JP2021536256A - Conditional active chimeric antigen receptor for modified T cells - Google Patents

Abstract

The present disclosure relates to chimeric antigen receptors for binding to tumor-specific target antigens. The chimeric antigen receptor contains at least one antigen-specific target region evolved from the parent protein antibody or fragment thereof and has reduced activity in analysis under normal physiological conditions compared to activity in analysis under abnormal conditions. including. Also provided is a method for producing a chimeric antigen receptor.

Description

Related Application Data This application is a partial continuation of US Patent Application No. 16/053166 filed on August 2, 2018 (currently pending), which in turn is now abandoned US Patent Application No. 1. A division of No. 15 / 052,487, which, in turn, is a partial continuation of PCT / US15 / 47197 filed on August 27, 2015, International Application No. 27, which designates the United States of America. In addition, it claims the benefit of US Provisional Application No. 62/043067 (now expired) filed August 28, 2014, all of which are incorporated herein by reference in their entirety. Will be done.

The present disclosure relates to the field of protein evolution. Specifically, the present disclosure relates to methods of producing conditional active chimeric antigen receptors from parental or wild-type proteins. This conditionally active chimeric antigen receptor is reversibly or irreversibly inactivated under wild-type normal physiological conditions, but is active under abnormal conditions.

There is a great deal of literature describing the potential for evolving various properties of proteins, especially enzymes. For example, the enzyme may be evolved so that its operation is stable under different conditions such as high temperature. In situations where activity increases at high temperatures, a significant portion of that improvement is generally higher kinetic as explained by the Q10 law (in the case of enzymes, it is estimated that a 10 ° C increase doubles the turnover). It can be due to activity.

In addition, there are examples of natural mutations that destabilize proteins under their normal operating conditions. Although certain variants are active at low temperatures, their levels can be low compared to parental or wild-type proteins. This is also typically explained by a decrease in activity as guided by Q10 or a similar law.

It is desirable to produce useful molecules that are conditionally activated. For example, it is virtually inactive under wild-type operating conditions, but is active at levels equal to or better than wild-type operating conditions, or activated or inactive in certain microenvironments, except under wild-type operating conditions. It is desirable to produce molecules that are activated or activated or inactivated over time. In addition to temperature, other conditions under which the protein can be evolved or optimized include pH, osmolality, osmolality by weight, oxidative stress and electrolyte concentration. Other desirable properties that can be optimized for evolution include chemical resistance and proteolysis resistance.

Many strategies for evolving or manipulating molecules have been published. However, while the protein is inactive or virtually inactive (less than 10% activity, preferably less than 1% activity) under wild-type operating conditions, its corresponding parent or wild under conditions other than wild-type operating conditions. In order to manipulate or evolve to maintain activity equal to or better than the type protein, the destabilizing mutation does not counteract its destabilizing effect, but increases activity. Must coexist with. It is expected that destabilization will reduce protein activity beyond the effects predicted by standard laws such as Q10. Therefore, if it becomes possible to evolve a protein that is inactivated under normal operating conditions for the corresponding parental or wild-type protein, but works efficiently at low temperatures, for example, an unexpected new class of protein will be produced. Be created.

Chimeric antigen receptors (CARs) have been used to treat cancer. U.S. Patent Application Publication No. 2013/0280220 discloses methods and compositions that provide improved cells encoding chimeric antigen receptors specific for two or more antigens, including tumor antigens. .. Cells expressing the chimeric antigen receptor can be used for cell therapy. Such cell therapies may be suitable for any medical condition, but in particular embodiments are cell therapies for cancers, including cancers involving solid tumors.

The present invention provides engineered, conditionally active chimeric antigen receptors that are inactive or hypoactive under normal physiological conditions but active under abnormal physiological conditions.

Various publications are referenced by author and date throughout this application. The disclosure of these publications as a whole is made herein to better explain the technical level of the art as known to those of skill in the art as of the date of the disclosure described and claimed in the present specification. Incorporated in this application by reference.

The schematic diagram of the chimeric antigen receptor in one Embodiment of this invention is shown. ASTR is an antigen-specific target region, L is a linker, ESD is an extracellular spacer domain, TM is a transmembrane domain, CSD is a co-stimulation domain, and ISD is an intracellular signaling domain. be.

It is shown that even if the conditional active antibody of Example 1 is expressed as a divalent or monovalent antibody, there is no significant change in the selectivity of these antibodies below pH 6.0 and above pH 7.4. It is shown that even if the conditional active antibody of Example 1 is expressed as a divalent or monovalent antibody, there is no significant change in the selectivity of these antibodies below pH 6.0 and above pH 7.4.

FIG. 6 is a profile of a size exclusion chromatograph showing that the conditionally active antibody of Example 2 does not aggregate.

The on and off velocities of the conditioned active antibody of Example 2 as measured by surface plasmon resonance (SPR) analysis are shown.

A to B show the selectivity of the conditioned active antibody as measured by the SPR analysis of Example 2.

It is shown that CAR-T cells had no effect on a population of CHO cells 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, but this antibody was not a conditionally active form (Comparative Example A).

It is shown that CAR-T cells reduced the population of CHO-63 cells expressing the target antigen X1 of CAR-T cells. These CAR-T cells are the same cells used to generate the data shown in FIG. 7A (Comparative Example A).

It is shown that CAR-T cells had no effect on a population of CHO cells that did not express the target antigen X1 of CAR-T cells. The CAR molecule in the CAR-T cells of Example 3 contained a conditionally active antibody against the target antigen X1.

It is shown that CAR-T cells reduced the population of CHO-63 cells expressing the target antigen X1 of CAR-T cells, as tested in Example 3. These CAR-T cells are the same cells used to generate the data shown in FIG. 8A.

A to B show cytokine release induced by the binding of CAR-T cells to the target antigen X1, as described in Example 3.

The conditional active antibody against the target antigen X2 is shown.

As described in Example 4, A to B have a cytotoxic effect induced by binding of CAR-T cells to Daudi cells expressing the target antigen X2 and CAR- on HEK293 cells not expressing the target antigen X2. Shows the cytotoxic effect induced by T cells.

A to B show cytokine release induced by the binding of CAR-T cells to the target antigen X1, as described in Example 5.

A to B show cytokine release induced by the binding of CAR-T cells to the target antigen X2, as described in Example 5.

The conditional active antibody against the target antigen X3 suitable for the construction of CAR-T cells is shown.

In one aspect, the invention provides a chimeric antigen receptor (CAR) for binding to a tumor-specific target antigen. The chimeric antigen receptor contains at least one antigen-specific target region that has evolved from the parent protein or its domain. CAR further contains a transmembrane domain and an intracellular signaling domain. At least one antigen-specific target region has reduced activity in analysis under normal physiological conditions compared to activity in analysis under abnormal conditions.

In another embodiment, the invention provides an expression vector comprising a polynucleotide sequence encoding a chimeric antigen receptor of the invention. The expression vector is a lentivirus vector, a γ retrovirus vector, a formy virus vector, an adeno-associated virus vector, an adenovirus vector, a poxvirus vector, a herpesvirus vector, a genetically engineered hybrid virus, and a transposon-mediated vector. Is selected from.

In yet another embodiment, the invention provides genetically engineered cytotoxic cells comprising a polynucleotide sequence encoding the chimeric antigen receptor of the invention. The cytotoxic cells may be T cells and can be selected from naive T cells, central memory T cells, and effector memory T cells.

In yet another embodiment, the invention comprises a pharmaceutical composition comprising the chimeric antigen receptor, expression vector, and / or genetically engineered cytotoxic cell of the invention and a pharmaceutically acceptable excipient. offer.

In yet another aspect, the invention provides a method of producing a chimeric antigen receptor comprising at least one antigen-specific target region, a transmembrane domain and an intracellular signaling domain. The method comprises producing at least one antigen-specific target region from a parent protein or domain thereof that specifically binds to a tumor-specific target antigen. These include (i) the step of evolving the DNA encoding the parent protein or wild-type protein or its domain using one or more evolutionary techniques to create mutant DNA, and (ii) expressing the mutant DNA. To obtain a mutant polypeptide, (iii) the mutant polypeptide is subjected to analysis under normal physiological conditions and abnormal conditions, and (iv) the mutant expressed in step (iii). It comprises the step of selecting at least one antigen-specific target region from the polypeptide that exhibits reduced activity in the analysis under normal physiological conditions by comparing the activity with the activity in the analysis under abnormal conditions.

Definitions To facilitate understanding of the examples provided herein, certain frequently-appearing methods and / or terms are described below.

As used herein in connection with a measure, the term "about" is the measure expected by those skilled in the art who make the measurement and exercise the level of attention and accuracy of the measuring instrument used for the purpose of the measurement. It is related to the normal fluctuations in. Unless otherwise stated, "about" is associated with a +/- 10% variation in a given value.

The term "drug" refers to a chemical compound, a mixture of chemical compounds, a spatially localized sequence of compounds (eg, VLSIPS peptide sequence, polynucleotide sequence, and / or combinatorial small molecule sequence), biopolymer, bacteriophage. Peptide display libraries, bacterial compound antibody (eg scFV) display libraries, polysome peptide display libraries, or extracts made from biological materials such as cells or tissues of bacteria, plants, fungi or animals (particularly mammals). Used to indicate. The agent is assessed for potential enzyme activity as a conditional active biotherapeutic enzyme by being included in the screening analysis described below. The agent is assessed for potential activity as a conditionally active biotherapeutic enzyme by being included in the screening analysis described below herein.

As used herein, the term "amino acid" is ^{any organic compound containing an amino group (-NH 2} ) and a carboxyl group (-COOH), preferably after condensation as part of a free group or peptide. Combine either. "20 naturally encoded polypeptides that form alpha-amino acids" are known in the art and are alanine (ala or A), arginine (arg or R), asparagine (asn or N), isoleucine. (Asp or D), cysteine (cys or C), glutamic acid (glu 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 it is related to V).

The term "amplification" as used herein means an increase in the number of copies of a polynucleotide.

The term "antibody", as used herein, is an immune that has the ability to bind to an epitope of an antigen, such as an intact immunoglobulin molecule and Fab, Fab', (Fab') 2, Fv, and SCA fragments. Refers to a fragment of a globulin molecule. These antibody fragments retain some ability to selectively bind to the antigen of the antibody from which they are derived (eg, polypeptide antigen) and should be prepared using methods well known in the art. (See, for example, Harlow and Lane, supra), which is further described as follows. The antibody can be used for isolation of the prepared dose of antigen by immunoaffinity chromatography. Various other uses of such antibodies include diagnosis and / or stage determination of diseases (eg, neoplasia) and treatment of diseases such as neoplasm formation, autoimmune diseases, AIDS, cardiovascular disease, infectious diseases and the like. It is a therapeutic application to do. Chimeric antibodies, human-like antibodies, humanized antibodies or fully human antibodies are particularly useful for administration to human patients.

Fab fragments consist of monovalent antigen-binding fragments of antibody molecules and can be made by digesting all antibody molecules with the enzyme papain to yield fragments consisting of intact light chains and parts of heavy chains.

Fab'fragments of antibody molecules can be obtained by treating all antibody molecules with pepsin and then reducing them to give rise to a molecule consisting of an intact light chain and a portion of a heavy chain. Two Fab'fragments are obtained for each antibody molecule thus treated.

Two (Fab') fragments of the antibody can be obtained by treating all antibody molecules with the enzyme pepsin without subsequent reduction. The (Fab') 2 fragment is a dimer of two Fab'fragments held together by two disulfide bonds.

An Fv fragment is defined as a genetically engineered fragment containing a variable region of a light chain expressed as two chains and a variable region of a heavy chain.

The term "antigen" or "Ag", as used herein, is defined as a molecule that elicits an immune response. This immune response may include antibody production, or activation of specific immune-qualified cells, or both. Those of skill in the art will appreciate that any macromolecule, including virtually any protein or peptide, can act as an antigen. In addition, the antigen can be derived from recombinant DNA or genomic DNA. Those of skill in the art will therefore appreciate that any DNA comprising a nucleotide sequence or a partial nucleotide sequence that encodes a protein that elicits an immune response encodes an "antigen" as the term is used herein. There will be. Moreover, one of ordinary skill in the art will appreciate that the antigen need not be limited to being encoded by the full-length nucleotide sequence of the gene. The invention includes, but is not limited to, the use of partial nucleotide sequences of two or more genes, and it is easy for these nucleotide sequences to be arranged in various combinations to elicit the desired immune response. It is clear to. Moreover, one of ordinary skill in the art will appreciate that the antigen does not need to be encoded by a "gene" at all. It is easy to see that the antigen may be produced from a biological sample and synthesized or derived from it. Such biological samples include, but are not limited to, tissue samples, tumor samples, cells or biological fluids.

"Antigen-deficient escape variant" as used herein refers to a cell that exhibits reduced or deleted expression of a target antigen, which antigen is targeted by the CAR of the present invention.

The term "autoimmune disease", as used herein, is defined as a disorder resulting from an autoimmune response. Autoimmune diseases are the result of inadequate and excessive responses to self-antigens. Examples of autoimmune diseases are, but are not limited to, Azison's disease, alopecia greata, tonic spondylitis, autoimmune hepatitis, autoimmune parotid inflammation, Crohn's disease, diabetes (1). Type), malnutrition type epidermal vesicular disease, supratestinal inflammation, glomerular nephritis, Graves' disease, Guillan-Barr syndrome, Hashimoto's disease, hemolytic anemia, systemic erythematosus, multiple sclerosis, severe muscle Asthenia, vulgaris vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthritis, thyroiditis, vasculitis, leukoplakia, mucoid edema, malignant anemia, ulcerative colitis. Will be.

The term "self", as used herein, refers to any material obtained from the same individual to which the material is later reintroduced. For example, T cells from a patient can be isolated, genetically engineered to express CAR, and then reintroduced into the patient.

The term "B cell-related disease" as used herein includes B cell immunodeficiency, B cell-related autoimmune disease and / or excessive / uncontrolled cell proliferation (including lymphoma and / or leukemia). )including. Examples of such diseases in which the bispecific CAR of the present invention can be used as a therapeutic method are, but are not limited to, systemic erythematosus (SLE), diabetes, rheumatoid arthritis (RA), reactive arthritis, and polysclerosis (multiple sclerosis). MS), scoliosis vulgaris, celiac disease, Crohn's disease, inflammatory bowel disease, ulcerative colitis, autoimmune thyroid disease, X-linked agammaglobulinaemis, pre-B acute lymphoblasts Diseases associated with sex leukemia, systemic erythematosus, unclassifiable immunodeficiency, chronic lymphocytic leukemia, selective IgA deficiency and / or IgG subclass deficiency, B-series lymphoma (hodgkin lymphoma and / or non-hodgkin lymphoma), Immunodeficiency with thoracic adenoma, transient hypogamma globulinemia and / or high IgM syndrome, and virus-mediated B-cell disease, such as EBV-mediated lymphoproliferative disease, and B-cells are involved in pathophysiology. Chronic infectious diseases can be mentioned.

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

As used herein, the terms "cancer" and "cancerous" refer to or describe a physiological condition in a mammal that is typically characterized by uncontrolled cell growth. Examples of cancers include, but are not limited to, B-cell lymphoma (hodgkin lymphoma and / or non-hodgkin lymphoma), brain tumor, breast cancer, colon cancer, lung cancer, hepatocellular carcinoma, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer. , Bladder cancer, urinary tract cancer, thyroid cancer, renal cancer, cancer, melanoma, head and neck cancer, brain cancer, and prostate cancer including but not limited to androgen-dependent and androgen-independent prostate cancer. Will be.

The terms "chimeric antigen receptor" or "CAR" or "CARs", as used herein, are engineered receptors that graft antigen specificity to cytotoxic cells such as T cells, NK cells and macrophages. Point to the body. The CARs of the invention include at least one antigen-specific target region (ASTR), extracellular spacer domain (ESD), transmembrane domain (TM), one or more co-stimulation domains (CSD), and intracellular signaling. It may include a domain (ISD). In certain embodiments, ESD and / or CSD are optional. In another embodiment, CAR is a bispecific CAR specific for two different antigens or epitopes. When ASTR specifically binds to the target antigen, ISD activates intracellular signaling. For example, ISDs can utilize the antigen-binding properties of antibodies to redirect T cell specificity and reactivity to selected targets in a manner that is not restricted by MHC. By antigen recognition not restricted by MHC, T cells expressing CAR gain antigen recognition ability independent of antigen processing and thus avoid major tumor escape mechanisms. Moreover, when expressed on T cells, CAR advantageously does not dimerize with the endogenous T cell receptor (TCR) α and β chains.

The term "co-expressed" as used herein refers to the simultaneous expression of two or more genes. The gene may be, for example, a nucleic acid encoding a chimeric protein as a single protein or a single polypeptide chain. For example, the CAR of the invention may be co-expressed with a therapeutic control (eg, truncated epidermal growth factor (EGFRt)), where the CAR is encoded by the first polynucleotide chain and the therapeutic control by the second polynucleotide chain. Coded. In certain embodiments, the first and second polynucleotide chains are linked by a nucleic acid sequence encoding a cleavable linker. Alternatively, the CAR and therapeutic control are not linked via a linker, instead are encoded by two different polynucleotides, eg, encoded by two different vectors.

As used herein, the term "homology" means a gene sequence that is evolutionary and functional with respect to the species. For example, in human genes, for example, the human CD4 gene is homologous to the mouse 3d4 gene, the sequences and structures of these two genes show high homology, and both genes are MHC class II. Encodes proteins that function in signaling T cell activation with restricted antigen recognition.

The term "conditionally active biological protein" refers to a variant or variant 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. This conditionally active protein also exhibits activity in specific areas of the body and / or exhibits increased or decreased activity under abnormal or acceptable physiological conditions. The term "normal physiological conditions", as used herein, refers to temperature, pH, osmotic pressure, weight osmolal concentration, oxidative stress, which can be considered to the subject within the normal range at the site of administration or in the tissue or organ of the site of action. , Electrolyte concentration, concentration of small organic molecules such as glucose, lactic acid, pyruvate, nutritional components, other metabolites, concentration of other molecules such as oxygen, carbonates, phosphates, and carbon dioxide. , And one of the cell types and nutrient availability.

In one embodiment, the normal physiological condition is the normal physiological pH in the plasma of a mammalian subject, which is higher than 7.0 to about 7.8, or from about 7.2 to about 7.8, or about 7. It is in the range of 2 to about 7.6, or about 7.3 to about 7.6, or about 7.3 to about 7.5. The abnormal condition is the pH of the tumor microenvironment, from about 6.0 to less than 7.0, or from about 6.2 to about 6.9, or from about 6.0 to about 6.8, or from about 6.2 to about. It is in the range of 6.8, or about 6.4 to about 6.8, or about 6.4 to about 6.6.

The term "abnormal condition" as used herein refers to a condition that is outside the range normally accepted for such condition. In one embodiment, the conditionally active biological protein is virtually inactive under normal physiological conditions, but under abnormal conditions it is active at a level equal to or better than the parental or wild-type protein from which it is derived. Is. For example, in one embodiment, the evolved conditionally active biological protein is effectively inactive at body temperature, but active at lower temperatures. In another embodiment, the conditionally active biological protein is reversibly or irreversibly inactivated under normal physiological conditions. In a further embodiment, the parental or wild-type protein is a therapeutic protein. In another embodiment, the conditional active biological protein is used as a drug, or therapeutic agent. In yet another embodiment, the protein is more or less active in the lower pH environment found in highly oxygenated blood or kidneys, for example after passage through the lungs.

"Conservative amino acid substitution" means compatibility of residues with similar side chains. For example, the group of amino acids having an aliphatic side chain includes glycine, alanine, valine, leucine, and isoleucine, the group of amino acids having a carboxylic acid side chain, the group of amino acids having a side chain containing serine and threonine, and amide. , Asparagine and glutamine, amino acids with aromatic side chains are phenylalanine, tyrosine, and tryptophan, amino acids with basic side chains are arginine, and histidine, and amino acids with sulfur-containing side chains. Are cysteine and methionine. Suitable conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

The term "corresponding" is used herein to indicate that a polynucleotide sequence is homologous to all or part of a reference polynucleotide sequence (ie, it is identical even if it is not strictly evolutionarily related). , Means that the polynucleotide sequence is identical to the reference polynucleotide sequence. In contrast, the term "complementary" is used herein to mean that the complementary sequence is homologous to all or part of the reference polynucleotide sequence. For example, the nucleotide sequence "TATAC" corresponds to the reference "TATAC" and is complementary to the reference sequence "GTATA".

The term "co-stimulatory ligand", as used herein, is a molecule on antigen-presenting cells (eg, dendritic cells, B cells, etc.) and is specific to a cognate co-stimulatory molecule on a T cell. T cell response including, but not limited to, proliferation, activation, differentiation, etc., in addition to the primary signal provided by binding, eg, binding of the TCR / CD3 complex to a peptide-carrying MHC molecule. Contains molecules that provide signals to mediate. Co-stimulatory ligands are, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible co-stimulatory ligands (ICOS-L). ), Cellular Adhesion Mole (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, Lymphotoxin β Receptor, 3 / TR6, ILT3, ILT4, HVEM, Toll Ligand Receptor It may include an agonist or antibody and a ligand that specifically binds to B7-H3. The co-stimulatory ligand is also not particularly limited, but is limited to CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-related antigen-1 (LFA-1), CD2, CD7. Also included are antibodies that specifically bind to costimulatory molecules present on T cells, such as ligands that specifically bind to LIGHT, NKG2C, B7-H3, and CD83.

As used herein, the term "co-stimulatory molecule", as used herein, specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response by a T cell, eg, but not limited to, proliferation. Refers to the cognate binding partner of. Co-stimulatory molecules include, but are not limited to, MHC class 1 molecules, BTLA and Toll ligand receptors.

As used herein, the term "co-stimulation signal" refers to a signal that, when used herein, results in T cell proliferation and / or upregulation or downregulation of a key molecule in combination with a primary signal such as TCR / CD3 ligation.

The term "cytotoxic cell" as used herein means a cell capable of damaging or destroying invading microorganisms, tumor cells or other affected tissue cells. The term refers to natural killer (NK) cells, activated NK cells, neutrophils, T cells, eosinocytes, basophils, B cells, macrophages and lymphokine-activated killer (LAK), among other cell types. ) Intended to contain cells. Cytotoxic cells form stable complexes by binding to target cells via antibodies, receptors, ligands or fragments / derivatives thereof, and stimulate cytotoxic cells to destroy the target cells.

Cytotoxic cells are also limited to, but are not limited to, natural killer T cells (Heczy et al., "Invariant NKT cells with chimeric antigen receptor protector provide a novel platterform for lover. .2824-2833, 2014) and other immune cells capable of tumor lysis, including granulocytes, may also be included. In addition, cytotoxic cells include, but are not limited to, macrophages and granulocytes, stem cells and / or cells with prodromal properties, such as, but not limited to, hematopoietic stem / progenitor cells (Zhen et al., "HIV". -Special Immunity Derivated From Chemical Antigen Receptor-enginered Stem Cells, "Mol Ther., Vol. 23, pp. 1358-1637, 2015", Embryonic Stem Cell (ESC), Embryonic Stem Cell (ESC), Umbilical Stem Cell (ESC), Umbilical Stem Cell ) (Themelli et al., "New cell cells for T cell enginering and adaptive immunotherapy," Cell Stem Cell., Vol. 16, pp. 357-366, 2015), including phasic cells, including phagocytosis. obtain. In addition, for cytotoxic cells, iPSC-derived T cells (TiPSC) (Themeli et al., "Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells cell. pp.928-933, 2013) or "synthetic cells" such as iPSC-derived NK cells are included.

The term "degradable" amount refers to the amount of enzyme required to process at least 50% of the substrate when compared to the case where the substrate is not contacted with the enzyme.

The term "directional ligation" refers to a ligation in which the 5'and 3'ends of the polynucleotide are sufficiently different to specify the preferred ligation direction. For example, an otherwise untreated and undigested PCR product with two blunt ends is typically preferred ligation when ligating to a cloning vector digested to give a blunt end at its multiple cloning site. It has no direction; therefore, directional ligation is typically not shown under these circumstances. In contrast, typically when digested PCR products with 5'EcoRI treated ends and 3'BamHI are ligated into a cloning vector with multiple cloning sites digested with EcoRI and BamHI, the direction The tional ligation is shown.

The term "disease targeted by genetically modified cytotoxic cells", as used herein, targets or targets affected cells so that the genetically modified cells have therapeutically beneficial results. It involves targeting any cell involved in any disease in any way, whether or not it targets healthy cells, with the genetically modified cells of the invention. Genetically modified cells include, but are not limited to, genetically modified T cells, NK cells, and macrophages. The genetically modified cell expresses the CAR of the invention, which can target any of the antigens expressed on the surface of the target cell. Examples of antigens that can be targeted include, but are not limited to, antigens expressed on B cells; antigens expressed on cancer, sarcoma, lymphoma, leukemia, embryonic cell tumors, and blastoma; on various immune cells. And antigens expressed on cells associated with various blood disorders, autoimmune disorders, and / or inflammatory disorders. Other antigens that can be targeted are apparent to those of skill in the art and can be targeted by the CARs of the invention in connection with alternative embodiments of the invention.

The terms "gene-modified cell," "redirected cell," "gene-manipulated cell," or "modified cell," as used herein, refer to a cell that expresses the CAR of the invention.

The term "DNA shuffling" is used herein to indicate recombination between sequences that are substantially homologous but not identical, and in some embodiments, DNA shuffling refers to cer / lox and. / Or may include crossover via non-homologous recombination, such as via the flap / frt system.

The term "drug" or "drug molecule" means a therapeutic agent comprising a substance having a beneficial effect on the human or animal body when administered to the human or animal body. Preferably, the agent treats, cures or alleviates one or more symptoms, illnesses, or abnormal conditions in the human or animal body, or enhances the health of the human or animal body. Can be done.

An "effective amount" is a condition that is effective in treating or preventing a condition in a living organism of a person who has been administered over a period of time, eg, providing a therapeutic effect during the desired dosing period. The amount of active biological protein or fragment.

The term "electrolyte" is used herein to define minerals in blood or other body fluids that carry charge. For example, in one embodiment, the normal physiological condition and the abnormal condition may be the condition of "electrolyte concentration". In one embodiment, the electrolyte concentration tested is selected from one or more of the ionized calcium, sodium, potassium, magnesium, chloride, bicarbonate, and phosphate concentrations. For example, in one embodiment, the usual range of serum calcium is 8.5 to 10.2 mg / dL. In this embodiment, the abnormal serum calcium concentration may be selected from above or below the normal range. In another embodiment, in one embodiment, serum chloride is 96-106 milligram equivalents (mEq / L) per liter. In this embodiment, the abnormal serum chloride concentration may be selected from above or below the normal range. In another embodiment, in one embodiment, the normal range of serum magnesium concentration is 1.7-2.2 mg / dL. In this embodiment, the abnormal serum magnesium concentration may be selected from above or below the usual range. In another example, in one embodiment, the usual range of serum phosphate is 2.4-4.1 mg / dL. In this embodiment, the abnormal serum phosphate concentration may be selected from above or below the usual range. In another embodiment, in one embodiment, the usual range of sodium in serum or blood is 135-145 mEq / L. In this embodiment, the abnormal serum or blood sodium concentration may be selected from above or below the normal range. In another embodiment, in one embodiment, the normal range of serum or blood potassium is 3.7-5.2 mEq / L. In this embodiment, the abnormal serum or blood potassium concentration may be selected from above or below the normal range. In a further embodiment, the usual range of serum bicarbonate is 20-29 mEq / L. In this embodiment, the abnormal serum or blood bicarbonate concentration may be selected from above or below the usual range. In different embodiments, bicarbonate levels may be used to indicate normal levels of acidity (pH) in blood. The term "electrolyte concentration" may also be used to define a particular electrolyte concentration in tissues or body fluids other than blood or plasma. In this case, normal physiological conditions are considered to be clinically normal in the tissue or body fluid. In this embodiment, the abnormal tissue or body fluid electrolyte concentration may be selected from above or below the normal range.

As used herein, the term "antigen determinant" relates to an antigenic determinant on an antigen, such as an enzyme polypeptide. Then, the antigen-binding site of the antibody such as an enzyme-specific antibody binds. Antigen determinants usually consist of chemically surface-active segments of molecules such as amino acids or side chains, and specific three-dimensional structural properties may also have specific chargeability. As used herein, an "antigen determinant" is another macromolecule capable of forming a binding interconnection that interacts with that portion of the antigen or the variable region that binds to the body of the antibody. means. Typically, such binding interactions are manifested as intermolecular interactions with one or more amino acid residues of the CDR.

As used herein, the term "evolving", or "evolving", refers to the use of one or more mutagenesis methods to generate a novel polynucleotide encoding a novel polypeptide. , This novel polypeptide is itself an improved biological molecule and / or contributes to the production of another improved biological molecule. In a detailed, non-limiting aspect, the present disclosure relates to the evolution of a conditionally active biological protein from a parental or wild-type protein. In one aspect, for example, evolution relates to a method of performing both non-probabilistic polynucleotide chimerization and non-probabilistic site-specific point mutation induction disclosed in US Patent Application Publication No. 2009/0130718. More specifically, the present disclosure exhibits reduced activity under normal physiological conditions compared to the parent or wild-type enzyme parent molecule, but under one or more abnormal conditions, the antigen-specific target region of the parent or wild-type enzyme. Provides a method for evolving a conditioned active biological enzyme that exhibits enhanced activity as compared to.

The terms "fragment", "derivative" and "similar organ" have at least one polypeptide having at least essentially the same physiological function or activity as the reference polypeptide when referring to the reference polypeptide. In addition, the terms "fragment," "derivative," and "similar organ" are exemplified by "preform" molecules, such as low-activity precursor protein, which can be modified by cleavage to produce a mature enzyme with significantly higher activity. Ru.

As used herein, the term "gene" means a DNA segment involved in the production of a polypeptide chain, a coding region (leader and trader) as well as an intervening sequence (nitrone) between individual coding segments (exons). Includes regions that precede and follow.

As used herein, the term "non-homologous" means that a single-stranded nucleic acid sequence cannot hybridize to another single-stranded nucleic acid sequence or a complementary sequence thereof. Thus, non-homologous moieties mean that the polynucleotide or polynucleotide has a moiety or region in a sequence that cannot hybridize to another nucleic acid or polynucleotide. Such regions or parts are, for example, parts of the mutation.

As used herein, the term "homology" or "homology" means that a single-stranded nucleic acid sequence can hybridize to a complementary single-stranded nucleic acid sequence. The degree of hybridization may depend on the amount of identity between sequences and the number of factors including hybridization conditions such as temperature and salt concentration as described below. Preferably, the same region is greater than about 5 bp, and more preferably, the same region is greater than 10 bp.

The benefits of this disclosure extend to "industrial applications" (or industrial processes), and the term is appropriate commercial as well as non-commercial industrial applications (eg, orthodox medical research in non-commercial institutions). Used to include industrial (or simply industrial) applications. Related applications include areas of the diagnostic, medical, agricultural, manufacturing and academic worlds.

As used herein, the term "immune cell" is used, but is not limited to, antigen-presenting cells, B cells, basal cells, cytotoxic T cells, dendritic cells, eosinocytes, granulocytes, helpers. Refers to cells of the mammalian immune system including T cells, leukocytes, lymphocytes, macrophages, mast cells, memory cells, monospheres, natural killer cells, neutrophils, phagocytic cells, plasma cells and T cells.

The term "immune response" as used herein is, but is not limited to, spontaneous immunity, humoral immunity, cell-mediated immunity, immunity, inflammatory response, adaptive (adaptive) immunity, autoimmunity and / or overreaction. Refers to immunity including immunity.

The term "isolated", as used herein, means that the material has been removed from its original environment (eg, the natural environment if it exists in nature). For example, the naturally occurring polynucleotides or enzymes of living animals have not been isolated, but the same polynucleotides or enzymes that have been isolated from some or all of the coexisting materials in the natural system are isolated. Has been done. Such a polynucleotide may be part of a vector and / or such polynucleotide or enzyme may be part of a composition and still in that such vector or composition is not part of its natural environment. Is also isolated.

As used herein, the term "isolated nucleic acid" is used to define a nucleic acid, eg, a DNA or RNA molecule. Nucleic acids such as DNA or RNA molecules are not directly adjacent to the 5'and 3'adjacent sequences that are normally directly adjacent when present in the spontaneous gene of the organism from which they are derived. Thus, the terms are, for example, plasmids or viral vectors, nucleic acids integrated within a gene of a heterologous cell (or a site that is a gene of a heterologous cell but naturally different from the raw one), and eg, PCR amplification or restriction. Represents a nucleic acid that is integrated into a vector, such as a nucleic acid that exists as a separated molecule, such as a DNA fragment made by enzymatic digestion or an RNA molecule made by in vitro transcription. The term also means recombinant nucleic acids that form part of a hybrid gene that encodes an additional protein that can be used, for example, in the production of molten proteins.

The term "lentivirus" as used herein refers to the genus of the family Retrovirus. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells; lentiviruses can deliver large amounts of genetic information to the DNA of host cells, so lentiviruses are the most efficient. It is one of the gene delivery vector delivery methods. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentivirus provide a means to achieve high levels of gene transfer in vivo.

The term "ligand" as used herein refers to a molecule recognized by a particular receptor, such as a random peptide or variable segment sequence. As one of skill in the art will recognize, a molecule (or macromolecular complex) can be both a receptor and a ligand. Generally, a binding partner having a smaller molecular weight is referred to as a ligand, and a binding partner having a larger molecular weight is referred to as a receptor.

As used herein, the term "ligation" refers to the process of formation of a phosphodiester bond between two double-stranded nucleic acid fragments (Sambrook et al., (1982). Molecular Cloning: A Laboratory Manual. ... .Cold Spring Harbour Laboratory, Cold Spring Harbor, NY, p.146; Sambrook et al, Molecular Cloning: a laboratory manual, 2 nd Ed, Cold Spring Harbor Laboratory Press, 1989). Unless otherwise provided, ligation can be achieved using known buffers and conditions of 10 units of T4 DNA ligase (“ligase”) per 0.5 microgram of DNA fragment to be ligated.

As used herein, the term "linker" or "spacer" is a molecule or group of molecules connecting two molecules, eg, a DNA binding protein and a random peptide, eg, the random peptide is from the DNA binding protein. Refers to a molecule or group of molecules that serves to place these two molecules in a favorable arrangement so that they can bind to the receptor with minimal steric damage. The "linker" (L) or "linker domain" or "linker region", as used herein, is an oligo of approximately 1-100 amino acids in length that integrally connects any domain / region of the CAR of the invention. Refers to a peptide or polypeptide region. The linker may be composed of mobile residues such as glycine and serine so that adjacent protein domains can move freely with each other. Longer linkers can be used if it is desirable to prevent two adjacent domains from sterically interfering with each other. The linker may be cleaveable or non-cleavable. Examples of cleavable linkers include 2A linkers (eg T2A), 2A-like linkers or their functional equivalents, and combinations thereof. In some embodiments, the linker may be a picornavirus 2A-like linker, a chuteschovirus CHYSEL sequence (P2A), a Tosea asigna virus (T2A) or a combination thereof, variants and functional equivalents. Things can be mentioned. Other linkers are apparent to those of skill in the art and can be used in connection with alternative embodiments of the invention.

The term "mammalian cell surface display", as used herein, for screening purposes (eg, by screening for specific antigen binding by a combination of magnetic beads and fluorescence activated cell sorting), is a protein or antibody. , Or a technique for expressing and presenting a portion of an antibody on the surface of a mammalian host cell. In one aspect, DuBridge et al. , US Patent Application Publication No. 2009/0136950 uses a mammalian expression vector to simultaneously express immunoglobulins as both secretory and cell surface bound. In another aspect, this technique is described by Gao et al. , US Patent Application Publication No. 2007/01/11260, used in the screening of viral vectors encoding a library of antibodies or antibody fragments presented on the cell membrane when expressed in cells. All IgG surface displays on mammalian cells are known. For example, Akamatsuu et al. Has developed a mammalian cell surface display vector suitable for direct isolation of IgG molecules based on their antigen binding affinity and biological activity. An antibody library was presented on the cell surface as a total IgG molecule using an episome vector derived from Epstein-Barr virus and screened for specific antigen binding by a combination of magnetic beads and fluorescence activated cell sorting. A plasmid encoding an antibody with the desired binding properties was recovered from the sorted cells and converted into a form suitable for the production of soluble IgG. Akamatsuu et al. J. Immunol. Methods, vol. See 327, pages 40-52, 2007. Ho et al. Used human fetal kidney 293T cells, which are widely used for transient protein expression, for the cell surface display of single-chain Fv antibodies for affinity maturation. Cells expressing rare mutant antibodies with higher affinities were 240-fold enriched by single-pass cell selection from large excess cells expressing WT antibodies with slightly lower affinities. In addition, after a single selection of a combinatorial library that randomizes unique antibody hotspots, highly enriched variants with increased binding affinity for CD22 were obtained. Ho et al. , "Isolation of anti-CD22 Fv with high affinity by Fv display on human cells," Proc Natl Accad Sci USA, vol. 103, pages 9637-9642, 2006.

Antigen-specific B cells can also be used. Such B cells can be isolated directly from human donor peripheral blood mononuclear cells (PBMCs). Recombinant antigen-specific single-chain Fv (scFv) libraries are generated from this B cell pool and screened by mammalian cell surface display using a Sindbis virus expression system. Variable regions (VR) of heavy chain (HC) and light chain (LC) can be isolated from positive clones to produce recombinant fully human antibodies as total IgG or Fab fragments. In this way, several hypervariant high-affinity antibodies that bind the model virus antigen Qβ virus-like particles (VLPs), as well as nicotine-specific antibodies, can be isolated. Beerli et al. , "Isolation of mammal monoclonal antibody by mammal cell display," Proc Natl Acad Sci USA, vol. 105, pages 14336-14341,2008.

Yeast cell surface displays can also be used in the present invention, for example, Kondo and Ueda, "Yeast cell-surface dispatch-applications of molecular display," Appl. Microbiol. Biotechnol. , Vol. 64, pages 28-40, 2004, which describes, for example, a cell surface engineering system using yeast Saccharomyces cerevisiae. Yeast S. Some representative display systems for expression in S. cerevisiae are described in Lee et al, "Microbial cell-surface display," TRENDS in Bitechnol. , Vol. 21, pages 45-52, 2003. In addition, Border and Wittrup, "Yeast surface display for screening combinatorial polypeptide libraries," Nature Biotechnology. , Vol. 15, pages 533, 1997.

The term "manufacturing", as used herein, in an amount sufficient to enable at least Phase I clinical trials of a Therapeutic protein, or in an amount sufficient for regulatory approval of a diagnostic protein. Refers to making a protein.

As used herein, the term "microenvironment" is used in any part of a tissue or body that has a permanent or temporary physical or chemical difference from other areas of the tissue or body. Or it means an area.

As used herein, the term "evolving molecular property" includes a molecule comprising a polynucleotide sequence, a molecule comprising a polypeptide sequence, and a partially polynucleotide sequence and partially a polypeptide sequence. Includes references to molecules containing. Examples of particularly relevant (but not limited to) molecular properties to be evolved are temperature; salt; osmotic pressure; pH; oxidative stress, and glycerol concentration, DMSO concentration, surfactant concentration, and /. Or protein activity under certain conditions, such as with respect to any other molecular species that comes into contact in the reaction environment. Further particularly relevant (but not limited to) examples of evolving molecular properties are stability-residues present after exposure to specific environments, such as those that may be encountered during storage, for a specific period of time. The amount of molecular properties can be mentioned.

As used herein, the term "mutation" means a change in the sequence of a wild-type nucleic acid sequence or a change in the sequence of a peptide. Such mutations can be point mutations such as metastasis or conversion. Mutations can be deletions, insertions or duplications.

The term "multispecific antibody", as used herein, is an antibody that has a binding affinity for at least two different epitopes. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments (eg, F (ab') ₂ -bispecific antibodies). The engineered antibody may bind to two, three or more (eg, four) antigens (see, eg, US Patent Application Publication No. 2002/0004587 A1). One conditionally active antibody may be engineered to be multispecific, or two antibodies may be engineered to contain a heterodimer that binds to two antigens. Multispecific antibodies can also be multifunctional.

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

The term "spontaneous" applies in connection with the fact that an object is found in nature, as used herein. For example, a polypeptide or polynucleotide sequence present in an organism (including a virus) that can be naturally isolated from a source material that is not intentionally modified by humans in the laboratory is spontaneous. In general, the term "spontaneous" relates to an object as it is present in a non-pathological (non-disease) individual, as is typical in a species.

As used herein, "normal physiological conditions" or "wild-type operating conditions" are temperatures, pH, osmolality, osmoles considered within normal limits at the site of administration or site of action to a subject. Conditions for concentration, oxidative stress and electrolyte concentration.

As used herein, a "nucleic acid molecule" consists of at least one base or one base pair, respectively, depending on whether it is single-stranded or double-stranded. In addition, nucleic acid molecules are any of nucleotides, including, but not limited to, molecules such as RNA, DNA, genetic nucleic acids, non-genetic nucleic acids, spontaneous and non-spontaneous nucleic acids, and groups of nucleic acid molecules such as synthetic nucleic acids. It can belong only to the group or chimerically. This is a non-limiting example for any organelle such as mitochondria, ribosomal RNA, and nucleic acid molecules that become chimeric from one or more naturally occurring and non-naturally occurring components. Contains related nucleic acids.

In addition, the "nucleic acid molecule" may include, but is not limited to, one or more non-nucleotide-based components, as exemplified by amino acids and sugars. Thus, by way of example, ribozymes that are, but are not limited to, partly nucleotide-based and partly protein-based are considered "nucleic acid molecules".

The terms "nucleic acid sequence encoding" or "DNA coding sequence of" or "nucleotide sequence encoding" are, as used herein, placed under the control of an appropriate regulatory sequence such as a promoter. Refers to a DNA sequence that is transcribed and translated into an enzyme when it is transcribed. A "promoter" is a DNA regulatory region capable of binding to RNA polymerase in a cell and initiating transcription of a downstream (3'direction) coding sequence. The promoter is part of the DNA sequence. This sequence region has a start codon at its 3'end. The promoter sequence contains a base that is the minimum number of elements required to initiate transcription at a detectable level above the background. However, after RNA polymerase binds to the sequence and transcription begins at the start codon (the 3'end with a promoter), transcription proceeds downstream in the 3'direction. Within the promoter sequence are transcription initiation sites (conveniently defined by mapping with nuclease S1) and protein binding domains (consensus sequences) involved in RNA polymerase binding.

The term "oligonucleotide" (or "oligo" as a synonym) refers to either a single-stranded polydeoxynucleotide or two complementary polydeoxynucleotide chains that can be chemically synthesized. Such synthetic oligonucleotides may or may not have 5'phosphoric acid. Those that do not have will not ligate to another oligonucleotide unless phosphate is added by ATP in the presence of kinase. Synthetic oligonucleotides ligate to non-dephosphorylated fragments.

As used herein, the term "manipulative binding" means the binding of polynucleotide elements in a functional relationship. Nucleic acids are "manipulatively bound" when placed in a functional relationship with other nucleic acid sequences. For example, if it affects the transcription of the coding sequence, the promoter or enhancer is operably linked to the coding sequence. The binding in an operable state is that the bound DNA sequences are typically adjacent and, if necessary to join the two protein coding regions, adjacent and within the reading frame. Means.

When RNA polymerase transcribes two coding sequences in a single mRNA, the coding sequence "manipulates" to the other coding sequence and is a single polypeptide having amino acids derived from both coding sequences. Translated within. The coding sequences need not be adjacent to each other as long as the expressed sequences are finally processed to produce the desired protein.

As used herein, the term "parental polynucleotide set" consists of one or more different polynucleotide species. Typically, the term is used to refer to a set of progeny polynucleotides, preferably obtained by mutagenesis of a set of parents, in which case the terms "parent", "launch" and "template" are used. Compatible.

The term "patient" or "subject" means an animal, such as a mammal, such as a human, for which it is intended for treatment. The subject or patient can be male or female.

As used herein, the term "physiological condition" is a temperature, pH, osmotic pressure, ionic strength that is compatible with a living organism and / or is typically present intracellularly in a living cultured yeast cell or mammalian cell. Refers to, viscosity, and similar biochemical parameters. For example, the intracellular conditions of yeast cells that grow under typical laboratory culture conditions are physiological conditions. Suitable in vitro reaction conditions for in vitro transcription cocktails are generally physiological conditions. Generally, in vitro physiological conditions are 50-200 mM NaCl or KCl, pH 6.5-8.5, 20-45 ° C. and 0.001-10 mM divalent cations (eg Mg ^{++ "} , Ca ⁺⁺ ); Preferably, it contains about 150 mM NaCl or KCl, pH 7.2-7.6, 5 mM divalent cations and is often 0.01-1.0% non-specific protein (eg bovine serum albumin (BSA)). )). Non-ionic surfactants (Tween, NP-40, Triton X-100) are usually about 0.001-2%, typically 0.05-0.2% (v / v). ). Detailed aqueous conditions can be selected by the practitioner by conventional methods. As a general guideline, the following buffered aqueous conditions can be applied: 10-250 mM NaCl, 5 Addition of ~ 50 mM Tris HCl, pH 5-8, optionally divalent cations and / or metal chelating agents and / or nonionic surfactants and / or membrane fractions and / or antifoaming agents and / or scintillants. Normal physiological conditions refer to conditions of temperature, pH, osmotic pressure, osmolal concentration, oxidative stress and electrolyte concentration at the site of administration or action in the patient or subject, which can be considered within the normal range of the patient.

Standard specifications (5'-3') are used herein to describe the sequence of double-stranded polynucleotides.

The term "population" means the collection of compositions such as polynucleotides, parts of polynucleotides or proteins. A "mixed population" is a collection of compositions that belong to the same genus (ie, are related) of a nucleic acid or protein, but differ (ie, are not) in their sequence and thus differ in their physiological and intellectual activity. be.

A molecule having a "substitute form" is one or more covalent and non-covalent to obtain a more mature molecular form with different properties (eg, increased activity) in comparison to a reference substitute molecule. During any combination of binding chemical modifications (eg, glycosylation, proteolytic cleavage, dimerization or oligomerization, temperature-induced or pH-induced higher-order structural changes, association with cofactors, etc.) It means a molecule that has passed through. Reference precursor molecules when two or more chemical modifications (eg, two proteolytic cleavages, or proteolytic cleavages and non-glycosylation) can be distinguished on the way to the production of mature molecules. Is referred to as a "precursor substitute" molecule.

As used herein, the term "receptor" refers to a molecule that has an affinity for a given ligand. The receptor can be a naturally occurring molecule or a synthetic molecule. Receptors may be used unmodified or as aggregates with other species. Receptors can covalently or non-covalently bind to a binding member, either directly or via a specific binding agent. Examples of receptors include, but are not limited to, antibodies including monoclonal antibodies that are reactive with specific antigenic determinants (such as those found in viruses, cells, or other materials) and anti-serum, cell membrane receptors. , Complex sugars and glycoproteins, enzymes, and hormone receptors.

The term "decreasing reassortment" as used herein refers to an increase in molecular diversity that occurs through repeat sequence-mediated deletion (and / or insertion) events.

As used herein, the term "restriction site" means a recognition sequence required for the expression of the action of a restriction enzyme and includes the site of contact cleavage. It is acknowledged that the site of cleavage may or may not be included in a portion of the restriction site consisting of a low ambiguity sequence (ie, a sequence containing a major determinant of the frequency of occurrence of the restriction site). It is understood that an enzyme (eg, a restriction enzyme) "cleaving" a polynucleotide means that the restriction enzyme catalyzes or facilitates the cleavage of the polynucleotide.

As used herein, the term "single-chain antibody" generally refers to a polypeptide comprising a VH domain and a VL domain in a polypeptide chain linked via a spacer peptide, which is an amino terminus and / Or may contain an additional amino acid sequence at the carboxy terminus. For example, a single chain antibody may include a mooring segment for linking to a coding polynucleotide. As an example, scFv is a single chain antibody. Single-chain antibodies are generally substantially encoded by genes in the immunoglobulin superfamily (eg, The Immunoglobulin Gene Superfamily, AF Williams and AN Barcry, in Immunoglobulin Genes, T. Honjo). W. Alt, and THE Labbits, eds., (1989) Academic press: San Diego, Calif., Pp. 361-368), most often rodents, non-human primates, birds. , Pig, bovine, sheep, goat, or a protein consisting of one or more polypeptide segments of at least 10 consecutive aminos encoded by the human heavy or light chain gene sequence. Functional single-chain antibodies generally contain a portion of an immunoglobulin superfamily gene product sufficient to retain binding properties to a specific target molecule, typically a receptor or antigen (epitope).

Members of a pair of molecules (eg, antibody-antigen pair and ligand-receptor pair) are said to "specifically bind" to each other if they bind to each other with a higher affinity than other non-specific molecules. Be told. For example, an antibody produced against an antigen to which it binds more efficiently than to a non-specific protein can be described as binding specifically to the antigen.

As used herein, the term "stimulation" means that a stimulating molecule (eg, a TCR / CD3 complex) binds to its cognate ligand, thereby, but without limitation, via the TCR / CD3 complex. It means a primary response caused by the mediation of a signal transduction event, such as signal transduction. Stimulation can mediate changes in the expression of certain molecules, such as downregulation of TGF-β and / or rearrangement of cytoskeletal structures.

As used herein, the term "stimulating molecule" means a molecule on a T cell that specifically binds to a cognate-stimulating ligand present on an antigen-presenting cell.

The term "stimulating ligand", as used herein, is a cognate binding partner on a T cell when present on an antigen presenting cell (eg, dendritic cell, B cell, etc.) ("stimulation" herein. A ligand that can specifically bind to (referred to as a molecule) and thereby mediate a primary response by T cells, including, but not limited to, activation, eliciting an immune response, proliferation, and the like. means. Stimulant ligands are well known in the art and include, in particular, peptide-carrying MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

As used herein, the term "target cell" is a cell involved in a disease and is a genetically modified cytotoxic cell of the invention, including but not limited to genetically modified T cells, NK cells, and. Refers to cells that can be targeted by (including macrophages). Other target cells will be apparent to those of skill in the art and may be used in connection with alternative embodiments of the invention.

The terms "T cell" and "T lymphocyte" are compatible and are used as synonyms herein. Examples include, but are not limited to, naive T cells, central memory T cells, effector memory T cells and combinations thereof.

The term "transduction", as used herein, refers to the introduction of a foreign nucleic acid into a cell using a viral vector. "Transfection" as used herein refers to the introduction of foreign nucleic acids into cells using recombinant DNA technology. The term "transformation" means the introduction of an "exogenous" (ie, extrinsic or extracellular) gene, DNA or RNA sequence into a host cell, so that the host cell expresses the introduced gene or sequence. It will produce the 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 "cloned" or "foreign" gene or sequence, depending on the genetic mechanism of the cell, such as initiation, arrest, promoter, signal, secretion, or other sequence. It may include the regulatory or control sequence used. The gene or sequence may include a non-functional sequence or a sequence of unknown function. The host cell that receives and expresses the introduced DNA or RNA is "transformed" and is a "transformant" or "clone". The DNA or RNA introduced into the host cell can be derived from any source, including cells of the same genus or species as the host cell, or cells of a different genus or species.

The term "treat" (1) suffers from or is susceptible to the clinical or potential symptoms of a condition, disease or condition, but is still experiencing the clinical or potential symptoms, disease or condition of the condition. Preventing or delaying the appearance of clinical manifestations of advanced conditions, diseases or conditions in animals that are absent or not shown, (2) Inhibiting conditions, diseases or conditions (ie, in the case of disease or maintenance therapy). Suppresses, reduces, or delays their reversion or progression of at least one clinical or latent sign) and / or (3) eases the condition (ie, condition, disease, or condition or To cause at least one relapse of clinical or latent symptoms). The benefits of the patient being treated are statistically significant, or at least recognizable to the patient or physician.

"Tumor" as used herein refers to any neoplastic cell growth and proliferation, whether malignant or benign, as well as any precancerous and cancerous cells and tissues.

As used herein, the term "tumor microenvironment" includes elements that create a structural and / or functional environment for a malignant process to survive and / or expand and / or spread. Refers to any element of the environment.

As used herein, the term "variable segment" means a portion of a developing peptide consisting of a random, pseudo-random, or defined jin sequence. "Variable segment" means a portion of a developing peptide consisting of a random, pseudo-random, or defined lysate sequence. The variability segment may consist of both variant and invariant residue positions, and the degree of mutated residues at variant residue positions may be limited, both options are at the discretion of the practitioner. Be selected. Typically, the variable segment is about 5-20 amino acid residues (eg, 8-10) in length, but the variable segment can be longer, and antibody fragments, protein-binding nucleic acids, and so on. It may also consist of an antibody protein such as a receptor protein or a receptor protein.

A "vector", "cloning vector" and "expression vector", as used herein, are sequences that have been introduced by introducing a polynucleotide sequence (eg, an exogenous gene) into a host cell to transform the host. Refers to a medium capable of promoting the expression of (eg, transcription and translation). Vectors include plasmids, phages, viruses and the like.

As used herein, the term "wild type" means that the polynucleotide does not contain any mutations. "Wild type protein", "wild type protein", "wild type biological protein", or "wild type biological protein" Refers to a protein that can be isolated from nature, is active at the level of activity found in nature, and contains an amino acid sequence found in nature. The terms "parent molecule" and "target protein" also refer to wild-type proteins. The "wild-type protein" preferably has some desired properties, such as higher binding affinity, or enzymatic activity, which provides better stability or improved selectivity in various temperature or pH environments. / Or can be obtained by screening a library of proteins for the desired properties, including solubility.

For example, the term "working" as in "working sample" is simply a sample that a person is working on. Similarly, for example, a "working molecule" is a molecule that a person is working on.

For purposes of illustration, the principles of the invention will be described by reference to various exemplary embodiments. Although specific embodiments of the invention are specifically described herein, one of ordinary skill in the art will readily recognize that the same principles are applicable and can be used in other systems and methods as well. There will be. Before elaborating on the disclosed embodiments of the invention, it should be understood that the invention is not limited to the details of any particular embodiment to which its application is shown. In addition, the terminology used herein is for illustration purposes only. In addition, some methods are described herein with reference to the steps presented in a particular order, but often, as one of ordinary skill in the art would understand, these steps are performed in any order. The novel method is therefore not limited to the particular process order disclosed herein.

As used herein and in the appended claims, the singular "a", "an", and "the" shall include plural references unless expressly specified in the context. You have to keep in mind. Further, the terms "a" (or "an"), "one or more" and "at least one" may be used interchangeably herein. The terms "comprising", "included", "having" and "construded from" can also be used synonymously.

The present disclosure is a chimeric antigen receptor (CAR) for binding to a target antigen, which has evolved from a parent or wild protein or its domain and has normal physiological conditions compared to its activity in analysis under aberrant conditions. With respect to a chimeric antigen receptor comprising at least one antigen-specific target region with reduced activity in the analysis in; 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 co-stimulating domain. The target antigen may be a tumor-specific antigen or may be Axl, ROR2 or CD22.

The CAR of the present invention is compared with (1) its affinity for a target antigen that is reversibly or irreversibly reduced under normal physiological conditions, and (2) the same CAR that does not have a conditionally active antigen-specific target region. , Increased affinity, may have at least one of them. These CARs can guide cytotoxic cells to diseased sites where abnormal conditions are present, such as tumor microenvironment or synovial fluid. As a result of these properties, CAR can preferentially direct cytotoxic cells to diseased sites, because of their low affinity for normal tissue. Such CAR can dramatically reduce side effects and increase therapeutic efficacy by allowing the use of higher doses of therapeutic agents. CAR is particularly effective in developing new therapeutic agents that require a shorter or limited duration in a subject's body. Examples of beneficial uses include systemic treatment at high dosages, as well as topical treatment at high concentrations.

The chimeric antigen receptor may include an antigen-specific target region having a reduced binding affinity for the target antigen as compared to the antigen-specific target region of the parent or wild-type protein or its domain under normal physiological conditions.

Chimeric antigen receptors have increased activity compared to the antigen-specific target region of the parent or wild protein or its domain in analysis under aberrant conditions, and the parent or wild protein or its domain under normal physiological conditions. It may include an antigen-specific target region having a reduced binding affinity for the target antigen as compared to the antigen-specific target region of the domain.

In any of the aforementioned chimeric antigen receptors, the antigen-specific target region also has increased selectivity in analysis under aberrant conditions compared to the antigen-specific target region of the parent or wild-type protein or its domain. May have.

In some embodiments, the antigen-specific target region has a ratio of activity under abnormal conditions to at least about 1.1, or at least about 1.2, or at least about 1.4, to the same activity under normal physiological conditions. , 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.

The CAR molecule contains a linker that connects two antigen-specific target regions (Fig. 1). This linker orients the two antigen-specific target regions on CAR-T cells such that they exhibit enhanced or optimal activity in binding to the target antigen (Jensen et al.,. "Design and Implementation of chimeric antigen receptor-modified T cells", Immunol Rev-1 Thus, the linker is preferably capable of taking specific conformations that allow for improved or optimal binding of the two antigen-specific target regions to the target antigen, thereby enabling CAR-T cells. Sex increases.

In some embodiments, the linker can be a Gly-Ser tandem repeat of 18-25 amino acids (Grada, "TanCAR: A Novel Bispecific Chimeric Antigen Receptor". for Cancer Immunotherapy) ”, Molecular Therapy Nuclear Antis, vol. 2, e105, 2013). This mobile linker is capable of taking many different conformations for improved or optimal presentation of two antigen-specific target regions for binding to the target antigen.

In some embodiments, the linker is capable of different conformations under normal and abnormal conditions. In particular, this linker has improved or optimal first conformation for the presentation of two antigen-specific target regions for binding to the target antigen under aberrant conditions, while the same linker has normal physiological conditions. In, it has a second conformation in which the presentation of two antigen-specific target regions is less effective in binding to the target antigen as compared to the first conformation of the linker under aberrant conditions. Such a linker can be referred to as a "conditional linker" that allows the two antigen-specific target regions to bind to the target antigen with higher binding activity under abnormal conditions than under normal physiological conditions. Therefore, CAR-T cells containing such a conditional linker are more active under abnormal conditions than the same CAR-T cells under normal physiological conditions.

Proteins whose conformation changes at different pH have previously been described, for example, by DiRusso et al. ("The effect on pH-dependent conformational changes in proteins and experimental pK (a): pH-Dependent conformational changes in protein and the effect on experimental pK (a) s: the case. 4) ”, PLos Comput Biol., Vol. 8, e1002761, 2012). In addition, proteins with different conformations at different temperatures are known as Caldwell, "Temperature-induced protein conformational change in barley root plasma membrane". .. , Vol. 84, pp. 924-929, 1989. Regarding the conformation of antibodies affected by pH and / or temperature, Gandhi, "Effect of pH and temperature on conformal change of humanized monoclonal" , Considered in the Master's Thesis of the University of Rhode Island, USA.

Within the scope of the invention is the selection of conditional linkers to use for CAR molecules. Conditional linkers have improved or optimal first conformation for binding of two antigen-specific target regions for binding to the target antigen under aberrant conditions and for binding to the target antigen under normal physiological conditions. The presentation of the two antigen-specific target regions can take a semi-optimal second conformation. In some embodiments, the conformation of the linker is suboptimal under normal physiological conditions, which results in approximately 90% of the binding activity of the CAR molecule with improved or optimal conformation of this linker 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 less than about 5% binding to the target antigen. An active CAR molecule is generated.

Conditional linkers include 2A linkers, 2A-like linkers, picornavirus 2A-like linkers, pig teschovirus 2A peptide (P2A), and Tosea asigna virus 2A peptide (T2A), as well as variants thereof. And may be generated from a starting linker selected from functional equivalents. This starting linker is evolved to produce mutant proteins; the mutant proteins are then subjected to analysis under normal physiological conditions and under abnormal conditions. From these variant proteins, a conditional linker, wherein the selected protein (a) has improved presentation of two antigen-specific target regions for binding to the target antigen under aberrant conditions or has an optimal first conformation. And (b) a protein with a conditioned linker is based on the fact that the presentation of two antigen-specific target regions for binding to the target antigen under normal physiological conditions presents a second conformation of the suboptimal conditioned linker. Be selected.

The CAR molecule also contains an extracellular spacer domain that connects the two antigen-specific target regions to the transmembrane domain, followed by the transmembrane domain into the co-stimulation domain and intracellular signaling domain inside the T cell. Connect (Fig. 1). The extracellular spacer domain is preferably capable of assisting the antigen-specific target region in recognizing and binding to the target antigen on the target cell (Hudesec et al., "Non-signaling of chimeric antigen receptor". The extracellular spacer domain is the decisive factor for in vivo antitumor activity. 2015). In some embodiments, the extracellular spacer domain is a mobile domain, so that the antigen-specific target region has a structure for optimally recognizing the specific structure and density of the target antigen on cells such as tumor cells. (Hudesec et al., "The non-signaling extracellular spucer domine of chimeric antigen receptor receptors", "The non-signaling extracellular scaper domine of chimeric antigen receptor receptors". Antigen activity) ”, Celler Immunol Res., Vol. 3, pp. 125-135, 2015). Due to the mobility of the extracellular spacer domain, the extracellular spacer domain can take many different conformations.

In some embodiments, the extracellular spacer domain is capable of taking different conformations under normal and abnormal conditions. In particular, the extracellular spacer domain has improved or optimal first conformation for the presentation of two antigen-specific target regions for binding to the target antigen under aberrant conditions, while the same extracellular spacer domain. However, under normal physiological conditions, the presentation of two antigen-specific target regions has a second conformation that is suboptimal for binding to the target antigen. Such extracellular spacer domains are referred to as "conditional extracellular spacer domains" because they allow the two antigen-specific target regions to bind to the target antigen with higher binding activity under abnormal conditions than in normal physiological conditions. be able to. Therefore, due to the conditional extracellular spacer domain, CAR-T cells may be more active under abnormal conditions than the same CAR-T cells under normal physiological conditions.

Within the scope of the invention is the selection of conditional extracellular spacer domains to be used for CAR molecules. In some embodiments, the conformation of the extracellular spacer domain is suboptimal under normal physiological conditions, so that approximately 90% of CAR molecules having the same extracellular spacer domain conformation 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 less than about 5% binding to the target antigen. An active CAR molecule is generated.

It has been discovered that ubiquitination-resistant morphology of regions containing extracellular spacer domains and transmembrane domains can enhance CAR-T cell signaling and thus increase antitumor activity (Kunii et la., "Ubiquitin". Redirected human T cell function enhancement that expresses a linker to activate ubiquitin-resistant T cells. , Vol. 24, pp. 27-37, 2013). Within this region, the extracellular spacer domain is outside the CAR-T cells and is therefore exposed to different conditions and may conditionally become ubiquitinated resistant.

Within the scope of the present invention, the extracellular spacer domain is conditionally resistant to ubiquitination. In particular, the extracellular spacer domain of the CAR molecule has higher ubiquitination resistance under abnormal conditions than under normal physiological conditions. Therefore, CAR-T cells that conditionally have a ubiquitination-resistant extracellular spacer domain will have enhanced cytotoxicity under abnormal conditions as compared to their cytotoxicity under normal physiological conditions.

Conditionally, the extracellular spacer domain that is resistant to ubiquitination is expected to be highly resistant to ubiquitination at abnormal pH or temperature, and to be low in resistance to ubiquitination at normal physiological pH or temperature. May be selected for. In one embodiment, the extracellular spacer domain that is conditionally resistant to ubiquitination is highly ubiquitinated at the pH of the tumor microenvironment and has normal physiology such as the pH of human plasma at pH 7.2-7.6. Ubiquitination resistance is low at the target pH.

To create a conditional extracellular spacer domain, a mutant protein is created by evolving a starting protein fragment selected from the Fc fragment of the antibody, the hinge region of the antibody, the CH2 region of the antibody, and the CH3 region of the antibody. This mutant protein is subjected to analysis under normal physiological conditions and analysis under abnormal conditions. The conditional extracellular spacer domain has (a) a first conformation in which the antigen-specific target region binds to the target antigen with higher binding activity under abnormal conditions, and the antigen-specific target region under normal physiological conditions. A variant protein that exhibits a conditional extracellular spacer domain with a second conformation of the conditional extracellular binding domain that binds to the target antigen with lower binding activity than under abnormal conditions, or (b) normal under abnormal conditions. It is selected from proteins that are more resistant to ubiquitination than physiological conditions.

Any of the aforementioned chimeric antigen receptors can be configured such that the protein containing this antigen receptor has an increased expression level as compared to the parental or wild-type protein or its domain.

In an alternative embodiment, the invention is a chimeric antigen receptor (CAR) for binding to a target antigen, evolved from a parent or wild protein or domain thereof, and in analysis under aberrant conditions. Or a chimeric antigen receptor having at least one antigen-specific target region with increased selectivity compared to the antigen-specific target region of the wild protein or its domain; a transmembrane domain; and an intracellular signaling domain. I will provide a. In some embodiments, the chimeric antigen receptor further comprises an extracellular spacer domain or at least one co-stimulating domain.

The disclosure also includes (a) reduced activity compared to the antigen-specific target region of the parent or wild-type protein or its domain in analysis under normal physiological conditions, and (b) parent or wild in analysis under abnormal conditions. It also relates to a method of evolving a parent or wild-type protein or its domain to produce a conditionally active protein having at least one of the increased activity compared to the antigen-specific target region of the type protein or its domain. Conditionally active proteins can be engineered to be CAR.

The chimeric antigen receptor produced by this method has an antigen-specific target region having a reduced binding affinity for the target antigen as compared to the antigen-specific target region of the parent or wild-type protein or its domain under normal physiological conditions. Can include.

The chimeric antigen receptor produced by this method has increased activity compared to the antigen-specific target region of the parent or wild protein or its domain in analysis under abnormal conditions, and is parent under normal physiological conditions. Alternatively, it may include an antigen-specific target region having a reduced binding affinity for the target antigen as compared to the antigen-specific target region of the wild protein or its domain.

In any of the aforementioned chimeric antigen receptors produced by the method, the antigen-specific target region is also compared to the antigen-specific target region of the parent or wild-type protein or its domain in analysis under aberrant conditions. It can also have increased selectivity.

Any of the aforementioned chimeric antigen receptors produced by the method can be configured such that the protein containing this antigen receptor has an increased expression level as compared to the parental or wild-type protein or its domain.

In an alternative embodiment of the method, the chimeric antigen receptor (CAR) produced by the method for binding to a target antigen has evolved from the parent or wild protein or its domain and under abnormal conditions. The analysis comprises at least one antigen-specific target region having increased selectivity compared to the antigen-specific target region of the parent or wild protein or its domain; 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 co-stimulating domain.

Chimeric antigen receptors The immune system of mammals, especially humans, has cytotoxic cells that target and destroy affected tissues and / or pathogens. Using these cytotoxic cells to remove unwanted tissue (ie, target tissue) such as tumors is a promising therapeutic technique. Other tissues that can be targeted for removal include glandular (eg, prostate) hyperplasia, warts, and unwanted adipose tissue. However, this relatively new treatment method has so far been of limited success. For example, tumor targeting and destruction using T cells has relatively long-term benefits because cancer cells can reduce the effectiveness of this therapy by reducing the expression of surface antigens and adapting to new therapies. few. Cancer cells may even dedifferentiate to escape detection in response to tumor-specific T cells. Maher, "Immunotherapy of Malignant Disease Using Chimeric Antigen Receptor Engrafted T Cells," ISRN Oncology, vol. 2012, article ID 278093, 2012.

Cytotoxic cells expressing the chimeric antigen receptor can significantly improve the specificity and susceptibility of these cytotoxic cells. For example, CAR-expressing T cells (CAR-T cells) have the ability to use CAR to direct T cells to tumor cells that express cell surface antigens that specifically bind to CAR. Such CAR-T cells can more selectively deliver cytotoxic agents to tumor cells. CAR-T cells are capable of directly recognizing target molecules and are therefore typically not restricted by polymorphic elements such as human leukocyte antigen (HLA). The advantages of this CAR targeting strategy consist of three points. First, because CAR-T cell function is independent of HLA status, in principle the same CAR-based approach can be used in any patient with a tumor expressing the same target surface antigen. Second, alterations in antigen processing and presentation mechanisms are common properties of tumor cells and can promote immune escape. However, this provides defense against CAR-T cells. Third, this system can be used to target a variety of macromolecules, including proteins, carbohydrates, and glycolipids.

The chimeric antigen receptor of the present invention is a chimeric artificial protein containing at least one antigen-specific target region (ASTR), a transmembrane domain (TM) and an intracellular signal transduction domain (ISD). In some embodiments, CAR may further comprise an extracellular spacer domain (ESD) and / or a costimulatory domain (CSD). See FIG.

ASTR is the extracellular space of CAR for binding to specific target antigens, including proteins, carbohydrates, and glycolipids. In some embodiments, the ASTR comprises an antibody, in particular a single chain antibody, or a fragment thereof. ASTRs can include full long heavy chains, Fab fragments, single chain Fv (scFv) fragments, divalent single chain antibodies or diabodies, each of which is specific for the target antigen.

ASTR may also include another protein functional domain for recognizing and binding to the target antigen. The target antigen may have other biological functions, such as acting as a receptor or ligand, or the ASTR may include a functional domain for specifically binding to the antigen. Some examples of proteins with functional domains include ligated cytokines, which lead to the recognition of cells carrying cytokine receptors, affibody, ligand-binding domains derived from naturally occurring receptors, Examples include soluble protein / peptide ligands for receptors on tumor cells. As one of ordinary skill in the art will understand, virtually any molecule capable of binding a given antigen with high affinity can be used for ASTR.

In one embodiment, the CAR of the invention comprises at least two ASTRs that target at least two different antigens or two epitopes on the same antigen. In certain embodiments, the CAR comprises three or more ASTRs that target at least three or more different antigens or epitopes. When multiple ASTRs are present in the CAR, the ASTRs may be arranged in tandem and separated by a linker peptide (FIG. 1).

In one embodiment, the ASTR is specific to the target antigen, and _{if the VH} domain alone is sufficient to confer antigen specificity (“single domain antibody”), _VH , CH1, hinge, and CH2 and Includes a full-length IgG heavy chain with a CH3 (Fc) Ig domain. _{If both V H} and _VL domains are required to create a fully active ASTR, _{both V H-} containing CARs and full-length λ light chains (IgL) are introduced into the same cytotoxic cells to produce active ASTR. Will be created. In another embodiment, each ASTR of CAR comprises at least two single chain antibody variable fragments (scFv), each specific for a different target antigen. scFv is for C-terminus of one variable domain (V _H or V _L) is anchored via a polypeptide linker to the N-terminus of the other variable domain (V _L or V _H, respectively), the antigen binding Alternatively, it has been developed without significantly impairing the binding specificity (Chaudhary et al., "A recombinant single-chain immunotoxin composited of antibody-Tac variable regitions and antigen. vol.87, page 9491, 1990; Bedzyk et al., "Immunological and structural characteristic of a high affinity anti-fluorescein single-chain, single-chain. These scFvs lack the constant regions (Fc) present in the heavy and light chains of native antibodies. ScFv specific for at least two different antigens is placed in tandem. In certain embodiments, an extracellular spacer domain may be linked between the ASTR and the transmembrane domain.

In another embodiment, the scFv fragment may be fused to all or part of the constant domain of the heavy chain. In a further embodiment, the ASTR of CAR comprises a divalent (or bivalent) single chain variable fragment (di-scFv, bi-scFv). In CARs containing di-scFVs, two scFvs, each antigen-specific, are integrally linked to form a single peptide chain containing _{two VH} regions and two _{VL regions (Xiong et al.} , "Development of tumor targeting antibody-MUC-1 multitimer: effects of di-scFv unpaired cylinder "A revival of bispecific antibodies," Trends in Biotechnology, vol.22, pages 238-244, 2004).

In yet another embodiment, the ASTR comprises a diabody. In the diabody, scFv is created with a linker peptide that drives the dimerization of scFv because the two variable regions are too short to be folded together. Shorter linkers (1 or 2 amino acids) lead to the formation of trimers, so-called tribodies or tribodies. Tetra bodies can also be used for ASTR.

When two or more ASTRs are present in the CAR, the ASTRs are covalently attached to each other on a single polypeptide chain via an oligopeptide or polypeptide linker, Fc hinge or membrane hinge region.

Antigens targeted by CAR are present on or inside the cells of tissues targeted for removal, such as tumors, glandular (eg, prostate) hyperplasia, warts, and unwanted adipose tissue. Surface antigens are more efficiently recognized and bound by the ASTR of CAR, but intracellular antigens can also be targeted by CAR. In some embodiments, the target antigen is preferably specific for cancer, inflammatory disease, neuropathy, diabetes, cardiovascular disease, or infectious disease. Examples of target antigens are expressed by various immune cells, carcinomas, sarcomas, lymphomas, leukemias, germ cell tumors, blastomas, and cells associated with various blood disorders, autoimmune disorders, and / or inflammatory disorders. Contains antigens that are used.

Cancer-specific antigens that can be targeted by ASTR include 4-IBB, 5T4, adenocarcinoma antigen, α-fetoprotein, Axl, BAFF, B lymphoma cells, C242 antigen, CA-125, carbon dioxide dehydration enzyme 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 extra domain-B, folic acid receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2 / neu, HGF, Human Scattering Factor Receptor Kinase, IGF-1 Receptor, IGF-I, IgGl, LI-CAM, IL-13, IL-6, Insulin-like Growth Factor I Receptor, Integulin α5β1, Integrin ανβ3, MORAb-009, MS4A1 , MUC1, Mutin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-R a, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, ROR1, ROR2, SCH 900105, SDC1, SLAMF7, TAG-72. , Teneicin C, TGFβ2, TGF-β, TRAIL-R1, TRAIL-R2, Tumor Antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2 or one or more of Vimentin.

Antigens specific for inflammatory diseases that can be targeted by ASTR include AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (basidin), CD154 (CD40L), CD2, CD20. , CD23 (IgE receptor), CD25 (IL-2 receptor a chain), CD3, CD4, CD5, IFN-a, 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, integulin a4, integulin α4β7, llama gramma ( Lama glama), LFA-1 (CD1 la), MEDI-528, myostatin, OX-40, rhumab β7, scleroscin, SOST, TGFβ1, TNF-a or VEGF-A. ..

Antigens specific for neuronal damage that can be targeted by the ASTR of the present invention include one or more of β-amyloid or MABT5102A. Diabetes-specific antigens that can be targeted by the ASTR of the present invention include one or more of L-Iβ or CD3. Cardiovascular disease-specific antigens that can be targeted by the ASTR of the present invention include C5, cardiac myosin, CD41 (integrin α-lib), fibrin II, β chain, ITGB2 (CD18) and sphingosine-1-phosphate. One or more of them may be mentioned.

Antigens specific for infectious diseases that can be targeted by the ASTR of the present invention include charcoal toxin, CCR5, CD4, clamping factor A, cytomegalovirus, cytomegalovirus glycoprotein B, endotoxin, Escherichia coli. , Hepatitis B surface antigen, Hepatitis B virus, HIV-1, Hsp90, influenza A hemagglutinin, lipotaicic acid, Pseudomonas aeruginosa, mad dog disease virus glycoprotein, respiratory follicles virus and TNF-a One or more are mentioned.

Further examples of target antigens are surface proteins found in specific or amplified form on cancer cells such as IL-14 receptors, CD19, CD20 and CD40 of B-cell lymphoma, Lewis Y of various cancers and CEA antigens, Tag72 antigens for breast cancer and colorectal cancer, EGF-R for lung cancer, folic acid-binding and HER-2 proteins often amplified in human breast cancer and ovarian cancer, or viral proteins such as HIV gp120 and gp41 envelopes. Examples include proteins, enveloped proteins from hepatitis B and C viruses, glycoproteins B and other enveloped glycoproteins of human cytomegalovirus, and enveloped proteins from oncoviruses such as Kaposi sarcoma-related herpesviruses. Other potential target antigens include CD4, whose ligand is the HIV gp120 envelope glycoprotein, and other viral receptors, such as ICAM, which is a receptor for hytreinovirus, and related receptor molecules for poliovirus. ..

In another embodiment, the CAR activates cancer-treated cells by targeting antigens associated with cancer-treated cells such as NK cells and other cells referred to herein and acting as immune effector cells. Can be transformed. One example of this is CAR, which targets the CD16A antigen and associates NK cells to combat malignant lesions expressing CD30. The bispecific tetravalent AFM13 antibody is an example of an antibody capable of delivering this effect. For more details on this type of embodiment, see, eg, Rose, A. et al. , Et al. , "A phase 1 study of the bispecific anti-CD30 / CD16A antibody construct AFM13 in patients with relaxed or refractory Hodgkin. 125, no. 26, pp. You can refer to 4024-4031.

In one embodiment, ASTR targets a tumor-specific antigen selected from Axl, ROR2 and CD22.

In some embodiments, ASTR is a single-chain antibody that targets the cancer antigen Axl and contains a nucleotide sequence selected from SEQ ID NOs: 2-5 or an amino acid sequence selected from SEQ ID NOs: 9-12. You may have. These single-chain antibodies targeting the cancer antigen Axl contain a human IgG Fc region having the nucleotide sequence of SEQ ID NO: 6 or 7 or the amino acid sequence of SEQ ID NO: 13 or 14. The single-chain antibody targeting the cancer antigen Axl has a higher binding activity to Axl at pH 6.0 as compared with a binding activity to Axl at pH 7.4.

In another embodiment, ASTR is a single-chain antibody that targets the cancer antigen ROR2 and may have the nucleotide sequence of SEQ ID NO: 16 or the amino acid sequence of SEQ ID NO: 15. In the single-chain antibody targeting the cancer antigen ROR2, the binding activity to ROR2 at pH 6.0 is increased as compared with the binding activity to ROR2 at pH 7.4.

Single-chain antibodies targeting Axl and ROR2 are suitable for linking with transmembrane domains and intracellular signaling domains to form CAR structures.

The extracellular spacer domain of CAR is a hydrophilic region located between the ASTR and the transmembrane domain. In some embodiments, this domain promotes protein folding appropriate for 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 the antibody, the hinge region of the antibody, the CH2 region of the antibody, the CH3 region of the antibody, the artificial spacer sequence or a combination thereof. Examples of extracellular spacer domains are CD8a hinges, artificial spacers made of polypeptides that can be as large as three glycines (Gly), and CH1 and CH3 domains of IgG (such as human IgG4). Can be mentioned.

The transmembrane domain of CAR is a region capable of spanning the cell membrane of cytotoxic cells. The transmembrane domain is selected from transmembrane regions of transmembrane proteins, such as type I transmembrane proteins, artificial hydrophobic sequences or combinations thereof. Examples of transmembrane domains include α, β or ζ chains of T cell receptors, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, Examples include transmembrane regions of CD134, CD137, and CD154. Synthetic transmembrane domains may include triplets of phenylalanine, tryptophan and valine. Optionally, a short, preferably 2-10 amino acid long oligopeptide or polypeptide linker may form a link between the transmembrane domain of CAR and the intracellular signaling domain. Glycine-serine doublets provide a particularly suitable linker between the transmembrane domain and the intracellular signaling domain.

The CAR of the present invention also includes an intracellular signal transduction domain. The intracellular signaling domain transmits effector function signals and directs cytotoxic cells to perform their specialized function: inhibition and / or destruction of target cells. Examples of intracellular signal transduction domains include ζ chains of T cell receptor complexes or their homologues, such as η chains, FcsRly and β chains, MB 1 (Iga) chains, B29 (Ig) chains, etc. , Human CD3ζ chain, CD3 polypeptides (Δ, δ and ε), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.) and others involved in T cell transduction. Examples include molecules such as CD2, CD5 and CD28. Specifically, the intracellular signaling domain is the human CD3ζ chain, FcyRIII, FcsRI, the cytoplasmic tail of the Fc receptor, the immunoreceptor tyrosine-based activation motif (ITAM) carrying the cytoplasmic receptor, and combinations thereof. possible.

The intracellular signaling domains used for CAR include several types of various other immune signaling receptors such as, but not limited to, CD3, B7 family co-stimulation, and tumor necrosis factor receptors (TNFR). Intracellular signaling domains of first-generation, second-generation, and third-generation T-cell signaling proteins, including superfamily receptors, may be included (Park et al., "Are all chimeric receptor receptors?" J. Clin Oncol., Vol.33, pp.651-653, 2015). In addition, the intracellular signaling domains include the signaling domains used by NK and NKT cells (Hermanson, et al., "Utilizing chimeric antigen receptors to direct natural killer cell activity," Front Immun. 195, 2015), for example, NKp30 (B7-H6) (Zhang et al., "An NKp30-based chimeric receptor receptor protectors T cell effector functionions and engine. 2290-2299, 2012), and DAP12 (Topfer et al., "DAP12-based activated chimeric antigen receptor for NK cell tumor immunotherapy," J Immunol. , NKp46, DAP10, and CD3z signaling domains and the like. In addition, the intracellular signaling domain also includes the signaling domains of human immunoglobulin receptors containing the immunoreceptor tyrosine-based activation motif (ITAM), such as FcγRI, FcγRIIA, FcγRIIC, FcγRIIIA, FcRL5 ( Gillis et al., "Contribution of Human FcγRs to Disease with Evidence from Human Polymorphisms and Transgenic Animal Studios," Front 25, 20.

In some embodiments, the intracellular signaling domain comprises the cytoplasmic signaling domain of TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, or CD66d. It is particularly preferred that the intracellular signaling domain of CAR comprises the cytoplasmic signaling domain of human CD3ζ.

The CAR of the present invention may contain a co-stimulation domain, which serves to promote cell proliferation, cell survival and memory cell development of cytotoxic cells expressing CAR. The CAR of the present invention is a protein of the TNFR superfamily, CD28, CD137 (4-1BB), CD134 (OX40), DappleO, CD27, CD2, CD7, CD5, ICAM-1, LFA-1 (CD1 la / CD18), One or more co-stimulation domains selected from the co-stimulation domains of Lck, TNFR-I, PD-1, TNFR-II, Fas, CD30, CD40, ICOS LIGHT, NKG2C, B7-H3, or combinations thereof. May include. If the CAR contains two or more co-stimulating domains, these domains can optionally be separated by a linker and placed in tandem. The co-stimulation domain is an intracellular domain that can be located between the transmembrane domain of CAR and the intracellular signaling domain.

In some embodiments, the two or more components of the CAR of the invention are separated by one or more linkers. For example, in a CAR containing at least two ASTRs, those two ASTRs may be separated by a linker. The linker is an oligopeptide or polypeptide region about 1-100 amino acids long. In some embodiments, the linker can be, for example, 5-12 amino acids long, 5-15 amino acids long or 5-20 amino acids long. The linker may be composed of mobile residues such as glycine and serine so that adjacent protein domains can move freely with each other. Longer linkers, such as those longer than 100 amino acids, may be used in connection with alternative embodiments of the invention, eg, two adjacent domains may be selected so that they do not sterically interfere with each other. Examples of linkers that can be used in the present invention include, but are not limited to, 2A linkers (eg, T2A), 2A-like linkers or functional equivalents thereof.

The conditional active antigen-specific target region CAR is a chimeric protein produced by fusing all of the various domains discussed above together to form a fusion protein. CAR is typically generated by an expression vector containing polynucleotide sequences encoding the various domains of CAR. The ASTR of the present invention, which functions to recognize and bind to an antigen on a target cell, is a conditioned active form. Specifically, ASTR is less active or inactive under normal physiological conditions and more active under abnormal conditions with respect to binding to the target antigen as compared to the corresponding parental or wild-type protein ASTR. The present invention provides a method for producing a conditional active ASTR from a parent or wild-type protein or a binding domain thereof (parent or wild-type ASTR).

Since the ASTR in the present invention can be discovered by creating a protein library and screening the library for proteins having the desired binding affinity for the target antigen, at least all or part of the binding thereof to the target antigen. A wild-type protein suitable for use in the domain. Wild-type proteins can be found by screening a cDNA library. A cDNA library is a combination of cloned cDNA (complementary DNA) fragments inserted into a collection of host cells that together form part of the transcriptome of an organism. The cDNA is made from fully transcribed mRNA and thus contains the coding sequence for the expressed protein of the organism. The information in the cDNA library is a powerful and useful tool for discovering proteins with the desired properties by screening the library for proteins with the desired binding affinity for the target antigen.

In some embodiments where the wild-type protein is an antibody, the wild-type antibody can be found by creating and screening an antibody library. The antibody library may be either a polyclonal antibody library or a monoclonal antibody library. Polyclonal antibody libraries against target antigens can be made by injecting the antigen directly into an animal or by administering the antigen to a non-human animal. The antibody thus obtained corresponds to a library of polyclonal antibodies that bind to the antigen. Any technique can be used to prepare the monoclonal antibody library to yield the antibodies produced by continuous cell line culture. Examples include hybridoma methods, trioma methods, human B cell hybridoma methods, and EBV-hybridoma methods (eg, Core (1985) in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-. See 96). The techniques described for making single-chain antibodies (see, eg, US Pat. No. 4,946,778) can be adapted for making single-chain antibody libraries.

There are other methods for creating and screening antibody libraries for the discovery of wild-type antibodies. For example, a fully human antibody display library can be utilized. Such a library is a collection of antibodies presented on the surface of one or more host cells. Preferably, the antibody library represents the human antibody repertoire in that it has the ability to bind a wide variety of antigens. Since the antibodies are presented on the surface of the cell, the effective affinity (due to avidity) of each antibody in the library is increased. Unlike other common library types such as phage display libraries (antibody avidity is less desirable for screening and identification purposes), the super-avidity provided by the cell surface display in the present invention is desirable. The cell surface display library allows the identification of antibodies with low, moderate and high binding affinities, as well as the identification of non-immunogenic and weak epitopes in the screening or selection process.

Generation of evolved molecules from the parent molecule When the parent or wild-type protein, or its binding domain (parent or wild-type ASTR) undergoes a mutagenesis process, a population of mutant polypeptide is produced, which is then screened. By doing so, the binding affinity for the target antigen is enhanced under abnormal conditions in comparison with the parent or wild-type ASTR, and optionally, the binding affinity for the target antigen is substantially the same or low under normal physiological conditions. The mutant ASTR can be identified.

Variant polypeptide populations can be generated using any chemical synthesis or recombinant mutagenesis method. Unless otherwise indicated, prior arts of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology can be used in the practice of the present invention. Is within the scope of the technology in the field. Such techniques are well described in the literature. For example, Molecular Cloning A Laboratory Manual, 2nd Ed. , Ed. by Sambrook, Fritzch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D.N. Grover ed., 1985); Oligonucleotide. et al. US Pat. No. 4,683,195; Nuclear Acid Hybridization (BD Hames & S.J. Higgins eds. 1984); Transscription And Transition (BD Hames & S. J. Higgins. 1984). ); Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In 1987 (Academic Press, Inc., NY); Gene Transfer , 1987, Cold Spring Harbor Laboratory); Methods In Enzymeology, Vols. 154 and 155 (Wu et al. Eds.), Immunochemical Materials In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987) and CC Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986).

The present disclosure provides a method of producing a nucleic acid variant encoding a conditional active variant polypeptide, in which (i) one or more nucleotides are replaced with different nucleotides (the nucleotides are). Including natural or unnatural nucleotides); (ii) deleting one or more nucleotides, (iii) adding one or more nucleotides, or (iv) modifying the nucleic acid by any combination of these. Includes steps. In one embodiment, the unnatural nucleotide comprises inosine. In another embodiment, the method is a step of analyzing a polypeptide encoded by a modified nucleic acid with respect to a change in enzymatic activity, thereby encoding one or more modified nucleic acids encoding a polypeptide with altered enzymatic activity. It further includes the step of identifying. In one embodiment, the modifications of step (a) include PCR, error-prone PCR, shuffling, oligonucleotide-specific mutagenesis, assembly PCR, sexual mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive 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, arbitrary DNA It is made by the generation technique and any combination thereof. In another embodiment, the method further comprises at least one iteration of the modification step.

This method further provides a method for producing a polynucleotide from two or more nucleic acids. This method comprises the following (a) to (c). (A) A step of identifying the same region and various regions among two or more nucleic acids. There, at least one of these nucleic acids comprises the nucleic acids of the present disclosure. (B) A step of providing a set of oligonucleotides corresponding to at least two sequences of two or more nucleic acids. And (c) a step of extending an oligonucleotide with a polymerase, thereby producing a polynucleotide.

Any mutagenesis technique may be used in various embodiments of the present disclosure. Stochastic or random mutagenesis is illustrated by the situation in which the parent molecule is mutated (modified or altered) to obtain a series of progeny molecules with unplanned mutations. Thus, an in vitro stochastic mutagenesis reaction is, for example, an unplanned product that is not intended for its production. Rather, it is uncertain and therefore random with respect to the exact nature of the resulting mutations and thus the product produced. Stochastic mutagenesis is demonstrated in random or unplanned mutagenesis methods such as error-prone PCR and stochastic shuffling. Variant forms can be obtained by error prone transcription, such as error prone PCR in the first variant form, by the use of polymerases lacking proofreading activity (see Liao (1990) Gene 88: 107-111), or by mutant strains (mutant hosts). Cells are discussed in more detail below, which are generally well known) and can be created by replication of the first form. Mutagenesis strains can include any variant that is deficient in the function of inconsistent repair. These include mutant gene products such as mutS, mutT, mutH, mutL, ovrD, dcm, vsr, umuC, umuD, sbcB, recJ. Defects are obtained by gene mutation, allelic exchange, microcompounds or expressed antisense RNA or other techniques. The defect can be the described gene or a homologous gene in any organism.

Other mutagenesis methods include oligonucleotide-specific mutagenesis techniques, error-prone polymerase chain reaction (error-prone PCR) and cassette mutagenesis, where specific regions of the parent polynucleotide are synthetic. Is replaced by a mutagenetic oligonucleotide. In such cases, a number of mutation sites are generated around a particular site in the parent sequence.

In oligonucleotide-designated mutations, short sequences are replaced with synthetically mutated oligonucleotides. In oligonucleotide-designated mutations, short sequences of polynucleotides are removed from synthetic polynucleotides using restriction enzyme digestion and various bases are replaced with synthetic polynucleotides modified from the original sequence. The polynucleotide sequence may also be altered by chemical mutagenesis. Chemical mutagens include, for example, sodium bisulfite, nitrite, hydroxylamine, hydrazine, or formic acid. Other agents similar to nucleotide precursors include nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine. Generally, these agents are added to the PCR reaction in place of the nucleotide precursors that thereby displace the sequence. Insertion of drugs such as proflavines, acriflavine, quinacrine, etc. may also be used. Random mutagenesis of polynucleotide sequences may be obtained by X-ray or UV irradiation. Generally, mutagenetic plasmid polynucleotides are introduced into E. coli and transmitted as a pool or library of crossed plasmids.

Errorprone PCR uses low fidelity polymerization conditions to randomly introduce low levels of point mutations over long sequences. In a mixture of fragments of unknown sequences, error-prone PCR may be used to mutate the mixture.

In cassette mutagenesis, single template sequence blockades are typically (partially) replaced by random sequences. Reidhar-Olson J F and Sauer RT: Combination cassette mutagenesis as a probe for the information content of a protein sequence. Science 241 (4861): 53-57, 1988.

Alternatively, any technique of non-stochastic or non-random gene abrupt induction can also be used in various embodiments of the present disclosure. Non-stochastic mutagenesis is illustrated by the situation in which the parent molecule is mutated (modified or altered) to obtain a molecule with one or more predetermined mutations. The presence of background products in certain amounts is present in many reactions where molecular processing occurs, and the presence of background products is the non-stochastic nature of mutagenesis steps with unplanned products. It is well understood that it does not diminish from. Site-saturated mutagenesis and synthetic ligation reassembly are examples of gene mutagenesis techniques, in which the exact chemical structure of the intended product is envisioned.

One method of site-saturation point mutagenesis is described in US Patent Application Publication No. 2009/0130718. This method provides a set of denatured primers corresponding to the codons of the template polynucleotide and performs polymerase extension to produce a progeny polynucleotide containing the sequence corresponding to the denatured primer. Progeny polynucleotides are expressed and screened in direct evolution. Specifically, this is a method for producing a series of progeny polypeptides, step (a) providing a copy of the template polypeptide consisting of a plurality of codons encoding the template polypeptide sequence. And (b) at each codon of the template polynucleotide, (1) a step of providing a series of denatured primers, wherein each primer consists of denatured codons corresponding to the codons of the template polynucleotide, at least one flanking sequence. The steps of (2) providing conditions under which the primer can anneal to a copy of the template polynucleotide, which is homologous to the sequence flanking the codon of the template polynucleotide, and (3) the polymerase extension reaction from the primer along the template. Steps to be performed, steps (1) to (3), thereby providing progeny polynucleotides, each containing a sequence corresponding to the denatured codon of the annealed primer, thereby a series of progeny polynucleotides. To manufacture. The site saturation mutagenesis method is a method related to the direct evolution of nucleic acid and screening clones containing the evolved nucleic acid to obtain the desired binding activity as a result.

Site saturation mutagenesis is generally 1) mutagenesis from one or more ancestral or parental generation templates to achieve at least one point mutation, addition, deletion, and / or chimerization. Prepare progeny of one or more molecules, including molecules containing a polypeptide sequence, molecules containing a polypeptide sequence, and molecules partially containing a polynucleotide sequence and partially containing a polypeptide sequence. 2) One or more progeny molecules-preferably using high-throughput methods-screened for the desired binding affinity for the target antigen; 3) optionally the structure and / or progeny molecules for the parent and / or progeny molecules. Or, and obtain and / or catalog functional information; and 4) optionally relate to a method of repeating any of steps 1) to 3).

In site saturation mutagenesis, the progeny of a polynucleotide called "codon site saturation mutagenesis" generated (eg, from a parent polynucleotide template) is a denatured codon that encodes all codons (or the same amino acid). Each has at least one set of three or more adjacent point mutations (ie, different bases consisting of new codons) so that the whole lineage of) appears at each codon position. Corresponding to the progeny generation of this polynucleotide, and encoded by the progeny generation of this polynucleotide, is a series of progeny polypeptides produced, each with at least one single amino acid point mutation. In a preferred embodiment, the generated example, referred to as "amino acid site saturation mutagenesis," is a naturally encoded 19 that forms an alpha amino acid substitution at each amino acid position and all amino acid positions along the polypeptide. It is a variant polypeptide in each of the polypeptides. This yield-all amino acid positions and all amino acid positions along the parent polypeptide-all of the 20 different progeny polypeptides, including the original amino acid, or additional amino acids replaces the 20 naturally encoded amino acids. , Or added, are potentially 21 or more different progeny polypeptides.

Other mutagenesis techniques may be used involving recombination, and more specifically, poly that encodes a polypeptide by a method of in vivo reassembly of a polynucleotide sequence containing a region of partial homology. Includes methods for preparing nucleotides, assembling polynucleotides to form at least one polynucleotide, and screening polynucleotides for the production of polypeptides with useful properties.

In other embodiments, the mutagenesis technique is intended to recombinate the molecule and / or to convey a reduced method that reduces the complexity of a range of repetitive or contiguous sequences with sequences and homologous regions. Take advantage of the nature.

Various mutagenesis techniques may be used alone or in combination to provide a method for producing a hybrid polypeptide that encodes a biologically active hybrid polypeptide. In achieving these and other objectives, according to one aspect of the present disclosure, there is provided a method of introducing a polypeptide into a suitable host cell and growing the host cell under conditions for producing a hybrid polypeptide. ing.

Chimeric genes are made by joining two polynucleotide fragments that use compatible sticky ends produced by restriction enzymes. Here, each fragment is derived from a separate ancestral (parent) molecule. Another embodiment is a single codon position mutagenesis (ie, codon substitution, in a parent polynucleotide for producing a single progeny polynucleotide that encodes for a single site mutagenetic polypeptide. To obtain additions and deletions).

In addition, the in vivo site-specific recombination system produces gene crosses and homologous but recombinants between genes cleaved on the plasmid, similar to random methods of in vivo recombination. Used for. Mutagenesis was also reported by duplication and PCR.

Non-random methods are used to obtain larger numbers of point mutations and / or chimerizations, eg, synthetic or comprehensive methods are specific structural groups (eg, specific singles) in the template molecule. All in a particular mutation grouping to functionally belong to an amino acid position or a sequence consisting of two or more amino acid positions) and to classify and compare specific groupings of mutations. Used to produce the molecular species of.

In the present disclosure, any of these or other evolutionary methods can be used to generate a novel population (library) of mutant polypeptides from parental or wild-type proteins.

Expression of evolutionary molecules The mutant polynucleotides produced from the evolutionary process are size fractionated on an agarose gel according to previously published protocols, inserted into an expression vector, and suitable for the production (expression) of the mutant polypeptide. It may or may not be transfected into the host cell. Expression can use routine molecular biology techniques. Therefore, various known methods can be used for the expression step.

For example, in brief, variant polynucleotides generated from evolutionary processes are then digested using standard molecular biology techniques and ligated into expression vectors such as plasmid DNA. The vector is then transformed into bacteria or other cells using standard protocols. This can be done on individual wells in a multi-well tray, such as a 96-well tray for high-throughput expression and screening. This process is repeated for each mutant polynucleotide.

The polynucleotide thus selected and isolated is introduced into a suitable host cell. Suitable host cells are any cells capable of promoting genetic recombination and / or reduced reassortment. The selected polynucleotide is preferably one already present in the vector containing the appropriate control sequence. The host cell may 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 may be a prokaryotic cell such as a bacterial cell. Introduction of constructs into host cells can be accomplished by calcium phosphate transfection, DEAE-dextran-mediated transfection, or electroporation (eg, Ecker and Davis, 1986, Inhibition of gene expression in plant cells by expression). of antisense RNA, Proc Natl Acad Sci USA, 83: 5372-5376).

Representative examples of available expression vectors are viral particles, baculovirus, phage, plasmid, phagemid, cosmid, phosmid, bacterial artificial chromosome, viral DNA (eg, vaccinia, adenovirus, foul poke virus, pseudomad dog disease, and SV40 faction). Organisms) pl-based artificial chromosomes, yeast plasmids, yeast artificial chromosomes, and any other vector specific for a particular host of interest (eg, rod fungus, Aspergillus, and yeast) can be referred to. Thus, for example, DNA can be included in any one of various expression vectors for expressing a polypeptide. Such vectors include chromosomal DNA sequences, non-chromosomal DNA sequences, and synthetic DNA sequences. Many suitable vectors are well known to those of skill in the art and are commercially available. The following vector is given as an example. Bacterial: pQE vector (Qiagen), pBluescrit plasmid, pNH vector, Lambda ZAP vector (Stratagene); ptrc99a, ρKK223-3, pDR540, pRIT2T (Pharmacia); Eukaryotic: pXTl, ρSG5 , PSVLSV40 (Pharmacia). However, any other plasmid or other vector can be used as long as they are replicable and viable in the host. A low copy count vector or a high copy count vector can be used in the present invention.

The variant polynucleotide sequence in the expression vector is operably linked to the appropriate expression control sequence (promoter) for deriving RNA synthesis. Specific named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda PR, PL, and trp. Eukaryotic promoters include pre-early CMV, HSV thymidine kinase, early and late SV40, retrovirus-derived LTR, and mouse metallothionein-1. Selection of suitable vectors and promoters is well within the standards of those of skill in the art. The expression vector also contains a ribosome binding site and a transcription terminator for translation initiation. This vector may contain suitable sequences for amplifying expression. The promoter region can be selected from any desired gene using a chloramphenicol transferase (CAT) vector with a selectable marker, or other vector. In addition, the expression vector is a phenotype for selection of transformed host cells, such as dihydrofolate reductase or neomycin resistance to eukaryotic cell culture, or tetracycline or ampicillin resistance in E. coli. To provide features, it preferably comprises one or more selectable marker genes.

Eukaryotic DNA transcription can be increased by inserting an enhancer sequence into the expression vector. Enhancers are 10-300 bp cis-acting sequences that increase transcription by promoters. The enhancer can effectively increase transcription when it is on either the 5'side or the 3'side of the transfer unit. Enhancers are also useful if they are located within the intron or within the coding sequence itself. Typically, virus enhancers including SV40 enhancers, cytomegalovirus enhancers, polyoma enhancers, and adenovirus enhancers are used. Enhancer sequences from mammalian descent, such as mouse immunoglobulin heavy chain enhancers, are also commonly used.

Mammalian expression vector systems also typically include selectable marker genes. Examples of suitable markers include dihydrofolate reductase gene (DHFR), thymidine kinase gene (TK), or prokaryotic genes that confer drug resistance. For the first two marker genes, it is preferred to use mutant cell lines that cannot grow without the addition of thymidine to the growth medium. Transformed cells can then be identified by their ability to grow in non-supplemented medium. Examples of prokaryotic drug resistance genes useful as markers include genes that confer resistance to G418, mycophenolic acid and hygromycin.

The expression vector containing the DNA segment of interest can be transferred to the host cell by a well-known method depending on the type of cell-producing host. For example, calcium chloride transfection is often used for prokaryotic host cells, while calcium phosphate treatment, lipofection, or electrical perforation can be used for eukaryotic host cells. Other methods used for transformation of mammalian cell-producing hosts include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection (generally see Sambrook et al., Supra).

When the expression vector is introduced into a suitable host, the host is maintained under suitable conditions such that the introduced mutant polynucleotide sequence is highly expressed to produce the mutant polypeptide. Expression vectors are typically replicable in the host organism as episomes or as an integral part of the host chromosomal DNA. In general, expression vectors may contain selectable markers such as tetracycline or neomycin to allow detection of cells transformed with the desired DNA sequence (see, eg, US Pat. No. 4,704,362). That).

Thus, in another aspect of the invention, the mutant polynucleotide can be produced by a method of decreasing reassortment. Methods include the generation of constructs, including contiguous sequences (original coding sequence sequences), their insertion into the appropriate vector, and subsequent introduction of them into the appropriate host cell. The reassortment of individual molecular identities occurs by a combination method between continuous sequences in constructs with homologous regions or between pseudo-repeating units. The recombination method recombines and / or reduces the complexity and range of repeats, resulting in the generation of new molecular species. Various treatments can be applied to increase the rate of reassortment. These can include UV treatment or DNA damaging chemicals and / or the use of host cell lines exhibiting enhanced levels of "genetic instability". Thus, reassortment methods can include the natural properties of homologous recombination or quasi-repeated sequences to guide their own evolution.

The cells then proliferate and "decreasing reassortment" is achieved. The proportion of the decreasing reassortment method can be stimulated by the introduction of DNA damage, if desired. In vivo reassortment is directed to an "intermolecular" method collectively referred to as "genetical recombination", which is commonly observed in bacteria as a "RecA-dependent" phenomenon. The present invention depends on the ability of the cell to regulate the method of genetic recombination of the host cell for recombination and rearrangement of the sequence, or the reduction method for reducing the complexity of quasi-repeated sequences in the cell by deletion. Can be done. This method of "reduced reassortment" occurs by a "intramolecular", RecA-independent method. The end result is the reassortment of molecules into all possible combinations.

In one embodiment, the host organism or cell comprises a Gram-negative bacterium, a Gram-positive bacterium or a eukaryote. In another aspect of the present disclosure, Gram-negative bacteria include Escherichia coli, or Pseudomonas fluororescens. In another aspect of the present disclosure, the gram-positive strains are Streptomyces diversa, Lactobacillus gasseri, Lactococcus lactis, Lactococcus lactis, Lactococcus cles Includes Bacillus subtilis. In another aspect of the present disclosure, the eukaryotic organisms are Saccharomyces cerevisiae, Saccharomyces pombe, Pichia pastoris, Pichia pastoris, and Pichia pastoris. -Includes Hansenula plymorpha, or Aspergillus niger. Representative examples of suitable hosts include bacterial cells such as E. coli, Streptomyces, Salmonella typurium; fungal cells such as yeast; insect cells such as fruit fly (Drosophila). ) S2 and Spodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma; adenovirus; and plant cells. The selection of a suitable host is considered to be within the skill of one of ordinary skill in the art from the teachings herein.

In addition to eukaryotic microorganisms such as yeast, mammalian tissue cell cultures can also be used to express the variant polypeptides of the invention (Winnacker, "From Genes to Clones," VCH Publishers, NY, N. . Y. (1987)). Eukaryotic cells are preferred because a number of suitable host cell lines capable of secreting intact immunoglobulins have been developed in the art, preferably CHO cell lines, various COS cell lines, HeLa cells, myeloma cell lines. , B cells or hybridomas. Expression vectors of these cells include expression control sequences such as origins of replication, promoters, enhancers (Queen et al., Immunol. Rev., vol. 89, page 49, 1986), and essential processing information sites, eg, It may include a ribosome binding site, an RNA splice site, a polyadenylation site, and a transcription termination sequence. Preferred expression control sequences are promoters derived from immunoglobulin genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus and the like.

In one embodiment, the eukaryotic host cells are CHO, HEK293, IM9, DS-1, THP-1, Hep G2, COS, NIH 3T3, C33a, A549, A375, SK-MEL-28, DU 145, PC. -3, HCT 116, Mia PACA-2, ACHN, Jarkat, MM1, Ovicar 3, HT 1080, Panc-1, U266, 769P, BT-474, Caco-2, HCC 1954, MDA-MB-468, LnCAP , NRK-49F, and SP2 / 0 cell line; and selected from mouse splenocytes and rabbit PBMC. In one embodiment, the mammalian host cell (mammalian hoist cell) is selected from the CHO or HEK293 cell lines. In a specific embodiment, the mammalian host cell is a CHO-S cell line. In another specific embodiment, the mammalian system is a HEK293 cell line. In another embodiment, the eukaryotic host is a yeast cell line. In one embodiment, the eukaryotic host is S. It is selected from S. cerevisiae yeast cells or Pichia yeast cells.

In another embodiment, the mammalian host cell may be commercially produced by a contract research organization or a contract research organization. For example, for recombinant antibodies or other proteins, Lonza (Lonza Group Ltd, Basel, Swisserland) created the vector and used GS Gene Expression System ™ technology with either the CHOK1SV or NS0 cell-producing host. These products can be expressed. Host cells containing the polynucleotide of interest can be cultured in conventional nutrient media appropriately modified for promoter activation, transformant selection or gene amplification. Culture conditions such as temperature, pH, etc. were previously used in the host cells selected for expression and will be apparent to those of skill in the art.

As discussed above, optimizing the expression of conditional active ASTR involves optimizing the vectors used (vector components such as promoters, splice sites, 5'and 3'ends and flanking sequences), genetic modification of host cells. By reducing gene deletion and rearrangement by, evolving host cell gene activity by methods of evolving related genes in vivo or in vitro, optimizing host glycosylating enzymes by evolving related genes. And / or chromosomal-wide host cells can be achieved by mutagenesis and selection strategies for selecting cells with enhanced expressive capacity.

Protein expression can be induced by a variety of well-known methods, and many genetic systems have been published for the induction of protein expression. For example, for suitable systems, the addition of inducers induces protein expression. The cells are then pelleted by centrifugation and the supernatant is removed. Periplasmic proteins can be enhanced by culturing cells with DNAse, RNAse and lysozyme. After centrifugation, the supernatant containing the novel protein is transferred to a new multi-well tray and stored prior to the assay.

Cells are generally harvested by centrifugation, separated by physical or chemical means, and the resulting crude extract is retained for further purification. Microbial cells used for protein expression can be isolated by any convenient method, including freeze-thaw cycles, sonication, mechanical separation, or the use of cytolytic agents. Such methods are well known to those of skill in the art. The expressed polypeptide or fragment thereof may be prepared by a method comprising ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. It can be recovered from the recombinant cell culture and purified. The protein refolding process can be used, if desired, in finishing the structure of the polypeptide. If desired, high performance liquid chromatography (HPLC) can be used for the final purification process. Screening for conditional active ASTR can be facilitated by the availability of convenient high-throughput screening or selection processes. Cell surface display expression and screening techniques (eg, as defined above) can be used to screen mutant proteins for conditional active ASTR.

Screening for Identifying Reversible or Irreversible Mutations Identification of the desired molecule is achieved very directly by measuring protein activity under acceptable and wild-type conditions. Mutants with the highest activity ratio (acceptable / wild-type) can then be selected and combined with individual mutations using standard methods to generate a permutation of point mutations. You can. The combined sorted protein library is then screened for the protein with the greatest difference in acceptable and wild-type activity.

The activity of the supernatant can be screened using a variety of methods, eg, high-throughput activity assays such as fluorescence assays, to identify protein variants that are sensitive to the desired properties (temperature, pH, etc.). It is possible. For example, to screen for time-sensitive variants, each individual variant is commercially available at low temperatures (such as 25 ° C) and at temperatures at which the original protein functions (such as 37 ° C). Enzyme activity or antibody activity is measured using possible substrates. Screening can be performed on a variety of media, especially serum and BSA. The reaction can be initially performed in a multi-well analytical format, such as 96-well analysis, and confirmed using another format, such as a 14 ml tube format.

In one aspect, the method further comprises modifying at least one nucleic acid or polypeptide prior to testing the candidate's conditional bioactivity. In another embodiment, the testing step (c) further comprises testing for elevated expression of the polypeptide in a host cell or host organism. In a further embodiment, the testing step (c) further comprises testing the enzyme activity in the pH range of about pH 3 to about pH 12. In a further embodiment, the testing step (c) further comprises testing the enzyme activity in the pH range of about pH 5 to about pH 10. In a further embodiment, the testing step (c) further comprises testing the enzyme activity in the pH range of about pH 6 to about pH 8. In a further embodiment, the testing step (c) further comprises testing the enzyme activity in the pH range of about pH 6.7 to about pH 7.5. In another embodiment, the testing step (c) further comprises testing the enzyme activity in the temperature range of about 4 ° C to about 55 ° C. In another embodiment, the testing step (c) further comprises testing the enzyme activity in the temperature range of about 15 ° C to about 47 ° C. In another embodiment, the testing step (c) further comprises testing the enzyme activity in the temperature range of about 20 ° C to about 40 ° C. In another embodiment, the testing step (c) further comprises testing the enzyme activity in the temperature range of about 25 ° C to about 37 ° C. In another embodiment, the step (c) to be tested further comprises testing the enzyme activity under normal osmotic pressure and (positively or negatively) abnormal osmotic pressure. be. In another embodiment, the step (c) of testing further comprises testing enzyme activity under normal electrolyte concentration and (positively or negatively) abnormal electrolyte concentration. It is a thing. The electrolyte concentration to be tested is selected from the concentrations of calcium, sodium, potassium, magnesium, chlorine, bicarbonate, and phosphoric acid. In another embodiment, the test step (c) further comprises a step of testing the enzyme activity resulting in a stable reaction product.

In another aspect, the disclosure provides a purified antibody that specifically binds to an enzymatically active polypeptide or fragment thereof of the present disclosure. In one aspect, the present disclosure provides a fragment of an antibody that specifically binds to a polypeptide having enzymatic activity.

Antibodies and Antibody-Based Screening Methods The present disclosure provides isolated or recombinant antibodies that specifically bind to the enzymes of the present disclosure. These antibodies can be used to isolate, identify, or quantify the enzymes of the present disclosure or related polypeptides. These antibodies can be used to isolate other polypeptides, or other related enzymes, within the scope of the present disclosure. Antibodies can be designed to bind to the active site of the enzyme. Accordingly, the present disclosure provides a method of suppressing an enzyme using the antibodies of the present disclosure.

Antibodies can be used in immunoprecipitation, staining, and immunoaffinity columns and the like. If required, the nucleic acid sequence encoding a particular antigen can be generated by immunological treatment followed by isolation, amplification or cloning of the polypeptide or nucleic acid, as well as immobilization of the polypeptide on the arrays of the present disclosure. Alternatively, the methods of the present disclosure can be used to modify the structure of the antibody produced by the cells, modifying the antibody, eg, increasing or decreasing the affinity of the antibody. In addition, the ability to produce or modify antibodies can be a phenotype designed intracellularly by the methods of the present disclosure.

Methods of immunotreatment, antibody production and isolation (polyclonal and monoclonal) are known to those of skill in the art and are described in the scientific and patent literature. For example, Colon, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley / Greene, NY (1991); ("States"); Goding, Monoclonal Antibodies: PRINCIPLES AND PRACTICE (2d ed.) Academic Press, New York, N. et al. Y. (1986); Kohler (1975) “Continuous cultures of fused cells secreting antibody of predefined specificity”, Nature 256: 495; Harlow (1988) ANTIBO checking ... Antibodies can also be produced in vitro, for example, by using a phage display library that expresses the binding site of the recombinant antibody, in addition to conventional animal-based in vivo methods. For example, Hoogenboom (1997) "Designing and optimizing library selection strategies for geneserating high-affinity antibodies", Trends Biotechnology. 15: 62-70; and Katz (1997) "Determinant and deterministic determinants of affinity and specialifecty of lidands diskovered or engineered by bacteriophage". Rev. Biophyss. Biomol. Struct. 26: 27-45. checking ...

The polypeptide or peptide can be used to specifically make a polypeptide of the present disclosure, eg, an antibody that binds to an enzyme. The resulting antibody may be used in immunoaffinity chromatography techniques to isolate or purify the polypeptide, or to determine if the polypeptide is present in a biological sample. .. In such a procedure, a protein preparation such as an extract, or a biological sample, is contacted with an antibody capable of specifically binding to one of the polypeptides of the present disclosure.

In immunoaffinity techniques, the antibody is attached to a solid support, such as beads or other column substrates. The protein preparation is placed in contact with the antibody under conditions where the antibody specifically binds to one of the polypeptides disclosed. After washing to remove non-specifically bound proteins, the specifically bound polypeptide is eluted.

The ability of a protein in a biological sample to bind to an antibody can be determined using various techniques known to those of skill in the art. Binding ability may be determined, for example, by labeling the antibody with a detectable label such as a fluorescent substance, enzyme label, radioisotope, etc. Alternatively, the ability of the antibody to bind to the sample may be determined by detection with a secondary antibody having such a detectable label on the surface. Specific assays include ELISA assays, sandwich assays, radioimmunoassays, and Western blots.

Polyclonal antibodies made against the polypeptides of the present disclosure can be obtained by direct injection of the polypeptide into animals or by administration of the polypeptide to non-human animals. The antibody thus obtained is then bound to the polypeptide itself. In this method, even a sequence that encodes only a fragment of the polypeptide can be used to generate an antibody that may bind to an undenatured full-length polypeptide. Such antibodies can then be used to isolate the polypeptide from cells expressing the polypeptide.

Any technique can be used to prepare the monoclonal antibody to provide the antibody produced by the subcultured cell line. See, for example, hybridoma methods, trioma methods, human B cell hybridoma methods, and EBV hybridoma methods (eg, Core (1985) in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96. ) Is included.

Adapting the techniques described for making single chain antibodies (see, eg, US Pat. No. 4,946,778) to make single chain antibodies to the polypeptides of the present disclosure. Can be done. Alternatively, transgenic mice may be used to express humanized antibodies against the polypeptide or fragments thereof. Antibodies made to the polypeptides of the present disclosure can be used to screen for similar polypeptides (eg, enzymes) from other organisms and samples. In such a procedure, a polypeptide of biological origin is brought into contact with an antibody to detect a polypeptide that specifically binds to the antibody. Any of the methods described above may be used to detect antibody binding.

Screening Methods and "Online" Monitoring Devices Various devices and methods can be used in conjunction with the polypeptides and nucleic acids of the present disclosure in the practice of the methods of the present disclosure. The devices and methods include, for example, for screening peptides by enzymatic activity, for screening compounds that are latent modulators such as activators or inhibitors for enzymatic activity, and binding to the polypeptides of the present disclosure. Examples include those for antibodies that are used, those for nucleic acids that hybridize to the nucleic acids of the present disclosure, those for screening cells that express the polypeptides of the present disclosure, and the like.

Array or "biochip"
The nucleic acids or polypeptides of the present disclosure can be immobilized or applied to an array. By using an array, a library of compositions (eg, small molecules, antibodies, nucleic acids, etc.) is screened or screened by the ability to bind to the nucleic acids or polypeptides of the present disclosure, or to regulate their activity. It can be monitored. For example, in one aspect of the present disclosure, the monitored parameter is transcriptional expression of the enzyme gene. Nucleic acid in which one or more, or all of a cell's transcript, a sample having a representative or complementary nucleic acid of that cell's transcript or cell's transcript, is immobilized on an array or "biochip". It is possible to measure by hybridizing to. By using an "array" of nucleic acids on a microchip, some or all of the transcripts can be quantified at the same time. Alternatively, an array with genomic nucleic acids can be used to genotype the newly designed strains made by the methods of the present disclosure. Multiple proteins can be quantified at the same time by using the polypeptide "array". The present disclosure can be carried out with any known "array", which is a "microarray" or "nucleic acid array" or "polypeptide array" or "antibody array" or "biochip". Or their variants. An array is generally a plurality of "spots" or "target elements", each target element having a defined amount of one or more biomolecules, such as an oligonucleotide, and a sample molecule. For example, those immobilized on a defined region on the surface of a substrate that specifically binds to an mRNA transcript.

In practicing the methods of the present disclosure, any known array and / or method of creating and using an array, as described in the following literature, may be in whole or in part thereof, or variants thereof. Can be used. For example, US 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; No. 5,965,452; No. 5,959,098; No. 5,856,174; No. 5,830,645; No. 5, 770,456; 5,632,957; 5,556,752; 5,143,854; 5,807,522; 5,807,522 No. 5,800,992; No. 5,744,305; No. 5,700,637; No. 5,556,752; No. 5,434; , 049; see, eg, International Publication No. 99/51773; International Publication No. 99/09217; International Publication No. 97/46313; International Publication No. 96/17958; see, 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 ", Genes, Chromosomes & Cancer 20: 399-407; Bowtell (1999) “Options Hybrid-From Start to Finish ￣ for Obtaining Expression Data by Microarray”, Nature Genomics SUP. See also 21: 25-32. US Patent Application Publication No. 2001018642; US Patent Application Publication No. 200100192827; US Patent Application Publication No. 2001016322; US Patent Application Publication No. 2001014449; US Patent Application Publication No. 2001014448; See also US Patent Application Publication No. 2001012537; US Patent Application Publication No. 2001098765.

Capillary Array Capillary arrays such as GIGAMATRIX ™ (Diversa Corporation, San Diego, Calif.) Can be used in the methods of the present disclosure. The nucleic acids or polypeptides of the present disclosure can be immobilized or applied to an array containing a capillary array. By using an array, a library of compositions (eg, small molecules, antibodies, nucleic acids, etc.) is screened or screened by the ability to bind to the nucleic acids or polypeptides of the present disclosure, or to regulate their activity. It can be monitored. Capillary arrays provide another system for holding and screening samples. For example, a sample screening device can include multiple capillaries formed as adjacent capillary arrays, where each capillary has at least one wall forming a lumen that holds the sample. The device may further include interstitium material placed between adjacent capillaries in the array, in which one or more reference indicators are formed. .. The capillary for screening the sample was adapted to bind to the capillary array and provided to the lumen to excite the sample with the first wall forming the lumen to hold the sample. It may include a second wall formed from a filter material that filters the excitation energy to be generated. A polypeptide or nucleic acid, such as a ligand, can be introduced into the first component of the capillary of at least a portion of the capillary array. Each capillary in the capillary array can have at least one wall forming a lumen that holds the first component. Bubbles can be introduced behind the first component in the capillary. It is possible to introduce the second component into the capillary, where the second component is separated from the first component by air bubbles. The target sample is introduced into one capillary in the capillary array as a first solution labeled with detectable particles, and each capillary in this capillary array is a tube for holding the first solution and the detectable particles. It has at least one wall forming the lumen, and at least one wall is coated with a binding material for binding the detectable particles. The method further comprises a step of removing the first liquid from the capillary tube holding the bound detectable particles and a step of introducing the second liquid into the capillary. A capillary array comprises a plurality of individual capillaries having at least one outer wall forming a lumen. The outer wall may be one or more walls fused to each other. Similarly, a wall can form a lumen of cylindrical, square, hexagonal, or other geometric form, as long as the wall forms a lumen that holds the liquid or sample. The capillaries in the capillary array can support each other in close proximity to form a planar structure. Capillaries can be bonded to each other by fusing, adhering, gluing, or fixing adjacent capillaries (eg, where the capillaries are made of glass). Capillary arrays can be formed from any number, eg 100-4,000,000, of individual capillaries. Capillary arrays can form microtiter plates with about 100,000 or more individual capillaries coupled to each other.

Manipulation of Conditional Active Antibodies Conditional active antibodies can be engineered to produce multispecific conditional active antibodies. The multispecific antibody can be an antibody having multiepitope specificity as described in WO 2013/170168. The multispecific antibodies include, but are not limited to, V _H V heavy chain variable _{domain L} units with polyepitopic specificity (V _H) and an antibody comprising a light chain variable domain (V _L), each V _H V _{L Antibodies with two or more VL} and _VH domains whose units bind to different epitopes, antibodies with two or more single variable domains where each single variable domain binds to different epitopes, and one or more. Included are antibodies containing antibody fragments of and antibodies containing antibody fragments that are covalently or non-covalently linked.

To construct multispecific antibodies, including bispecific antibodies, antibody fragments with at least one free sulfhydryl group are obtained. The antibody fragment may be obtained from a full-length conditionally active antibody. Enzymatic digestion of conditional active antibodies can yield 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, diabody (Db); tandem diabody (taDb), linear antibody (US Pat. No. 5,641, 870, Example 2; Zapata et al., Protein Eng., Vol. 8, pages 1057-1062 (1995)); 1-arm antibody, single variable domain antibody, minibody (Orafsen et al (see). 2004) Protein Eng. Design & Sel., Vol. 17, pages 315-323), single-stranded antibody molecule, fragment made by Fab expression library, anti-idiotype (anti-Id) antibody, complementarity determining region (CDR). ), And epitope binding fragments. Antibody fragments may also be made using DNA recombination techniques. The DNA encoding the antibody fragment may be cloned into a plasmid expression vector or a phagemid vector and expressed directly in E. coli. Methods of digesting antibody enzymes, DNA cloning and recombinant protein expression are well known to those of skill in the art.

The antibody fragment may be purified using conventional techniques and may be subjected to reduction to produce free thiol groups. Antibody fragments with free thiol groups can react with cross-linking agents such as bismaleimide. The crosslinked antibody fragment is purified and then reacted with a second antibody fragment having a free thiol group. The end product, in which the two antibody fragments are crosslinked, is purified. In certain embodiments, each antibody fragment is a Fab, and the end product in which the two Fabs are linked via bismaleimide is referred to herein as bismaleimide- (thio-Fab) 2 or bis-Fab. To. Such multispecific antibodies and antibody analogs can be utilized to rapidly synthesize combinations of multiple antibody fragments, including bis-Fab, or structural variants of native antibodies or specific antibody / fragment combinations. can.

The multispecific antibody can be synthesized with a modified cross-linking agent so that additional functional moieties can be added to the multispecific antibody. The modified cross-linking agent allows the binding of any sulfhydryl reactive moiety. In one embodiment, N-succinimidyl-S-acetylthioacetate (SATA) binds to bismaleimide to form bismaleimide acetylthioacetate (BMata). After deprotection of the masked thiol group, any functional group having a sulfhydryl-reactive (or thiol-reactive) moiety can be attached to the multispecific antibody.

Exemplary thiol-reactive reagents include multifunctional linker reagents, capture or affinity labeling reagents (eg biotin-linker reagents), detection labels (eg fluorophore reagents), solid phase immobilization reagents (eg SEPHAROSE ™). Polystyrene, or glass), or drug-linker intermediates. An example of a thiol-reactive reagent is N-Ethylmaleimide (NEM). Such multispecific antibodies or antibody analogs with modified cross-linking agents may be further reacted with drug partial reagents or other labels. Reactions of a multispecific antibody or antibody analog with a drug-linker intermediate result in a multispecific antibody-drug conjugate or antibody analog-drug conjugate, respectively.

Other techniques for making multispecific antibodies can also be used in the present invention. References describing these techniques include: (1) Milstein and Cuello, Nature, vol. For recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities. 305, page 537 (1983)), International Publication No. 93/08829, and Traunecker et al. , EMBO J. , Vol. 10, page 3655 (1991); (2) US Pat. No. 5,731,168 with respect to "knob-in-hole" operation; (3) antibody Fc-heterogeneous by manipulating the electrostatic steering effect. US Pat. No. 4,676,980, and Brennan et al. For cross-linking of two or more antibodies or fragments. , Science, vol. 229, page 81 (1985); (5) with respect to the production of bispecific antibodies using leucine zippers, Kostelny et al. , J. Immunol. , Vol. 148, pages 1547-1553 (1992); (6) with respect to the preparation of bispecific antibody fragments using the "diabody" technique Hollinger et al. , Proc. Natl. Acad. Sci. USA, vol. 90, pages 6444-6448 (1993); (7) with respect to the use of single chain Fv (sFv) dimers, Gruber et al. , J. Immunol. , Vol. 152, page 5368 (1994); (8) with respect to the preparation of trispecific antibodies, Tutt et al. J. Immunol. 147: 60 (1991); and (9) US patent application for engineered antibodies with three or more functional antigen binding sites, including "Octopus antibody" or "dual variable domain immunoglobulin" (DVD). Publication No. 2006/0025576A1 and Wu et al. Nature Biotechnology, vol. 25, pages 1290-1297 (2007).

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

In one embodiment, conditionally active antibodies to cross the blood-brain barrier (BBB) are engineered to generate multispecific antibodies (eg, bispecific antibodies). This 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 the very least, the first antigen binding site for BBB-R is a conditionally active form. Brain antigens are antigens that are expressed in the brain and can be targeted by antibodies or small molecules. Examples of such antigens include, without limitation: β-secretase 1 (BACE1), amyloid β (Aβ), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), and the like. Tau, apolypoprotein E4 (ApoE4), α-sinucrane, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), perkin, presenirin 1, presenirin 2, γ-secretase, desreceptor 6 (DR6) , Amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), and caspase 6. In one embodiment, the antigen is BACE1.

BBB has an endogenous transport system mediated by the BBB receptor (BBBB-R), which is a specific receptor that allows the transport of macromolecules through the BBB. For example, an antibody capable of binding to BBB-R can be transported through the BBB using this endogenous transport system. Such antibodies can serve as a means of transport for transporting drugs or other agents through the BBB by using an endogenous BBB receptor-mediated transport system through the BBB. Such antibodies need not have high affinity for BBB-R. As described in U.S. Patent Application Publication No. 2012/0171201, an antibody that is not a conditioned active antibody with a low affinity for BBB-R is described as passing through the BBB with higher efficiency than a high affinity antibody. ing.

Another way to manipulate an antibody to enter the brain is to manipulate the antibody so that it is delivered to the brain via the central nervous system lymphatic vessels. Thus, antibodies can be engineered to bind to or mimic immune cells such as T cells that migrate through lymphatic vessels to the central nervous system, or synovial or cerebrospinal fluid. For more information on the lymphatic vessels of the central nervous system, see, eg, Louveau, A. et al. , Et al. , "Structural and functional facials of central nervous system lymphatic vessels," Nature 523, pp. It is publicly available as of the filing date of 337-341,16 July 2015 and the present application.

Unlike conventional antibodies, conditional active antibodies do not need to have a low affinity for BBB-R because they cross the BBB and remain inside the brain. Conditionally active antibodies may have a high affinity for BBB-R on the blood side of the BBB and little or no affinity on the brain side of the BBB. Drugs such as drug conjugates can be transported with the antibody through the BBB into the brain by coupling to a conditioned active antibody.

BBB-R is a transmembrane receptor protein expressed on brain endothelial cells and has the ability to transport molecules through the blood-brain barrier. Examples of BBB-R include transferase receptor (TfR), insulin receptor, insulin-like growth factor receptor (IGF-R), low density lipoprotein receptor, eg, low density lipoprotein receptor-related without limitation. Examples include protein 1 (LRP1) and low density lipoprotein receptor-related protein 8 (LRP8), and heparin-binding epidermal growth factor-like growth factor (HB-EGF). An exemplary BBB-R herein is the transferrin receptor (TfR). TfR is a transmembrane glycoprotein (molecular weight of about 180,000) composed of two disulfide-bonded subunits (each apparent molecular weight of about 90,000) involved in iron uptake in vertebrates.

In some embodiments, the invention provides a conditionally active antibody produced from a parental antibody or wild-type antibody against BBB-R. This conditionally active antibody binds to BBB-R on the blood side of BBB and has a lower affinity for BBB-R on the brain side of BBB than the parent antibody or wild-type antibody. In some other embodiments, this conditionally active antibody has an affinity for BBB-R on the blood side of BBB as compared to wild-type or parental antibodies and on the brain side of BBB against BBB-R. Has no affinity.

Plasma is a body fluid that is significantly different from extracellular fluid (ECF). Somjen ( "Ions in the Brain: Normal Function, Seizures, and Stroke," Oxford University Press, 2004, pages 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), Cerebrospinal fluid has significantly less ^{K + and more Mg 2+} and H ⁺ than plasma. Differences in ion concentration between plasma and brain ECF result in significant differences in osmolality and weight osmolality between these two body fluids. Table 1 shows the concentrations (in millimoles) of common ions in both plasma and brain ECF.

Brain ECF also contains significantly more lactate than plasma and significantly less glucose compared to plasma (Abi-Saab et al., "Striking Differenses in Glucose and Lactate Labels Between Brain Extracell". Conscients Human Substances: Effects of Hyperglycemia and Hypoglycemia, "Journal of Cerebral Blood Flow & Metabolism, vol.22, page2, 2-2, pages27.

Therefore, there are several different physiological conditions on both sides of the BBB, such as pH, concentrations of various substances (lactose, glucose, K +, Mg2 +, etc.), osmotic pressure and osmolality by weight. In terms of pH physiology, human plasma has a higher pH than human brain ECF. With respect to the physiological conditions of K + concentration, brain ECF has a lower K + concentration than human plasma. With respect to the physiological conditions of Mg2 + concentration, human brain ECF has significantly more Mg2 + than human plasma. With respect to the physiological conditions of osmotic pressure, human brain ECF has different osmotic pressure from human plasma. In some embodiments, the physiology of the brain ECF may be the composition, pH, osmolality and weight osmolality of the brain ECF of a patient with a particular neurological disorder, which is the physiology of the general population of brain ECF. Can be different from the conditions.

Therefore, the present invention provides a method for creating a mutant DNA library by evolving the DNA encoding the template antibody against BBB-R. Next, when this mutant DNA library is expressed, a mutant antibody is obtained. Are these variant antibodies bound to BBB-R under at least one plasma physiological condition and have a lower affinity for BBB-R than template antibodies under at least one brain physiological condition in the brain ECF? , Or conditionally active antibodies that do not have it. Therefore, the selected mutant antibody has low or high affinity for BBB-R on the plasma side and low or no affinity for BBB-R on the brain ECF side. This selected variant antibody is useful as a conditionally active antibody for transport through the BBB.

Such conditionally active antibodies are advantageous for passage through the BBB and retention in the brain ECF. Due to its low affinity for BBB-R on the brain side, the rate at which conditionally active antibodies are transported back from the brain through the BBB and back into the blood is slower (or such) than template antibodies. Will disappear).

In some other embodiments, the invention provides a method of evolving DNA encoding a template antibody against BBB-R to create a mutant DNA library. Next, when this mutant DNA library is expressed, a mutant antibody is obtained. These mutant antibodies are screened for conditionally active antibodies that bind to BBB-R under at least one plasma physiological condition and have little or no affinity for BBB-R under at least one brain physiological condition. To. Therefore, the selected mutant antibody has an affinity for BBB-R on the plasma side and little or no affinity for BBB-R on the brain ECF side. This selected variant antibody is a conditionally active antibody.

Such conditionally active antibodies are advantageous for passage through the BBB and retention in the brain ECF. After binding to BBB-R on the plasma side, the conditionally active antibody is transported through the BBB, and on the brain ECF side there is little or no affinity for BBB-R, which means that the conditionally active antibody. Means that is unlikely to be carried out of the brain.

The affinity of a conditionally active antibody for BBB-R can be measured by its maximum half-inhibition concentration (IC50), because the IC50 inhibits 50% of the binding of known BBB-R ligands to BBB-R. It is a measure of how much antibody is needed. A common technique is to perform competitive binding analysis such as competitive ELISA analysis (assy). Exemplary competition ELISA assay measuring the IC50 about TfR (BBB-R) are those anti-TfR antibodies of increasing concentrations competes with biotinylated TfR ^A for binding to TfR. Anti-TfR antibody competing ELISA can be performed overnight at 4 ° C. on Maxisorp plates (Neptune, NJ) coated with 2.5 μg / ml purified mouse TfR extracellular domain in PBS. Plates are washed with PBS / 0.05% Tween 20 and blocked with Superblock blocking buffer (Thermo Scientific, Hudson, NH) in PBS. Titration of each individual anti-TfR antibody (1: 3-step dilution ^{) is combined with biotinylated anti-TfRA A} (0.5 nM final concentration) and added to the plate at room temperature for 1 hour. The plate is washed with PBS / 0.05% Tween 20, HRP-streptavidin (Southern Biotech, Birmingham) is added to the plate, and the mixture is incubated at room temperature for 1 hour. The plates were washed with PBS / 0.05% Tween 20, biotinylated anti-TfR ^A bound to the plate detected using TMB substrate (BioFX Laboratories, Owings Mills).

A high IC50 indicates that more conditionally active antibodies are needed to inhibit the binding of known ligands for BBB-R, thus indicating that the antibody's affinity for the BBB-R is relatively low. Conversely, a low IC50 indicates that less conditional active antibody is required to inhibit the binding of known ligands, and thus the antibody's affinity for the BBB-R in question is relatively high.

In some embodiments, the IC50 of the conditionally active antibody from BBB-R in plasma is about 1 nM to about 100 μM, or about 5 nM to about 100 μM, or about 50 nM to about 100 μM, or about 100 nM to about 100 μM. , Or about 5 nM to about 10 μM, or about 30 nM to about 1 μM, or about 50 nM to about 1 μM.

Conditional active biological proteins for synovial fluid Joint disease is a major cause of disability and premature retirement in industrialized countries. Joint disorders often result in joint damage, which is difficult to repair. Synovial fluid is the fluid found between the cartilage and the synovium on the opposite joint surface in the synovial space of joints (eg, knees, hips, shoulders) of the human or animal body. Synovial fluid nourishes cartilage and also acts as a lubricant for joints. Cartilage and synovial cells secrete fluid that acts as a lubricant between joint surfaces. Human synovial fluid contains about 85% water. It is derived from plasma dialysate, which itself consists of water, dissolved proteins, glucose, coagulation factors, inorganic ions, hormones and the like. Proteins such as albumin and globulin are present in synovial fluid and are thought to play an important role in lubrication of joint areas. Several other proteins are also found in human synovial fluid, including glycoproteins such as α-1-acid glycoprotein (AGP), α-1-antitrypsin (A1AT) and rubricin.

Synovial fluid has a composition that is very different from other parts of the body. Therefore, synovial fluid has different physiological conditions than other parts of the body such as plasma. For example, synovial fluid has less than about 10 mg / dL of glucose, but the average normal glucose value of human plasma is about 100 mg / dL and varies within the range of 70-100 mg / dL throughout the day. In addition, it is not easy for large molecules such as proteins to cross the synovial membrane and enter the synovial fluid, so the total protein level in the synovial fluid is about one-third that of plasma protein. It has also been found that the pH of human synovial fluid is higher than that of human synovial fluid (Jevens et al., "On the viscosity and pH of synovial fluid and the pH of blood," The Journal of B. .41 B, pages 388-400, 1959; Farr et al., "Synovial of the rhematoid arthritis, synovial fluid in Rheumatoid Arthritis," Clinical.

Therefore, synovial fluid has some physiological conditions that differ from those of other parts of the body, such as those in plasma. Synovial fluid has a higher pH than other parts of the body, especially plasma. Synovial fluid has a lower glucose concentration than other parts of the body, such as plasma. Synovial fluid also has a lower concentration of protein than other parts of the body, such as plasma.

Several antibodies have been used by introducing antibodies into synovial fluid to treat joint disease. For example, synovial fluid in an injured joint is known to contain many factors that influence the progression of osteoarthritis (eg, Fernandes, et al, "The Roll of Cytokines in Osteoarthritis Pathophysiology," Biotherapy. , Vol. 39, pages 237-246, 2002). Cytokines such as interleukin-1 (IL-I) and tumor necrosis factor-α (TNF-α) are produced by activated synovial cells and upregulate matrix metalloproteinase (MMP) gene expression. It is known to do. Upregulation of MMP causes degradation of matrix and non-matrix proteins in joints. Antibodies that neutralize cytokines can stop the progression of osteoarthritis.

The use of antibodies as drugs is a promising strategy for the treatment of joint diseases. For example, antibodies (such as antibodies to agrecan or aggrecanase) have been developed to treat osteoarthritis, which has by far the highest prevalence among joint diseases (International Publication No. 1993/022429A1 pamphlet). Antibodies to acetylated high mobility group box 1 (HMGB1) have been developed for the diagnosis or treatment of inflammatory, autoimmune, neurodegenerative or malignant / disordered joint diseases such as arthritis. This antibody can be used to detect the acetylated form of HMGB1 in synovial fluid (International Publication No. 2011/157905A1 Pamphlet). Another antibody (CD20 antibody) has also been developed to treat damage to connective tissue and cartilage in joints.

However, the antigens of these antibodies are often expressed in other parts of the body that carry out important physiological functions. Although antibodies to these antigens are effective in treating joint disease, they can also significantly interfere with the normal physiological function of these antigens in other parts of the body. Therefore, severe side effects can occur in the patient. Thus, to reduce side effects, they may preferentially bind to those antigens (proteins or other macromolecules) in synovial fluid with higher affinity, while not binding to the same antigen in other parts of the body. It is desirable to develop antibodies against therapeutic agents that either or only weakly bind, such as cytokines or other antigens.

Such conditionally active biological proteins can be conditionally active antibodies. In some embodiments, the invention also provides a conditionally active biological protein that is a protein other than an antibody. For example, the present invention develops conditional active immunomodulators for preferentially regulating an immune response in synovial fluid, which may or may not have a low or no effect on the immune response in other parts of the body. Can be done.

The conditionally active biological protein can be a conditionally active cytokine signaling suppressor (SOCS). Many of these SOCS are involved in the inhibition of the JAK-STAT signaling pathway. Conditionally active cytokine signaling suppressors may preferentially suppress cytokine signaling in synovial fluid, while not or to a lesser extent suppress cytokine signaling in other parts of the body.

In some embodiments, the invention provides a conditionally active biological protein derived from a parental or wild-type biological protein. Conditionally active biological proteins have lower activity than parental or wild-type biological proteins under at least one physiological condition in a particular part of the body, such as plasma, and at least one physiological condition in the lubricant. Has higher activity than parental or wild-type biological proteins. Such conditionally active biological proteins may function preferentially in synovial fluid, but do not or have a lesser degree of action on other parts of the body. As a result, such conditioned active biological proteins may have reduced side effects.

In some embodiments, the conditional active biological protein is an antibody against an antigen that is in or exposed to synovial fluid. Such antigens may be any protein involved in the immune response / inflammation in joint disease, but the antigens are often cytokines. Conditionally active antibodies have a lower affinity for the antigen under at least one physiological condition in other parts of the body (such as plasma) than a parental or wild antibody against the same antigen, but at least one synovial fluid. Under conditions, the affinity for the antigen is higher than that of the parental or wild-type antibody. Such conditional active antibodies may bind weakly or not at all to the antigen in other parts of the body, but bind to the antigen in synovial fluid, eg, strongly and tightly, or more strongly.

Conditional Active Biological Proteins for Tumors Cancer cells in solid tumors have the ability to form a tumor microenvironment around them and support the growth and metastasis of the cancer cells. The tumor microenvironment promotes surrounding blood vessels, immune cells, fibroblasts, other cells, soluble factors, signaling molecules, extracellular matrix, and neoplasmic transformation, supports tumor growth and invasion, and hosts tumors. A cellular environment in which a tumor is present, including a mechanical queue that can protect against immunity, promote resistance to treatment, and provide a niche for the development of latent metastases. Tumors and their surrounding microenvironments are closely related and always interacting. Tumors can affect their microenvironment by emitting extracellular signals, promoting tumor angiogenesis, and inducing peripheral immune tolerance, while immune cells in the microenvironment grow and evolve cancerous cells. Can affect. Swarts et al. "Tumor Complexity: Emerging Roles in Cancer Therapy," Cancer Res, vol. , 72, pages 2473-2480, 2012.

The tumor microenvironment is often hypoxic. As the tumor mass grows, the inside of the tumor grows further away from the existing blood supply, making it difficult to completely supply oxygen to the tumor microenvironment. The oxygen partial pressure in the tumor environment is more than 50% of locally advanced solid tumors and less than 5 mmHg, whereas in plasma it is about 40 mmHg. In contrast, other parts of the body are not hypoxic. The hypoxic environment leads to genetic instability through nucleotide excision repair and downregulation of mismatch repair pathways, which is associated with cancer progression. Hypoxia also causes upregulation of hypoxia-inducible factor 1α (HIF1-α), which induces angiogenesis and is associated with poor prognosis and activation of genes associated with metastasis. Weber et al. , "The tumor microenvironment," Surgical Oncology, vol. 21, pages 172-177, 2012 and Bragosklonny, "Angiogenic therapy and tumor progression," Cancer Cell, vol. 5, pages 13-17, 2004.

In addition, tumor cells tend to rely on the energy produced by lactic acid fermentation, which does not require oxygen. Therefore, tumor cells rarely use normal aerobic respiration, which requires oxygen. As a result of using lactic acid fermentation, the tumor microenvironment becomes acidic (pH 6.5-6.9), in contrast to other parts of the body, which are typically either neutral or weakly basic. For example, human plasma has a pH of about 7.4. Estrella et al. , "Acidity Generated by the Tumor Microenvironment Drivees Local Invasion," Cancer Research, vol. 73, pages 1524-1535, 2013. In the tumor microenvironment, the availability of nutrients is low due to the relatively high demand for nutrients in proliferating cancer cells compared to cells located in other parts of the body.

In addition, the tumor microenvironment also contains many characteristic cell types that are rarely found in other parts of the body. These cell types include endothelial cells and their precursors, peripheral cells, smooth muscle cells, fibroblasts, cancer-related fibroblasts, myofibroblasts, neutrophils, eosinophils, basal spheres, and masts. Includes cells, T and B lymphocytes, natural killer cells and antigen presenting cells (APCs), such as macrophages and dendritic cells (Lorusso et al., "The tumor microenvivronnent and it control to tumor evolution cell" Biol, vol. 130, pages 1091-1103, 2008).

Thus, the tumor microenvironment has at least some physiological conditions that differ from those of other parts of the body, such as physiological conditions in plasma. The tumor microenvironment has a lower pH (acidic) than other parts of the body, especially plasma (pH 7.4). The tumor microenvironment has lower oxygen levels than other parts of the body, such as plasma. Tumor microenvironments also have lower availability of nutrients than other parts of the body, especially plasma. The tumor microenvironment also has some characteristic cell types that are rarely found in other parts of the body, especially plasma.

Certain anti-cancer drugs include antibodies that can invade the tumor microenvironment and act on cancer cells there. Antibody-based cancer therapies are well established and have become one of the most successful and important strategies for the treatment of patients with hematological malignancies and solid tumors. A wide variety of cell surface antigens expressed by human cancer cells, which are overexpressed, mutated, or selectively expressed in cancer cells as compared with normal tissues. These cell surface antigens are excellent targets for antibody cancer therapy.

Cancer cell surface antigens that antibodies can target are classified into several different types. Hematopoietic differentiation antigens are glycoproteins that are usually associated with differentiation cluster (CD) classification and include CD20, CD30, CD33 and CD52. Cell surface differentiation antigens are a diverse set of glycoproteins and carbohydrates found on the surface of both normal and tumor cells. Antigens involved in growth and differentiation signaling are often growth factors and growth factor receptors. Growth factors targeted by antibodies in cancer patients include CEA2, epidermal growth factor receptor (EGFR; also known as ERBB1) 12, ERBB2 (also known as HER2) 13, ERBB3 (Reference 18), MET (HGFR). (Also known as) 19, insulin-like growth factor 1 receptor (IGF1R) 20, effrin 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 a receptor activator ligand for nuclear factor κB (RANKL; also known as TNFSF11) 22. Antigens involved in angiogenesis are usually proteins or growth factors that assist in the formation of new microvasculature, including vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR), integulin αVβ3 and integrin α5β1 ( Document 10). Tumor stroma and extracellular matrix are essential supporting structures for tumors. Interstitial and extracellular matrix antigens that are therapeutic targets include fibroblast-activating proteins (FAPs) and tenascin. Scott et al. , "Antibody therapy of cancer," Nature Reviews Cancer, vol. 12, pages 278-287, 2012.

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

Cancer stem cells are cancer cells that are capable of producing all cell types found in a particular cancer sample and are therefore tumorigenic. Cancer stem cells can develop tumors through a stem cell process that self-regenerates and differentiates into multiple cell types. Cancer stem cells continue to exist as a characteristic population in tumors and are thought to cause recurrence and metastasis by giving rise to new tumors. The development of specific therapies targeting cancer stem cells can improve the survival and quality of life of cancer patients, especially those with metastatic disease.

These tumor treatments often interfere with normal physiologic function in other parts of the body other than the tumor. For example, proteins that induce apoptosis in tumors can also induce apoptosis in some other part of the body, thus causing side effects. In embodiments where the antibody is used to treat a tumor, the antigen of the antibody can also be expressed in other parts of the body that perform normal physiological functions. For example, the monoclonal antibody bevacizumab, which stops tumor vascular growth (targets vascular endothelial growth factor). This antibody can also block vascular growth and repair in other parts of the body, thus causing bleeding, poor wound healing, blood clots, and kidney damage. For more effective tumor treatment, it is highly desirable to develop conditional active biological proteins that focus primarily or exclusively on tumor targeting.

In some embodiments, the invention provides a conditionally active biological protein produced from a parental or wild-type biological protein that may be a candidate for tumor treatment. This conditionally active biological protein has less activity than the parental or wild-type biological protein under at least one physiological condition in parts of the body other than the tumor microenvironment, such as plasma, while the tumor microenvironment. Has higher activity than parental or wild-type biological proteins under at least one physiological condition in. Such conditional active biological proteins can preferentially act on cancer cells in the tumor microenvironment to treat the tumor 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 the tumor cell (the antigen is exposed to the tumor microenvironment), the conditional active antibody is the other part of the body, eg, the non-tumor microenvironment. The affinity for the antigen is lower than that of the parent or wild antibody, while the affinity for the antigen is higher than that of the parent or wild antibody in the tumor microenvironment. Such conditionally active antibodies may or may not bind weakly to the antigen elsewhere in the body, but have higher binding or stronger and tighter binding to the antigen in the tumor microenvironment. ..

In some embodiments, the conditioned active antibody is an antibody against an immune checkpoint protein, resulting in inhibition of the immune checkpoint. Such conditionally active antibodies are (1) increased binding affinity for immune checkpoint proteins in the tumor microenvironment compared to the parental or wild antibody from which the conditionally active antibody is derived, and (2) the conditions. It has at least one of a reduced binding affinity for immune checkpoint proteins in a non-tumor microenvironment compared to the parental or wild antibody from which the active antibody is derived.

Immune checkpoints serve as an endogenous inhibition pathway of the immune system that maintains self-tolerance and regulates antigen stimulation, the duration and extent of an immune response to exogenous molecules, cells and tissues. Pardol, Nature Reviews Cancer, vol. 12, pages 252-264, 2012. Inhibition of immune checkpoints by suppressing one or more checkpoint proteins can lead to excessive activation of the immune system, especially T cells, which in turn can induce the immune system to attack tumors. Suitable checkpoint proteins for the present invention include CTLA4 and its ligands CD80 and CD86, PD1 and its ligands PDL1 and PDL2, T cell immunoglobulin and mutin protein-3 (TIM3) and their ligands GAL9, B and T lymphocytes. Nuator (BTLA) and its ligand HVEM (herpesvirus invading mediator), receptors such as killer cell immunoglobulin-like receptor (KIR), lymphocyte activation gene-3 (LAG3) and adenosine A2a receptor (A2aR), Also mentioned are ligands B7-H3 and B7-H4. Further suitable immune checkpoint proteins are described in Pardol, Nature Reviews Cancer, vol. 12, pages 252-264, 2012 and Nirschl & Drake, Clin Cancer Res, vol. 19, pages 4917-494, 2013.

CTLA-4 and PD1 are two of the most well-known immune checkpoint proteins. CTLA-4 may downregulate the T cell activation pathway (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 increase T cell activation and proliferation. Examples of CTLA-4 inhibitors include anti-CTLA-4 antibodies. The anti-CTLA-4 antibody binds to CTLA-4 and blocks the interaction of CTLA-4 with its ligand CD80 or CD86, thereby lowering the immune response evoked by the interaction of CTLA-4 with its ligand. Cut off control.

The checkpoint protein PD1 is known to suppress T cell activity in peripheral tissues and suppress autoimmunity when an inflammatory response to infection occurs. Blocking PD1 in vitro can enhance T cell proliferation and cytokine production by specific antigen targets or in response to allogeneic cell stimulation in mixed lymphocyte reactions. A strong correlation between PD1 expression and decreased immune response has been shown to result from PD1 inhibitory function, i.e. induction of immune checkpoints (Pardall, Nature Reviews Cancer, 12: 252-264, 2012). Blocking of PD1 can be achieved by a variety of mechanisms, including antibodies that bind PD1 or its ligands PDL1 or PDL2.

In previous studies, antibodies against several checkpoint proteins (CTLA4, PD1, PD-L1) have been discovered. These antibodies are effective in treating tumors by inhibiting immune checkpoints and thereby overactivating the immune system, especially T cells, to attack the tumor (Pardol, Nature Reviews Cancer, vol). .12, pages 252-264, 2012). However, overactivated T cells can also attack host cells and / or tissues, resulting in concomitant damage to the patient's body. Therefore, treatments based on the use of these known antibodies that inhibit immune checkpoints are difficult to manage and patient risk is a major concern. For example, FDA-approved antibodies against CTLA-4 have a black box warning due to their high toxicity.

The present invention addresses the problem of concomitant damage by overactivated T cells by providing conditionally active antibodies against immune checkpoint proteins. These conditionally active antibodies preferentially activate immune checkpoints in the tumor microenvironment. At the same time, immune checkpoints in non-tumor microenvironments, such as normal living tissues, are not or less inhibited by conditioned active antibodies, thus reducing the potential for concomitant damage to the body in non-tumor microenvironments. To. This goal is achieved by manipulating conditionally active antibodies to be more active in the tumor microenvironment compared to the non-tumor microenvironment.

In some embodiments, the conditionally active antibody against the immune checkpoint protein has a ratio of binding activity to the immune checkpoint protein in the non-tumor microenvironment to binding activity to the same immune checkpoint protein in the tumor microenvironment. 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 analysis for measuring antibody binding activity is an ELISA analysis.

Highly immunogenic tumors, such as malignant melanoma, are the most vulnerable to the overactivated immune system achieved by manipulation of the immune system. Therefore, conditionally active antibodies against immune checkpoint proteins may be particularly effective in treating such hyperimmuneogenic tumors. However, other types of tumors are also vulnerable to the overactivated immune system.

In some embodiments, conditionally active antibodies against immune checkpoint proteins can be used in combination therapy. For example, combination therapy may include conditionally active antibodies against tumor cell surface molecules (tumor-specific antigens) and conditionally active antibodies against immune checkpoint proteins. In one embodiment, both the binding activity of the conditionally active antibody to the tumor cell surface molecule and the binding activity of the conditionally active antibody to the immune checkpoint protein may be present in a single protein, i.e. It may be a bispecific conditionally activated antibody as disclosed herein. In some further embodiments, the combination therapy comprises a conditionally active antibody against a tumor cell surface molecule (tumor-specific antigen) and two or more conditionally active antibodies against two or more different immune checkpoint proteins. Can include. In one embodiment, all of these binding activities may be present in a single protein, i.e., a multispecific antibody as disclosed herein.

Conditionally active antibodies are more active in the tumor microenvironment against the same tumor cell surface molecule or checkpoint protein as compared to the activity of the parental or wild-type antibody from which the conditionally active antibody is derived. These combination therapies can result in both increased efficacy and significantly reduced toxicity. The reduced toxicity of these conditionally active antibodies, especially to immune checkpoint proteins, allows the safe use of potent antibodies, such as ADC antibodies as described herein, as well as higher doses of the antibody. Can be.

In some embodiments, the conditionally active antibody against the checkpoint protein may be in prodrug form. For example, a conditioned active antibody can be a prodrug that does not have the desired drug activity before it is cleaved and transformed into a drug form. Prodrugs have tumor microenvironments in which the enzyme that catalyzes such cleavage is predominantly present in the tumor microenvironment or that conditional active antibodies are accessible to the cleavage site in the non-tumor microenvironment. In the environment, the tumor may preferentially cleave in the microenvironment, either for any reason that makes it easier to reach the site of cleavage.

Conditional active biological proteins for stem cell niches, including tumor stem cells Stem cells reside in an environment called the body's stem cell niche, which forms the basic unit of tissue physiology and is the response of stem cells to biological demands. Integrate the signals that mediate. However, niches can also induce pathology by imposing abnormal functions on stem cells or other targets. The interaction between stem cells and their niche creates the dynamic systems needed for tissue persistence and the final design of stem cell therapy (Scadden, "The stem-cell niche as an entity of action," Nature, vol. .441, pages 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.

The stem cell niche is a specialized environment that differs from other parts of the body (eg, plasma) (Drummond-Barbosa, "Stem Cells, Their Niches and the Systemic Environment: An Agging Network," Genetics. -1797, 2008; Fuchs, "Socializing with the Nightbors: Stem Cells and Their Niche," Cell, vol. 116, pages 769-778, 2004). The stem cell niche is hypoxic and has reduced oxidative DNA damage. Direct measurement of oxygen levels has generally revealed that bone marrow is virtually hypoxic (about 1% to 2% O2) compared to plasma (Keith et al., "Hypoxia-Industrial Factors"). , Stem Cell, and Celler, "Cell, vol. 129, pages 465-472, 2007; Mohyeldin et al.," Oxygen in Stem Cell Body: A Critical Component of the Cell. , Pages 150-161, 2010). In addition, the stem cell niche has several other factors for regulating stem cell properties within the niche: extracellular matrix components, growth factors, cytokines, and factors of environmental physiochemical properties such as pH, ionic intensity (eg, pH, ionic intensity ( For example, ^{it must have Ca 2+} concentration) and metabolites.

Thus, the stem cell niche has at least some physiological conditions that differ from those of other parts of the body, such as those in plasma. Stem cell niches have lower oxygen levels (1-2%) compared to other parts of the body, especially plasma. Other physiological conditions of the stem cell niche, including pH and ionic strength, can also differ from other parts of the body.

Stem cell therapy is an interventional strategy that introduces new adult stem cells into injured tissue to treat a disease or injury. This strategy depends on the ability of stem cells to self-regenerate and produce progeny with varying degrees of differentiation potential. Stem cell therapy offers great potential for tissue regeneration, with the potential to replace the extent of morbidity and damage to the body while minimizing the risk of rejection and side effects. Therefore, delivering a drug (biological protein (eg, antibody) or chemical compound) to the stem cell niche to affect stem cell regeneration and differentiation is an important part of stem cell therapy.

There are several examples of how the stem cell niche affects stem cell regeneration and / or differentiation in mammals. The first example is in the skin, where β-1 integrin is differentially expressed on primitive cells and is involved in the constraining localization of stem cell populations through interaction with matrix glycoprotein ligands. Known to be involved. Second, in the nervous system, the absence of tenascin C alters the number and function of neural stem cells in the subventricular zone. Tenascin C appears to regulate stem cell susceptibility to fibroblast growth factor 2 (FGF2) and bone morphogenetic protein 4 (BMP4), resulting in an increase in stem cell propensity. Third, another matrix protein, osteopontin (OPN), a sialoprotein containing Arg-Gly-Asp, has now been demonstrated to contribute to hematopoietic stem cell regulation. OPN interacts with hematopoietic stem cells CD44, as well as several receptors known to be on α4 and α5β1 integrins. OPN production can be significantly altered, especially by activation of osteoblasts. OPN-deficient animals have an increased number of HS cells due to hyperphysiological stem cell growth under stimulus conditions due to the absence of OPN. Therefore, OPN appears to act as a constraint on the number of hematopoietic stem cells that limits the number of stem cells under constant conditions or stimulation. Scadden, "The stem-cell niche as an entity of action," Nature, vol. See 441, pages 1075-1079, 2006.

Xie et al. "Autocrine sensing based selection of combinatorial antibodies the transdifferentiation human stem cells," Proc Natl Accad Sci USA, vol. 110, pages 8099-8104, 2013) disclose methods of using antibodies to influence stem cell differentiation. This antibody is an agonist of granulocyte colony stimulating factor receptor. Unlike the natural granulocyte colony-stimulating factor, which activates cells and differentiates along a predetermined pathway, this isolated agonist antibody transdifferentiates human bone marrow lineage CD34 + bone marrow cells into neural progenitor cells. Melidoni et al. ("Selecting antibodies the control differentiation thrust induce expression in embryonic stem cells," Proc NatlA c20 Discloses a method of interfering with and thus blocking autocrine FGF4-mediated embryonic stem cell differentiation.

Understanding the function of ligands / receptors in stem cell differentiation allows for strategies that utilize biological proteins to regulate or even direct stem cell differentiation to interfere with these ligands / receptors. The ability to control the differentiation of unmodified human stem cells by administration of antibodies to the stem cell niche may result in novel ex vivo or in vivo approaches to stem cell-based therapies. In some embodiments, the invention is a conditionally active organism produced from a parental or wild-type biological protein capable of invading a stem cell niche and regulating the growth of the stem cell or tumor, including cancer stem cells. Provides a scientific protein. This conditionally active biological protein has lower activity than the parental or wild-type biological protein under at least one physiological condition in other parts of the body, while at least in a stem cell niche, eg, a cancer stem cell environment. It has higher activity than parental or wild-type biological proteins under one physiological condition. Such conditioned active biological proteins are less prone to side effects and act preferentially in the stem cell niche to regulate stem cell regeneration and differentiation. In some embodiments, the conditional active biological protein is an antibody. Such conditionally active antibodies can bind weakly or not to the antigen in other parts of the body, but strongly and tightly to the antigen in the stem cell niche.

Conditional active biological proteins for synovial fluid, tumor microenvironment and stem cell niche of the present invention are produced by a method of evolving DNA encoding a parental or wild-type biological protein to create a mutant DNA library. To. Next, when this mutant DNA library is expressed, a mutant protein is obtained. This variant protein has higher activity than the parental or wild-type biological protein under at least one physiological condition of the first part of the body selected from the group consisting of salivary fluid, tumor microenvironment, and stem cell niche. It is screened for conditionally active biological proteins that have lower activity than the parental or wild-type biological proteins under at least one physiological condition in the second part of the body that is different from the first part of the body. The second part of the body may be plasma. Such selected mutant biological proteins are conditionally active biological proteins that have high activity in the first part of the body but low activity in the second part of the body.

Such a conditionally active biological protein is a parental or wild type because the activity of the conditionally active biological protein is lower in other parts of the body where the conditionally active biological protein is not intended to act. It is beneficial in reducing the side effects of proteins. For example, if it is intended to introduce a conditionally active biological protein into the tumor microenvironment, the low activity of the conditionally active biological protein in any part of the body other than the tumor microenvironment is such. It means that conditioned active biological proteins are less likely to interfere with normal physiologic function in parts of the body other than the tumor microenvironment. At the same time, conditioned active biological proteins are highly active in the tumor microenvironment, which makes conditioned active biological proteins more effective in treating tumors.

Due to the low side effects, this conditionally active biological protein allows the safe use of significantly higher doses of the protein as compared to the parental or wild-type biological protein. This is particularly beneficial for antibodies to cytokines or growth factors, as antibodies to cytokines or growth factors can interfere with the normal physiological function of cytokines or growth factors in other parts of the body. Higher efficacy can be achieved with higher doses by using conditioned active biological proteins with lower side effects.

Conditionally active biological proteins that act in synovial fluid, tumor microenvironment, or one of the stem cell niches may also allow the use of novel drug targets. The use of conventional biological proteins as therapeutic agents can have unacceptable side effects. For example, inhibition of the epidermal growth factor receptor (EGFR) can be highly effective in suppressing tumor growth. However, drugs that inhibit EGFR can also suppress growth in the skin and gastrointestinal (GI) tracts. This side effect makes EGFR unsuitable as a tumor drug target. Conditionally active antibodies that bind EGFR with high affinity only in the tumor microenvironment but with no or very low affinity in any other part of the body can significantly reduce side effects while at the same time tumor. Can suppress growth. In this case, by using a conditional active antibody, EGFR can be an effective novel tumor drug target.

In another example, cytokine suppression is often beneficial in repairing joint injuries. However, suppression of cytokines in other parts of the body can also suppress the body's immune response and cause immunodeficiency. Therefore, cytokines in synovial fluid are not ideal targets for the development of conventional antibody drugs for the treatment of joint injuries. However, the side effects of immunodeficiency can be dramatically reduced by using conditional active antibodies that bind preferentially to cytokines in synovial fluid while binding to the same cytokines entirely or weakly in other parts of the body. Can be reduced. Therefore, by using conditional active antibodies, cytokines in synovial fluid can be a good target for repairing joint damage.

Conditional Active Biological Proteins for Inflammable Organs / Tissues In some embodiments, conditional active biological proteins cause inflammation, such as lymph nodes, tonsils, adenoids, and sinuses. Designed to act preferentially in easy organs or tissues. For additional organs and tissues that are prone to inflammation, ^{anatomy textbooks such as Gray's Anatomy by Henry Gray, 41 st} edition, 2015, publisher Elsevier may be referenced.

These organs and tissues typically exhibit at least one abnormal condition once inflamed. For example, these inflamed organs and tissues have higher osmotic pressure and / or lower concentrations of one or more ions compared to normal physiological conditions of other parts of the body, such as human plasma. Can be. Moreover, such inflamed organs and tissues can have high concentrations of small molecules, lactic acid, cytokines and leukocytes when compared to normal physiological conditions of other parts of the body such as human plasma.

In some embodiments, the present invention provides a conditioned active biological protein using anomalous conditions selected from one or more anomalous conditions faced in the area of inflammation and normal physiological conditions in human plasma. Can be made. Thus, such conditional active biological proteins have higher activity in inflamed organs / tissues as compared to the activity of parental or wild-type biological proteins, and parental or wild-type biological proteins in human plasma. It may have lower activity compared to the activity of the protein. Such conditional active biological proteins may act preferentially in the inflamed areas of the body, but have little or no activity in the non-inflamed areas of the body.

Conditional Active Viral Particles Viral particles have long been used as delivery vehicles for transporting proteins, nucleic acid molecules, chemical compounds or radioactive isotopes to target cells or tissues. Virus particles commonly used as delivery media include retroviruses, adenoviruses, lentiviruses, herpesviruses, and adeno-associated viruses. Virus particles often recognize their target cells in a ligand-receptor binding system via a surface protein that acts as a recognition protein for specific binding to the cellular protein that acts as the target protein of the target cell (Lentz). , "The recognition event protein virus and host cell receptor: a target for protein viral agents," J. of Gen. Virol., Vol. 71, pages 195, 195. For example, a virus recognition protein can be a ligand for a receptor on a target cell. The specificity between the ligand and the receptor allows the viral particles to specifically recognize the target cell and deliver its contents to it.

Techniques for making artificial virus particles from wild-type viruses are well known to those of skill in the art. Artificial virus particles known as delivery media include retroviruses (eg, International Publication No. 90/07936; International Publication No. 94/03622; International Publication No. 93/25698; International Publication No. 93/25234; Pamphlet; US Pat. No. 5,219,740; International Publication No. 93/11230; International Publication No. 93/10218; US Pat. No. 4,777,127; British Patent No. 2,200 , 651; see European Patent No. 0345 242; and WO 91/02805; alpha virus (eg, Sindbis virus vector, Semuliki forest virus (ATCC VR-67;); ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246), Venezuelan encephalitis encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), and adeno-related Viruses (eg, International Publication No. 94/12649, International Publication No. 93/03769, International Publication No. 93/19191; International Publication No. 94/28938; International Publication No. 95/11984 and International Includes those based on Publication No. 95/00655 (see Pamphlet No. 95/00655).

In general, artificial virus particles are constructed by inserting foreign recognition proteins into the virus particles, often by substitution of naturally recognized proteins by recombinant techniques. The exogenous recognition protein can be, for example, an antibody, receptor, ligand or collagen binding domain. The present invention provides a conditionally active recognition protein in which the binding to cells is inactive or inactive under normal physiological conditions, and the binding to cells is active or highly active under abnormal conditions. Therefore, the conditional active recognition protein preferentially binds to the target cells of the affected tissue and / or the diseased site based on the presence of abnormal conditions at the site, and with the cells of the normal tissue having normal physiological conditions. Bindings can be avoided or minimal. Conditional active recognition proteins can be expressed and presented on the surface of viral particles.

In some embodiments, the invention provides a method of evolving a parental or wild-type recognition protein to screen for a conditional active recognition protein. Conditional active recognition proteins have lower cell-binding activity under normal physiological conditions than parental or wild-type recognition proteins, and higher cell-binding activity than parental or wild-type recognition proteins under abnormal conditions. Insertion of such a conditionally active recognition protein into virus particles by a well-known recombination technique can produce conditionally active virus particles.

In another embodiment, the invention provides a conditionally active recognition protein comprising a conditionally active recognition protein, wherein the conditionally active recognition protein is a target of the affected tissue or disease site. Allows cells to recognize and bind to it, but cells in normal tissue do not recognize and bind. Such conditionally active viral particles can preferentially deliver therapeutic agents within the viral particles to diseased tissue or site, whereas the conditional active viral particles deliver less therapeutic agent to cells of normal tissue. Deliver or not deliver.

In some embodiments, the target cells at the diseased site have an abnormal pH (eg, pH 6.5) compared to the pH or temperature at other parts of the body that are healthy or not affected by a particular disease or condition. ) Or in a region with an abnormal temperature or inside a microenvironment. In this embodiment, the conditional active recognition protein has a lower binding activity to the target protein of the target cell than the parent or wild-type recognition protein at normal physiological pH or temperature, and the parent or at abnormal pH or temperature. The binding activity of the target cell to the target protein is higher than that of the wild-type recognition protein. Thus, this recognition protein preferentially binds to sites where abnormal pH or temperature occurs, thereby delivering treatment to disease sites.

In one embodiment, the viral particles may comprise conditionally active antibodies of the invention, particularly variable regions of the antibody (eg, Fab, Fab', Fv). Such conditional active antibodies have a lower affinity than parental or wild-type antibodies under normal physiological conditions that can occur at sites containing normal tissues, and parental or wild-type antibodies under abnormal conditions that can occur at diseased sites or affected tissues. With higher affinity, it can bind to the target protein (as an antigen) of the target cell. Conditionally active antibodies can be derived from parental or wild-type antibodies according to the methods of the invention.

In certain embodiments, the target protein on the target cell includes, for example, a tyrosine kinase growth factor receptor that is overexpressed on the cell surface in many tumors. Exemplary tyrosine kinase growth factors include 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.

Conditionally Active DNA / RNA Modified Proteins DNA / RNA modified proteins, especially those called CRISPR, have been discovered as a form of new genome manipulation tool, according to which researchers apply microsurgery to genes. , The DNA sequence at the exact location on the chromosome can be altered precisely and easily (Genome Editing, Mari et al., "Cas9 as a versaille tool for geneering biologic," Nature Methods, vol.10, pages 957-963, 2013). For example, sickle cell anemia is caused by a single nucleotide mutation, which may be corrected using DNA / RNA modification proteins. This technique allows precise deletion or editing of chromosomal fragments, even by single base pair alterations (Makarova et al., "Evolution and microbiology of the CRISPR-Cas systems," Nature. Reviews Microbiology, vol. 9, pages 467-477, 2011).

Genome editing with CRISPR has the ability to rapidly and simultaneously generate multiple genetic changes in cells. Many human illnesses are affected by mutations in multiple genes, including heart disease, diabetes, and nervous system diseases. This CRISPR-based technique has the potential to restore disease-causing mutations, cure these diseases, or at least reduce the severity of these diseases. Genome editing relies on CRISPR-related (Cas) proteins (enzyme families) for cleavage of genomic DNA. Typically, the Cas protein is guided to a target region within the genome by a small guide RNA, where the guide RNA coincides with the target region. Since the Cas protein has little or no sequence specificity, the guide RNA acts as an indicator for the Cas protein to achieve accurate genome editing. In one embodiment, one Cas protein can be used with multiple guide RNAs to simultaneously correct multiple gene mutations.

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. , Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx1 , Csf1, Csf2, Csf3, and Csf4 ((Makarova et al., "Evolution and culture of the CRISPR-Cas systems," Nature Review, Micro

In order to perform genome editing, the Cas protein needs to invade the target cell. The cells of interest may have different intracellular pH inside the cells. Some cells in the affected tissue have an abnormal intracellular pH. For example, some tumor cells tend to have an alkaline intracellular pH of about 7.12 to 7.65, while cells of normal tissue have a neutral intracellular pH in the range of 6.99 to 7.20. Has pH. Cardone et al. , "The roll of disturbed pH dynamics and the Na (+) / H (+) exchanger in metastasis," Nat. Rev. Cancer, vol. 5, pages 786-795, 2005. In chronic hypoxia, cells in affected tissue have an intracellular pH of approximately 7.2-7.5, which is also higher than the intracellular pH in normal tissue (Rios et al., "Chronic physioxia elevates intracellular pH and". activates Na + / H + exchang in pulponary artificial cells cells, "American Journal of Physiology-Lung Cellular and Molecular Phyla.7 In addition, in ischemic cells, the intracellular pH is typically in the range of 6.55-6.65, which is lower than the intracellular pH of normal tissues (Haqberg, "Intracellular pH daring ischemia in skeletal muscle: resonance to membrane". positive, extracellular pH, tissue lactic acid and ATP, "Pflugers Arch., Vol. 404, pages 342-347, 1985). Further examples of abnormal intracellular pH in affected tissue are described in Han et al. , "Fluorescent Indicators for Intracellular pH," Chem Rev. , Vol. 110, pages 2709-2728, 2010.

The present invention provides a method for producing a conditionally active Cas protein from a parent or wild Cas protein, wherein the conditionally active Cas protein is (1) parent or wild under normal physiological conditions inside normal cells. Enzyme activity decreased compared to the activity of the type Cas protein, and (2) Enzyme increased compared to the activity of the parental or wild Cas protein under abnormal conditions inside the target cell, such as one of the affected cells discussed above. Has at least one of the activities. In some embodiments, the normal physiological condition is a nearly neutral intracellular pH, and the abnormal condition is a different intracellular pH that is higher or lower than neutral. In certain embodiments, the anomalous condition is an intracellular pH of 7.2-7.65 or an intracellular pH of 6.5-6.8.

In some embodiments, the conditionally active Cas protein can be delivered to target cells using the conditionally active virus particles of the invention. Conditionally active viral particles include a conditionally active Cas protein and at least one guide RNA for directing the Cas protein to a location where the Cas protein will edit genomic DNA.

Multispecific antibodies have high selectivity in the preferred target tissue containing all or most of the targets (antigens) to which the multispecific antibody can bind. For example, bispecific antibodies show higher preference to target cells that express both of the antigens recognized by the bispecific antibody compared to non-target cells that can express only one of the antigens. This provides selectivity for target cells. Therefore, due to the dynamism of this system, more bispecific antibodies bind to target cells compared to non-target cells in equilibrium.

Multispecific antibodies, or antigen recognition fragments thereof, engineered herein can be used as ASTRs for the chimeric antigen receptors of the invention.

Manipulation of cytotoxic cells Once a conditionally active ASTR has been identified, a single polynucleotide sequence (CAR gene, which is a conditionally active CAR) is ligated to the polynucleotide sequence encoding the individual domain. The chimeric antigen receptor can be assembled by forming). Individual domains include conditional active ASTRs, TMs, and ISDs. In some embodiments, other domains, including ab ESD and CSD, can also be introduced into CAR (FIG. 1). When the conditional active CAR is a bispecific CAR, the CAR gene is composed, for example, from the N-terminus to the C-terminus with the following composition: N-terminal signal sequence-ASTR1-linker-ASTR2-extracellular spacer domain-membrane. It may be a transmembrane domain-a co-stimulation domain-an intracellular signal transduction domain. In one embodiment, such CAR gene may contain more than one costimulatory domain.

Alternatively, the polynucleotide sequence encoding the conditioned active CAR has the following configuration in the N-terminal to C-terminal orientation: N-terminal signal sequence-ASTR1-linker-ASTR2-transmembrane domain-costimulation domain-intracellular signaling. It may be a domain. In certain embodiments, such CAR may include more than one co-stimulation domain. When the CAR contains more than one ASTR, the polynucleotide sequence encoding the CAR has the following configuration from the N-terminus to the C-terminus: N-terminus signal sequence-ASTR1-linker-ASTR2-linker- (antigen-specific target). Region) _n- transmembrane domain-costimulation domain-intracellular signaling domain. Such CAR may further comprise an extracellular spacer domain. Each ASTR may be separated by a linker. In certain embodiments, such CAR may include more than one co-stimulation domain.

Conditionally active CAR is introduced into cytotoxic cells by an expression vector. Expression vectors containing the polynucleotide sequences encoding the conditionally active CARs of the invention are also provided herein. Suitable expression vectors include, but are not limited to, lentivirus vector, γ retrovirus vector, formy virus vector, adeno-associated virus (AAV) vector, adenovirus vector, genetically engineered hybrid virus, naked DNA, and the like. , Transposon-mediated vectors such as Sleeping Beauty, Piggybak, and integrases such as Phi31. Some other suitable expression vectors include herpes simplex virus (HSV) and retrovirus expression vectors.

The adenovirus expression vector is based on adenovirus, which has low ability to integrate into genomic DNA, but has high host cell transfection efficiency. The adenovirus expression vector contains (a) an adjunct to the packaging of the expression vector and (b) an adenovirus sequence sufficient for the final expression of the CAR gene in the host cell. The adenovirus genome is a 36 kb linear double-stranded DNA, where an exogenous DNA sequence (such as the CAR gene) can be inserted to replace a large fragment of adenovirus DNA to create the expression vector of the invention. (Grunhaus and Hornitz, "Adenoviruses as cloning vectors," Seminars Virol., Vol. 3, pages 237-252, 1992).

Another expression vector is based on adeno-associated virus, 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 and is therefore useful for, for example, tissue culture or gene delivery to mammalian cells in vivo. AAV vectors have a wide host range with respect to infectivity. Details regarding the preparation and use of AAV vectors are described in US Pat. Nos. 5,139,941 and 4,797,368.

Retrovirus expression vectors have the ability to integrate into the host genome, deliver large amounts of exogenous genetic material, infect a wide range of species and cell types, and be packaged in specific cell lines. Retroviral vectors are constructed by inserting nucleic acids (eg, those encoding CAR) at specific locations in the viral genome to create replication-deficient viruses. Retroviral vectors can infect a wide variety of cell types, but host cell division is required for CAR gene integration and stable expression.

The lentiviral vector is derived from lentivirus, which is a complex retrovirus containing the common retroviral genes gag, pol, and envelope, as well as other genes with regulatory or structural functions ( US Pat. Nos. 6,013,516 and 5,994,136). Some examples of lentiviruses include human immunodeficiency viruses (HIV-1, HIV-2) and simian immunodeficiency viruses (SIV). Lentiviral vectors have been created by multiple attenuation of HIV pathogenic genes, for example by deleting the genes env, viv, vpr, vpu and nef to make the vector biologically safe. Lentiviral vectors have the ability to infect non-dividing cells and can be used for gene transfer and expression of the CAR gene both in vivo and in exobibo (US Pat. No. 5,994,136).

Expression vectors containing the conditional active CAR gene can be introduced into host cells by any means known to those of skill in the art. The expression vector may contain a viral sequence for transfection, if desired. Alternatively, the expression vector may be introduced by fusion, electroporation, fine particle gun, transfection, lipofection and the like. Host cells may be grown and expanded in culture prior to introduction of the expression vector, followed by appropriate treatment for vector introduction and integration. The host cells are then expanded and screened by the markers present in the vector. Various markers that can be used include hprt, neomycin resistance, thymidine kinase, hygromycin resistance and the like. As used herein, the terms "cell", "cell line", and "cell culture" may be used interchangeably. In some embodiments, the host cells are T cells, NK cells and NKT cells.

In another aspect, the invention also provides genetically engineered cytotoxic cells comprising the conditionally active CAR of the invention and stably expressing it. In one embodiment, the genetically engineered cells include T lymphocytes (T cells), naive T cells ( _TN ), memory T cells (eg, central memory T cells) that have the ability to produce therapeutically relevant progeny. (T _CM), effector memory cells (T _EM)), natural killer cells, and macrophages and the like. In another embodiment, the genetically engineered cell is an autologous cell. Examples of suitable T ^{cells, CD4 + / CD8 -, CD4} - / CD8 +, CD4 - / CD8 - or CD4 ⁺ / CD8 ⁺ T cells. T cells, CD4 ⁺ / CD8 ^- and CD4 ^- / CD8 ⁺ may be a population of mixed population or a single clone of cells. The CD4 ⁺ T cells of the invention can also be co-cultured in vitro with cells expressing the target antigen (eg, CD20 ⁺ and / or CD19 ⁺ tumor cells) to IL-2, IFN-γ, TNF-α and other T cells. Cellular effector cytokines can also be produced. The CD8 ⁺ T cells of the present invention can lyse cells expressing the target antigen. In some embodiments, the T cells can be CD45RA ⁺ CD62L ⁺ naive cells, CD45RO CD62I7 central memory cells, CD62L ^" effector memory cells" or any one or more of these (Berger et al). ., "Adaptive transfer of virus-specific and tumor-special T cell immunity," Curr. Opin. Immunol., Vol. 21, pages 224-232, 2009).

Genetically engineered cytotoxic cells can be produced by stably transfecting a host cell with an expression vector containing the CAR gene of the present invention. Further methods of genetically engineering cytotoxic cells using the expression vector include chemical transformation methods (eg, the use of calcium phosphate, dendrimers, liposomes and / or cationic polymers), non-chemical transformation methods (eg, use). , Electrical perforation, optical transformation, gene electrotransfer and / or hydrodynamic delivery) and / or particle-based methods (eg, impalefection, use of gene guns and / or magnetism). Is done. Transfected cells showing the presence of a single unrearranged integrated vector and expressing conditional active CAR can be expanded in exobibo.

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

After introducing the expression vector containing the CAR gene into the host cell, the CAR gene is expressed, thereby producing a CAR molecule capable of binding to the target antigen. Since the produced CAR molecule has a transmembrane domain, it becomes a transmembrane protein. The host cell will then be converted to CAR cells such as CAR-T cells. For methods of making engineered cytotoxic cells containing CAR molecules, such as CAR-T cells, see, for example, (Cartellieri et al., T cells engineered with a chimeric antigen receptor for cancer immunotherapy (Cartellieri et al.). Chimeric antigen receptor-engagered T cells for immunotherapy of cancer) ”, Journal of Biomedicine and Biotechnology, vol.2010, vol.2010, Art. Versatile strategy for controlling the specificity and activity of engineered T cells, PNAS, vol. 113, E450-E458, 2016).

Whether prior to or after genetic modification of cytotoxic cells to express the desired conditional active CAR, for example, US Pat. No. 6,352,694; 6.543, U.S.A. 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. No. 7,175,843; No. 5,883,223; No. 6,905,874; No. 6,797,514; No. Cells can be activated and expanded in numbers using methods as described in 6,867,041; and US Patent Application Publication No. 20060121005. For example, the T cells of the invention can be expanded by contacting a surface to which a drug that stimulates a CD3 / TCR complex-related signal and a ligand that stimulates a co-stimulator molecule on the surface of the T cell are bound. Specifically, the T cell population is contacted with a surface-immobilized anti-CD3 antibody, or an antigen-binding fragment thereof, or an anti-CD2 antibody, or with a calcium ionophore and a protein kinase C activator (eg, bryostatin). ) Can be stimulated. A ligand that binds to the accessory molecule is used to co-stimulate the accessory molecule on the surface of the T cell. For example, T cells can be contacted with anti-CD3 and anti-CD28 antibodies under conditions appropriate to stimulate T cell proliferation. Anti-CD3 and anti-CD28 antibodies to stimulate the proliferation of either CD4 ⁺ T cells or CD8 ^{+ T cells.} Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France), which, like other methods commonly known in the art, are the present invention. It can be used in the 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).

In various embodiments, the invention provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a therapeutically effective amount of the conditionally active CAR of the invention. The conditionally active CAR in this composition can be any one or more of the polynucleotides encoding the CAR, the protein containing the CAR or the genetically modified cells expressing the CAR protein. The CAR protein may be in the form of a pharmaceutically acceptable salt. Pharmaceutically acceptable salts include, for example, salts such as sodium, potassium, calcium and amine salts such as procaine, dibenzylamine, ethylenediamine, ethanolamine, methylglucamine, taurine, and acid additions such as hydrochloride. Refers to salts that can be used as salts of therapeutic proteins in the pharmaceutical industry, including salts and basic amino acids.

Pharmaceutically acceptable excipients include any excipient useful in the preparation of desirable pharmaceutical compositions that are generally safe and non-toxic and are acceptable for use in animals as well as in human pharmaceuticals. Excipients to be used are mentioned. Such excipients can be solids, liquids, semi-solids, or gases in the case of aerosol compositions. Certain excipients include a pharmaceutically acceptable carrier, which can be added to enhance or stabilize the composition or facilitate the preparation of the composition. Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline, alcohol and water. Solid carriers include starch, lactose, calcium sulphate, dihydrate, clay, magnesium stearate or stearic acid, talc, pectin, acacia, agar and gelatin. The carrier may also contain sustained release materials such as glyceryl monostearate or glyceryl distearate alone or with wax.

The pharmaceutically acceptable carrier is, in part, determined by the detailed composition administered and also by the detailed method used to administer the composition. Therefore, there are a wide variety of suitable formulations of the pharmaceutical composition of the present invention. Various aqueous carriers such as buffered saline can be used. These solutions are sterile and generally free of unwanted substances. These compositions can be sterilized by conventional well known sterilization techniques. The composition comprises an appropriately pharmaceutically acceptable auxiliary substance such as a pH adjuster and a buffer, a toxicity adjuster, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. to approximate physiological conditions. May contain. The concentration of CAR in these formulations can vary widely and will be selected according to the detailed dosing method selected and the patient's needs, primarily based on fluid volume, viscosity, and body weight.

In various embodiments, the pharmaceutical composition according to the invention can be formulated for delivery by any suitable route of administration. "Routes of administration" are, but are not limited to, aerosol, nasal, oral, intravenous, intramuscular, intraperitoneal, inhalation, transmucosal, transdermal, parenteral, implantable pump, continuous infusion, topical application, capsules and / Or can refer to any route of administration known in the art, including injection.

The pharmaceutical composition according to the present invention may be encapsulated for oral administration, tableted, or prepared in an emulsion or syrup. Pharmaceutical compositions are made according to conventional pharmaceutical techniques involving grinding, mixing, granulation and compression (if required) in tablet form; or grinding, mixing and filling in hard gelatin capsule form. When liquid carriers are used, the formulations can be in the form of syrups, elixirs, emulsions or aqueous or non-aqueous suspensions. Such a liquid preparation may be directly orally administered or may be filled in a soft gelatin capsule.

The pharmaceutical composition is (a) an effective amount of packaged nucleic acid suspended in a liquid solution, eg, water, physiological saline or a diluent such as PEG 400; (b) liquid, solid, granules, respectively. Alternatively, it can be formulated as a capsule, sachet or tablet containing a predetermined amount of active ingredient as gelatin; (c) a suspension in a suitable liquid; and (d) a suitable emulsion. In particular, suitable dosage forms include, but are not limited to, tablets, pills, powders, sugar coatings, capsules, liquids, lozenges, gels, syrups, slurries, suspensions and the like.

Solid formulations include, for example, sugars such as lactose, sucrose, mannitol, or sorbitol; starches derived from corn, wheat, rice, potatoes, or other plants; methylcellulose, hydroxypropylmethylcellulose, and sodium carboxymethylcellulose. Cellulose; and rubber, including Arabia and tragacanto; and suitable solid excipients such as carbohydrates or protein fillers, including proteins such as gelatin and collagen. A disintegrant or solubilizer may be added, such as a crosslinked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof such as sodium alginate. Tablet forms include lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, potato starch, tragacanth, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscannelose sodium, talc, magnesium stearate, Of stearate and other excipients, colorants, fillers, binders, diluents, buffers, wetting agents, preservatives, flavoring agents, pigments, disintegrants, and pharmaceutically acceptable carriers. Can include one or more.

Liquid suspensions have a conditionally active CAR in a mixture with excipients suitable for the production of aqueous suspensions. Such excipients include suspending agents such as sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, sodium alginate, polyvinylpyrrolidone, tragacant gum and arabic gum, and naturally occurring phosphatides (eg lecithin), alkylene oxides. Condensation product of fatty acid with fatty acid (eg polyoxyethylene stearate), condensation product of ethylene oxide with long-chain aliphatic alcohol (eg heptadecaethyleneoxysetanol), ethylene oxide with fatty acid and partial ester derived from fatty acid and hexitol. May include nucleic acid or wetting agents such as condensation products (eg polyethylene sorbitol monoolate) or condensation products of ethylene oxide with fatty acids and partial esters derived from fatty acids and partial esters derived from anhydrous hexitol (eg polyoxyethylene sorbitol monooleate). I can. Liquid suspensions are also one or more preservatives such as ethyl or n-propyl-p-hydroxybenzoate, one or more colorants, one or more flavorings, and sucrose, aspartame or saccharin and the like. Can contain one or more of the sweeteners. The pharmaceutical product may be adjusted for osmotic pressure.

The troche dosage form usually has the active ingredient in a flavoring agent such as sucrose and acacia or tragacanth, and the incense tablet is active in an inert substrate such as gelatin and glycerin or sucrose and acacia emulsion, gel. It has an ingredient and can have a carrier known in the art in addition to these active ingredients. It is understood that conditioned active CAR must be protected from digestion when administered orally. This is generally done by forming a complex of conditional CAR with a composition that confer resistance to acidic and enzymatic hydrolysis, or by making conditional active CAR suitable, such as liposomes. Achieved by packaging in a resistant carrier. Methods of protecting proteins from digestion are known in the art. The pharmaceutical composition can be encapsulated, for example, in liposomes or in a formulation that allows sustained release of the active ingredient.

The pharmaceutical compositions may be formulated as aerosol formulations for administration by inhalation (eg, they can be "sprayed"). The aerosol formulation can be placed in a pressurized and acceptable propellant, such as dichlorodifluoromethane, propane, nitrogen and the like. Suitable formulations for rectal administration include, for example, a suppository consisting of a packaging nucleic acid with a suppository base. Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons, plus gelatin consisting of a base containing, for example, liquid triglycerides, polyethylene glycol, and paraffin hydrocarbons and a packaging nucleic acid. It is also possible to use rectal capsules.

The pharmaceutical composition is formulated for parenteral administration, for example, by intra-articular (injections), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes. Aqueous and non-aqueous isotonic sterile injectable solutions, which may contain antioxidants, buffers, bacteriostatic agents, and solutes that make the formulation isotonic with the intended recipient's blood, as well as suspensions. Aqueous and non-aqueous sterile suspensions, which may include turbinants, solubilizers, thickeners, stabilizers, and preservatives, may be included, and in the practice of the invention, the composition is described, for example, by intravenous injection. Can be administered orally, topically, intraperitoneally, intravesically or intrathecally. In one aspect, the parenteral administration method is the preferred method of administration for compositions comprising CAR protein or genetically engineered cytotoxic cells. The composition may be conveniently administered in unit dosage form, eg, Remington's Pharmaceutical Sciences, Mac Publishing Co., Ltd. Easton Pa. , 18 ^th Ed. , 1990, may be prepared by any of the methods well known in the pharmaceutical art. The pharmaceutical product for intravenous administration may contain a pharmaceutically acceptable carrier such as sterile water or saline, polyalkylene glycols such as polyethylene glycol, plant-derived oil, and hydrogenated naphthalene.

The pharmaceutical composition is parenteral, subcutaneous, intramuscular, intravenous, intra-articular, intrabronchial, intraperitoneal, intracapsular, intrachondral, sinus, intracavitary, intracerebral, intraventricular, colon. Intracervical, intragastric, intrahepatic, myocardial, intraosseous, pelvic, intraperitoneal, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, lumbar It can be administered by at least one method selected from intracapsular, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, or percutaneous. This method is optional, before, simultaneously with, or after conditionally active CAR, detectable labels or reporters, TNF antagonists, anti-rheumatic drugs, muscle relaxants, narcotic drugs, non-steroidal anti-inflammatory drugs (NSAK). )), Painkillers, anesthetics, sedatives, local anesthetics, neuromuscular blockers, antibacterial agents, anti-asthmatic agents, corticostiod, anabolic steroids, erythropoetin, immunization, immunoglobulins, immunosuppressants, Metabolism of growth hormones, hormone replacement drugs, radiopharmaceuticals, antidepressants, antipsychotics, stimulants, asthma drugs, β-agonists, inhaled steroids, epinephrine or its analogs, cytotoxic drugs or other anticancer drugs, methotrexate, etc. Further comprising administering at least one composition comprising an effective amount of at least one compound or protein selected from at least one of an antagonist or anti-proliferative agent.

The types of cancer treated with the genetically engineered cytotoxic cells or pharmaceutical compositions of the invention include carcinomas, blastomas, and sarcomas, and specific leukemia or lymphocytic malignancies, benign and malignant tumors, and. Malignant diseases include, for example, sarcomas, carcinomas, and melanomas. The cancer may be a non-solid tumor (such as a hematological tumor) or a solid tumor. Adult tumors / cancers and childhood tumors / cancers are also included.

Blood cancer is cancer of the blood or bone marrow. Examples of hematological cancers (or hematogenous cancers) include leukemia, eg, acute leukemia (acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, premyelocytic, myelomonocytic). , Monocytic and erythroid leukemia, etc.), Chronic leukemia (chronic myelocytic (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia, etc.), true erythrocytosis, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (Painless and high-grade types), multiple myeloma, Waldenstrem macroglobulinemia, heavy chain disease, myelodysplasia syndrome, hairy cell leukemia and myelodystrophy.

Solid tumors are abnormal tissue masses that usually do not contain cysts or fluid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcoma and cancer are fibrosarcoma, mucocele, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovial, mesopharyngeal, Ewing tumors, smooth myomas, rhizome muscles. Tumor, colon cancer, lymphatic malignant tumor, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous epithelial cancer, basal cell cancer, adenocarcinoma, sweat adenocarcinoma, thyroid medullary cancer, papillary thyroid cancer, brown Cell tumor, sebaceous adenocarcinoma, papillary adenocarcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial cancer, renal cell carcinoma, hepatocellular carcinoma, bile duct cancer, chorionic villus cancer, Wilms tumor, cervical cancer, testis tumor, seminoma, bladder cancer , Chloroma, and CNS tumors (such as glioma (such as brain stem glioma and mixed glioma), glioma (also known as polymorphic glioma), stellate cell tumor, CNS lymphoma, embryo Celloma, medullary blastoma, Schwan's tumor, craniopharyogima, lining tumor, pine fruit tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, Retinal blastoma and brain metastasis, etc.).

The invention also provides a medical device comprising at least one CAR protein, a polynucleotide sequence encoding CAR, or a host cell expressing CAR, which is parenteral, subcutaneous, intramuscular, intravenous, joint. Intratricular, intrabronchial, intraperitoneal, intracapsular, intrachondral, sinus, intraluminal, intracerebral, intraventricular, intracolonic, cervical, intragastric, intrahepatic, intramyocardial, intraosseous, pelvic. Intracardiac, intraperitoneal, intraperitoneal, intrathoracic, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasulphal, intrathoracic, intrauterine, intravesical, bolus, vagina, rectum, cheek It is suitable for administering at least one conditionally active CAR by at least one method selected from lateral, sublingual, intranasal, or transdermal.

In a further embodiment, the invention comprises a host cell expressing at least one CAR protein in lyophilized form, a polynucleotide sequence encoding CAR, or CAR in a first container, and sterile water or sterile buffer water. Or phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercury nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride, alkylparaben, benzalkonium chloride in aqueous diluents , Benzalkonium chloride, sodium dehydroacetate and thimerosal, or an optional second container comprising at least one preservative selected from the group consisting of mixtures thereof. In one aspect, in this kit, the conditionally active CAR or the concentrate of a particular moiety or variant in the first container is from about 0.1 mg / ml to about 500 mg / ml in the contents of the second container. Is reconstituted in the concentration of. In another embodiment, the second container further comprises an isotonic agent. In another embodiment, the second container further comprises a physiologically acceptable buffer. In one aspect, the present disclosure is a method of treating a pathology mediated by at least one wild-type protein, wherein the patient in need thereof is administered with the formulation provided in the kit and reconstituted prior to administration. A method including a process is provided.

Also provided are products for human pharmaceutical or diagnostic use comprising a container and packaging material containing at least one CAR protein in solution or lyophilized form, a polynucleotide sequence encoding CAR, or a host cell expressing CAR. .. This product is optional, parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraperitoneal, intracapsular, intrachondral, sinus, intracavitary, intracerebral, intraventricular. Intracolon, cervical, gastric, hepatic, myocardial, bone, pelvic, peritoneal, abdominal cavity, pleura, prostate, lung, rectum, kidney, retina, spinal cord, It may include intrathecal, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, or having a container as a component of a percutaneous delivery device or system.

In some embodiments, the invention comprises a step of recovering a cytotoxic cell from a subject, a step of genetically manipulating the cytotoxic cell by introducing the CAR gene of the present invention into the cytotoxic cell, and a gene. Provided are methods comprising the step of administering to a subject an engineered cytotoxic cell. In some embodiments, the cytotoxic cells are selected from T cells, naive T cells, memory T cells, effector T cells, natural killer cells, and macrophages. In one embodiment, the cytotoxic cell is a T cell.

In one embodiment, T cells are obtained from the subject. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymic tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the invention, any T cell line available in the art can be used. In certain embodiments of the invention, T cells can be obtained from blood drawn from a subject using any technique known to those of skill in the art, such as Ficoll ™ isolation.

In a preferred embodiment, cells from the individual's circulating blood are obtained by apheresis. Apheresis products typically contain lymphocytes such as T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one embodiment, the cells collected by apheresis can be washed to remove the plasma fraction and the cells can be placed in a suitable buffer or medium for subsequent processing steps. In one embodiment of the invention, cells are washed with phosphate buffered saline (PBS). In an alternative embodiment, the wash solution may be calcium-free and magnesium-free, or may be free of many, if not all, divalent cations. Again, surprisingly, the initial activation step in the absence of calcium leads to increased activation. As will be readily appreciated by those skilled in the art, the washing process may include using a semi-automatic "flow-through" centrifuge (eg, Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) according to the manufacturer's instructions. It can be achieved by a method known to those skilled in the art. After washing, cells may be resuspended in a variety of biocompatible buffers, such as Ca ²⁺ -free, Mg ²⁺ -free PBS, PlasmaLite A, or another buffer-containing or non-buffered saline solution. .. Alternatively, the undesired components of the apheresis sample may be removed and the cells resuspended directly in the culture medium.

In another embodiment, T cells are isolated from peripheral blood by lysing red blood cells and centrifuging, for example, with a PERCOLL ™ gradient, or by depleting monocytes by countercurrent centrifugal eltriation. Certain partial T cell populations such as CD3 ⁺ , CD28 ⁺ , CD4 ⁺ , CD8 ⁺ , CD45RA ⁺ , and CD45RO ⁺ T cells can be further isolated by positive or negative selection methods. For example, enrichment of T cell populations by negative selection can be achieved in combination with antibodies to surface markers that are unique to the negatively selected cells. One method is cell fractionation and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on negatively selected cells. ^{To enrich CD4 +} cells by negative selection, monoclonal antibody cocktails typically include antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In certain embodiments, it may be desirable to enrich or positively select regulatory T cells that ^{typically express CD4 +} , CD25 ⁺ , CD62L ^hi , GITR ⁺ , and FoxP3 ^+.

For example, in one embodiment, T cells, along with anti-CD3 / anti-CD28 (ie, 3x28) conjugated beads, such as DYNABEADS® M-450 CD3 / CD28 T, are used for positive selection of desired T cells. It is isolated by incubating for a sufficient period of time. In one embodiment, this time is about 30 minutes. In a further embodiment, this time is in the range of 30 minutes to 36 hours or more, and all integer values in between. In a further embodiment, this time is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, this time is 10 to 24 hours. In one preferred embodiment, the incubation time is 24 hours. For isolation of T cells from leukemia patients, longer incubation times, such as 24 hours, can increase cell yields. Longer incubation times are for isolating T cells in any situation where there are fewer T cells when compared to other cell types, such as isolation of tumor infiltrating lymphocytes (TIL) from tumor tissue or immunocompromised individuals. Can be used. In addition, longer incubation times can increase the efficiency of CD8 + T cell capture. Thus, simply by shortening or prolonging the time it takes for T cells to bind to CD3 / CD28 beads and / or by increasing or decreasing the ratio of beads to T cells (as further described herein). ), Preferred positive or negative selection of some T cell populations can be made at the start of culture or at other times during the process. In addition, by increasing or decreasing the proportion of anti-CD3 and / or anti-CD28 antibodies on beads or other surfaces, preferential positives of some T cell populations at the start of culture or at other times during the process. Or you can make a negative choice. Those skilled in the art will recognize that multiple selection rounds may also be used in the context of the present invention. In certain embodiments, it may be desirable to carry out a selection procedure and use "unselected" cells for the activation and expansion process. "Unselected" cells can also be subjected to further selection rounds.

The resulting cytotoxic cells are then genetically engineered as described herein. The polynucleotide encoding the CAR (typically located in the expression vector) is introduced into the cytotoxic cell so that the cytotoxic cell expresses the CAR, preferably stably. The polynucleotide encoding CAR is typically integrated into the cytotoxic cell host genome. In some embodiments, the introduction of the polynucleotide does not need to result in integration, but rather it may be sufficient for the introduced polynucleotide to be transiently maintained. In this way, short-term effects can be obtained, where cytotoxic cells can be introduced into the host and then acted after a predetermined time, eg, after the cells have been able to migrate to a particular treatment site.

Depending on the nature of the cytotoxic cells and the disease being treated, the genetically engineered cytotoxic cells can be introduced into the subject, eg, a mammal, in a wide variety of ways. Genetically engineered cytotoxic cells can be introduced into the site of the tumor. In one embodiment, the genetically engineered cytotoxic cells progress to or are modified to progress to cancer. The number of genetically engineered cytotoxic cells used will depend on a number of factors, including circumstances, purpose of introduction, cell lifespan, protocol used, and many other factors. For example, the number of doses, the ability of cells to proliferate, and the stability of recombinant constructs. The genetically engineered cytotoxic cells can be applied as a dispersion injected at or near the site of interest. The cells can be in a physiologically acceptable medium.

Treatment methods include the cellular response to CAR, the efficiency of CAR expression by cytotoxic cells, and, as appropriate, the level of secretion, activity, and specific needs of the subject (which may vary over time and circumstances). It must be understood that it is affected by many variables, such as the rate of loss of cell activity due to the loss of genetically engineered cytotoxic cells or the expression activity of individual cells. Therefore, for each individual patient, even if there are universal cells that can be administered to the population as a whole, it is expected that each patient will be monitored for the appropriate dosage for that person, such patients. The task of monitoring is routine in the art.

The following examples illustrate, but are not limited to, the methods of the present disclosure. Other suitable changes and adaptations of the various conditions and parameters normally encountered in the field to those of skill in the art are within the scope of this disclosure.

Example 1: Generation of scFv Conditionally Active Single-Chain Antibodies Against Axl Two Conditionally Active Single-Chains Antibodies (CAB-scFv-63.9-4 and CAB-scFv-63.9-6) Is expressed as a homodimer with wild-type human IgG1 Fc, SEQ ID NO: 13 or 14 (divalent antibody CAB-scFv-63.9-4-01, SEQ ID NO: 9 and CAB in FIGS. 2 to 3). -ScFv-63.9-6-01 (SEQ ID NO: 10) was obtained), and expressed as a heterodimer of the knob-in-hole system to obtain a monovalent scFv (FIGS. 2 to 3). The monovalent antibody scFv CAB-scFv-63.9-4-02, SEQ ID NO: 11, and CAB-scFv-63.9-6-02, SEQ ID NO: 12 were obtained).

The binding affinity of these antibodies for the drug target antigen Axl at pH 6.0 and pH 7.4 was measured by ELISA analysis. As shown in FIG. 2, these scFv antibodies showed affinity for the drug target antigen Axl at both pH 6.0 and pH 7.4, and these affinities were comparable to those of fully divalent antibodies. Furthermore, as shown in FIG. 3, the selectivity of these scFv antibodies at pH 6.0 compared to pH 7.4 was also comparable to that of fully divalent antibodies. In this example, it was demonstrated that the conditionally active antibody of the invention has comparable affinity and selectivity for either scFv or fully divalent antibodies. Therefore, the conditional active antibody of the present invention can be inserted as a single DNA strand into the DNA molecule encoding CAR in the CAR-T platform of the present invention.

Example 2: scFv antibody against target antigen Axl for constructing CAR-T cells In one embodiment of the invention, simultaneous screening for selectivity and affinity, as well as expression levels at both pH 6.0 and pH 7.4. By doing so, a conditionally active antibody against the drug target antigen Axl was generated. Screening was performed using the FLAG tag in serum because there were human antibodies in the serum that could give false positives for screening. The screening buffer was carbonate buffer (a Krebs buffer that contains Ringer's standard buffer but is different from PBS). Both of the produced conditional active antibodies have a higher affinity for the drug-targeted antigen Axl at pH 6.0 compared to the wild-type antibody, but have a pH of 7.4 for the same drug-targeted antigen Axl. It turned out that the affinity was lower. In addition, all of these conditionally active antibodies have high expression levels, as shown in Table 2 below (column "clone" indicates antibody, and expression level "mg / ml" is indicated in the second column). Have.

Clones of these antibodies were sent to the service provider along with the requested expression level (“order volume”, expected expression level). However, the actual expression level (“delivery amount”) of these antibodies was extremely high and exceeded the expected expression level.

As shown in FIG. 4 using the BAP063.9-13-1 antibody as an example, the conditioned active antibody did not show aggregation in buffer. BAP063.9-13-1 antibody was analyzed by size exclusion chromatography. Only one peak was detected in FIG. 4, demonstrating little or no antibody aggregation.

Conditionally active antibodies were also analyzed using surface plasmon resonance (SPR) and their on and off rates for the drug target antigen Axl were measured. SPR analysis is known to measure on and off rates of conditioned active antibodies. SPR analysis was performed in the presence of bicarbonate. The in vivo on and off rates of conditionally active antibodies (in animals and humans) are crucial features for conditionally active antibodies.

Conditionally active antibodies have a faster on-rate at pH 6.0 and a faster on-rate at pH 7.4 in comparison to the negative control (BAP063 10F10, which has similar on-rates at both pH 6.0 and pH 7.4). A slower on-speed was observed (Fig. 5). In addition, raising the temperature from room temperature to 60 ° C. does not significantly change the SPR analysis results (Fig. 5). SPR analysis also showed that these conditionally active antibodies were highly selective at pH 6.0 when compared to pH 7.4 (FIGS. 6A-6B show one antibody as an example).

Conditional active biological antibodies are summarized in Table 3. Two of these antibodies were expressed as scFv (BAP063.9-13.3 and BAP063.9-48.3) and were ready for immediate insertion into CAR on the CAR-T platform. rice field. Incubation of the antibody at 60 ° C. for 1 hour did not change much of the antibody's affinity (“thermostability”). In the two columns reporting measurement data of binding activity at pH 6.0 and pH 7.4 using SPR (last two columns in Table 3), "BAP063.6-hum10F10-FLAG" (negative control, Table 3). The second column) was compared with. The selectivity of these antibodies can be determined by the difference between the data in the two final columns. The two scFv antibodies had extremely high selectivity (0% at pH 7.4 vs. 75% and 50% at pH 6).

比較例Ａ：標的抗原Ａｘｌに対する非条件的活性型抗体を有するＣＡＲ−Ｔ細胞
標的抗原Ａｘｌに対する非条件的活性型ｓｃＦｖ抗体を使用して、標的抗原Ａｘｌ又は細胞表面上に標的抗原Ａｘｌを発現するＣＨＯ細胞（ＣＨＯ−Ａｘｌ）に結合するＣＡＲ−Ｔ細胞を構築した、図７Ａ〜図７Ｂ。非条件的活性型抗体をＣＡＲ分子のＡＳＴＲとして使用し、このＣＡＲ分子をＴ細胞に挿入して、標的抗原Ａｘｌに結合するＣＡＲ−Ｔ細胞を構築した。

Comparative Example A: CAR-T cell having a non-conditionally active antibody against the target antigen Axl A non-conditionally active scFv antibody against the target antigen Axl is used to express the target antigen Axl or the target antigen Axl on the cell surface. 7A-7B, which constructed CAR-T cells that bind to CHO cells (CHO-Axl). A non-conditionally active antibody was used as the ASTR of the CAR molecule and the CAR molecule was inserted into T cells to construct CAR-T cells that bind to the target antigen Axl.

For comparison, CHO cells that do not express the target antigen Axl are (1) T cells that have not been transfected with a CAR molecule, (2) T cells that have been transfected with a CAR molecule that does not bind to the target antigen Axl, and (3) a target. Treated with T cells transfected with a CAR molecule having a non-conditionally active antibody against the antigen Axl (FIG. 7A). The CHO cell population is indicated by the cell index (Y-axis in FIG. 7A), where a decrease in the cell index indicates cytotoxicity (cell death) by CAR-T cells.

Referring to FIG. 7A, CHO cells showed growth prior to the addition of T cells. After the addition of CAR-T cells that bind to the target antigen Axl, the cell index was initially reduced, suggesting non-specific cytotoxicity of the T cells. However, shortly thereafter, CHO cells resumed growth. More importantly, the difference between the three treatments is not significant, and significant cytotoxicity against CHO cells that do not express the target antigen Axl in CAR-T cells with non-conditionally active antibodies against the target antigen Axl. It was shown that there is no.

Next, in the same manner as above, CHO cells expressing the target antigen Axl are (1) T cells that have not been transfected with a CAR molecule, (2) T cells that have been transfected with a CAR molecule that does not bind to the target antigen Axl, and (3) Treatment was performed with T cells transfected with a CAR molecule having a non-conditionally active antibody against the target antigen Axl (FIG. 7B). After adding T cells, treatment with CAR-T cells having a non-conditionally active antibody against the target antigen Axl significantly reduced the cell index, but not with the other two treatments, and the target antigen was not reduced. Cytotoxicity to CHO-Axl cells expressing the target antigen Axl by CAR-T cells with non-conditionally active antibodies to Axl is shown.

Example 3: CAR-T cells having a conditionally active scFv antibody against the target antigen Axl A CAR molecule was constructed using the conditionally active scFv antibody against the target antigen Axl. T cells were transduced with this CAR molecule so that the T cells expressed the CAR molecule (CAR-T cells). CHO cells expressing the target antigen Axl (CHO-63 cells) or normal CHO cells not expressing the target antigen Axl (CHO cells) were treated separately with CAR-T cells. Non-transduced T cells (without CAR molecule) were used as controls (FIGS. 8A-8B).

Referring to FIG. 8A, CHO cells not expressing the target antigen Axl were treated with CAR-T cells and non-transduced T cells. There was no significant difference between these two treatments, indicating that CAR-T cells are not cytotoxic to CHO cells. Referring to FIG. 8B in which CHO cells expressing the target antigen Axl (CHO-63) were similarly treated, CAR-T cells having a conditionally active antibody against the target antigen Axl were CHO compared to non-transduced T cells. The -63 cell population was significantly reduced. This showed that CAR-T cells having a conditionally active antibody against the target antigen Axl were cytotoxic to CHO-63 cells.

CAR-T cells induced cytotoxicity after binding to the target antigen Axl. This effect was confirmed by measuring the levels of the cytokines interferon gamma (INFg) and IL2. Cytokine data are shown in FIGS. 9A-9B. In FIG. 9A, the binding of CAR-T cells carrying the target antigen Axl to CHO-63 cells was significant as indicated by the observed increase in cytokine levels compared to non-transduced T cells. Caused the release. Similarly, in FIG. 9B, the binding of CAR-T cells carrying the target antigen Axl to CHO-63 cells is IL2 as indicated by the observed increase in cytokine levels compared to non-transduced T cells. Caused a significant release of.

Example 4: CAR-T cells having a conditionally active scFv antibody against the target antigen RR2 A conditionally active scFv antibody against the target antigen RR2 was prepared. Their binding activity to the target antigen ROR2 was measured using an ELISA assay (essay) (FIG. 10).

A CAR molecule was constructed using one of the scFv antibodies shown in FIG. 10, scFv-116101, to generate CAR-T cells (116101 CAR-T). The constructed CAR-T cells were used to target Daudi cells expressing the target antigen ROR2. Negative controls are T cells that have not been transduced with a CAR molecule (non-transduced T cells) and CAR-T cells that have been transduced with a CAR molecule that does not have the ability to bind to the target antigen ROR2 (non-TR2 scFv CAR-T). Met. The results are shown in FIG. 11A. The ratio of the number of T cells to the number of Daudi cells in these treatments was 10: 1. CAR-T cells (116101 CAR-T) with scFv antibodies targeting the target antigen ROR2 on Daudi cells showed significant cell death of Daudi cells, as shown by the higher dead / live cell ratio in FIG. 11A. Was induced.

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 (116101 CAR-T) having an scFv antibody targeting the target antigen ROR2 have significant cell death in HEK293 cells when compared to negative controls. Was not induced (Fig. 11B).

Example 5: Cytokine release of CAR-T cells having antibodies against the target antigens Axl and RR2 of Examples 1 to 4 In this example, cytokine release induced by binding of CAR-T cells to the target antigen was measured. 12A-12B show INFg and IL2 release after CAR-T cells containing a conditionally active scFv antibody against the target antigen Axl bind to CHO-63 cells expressing the target antigen Axl. After treating CAR-T cells with these CHO-63 cells for 24 hours, there is a significant increase in cytokine levels of both INFg and IL2, and CHO cells that do not express the target antigen Axl using the same CAR-T cells. The release of both INFg and IL2 cytokines was shown compared to controls treated with. Furthermore, T cells that were not transduced with CAR molecules and CAR-T cells that did not bind to the target antigen Axl did not result in significant release of INFg and IL2 cytokines.

13A-13B show INFg and IL cytokine levels after CAR-T cells containing a conditionally active scFv antibody against target antigen ROR2 bind to large B cells and Daudi cells, both expressing target antigen ROR2. Is shown. Large B cells and Daudi cells were treated with CAR-T cells for 24 hours and then the same CAR-T cells were used to treat HEK293 cells that do not express the target antigen ROR2, compared to controls with INFg and IL2 cytokine levels. A significant increase was observed. Furthermore, there was no significant increase in cytokine levels in T cells that were not transduced with CAR molecules and CAR-T cells that did not bind to the target antigen ROR2, which failed to induce significant release of INFg and IL2 cytokines. Was shown.

Example 6: Conditionally active scFv antibody against target antigen CD22 Five conditionally active scFv antibodies against target antigen CD22 were selected. The selected conditional active scFv antibody is 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.

However, even though many features and advantages of the invention are shown in the above description along with details of the structure and function of the invention, the present disclosure is merely exemplary and details, in particular the shape and size of the elements. And in terms of placement, it should be understood that changes may be made within the principles of the invention to the maximum extent indicated by the broad general meaning of the terms relating to the appended claims. be.

Claims (23)

A chimeric antigen receptor for binding to tumor-specific target antigens,
i. At least one antigen-specific target region evolved from the parent protein or wild-type protein or its domain, under abnormal conditions in which the activity of the antigen-specific target region in analysis under normal physiological conditions deviates from normal physiological conditions. At least one antigen-specific target region, which is less active compared to its activity in the analysis.
ii. Transmembrane domain; and iii. A chimeric antigen receptor that contains an intracellular signaling domain. The chimeric antigen receptor according to claim 1, wherein the tumor-specific target antigen is selected from Axl, ROR2 and CD22. The at least one antigen-specific target region is an antibody, antibody fragment, single-stranded antibody, bivalent single-stranded antibody or diabody, ligand, acceptor-binding domain of ligand, acceptor, ligand-binding domain of receptor. , And the chimeric antigen receptor according to claim 1, which is selected from Affibody. The chimeric antigen receptor according to claim 1, wherein the tumor-specific target antigen is Axl, and at least one antigen-specific target region is a single-chain antibody having an amino acid sequence selected from SEQ ID NOs: 9 to 12. The chimeric antigen receptor according to claim 1, wherein the tumor-specific target antigen is ROR2, and at least one antigen-specific target region is a single-chain antibody having the amino acid sequence of SEQ ID NO: 15. The normal and abnormal conditions are the same conditions selected from temperature, pH, osmotic pressure, osmolality, oxidative stress, electrolyte concentration, small organic molecule concentration, inorganic molecule concentration, cell type, nutrient availability, claim. Item 1. The chimeric antigen receptor according to Item 1. The chimeric antigen receptor according to claim 1, wherein the normal physiological condition is the normal physiological pH in the plasma of a subject which is a mammal, and the abnormal condition is the pH in the tumor microenvironment. The chimeric antigen receptor according to claim 7, wherein the normal physiological pH is higher than 7.0 and is in the range up to about 7.8. The chimeric antigen receptor according to claim 8, wherein the normal physiological pH is in the range of about 7.2 to about 7.6. The chimeric antigen receptor according to claim 7, wherein the abnormal pH is in the range of 6.0 to less than 7.0. The chimeric antigen receptor according to claim 10, wherein the abnormal pH is in the range of 6.0 to about 6.8. The chimeric antigen receptor according to claim 1, further comprising an extracellular spacer domain or at least one costimulatory domain. The chimeric antigen according to claim 12, wherein the extracellular spacer domain is selected from the group consisting of an 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, and a combination thereof. Receptor. The chimeric antigen receptor according to claim 1, wherein at least one antigen-specific target region has a ratio of activity under abnormal conditions to at least about 2 to the same activity under normal physiological conditions. The chimeric antigen receptor according to claim 1, wherein at least one antigen-specific target region comprises two antigen-specific target regions linked by a linker. The chimeric antigen receptor according to claim 15, wherein the two antigen-specific target regions bind to different target antigens or different epitopes of the same target antigen. Transmembrane domains are artificial hydrophobic sequences of type I transmembrane proteins and transmembrane domains, alpha, beta, or zeta chains of T cell receptors, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, The chimeric antigen receptor according to claim 1, which is selected from the group consisting of CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. The intracellular signaling domain is the cytoplasmic signaling domain of the human CD3 zeta chain, FcyRIII, FcsRI, the cytoplasmic tail of the Fc receptor, the cytoplasmic receptor with an immunoreceptor tyrosine-based activation motif (ITAM), the TCR zeta, FcR. The chimeric antigen receptor according to claim 1, which is selected from the group consisting of gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. TNFR superfamily proteins, CD28, CD137, CD134, DappleO, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS LIGHT, NKG2C, and The chimeric antigen receptor according to claim 1, further comprising a co-stimulatory domain selected from the group consisting of the co-stimulatory domain of B7-H3. An expression vector comprising the polynucleotide sequence encoding the chimeric antigen receptor according to claim 1. Expression vectors are from lentivirus vectors, gamma retrovirus vectors, formy virus vectors, adeno-associated virus vectors, adenovirus vectors, poxvirus vectors, herpesvirus vectors, genetically engineered hybrid viruses, and transposon-mediated vectors. 20. The expression vector according to claim 20, which is selected from the group consisting of. A genetically engineered cytotoxic cell comprising the polynucleotide sequence encoding the chimeric antigen receptor according to claim 1. A method for producing a chimeric antigen receptor comprising at least one antigen-specific target region, a transmembrane domain and an intracellular signal transduction domain.
i. Evolving the DNA encoding the parent protein, wild-type protein, or its domain using one or more evolutionary techniques to create mutant DNA,
ii. Expressing mutant DNA to obtain a mutant polypeptide,
iii. The mutant polypeptide is subjected to analysis under normal physiological conditions and under abnormal conditions deviating from normal physiological conditions; and iv. From the mutant polypeptide expressed in step (ii), select at least one antigen-specific target region that exhibits reduced activity in analysis under normal physiological conditions compared to activity in analysis under abnormal conditions. By
A method comprising the step of producing at least one antigen-specific target region from a parent protein or wild-type protein or domain thereof that specifically binds to a tumor-specific target antigen.

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