically acceptable salts refer to salts that can be used as salts of therapeutic proteins in the pharmaceutical industry, including salts of, for example, sodium, potassium, calcium, and the like, as well as ammonium salts of procaine, dibenzylamine, ethylenediamine, ethanolamine, methylglucosamine, taurine, and acid addition salts such as hydrochlorides and basic amino acids.

[0362] Pharmaceutically acceptable excipients can include any excipient for preparing pharmaceutical compositions, which are generally safe, nontoxic and desirable, and include veterinary use and acceptable excipients for human medicine. Such excipients can be solid, liquid, semisolid, or gaseous in the case of aerosol compositions. One type of excipient includes a pharmaceutically acceptable carrier, which can be added to enhance or stabilize the composition, or to facilitate the preparation of the composition. Liquid carriers include syrup, peanut oil, olive oil, glycerine, saline, alcohol and water. Solid carriers include starch, lactose, calcium sulfate, dihydrate, carclazyte, magnesium stearate or stearic acid, talc, pectin, gum arabic, agar and gelatin. Carriers can also include sustained-release materials, such as glyceryl monostearate or glyceryl distearate, which can be used alone or together with wax.

[0363] The pharmaceutically acceptable carrier is determined in part by the specific composition administered and the specific method used to administer the composition. Therefore, the pharmaceutical composition of the present invention has a variety of suitable formulations. A variety of aqueous carriers can be used, such as buffered saline, etc. These solutions are sterile and generally free of undesirable substances. These compositions can be sterilized by conventional, well-known sterilization techniques. The composition may contain pharmaceutically acceptable auxiliary substances required for approximate physiological conditions, such as pH adjusters and buffers, toxicity regulators, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The CAR concentration in these preparations can vary widely, and the CAR concentration is selected mainly based on the liquid volume, viscosity and body weight and according to the specific mode of administration selected and required by the patient.

[0364] In various embodiments, the pharmaceutical compositions of the present invention can be formulated for delivery by any suitable route of administration. "Route of administration" can refer to any route of administration known in the art, including but not limited to aerosol, nasal administration, oral administration, intravenous administration, intramuscular administration, intraperitoneal administration, inhalation, transmucosal administration, transdermal administration, parenteral administration, implantable pumps, continuous infusion, topical application, capsules and / or injections.

[0365] The pharmaceutical composition of the present invention can be encapsulated, tableted or prepared as an emulsion or syrup for oral administration. The pharmaceutical composition is prepared according to conventional techniques of pharmacy, which include grinding, mixing, granulation and tableting (when necessary) for tablet form; or grinding, mixing and filling for hard capsule form. When a liquid carrier is used, the preparation can be in the form of a syrup, elixir, emulsion or aqueous or non-aqueous suspension. Such liquid preparations can be directly administered orally or filled into soft gelatin capsules.

[0366] The pharmaceutical composition can be formulated as: (a) a liquid solution, such as an effective amount of encapsulated nucleic acid suspended in a diluent (e.g., water, saline or PEG400); (b) a capsule, sachet or tablet, each containing a predetermined amount of active ingredient, such as a liquid, solid, granules or gelatin; (c) a suspension in an appropriate liquid; and (d) a suitable emulsion. In particular, suitable dosage forms include, but are not limited to, tablets, pills, powders, dragees, capsules, liquids, lozenges, gels, syrups, slurries, suspensions, etc.

[0367] Solid preparations contain suitable solid excipients, such as carbohydrates or protein fillers, including, for example, sugars such as lactose, sucrose, mannitol or sorbitol; starch from corn, wheat, rice, potato or other plants; cellulose such as methylcellulose, hydroxypropyl methylcellulose and sodium carboxymethylcellulose; and gums, including gum arabic and tragacanth; and proteins, such as gelatin and collagen. Disintegrants or solubilizers may be added, such as cross-linked polyvinyl pyrrolidone, agar, alginic acid or its salts, such as sodium alginate. Tablet forms may include lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, potato starch, tragacanth, microcrystalline cellulose, gum arabic, gelatin, colloidal silicon dioxide, cross-linked sodium carboxymethylcellulose, talc, magnesium stearate, stearic acid and other excipients, colorants, fillers, binders, diluents, buffers, wetting agents, preservatives, flavorings, dyes, disintegrants and pharmaceutically acceptable carriers.

[0368] Liquid suspensions include conditionally active CAR, mixed with excipients suitable for preparing aqueous suspensions. Such excipients include suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth and gum arabic, and dispersants or wetting agents, such as naturally occurring phosphatides (such as lecithin), condensation products of alkylene oxide and fatty acids (such as polyoxyethylene stearate), condensation products of ethylene oxide and long-chain fatty alcohols (such as heptadecaethylene oxycetanol), condensation products of ethylene oxide and partial esters derived from fatty acids and hexitol (such as polyoxyethylene sorbitol monooleate) or condensation products of ethylene oxide and partial esters derived from fatty acids and hexitol anhydrides (such as polyoxyethylene sorbitan monooleate). Liquid suspensions may also contain one or more preservatives (e.g., ethyl or n-propyl p-hydroxybenzoate), one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, aspartame, or saccharin. The osmotic pressure of the formulation may be adjusted.

[0369] The lozenge form may include flavored active ingredients, typically sucrose and gum arabic or tragacanth, and lozenges containing in an inert matrix such as gelatin and glycerin or sucrose and gum arabic emulsions, gels, etc., in addition to the active ingredient, also contain carriers known in the art. It is known that when conditionally active CAR is administered orally, it must be protected from digestion. This is generally achieved by mixing the conditionally active CAR with a composition that makes it resistant to acid or enzyme hydrolysis or by encapsulating the conditionally active CAR in a suitable resistant carrier such as a liposome. Methods for protecting proteins from digestion are well known in the art. The pharmaceutical composition can be encapsulated, for example, in a liposome, or in a formulation, to slowly release the active ingredient.

[0370] Pharmaceutical compositions can be formulated as aerosols (e.g., they can be "nebulized") for administration by inhalation. Aerosols can be placed in pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, etc. Suitable rectal administration agents include, for example, suppositories consisting of nucleic acids encapsulated by a suppository base. Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons. In addition, it is also possible to use gelatin rectal capsules consisting of a combination of nucleic acids encapsulated by a base, the base including, for example, liquid triglycerides, polyethylene glycol, and paraffin hydrocarbons.

[0371] Pharmaceutical compositions can be formulated for parenteral administration, for example, by intraarticular (in the joint), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, including aqueous and non-aqueous isotonic sterile injections, which may contain antioxidants, buffers, thiobisdichlorophenol, and solutes that make the preparation isotonic with the blood received, as well as aqueous and non-aqueous sterile suspensions, which may include suspending agents, solubilizers, thickeners, stabilizers, and preservatives. In the operation of the present invention, the composition can be administered, for example, by intravenous infusion, oral, topical, intraperitoneal, intravesical, or intrathecal administration. In one aspect, the parenteral administration mode is a preferred method of administration for administering a composition containing CAR protein and genetically engineered cytotoxic cells. The composition can be conveniently administered in unit dosage form and can be prepared by any well-known method in the pharmaceutical field, for example, as described in "Remington's Pharmaceutical Sciences", Mack Publishing Co. Easton Pa., 18 th Ed., 1990. Preparations for intravenous administration may contain pharmaceutical carriers such as sterile water or saline, polyalkylene glycols such as polyethylene glycol, vegetable oils, hydrogenated naphthalene, and the like.

[0372] The pharmaceutical composition can be administered by at least one mode selected from the group consisting of parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraperitoneal, intracapsular, intracartilaginous, intracavitary, intracavitary, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, pill, vaginal, rectal, oral, sublingual, intranasal or transdermal administration. The method may optionally further include administering at least one composition containing an effective amount of at least one compound or protein selected from the following substances before, simultaneously with, or after administering the conditionally active CAR: a detectable marker or reporter factor, a TNF antagonist, an anti-rheumatic drug, a muscle relaxant, an anesthetic, a non-steroidal anti-inflammatory drug (NSAK), an analgesic, an anesthetic, a sedative, a local anesthetic, a neuromuscular blocker, an antibacterial agent, an antipsoriatic agent, a corticosteroid, an anabolic steroid, erythropoietin, an immunization, an immunoglobulin, an immunosuppressive agent, a growth hormone, a hormone replacement drug, a radiopharmaceutical, an antidepressant, an antipsychotic drug, a stimulant, an asthma drug, a beta agonist, an inhaled steroid, epinephrine or its analogs, a cytotoxic agent or other anticancer agent, an antimetabolite such as methotrexate or an antiproliferative agent.

[0373] The types of cancers treated with the genetically engineered cytotoxic cells or pharmaceutical compositions of the present invention include carcinomas, blastomas, and sarcomas, as well as certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignancies, such as sarcomas, carcinomas, and melanomas. Cancers can be non-solid tumors (e.g., hematological tumors) or solid tumors. Also included are adult tumors / cancers and pediatric tumors / cancers.

[0374] Hematological cancer is a cancer of the blood or bone marrow. Examples of hematological (or hematological) cancers include leukemias, including acute leukemias (e.g., acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia), chronic leukemias (e.g., chronic myeloid leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and advanced forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0375] Solid tumors are abnormal masses of tissue that usually do not contain cysts or areas of fluid. Solid tumors can be benign or malignant. Different types of solid tumors (such as sarcomas, carcinomas, and lymphomas) are named after the type of cells that form the solid tumor. Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumors, seminoma, bladder cancer, melanoma, and CNS tumors (e.g., gliomas (such as brain stem gliomas and mixed gliomas), glioblastomas (also known as glioblastomas) multiforme), astrocytoma, CNS lymphoma, germ cell tumor, medulloblastoma, schwannoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastases).

[0376] The present invention also provides a medical device comprising at least one CAR protein, a polynucleotide sequence encoding CAR, or a host cell expressing CAR, wherein the device is suitable for administering the at least one conditionally active CAR by at least one selected from the following modes: parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraperitoneal, intracapsular, intracartilaginous, intracavitary, intracavitary, intracerebellar, intraventricular, intracolonic, intrauterine, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, oral, sublingual, intranasal or transdermal.

[0377] On the other hand, the present invention provides a kit, which comprises at least one CAR protein in a lyophilized form in a first container, a polynucleotide sequence encoding CAR or a host cell expressing CAR, and an optional second container, which comprises sterile water, sterile buffered water or at least one preservative, selected from phenol, meta-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride, alkyl parabens, benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal or its mixture in an aqueous diluent. On the one hand, in the kit, the concentration of the conditionally active CAR or a specific part or variant in the first container is restored (reconstituted) to a concentration of about 0.1 mg / ml to about 500 mg / ml using the contents of the second container. On the other hand, the second container also includes an isotonic agent. On the other hand, the second container also includes a physiologically acceptable buffer. On the one hand, the present invention provides a method for treating at least one wild-type protein-mediated disease, including administering to a patient in need thereof a preparation provided in a kit and restored before administration.

[0378] Also provided is a product for pharmaceutical use or diagnostic use, comprising packaging materials and containers, the container comprising at least one CAR protein, a polynucleotide sequence encoding CAR, or a solution or lyophilized form of a host cell expressing CAR. The product may optionally include using the container as a component of the following delivery device or system: parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracavitary, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intrabone, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial cavity, intrathoracic, intrauterine, intravesical, pill, vaginal, rectal, oral, sublingual, intranasal or transdermal delivery device or system.

[0379] In some embodiments, the present invention provides a method comprising obtaining cytotoxic cells from an individual, engineering the cytotoxic cells by introducing the CAR gene of the present invention into the cytotoxic cell gene, and administering the genetically engineered cytotoxic cells to the individual. In some embodiments, the cytotoxic cells are selected from T cells, initial T cells, memory T cells, effector T cells, natural killer cells and macrophages. In one embodiment, the cytotoxic cells are T cells.

[0380] In one embodiment, T cells are obtained from individuals. T cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue and tumor. In certain embodiments of the present invention, any number of T cell lines available in the art can be used. In certain embodiments of the present invention, any number of techniques known to those skilled in the art (e.g., Ficoll TM Isolation) obtains T cells from blood obtained from an individual.

[0381] In a preferred embodiment, cells from the circulating blood of an individual are obtained by apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one embodiment, cells collected by apheresis can be washed to remove the plasma portion and the cells are placed in an appropriate buffer or culture medium for subsequent processing steps. In one embodiment of the invention, cells are washed with phosphate buffered saline (PBS). In alternative embodiments, the washing solution lacks calcium and may lack magnesium or may lack many (if not all) divalent cations. In addition, surprisingly, the initial activation step in the absence of calcium leads to amplified activation. As those of ordinary skill in the art will readily appreciate, the washing step can be performed by methods known to those skilled in the art, such as by using a semi-automatic "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter CytoMate or blood cell recovery instrument (Haemonetics Cell Saver) 5) and in accordance with the manufacturer's instructions. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as those without Ca. 2+ And does not contain Mg 2+ Alternatively, the apheresis sample can be freed of undesirable components and the cells resuspended directly in culture medium.

[0382] In another embodiment, the erythrocytes are lysed and the mononuclear cells are depleted (e.g., by PERCOLL TM T cells can be isolated from peripheral blood by gradient centrifugation or by counterflow elutriation centrifugation. Specific T cell subsets, such as CD3 + 、CD28 + 、CD4 + 、CD8 + 、CD45RA + and CD45RO +T cells. For example, enrichment of a T cell population by negative selection can be achieved using a combination of antibodies directed against surface markers unique to the negatively selected cells. One approach is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on the negatively selected cells. To enrich for CD4 + For cells, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In certain embodiments, it may be desirable to enrich or positively select for cells that normally express CD4 + 、CD25 + 、CD62L hi GITR + and FoxP3 + of regulatory T cells.

[0383] For example, in one embodiment, beads (eg, M-450 CD3 / CD28 T) is incubated for a period of time sufficient to positively select the desired T cells to separate T cells. In one embodiment, the period is about 30 minutes. In another embodiment, the period ranges from 30 minutes to 36 hours or longer, and all integer values ​​therebetween. In another embodiment, the period is at least 1, 2, 3, 4, 5 or 6 hours. In another preferred embodiment, the period is 10-24 hours. In a preferred embodiment, the incubation period is 24 hours. In order to separate T cells from patients with leukemia, using a longer incubation time (e.g., 24 hours) can increase the cell yield. In any case where T cells are less than other cell types, such as when separating tumor infiltrating lymphocytes (TIL) from tumor tissue or immunocompromised individuals, longer incubation times can be used to separate T cells. In addition, using longer incubation times can increase the capture of CD8 + The efficiency of T cell.Therefore, by simply shortening or extending the time when T cell is combined with CD3 / CD28 beads and / or by increasing or reducing the ratio of beads and T cells (as further described in this article), at the beginning of cultivation or at other time points in the process, T cell subpopulations for which can be preferentially selected.In addition, by increasing or reducing the ratio of CD3 antibodies and / or CD28 antibodies on beads or other surfaces, at the beginning of cultivation or at other required time points, T cell subpopulations for which can be preferentially selected.Technicians will recognize that multiple rounds of selection can also be adopted in the case of the present invention.In certain embodiments, it can be expected to perform a selection process and use "unselected" cells in activation and amplification processes.Also "unselected" cells can be experienced in other rounds of selection.

[0384] The obtained cytotoxic cells are then genetically engineered as described herein. The polynucleotides encoding conditional active CAR (generally located in an expression vector) are introduced into cytotoxic cells so that the cytotoxic cells will express, preferably stably express CAR. The polynucleotides encoding CAR are generally integrated into the cytotoxic cell host genome. In some embodiments, the polynucleotide introduction does not need to cause integration, but it is sufficient to only instantaneously keep the introduced polynucleotides. In this way, it is possible to have a short-term effect, wherein cytotoxic cells can be introduced into the host, and then opened after a predetermined time, for example, after the cell has been able to migrate to a specific treatment site.

[0385] According to the properties of cytotoxic cells and the disease to be treated, genetically engineered cytotoxic cells can be introduced into individuals (e.g., mammals) in various ways. Genetically engineered cytotoxic cells can be introduced into tumor sites. In one embodiment, genetically engineered cytotoxic cells guide cancer or are modified to guide cancer. The quantity of genetically engineered cytotoxic cells used will depend on many factors, such as environment, the purpose of introduction, the life span of cells, the scheme used (e.g., the number of administrations), the ability of cell proliferation and the stability of the recombinant construct. Genetically engineered cytotoxic cells can be used as dispersions, injected at or near the site of interest. The cell can be in a physiologically acceptable culture medium.

[0386] It will be appreciated that treatment methods are affected by many variables, such as cellular response to the CAR, efficiency of CAR expression by cytotoxic cells and, as appropriate, secretion levels, activity of the expressed CAR, specific needs of the individual (which may vary over time and circumstances), rate of cell activity loss due to loss of genetically engineered cytotoxic cells or loss of expression activity by individual cells, etc. Therefore, it is desirable to monitor each individual patient for appropriate dosages for each individual, even if there are universal cells that can be administered to the entire population, and the practice of monitoring patients is routine in the art.

[0387] The following examples are illustrative of the methods of the present invention rather than limiting. Other suitable modifications and adjustments of the various conditions and parameters normally encountered in the art and obvious to those skilled in the art are within the scope of the present invention. Example

[0388] Example 1: Generation of scFv conditionally active antibodies against Axl

[0389] Two conditionally active single-chain antibodies (CAB-scFv-63.9-4 and CAB-scFv-63.9-6) against the drug target antigen Axl were expressed as homodimers with wild-type human IgG1 Fc, SEQ ID NO: 13 or 14, (obtained Figure 2 and Figure 3 The bivalent antibodies CAB-scFv-63.9-4-01, SEQ ID NO: 9 and CAB-scFv-63.9-6-01, SEQ ID NO: 10) in the "knob-and-mortar" system were expressed as heterodimers to produce monovalent scFvs (obtained Figure 2 and Figure 3 The monovalent antibody scFvs CAB-scFv-63.9-4-02, SEQ ID NO: 11 and CAB-scFv-63.9-6-02, SEQ ID NO: 12).

[0390] The binding affinity of these antibodies to the drug target antigen Axl at pH 6.0 and pH 7.4 was measured by ELISA assay. Figure 2 As shown in Figure 5, the scFv antibody showed comparable affinity to the drug target antigen Axl as full bivalent antibodies at both pH 6.0 and pH 7.4. Figure 3 The selectivity of these scFv antibodies shown at pH 6.0 and pH 7.4 is also comparable to that of the full bivalent antibody. This embodiment demonstrates that the conditionally active antibodies of the present invention have affinity and selectivity comparable to that of scFv antibodies or full bivalent antibodies. Therefore, the conditionally active antibodies of the present invention can be inserted as a single DNA chain into the DNA molecule encoding the CAR in the CAR-T platform of the present invention.

[0391] Example 2: scFv antibody against target antigen Axl for constructing CAR-T cells

[0392] According to one embodiment of the present invention, selectivity and affinity and expression level under pH 6.0 and pH 7.4 are screened simultaneously to produce conditional active antibodies for drug target antigen Axl. FLAG tags are used to screen in serum, because there are human antibodies that may cause false positive screening in serum. The screening buffer is carbonate buffer (krebs buffer with Ringer-standard buffer, but different from PBS). It is found that the conditional active antibodies produced have higher affinity to drug target antigen Axl compared with wild-type antibodies at pH 6.0, but have lower affinity to the same drug target antigen Axl compared with wild-type antibodies at pH 7.4. In addition, these conditional active antibodies all have high expression levels as shown in Table 2 below, wherein "clone" column shows antibody, and expression level "mg / ml" is displayed in the second column.

[0393] The clones of these antibodies were sent to the service provider along with the required expression levels ("amount ordered", expected expression levels). However, the actual expression levels of these antibodies ("amount delivered") were very high and exceeded the expected expression levels.

[0394] Table 2 Conditionally active antibodies with high expression levels

[0395]

[0396] Taking the BAP063.9-13-1 antibody as an example, the conditionally active antibody does not show aggregation in the buffer, e.g. Figure 4 As shown. BAP063.9-13-1 antibody was analyzed by size exclusion chromatography. Figure 4 In the assay, only one peak was detected, indicating little or no aggregation of the antibody.

[0397] Conditionally active antibodies are also measured using surface plasmon resonance (SPR) to measure their binding and dissociation rates to the drug target antigen Axl. Known SPR assays are used to measure the binding and dissociation rates of conditionally active antibodies. SPR assays are performed in the presence of bicarbonate. The in vivo binding and dissociation rates of conditionally active antibodies (in animals and humans) are very important features of conditionally active antibodies.

[0398] It was observed that the conditionally active antibody had a fast on-rate at pH 6.0 and a slower on-rate at pH 7.4 compared to the negative control (BAP06310F10, which had similar on-rates at pH 6.0 and pH 7.4) ( Figure 5 ). In addition, increasing the temperature from room temperature to 60°C did not significantly change the SPR measurement results ( Figure 5SPR assays also showed that these conditionally active antibodies were highly selective at pH 6.0 compared to pH 7.4 ( Figure 6A-6B One antibody is shown as an example).

[0399] The conditionally active biological antibodies are summarized in Table 3. Two of the antibodies are expressed as scFv (BAP063.9-13.3 and BAP063.9-48.3), which are ready to be inserted into the CAR in the CAR-T platform. The antibodies were incubated at 60 ° C for 1 hour without changing the affinity ("thermal stability") of most antibodies. In the two columns of reported data (the last two columns of Table 3) measuring binding activity at pH 6.0 and pH 7.4 using SPR, it was compared with "BAP063.6-hum10F10-FLAG" (negative control, second row of Table 3). The selectivity of these antibodies can be determined by the difference between the data in the last two columns. Two scFv antibodies have very high selectivity (75% and 50% at pH 6 compared to 0% at pH 7.4).

[0400]

[0401] Comparative Example A: CAR-T cells with non-conditionally active antibodies against target antigen Axl

[0402] Use non-conditionally active scFv antibodies against the target antigen Axl to construct CAR-T cells that bind to the target antigen Axl or CHO cells (CHO-Axl) that express the target antigen Axl on the cell surface. Figure 7A-7B ASTR using non-conditionally active antibodies as CAR molecules was inserted into T cells to construct CAR-T cells that can bind to the target antigen Axl.

[0403] For comparison, CHO cells that do not express the target antigen Axl were treated with: (1) T cells not transduced with a CAR molecule, (2) T cells transduced with a CAR molecule that does not bind to the target antigen Axl, and (3) T cells transduced with a CAR molecule that has an unconditionally active antibody against the target antigen Axl ( Fig. 7A ). CHO cell populations are characterized by the Cell Index ( Fig. 7A ), a decrease in the Cell Index indicates cytotoxicity (cell killing) caused by CAR-T cells.

[0404] refer to Fig. 7A, before the addition of T cells, CHO cells showed growth. After the addition of CAR-T cells that bind to the target antigen Axl, the cell index initially decreased, indicating nonspecific cytotoxicity of T cells. However, shortly thereafter, CHO cells resumed growth. More importantly, the differences between the three treatments were negligible, indicating that CAR-T cells with non-conditionally active antibodies against the target antigen Axl had no significant cytotoxicity against CHO cells that did not express the target antigen Axl.

[0405] CHO cells expressing the target antigen Axl were then treated in the same manner as above: (1) T cells not transduced with CAR molecules, (2) T cells transduced with CAR molecules that do not bind to the target antigen Axl, and (3) T cells transduced with CAR molecules with non-conditionally active antibodies against the target antigen Axl ( Figure 7B ). After the addition of T cells, the cell index was significantly reduced by treatment with CAR cells with unconditional active antibodies against the target antigen Axl, while the cell index was not reduced by the other two treatments, indicating that CAR cells with unconditional active antibodies against the target antigen Axl were cytotoxic to CHO-X1 cells expressing the target antigen Axl.

[0406] Example 3: CAR-T cells with conditionally active scFv antibodies against target antigen Axl

[0407] Conditionally active scFv antibodies against target antigen Axl are used to construct CAR molecules. T cells are transduced with CAR molecules so that T cells express CAR molecules (CAR-T cells). CHO cells (CHO-63 cells) expressing target antigen Axl or conventional CHO cells (CHO cells) not expressing target antigen Axl are treated with CAR-T cells. Untransduced T cells (without CAR molecules) are used as controls ( Figures 8A-8B ).

[0408] refer to Fig. 8A , CHO cells that do not express the target antigen Axl were treated with CAR-T cells and non-transduced T cells. There was no significant difference between the two treatments, indicating that CAR-T cells have no cytotoxicity to CHO cells. Figure 8B . In which CHO cells (CHO-63) expressing the target antigen Axl were treated in a similar manner, CAR-T cells with conditionally active antibodies against the target antigen Axl significantly reduced the CHO-63 cell population compared to untransduced T cells. This suggests that CAR-T cells with conditionally active antibodies against the target antigen Axl are cytotoxic to CHO-63 cells.

[0409] Once bound to the target antigen Axl, CAR-T cells induced cytotoxicity. This effect was confirmed by measuring the levels of cytokines interferon gamma (INFg) and IL2. Cytokine data are shown in Figures 9A-9B In. Fig. 9A In the study, the binding of CAR-T cells to the target antigen Axl on CHO-63 cells triggered a significant release of INFg, as indicated by the increased cytokine levels observed, compared with non-transduced T cells. Fig. 9B In the present study, the binding of CAR-T cells to the target antigen Axl on CHO-63 cells triggered a significant release of IL2, as indicated by the observed increased cytokine levels, compared with non-transduced T cells.

[0410] Example 4: CAR-T cells with conditionally active scFv antibodies against target antigen ROR2

[0411] Conditionally active scFv antibodies against the target antigen ROR2 were generated. Their binding activity to the target antigen ROR2 was measured using an ELISA assay ( Fig.10 ).

[0412] Fig.10 One of the scFv antibodies shown in , scFv-116101, was used to construct a CAR molecule for generating CAR-T cells (116101CAR-T). The constructed CAR-T cells were used to target Daudi cells expressing the target antigen ROR2. Negative controls were T cells that were not transduced with CAR molecules (non-transduced T cells) and CAR-T cells that were transduced with CAR molecules that could not bind to the target antigen ROR2 (non-ROR2 scFv CAR-T). The results are shown in Fig.11A The ratio of T cell numbers to Daudi cell numbers in these treatments was 10:1. CAR-T cells (116101CAR-T) with scFv antibodies targeting the target antigen ROR2 on Daudi cells induced significant cell death of Daudi cells, as shown in Fig.11A This is shown by the higher dead cell / live cell ratio in the

[0413] HEK293 cells were treated with the same T cells used to treat Daudi cells. The results are shown in Fig. 11B Since HEK293 cells do not express the target antigen ROR2 on the cell surface, CAR-T cells (116101CAR-T) with scFv antibodies against the target antigen ROR2 do not induce significant cell death in HEK293 cells compared with the negative control ( Fig. 11B ).

[0414] Example 5: Cytokine release of CAR-T cells with antibodies against target antigens Axl and ROR2 in Examples 1-4

[0415] In this example, cytokine release induced by binding of CAR-T cells to target antigens was measured. Figures 12A-12B The release of INFg and IL2 after binding of CAR-T cells containing conditionally active scFv antibodies to the target antigen Axl to CHO-63 cells expressing the target antigen Axl is shown. After 24 hours of treatment of CAR-T cells with these CHO-63 cells, the levels of INFg and IL2 cytokines were significantly increased compared to the control in which the same CAR-T cells were used to treat CHO cells that did not express the target antigen Axl, indicating the release of INFg and IL2 cytokines. In addition, T cells not transduced with CAR molecules and CAR-T cells not bound to the target antigen Axl did not result in significant release of INFg and IL2 cytokines.

[0416] Figures 13A-13B The INFg and IL cytokine levels after CAR-T cells containing conditionally active scFv antibodies against the target antigen ROR2 are combined with Rajib cells and Daudi cells expressing the target antigen ROR2 are shown. After 24 hours of treatment of Rajib cells and Daudi cells with CAR-T cells, a significant increase in INFg and IL2 cytokine levels was observed compared to the control in which the same CAR-T cells were used to treat HEK293 cells that did not express the target antigen ROR2. In addition, T cells that were not transduced with CAR molecules and CAR-T cells that were not combined with the target antigen ROR2 did not significantly increase cytokine levels, indicating that the significant release of INFg and IL2 cytokines could not be induced.

[0417] Example 6: Conditionally active scFv antibody against target antigen CD22

[0418] Five conditionally active scFv antibodies against the target antigen CD22 were selected. The selected conditionally active scFv antibodies were more active at pH 6.0 than at pH 7.4. These conditionally active scFv antibodies can be used to construct CAR-T cells that bind to cells expressing the target antigen CD22.

[0419] It should be understood that although numerous features and advantages of the present invention and details of its structure and function have been set forth in the foregoing description, this disclosure is illustrative only and that detailed changes may be made within the principles of the present invention, especially in the shape, size and arrangement of parts, within the full scope indicated by the broad general meaning of the terms as expressed by the appended claims. Sequence Listing <110> BioAtla, LLC <120> Conditionally active chimeric antigen receptors for modified T cells <130> KHP212110206.5 <160> 16 <170> PatentIn version 3.5 <210> 1 <211> 1437 <212> DNA <213> Artificial <220> <223> Synthetic sequence <400> 1 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60 acctgcactg tctctggcta cgtttttat agctactggc tgcactggat ccgccagccc 120 ccagggaagg ggctggagtg gattggttac attaatccta ggaatgatta tactgagtac 180 aatcggattt tcaaggggag agtcacgatt accgcggaca aatccacgag cacagcctac 240 atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagaaggggg 300 attactacgt tctactgggg ccagggaacg ctggtcaccg tcagctcagg cggcggagga 360 agcggcggtg gatccggagg aggaggctct gaaattgtgt tgacacagtc tccagccacc 420 ctgtctttgt ctccagggga aagagccacc ctctcctgca agtccagtca aagtgtttta 480 tacagtgcag tggagaagaa ctacttggcc tggtaccagc agaaacctgg ccaggctccc 540 aggctcctca tctattgggc atccactagg gaaagggga tcccagacag gttcagtggc 600 agtgggtctg ggacagactt cactctcacc atcagcagac tggagcctga agattttgca 660 720 atcaaacgta cggacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg 780 ggaccgtcag tcttctctctt ccccccaaaaa cccaaggaca ccctcatgat ctcccggacc 840 cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 900 tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 960 aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1020 aaggagtca agtgcaaggt cagcaacaaa gccctcccag cccccatcga gaaaccatc 1080 tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 1140 gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1200 atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1260 gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1320 tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1380 acgcagaaga gcctctccct gtctccgggt aaagattaca aggatgacga cgataag 1437 <210> 2 <211> 1437 <212> DNA <213> Artificial <220> <223> Synthetic sequence <400> 2 gaggtccagc tggtacagtc tggggctgag gtgaagaagc ctggggctac agtgaaaatc 60 tcctgcaagg tttctggtta ctcattcact ggcgctacca tgaactggat ccgccagccc 120 ccagggaagg ggctggagtg gattggtctt attaaacctt ccaatggtgg tactagttac 180 aaccagaagt tcaagggcag agtcaccatc tcagccgaca agtccatcag caccgcctac 240 ctgcagtgga gcagcctgaa ggcctcggac accgccatgt attactgtgc acatggtcac 300 tacgagagtt acgaggctat ggactactgg ggccagggaa cgctggtcac cgtcagctca 360 ggcggcggag gaagcggcgg tggatccgga ggaggaggct ctgacatcca gatgacccag 420 tctccatcct ccctgtctgc atctgtagga gacagagtca ccatcacttg caaggccagt 480 caggatgtgg tttctgctgt agcctggtac cagcagaaac ctggccaggc tcccaggctc 540 ctcatctatt ggcaggatac ccggcacact ggagtcccat caaggttcag cggcagtgga 600 tctgggacag aattcactct caccatcagc agcctgcagc ctgatgattt tgcaacttat 660 tactgtcagg aacattttag cactccgctc acgttcggcc aagggaccaa ggtggaaatc 720 aaacgtacga cggacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg 780 ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 840 cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 900 tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 960 aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1020 aaggagtaca agtgcaaggt cagcaacaaa gccctcccag cccccatcga gaaaaccatc 1080 tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 1140 gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1200 atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1260 gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1320 tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1380 acgcagaaga gcctctccct gtctccgggt aaagattaca aggatgacga cgataag 1437 <210> 3 <211> 1437 <212> DNA <213> Artificial <220> <223> Synthetic sequence <400> 3 gaggtccagc tggtacagtc tggggctgag gtgaagaagc ctggggctac agtgaaaatc 60 tcctgcaagg tttctggtta ctcattctgg ggcgctacca tgaactggat ccgccagccc 120 ccagggaagg ggctggagtg gattggtctt attaaacctt ccaatggtgg tactagttac 180 aaccagaagt tcaagggcag agtcaccatc tcagccgaca agtccatcag caccgcctac 240 ctgcagtgga gcagcctgaa ggcctcggac accgccatgt attactgtgc acatggtcac 300 tacgagagtt acgaggctat ggactactgg ggccagggaa cgctggtcac cgtcagctca 360 ggcggcggag gaagcggcgg tggatccgga ggaggaggct ctgacatcca gatgacccag 420 tctccatcct ccctgtctgc atctgtagga gacagagtca ccatcacttg caaggccagt 480 caggatgtgg tttctgctgt agcctggtac cagcagaaac ctggccaggc tcccaggctc 540 ctcatctatt ggcaggatac ccggcacact ggagtcccat caaggttcag cggcagtgga 600 tctgggacag aattcactct caccatcagc agcctgcagc ctgatgattt tgcaacttat 660 tactgtcagg aacattttag ccctccgctc acgttcggcc aagggaccaa ggtggaaatc 720 aaacgtacga cggacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg 780 ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 840 cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 900 tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 960 aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1020 aaggagtaca agtgcaaggt cagcaacaaa gccctcccag cccccatcga gaaaaccatc 1080 tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 1140 gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1200 atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1260 gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1320 tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1380 acgcagaaga gcctctccct gtctccgggt aaagattaca aggatgacga cgataag 1437 <210> 4 <211> 1437 <212> DNA <213> Artificial <220> <223> Synthetic sequence <400> 4 gaggtccagc tggtacagtc tggggctgag gtgaagaagc ctggggctac agtgaaaatc 60 tcctgcaagg tttctggtta ctcattcact ggcgctacca tgaactggat ccgccagccc 120 ccagggaagg ggctggagtg gattggtctt attaaacctt ccaatggtgg tactagttac 180 aaccagaagt tcaagggcag agtcaccatc tcagccgaca agtccatcag caccgcctac 240 ctgcagtgga gcagcctgaa ggcctcggac accgccatgt attactgtgc acatggtcac 300 tacgagagtt acgaggctat ggactactgg ggccagggaa cgctggtcac cgtcagctca 360 ggcggcggag gaagcggcgg tggatccgga ggaggaggct ctgacatcca gatgacccag 420 tctccatcct ccctgtctgc atctgtagga gacagagtca ccatcacttg caaggccagt 480 caggatgtgg tttctgctgt agcctggtac cagcagaaac ctggccaggc tcccaggctc 540 ctcatctatt ggcaggatac ccggcacact ggagtcccat caaggttcag cggcagtgga 600 tctgggacag aattcactct caccatcagc agcctgcagc ctgatgattt tgcaacttat 660 tactgtcagg aacattttag cactccgctc acgttcggcc aagggaccaa ggtggaaatc 720 aaacgtacga cggacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg 780 ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 840 cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 900 tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 960 aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1020 aaggagtaca agtgcaaggt cagcaacaaa gccctcccag cccccatcga gaaaaccatc 1080 tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 1140 gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1200 atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1260 gtgctggact ccgacggctc cttcttcctc accagcaagc tcaccgtgga caagagcagg 1320 tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1380 acgcagaaga gcctctccct gtctccgggt aaagattaca aggatgacga cgataag 1437 <210> 5 <211> 1437 <212> DNA <213> Artificial <220> <223> Synthetic sequence <400> 5 gaggtccagc tggtacagtc tggggctgag gtgaagaagc ctggggctac agtgaaaatc 60 tcctgcaagg tttctggtta ctcattctgg ggcgctacca tgaactggat ccgccagccc 120 ccagggaagg ggctggagtg gattggtctt attaaacctt ccaatggtgg tactagttac 180 aaccagaagt tcaagggcag agtcaccatc tcagccgaca agtccatcag caccgcctac 240 ctgcagtgga gcagcctgaa ggcctcggac accgccatgt attactgtgc acatggtcac 300 tacgagagtt acgaggctat ggactactgg ggccagggaa cgctggtcac cgtcagctca 360 ggcggcggag gaagcggcgg tggatccgga ggaggaggct ctgacatcca gatgacccag 420 tctccatcct ccctgtctgc atctgtagga gacagagtca ccatcacttg caaggccagt 480 caggatgtgg tttctgctgt agcctggtac cagcagaaac ctggccaggc tcccaggctc 540 ctcatctatt ggcaggatac ccggcacact ggagtcccat caaggttcag cggcagtgga 600 tctgggacag aattcactct caccatcagc agcctgcagc ctgatgattt tgcaacttat 660 tactgtcagg aacattttag ccctccgctc acgttcggcc aagggaccaa ggtggaaatc 720 aaacgtacga cggacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg 780 ggaccgtcag tcttctctctt ccccccaaaaa cccaaggaca ccctcatgat ctcccggacc 840 cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 900 tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 960 aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1020 aaggagtca agtgcaaggt cagcaacaaa gccctcccag cccccatcga gaaaccatc 1080 tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 1140 gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1200 atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1260 gtgctggact ccgacggctc cttcttcctc accagcaagc tcaccgtgga caagagcagg 1320 tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1380 1437 <210> 6 <211> 681 <212> DNA <213> Artificial <220> <223> Synthetic sequence <400> 6 gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 60 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300 tgcaaggtca gcaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 420 aaccaggtca gcctgtactg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 600 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 660 ctctccctgt ctccgggtaa a 681 <210> 7 <211> 681 <212> DNA <213> Artificial <220> <223> Synthetic sequence <400> 7 gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 60 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300 tgcaaggtca gcaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 420 aaccaggtca gcctgtactg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 600 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 660 ctctccctgt ctccgggtaa a 681 <210> 8 <211> 479 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 8 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Val Phe Thr Ser Tyr 20 25 30 Trp Leu His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Arg Asn Asp Tyr Thr Glu Tyr Asn Arg Ile Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Gly Ile Thr Thr Phe Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser 130 135 140 Pro Gly Glu Arg Ala Thr Leu Ser Cys Lys Ser Ser Gln Ser Val Leu 145 150 155 160 Tyr Ser Ala Val Glu Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro 165 170 175 Gly Gln Ala Pro Arg Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Arg 180 185 190 Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 195 200 205 Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys 210 215 220 Lys Gln Tyr Leu Ser Ser Trp Thr Phe Gly Gln Gly Thr Lys Val Glu 225 230 235 240 Ile Lys Arg Thr Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 370 375 380 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys Asp Tyr Lys Asp Asp Asp Asp Lys 465 470 475 <210> 9 <211> 479 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 9 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Ser Phe Thr Gly Ala 20 25 30 Thr Met Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Lys Pro Ser Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala His Gly His Tyr Glu Ser Tyr Glu Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 130 135 140 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser 145 150 155 160 Gln Asp Val Val Ser Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln 165 170 175 Ala Pro Arg Leu Leu Ile Tyr Trp Gln Asp Thr Arg His Thr Gly Val 180 185 190 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr 195 200 205 Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Glu 210 215 220 His Phe Ser Thr Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 225 230 235 240 Lys Arg Thr Thr Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 370 375 380 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys Asp Tyr Lys Asp Asp Asp Asp Lys 465 470 475 <210> 10 <211> 479 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 10 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Ser Phe Trp Gly Ala 20 25 30 Thr Met Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Lys Pro Ser Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala His Gly His Tyr Glu Ser Tyr Glu Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 130 135 140 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser 145 150 155 160 Gln Asp Val Val Ser Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln 165 170 175 Ala Pro Arg Leu Leu Ile Tyr Trp Gln Asp Thr Arg His Thr Gly Val 180 185 190 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr 195 200 205 Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Glu 210 215 220 His Phe Ser Pro Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 225 230 235 240 Lys Arg Thr Thr Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 370 375 380 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys Asp Tyr Lys Asp Asp Asp Asp Lys 465 470 475 <210> 11 <211> 479 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 11 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Ser Phe Thr Gly Ala 20 25 30 Thr Met Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Lys Pro Ser Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala His Gly His Tyr Glu Ser Tyr Glu Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 130 135 140 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser 145 150 155 160 Gln Asp Val Val Ser Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln 165 170 175 Ala Pro Arg Leu Leu Ile Tyr Trp Gln Asp Thr Arg His Thr Gly Val 180 185 190 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr 195 200 205 Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Glu 210 215 220 His Phe Ser Thr Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 225 230 235 240 Lys Arg Thr Thr Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 370 375 380 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Thr Ser 420 425 430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys Asp Tyr Lys Asp Asp Asp Asp Lys 465 470 475 <210> 12 <211> 479 <212> PRT <213> Artificial <220> <223> Synthetic sequence <400> 12 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Ser Phe Trp Gly Ala 20 25 30 Thr Met Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Lys Pro Ser Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala His Gly His Tyr Glu Ser Tyr Glu Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 130 135 140 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser 145 150 155 160 Gln Asp Val Val Ser Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln 165 170 175 Ala Pro Arg Leu Leu Ile Tyr Trp Gln Asp Thr Arg His Thr Gly Val 180 185 190 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr 195 200 205 Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Glu 210 215 220 His Phe Ser Pro Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 225 230 235 240 Lys Arg Thr Thr Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val ...

Claims

1. A chimeric antigen receptor for binding to a tumor-specific target antigen, comprising: i. at least one antigen-specific targeting region, the antigen-specific targeting region evolved from a parent or wild-type protein or a domain thereof, and having the following properties: the activity of the antigen-specific targeting region in a determination under normal physiological conditions is decreased compared to the activity in a determination under abnormal conditions that deviate from the normal physiological conditions; ii. a transmembrane domain; and iii. Intracellular signaling domain, wherein the tumor-specific target antigen is Axl, and the at least one antigen-specific targeting region is a single-chain antibody having an amino acid sequence selected from SEQ ID NOs: 9-12, or The tumor-specific target antigen is ROR2, and the at least one antigen-specific targeting region is a single-chain antibody having an amino acid sequence of SEQ ID NO:

15.

2. The chimeric antigen receptor of claim 1, wherein the tumor-specific target antigen is Axl, and the at least one antigen-specific targeting region is a single-chain antibody having an amino acid sequence selected from SEQ ID NOs: 9-12.

3. The chimeric antigen receptor of claim 1, wherein the tumor-specific target antigen is ROR2, and the at least one antigen-specific targeting region is a single-chain antibody having the amino acid sequence of SEQ ID NO:

15.

4. The chimeric antigen receptor of claim 1, wherein the normal physiological condition is the normal physiological pH in the plasma of a mammalian individual, and the abnormal condition is the pH in a tumor microenvironment.

5. The chimeric antigen receptor of claim 4, wherein the normal physiological pH is in the range of greater than 7.0 to 7.

8.

6. The chimeric antigen receptor of claim 5, wherein the normal physiological pH is in the range of 7.2 to 7.

6.

7. The chimeric antigen receptor of claim 4, wherein the abnormal pH is in the range of 6.0 to less than 7.

0.

8. The chimeric antigen receptor according to claim 7, wherein the abnormal pH is in the range of 6.0 to 6.

8.

9. The chimeric antigen receptor of claim 1, further comprising an extracellular spacer domain or at least one co-stimulatory domain.

10. The chimeric antigen receptor according to claim 9, wherein the extracellular spacer domain is selected from the group consisting of an antibody Fc fragment, an antibody hinge region, an antibody CH2 region, an antibody CH3 region, an artificial spacer sequence, and a combination thereof.

11. The chimeric antigen receptor of claim 1, wherein the ratio of the activity of the at least one antigen-specific targeting region under abnormal conditions to the same activity under normal physiological conditions is at least 2.

12. The chimeric antigen receptor of claim 1, wherein the at least one antigen-specific targeting region comprises two antigen-specific targeting regions connected to a linker.

13. The chimeric antigen receptor of claim 12, wherein the two antigen-specific targeting regions each bind to a different target antigen or a different epitope of the same target antigen.

14. The chimeric antigen receptor of claim 1, wherein the transmembrane domain is selected from the group consisting of an artificial hydrophobic sequence; and a type I transmembrane protein, an α, β or ζ chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and a transmembrane domain of CD154.

15. The chimeric antigen receptor of claim 1, wherein the intracellular signaling domain is selected from the group consisting of the cytoplasmic signaling domains of human CD3 zeta chain, FcyRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor with an immunoreceptor tyrosine-based activation motif (ITAM), TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.

16. The chimeric antigen receptor of claim 1, further comprising a co-stimulatory domain selected from the co-stimulatory domains of proteins in the TNFR superfamily, CD28, CD137, CD134, DaplO, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS LIGHT, NKG2C, and B7-H3.

17. An expression vector comprising a polynucleotide sequence encoding the chimeric antigen receptor of claim 1.

18. The expression vector according to claim 17, wherein the expression vector is selected from the group consisting of a lentiviral vector, a gamma-retroviral vector, a foamy virus vector, an adeno-associated virus vector, an adenovirus vector, a poxvirus vector, a herpesvirus vector, an engineered hybrid virus, and a transposon-mediated vector.

19. A genetically engineered cytotoxic cell comprising a polynucleotide sequence encoding the chimeric antigen receptor of claim 1.

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