JP7432250B2 - Conditionally activated chimeric antigen receptor for modified T cells - Google Patents

Description

RELATED APPLICATION DATA This application is a continuation-in-part of U.S. patent application Ser. No. 15/052,487, which in turn is a continuation-in-part of PCT/US15/47197, filed August 27, 2015, designating the United States of America. , further claims the benefit of U.S. Provisional Application No. 62/043067, filed August 28, 2014 (now expired), all of which are hereby incorporated by reference in their entirety. be done.

TECHNICAL FIELD This disclosure relates to the field of protein evolution. Specifically, the present disclosure relates to methods of generating conditionally active chimeric antigen receptors from parental or wild-type proteins. The 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 large body of literature describing the possibility of evolving various properties of proteins, especially enzymes. For example, enzymes may be evolved to be stable under different conditions, such as high temperatures. In situations where activity increases at higher temperatures, a significant portion of the increase is generally due to the higher kinetics explained by the Q10 law (which estimates that a 10°C increase in enzymes doubles turnover). This may be due to activity.

In addition, there are examples of natural mutations that destabilize a protein under its normal operating conditions. Although certain variants are active at low temperatures, the levels may be lower compared to the parent or wild-type protein. This is also typically explained by a decrease in activity as guided by Q10 or similar laws.

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

Many strategies have been published for evolving or manipulating molecules. However, while a protein is inactive or virtually inactive (less than 10% active, preferably less than 1% active) under wild-type operating conditions, its corresponding parent or wild-type protein under conditions other than wild-type operating conditions In order to engineer or evolve the type protein to maintain an activity equal to or better than that of the type protein, the destabilizing mutation does not counteract the destabilizing effect and the activity-increasing mutation. It is necessary to coexist with It is expected that destabilization will reduce the activity of the protein beyond the effect predicted by standard laws such as Q10. Therefore, if it were possible to evolve proteins that are inactive under normal operating conditions for the corresponding parent or wild-type protein, but which work efficiently at low temperatures, for example, an unexpected new class of proteins could emerge. produced.

Chimeric antigen receptors (CARs) have been used in the treatment of cancer. U.S. Patent Application Publication No. 2013/0280220 discloses methods and compositions for providing improved cells encoding chimeric antigen receptors specific for two or more antigens, including tumor antigens. . Cells expressing chimeric antigen receptors can be used for cell therapy. Although such cell therapy may be suitable for any medical condition, a specific embodiment is cell therapy of cancer, including cancers involving solid tumors.

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

Throughout this application, various publications are referenced by author and date. The disclosures of these publications are herein incorporated by reference in their entirety to more fully describe the state of the art as known to those skilled in the art as of the date of the disclosure described and claimed herein. Incorporated into this application by reference.

1 shows a schematic diagram of a chimeric antigen receptor in one embodiment of the present invention. ASTR is the antigen-specific targeting region, L is the linker, ESD is the extracellular spacer domain, TM is the transmembrane domain, CSD is the costimulatory domain, and ISD is the intracellular signaling domain. be.

It is shown that whether the conditionally activated antibodies of Example 1 are expressed as divalent or monovalent antibodies, there is no significant change in the selectivity of these antibodies at pH below 6.0 and above pH 7.4. It is shown that whether the conditionally activated antibodies of Example 1 are expressed as divalent or monovalent antibodies, there is no significant change in the selectivity of these antibodies at pH below 6.0 and above pH 7.4.

1 is a size exclusion chromatography profile showing that the conditionally activated antibody of Example 2 does not aggregate.

Figure 2 shows the on and off kinetics of the conditionally activated antibody of Example 2 as measured by surface plasmon resonance (SPR) analysis.

AB show selectivity of conditionally active antibodies as measured by SPR analysis of Example 2.

It is shown that CAR-T cells had no effect on the population of CHO cells that do not express the CAR-T cell target antigen X1. The CAR molecules in the CAR-T cells of this example contained an antibody against target antigen X1, but this antibody was not conditionally active (Comparative Example A).

CAR-T cells are shown to reduce the population of CHO-63 cells expressing CAR-T cell target antigen X1. These CAR-T cells are the same cells used to generate the data shown in Figure 7A (Comparative Example A).

This shows that CAR-T cells had no effect on the population of CHO cells that do not express the CAR-T cell target antigen X1. The CAR molecules in the CAR-T cells of this Example 3 contained conditionally activated antibodies against target antigen X1.

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

AB show cytokine release induced by CAR-T cell binding to target antigen X1, as described in Example 3.

A conditionally active antibody against target antigen X2 is shown.

A-B shows the cytotoxic effect induced by binding of CAR-T cells to Daudi cells expressing target antigen X2 and CAR- on HEK293 cells not expressing target antigen X2, as described in Example 4. Figure 3 shows the cytotoxic effect induced by T cells.

AB show cytokine release induced by CAR-T cell binding to target antigen X1, as described in Example 5.

AB show cytokine release induced by CAR-T cell binding to target antigen X2, as described in Example 5.

A conditionally activated antibody against target antigen X3 suitable for the construction of CAR-T cells is shown.

In one aspect, the invention provides chimeric antigen receptors (CARs) for binding tumor-specific target antigens. A chimeric antigen receptor contains at least one antigen-specific target region evolved from a parent protein or domain thereof. CAR further contains a transmembrane domain and an intracellular signaling domain. At least one antigen-specific target region has reduced activity when assayed under normal physiological conditions as compared to activity when assayed under abnormal conditions.

In another aspect, the invention provides an expression vector comprising a polynucleotide sequence encoding a chimeric antigen receptor of the invention. The expression vectors include lentivirus vectors, gamma retrovirus vectors, foamy virus vectors, adeno-associated virus vectors, adenovirus vectors, poxvirus vectors, herpesvirus vectors, genetically engineered hybrid viruses, and transposon-mediated vectors. selected from.

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

In yet another aspect, the invention provides a pharmaceutical composition comprising a chimeric antigen receptor, an expression vector, and/or a genetically engineered cytotoxic cell of the invention, and a pharmaceutically acceptable excipient. provide.

In yet another aspect, the invention provides a method of producing a chimeric antigen receptor that includes at least one antigen-specific targeting region, a transmembrane domain, and an intracellular signaling domain. The method includes generating at least one antigen-specific target region from a parent protein or domain thereof that specifically binds a tumor-specific target antigen. These include (i) evolving DNA encoding the parent or wild-type protein or its domains using one or more evolution techniques to create mutant DNA; (ii) expressing the mutant DNA; (iii) subjecting the mutant polypeptide to analysis under normal physiological conditions and analysis under abnormal conditions; and (iv) the mutant expressed in step (iii). Selecting from the polypeptide at least one antigen-specific target region that exhibits decreased activity when assayed under normal physiological conditions compared to activity when assayed under abnormal conditions.

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

As used herein in connection with a measurand, the term "about" means the measurand that would be expected by a person skilled in the art making the measurement and exercising a level of care and precision of the measuring equipment used commensurate with the purpose of the measurement. associated with normal fluctuations in Unless otherwise specified, "about" refers to a +/-10% variation of the given value.

The term "agent" refers to a chemical compound, a mixture of chemical compounds, a spatially localized array of compounds (e.g., a VLSIPS peptide sequence, a polynucleotide sequence, and/or a combinatorial small molecule array), a biological macromolecule, a bacteriophage. Peptide display libraries, bacteriophage antibody (e.g. scFV) display libraries, polysomal peptide display libraries, or extracts made from biological materials such as bacteria, plants, fungi or animal (especially mammalian) cells or tissues. used to indicate Agents are evaluated for potential enzymatic activity as conditionally active biological therapeutic enzymes by inclusion in the screening assays described herein below. Agents are evaluated for potential activity as conditionally active biological therapeutic enzymes by inclusion in the screening assays described herein below.

The term "amino acid" as used herein is any organic compound containing an amino group ( ^-NH2 ) and a carboxyl group (-COOH), preferably after condensation as a free group or as part of a peptide. Combine either. The "20 naturally encoded polypeptides forming alpha-amino acids" are known in the art and include alanine (ala or A), arginine (arg or R), asparagine (asn or N), aspartic acid (asp or D), cysteine (cys or C), glutamic acid (glu or G), histidine (his or H), isoleucine (ile or I), leucine (leu or L), lysine (lys or K), methionine ( met or M), phenylalanine (phe or F), proline (Pro or P), serine (ser or S), threonine (thr or T), tryptophan (tip or W), tyrosine (tyr or Y) and valine (val or V).

The term "amplification" as used herein means that the number of copies of a polynucleotide is increased.

The term "antibody" as used herein refers to intact immunoglobulin molecules as well as immunoglobulin molecules capable of binding an epitope of an antigen, such as Fab, Fab', (Fab')2, Fv, and SCA fragments. Refers to a fragment of a globulin molecule. These antibody fragments, which retain some ability to selectively bind to the antigen (e.g., a polypeptide antigen) of the antibody from which they are derived, can be produced using methods well known in the art. (see, eg, Harlow and Lane, supra) and is further explained as follows. Antibodies can be used to isolate preparative amounts of antigen by immunoaffinity chromatography. Various other uses of such antibodies include the diagnosis and/or staging of diseases (e.g. neoplasia) and the treatment of diseases such as, e.g. neoplasia, autoimmune diseases, AIDS, cardiovascular diseases, infectious diseases, etc. It is a therapeutic application for Chimeric, human-like, humanized 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 produced by digesting whole antibody molecules with the enzyme papain to generate fragments consisting of an intact light chain and a portion of the heavy chain.

Fab' fragments of antibody molecules can be obtained by treating whole antibody molecules with pepsin followed by reduction to yield molecules consisting of an intact light chain and a portion of the heavy chain. Two Fab' fragments are obtained per antibody molecule treated in this way.

(Fab')2 fragments of antibodies can be obtained by treating whole antibody molecules with the enzyme pepsin without subsequent reduction. (Fab')2 fragments are dimers of two Fab' fragments held together by two disulfide bonds.

Fv fragments are defined as genetically engineered fragments that include a light chain variable region and a heavy chain variable region expressed as two chains.

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 immunocompetent cells, or both. Those skilled in the art will appreciate that any macromolecule can serve as an antigen, including virtually any protein or peptide. Additionally, the antigen can be derived from recombinant or genomic DNA. Those skilled in the art will therefore understand that any DNA that contains a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an immune response encodes an "antigen" as that term is used herein. Probably. Furthermore, those skilled in the art will understand that antigens need not be limited to being encoded by the full-length nucleotide sequence of a gene. The invention includes, but is not limited to, the use of partial nucleotide sequences of two or more genes, and these nucleotide sequences can easily be arranged in various combinations to elicit the desired immune response. It is clear that Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that antigens may be produced, synthesized, or derived from biological samples. Such biological samples can 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 invention.

The term "autoimmune disease" as used herein is defined as a disorder resulting from an autoimmune response. Autoimmune diseases are the result of inappropriate and exaggerated responses to self-antigens. Examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia greata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes mellitus (1), among others. epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, muscle gravis Asthenia, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, and ulcerative colitis are listed. It will be done.

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

The term "B cell related disease" as used herein refers to B cell immunodeficiency, B cell related autoimmune disease and/or excessive/uncontrolled cell proliferation (including lymphoma and/or leukemia). )including. Examples of such diseases for which the bispecific CARs of the present invention may be used in therapeutic approaches include, but are not limited to, systemic lupus erythematosus (SLE), diabetes, rheumatoid arthritis (RA), reactive arthritis, multiple sclerosis ( MS), pemphigus vulgaris, celiac disease, Crohn's disease, inflammatory bowel disease, ulcerative colitis, autoimmune thyroid disease, X-linked agammaglobulinaemia, pre-B acute lymphoblasts sexual leukemia, systemic lupus erythematosus, unclassifiable immunodeficiency, chronic lymphocytic leukemia, diseases associated with selective IgA deficiency and/or IgG subclass deficiency, B-lineage lymphoma (Hodgkin lymphoma and/or non-Hodgkin lymphoma), Immunodeficiency with thymoma, transient hypogammaglobulinemia and/or hyperIgM syndromes, and virus-mediated B-cell diseases, such as EBV-mediated lymphoproliferative disease, and B-cells are involved in the pathophysiology Chronic infections include.

The term "blood-brain barrier" or "BBB" refers to the tight barrier within the brain capillary endothelial cell membranes that creates a tight barrier that restricts the transport of molecules to the brain, even extremely small molecules such as urea (60 daltons). Refers to the physiological barrier between the peripheral circulation and the brain and spinal cord formed by junctions. The blood-brain barrier in the brain, the blood-spinal 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-cerebrospinal fluid barrier (choroid plexus), which contains ependymal cells rather than capillary endothelial cells.

The terms "cancer" and "cancerous" as used herein refer to or describe the physiological condition in mammals that is typically characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, B-cell lymphoma (Hodgkin's lymphoma and/or non-Hodgkin's lymphoma), brain cancer, 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, carcinoma, melanoma, head and neck cancer, brain cancer, and prostate cancer, including but not limited to androgen-dependent prostate cancer and androgen-independent prostate cancer. It will be done.

The term "chimeric antigen receptor" or "CAR" or "CARs" as used herein refers to engineered receptors that graft antigen specificity onto cytotoxic cells, such as T cells, NK cells and macrophages. Point to the body. The CAR of the present invention comprises at least one antigen-specific targeting region (ASTR), an extracellular spacer domain (ESD), a transmembrane domain (TM), one or more costimulatory domains (CSD), and an intracellular signaling domain. domain (ISD). In some embodiments, ESD and/or CSD are optional. In another embodiment, the CAR is a bispecific CAR that is specific for two different antigens or epitopes. When ASTR specifically binds to a target antigen, ISD activates intracellular signaling. For example, ISDs can take advantage of the antigen binding properties of antibodies to redirect T cell specificity and reactivity to targets of choice in a manner free from MHC restrictions. Due to MHC-free antigen recognition, CAR-expressing T cells gain antigen recognition capabilities that are independent of antigen processing, thus circumventing major tumor escape mechanisms. Furthermore, when expressed in T cells, CAR advantageously does not dimerize with endogenous T cell receptor (TCR) α and β chains.

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

The term "homologous" as used herein refers to gene sequences that are evolutionary and functional between species. For example, but not limited to, in human genes, the human CD4 gene is a homologous gene to the mouse 3d4 gene, and the sequences and structures of these two genes show high homology, and both genes conform to MHC class II. Encodes a protein that functions in signaling T cell activation through restricted antigen recognition.

The term "conditionally active biological protein" refers to a variant, or mutant, 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 regions of the body and/or exhibits increased or decreased activity under abnormal or permissive physiological conditions. The term "normal physiological conditions" as used herein refers to temperature, pH, osmolality, oxidative stress, and other conditions that may be considered within normal ranges for a subject at the site of administration or in a tissue or organ at the site of action. , electrolyte concentrations, concentrations of small organic molecules such as glucose, lactate, pyruvate, nutrients, other metabolites, and concentrations of other molecules such as oxygen, carbonate, phosphate, and carbon dioxide. , cell type, and nutrient availability.

In one embodiment, the normal physiological condition is a normal physiological pH in the plasma of a mammalian subject, greater than 7.0 and up to about 7.8, or from about 7.2 to about 7.8, or about 7.0. 2 to about 7.6, or from about 7.3 to about 7.6, or from 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 6.8, or within the range of 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 normally accepted range for the condition in question. In one embodiment, a conditionally active biological protein is substantially inactive under normal physiological conditions, but active under abnormal conditions at a level equal to or better than the parent or wild-type protein from which it is derived. It is. For example, in one embodiment, an evolved conditionally active biological protein is effectively inactive at body temperature, but active at lower temperatures. In another embodiment, a conditionally active biological protein is reversibly or irreversibly inactivated under normal physiological conditions. In further embodiments, the parent or wild type protein is a therapeutic protein. In another embodiment, the conditionally 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, such as after passage through the lungs.

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

The term "corresponding" as used herein means that a polynucleotide sequence is homologous (i.e., identical without being strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence; , means that the polynucleotide sequence is identical to the reference polynucleotide sequence. In contrast, the term "complementary" as used herein means that a complementary sequence is homologous to all or a portion of a 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 an antigen-presenting cell (e.g., dendritic cell, B cell, etc.) that specifically targets a cognate costimulatory molecule on a T cell. In addition to the primary signals provided by the binding of the TCR/CD3 complex to, e.g., peptide-carrying MHC molecules, T cell responses including, but not limited to, proliferation, activation, differentiation, etc. contains molecules that provide signals that mediate Co-stimulatory ligands include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L). ), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin β receptor, 3/TR6, ILT3, ILT4, HVEM, Toll ligand receptor Agonists or antibodies and ligands that specifically bind B7-H3 may be included. Co-stimulatory ligands also include, but are not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7. Also included are antibodies that specifically bind costimulatory molecules present on T cells, such as ligands that specifically bind to , LIGHT, NKG2C, B7-H3, and CD83.

The term "co-stimulatory molecule" as used herein refers to a compound on a T cell that specifically binds a costimulatory ligand and thereby mediates a costimulatory response by the T cell, such as, 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.

The term "co-stimulatory signal" as used herein refers to a signal that, in combination with a primary signal such as TCR/CD3 ligation, results in T cell proliferation and/or up- or down-regulation of key molecules.

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

Cytotoxic cells also include, but are not limited to, natural killer T cells (Heczey et al., “Invariant NKT cells with chimeric antigen receptors provide a novel platform for safe and Effective cancer immunotherapy, “Blood, vol. 124, pp. 2824-2833, 2014) and granulocytes, and granulocytes, may also be included. Additionally, cytotoxic cells include, but are not limited to, macrophages and granulocytes, cells with stem cell and/or progenitor cell properties, such as, but not limited to, hematopoietic stem/progenitor cells (Zhen et al., “HIV -specific Immunity Derived From Chimeric Antigen Receptor-engineered Stem Cells, "Mol Ther., vol. 23, pp. 1358-1367, 2015), embryonic stem cells (E SC), cord blood stem cells, and induced pluripotent stem cells (iPSC) ) (Themeli et al., “New cell sources for T cell engineering and adaptive immunotherapy,” Cell Stem Cell., vol. 16, pp. 357-366, 201 Contains immune cells with phagocytic ability, including 5) obtain. In addition, cytotoxic cells include iPSC-derived T cells (TiPSCs) (Themeli et al., “Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells. s for cancer therapy,” Nat Biotechnol., vol. 31, pp. 928-933, 2013) or “synthetic cells” such as iPSC-derived NK cells.

The term "degradation effective" amount refers to the amount of enzyme required to process at least 50% of the substrate when compared to not contacting the substrate with the enzyme.

The term "directional ligation" refers to a ligation in which the 5' and 3' ends of a polynucleotide are sufficiently different to specify the preferred ligation direction. For example, when an otherwise untreated and undigested PCR product with two blunt ends is typically ligated into a cloning vector that has been digested to produce blunt ends at its multiple cloning site, the preferred ligation It has no direction; therefore, directional ligation is typically not indicated under such circumstances. In contrast, typically when a digested PCR product with 5'EcoR I treated ends and 3'BamH I is ligated into a cloning vector that has multiple cloning sites that are digested with EcoR I and BamH I, the direct National ligation is shown.

The term "disease targeted by genetically modified cytotoxic cells" as used herein refers to diseases in which the genetically modified cells target diseased cells to produce therapeutically beneficial results; It encompasses targeting by the genetically modified cells of the invention any cell that is involved in any disease in any way, whether targeting healthy cells or otherwise. Genetically modified cells include, but are not limited to, genetically modified T cells, NK cells, and macrophages. Genetically modified cells express a CAR of the invention, and the CAR can target any antigen 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 carcinomas, sarcomas, lymphomas, leukemias, germ cell tumors, and blastomas; and antigens expressed on various immune cells. and antigens expressed on cells associated with various hematological, autoimmune, and/or inflammatory diseases. Other antigens that can be targeted will be apparent to those skilled in the art and can be targeted by the CARs of the invention in connection with alternative embodiments of the invention.

The term "genetically modified cell", "redirect cell", "genetically engineered cell" or "modified cell" as used herein refers to a cell that expresses a CAR of the invention.

The term "DNA shuffling" is used herein to refer to recombination between substantially homologous, but not identical, sequences; in some embodiments, DNA shuffling includes cer/lox and / or may include crossover via non-homologous recombination, such as via the flp/frt system.

The term "drug" or "drug molecule" refers to a therapeutic agent comprising a substance that has a beneficial effect on the human or animal body when administered to the human or animal body. Preferably, the medicament treats, cures or alleviates one or more symptoms, diseases or abnormal conditions in the human or animal body, or promotes the health of the human or animal body. It is something that can be done.

An "effective amount" is a condition that is effective to treat or prevent a condition in the living organism of the person to whom it is administered over a period of time, e.g., to provide a therapeutic effect during the desired period of administration. The amount of biologically active biological protein or fragment.

The term "electrolyte" is used herein to define minerals within blood or other body fluids that transport electrical charge. For example, in one embodiment, the normal physiological condition and the abnormal condition may be an "electrolyte concentration" condition. In one embodiment, the electrolyte concentration tested is selected from one or more of ionized calcium, sodium, potassium, magnesium, chloride, bicarbonate, and phosphate concentrations. For example, in one embodiment, the normal range for serum calcium is 8.5-10.2 mg/dL. In this embodiment, the abnormal serum calcium concentration may be selected from above or below the normal range. In another example, in one aspect, the serum chloride is between 96 and 106 milliequivalents per liter (mEq/L). In this embodiment, the abnormal serum chloride concentration may be selected from above or below the normal range. In another example, in one aspect, 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 normal range. In another example, in one aspect, the normal 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 normal range. In another example, in one aspect, the normal range of serum or blood sodium 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 example, in one aspect, the normal range for 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 further embodiments, the normal 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 normal range. In different embodiments, bicarbonate levels may be used to indicate the normal level of acidity (pH) in the blood. The term "electrolyte concentration" may also be used to define specific electrolyte concentrations in tissues or body fluids other than blood or plasma. In this case, normal physiological conditions are considered to be the clinically normal range for that tissue or body fluid. In this embodiment, abnormal tissue or body fluid electrolyte concentrations may be selected from above or below the normal range.

The term "antigenic determinant" as used herein refers to an antigenic determinant on an antigen, such as an enzymatic polypeptide. The antigen-binding site of an antibody such as an enzyme-specific antibody then binds. Antigenic determinants usually consist of chemical surface active moieties of molecules such as amino acids or side chains, and may also have specific three-dimensional structural properties, specific charge characteristics. As used herein, an "antigenic determinant" refers to that portion of an antigen or other macromolecule capable of forming a binding interaction with the variable region that binds the body of an antibody. means. Typically, such binding interactions are manifested as intermolecular interactions with amino acid residues of one or more CDRs.

As used herein, the term "evolution" or "evolving" refers to the use of one or more mutagenesis methods to generate a new polynucleotide that encodes a new polypeptide. , this new 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 conditionally active biological proteins from parent or wild-type proteins. In one aspect, for example, evolution relates to methods of performing both non-stochastic polynucleotide chimerization and non-stochastic site-directed point mutagenesis as disclosed in US Patent Application Publication No. 2009/0130718. More particularly, the present disclosure provides that an antigen-specific target region of a parent or wild-type enzyme exhibits reduced activity compared to the parent or wild-type enzyme parent molecule under normal physiological conditions, but under one or more abnormal conditions. A method is provided for evolving a conditionally active biological enzyme that exhibits enhanced activity compared to a biological enzyme.

The terms "fragment," "derivative," and "analogue," when referring to a reference polypeptide, have a polypeptide that retains at least one, at least essentially the same physiological function or activity as the reference polypeptide. Additionally, the terms "fragment," "derivative," and "analogous organ" are exemplified by "preform" molecules such as low-activity precursor proteins that can be modified by cleavage to produce mature enzymes with significantly higher activity. Ru.

The term "gene" as used herein refers to a DNA segment that is involved in the production of a polypeptide chain and includes intervening sequences (nitrons) between individual coding segments (exons) as well as coding regions (leaders and traders). includes the areas preceding and following.

The term "heterologous," as used herein, means that a single-stranded nucleic acid sequence cannot hybridize to other single-stranded nucleic acid sequences or their complementary sequences. Thus, a heterologous portion means that a polynucleotide or polynucleotide has a portion or region in its sequence that cannot hybridize to other nucleic acids or polynucleotides. Such a region or portion is, for example, a portion of the mutation.

The term "homologous" or "homologous" as used herein means that a single-stranded nucleic acid sequence is capable of hybridizing to a complementary single-stranded nucleic acid sequence. The degree of hybridization may depend on a number of factors, including the amount of identity between the sequences and hybridization conditions such as temperature and salt concentration, as discussed below. Preferably, the identical region is larger than about 5 bp, and even more preferably, the identical region is larger than 10 bp.

The benefits of this disclosure extend to "industrial applications" (or industrial processes), and the term is used in appropriate commercial applications, as well as in non-commercial industrial applications (e.g., formal medical research in non-profit institutions). used to include industrial (or simply industrial) applications. Relevant applications include the fields of diagnostics, medicine, agriculture, manufacturing and academia.

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

The term "immune response" as used herein includes, but is not limited to, innate immunity, humoral immunity, cell-mediated immunity, immunity, inflammatory response, acquired (adaptive) immunity, autoimmunity and/or hyperreactivity. 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 occurs naturally). For example, a naturally occurring polynucleotide or enzyme in a living animal is not isolated, but the same polynucleotide or enzyme that is separated from some or all of the materials with which it coexists in a natural system is isolated. has been done. Such polynucleotides may be part of a vector, and/or such polynucleotides or enzymes may be part of a composition, provided that such a vector or composition is not part of its natural environment. has also been isolated.

As used herein, the term "isolated nucleic acid" is used to define a nucleic acid, eg, a DNA or RNA molecule. A nucleic acid, such as a DNA or RNA molecule, is not directly adjacent to the 5' and 3' flanking sequences that normally would be immediately adjacent when present in the naturally occurring gene of the organism from which it is derived. The term thus includes, for example, a plasmid or a viral vector, a nucleic acid integrated into a gene of a foreign cell (or a gene of a foreign cell, but at a site different from that in nature), and, for example, by PCR amplification or restriction. Refers to a nucleic acid that is incorporated into a vector, such as a DNA fragment created by enzymatic digestion, or a nucleic acid that exists as a separate molecule, such as an RNA molecule created by in vitro transcription. The term also refers to recombinant nucleic acids that form part of hybrid genes encoding additional proteins that can be used, for example, in the production of fusion proteins.

The term "lentivirus" as used herein refers to the genus of the family Retroviridae. Lentiviruses are unique among retroviruses in their ability to infect nondividing cells; lentiviruses can deliver large amounts of genetic information into the host cell's DNA, and therefore lentiviruses are the most efficient This is one of the most popular gene delivery vector delivery methods. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses provide a means to achieve high levels of gene transfer in vivo.

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

The term "ligation" as used herein refers to the process of forming phosphodiester bonds between two double-stranded nucleic acid fragments (Sambrook et al., (1982). Molecular Cloning: A Laboratory Manual .Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., p.146; Sambrook et al., Molecular Cloning: a laboratory manual, ^2nd Ed., Cold Spring Harbor Laboratory Press, 1989). Unless otherwise provided, ligation may be accomplished using known buffers and conditions, such as 10 units of T4 DNA ligase ("ligase") per approximately equimolar amount of DNA fragments of 0.5 micrograms to be ligated.

The term "linker" or "spacer" as used herein is a molecule or group of molecules that connects two molecules, e.g. a DNA binding protein and a random peptide, e.g. Refers to a molecule or group of molecules that serves to place two molecules in a preferred configuration so that they can bind to a receptor with minimal steric hindrance. "Linker" (L) or "linker domain" or "linker region" as used herein refers to an oligonucleotide of about 1-100 amino acids in length that connects together any domain/region of a CAR of the invention. Refers to a peptide or polypeptide region. Linkers may be composed of flexible residues such as glycine and serine so that adjacent protein domains can move freely relative to each other. Longer linkers may be used if it is desired that two adjacent domains do not sterically interfere with each other. Linkers may be cleavable or non-cleavable. Examples of cleavable linkers include 2A linkers (eg, T2A), 2A-like linkers or functional equivalents thereof, and combinations thereof. In some embodiments, the linker includes a Picornavirus 2A-like linker, the CHYSEL sequence of Porcine Teschovirus (P2A), Thosea asigna virus (T2A), or combinations, variants and functional equivalents thereof. Things can be mentioned. Other linkers will be apparent to those skilled in the art and may be used in conjunction with alternative embodiments of the invention.

The term "mammalian cell surface display" as used herein refers to the use of proteins or antibodies for screening purposes (e.g., by screening for specific antigen binding by a combination of magnetic beads and fluorescence-activated cell sorting). , or a technique in which a portion of an antibody is expressed and displayed on the surface of a mammalian host cell. In one aspect, DuBridge et al. , US Patent Application Publication No. 2009/0136950, mammalian expression vectors are used to simultaneously express immunoglobulins as both secreted and cell surface bound forms. In another aspect, this technique is described by Gao et al. , US Patent Application Publication No. 2007/0111260, it is used to screen viral vectors encoding libraries of antibodies or antibody fragments that, when expressed in cells, are displayed on the cell membrane. Total IgG surface display on mammalian cells is known. For example, Akamatsuu et al. developed a mammalian cell surface display vector suitable for directly isolating IgG molecules based on their antigen binding affinity and biological activity. Antibody libraries were displayed as whole IgG molecules on the cell surface using Epstein-Barr virus-derived episomal vectors and screened for specific antigen binding by a combination of magnetic beads and fluorescence-activated cell sorting. Plasmids encoding antibodies with the desired binding properties were recovered from the sorted cells and converted into a form suitable for the production of soluble IgG. Akamatsuu et al. J. Immunol. Methods, vol. 327, pages 40-52, 2007. Ho et al. used human embryonic kidney 293T cells, which are widely used for transient protein expression, for cell surface display of single chain Fv antibodies for affinity maturation. From a large excess of cells expressing WT antibodies with slightly lower affinity, cells expressing rare variant antibodies with higher affinity were enriched 240-fold by single-pass cell sorting. Moreover, highly enriched variants with increased binding affinity for CD22 were obtained after a single selection of the combinatorial library randomizing unique antibody hotspots. Ho et al. , “Isolation of anti-CD22 Fv with high affinity by Fv display on human cells,” Proc Natl Acad Sci USA, vol. 103, pages 9637-9642, 2006.

B cells specific for the antigen can also be used. Such B cells can be isolated directly from peripheral blood mononuclear cells (PBMC) of human donors. A recombinant antigen-specific single chain Fv (scFv) library is generated from this B cell pool and screened by mammalian cell surface display by using the Sindbis virus expression system. Heavy chain (HC) and light chain (LC) variable regions (VR) can be isolated from positive clones to produce recombinant fully human antibodies as whole IgG or Fab fragments. In this way, several hypermutated high affinity antibodies that bind the model viral antigen Qβ virus-like particles (VLPs) as well as antibodies specific for nicotine can be isolated. Beerli et al. , “Isolation of human monoclonal antibodies by mammalian 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 and are described, for example, in Kondo and Ueda, “Yeast cell-surface display-applications of molecular display,” Appl. Microbiol. Biotechnol. , vol. 64, pages 28-40, 2004, which describes, for example, a cell surface engineering system using the yeast Saccharomyces cerevisiae. Yeast S. Some representative display systems for expression in S. cerevisiae are described by Lee et al, “Microbial cell-surface display,” TRENDS in Bitechnol. , vol. 21, pages 45-52, 2003. Also, see Boder and Wittrup, “Yeast surface display for screening combinatorial polypeptide libraries,” Nature Biotechnol. , vol. 15, pages 553, 1997.

The term "manufacture," as used herein, refers to at least a quantity sufficient to permit Phase I clinical trials of a therapeutic protein, or a quantity sufficient for regulatory approval of a diagnostic protein. Refers to the production of proteins.

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

As used herein, the term "molecular property to evolve" includes molecules that include polynucleotide sequences, molecules that include polypeptide sequences, and molecules that include partially polynucleotide sequences and partially include polypeptide sequences. Contains references to molecules containing. Particularly relevant (but by no means limiting) examples of molecular properties to evolve include temperature; salinity; osmolarity; pH; oxidative stress; and glycerol concentration, DMSO concentration, surfactant concentration, and/or or protein activity under specific conditions, such as with respect to any other molecular species contacted in the reaction environment. Further particularly relevant (but by no means limiting) examples of molecular properties to evolve include: stability - for example, the presence of residues after exposure to a particular environment for a particular time, such as may be encountered during storage; Includes quantities of molecular properties.

The term "mutation" as used herein refers to a change in the sequence of a wild-type nucleic acid sequence or a change in the sequence of a peptide. Such mutations may be point mutations such as transpositions or transversions. Mutations can be deletions, insertions or duplications.

The term "multispecific antibody" as used herein is an antibody that has 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). Engineered antibodies may bind 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 two antigens. Multispecific antibodies can also be multifunctional.

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

The term "naturally occurring," as used herein, is applied in reference to the fact that a subject is found in nature. For example, a polypeptide or polynucleotide sequence that occurs in an organism (including a virus) that can be isolated from its source in nature and that has not been intentionally modified by humans in a laboratory is naturally occurring. Generally, the term "naturally occurring" refers to subjects as present in non-pathological (not diseased) individuals, as is typical of the species.

As used herein, "normal physiological conditions" or "wild-type operating conditions" refer to temperature, pH, osmolality, and concentration, oxidative stress and electrolyte concentration conditions.

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

In addition, a "nucleic acid molecule" may include in part one or more non-nucleotide-based components, as exemplified by, but not limited to, amino acids and saccharides. Thus, by way of example and without limitation, ribozymes that are partially nucleotide-based and partially protein-based are considered "nucleic acid molecules."

As used herein, the term "nucleic acid sequence encoding" or "DNA coding sequence for" or "nucleotide sequence encoding" refers to Refers to a DNA sequence that is transcribed and translated into an enzyme. A "promoter" is a DNA regulatory region that has the ability to bind RNA polymerase and initiate transcription of a downstream (3' direction) coding sequence in a cell. A promoter is part of a DNA sequence. This sequence region has an initiation codon at its 3' end. A promoter sequence contains bases that are the minimum number of elements necessary to initiate transcription at a detectable level above background. However, after RNA polymerase binds to the sequence and begins transcription at the start codon (the 3' end with the promoter), transcription proceeds downstream in the 3' direction. Within the promoter sequence is found a transcription initiation site (conveniently defined by mapping with nuclease S1) as well as protein binding domains (consensus sequences) responsible for binding of RNA polymerase.

The term "oligonucleotide" (or, synonymously, "oligo") refers to either a single-stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands that can be chemically synthesized. Such synthetic oligonucleotides may or may not have a 5' phosphate. Those that do not have ligation to another oligonucleotide without the addition of phosphate by ATP in the presence of a kinase. Synthetic oligonucleotides are ligated to the non-dephosphorylated fragment.

As used herein, the term "operably linked" refers to the association of polynucleotide elements in a functional relationship. Nucleic acids are "operably linked" when placed into a functional relationship with other nucleic acid sequences. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operable linkage means that the DNA sequences being joined are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame. means.

When RNA polymerase transcribes two coding sequences in a single mRNA, the coding sequences are "operably linked" to the other coding sequence, creating a single polypeptide with 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 ultimately processed to create 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 parental set, in which case the terms "parental", "priming" and "template" refer to Compatible.

The term "patient" or "subject" refers to an animal, eg, a mammal, such as a human, who is the object of treatment. The subject or patient can be male or female.

As used herein, the term "physiological conditions" refers to the temperature, pH, osmotic pressure, ionic strength that is compatible with a living organism and/or that typically exists intracellularly in a living cultured yeast cell or mammalian cell. , viscosity, and similar biochemical parameters. For example, the intracellular conditions of yeast cells growing under typical laboratory culture conditions are physiological conditions. In vitro reaction conditions suitable for in vitro transcription cocktails are generally physiological conditions. Generally, in vitro physiological conditions include 50-200mM NaCl or KCl, pH 6.5-8.5, 20-45°C and 0.001-10mM divalent cations (e.g. Mg ^++" , Ca ⁺⁺ ); Preferably, it contains about 150mM NaCl or KCl, pH 7.2-7.6, 5mM divalent cations, and often 0.01-1.0% non-specific proteins such as bovine serum albumin (BSA). )).Nonionic surfactants (Tween, NP-40, Triton ). The detailed aqueous conditions can be selected by the practitioner using conventional methods. As a general guideline, the following buffered aqueous conditions can be applied: 10-250 mM NaCl, 5 ~50mM Tris HCl, pH 5-8, optionally with addition of 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, osmolality, osmolarity, oxidative stress, and electrolyte concentration at the site of administration or action within the body of a patient or subject that can be considered within the normal range for the patient.

The standard convention (5'-3') is used herein to describe the sequence of double-stranded polynucleotides.

The term "population" refers to a collection of compositions such as polynucleotides, portions of polynucleotides, or proteins. A "mixed population" is a collection of compositions of nucleic acids or proteins that are of the same genus (i.e., related) but differ in their sequence (i.e., are not identical) and therefore differ in their physiological and intellectual activity. be.

A molecule with a "surrogate form" is one in which one or more covalent and non-covalent bonds are added to obtain a more mature molecular form with different properties, e.g. Any combination of bond chemical modifications (e.g., glycosylation, proteolytic cleavage, dimerization or oligomerization, temperature-induced or pH-induced conformational changes, association with cofactors, etc.) means a molecule that has passed through. A reference precursor molecule is used when two or more chemical modifications (e.g. two proteolytic cleavages, or a proteolytic cleavage and a non-glycosylation) can be distinguished en route to the production of the mature molecule. are referred to as "precursor surrogate" molecules.

As used herein, the term "receptor" refers to a molecule that has affinity for a given ligand. Receptors can be naturally occurring or synthetic molecules. The receptor may be used in its unmodified state or as an aggregate with other species. A receptor can be bound covalently or non-covalently to a binding member directly or via a specific binding agent. Examples of receptors include, but are not limited to, antibodies and antisera, including monoclonal antibodies that exhibit reactivity with specific antigenic determinants (such as those on viruses, cells, or other materials); cell membrane receptors; , complex carbohydrates and glycoproteins, enzymes, and hormone receptors.

The term "deductive reassortment" as used herein refers to an increase in molecular diversity that occurs through deletion (and/or insertion) events mediated by repetitive sequences.

The term "restriction site" as used herein refers to the recognition sequence necessary for expression of the action of a restriction enzyme, and includes the site of catalytic cleavage. It is recognized that the site of cleavage may or may not be included in a portion of the restriction site that consists of low ambiguity sequences (ie, sequences that contain the major determinants of the frequency of occurrence of the restriction site). When an enzyme (eg, a restriction enzyme) "cleaves" a polynucleotide, it is understood to mean that the restriction enzyme catalyzes or facilitates the cleavage of the polynucleotide.

As used herein, the term "single chain antibody" refers to a polypeptide comprising a VH domain and a VL domain in a polypeptide chain, generally linked via a spacer peptide, which includes an amino-terminal and a VL domain. /or may contain additional amino acid sequences at the carboxy terminus. For example, a single chain antibody can include a tethering segment for linking to the encoding polynucleotide. As an example, a scFv is a single chain antibody. One -stranded antibody is generally coded by the gene of the immunoglobulin super family (for example, the IMMUNOGLOBUBULIN GENE SUPERFAMILY, A.Williams And a. Barclay, IIN IMUNOG. LOBULIN GENES, T.HONJO, F W. Alt, and THE. Rabbits, eds., (1989) Academic press: San Diego, Calif., pp. 361-368), most often rodents, non-human primates, and birds. A protein consisting of one or more polypeptide segments of at least 10 consecutive amino acids encoded by a porcine, bovine, ovine, caprine, or human heavy or light chain gene sequence. Functional single chain antibodies generally contain a sufficient portion of an immunoglobulin superfamily gene product to retain binding properties with a specific target molecule, typically a receptor or antigen (epitope).

Members of a pair of molecules (e.g., an antibody-antigen pair and a ligand-receptor pair) "bind specifically" to each other if they bind to each other with high affinity compared to other non-specific molecules. It is said. For example, an antibody produced against an antigen to which it binds more efficiently compared to binding to a non-specific protein can be described as specifically binding to the antigen.

The term "stimulation," as used herein, refers to the binding of a stimulatory molecule (e.g., TCR/CD3 complex) to its cognate ligand, thereby mediated by, but not limited to, the TCR/CD3 complex. Refers to a primary response caused by a mediated signal transduction event, such as signal transduction. Stimulation may mediate changes in the expression of certain molecules, such as downregulation of TGF-β and/or reorganization of cytoskeletal structures.

The term "stimulatory molecule" as used herein refers to a molecule on a T cell that specifically binds a cognate stimulating ligand present on an antigen presenting cell.

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

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

The terms "T cell" and "T lymphocyte" are interchangeable and are used synonymously 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 acid into cells using recombinant DNA technology. The term "transformation" refers to the introduction of a "foreign" (i.e. exogenous or extracellular) gene, DNA or RNA sequence into a host cell so that the host cell expresses the introduced gene or sequence. This will produce the desired substance, such as a protein or enzyme, encoded by the introduced gene or sequence. An introduced gene or sequence is also sometimes referred to as a "cloned" or "exogenous" gene or sequence and is modified by the cell's genetic machinery, such as start, stop, promoter, signal, secretion, or other sequences. may include regulatory or control sequences used. A gene or sequence may include non-functional sequences or sequences of unknown function. A host cell that accepts and expresses introduced DNA or RNA has been "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" means: (1) suffering from or susceptible to clinical or subclinical symptoms of a condition, disease or condition, but not yet experiencing clinical or subclinical symptoms of the condition, disease or condition; (2) inhibiting the condition, disease, or condition (i.e., in the case of a disease or maintenance therapy); (3) inhibiting, reducing, or delaying their reversal or progression of at least one clinical or subclinical symptom) and/or (3) alleviating the condition (i.e., the condition, disease, or condition or causing regression of at least one clinical or subclinical symptom). The benefit to the treated patient is statistically significant or at least perceivable to the patient or physician.

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

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

As used herein, the term "variable segment" refers to a portion of a nascent peptide that consists of a random, pseudorandom, or defined sequence. "Variable segment" means a portion of a nascent peptide that consists of a random, pseudorandom, or defined sequence. Variable segments may consist of both variant and invariant residue positions, and the degree of variant residues at variant residue positions may be limited, both options at the discretion of the practitioner. selected. Typically, variable segments are about 5-20 amino acid residues (e.g., 8-10) in length, but variable segments can be longer and include antibody fragments, protein-binding nucleic acids, It may also consist of an antibody protein or receptor protein, such as a receptor protein.

"Vector," "cloning vector," and "expression vector," as used herein, refer to vectors that transform a host cell by introducing a polynucleotide sequence (e.g., a foreign gene) into the host cell, and refers to a medium capable of promoting the expression (e.g. transcription and translation) of 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 includes an amino acid sequence found in nature. The terms "parent molecule" and "target protein" also refer to the wild type protein. A "wild type protein" preferably has some desired property, such as higher binding affinity, or enzymatic activity, which may result in better stability in various temperature or pH environments, or improved selectivity and and/or may be obtained by screening libraries of proteins for desired properties, including solubility.

The term "working", as in "working sample", is simply a sample on which a person is working. Similarly, for example, a "working molecule" is a molecule that is being worked on by a person.

For purposes of illustration, the principles of the invention are explained by reference to various exemplary embodiments. Although particular embodiments of the invention are specifically described herein, those skilled in the art will readily recognize that the same principles are equally applicable and may be used in other systems and methods. Probably. Before describing disclosed embodiments of the invention in detail, it is to be understood that the invention is not limited to the details of any particular embodiments to which the invention is shown. Additionally, the terminology used herein is for purposes of explanation rather than limitation. Additionally, although some methods are described herein with reference to steps presented in a particular order, in many cases these steps may be performed in any order, as one of ordinary skill in the art would appreciate. therefore, the novel method is not limited to the particular order of steps disclosed herein.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Must be kept in mind. Additionally, the terms "a" (or "an"), "one or more" and "at least one" may be used interchangeably herein. The terms "comprising," "including," "having," and "constructed from" may also be used interchangeably.

The present disclosure describes a chimeric antigen receptor (CAR) for binding a target antigen evolved from a parent or wild-type protein or domain thereof and comparing its activity in assays under abnormal conditions under normal physiological conditions. The present invention relates to a chimeric antigen receptor comprising: at least one antigen-specific target region; a transmembrane domain; and an intracellular signaling domain, having reduced activity in assays. In some embodiments, the chimeric antigen receptor further comprises an extracellular spacer domain or at least one costimulatory domain. The target antigen may be a tumor-specific antigen and may be Axl, ROR2 or CD22.

The CARs of the invention are characterized by: (1) their affinity for a target antigen that is reversibly or irreversibly reduced under normal physiological conditions; , increased affinity. These CARs can direct cytotoxic cells to disease sites where abnormal conditions exist, such as the tumor microenvironment or synovial fluid. As a result of these properties, CAR can preferentially direct cytotoxic cells to disease sites due to its low affinity for normal tissues. Such CARs can dramatically reduce side effects and allow the use of higher doses of therapeutic agents, thereby increasing therapeutic efficacy. CAR is particularly useful in the development of new therapeutic agents that need to remain in the subject's body for a shorter or limited period of time. Examples of beneficial uses include systemic treatment at high dosages, as well as local treatment at high concentrations.

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

A chimeric antigen receptor has an increased activity compared to the antigen-specific target region of the parent or wild-type protein or its domains when analyzed under abnormal conditions, and which has an increased activity compared to the antigen-specific target region of the parent or wild-type protein or its domains under normal physiological conditions. It may include an antigen-specific target region that has reduced binding affinity for the target antigen compared to the antigen-specific target region of the domain.

In any of the aforementioned chimeric antigen receptors, the present antigen-specific target region also exhibits increased selectivity compared to the antigen-specific target region of the parent or wild-type protein or domain thereof in assays under abnormal conditions. may have.

In some embodiments, the antigen-specific target region has a ratio of activity under abnormal conditions to the same activity under normal physiological conditions of 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.

The CAR molecule contains a linker connecting two antigen-specific target regions (Figure 1). This linker orients the two antigen-specific target regions on the CAR-T cell such that they exhibit enhanced or optimal activity in binding the target antigen (Jensen et al., “Design and implementation of adaptive therapy with chimeric antigen receptor-modified T cells”, Immunol Rev. ., vol. 257, pp. 127-144, 2014). The linker is therefore preferably capable of assuming a specific conformation that allows for improved or optimal binding of the two antigen-specific target regions to the target antigen, thereby enabling the effectiveness of 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"). 2, e105, 2013). This flexible linker is capable of assuming many different conformations for enhanced or optimal presentation of the two antigen-specific target regions for binding to the target antigen.

In some embodiments, the linker is capable of assuming different conformations under normal and abnormal physiological conditions. In particular, the linker has a first conformation that enhances or optimally presents the two antigen-specific target regions for binding to the target antigen under abnormal conditions, whereas the same linker has a first conformation that enhances or optimally presents the two antigen-specific target regions for binding to the target antigen. The linker has a second conformation in which the presentation of the two antigen-specific target regions is less effective in binding to the target antigen than the first conformation of the linker under abnormal conditions. Such linkers can be referred to as "conditional linkers" that allow two antigen-specific target regions to bind a target antigen with higher avidity under abnormal conditions than under normal physiological conditions. Therefore, CAR-T cells containing such conditional linkers are more active under abnormal conditions than the same CAR-T cells under normal physiological conditions.

Proteins that change conformation at different pH have been previously described, eg, by Di Russo et al. "pH-Dependent conformational changes in proteins and their effect on experimental pK(a)s: the case of nitrophorin 4" case of Nitrophorin 4), PLoS Comput Biol., vol. 8, e1002761, 2012). Furthermore, proteins that have different conformations at different temperatures have been described by Caldwell, “Temperature-induced protein conformational changes in barley root plasma membrane-enriched microsomes. "rane-enriched microsomes)", Plant Physiol ．． , vol. 84, pp. 924-929, 1989. Regarding the conformation of antibodies affected by pH and/or temperature, see Gandhi, "Effect of pH and temperature on conformational changes of a humanized monoclonal antibody. lonal antibody) ”, discussed in a master's thesis at the University of Rhode Island in the United States.

Included within the scope of this invention is the selection of conditional linkers for use in CAR molecules. A conditional linker assumes a first conformation that enhances or optimally presents the two antigen-specific target regions for binding to the target antigen under abnormal conditions and that under normal physiological conditions assumes a first conformation that is optimal for binding to the target antigen. Presentation of the two antigen-specific target regions can assume a suboptimal second conformation. In some embodiments, a suboptimal conformation of the linker under normal physiological conditions results in about 90% of the binding activity of a CAR molecule having an enhanced or optimal conformation of the 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 the 2A linker, the 2A-like linker, the picornavirus 2A-like linker, the 2A peptide of porcine teschovirus (P2A), and the 2A peptide of Thosea asigna virus (T2A), and variants thereof. and functional equivalents. This starting linker is evolved to create a mutant protein; the mutant protein is then subjected to analysis under normal physiological conditions and under abnormal conditions. From these variant proteins, selected proteins are selected from (a) conditional linkers having a first conformation with improved or optimal presentation of the two antigen-specific target regions for binding to the target antigen under abnormal conditions; and (b) the protein with the conditional linker assumes a second conformation of the conditional linker, on the basis that under normal physiological conditions the presentation of the two antigen-specific target regions assumes a second conformation of the conditional linker that is suboptimal for binding to the target antigen. selected.

The CAR molecule also contains an extracellular spacer domain that connects two antigen-specific targeting regions with a transmembrane domain, which in turn connects to a costimulatory domain and an intracellular signaling domain inside the T cell. Connect (Figure 1). The extracellular spacer domain is preferably capable of assisting the antigen-specific targeting region in recognizing and binding the target antigen on the target cell (Hudecek et al., "Non-Signal Transduction of Chimeric Antigen Receptors"). The non-signaling extracellular spacer domain of chimeric antigen receptors is decisive for in vivo antitumor activity. Cancer Immunol Res., vol. 3, pp. 125-135, 2015). In some embodiments, the extracellular spacer domain is a flexible domain such that the antigen-specific targeting region has a structure for optimal recognition of the specific structure and density of the target antigen on cells, such as tumor cells. (Hudecek et al., “The non-signaling extracellular spacer domain of chimeric antigen receptor is the key to in vivo antitumor activity.” It is decided for in vivo Cancer Immunol Res., vol. 3, pp. 125-135, 2015). The flexibility of extracellular spacer domains allows them to assume many different conformations.

In some embodiments, the extracellular spacer domain is capable of assuming different conformations during normal and abnormal physiological conditions. In particular, the extracellular spacer domain has a first conformation that enhances or optimally presents two antigen-specific target regions for binding to the target antigen under abnormal conditions, whereas the same extracellular spacer domain However, under normal physiological conditions, it has a second conformation in which the presentation of the two antigen-specific target regions is suboptimal for binding to the target antigen. Such extracellular spacer domains are referred to as "conditional extracellular spacer domains" because they allow two antigen-specific target regions to bind the target antigen with higher avidity under abnormal conditions than under normal physiological conditions. be able to. Thus, 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.

Included within the scope of the invention is the selection of conditional extracellular spacer domains for use in CAR molecules. In some embodiments, the conformation of the extracellular spacer domain is suboptimal under normal physiological conditions, such that about 90% of the CAR molecules have the same optimal conformation of the extracellular spacer domain under abnormal conditions; or about 80%, or about 70%, or about 60%, or about 50%, or about 40%, or about 30%, or about 20%, or about 10%, or less than about 5% binding to the target antigen. An active CAR molecule is generated.

It has been discovered that ubiquitination-resistant forms of regions containing extracellular spacer domains and transmembrane domains can enhance CAR-T cell signaling and thus increase antitumor activity (Kunii et la., “Ubiquitin Enhanced function of redirected human cells expressing linker for activation of t cells that s resistant to ubiquitylation), Human Gene Therapy , vol. 24, pp. 27-37, 2013). Within this region, the extracellular spacer domain is outside of the CAR-T cell and thus exposed to different conditions, potentially rendering it conditionally ubiquitination-resistant.

It is included within the scope of the invention that the extracellular spacer domain is conditionally resistant to ubiquitination. In particular, the extracellular spacer domain of the CAR molecule is more resistant to ubiquitination under abnormal conditions than under normal physiological conditions. Therefore, CAR-T cells with extracellular spacer domains that are conditionally resistant to ubiquitination will have enhanced cytotoxicity under abnormal conditions compared to their cytotoxicity under normal physiological conditions.

A conditionally ubiquitination-resistant extracellular spacer domain has high ubiquitination resistance at abnormal pH or abnormal temperature, and low ubiquitination resistance at normal physiological pH or normal physiological temperature. may be selected. In one embodiment, the conditionally ubiquitination-resistant extracellular spacer domain is ubiquitination-resistant at the pH of the tumor microenvironment and at normal physiological pH, such as human plasma pH between 7.2 and 7.6. ubiquitination resistance is low at normal pH.

To create a conditional extracellular spacer domain, a starting protein fragment selected from an antibody Fc fragment, an antibody hinge region, an antibody CH2 region, and an antibody CH3 region is evolved to create a variant protein. This mutant protein is subjected to analysis under normal physiological conditions and analysis under abnormal conditions. A conditional extracellular spacer domain (a) has a first conformation in which the antigen-specific target region binds to the target antigen with higher avidity under abnormal conditions, and which binds to the target antigen with higher avidity under normal physiological conditions; (b) a mutant protein exhibiting a conditional extracellular spacer domain with a second conformation of the conditional extracellular binding domain that binds to the target antigen with lower avidity than in abnormal conditions; or (b) normal in abnormal conditions. Selected from proteins with higher ubiquitination resistance than under physiological conditions.

Any of the aforementioned chimeric antigen receptors may be constructed such that the protein containing the antigen receptor has an increased level of expression compared to the parent or wild type protein or domain thereof.

In an alternative embodiment, the invention provides a chimeric antigen receptor (CAR) for binding a target antigen that is evolved from a parent or wild-type protein or domain thereof and that 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-type protein or domain thereof; 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 costimulatory domain.

The present disclosure also discloses (a) reduced activity compared to the antigen-specific target region of the parent or wild-type protein or domain thereof when analyzed under normal physiological conditions; and (b) when the parent or wild-type protein or domain thereof is analyzed under abnormal conditions. The present invention also relates to a method of evolving a parent or wild-type protein or domain thereof to produce a conditionally active protein having at least one of: increased activity relative to an antigen-specific target region of the protein or domain thereof. Conditionally active proteins can be engineered to be CARs.

Chimeric antigen receptors produced by this method have an antigen-specific target region that has reduced binding affinity for the target antigen compared to the antigen-specific target region of the parent or wild-type protein or domain thereof under normal physiological conditions. may be included.

Chimeric antigen receptors produced by this method have increased activity compared to the antigen-specific target region of the parent or wild-type protein or domain thereof in assays under abnormal conditions and or may contain an antigen-specific target region that has reduced binding affinity for the target antigen compared to the antigen-specific target region of the wild-type protein or domain thereof.

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

Any of the aforementioned chimeric antigen receptors produced by the present methods can be configured such that the protein containing the antigen receptor has an increased level of expression compared to the parent or wild type protein or domain thereof.

In an alternative embodiment of the method, a chimeric antigen receptor (CAR) produced by the method for binding a target antigen is evolved from a parent or wild-type protein or domain thereof and under abnormal conditions. at least one antigen-specific target region that has increased selectivity in the assay compared to the antigen-specific target region of the parent or wild-type protein or domain thereof; a transmembrane domain; and an intracellular signaling domain. In some embodiments, the chimeric antigen receptor further comprises an extracellular spacer domain or at least one costimulatory domain.

Chimeric Antigen Receptors The immune system of mammals, particularly humans, possesses cytotoxic cells that target and destroy diseased tissues and/or pathogens. The use of these cytotoxic cells to remove unwanted tissue (ie, target tissue) such as tumors is a promising therapeutic approach. Other tissues that may be targeted for removal include glandular (eg, prostate) hyperplasia, warts, and unwanted fatty tissue. However, this relatively new treatment approach has so far met with limited success. For example, targeting and destroying tumors using T cells has relatively little long-term benefit because cancer cells may reduce surface antigen expression and adapt to new therapies, reducing the effectiveness of this therapy. 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 chimeric antigen receptors can greatly improve the specificity and sensitivity of these cytotoxic cells. For example, T cells that express CAR (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 can directly recognize target molecules and are therefore typically not limited by polymorphic elements such as human leukocyte antigens (HLA). The advantages of this CAR targeting strategy are threefold. First, because CAR-T cell function is independent of HLA status, the same CAR-based approach can in principle be used in any patient with a tumor expressing the same target surface antigen. Second, altered antigen processing and presentation machinery is a common feature of tumor cells and may facilitate immune escape. However, this leaves them vulnerable to 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 engineered protein that includes at least one antigen-specific targeting region (ASTR), a transmembrane domain (TM), and an intracellular signaling domain (ISD). In some embodiments, the CAR may further include an extracellular spacer domain (ESD) and/or a costimulatory domain (CSD). See Figure 1.

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

ASTR may also contain another protein functional domain for recognizing and binding target antigens. The target antigen may have other biological functions, such as serving as a receptor or ligand, or the ASTR may contain a functional domain for specifically binding the antigen. Some examples of proteins with functional domains include linked cytokines (which lead to recognition of cells bearing cytokine receptors), affibodies, ligand binding domains from naturally occurring receptors, Examples include soluble protein/peptide ligands for receptors on tumor cells. As one of skill in the art will appreciate, virtually any molecule that has the ability to bind a given antigen with high affinity can be used for ASTR.

In one embodiment, a 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, a CAR comprises three or more ASTRs that target at least three or more different antigens or epitopes. When multiple ASTRs are present in a CAR, the ASTRs may be arranged in tandem and separated by a linker peptide (Figure 1).

In one embodiment, ASTR is specific for the target antigen, and where the V _H domain alone is sufficient to confer antigen specificity (a "single domain antibody"), the ASTR comprises V _H , CH1, hinge, and CH2 and Contains a full-length IgG heavy chain with a CH3 (Fc) Ig domain. If both the V _H and V _L domains are required to create fully active ASTR, both the V _H -containing CAR and full-length lambda light chain (IgL) can be introduced into the same cytotoxic cell to generate active ASTR. Created. In another embodiment, each ASTR of the CAR comprises at least two single chain antibody variable fragments (scFv), each specific for a different target antigen. scFv has the C-terminus of one variable domain (V _H or V _L ) tethered to the N-terminus of the other variable domain (V _L or V _H , respectively) via a polypeptide linker, and has antigen-binding properties. or have been developed without significantly interfering with binding specificity (Chaudhary et al., “A recombinant single-chain immunotoxin composite of anti-Tac variable regions and a truncate d diphtheria toxin,”Proc. Natl. Acad. Sci., vol. 87, page 9491, 1990; Bedzyk et al., “Immunological and structural characterization of a high affinity anti-fluorescein single-chain antibody,” J. Biol. Chem., vol. 265, page 18615, 1990). These scFvs lack the constant region (Fc) present in the heavy and light chains of natural antibodies. The scFvs specific for at least two different antigens are arranged in tandem. In certain embodiments, an extracellular spacer domain can be linked between ASTR and the transmembrane domain.

In another embodiment, the scFv fragment may be fused to all or a portion of the constant domain of the heavy chain. In a further embodiment, the ASTR of the CAR comprises a divalent (or bivalent) single chain variable fragment (di-scFv, bi-scFv). In a CAR containing di-scFV, two scFvs, each specific for an antigen, are linked together to form a single peptide chain containing two V _H regions and two V _L regions (Xiong et al. , “Development of tumor targeting anti-MUC-1 multimer: effects of di-scFv unpaired cysteine location on PEGylation and tumor binding,” Protein Engineering Design and Selection, vol. 19, pages 359-367, 2006; Kufer et al., “A revival of bispecific antibodies,” Trends in Biotechnology, vol. 22, pages 238-244, 2004).

In yet another embodiment, the ASTR comprises a diabody. In diabodies, the scFv is created with a linker peptide that is too short for the two variable regions to fold together and thus drives dimerization of the scFv. Even shorter linkers (1 or 2 amino acids) lead to the formation of trimers, so-called triabodies or tribodies. Tetrabodies may also be used for ASTR.

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

Antigens targeted by CARs are present on or within the cells of tissues targeted for removal, such as tumors, glandular (eg, prostate) hyperplasia, warts, and unwanted adipose tissue. Although surface antigens are more efficiently recognized and bound by the CAR's ASTR, intracellular antigens can also be targeted by the CAR. In some embodiments, the target antigen is preferably specific for cancer, inflammatory diseases, neuronal disorders, diabetes, cardiovascular diseases, or infectious diseases. Examples of target antigens include those expressed by various immune cells, carcinomas, sarcomas, lymphomas, leukemias, germ cell tumors, blastomas, and cells associated with various hematological, autoimmune, and/or inflammatory diseases. Contains antigens that are

Cancer-specific antigens that can be targeted by ASTR include 4-IBB, 5T4, adenocarcinoma antigen, alpha fetoprotein, Axl, BAFF, B lymphoma cells, C242 antigen, CA-125, carbonic anhydrase 9 (CA- IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extra domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2/neu, HGF, Human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgGl, LI-CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin α5β1, integrin ανβ3, MORAb-009, MS4A1 , MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-R a, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, ROR1, ROR2, SCH 900105, SDC1, SLAMF7, TAG-72 , tenascin C, TGFβ2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2 or vimentin.

Inflammatory disease-specific antigens that can be targeted by ASTR include AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (Basigin), CD154 (CD40L), CD2, CD20. , CD23 (IgE receptor), CD25 (a chain of IL-2 receptor), 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, integrin a4, integrin α4β7, Llama grama ( 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 disorders that can be targeted by the ASTRs of the invention include one or more of beta amyloid or MABT5102A. Diabetes-specific antigens that can be targeted by the ASTRs of the invention include one or more of LIβ or CD3. Cardiovascular disease-specific antigens that can be targeted by the ASTR of the invention include C5, cardiac myosin, CD41 (integrin alpha-lib), fibulin II, beta chain, ITGB2 (CD18) and sphingosine-1-phosphate. One or more of the following may be mentioned.

Infectious disease-specific antigens that can be targeted by the ASTR of the invention include anthrax toxin, CCR5, CD4, clumping factor A, cytomegalovirus, cytomegalovirus glycoprotein B, endotoxin, Escherichia coli. , hepatitis B surface antigen, hepatitis B virus, HIV-1, Hsp90, influenza A hemagglutinin, lipoteichoic acid, Pseudomonas aeruginosa, rabies virus glycoprotein, respiratory syncytial virus, and TNF-a. one or more.

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

In another embodiment, the CAR targets antigens associated with cancer therapeutic cells, such as NK cells and other cells mentioned herein, to activate cancer therapeutic cells by acting as immune effector cells. can be made into An example of this is a CAR that targets the CD16A antigen to engage NK cells to fight malignant lesions that express CD30. The bispecific tetravalent AFM13 antibody is an example of an antibody that can deliver this effect. For further details of this type of embodiment, see, eg, Rothe, A.; , et al. , “A phase 1 study of the bispecific anti-CD30/CD16A antibody construct AFM13 in patients with collapsed or refractory Hodgki n lymphoma, “Blood, 25 June 2015, Vl. 125, no. 26, pp. 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 comprises a nucleotide sequence selected from SEQ ID NOs: 2-5, or an amino acid sequence selected from SEQ ID NOs: 9-12. may have. These single chain antibodies targeting the cancer antigen Axl contain human IgG Fc regions having the nucleotide sequence of SEQ ID NO: 6 or 7, or the amino acid sequence of SEQ ID NO: 13 or 14. This single chain antibody targeting the cancer antigen Axl has a higher binding activity to Axl at pH 6.0 than that 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. This single chain antibody targeting cancer antigen ROR2 has increased ROR2 binding activity at pH 6.0 compared to ROR2 binding activity at pH 7.4.

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

The extracellular spacer domain of CAR is a hydrophilic region located between ASTR and the transmembrane domain. In some embodiments, this domain promotes CAR-proper protein folding. The extracellular spacer domain is an optional component of the CAR. The extracellular spacer domain can include a domain selected from 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, or a combination thereof. Examples of extracellular spacer domains include the CD8a hinge, artificial spacers made of polypeptides that can be as large as three glycines (Gly), and the CH1 and CH3 domains of IgG (such as human IgG4). can be mentioned.

The transmembrane domain of CAR is a region that has the ability to span the cell membrane of cytotoxic cells. The transmembrane domain is selected from a transmembrane region of a transmembrane protein, such as a type I transmembrane protein, an artificial hydrophobic sequence, or a combination thereof. Examples of transmembrane domains include T cell receptor α, β or ζ chains, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, Examples include the transmembrane regions of CD134, CD137, and CD154. The synthetic transmembrane domain can include a triplet of phenylalanine, tryptophan, and valine. Optionally, a short oligopeptide or polypeptide linker, preferably 2-10 amino acids in length, may form the link between the transmembrane domain and the intracellular signaling domain of the CAR. Glycine-serine doublets provide a particularly suitable linker between the transmembrane domain and the intracellular signaling domain.

CARs of the invention also include intracellular signaling domains. Intracellular signaling domains transmit effector function signals, directing the cytotoxic cell to perform its specialized function, namely inhibition and/or destruction of target cells. Examples of intracellular signaling domains include the ζ chain of the T cell receptor complex or any of its homologues, such as the η chain, FcsRly and β chains, the MB 1 (Iga) chain, the B29 (Ig) chain, 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. Molecules such as CD2, CD5 and CD28 are mentioned. Specifically, the intracellular signaling domain includes the human CD3ζ chain, FcyRIII, FcsRI, the cytoplasmic tail of the Fc receptor, the immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptor, and combinations thereof. could be.

Intracellular signaling domains used in CAR include several types of various other immune signaling receptors, such as, but not limited to, CD3, B7 family costimulation, and tumor necrosis factor receptor (TNFR). Intracellular signaling domains of first, second, and third generation T cell signaling proteins, including superfamily receptors, may be included (Park et al., "Are all chimeric antigen receptors created equal?" J Clin Oncol., vol. 33, pp. 651-653, 2015). In addition, intracellular signaling domains include signaling domains used by NK and NKT cells (Hermanson, et al., "Utilizing chimeric antigen receptors to direct natural killer cell activity," nt Immunol., vol. 6, p. .195, 2015), for example, NKp30(B7-H6) (Zhang et al., “An NKp30-based chimeric antigen receptor promotes T cell effector functions and a antitumor efficacy in vivo,” J Immunol., vol. 189, pp. 2290-2299, 2012), and DAP12 (Topfer et al., “DAP12-based activating chimeric antigen receptor for NK cell tumor immunotherapy,” J I mmunol., vol. 194, pp. 3201-3212, 2015), NKG2D, NKp44 , NKp46, DAP10, and CD3z signaling domains. In addition, intracellular signaling domains also include the signaling domains of human immunoglobulin receptors that contain immunoreceptor tyrosine-based activation motifs (ITAMs), such as FcγRI, FcγRIIA, FcγRIIC, FcγRIIIA, FcRL5 ( Gillis et al., “Contribution of Human FcγRs to Disease with Evidence from Human Polymorphisms and Transgenic Animal Studies,” "Front Immunol., vol. 5, p. 254, 2014).

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

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

In some embodiments, two or more components of a CAR of the invention are separated by one or more linkers. For example, in a CAR that includes at least two ASTRs, the two ASTRs may be separated by a linker. A 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. Linkers may be composed of flexible residues such as glycine and serine so that adjacent protein domains can move freely relative to each other. Longer linkers, eg, longer than 100 amino acids, may be used in conjunction with alternative embodiments of the invention, and may be selected, eg, such that two adjacent domains 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.

Conditionally Active Antigen-Specific Targeting Regions CARs are chimeric proteins produced by fusing the various domains discussed above all together to form a fusion protein. CARs are typically produced by expression vectors containing polynucleotide sequences encoding various domains of CARs. The ASTR of the present invention, which functions to recognize and bind to antigens on target cells, is conditionally active. Specifically, ASTR is less active or inactive in binding to a target antigen under normal physiological conditions and active under abnormal conditions compared to the corresponding parent or wild-type protein ASTR. The present invention provides methods for generating conditionally active ASTR from a parent or wild type protein or its binding domain (parent or wild type ASTR).

ASTR in the present invention can be discovered by creating a protein library and screening the library for proteins that have the desired binding affinity for the target antigen, so that ASTR can be determined at least in whole or in part by its binding to the target antigen. A wild type protein suitable for use in the domain. Wild-type proteins can be discovered by screening cDNA libraries. A cDNA library is a combination of cloned cDNA (complementary DNA) fragments that are inserted into a collection of host cells and together make up some portion of an organism's transcriptome. cDNA is made from completely transcribed mRNA and therefore contains the coding sequence for an organism's expressed protein. cDNA library information is a powerful and useful tool in the discovery of proteins with desired properties by screening libraries for proteins with the desired binding affinity for the target antigen.

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

There are other methods for creating and screening antibody libraries to discover wild-type antibodies. For example, fully human antibody display libraries can be utilized. Such a library is a collection of antibodies displayed on the surface of one or more host cells. Preferably, the antibody library is representative of the human antibody repertoire in that it has the ability to bind a wide variety of antigens. Because the antibodies are displayed on the surface of cells, the effective affinity (due to avidity) of each antibody in the library is increased. Unlike other common library types such as phage display libraries, where antibody avidity is less desirable for screening and identification purposes, the superavidity provided by cell surface display in the present invention is desirable. Cell surface display libraries allow the identification of antibodies with low, medium, and high binding affinities, as well as non- and weakly immunogenic epitopes, during screening or selection steps.

Generation of Evolved Molecules from Parent Molecules When the parent or wild-type protein, or its binding domain (parent or wild-type ASTR), undergoes a process of mutagenesis, a population of mutant polypeptides is produced, which is then screened. has enhanced binding affinity for the target antigen under abnormal conditions as compared to the parent or wild-type ASTR, and optionally has substantially the same or lower binding affinity for the target antigen under normal physiological conditions. Mutant ASTRs can be identified.

Any chemical synthesis or recombinant mutagenesis method may be used to generate a population of variant polypeptides. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology; is within the skill of the art. Such techniques are well explained in the literature. For example, Molecular Cloning A Laboratory Manual, 2nd Ed. , ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D.N. Glover ed., 1985 ); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Patent No. 4,683,195; Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. 1984); Transcription And Translation (B.D. Hames & S.J. .Higgins eds.1984 ); Culture Of Animal Cells (R.I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Tra nsfer Vectors For Mammalian Cells (J.H. Miller and M.P. Cabs eds ., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London , 1987); Handbook of Experimental Immunology, Volumes l-IV (DM. Weir and C.C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986); See 6).

The present disclosure provides a method of producing a nucleic acid variant encoding a conditionally active variant polypeptide, the method comprising: (i) substituting one or more nucleotides with a different nucleotide; modifying the nucleic acid by (ii) deleting one or more nucleotides, (iii) adding one or more nucleotides, or (iv) any combination thereof; Including process. In one embodiment, the non-natural nucleotide comprises inosine. In another embodiment, the method comprises analyzing a polypeptide encoded by a modified nucleic acid for changes in enzymatic activity, thereby detecting one or more modified nucleic acids encoding a polypeptide with altered enzymatic activity. The method further includes the step of identifying. In one embodiment, the modification of step (a) comprises PCR, error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-directed mutagenesis, gene reassembly, gene site saturation mutagenesis, ligase chain reaction, in vitro mutagenesis, ligase chain reaction, oligonucleotide synthesis, arbitrary DNA produced by generation techniques and any combination thereof. In another embodiment, the method further comprises at least one repetition of the modification step.

The method further provides a method for producing a polynucleotide from two or more nucleic acids. This method consists of the following (a) to (c). (a) Identifying regions of identity and regions of diversity between two or more nucleic acids. There, at least one of the nucleic acids consists of a nucleic acid of the present disclosure. (b) providing a set of oligonucleotides corresponding to at least two sequences of the two or more nucleic acids. and (c) extending the oligonucleotide with a polymerase, thereby producing a polynucleotide.

Any mutagenesis technique may be used in various embodiments of this disclosure. Stochastic or random mutagenesis is exemplified by a situation in which a parent molecule is mutated (modified or changed) to obtain a series of progeny molecules with unplanned mutations. Thus, an in vitro stochastic mutagenesis reaction, for example, is a product that is not specifically intended for its production. Rather, it is uncertain and therefore random as to the exact nature of the mutations obtained and thus as to the products produced. Stochastic mutagenesis is demonstrated in methods where mutations are random or unplanned, such as error-prone PCR and stochastic shuffling. Variant forms can be generated by error-prone transcription such as error-prone PCR of 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 can be created by a first form of replication (discussed in further detail below and which is generally well known). A mutagen strain can include any mutant that is deficient in the function of mismatched repair. These include mutant gene products such as mutS, mutT, mutH, mutL, ovrD, dcm, vsr, umuC, umuD, sbcB, recJ. Defects may be obtained by genetic mutation, allelic exchange, small chemical compounds or expressed antisense RNA or other techniques. The defect may be in the described gene or a homologous gene in any organism.

Other mutagenesis methods include oligonucleotide-directed mutagenesis techniques, error-prone polymerase chain reaction (error-prone PCR), and cassette mutagenesis, in which specific regions of the parent polynucleotide are synthetically is replaced by a mutagenized oligonucleotide. In such cases, multiple mutation sites are generated around a particular site in the parent sequence.

In oligonucleotide-directed mutagenesis, short sequences are replaced with synthetically mutagenized oligonucleotides. In oligonucleotide-directed mutagenesis, a short sequence of polynucleotides is removed from a synthetic polynucleotide using restriction enzyme digestion and replaced with a synthetic polynucleotide in which various bases have been changed from the original sequence. Polynucleotide sequences may also be altered by chemical mutagenesis. Chemical mutagens include, for example, sodium bisulfite, nitrous acid, 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 thereby displacing the sequence. Insertion of drugs such as proflavin, acriflavine, quinacrine, etc. may also be used. Random mutagenesis of polynucleotide sequences may be obtained by X-ray or ultraviolet radiation. Generally, mutagenized plasmid polynucleotides are introduced into E. coli and propagated as a pool or library of hybrid plasmids.

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

In cassette mutagenesis, a single template sequence block is typically (partially) replaced by a random sequence. Reidhaar-Olson J F and Sauer R T: Combinatorial cassette mutagenesis as a probe of the information content of protein sequences. Science 241(4861):53-57, 1988.

Alternatively, any technique of non-stochastic or non-random gene mutagenesis can also be used in various embodiments of this disclosure. Non-stochastic mutagenesis is exemplified by a situation where a parent molecule is mutated (modified or changed) to obtain a molecule with one or more predetermined mutations. The presence of background products in some amount is real in many reactions where molecular processing occurs, and the presence of background products reflects the non-stochastic nature of the mutagenesis process with unpredicted products. It is well understood that it does not subtract from Site-saturation mutagenesis and synthetic ligation reassembly are examples of genetic mutagenesis techniques in which the precise chemical structure of the intended product is predetermined.

One method of site-saturation point mutagenesis is described in US Patent Application Publication No. 2009/0130718. The method provides a series of degenerate primers corresponding to codons of a template polynucleotide and performs polymerase extension to produce progeny polynucleotides containing sequences corresponding to the degenerate primers. Progeny polynucleotides are expressed and screened in direct evolution. Specifically, this is a method for producing a series of progeny polypeptides, comprising the steps of: (a) providing copies of a template polypeptide, each consisting of a plurality of codons encoding a template polypeptide sequence; and (b) at each codon of the template polynucleotide, (1) providing a series of degenerate primers, where each primer consists of a degenerate codon corresponding to a codon of the template polynucleotide, and at least one flanking sequence comprises: (2) providing conditions that allow the primer to anneal to a copy of the template polynucleotide; and (3) directing a polymerase extension reaction from the primer along the template. performing steps (1) to (3) of the step of performing, thereby providing progeny polynucleotides, each comprising a sequence corresponding to a degenerate codon of the annealed primer, thereby providing a series of progeny polynucleotides; Manufacture. Site-saturation mutagenesis is a method that involves the direct evolution of nucleic acids and screens clones containing the evolved nucleic acids, resulting in the desired binding activity.

Site-saturation mutagenesis generally involves: 1) mutagenesis of one or more ancestral or parental generation templates to achieve at least one point mutation, addition, deletion, and/or chimerism; preparing a progeny generation of one or more molecules (including molecules comprising a polynucleotide sequence, molecules comprising a polypeptide sequence, and molecules partly comprising a polynucleotide sequence and partly comprising a polypeptide sequence); 2) screening one or more progeny molecules - preferably using high-throughput methods - for the desired binding affinity for the target antigen; 3) optionally screening the structure and/or progeny molecules for the parent and/or progeny molecules; or, and 4) optionally repeating any of steps 1) to 3).

In site-saturation mutagenesis, the progeny generation of polynucleotides generated (e.g., from a parent polynucleotide template), referred to as "codon site-saturation mutagenesis," has all codons (or degenerated codons encoding the same amino acid) Each has at least one set of three or more contiguous point mutations (i.e., different bases consisting of a new codon) such that a full lineage of codons) is represented at each codon position. Corresponding to and encoded by the progeny generations of this polynucleotide are the series of progeny polypeptides produced, each having at least one single amino acid point mutation. In a preferred embodiment, an example of so-called "amino acid site saturation mutagenesis" is generated that creates alpha amino acid substitutions at each and every amino acid position along the polypeptide. A variant polypeptide in each of the polypeptides. This yields - for each and every amino acid position along the parent polypeptide - all 20 different progeny polypeptides containing the original amino acid, or the added amino acid, in place of the 20 naturally encoded amino acids. , or in addition, potentially 21 or more different progeny polypeptides.

Other mutagenesis techniques may be used, including recombination, and more specifically, by methods of in vivo reassortment of polynucleotide sequences containing regions of partial homology to generate polynucleotides encoding polypeptides. Includes methods for preparing nucleotides, methods for assembling polynucleotides to form at least one polynucleotide, and methods for screening polynucleotides for the production of polypeptides with useful properties.

In other embodiments, mutagenesis techniques are used to recombine molecules and/or to convey sequences and reduced clutter in a range of repetitive or contiguous sequences with regions of homology. Take advantage of nature.

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

Chimeric genes are created by joining two polynucleotide fragments using compatible sticky ends generated by restriction enzymes. Here, each fragment is derived from a separate ancestral (parent) molecule. Other examples involve mutagenesis of a single codon position (i.e., codon substitution, (to obtain additions, deletions).

Additionally, in vivo site-specific recombination systems, as well as random methods of in vivo recombination, can generate hybridization of genes and recombination between homologous but cleaved genes on plasmids. used for. Mutagenesis was also reported by duplication expansion and PCR.

Non-random methods are used to obtain larger numbers of point mutations and/or chimerizations; for example, synthetic or comprehensive methods are used to obtain specific structural groups (e.g. specific single molecules) in the template molecule. in order 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 generate molecular species.

Any of these or other evolution methods can be used in the present disclosure to generate new populations (libraries) of variant polypeptides from parent or wild-type proteins.

Expression of evolved molecules The variant polynucleotides generated from the evolution process are size fractionated on agarose gels according to previously published protocols, inserted into expression vectors, and inserted into appropriate expression vectors for the production (expression) of variant polypeptides. The host cell may or may not be transfected. Expression can use routine molecular biology techniques. Therefore, various known methods can be used for the expression step.

For example, briefly, the variant polynucleotides generated from the evolution process are then digested using standard molecular biology techniques and ligated into an expression vector such as plasmid DNA. The vector is then transformed into bacteria or other cells using standard protocols. This can be done in individual wells of multi-well trays, such as 96-well trays for high-throughput expression and screening. This process is repeated for each variant polynucleotide.

The polynucleotides thus selected and isolated are introduced into appropriate host cells. A suitable host cell is any cell capable of facilitating genetic recombination and/or reductive reassortment. The selected polynucleotide is preferably one already present in a vector containing appropriate control sequences. 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. The introduction of constructs into the host cell can be performed by calcium phosphate, DEAE -dextran, or electroporation (for example, ECKER and DAVIS, 1986, INHIBITION OF GENE EXPR ESSION IN Plant Cells by Expression of antisense RNA, Proc Natl Acad Sci USA, 83:5372-5376).

Representative examples of expression vectors that can be used include viral particles, baculoviruses, phages, plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes, viral DNA (e.g., vaccinia, adenovirus, foul pox viruses, pseudorabies, and SV40). Artificial chromosomes based on organisms) pl, yeast plasmids, yeast artificial chromosomes, and any other vectors specific for a particular target host (eg Bacillus, Aspergillus, and yeast) may be mentioned. Thus, for example, the DNA can be included in any one of a variety of expression vectors for expressing the polypeptide. Such vectors include chromosomal, non-chromosomal, and synthetic DNA sequences. Large numbers of suitable vectors are well known to those skilled in the art and are commercially available. The following vectors are given as examples. Bacterial: pQE vector (Qiagen), pBluescript plasmid, pNH vector, lambda ZAP vector (Stratagene); ptrc99a, ρKK223-3, pDR540, pRIT2T (Pharmacia); Eukaryotic: pXTl, ρSG5 (Stratagene) p SVK3, pBPV, pMSG , pSVLSV40 (Pharmacia). However, any other plasmids or other vectors can be used as long as they are replicable and viable in the host. Low copy number vectors or high copy number vectors can be used in the present invention.

The variant polynucleotide sequence in the expression vector is operably linked to an appropriate expression control sequence (promoter) to direct RNA synthesis. Particularly named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda PR, PL, and trp. Eukaryotic promoters include immediate early CMV, HSV thymidine kinase, early and late SV40, LTRs from retroviruses, and mouse metallothionein-1. Selection of an appropriate vector and promoter is well within the level of skill in the art. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. The vector may contain appropriate sequences to amplify expression. Promoter regions can be selected from any desired gene using chloramphenicol transferase (CAT) vectors, or other vectors, with selectable markers. In addition, the expression vector can have phenotypic properties for selection of transformed host cells, such as dihydrofolate reductase or neomycin resistance in eukaryotic cell culture, or tetracycline or ampicillin resistance in E. coli. Preferably, one or more selectable marker genes are included to provide the characteristics.

Eukaryotic DNA transcription can be increased by inserting enhancer sequences into the expression vector. Enhancers are 10-300 bp cis-acting sequences that increase transcription by a promoter. Enhancers can effectively increase transcription when located on either the 5' or 3' side of the transcription unit. Enhancers are also useful when located within introns or within the coding sequence itself. Typically, viral enhancers are used, including the SV40 enhancer, cytomegalovirus enhancer, polyoma enhancer, and adenovirus enhancer. Enhancer sequences from mammalian systems, such as the mouse immunoglobulin heavy chain enhancer, are also commonly used.

Mammalian expression vector systems also typically include a selectable marker gene. Examples of suitable markers include the dihydrofolate reductase gene (DHFR), the thymidine kinase gene (TK), or a prokaryotic gene that confers drug resistance. For the first two marker genes, the use of mutant cell lines that are unable to grow without the addition of thymidine to the growth medium is preferred. Transformed cells can then be identified by their ability to grow in unsupplemented medium. Examples of prokaryotic drug resistance genes useful as markers include G418, genes that confer resistance to mycophenolic acid and hygromycin.

Expression vectors containing the DNA segment of interest can be introduced into host cells by well-known methods depending on the type of cell production host. For example, calcium chloride transfection is commonly utilized for prokaryotic host cells, while calcium phosphate treatment, lipofection, or electroporation may be used for eukaryotic host cells. Other methods used to transform mammalian cell production hosts include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection (see generally Sambrook et al., supra).

Once the expression vector is introduced into a suitable host, the host is maintained under suitable conditions such that the introduced variant polynucleotide sequence is highly expressed and the variant polypeptide is produced. Expression vectors are typically replicable in the host organism either as episomes or as an integral part of the host chromosomal DNA. Generally, the expression vector may contain a selectable marker, such as tetracycline or neomycin, to allow detection of cells transformed with the desired DNA sequence (see, e.g., U.S. Pat. No. 4,704,362). ).

Accordingly, in other aspects of the invention, variant polynucleotides can be generated by methods of reductive reassortment. The method involves the generation of constructs containing contiguous sequences (original coding sequence sequences), their insertion into suitable vectors, and their subsequent introduction into suitable host cells. Reassortment of individual molecular identities occurs by combinatorial methods between consecutive sequences in constructs with regions of homology or between quasi-repeat units. Reassortment methods recombine and/or reduce the complexity and extent of repetitive sequences, resulting in the generation of new molecular species. Various treatments can be applied to enhance the rate of reassembly. These may include the use of ultraviolet light treatment or DNA damaging chemicals, and/or host cell lines that exhibit enhanced levels of "genetic instability." Thus, reassortment methods can involve homologous recombination or the natural properties of quasi-repetitive sequences to guide their own evolution.

The cells then proliferate and "decreasive repopulation" is achieved. The rate of decreasing reassortment methods can optionally be stimulated by the introduction of DNA damage. In vivo reassortment is directed to an "intermolecular" method, collectively referred to as "genetic recombination", which is commonly observed in bacteria as a "RecA-dependent" phenomenon. The present invention relies on the ability of cells to modulate host cell genetic modification methods to recombine and reassemble sequences, or reduction methods to reduce the complexity of quasi-repetitive sequences in cells by deletion. Can be done. This method of "reduction reassembly" occurs by an "intra-molecular", RecA-independent method. The final result is the reassembly of the 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 disclosure, the Gram-negative bacteria include Escherichia coli or Pseudomonas fluorescens. In another aspect of the disclosure, the Gram-positive bacterium is Streptomyces diversa, Lactobacillus gasseri, Lactococcus lactis, Lactococcus cremoris. occus cremoris), or Contains Bacillus subtilis. In another aspect of the disclosure, the eukaryote is Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Kluyveromyces lactis ( Kluyveromyces lactis), Hansenula - Contains Hansenula plymorpha, or Aspergillus niger. Representative examples of suitable hosts include bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium; fungal cells, such as yeast; insect cells, such as Drosophila. ) S2 and Spodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma; adenoviruses; and plant cells. Selection of an appropriate host is considered to be within the 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 as a number of suitable host cell lines capable of secreting intact immunoglobulins have been developed in the art, including CHO cell lines, various COS cell lines, HeLa cells, myeloma cell lines. , B cells or hybridomas. Expression vectors for these cells contain 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, such as It may include ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription termination sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, and the like.

In one embodiment, the eukaryotic host cell is 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, Jurkat, MM1, Ovcar 3, HT 1080, Panc-1, U266, 769P, BT-474, Caco-2, HCC 1954, MDA-MB-468, LnCAP , NRK-49F, and SP2/0 cell lines; and mouse splenocytes and rabbit PBMC. In one embodiment, the mammalian host cell is selected from the CHO or HEK293 cell lines. In one specific embodiment, the mammalian host cell is a CHO-S cell line. In another specific embodiment, the mammalian system is the HEK293 cell line. In another embodiment, the eukaryotic host is a yeast cell system. In one embodiment, the eukaryotic host is S. S. cerevisiae yeast cells or picchia yeast cells.

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

As discussed above, optimization of expression of conditionally active ASTR involves optimization of the vector used (vector components, e.g., promoter, splice site, 5' and 3' ends and flanking sequences), genetic modification of the host cell. by reducing gene deletions and rearrangements by evolving relevant genes, evolving host cell gene activity by evolving relevant genes in vivo or in vitro, and optimizing host glycosylation enzymes by evolving relevant genes. and/or by chromosome-wide host cell mutagenesis and selection strategies to select cells with enhanced expression capacity.

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

Cells are typically 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 cell lysing agents. Such methods are well known to those skilled in the art. The expressed polypeptides or fragments thereof are prepared by methods including 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 and purified from recombinant cell culture. Protein refolding processes can be used, if necessary, in finalizing the structure of the polypeptide. If desired, high performance liquid chromatography (HPLC) can be used for the final purification step. Screening for conditionally 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 variant proteins for conditionally active ASTR.

Screening to identify reversible or irreversible mutations Identification of desirable molecules is accomplished very directly by measuring protein activity under permissive and wild-type conditions. The mutant showing the highest ratio of activity (permissive/wild type) can then be selected and point mutation permutations can be generated by combining individual mutations using standard methods. I can do it. This combined, sorted protein library is then screened for proteins that exhibit the greatest difference in permissive and wild-type activity.

The activity of the supernatant can be screened using various methods, e.g., high-throughput activity assays such as fluorescence assays, to identify protein variants that are sensitive to desired properties (temperature, pH, etc.). It is possible. For example, to screen for time-sensitive mutants, commercially available Measurement of enzyme or antibody activity is performed using possible substrates. Screening can be performed in a variety of media, including serum and BSA, among others. Reactions can be initially performed in a multi-well analysis format, such as a 96-well analysis, and confirmed using another format, such as a 14 ml tube format.

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

In another aspect, the disclosure provides purified antibodies that specifically bind to a polypeptide of the disclosure or a fragment thereof that has enzymatic activity. In one aspect, the present disclosure provides fragments of antibodies that specifically bind to polypeptides with 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 enzymes of the present disclosure, or related polypeptides. These antibodies can be used to isolate other polypeptides within the scope of this disclosure, or other related enzymes. Antibodies can be designed to bind to the active site of an enzyme. Accordingly, the present disclosure provides methods of inhibiting enzymes using the antibodies of the present disclosure.

Antibodies can be used in immunoprecipitation, staining, immunoaffinity columns, and the like. If desired, nucleic acid sequences encoding particular antigens can be generated by immunization followed by isolation, amplification or cloning of the polypeptides or nucleic acids, as well as immobilization of the polypeptides to the arrays of the present disclosure. Alternatively, the methods of the present disclosure can be used to modify the structure of antibodies produced by cells, and it is possible to modify antibodies, eg, to increase or decrease their affinity. Additionally, the ability to create or modify antibodies can be phenotypically engineered into cells by the methods of the present disclosure.

Methods of immunization, antibody production and isolation (polyclonal and monoclonal) are known to those skilled in the art and are described in the scientific and patent literature. For example, Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY (1991); Stites (eds.) BASIC AND CLINICAL IMMUNOLOGY (7th ed.) Lange Medi. cal Publications, Los Altos, Calif. (“Stites”); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2d ed.) Academic Press, New York, N. Y. (1986); Kohler (1975) “Continuous cultures of fused cells secreting antibodies of predefined specificity”, Nature 256:495; Harlow (1 988) ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Publications, New York. checking ... Antibodies can also be produced in vitro, for example, by using phage display libraries expressing recombinant antibody binding sites, in addition to traditional in vivo animal methods. For example, Hoogenboom (1997) “Designing and optimizing library selection strategies for generating high-affinity antibodies”, Trends B. iotechnol. 15:62-70; and Katz (1997) “Structural and mechanical determinants of affinity and specificity of ligands discovered or engine. red by phage display”, Annu. Rev. Biophys. Biomol. Struct. 26:27-45. checking ...

The polypeptides or peptides can be used to generate antibodies that specifically bind to the polypeptides of the present disclosure, such as enzymes. The resulting antibodies may be used in immunoaffinity chromatography techniques to isolate or purify the polypeptide or to determine whether the polypeptide is present in a biological sample. . In such techniques, a protein preparation, such as an extract, or a biological sample is contacted with an antibody capable of specifically binding one of the polypeptides of the disclosure.

In immunoaffinity techniques, antibodies are bound to a solid support, such as beads or other column substrates. The protein preparation is placed in contact with the antibody under conditions that cause the antibody to specifically bind to one of the polypeptides of the disclosure. After washing to remove non-specifically bound proteins, specifically bound polypeptides are eluted.

The ability of proteins in a biological sample to bind to antibodies can be determined using a variety of techniques known to those skilled in the art. For example, binding capacity may be determined by labeling the antibody with a detectable label, such as a fluorescent material, an enzyme label, a radioisotope, or the like. Alternatively, the ability of the antibody to bind to the sample may be determined by detection using a second antibody having such a detectable label on its surface. Specific assays include ELISA assays, sandwich assays, radioimmunoassays, and Western blots.

Polyclonal antibodies raised against polypeptides of the present disclosure can be obtained by direct injection of the polypeptide into an animal or by administration of the polypeptide to a non-human animal. The antibody so obtained is then coupled to the polypeptide itself. In this manner, even sequences encoding only fragments of a polypeptide can be used to generate antibodies that are capable of binding the native, full-length polypeptide. Such antibodies can then be used to isolate the polypeptide from cells expressing the polypeptide.

Any technique that provides antibodies produced by subcultured cell lines can be used to prepare monoclonal antibodies. Examples include hybridoma methods, trioma methods, human B cell hybridoma methods, and EBV hybridoma methods (see, e.g., Cole (1985) in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). ) is included.

Adapting techniques described for producing single chain antibodies (see, e.g., U.S. Pat. No. 4,946,778) to produce single chain antibodies to polypeptides of the present disclosure. I can do it. Alternatively, transgenic mice may be used to express humanized antibodies against the polypeptide or fragment thereof. Antibodies raised against polypeptides of the present disclosure can be used to screen similar polypeptides (eg, enzymes) from other organisms and samples. In such techniques, polypeptides of biological origin are brought into contact with antibodies, and polypeptides that specifically bind to the antibodies are detected. Any of the techniques described above may be used to detect antibody binding.

Screening Methods and "On-line" Monitoring Devices A variety of devices and methods can be used in conjunction with the polypeptides and nucleic acids of the present disclosure in practicing the methods of the present disclosure. The apparatus and methods include, for example, those for screening peptides for enzymatic activity, those for screening for compounds that are potential modulators, such as activators or inhibitors, of enzymatic activity, and those that bind to polypeptides of the present disclosure. Examples include those for antibodies that hybridize with the nucleic acids of the present disclosure, those for screening cells expressing the polypeptides of the present disclosure, and the like.

array or “biochip”
Nucleic acids or polypeptides of the present disclosure can be immobilized or applied to arrays. Arrays can be used to screen or screen libraries of compositions (e.g., small molecules, antibodies, nucleic acids, etc.) for their ability to bind to, or modulate the activity of, the nucleic acids or polypeptides of the present disclosure. It can be monitored. For example, in one aspect of the present disclosure, the parameter monitored is transcriptional expression of an enzyme gene. Nucleic acid immobilized on an array or "biochip" of one, more than one, or all of the transcripts of a given cell, or a sample containing representative or complementary nucleic acids of the transcripts of that cell or the transcripts of a cell. It is possible to measure by hybridizing with. By using an "array" of nucleic acids on a microchip, some or all of the transcripts can be quantified simultaneously. Alternatively, arrays with genomic nucleic acids can be used to determine the genotype of newly designed strains created by the methods of the present disclosure. By using polypeptide "arrays", it is also possible to quantify multiple proteins at the same time. The present disclosure can be practiced with any known "array," including a "microarray," or "nucleic acid array," or "polypeptide array," or "antibody array," or "biochip array." ” or variations thereof. Arrays generally include a plurality of "spots" or "target elements," each target element having a defined amount of one or more biomolecules, e.g., oligonucleotides, and a sample molecule. For example, it is immobilized on a defined region on the surface of a substrate that specifically binds to mRNA transcripts.

In practicing the methods of the present disclosure, any known arrays and/or methods of making and using arrays may be used, in whole or in part, or variations thereof, such as those described in the following documents: may be used. For example, US Pat. No. 6,277,628; US Pat. No. 6,277,489; US Pat. No. 6,261,776; US Pat. No. 6,258,606; , 054,270; 6,048,695; 6,045,996; 6,022,963; 6,013,440 Specification No. 5,965,452; Specification No. 5,959,098; Specification No. 5,856,174; Specification No. 5,830,645; Specification No. 5, No. 770,456; No. 5,632,957; No. 5,556,752; No. 5,143,854; No. 5,807,522 5,800,992; 5,744,305; 5,700,637; 5,556,752; 5,434 ,049 specification; see also, for example, WO 99/51773 pamphlet; WO 99/09217 pamphlet; WO 97/46313 pamphlet; WO 96/17958 pamphlet; 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”, Biotechni ques 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 Available-From Start to Finish￣for Obtaining Expression Data by Microarray”, Nature Genetics Support. See also 21:25-32. US Patent Application Publication No. 20010018642; US Patent Application Publication No. 20010019827; US Patent Application Publication No. 20010016322; US Patent Application Publication No. 20010014449; US Patent Application Publication No. 20010014448; See also US Patent Application Publication No. 20010012537; US Patent Application Publication No. 20010008765.

Capillary Arrays Capillary arrays such as GIGAMATRIX™ (Diversa Corporation, San Diego, Calif.) can be used in the methods of the present disclosure. Nucleic acids or polypeptides of the present disclosure can be immobilized or applied to arrays, including capillary arrays. Arrays can be used to screen or screen libraries of compositions (e.g., small molecules, antibodies, nucleic acids, etc.) for their ability to bind to, or modulate the activity of, the nucleic acids or polypeptides of the present disclosure. It can be monitored. Capillary arrays provide another system for holding and screening samples. For example, a sample screening device can include a plurality of capillaries formed as an adjacent capillary array, where each capillary has a wall defining at least one lumen that retains a sample. The device may further include interstitial material disposed between adjacent capillaries in the array, with one or more reference indicia formed within the interstitial material. . A capillary for screening a sample is one that is adapted to couple to a capillary array and has a first wall forming a lumen for holding the sample and a first wall for exciting the sample. The second wall may be formed from a filter material that filters the excitation energy generated. A polypeptide or nucleic acid, eg, a ligand, can be introduced into the first component of at least some of the capillaries of the capillary array. Each capillary of the capillary array can have walls defining at least one lumen that retains the first component. An air bubble can be introduced after the first component in the capillary. A second component can be introduced into the capillary, where it is separated from the first component by an air bubble. The sample of interest is introduced into one capillary in a capillary array as a first fluid labeled with detectable particles, each capillary in the capillary array having a tube for holding the first fluid and the detectable particles. having at least one wall defining a cavity, the at least one wall being coated with a binding material for binding detectable particles. The method further includes removing the first liquid from the capillary tube in which bound detectable particles are retained and introducing a second liquid into the capillary. A capillary array includes a plurality of individual capillaries having at least one outer wall defining a lumen. The outer wall may be one or more walls fused together. Similarly, the wall can form a lumen of cylindrical, square, hexagonal, or other geometric form, so long as the wall forms a lumen that retains liquid or sample. The capillaries in a capillary array can be supported closely together to form a planar structure. The capillaries can be joined together by fusing them next to each other (eg, here the capillaries are made of glass), gluing, caking, or fixing them. A capillary array can be formed from any number of individual capillaries, for example from 100 to 4,000,000. A capillary array can form a microtiter plate with about 100,000 or more individual capillaries connected to each other.

Engineering Conditionally Active Antibodies Conditionally active antibodies can be engineered to produce multispecific conditionally active antibodies. A multispecific antibody can be an antibody with multi-epitope specificity, as described in WO 2013/170168. Multispecific antibodies include, but are not limited to, antibodies in which the V _H V _L unit comprises a heavy chain variable domain (V _H ) and a light chain variable domain (V _L ) with multiple epitope specificities, each V _H V _L Antibodies with two or more V _L and V _H domains that bind different epitopes, antibodies with two or more single variable domains where each single variable domain binds a different epitope, and one or more as well as 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. Antibody fragments may be obtained from full-length conditionally activated antibodies. Enzymatic digestion of conditionally active antibodies can generate antibody fragments. Exemplary enzymatic digestion methods include, but are not limited to, pepsin, papain, and Lys-C. Exemplary antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, diabodies (Db); tandem diabodies (taDb), linear antibodies (U.S. Pat. No. 5,641, No. 870, Example 2; Zapata et al., Protein Eng., vol. 8, pages 1057-1062 (1995)); one-arm antibodies, single variable domain antibodies, minibodies (Olafsen et al. 2004) Protein Eng. Design & Sel., vol. 17, pages 315-323), single chain antibody molecules, fragments produced by Fab expression libraries, anti-idiotypic (anti-Id) antibodies, complementarity determining region (CDR) ), and epitope-binding fragments. Antibody fragments may also be produced using recombinant DNA techniques. DNA encoding antibody fragments may be cloned into plasmid expression vectors or phagemid vectors and expressed directly in E. coli. Methods of antibody enzymatic digestion, DNA cloning, and recombinant protein expression are well known to those skilled in the art.

Antibody fragments may be purified using conventional techniques and subjected to reduction to generate free thiol groups. Antibody fragments with free thiol groups can be reacted with a crosslinking agent, such as bismaleimide. Such cross-linked antibody fragments are purified and then reacted with a second antibody fragment having free thiol groups. The final product, in which the two antibody fragments are cross-linked, is purified. In certain embodiments, each antibody fragment is a Fab, and the final product of two Fabs linked via bismaleimide is referred to herein as bismaleimide-(thio-Fab)2, or bis-Fab. Ru. Such multispecific antibodies and antibody analogs can be utilized to rapidly synthesize combinations of large numbers of antibody fragments, including bis-Fabs, or structural variants of natural antibodies or specific antibody/fragment combinations. can.

Multispecific antibodies can be synthesized with modified crosslinkers so that additional functional moieties can be added to the multispecific antibody. Modified crosslinkers allow attachment of any sulfhydryl-reactive moiety. In one embodiment, N-succinimidyl-S-acetylthioacetate (SATA) is attached to bismaleimide to form bismaleimide acetylthioacetate (BMata). After deprotection of the masked thiol groups, 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 (e.g., biotin-linker reagents), detection labels (e.g., fluorophore reagents), solid phase immobilization reagents (e.g., 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 crosslinkers may be further reacted with drug moiety reagents or other labels. Reaction of a multispecific antibody or antibody analog with a drug-linker intermediate results in a multispecific antibody-drug conjugate or antibody analog-drug conjugate, respectively.

Other techniques for producing multispecific antibodies can also be used in the present invention. References describing these techniques include: (1) Milstein and Cuello, Nature, vol. 305, page 537 (1983)), WO 93/08829 pamphlet, and Traunecker et al. , EMBO J. , vol. 10, page 3655 (1991); (2) for "knob-in-hole" operation, U.S. Pat. WO 2009/089004A1 for the production of polymeric molecules; (4) US Pat. No. 4,676,980 for cross-linking two or more antibodies or fragments, and Brennan et al. , Science, vol. 229, page 81 (1985); (5) Regarding the production of bispecific antibodies using leucine zippers, see Kostelny et al. , J. Immunol. , vol. 148, pages 1547-1553 (1992); (6) Regarding the production of bispecific antibody fragments using "diabody" technology, see Hollinger et al. , Proc. Natl. Acad. Sci. USA, vol. 90, pages 6444-6448 (1993); (7) Regarding the use of single chain Fv (sFv) dimers, see Gruber et al. , J. Immunol. , vol. 152, page 5368 (1994); (8) Regarding the preparation of trispecific antibodies, Tutt et al. J. Immunol. 147:60 (1991); and (9) U.S. patent applications for engineered antibodies with three or more functional antigen binding sites, including "Octopus antibodies" or "dual variable domain immunoglobulins" (DVDs). Publication No. 2006/0025576A1 and Wu et al. Nature Biotechnology, vol. 25, pages 1290-1297 (2007).

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

In one embodiment, multispecific antibodies (eg, bispecific antibodies) are generated by engineering conditionally active antibodies to cross the blood-brain barrier (BBB). This multispecific antibody includes a first antigen binding site that binds to the BBB-R and a second antigen binding site that binds to a brain antigen. At least the first antigen binding site for the BBB-R is conditionally active. Brain antigens are antigens 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), Tau, apolipoprotein E4 (ApoE4), α-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, γ-secretase, death receptor 6 (DR6) , amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), and caspase-6. In one embodiment, the antigen is BACE1.

The BBB has an endogenous transport system mediated by BBB receptors (BBB-R), which are specific receptors that allow the transport of macromolecules across the BBB. For example, antibodies capable of binding to the BBB-R can be transported across the BBB using this endogenous transport system. Such antibodies can serve as vehicles for transporting drugs or other agents across the BBB by using the endogenous BBB receptor-mediated transport system across the BBB. Such antibodies need not have high affinity for the BBB-R. As described in US Pat. ing.

Another way to engineer antibodies to enter the brain is to engineer them to be delivered to the brain via the central nervous system lymphatics. 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. , “Structural and functional features of central nervous system lymphatic vessels,” Nature 523, pp. 337-341, 16 July 2015 and is publicly available as of the filing date of this application.

Unlike conventional antibodies, conditionally active antibodies do not need to have low affinity for the BBB-R in order to cross the BBB and remain inside the brain. A conditionally active antibody can be one that has high affinity for the 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 coupled to conditionally activated antibodies and transported along with the antibodies across the BBB into the brain.

BBB-R is a transmembrane receptor protein expressed on brain endothelial cells and has the ability to transport molecules across the blood-brain barrier. Examples of BBB-Rs include transferrin receptor (TfR), insulin receptor, insulin-like growth factor receptor (IGF-R), low-density lipoprotein receptors, such as, without limitation, low-density lipoprotein receptor-related These 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 approximately 180,000) composed of two disulfide-linked subunits (each with an apparent molecular weight approximately 90,000) that is involved in iron uptake in vertebrates.

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

Plasma is a body fluid that is very different from extracellular brain 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), brain extracellular fluid is significantly lower in K ⁺ and higher in Mg ²⁺ and H ⁺ compared to plasma. Differences in ionic concentrations between plasma and brain ECF result in significant differences in osmolality and osmolality between these two body fluids. Table 1 shows the concentrations (in millimolar units) of common ions in both plasma and brain ECF.

Brain ECF also contains significantly more lactate compared to plasma and significantly less glucose compared to plasma (Abi-Saab et al., “Striking Differences in Glucose and Lactate Levels Between Brain Extracellular ar Fluid and Plasma in Conscious Human Subjects: Effects of Hyperglycemia and Hypoglycemia, “Journal of Cerebral Blood Flow & Metabolism, vol. 22, pa ges 271-279, 2002).

Therefore, there are several physiological conditions that are different on either side of the BBB, such as pH, concentration of various substances (lactose, glucose, K+, Mg2+, etc.), osmolality and osmolarity. Regarding pH physiological conditions, human plasma has a higher pH than human brain ECF. Regarding the physiological conditions of K+ concentration, brain ECF has a lower K+ concentration than human plasma. Regarding the physiological conditions of Mg2+ concentration, human brain ECF has significantly more Mg2+ than human plasma. Regarding the physiological conditions of osmolarity, human brain ECF has a different osmolality than human plasma. In some embodiments, the physiological conditions of the brain ECF may be the composition, pH, osmolality and osmolarity of the brain ECF in patients with a particular neurological disorder, which is similar to the physiology of the brain ECF in the general population. conditions may differ.

Accordingly, the present invention provides methods for evolving DNA encoding template antibodies against BBB-R to create mutant DNA libraries. This mutant DNA library is then expressed to obtain mutant antibodies. These variant antibodies bind to the BBB-R under at least one plasma physiological condition and have reduced affinity for the BBB-R compared to the template antibody under at least one brain physiological condition in brain ECF. screened for conditionally active antibodies with or without it. Thus, the selected variant antibodies have low or high affinity for the BBB-R on the plasma side and low or no affinity for the BBB-R on the brain ECF side. The selected variant antibodies are useful as conditionally active antibodies for transport across the BBB.

Such conditionally activated antibodies have an advantage in crossing the BBB and retention in the brain ECF. Because the brain has a lower affinity for the BBB-R, conditionally active antibodies are transported across the BBB out of the brain and back into the blood at a slower rate than the template antibody (or something like that). (will disappear).

In some other embodiments, the invention provides methods for evolving DNA encoding template antibodies against BBB-R to create variant DNA libraries. This mutant DNA library is then expressed to obtain mutant antibodies. These variant antibodies are screened for conditionally active antibodies that bind to the BBB-R under at least one plasma physiological condition and have little or no affinity for the BBB-R under at least one brain physiological condition. Ru. Therefore, the selected variant antibodies have affinity for the BBB-R on the plasma side and little or no affinity for the BBB-R on the brain ECF side. This selected variant antibody is a conditionally activated antibody.

Such conditionally activated antibodies have an advantage in crossing the BBB and retention in the brain ECF. After binding to the BBB-R on the plasma side, the conditionally activated antibody is transported across the BBB and has little to no affinity for the BBB-R on the brain ECF side, meaning that the conditionally activated antibody This means that the possibility of being transported out of the brain is low.

The affinity of a conditionally activated antibody for the BBB-R can be measured by its half-maximal inhibitory concentration (IC50), which is defined as the 50% inhibition of the binding of a known BBB-R ligand to the BBB-R. It is a measure of how much antibody is needed for A common approach is to perform a competitive binding assay, such as a competitive ELISA assay (assy). An exemplary competitive ELISA assay to measure IC50 for TfR (BBB-R) is where increasing concentrations of anti-TfR antibody compete with biotinylated TfR ^A for binding to TfR. Anti-TfR antibody competition ELISA can be performed overnight at 4°C in Maxisorp plates (Neptune, N.J.) coated with 2.5 μg/ml purified mouse TfR extracellular domain in PBS. Plates are washed with PBS/0.05% Tween 20 and blocked using Superblock blocking buffer (Thermo Scientific, Hudson, N.H.) in PBS. Each individual anti-TfR antibody titration (1:3 serial dilution) is combined with biotinylated anti-TfR ^A (0.5 nM final concentration) and added to the plate for 1 hour at room temperature. Wash the plates with PBS/0.05% Tween 20, add HRP-streptavidin (Southern Biotech, Birmingham) to the plates and incubate for 1 hour at room temperature. Plates are washed with PBS/0.05% Tween 20 and biotinylated anti-TfR ^A bound to the plate is detected using TMB substrate (BioFX Laboratories, Owings Mills).

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

In some embodiments, the IC50 of a conditionally activated antibody from the 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.

Conditionally Active Biological Proteins for Synovial Fluid Joint diseases are a major cause of disability and early retirement in industrialized countries. Joint diseases often result in joint damage that is difficult to repair. Synovial fluid is a fluid found between the cartilage and synovium of opposing joint surfaces in the synovial cavity 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 a fluid that acts as a lubricant between joint surfaces. Human synovial fluid contains approximately 85% water. It is derived from the dialysate of plasma, which itself consists of water, dissolved proteins, glucose, clotting factors, inorganic ions, hormones, etc. Proteins such as albumin and globulin are present in synovial fluid and are thought to play an important role in the lubrication of joint areas. Several other proteins are also found in human synovial fluid, including glycoproteins such as alpha-1-acid glycoprotein (AGP), alpha-1-antitrypsin (A1AT) and lubricin.

Synovial fluid has a very different composition 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 glucose, whereas the average normal glucose value in human plasma is about 100 mg/dL, varying within a range of 70-100 mg/dL throughout the day. In addition, large molecules such as proteins do not easily cross the synovial membrane into the synovial fluid, so total protein levels in the synovial fluid are approximately one-third of plasma protein levels. It is also known that the pH of human synovial fluid is higher than that of human plasma (Jebens et al., “On the viscosity and pH of synovial fluid and the pH of blood,” The Journal of Bone and Joint Surgery, vol. .41 B, pages 388-400, 1959; Farr et al., “Significance of the hydrogen ion concentration in synovial fluid in Rheumatoid Arthri tis, "Clinical and Experimental Rheumatology, vol. 3, pages 99-104, 1985).

Therefore, synovial fluid has some physiological conditions that are different from those of other parts of the body, such as those of plasma. Synovial fluid has a high pH compared to other parts of the body, especially blood plasma. Synovial fluid has a low concentration of glucose compared to other parts of the body, such as plasma. Synovial fluid also has a low concentration of proteins compared to other parts of the body, such as plasma.

Several antibodies have been used to treat joint diseases by introducing the antibodies into synovial fluid. For example, the synovial fluid of injured joints is known to contain many factors that influence the progression of osteoarthritis (e.g., Fernandes, et al, “The Role of Cytokines in Osteoarthritis Pathophysiology,” Biorheology , 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 MMPs causes degradation of matrix and non-matrix proteins in the joint. Antibodies that neutralize cytokines can halt 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 against aggrecan or aggrecanase) have been developed to treat osteoarthritis, which has by far the highest prevalence among joint diseases (International Publication No. 1993/022429A1). Antibodies against acetylated high mobility group box 1 (HMGB1) have been developed for the diagnosis or treatment of joint diseases that are inflammatory, autoimmune, neurodegenerative or malignant diseases/disorders, such as arthritis. This antibody can be used to detect the acetylated form of HMGB1 in synovial fluid (WO 2011/157905A1). Another antibody (CD20 antibody) has also been developed to treat joint connective tissue and cartilage damage.

However, the antigens of these antibodies are often expressed in other parts of the body that are responsible for important physiological functions. Although antibodies against these antigens are effective in treating joint diseases, the normal physiological functions of these antigens in other parts of the body can also be significantly interfered with. Therefore, severe side effects may occur to the patient. In this way, they can preferentially bind to those antigens (proteins or other macromolecules) with higher affinity in synovial fluid, while not binding to the same antigens in other parts of the body, reducing side effects. It would be desirable to develop therapeutic agents, such as antibodies to cytokines or other antigens, that bind only weakly or weakly to antigens.

Such a conditionally active biological protein may be a conditionally active antibody. In some embodiments, the invention also provides conditionally active biological proteins that are proteins other than antibodies. For example, the present invention develops conditionally active immunomodulatory factors to preferentially modulate immune responses in synovial fluid, which may have less or no effect on immune responses in other parts of the body. can be done.

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

In some embodiments, the invention provides a conditionally active biological protein derived from a parent or wild type biological protein. A conditionally active biological protein has lower activity than the parent or wild-type biological protein under at least one physiological condition in a particular part of the body, such as blood plasma, and under at least one physiological condition in synovial fluid. has higher activity than the parent or wild type biological protein. Such conditionally active biological proteins may function preferentially in synovial fluid, but not or to a lesser extent in other parts of the body. Consequently, such conditionally active biological proteins may have reduced side effects.

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

Conditionally Active Biological Proteins for Tumors Cancer cells in solid tumors have the ability to form a tumor microenvironment around them that supports cancer cell growth and metastasis. The tumor microenvironment includes surrounding blood vessels, immune cells, fibroblasts, other cells, soluble factors, signaling molecules, extracellular matrix, and other components that promote neoplastic transformation, support tumor growth and invasion, and support tumor host cells. It is the cellular environment in which tumors reside that includes mechanical cues that can protect against immunity, promote therapeutic resistance, and provide a niche for latent metastases to develop. The tumor and its surrounding microenvironment are closely related and constantly interacting. A tumor can influence its microenvironment by releasing extracellular signals, promoting tumor angiogenesis, and inducing peripheral immune tolerance, while immune cells in the microenvironment can influence the growth and evolution of cancerous cells. can have an impact on Swarts et al. “Tumor Microenvironment Complexity: Emerging Roles in Cancer Therapy,” Cancer Res, vol. , 72, pages 2473-2480, 2012.

The tumor microenvironment is often hypoxic. As the tumor mass increases, the interior of the tumor grows further away from the existing blood supply, making it difficult to fully oxygenate the tumor microenvironment. The partial pressure of oxygen in the tumor environment is less than 5 mm Hg in more than 50% of locally advanced solid tumors, compared to an oxygen partial pressure of approximately 40 mm Hg in plasma. In contrast, other parts of the body are not hypoxic. A hypoxic environment leads to genetic instability through downregulation of nucleotide excision repair and 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 activation of genes associated with poor prognosis and metastasis. Weber et al. , “The tumor microenvironment,” Surgical Oncology, vol. 21, pages 172-177, 2012 and Blagosklonny, “Antiangiogenic therapy and tumor progression,” Cancer Cell, vol. 5, pages 13-17, 2004.

In addition, tumor cells tend to rely on energy produced by lactic acid fermentation, which does not require oxygen. Tumor cells are therefore less likely to use normal aerobic respiration, which requires oxygen. As a result of using lactic acid fermentation, the tumor microenvironment is 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 Drives Local Invasion,” Cancer Research, vol. 73, pages 1524-1535, 2013. In the tumor microenvironment, nutrient availability is also low due to the relatively high nutrient requirements of proliferating cancer cells compared to cells located in other parts of the body.

Furthermore, the tumor microenvironment also contains many characteristic cell types that are not commonly found in other parts of the body. These cell types include endothelial cells and their precursors, pericytes, smooth muscle cells, fibroblasts, cancer-associated fibroblasts, myofibroblasts, neutrophils, eosinophils, basophils, and mast cells. cells, T and B lymphocytes, natural killer cells and antigen-presenting cells (APC), such as macrophages and dendritic cells (Lorusso et al., “The tumor microenvironment and its contribution to tumor evolution toward met astasis,”Histochem Cell Biol, vol. 130, pages 1091-1103, 2008).

Thus, the tumor microenvironment has at least some physiological conditions that are different from physiological conditions in other parts of the body, such as physiological conditions in plasma. The tumor microenvironment has a low pH (acidic) compared to other parts of the body, especially plasma (pH 7.4). The tumor microenvironment has low oxygen concentrations compared to other parts of the body, such as blood plasma. The tumor microenvironment also has low nutrient availability compared to other parts of the body, especially plasma. The tumor microenvironment also has some characteristic cell types that are not commonly found in other parts of the body, especially the plasma.

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

Cancer cell surface antigens that can be targeted by antibodies fall into several different types. Hematopoietic differentiation antigens are glycoproteins commonly associated with the cluster of differentiation (CD) classification and include CD20, CD30, CD33 and CD52. Cell surface differentiation antigens are a diverse group 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), ERBB2 (also known as HER2), ERBB3 (Reference 18), and MET (HGFR). 19, insulin-like growth factor 1 receptor (IGF1R) 20, ephrin receptor A3 (EPHA3) 21, tumor necrosis factor (TNF)-related apoptosis-inducing ligand receptor 1 (TRAILR1; also known as TNFRSF10A) , TRAILR2 (also known as TNFRSF10B) and receptor activator ligand for nuclear factor kappa B (RANKL; also known as TNFSF11) 22. Antigens involved in angiogenesis are usually proteins or growth factors that assist in the formation of new microvasculature and include vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR), integrin αVβ3 and integrin α5β1 ( Reference 10). Tumor stroma and extracellular matrix are essential supporting structures for tumors. Interstitial and extracellular matrix antigens that are therapeutic targets include fibroblast activation protein (FAP) 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 showing promise in the treatment of cancer. Examples include tumor suppressors such as retinoblastoma protein (pRb), p53, pVHL, APC, CD95, ST5, YPEL3, ST7, and ST14. Some proteins that induce apoptosis in cancer cells can also be introduced into tumors to reduce tumor size. There are at least two mechanisms that can induce apoptosis in tumors: a mechanism induced by tumor necrosis factor and a mechanism mediated by Fas-Fas ligand. At least some of the proteins involved in either of these two apoptotic mechanisms can be introduced into tumors for therapy.

Cancer stem cells are cancer cells that have the ability to give rise to any cell type found in a particular cancer sample and are therefore tumorigenic. Cancer stem cells can give rise to tumors through the stem cell process of self-renewal and differentiation into multiple cell types. Cancer stem cells continue to exist as a distinct population in tumors and are thought to cause recurrence and metastasis by giving rise to new tumors. The development of specific therapies that target cancer stem cells can improve the survival and quality of life of cancer patients, especially those with metastatic disease.

These tumor therapeutics often interfere with normal physiological functions in other parts of the body other than the tumor. For example, a protein that induces apoptosis in a tumor may also induce apoptosis in some other part of the body, thus causing side effects. In embodiments where antibodies are used to treat tumors, the antigens of the antibodies may also be expressed in other parts of the body that perform normal physiological functions. For example, the monoclonal antibody bevacizumab (targets vascular endothelial growth factor), which stops tumor blood vessel growth. The antibodies can also block blood vessel growth and repair in other parts of the body, leading to bleeding, poor wound healing, blood clots, and kidney damage. For more effective tumor therapy, the development of conditionally active biological proteins that focus primarily or exclusively on tumor targeting is highly desirable.

In some embodiments, the invention provides conditionally active biological proteins produced from parent or wild-type biological proteins that can be tumor therapeutic candidates. This conditionally active biological protein has lower activity than the parent or wild-type biological protein under at least one physiological condition in a part of the body other than the tumor microenvironment, such as plasma, while the tumor microenvironment has higher activity than the parent or wild-type biological protein under at least one physiological condition at . Such conditionally active biological proteins can preferentially act on cancer cells in the tumor microenvironment to treat tumors and are therefore less likely to cause side effects. In embodiments where the biological protein is an antibody against an antigen on the surface of a tumor cell (where the antigen is exposed to the tumor microenvironment), the conditionally active antibody is directed against an antigen in other parts of the body, e.g., in the non-tumor microenvironment. In the tumor microenvironment, the affinity for the antigen is lower than the parent or wild type antibody, whereas in the tumor microenvironment the affinity for the antigen is higher than the parent or wild type antibody. Such conditionally activated antibodies may bind antigen weakly or not at all in other parts of the body, but have higher avidity or bind strongly and tightly to antigen in the tumor microenvironment. .

In some embodiments, the conditionally active antibody is an antibody against an immune checkpoint protein, resulting in inhibition of the immune checkpoint. Such conditionally activated antibodies have (1) increased binding affinity for immune checkpoint proteins in the tumor microenvironment compared to the parent or wild-type antibody from which they are derived; and (2) the conditions. a reduced binding affinity for an immune checkpoint protein in a non-tumor microenvironment compared to the parent or wild-type antibody from which the sexually active antibody is derived.

Immune checkpoints function as endogenous inhibitory pathways of the immune system that maintain self-tolerance and regulate the duration and extent of immune responses to antigenic stimuli, ie, foreign molecules, cells, and tissues. Pardoll, 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, particularly T cells, which can then be induced to attack tumors. Checkpoint proteins suitable for the present invention include CTLA4 and its ligands CD80 and CD86, PD1 and its ligands PDL1 and PDL2, T cell immunoglobulin and mucin protein-3 (TIM3) and its ligand GAL9, B and T lymphocyte attenuator. receptors such as nuator (BTLA) and its ligand HVEM (herpesvirus entry mediator), killer cell immunoglobulin-like receptor (KIR), lymphocyte activation gene-3 (LAG3) and adenosine A2a receptor (A2aR), and the ligands B7-H3 and B7-H4. Additional suitable immune checkpoint proteins are described by Pardoll, Nature Reviews Cancer, vol. 12, pages 252-264, 2012 and Nirschl & Drake, Clin Cancer Res, vol. 19, pages 4917-4924, 2013.

CTLA-4 and PD1 are two of the best known immune checkpoint proteins. CTLA-4 can downregulate T cell activation pathways (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. CTLA-4 inhibitors include anti-CTLA-4 antibodies. Anti-CTLA-4 antibodies bind to CTLA-4 and block the interaction of CTLA-4 with its ligand CD80 or CD86, thereby reducing the immune response elicited by the interaction of CTLA-4 with its ligand. Cut off control.

Checkpoint protein PD1 is known to suppress T cell activity in peripheral tissues when an inflammatory response to infection occurs, thereby suppressing autoimmunity. Blocking PD1 in vitro can enhance T cell proliferation and cytokine production in response to stimulation by specific antigen targets or by allogeneic cells in mixed lymphocyte reactions. The strong correlation between PD1 expression and decreased immune response was shown to be caused by the inhibitory function of PD1, ie, the induction of immune checkpoints (Pardoll, Nature Reviews Cancer, 12:252-264, 2012). Blocking 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, thereby overactivating the immune system, particularly T cells, to attack tumors (Pardoll, Nature Reviews Cancer, vol. .12, pages 252-264, 2012). However, hyperactivated T cells may also attack host cells and/or tissues, resulting in collateral damage to the patient's body. Therefore, therapies 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, the FDA-approved antibody against CTLA-4 has a black box warning due to its high toxicity.

The present invention addresses the problem of collateral damage from 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 the non-tumor microenvironment, e.g. normal biological tissues, are not inhibited or are inhibited to a lesser extent by conditionally active antibodies, thus reducing the possibility of collateral damage to the body in the non-tumor microenvironment. Ru. This goal is achieved by engineering conditionally active antibodies to be more active in the tumor microenvironment compared to the non-tumor microenvironment.

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

Highly immunogenic tumors, such as malignant melanoma, are most vulnerable to a hyperactivated immune system achieved by immune system manipulation. Conditionally active antibodies against immune checkpoint proteins may therefore be particularly effective in treating such highly immunogenic tumors. However, other types of tumors are also vulnerable to an overactive immune system.

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

A conditionally active antibody is more active in the tumor microenvironment against the same tumor cell surface molecule or checkpoint protein compared to the activity of the parent or wild type antibody from which the conditionally active antibody is derived. These combination therapies can result in both enhanced efficacy and significantly reduced toxicity. The reduced toxicity of these conditionally active antibodies, particularly those directed against immune checkpoint proteins, allows for the safe use of potent antibodies, such as ADC antibodies as described herein, as well as higher doses of antibodies. It can be done.

In some embodiments, conditionally active antibodies against checkpoint proteins may be in prodrug form. For example, a conditionally active antibody can be a prodrug that does not have the desired drug activity before being cleaved into the drug form. The prodrug may be present in the tumor microenvironment because the enzyme that catalyzes such cleavage is preferentially present in the tumor microenvironment, or if the conditionally active antibody has a higher accessibility to the cleavage site in the non-tumor microenvironment. They may preferentially cleave in the tumor microenvironment, either because the environment makes it easier to reach the cleavage site.

Conditionally Active Biological Proteins for Stem Cell Niches, Including Tumor Stem Cells Stem cells exist in an environment called the stem cell niche of the body, and the stem cell niche constitutes a fundamental unit of tissue physiology, and the response of stem cells to the demands of the organism. integrate 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 niches creates a dynamic system necessary for tissue persistence and the ultimate design of stem cell therapies (Scadden, “The stem-cell niche as an entity of action,” Nature, vol. .441, pages 1075-1079, 2006). Common stem cell niches in vertebrates include the germline stem cell niche, hematopoietic stem cell niche, hair follicle stem cell niche, intestinal stem cell niche, and cardiovascular stem cell niche.

Stem cell niches are specialized environments that are distinct from other parts of the body (e.g., plasma) (Drummond-Barbosa, “Stem Cells, Their Niches and the Systemic Environment: An Aging Network,” Genetics, vol. .180, pages 1787 -1797, 2008; Fuchs, “Socializing with the Neighbors: 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 shown that bone marrow is generally hypoxic in nature (approximately 1% to 2% O2) compared to plasma (Keith et al., "Hypoxia-Inducible Factors"). , Stem Cells, and Cancer,” Cell, vol. 129, pages 465-472, 2007; Mohyeldin et al., “Oxygen in Stem Cell Biology: A Critical Component of the Stem Cell Niche,”Cell Stem Cell, vol.7 , pages 150-161, 2010). In addition, the stem cell niche is controlled by several other factors that regulate stem cell properties within the niche: extracellular matrix components, growth factors, cytokines, and factors of the physiochemical nature of the environment, e.g., pH, ionic strength ( e.g. Ca ²⁺ concentration) and metabolites.

Therefore, the stem cell niche has at least some physiological conditions that are different from physiological conditions in other parts of the body, such as physiological conditions in plasma. Stem cell niches have low oxygen concentrations (1-2%) compared to other parts of the body, especially plasma. Other physiological conditions of the stem cell niche, including pH and ionic strength, may also differ from other parts of the body.

Stem cell therapy is an interventional strategy that introduces new adult stem cells into damaged tissue to treat disease or injury. This strategy relies on the ability of stem cells to self-renew and give rise to subsequent progeny with varying degrees of differentiation potential. Stem cell therapy offers great potential for tissue regeneration, potentially capable of replacing diseased and damaged areas of the body while minimizing the risk of rejection and side effects. Therefore, delivering drugs (biological proteins (eg, antibodies) or chemical compounds) to the stem cell niche to influence stem cell regeneration and differentiation is an important part of stem cell therapy.

There are several examples of how stem cell niches affect 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 responsible for the restricted localization of stem cell populations through interactions 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 modulate stem cell sensitivity to fibroblast growth factor 2 (FGF2) and bone morphogenetic protein 4 (BMP4) resulting in increased stem cell propensity. Third, another matrix protein, the Arg-Gly-Asp-containing sialoprotein osteopontin (OPN), has now been demonstrated to contribute to hematopoietic stem cell regulation. OPN interacts with hematopoietic stem cell CD44 and 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 increased HS cell numbers because the absence of OPN results in supraphysiological stem cell expansion under stimulated conditions. Therefore, OPN appears to act as a hematopoietic stem cell number limiting factor that limits the number of stem cells under homeostatic conditions or under stimulation. Scadden, “The stem-cell niche as an entity of action,” Nature, vol. 441, pages 1075-1079, 2006.

Xie et al. “Autocrine signaling based selection of combinatorial antibodies that transdifferentiate human stem cells,” Proc Natl Acad S ci USA, vol. 110, pages 8099-8104, 2013) disclose methods of using antibodies to influence stem cell differentiation. This antibody is an agonist of the granulocyte colony stimulating factor receptor. Unlike natural granulocyte colony stimulating factor, which activates cells to differentiate along a defined pathway, this isolated agonist antibody transdifferentiated human myeloid lineage CD34+ bone marrow cells into neural progenitor cells. Melidoni et al. (“Selecting antagonistic antibodies that control differentiation through inducible expression in embryonic stem cells,”Proc Natl Acad Sci USA, vol. 110, pages 17802-17807, 2013) also used antibodies to detect FGF4 and its receptor FGFR1β. discloses a method of interfering with the interaction between FGF4 and, thus, blocking autocrine FGF4-mediated embryonic stem cell differentiation.

The understanding of the function of ligands/receptors in stem cell differentiation has enabled strategies to interfere with these ligands/receptors using biological proteins to regulate or even direct stem cell differentiation. The ability to control the differentiation of non-genetically modified human stem cells by administration of antibodies into the stem cell niche may provide novel ex vivo or in vivo approaches to stem cell-based therapy. In some embodiments, the present invention provides conditionally activated organisms produced from parent or wild-type biological proteins that have the ability to enter stem cell niches and modulate stem cell or tumor development, including cancer stem cells. provides biological proteins. This conditionally active biological protein has lower activity than the parent or wild-type biological protein under at least one physiological condition in other parts of the body, while at least one in the stem cell niche, e.g. cancer stem cell environment. It has higher activity than the parent or wild type biological protein under one physiological condition. Such conditionally active biological proteins are less likely to cause side effects and preferentially act in the stem cell niche to regulate stem cell regeneration and differentiation. In some embodiments, the conditionally active biological protein is an antibody. Such conditionally activated antibodies bind weakly or not at all to their antigen in other parts of the body, but may bind strongly and tightly to the antigen in the stem cell niche.

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

Because the activity of a conditionally active biological protein is lower in other parts of the body where it is not intended to act, such a conditionally active biological protein may be less active than the parent or wild type. It is advantageous to reduce 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 parts of the body other than the tumor microenvironment means that such This means that conditionally active biological proteins are less likely to interfere with normal physiological functions in parts of the body other than the tumor microenvironment. At the same time, conditionally active biological proteins are more active in the tumor microenvironment, which makes them more effective in treating tumors.

Because of the low side effects, this conditionally active biological protein allows for the safe use of significantly higher doses of the protein compared to the parent or wild type biological protein. This is particularly beneficial for antibodies to cytokines or growth factors, since antibodies to cytokines or growth factors can interfere with the normal physiological function of the cytokine or growth factor in other parts of the body. By using conditionally active biological proteins with lower side effects, higher doses can be used to achieve higher efficacy.

Conditionally active biological proteins acting in one of the synovial fluid, tumor microenvironment, or stem cell niches may also enable the use of novel drug targets. The use of conventional biological proteins as therapeutic agents can result in unacceptable side effects. For example, inhibition of epidermal growth factor receptor (EGFR) can be highly effective in suppressing tumor growth. However, drugs that inhibit EGFR can also inhibit growth in the skin and gastrointestinal (GI) tract. This side effect makes EGFR unsuitable as a tumor drug target. The use of conditionally activated antibodies that bind with high affinity to EGFR 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 Growth can be inhibited. In this case, by using conditionally active antibodies, EGFR may become an effective new tumor drug target.

In another example, inhibition of cytokines is often beneficial in repairing joint damage. However, suppression of cytokines in other parts of the body may also suppress the body's immune response, causing 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 use of conditionally activated antibodies that bind preferentially to cytokines in synovial fluid, but not or only weakly to the same cytokines in other parts of the body, can dramatically reduce the side effects of immunodeficiency. can be reduced. Therefore, by using conditionally active antibodies, cytokines in the synovial fluid may become suitable targets for repairing joint damage.

Conditionally Active Biological Proteins for Inflammation Prone Organs/Tissues In some embodiments, the conditionally active biological proteins are for organs/tissues that are prone to inflammation, such as lymph nodes, tonsils, adenoids, and sinuses. Designed to preferentially act on susceptible organs or tissues. For additional organs and tissues prone to inflammation, reference may be made to anatomy textbooks such as Gray's Anatomy by Henry Gray, 41 ^st edition, 2015, published by Elsevier.

These organs and tissues typically exhibit at least one abnormal condition once inflamed. For example, these inflamed organs and tissues have increased osmolarity and/or lower concentrations of one or more ions compared to normal physiological conditions in other parts of the body, such as human plasma. It can be. Furthermore, such inflamed organs and tissues may have elevated concentrations of small molecules, lactic acid, cytokines, and white blood cells when compared to normal physiological conditions in other parts of the body, such as human plasma.

In some embodiments, the present invention provides a conditionally activated biological protein using abnormal conditions selected from one or more abnormal conditions encountered in the inflammatory spectrum and normal physiological conditions in human plasma. can be made. Such a conditionally active biological protein therefore has a high activity compared to that of the parent or wild type biological protein in organs/tissues under inflammatory conditions and It may have a low activity compared to that of the protein. Such conditionally active biological proteins may act preferentially in inflamed areas of the body, but will have little or no activity in areas of the body that are not inflamed.

Conditionally Active Viral Particles Viral particles have been used for many years as delivery vehicles to transport proteins, nucleic acid molecules, chemical compounds, or radioactive isotopes to target cells or tissues. Viral particles commonly used as delivery vehicles include retroviruses, adenoviruses, lentiviruses, herpesviruses, and adeno-associated viruses. A virus particle recognizes its target cell through a surface protein that acts as a recognition protein for specific binding with a cellular protein that acts as a target protein for the target cell, often in a ligand-receptor binding system (Lentz , “The recognition event between viruses and host cell receptor: a target for antiviral agents,” J. of Gen. Virol., vol. 71, pages 75 1-765, 1990). For example, a viral recognition protein can be a ligand for a receptor on a target cell. The specificity between the ligand and the receptor allows the virus particle to specifically recognize the target cell and deliver its contents there.

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

Generally, artificial virus particles are constructed by inserting foreign recognition proteins into the virus particle, often replacing natural recognition proteins by recombinant techniques. The foreign recognition protein can be, for example, an antibody, a receptor, a ligand or a collagen binding domain. The present invention provides conditionally active recognition proteins that are inactive or have low activity in binding to cells under normal physiological conditions, and active or highly active in binding to cells under abnormal conditions. Therefore, conditionally activated recognition proteins bind preferentially to target cells in diseased tissues and/or diseased sites based on the presence of abnormal conditions at the site, and to cells in normal tissues where normal physiological conditions exist. Coupling may be avoided or minimized. Conditionally active recognition proteins can be expressed and displayed on the surface of viral particles.

In some embodiments, the invention provides methods for evolving a parental or wild-type recognition protein and screening for conditionally active recognition proteins. A conditionally active recognition protein has a lower cell-binding activity than the parent or wild-type recognition protein under normal physiological conditions, and a higher cell-binding activity than the parent or wild-type recognition protein under abnormal conditions. Insertion of such conditionally active recognition proteins into virus particles by well-known recombinant techniques can produce conditionally active virus particles.

In another embodiment, the invention provides a conditionally active virus particle that includes a conditionally active recognition protein, the conditionally active recognition protein is a conditional active virus particle that targets the conditionally active virus particle to a diseased tissue or disease site. It recognizes and binds to cells, but allows cells of normal tissue not to recognize and bind. Such conditionally active virus particles can preferentially deliver the therapeutic agent within the virus particle to diseased tissues or diseased sites, while the conditionally active virus particles deliver less therapeutic agent to cells of normal tissues. To serve or not to serve.

In some embodiments, the target cells at the disease site have an abnormal pH or temperature (e.g., pH 6.5 ) or within an area or microenvironment with abnormal temperatures. In this embodiment, the conditionally activated recognition protein has reduced binding activity with the target protein of the target cell compared to the parent or wild-type recognition protein at normal physiological pH or temperature, and at abnormal pH or temperature. It has higher binding activity with the target protein of target cells than the wild type recognition protein. This recognition protein may thus preferentially bind to sites where abnormal pH or temperature occurs, thereby delivering therapy to the disease site.

In one embodiment, the viral particle may comprise a conditionally active antibody of the invention, particularly an antibody variable region (eg, Fab, Fab', Fv). Such conditionally activated antibodies have lower affinity than the parental or wild-type antibody under normal physiological conditions, which may occur at sites including normal tissues, and with lower affinity than the parental or wild-type antibodies under abnormal conditions, which may occur at disease sites or diseased tissues. It can bind to the target protein (as an antigen) of the target cell with higher affinity. Conditionally active antibodies may 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, the tyrosine kinase growth factor receptor, which 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 Modifying Proteins DNA/RNA modifying proteins, specifically CRISPR, have been discovered as a form of novel genome manipulation tool, allowing researchers to perform microsurgery on genes. , it becomes possible to precisely and easily change the DNA sequence at a precise location on a chromosome (genome editing, Mali et al., “Cas9 as a versatile tool for engineering biology,” Nature Methods, vol. 10, pages 957-963, 2013). For example, sickle cell anemia is caused by a single base mutation, which could potentially be corrected using DNA/RNA modification proteins. This technology can precisely delete or edit small pieces of chromosomes, even by changing a single base pair (Makarova et al., “Evolution and classification 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 create multiple genetic changes in cells. Many human diseases are affected by mutations in multiple genes, including heart disease, diabetes, and neurological diseases. This CRISPR-based technology has the potential to reverse disease-causing mutations, cure these diseases, or at least reduce the severity of these diseases. Genome editing relies on CRISPR-associated (Cas) proteins (a family of enzymes) to cut genomic DNA. Typically, Cas proteins are guided to a target region within the genome by a small guide RNA, where the guide RNA is coincident with the target region. Because Cas proteins have little or no sequence specificity, guide RNAs serve as instructions for Cas proteins to achieve precise genome editing. In one embodiment, one Cas protein may be used with multiple guide RNAs to correct multiple genetic mutations simultaneously.

There are many Cas proteins. Examples include Cas1, Cas2, Cas3', Cas3'', Cas4, Cas5, Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cas10d, Csy1, Csy2, Csy3, Cse1, Cs e2 , Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx 1, Csx15 , Csf1, Csf2, Csf3, and Csf4 ((Makarova et al., “Evolution and classification of the CRISPR-Cas systems,” Nature Reviews Microbiol ogy, vol. 9, pages 467-477, 2011).

Genome editing requires Cas proteins to invade target cells. The cells of interest may have different intracellular pHs inside the cells. Some cells in the affected tissue have abnormal intracellular pH. For example, some tumor cells tend to have an alkaline intracellular pH of about 7.12 to 7.65, whereas normal tissue cells have a neutral intracellular pH in the range of 6.99 to 7.20. It has a pH. Cardone et al. , “The role of disturbed pH dynamics and the Na(+)/H(+) exchanger in metastasis,” Nat. Rev. Cancer, vol. 5, pages 786-795, 2005. In chronic hypoxia, the cells of the affected tissue have an intracellular pH of approximately 7.2-7.5, which is also higher than the intracellular pH of normal tissue (Rios et al., “Chronic hypoxia elevates intracellular pH and activates Na+/H+ exchange in pulmonary arterial smooth muscle cells,” American Journal of Physiology-Lung Cellular and Molec ular Physiology, vol. 289, pages L867-L874, 2005). Furthermore, in ischemic cells, the intracellular pH typically ranges from 6.55 to 6.65, which is lower than the intracellular pH of normal tissues (Haqberg, “Intracellular pH during ischemia in skeletal muscle: relationship to membrane ne potential, extracellular pH, tissue lactic acid and ATP, "Pflugers Arch., vol. 404, pages 342-347, 1985). A further example of abnormal intracellular pH in affected tissues is given by 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 type Cas protein, wherein the conditionally active Cas protein is: (1) under normal physiological conditions inside a normal cell; (2) an enzyme activity that is decreased compared to that of the parental or wild-type Cas protein under abnormal conditions inside the target cell, such as one of the affected cells discussed above. activity. In some embodiments, the normal physiological condition is an approximately neutral intracellular pH, and the abnormal condition is a different intracellular pH that is above or below neutral. In certain embodiments, the abnormal condition is an intracellular pH of 7.2-7.65 or an intracellular pH of 6.5-6.8.

In some embodiments, conditionally active Cas proteins can be delivered to target cells using conditionally active viral particles of the invention. A conditionally active viral particle comprises a conditionally active Cas protein and at least one guide RNA to direct the Cas protein to a location where the Cas protein will edit genomic DNA.

Multispecific antibodies have high selectivity in preferential target tissues that contain all or most of the target (antigen) to which the multispecific antibody can bind. For example, a bispecific antibody exhibits a higher preference for target cells that express both antigens recognized by the bispecific antibody compared to non-target cells that may express only one of the antigens. This provides selectivity for target cells. Therefore, due to the dynamism of this system, at equilibrium more bispecific antibodies bind to target cells compared to non-target cells.

The multispecific antibodies engineered herein, or antigen-recognizing fragments thereof, can be used as ASTRs of the chimeric antigen receptors of the invention.

Cytotoxic Cell Engineering Once a conditionally active ASTR is identified by the screening process, the polynucleotide sequences encoding the individual domains are ligated to form a single polynucleotide sequence (the CAR gene, which is a conditionally active CAR A chimeric antigen receptor can be assembled by forming a protein (encoding). Individual domains include conditionally active ASTR, TM, and ISD. In some embodiments, other domains may also be introduced into the CAR, including the ab ESD and CSD (Figure 1). If the conditionally active CAR is a bispecific CAR, the CAR gene has the following configuration, for example, in the N-terminal to C-terminal direction: N-terminal signal sequence-ASTR1-linker-ASTR2-extracellular spacer domain-membrane. It may be a transmembrane domain-co-stimulatory domain-intracellular signaling domain. In one embodiment, such a CAR gene may contain two or more costimulatory domains.

Alternatively, a polynucleotide sequence encoding a conditionally active CAR has the following configuration in N-terminal to C-terminal orientation: N-terminal signal sequence-ASTR1-linker-ASTR2-transmembrane domain-co-stimulatory domain-intracellular signaling. It may be a domain. In certain embodiments, such a CAR may include two or more co-stimulatory domains. When a CAR contains three or more ASTRs, the polynucleotide sequence encoding the CAR has the following configuration from N-terminus to C-terminus: N-terminal signal sequence - ASTR1 - linker - ASTR2 - linker - (antigen-specific target region) _n - transmembrane domain - costimulatory domain - intracellular signal transduction domain. Such CARs may further include an extracellular spacer domain. Each ASTR may be separated by a linker. In certain embodiments, such a CAR may include two or more co-stimulatory domains.

Conditionally active CAR is introduced into cytotoxic cells by an expression vector. Expression vectors containing polynucleotide sequences encoding conditionally active CARs of the invention are also provided herein. Suitable expression vectors include, but are not limited to, lentiviral vectors, gamma retroviral vectors, foamy viral vectors, adeno-associated virus (AAV) vectors, adenoviral vectors, genetically engineered hybrid viruses, naked DNA, etc. , transposon-mediated vectors such as Sleeping Beauty, Piggybak, and integrases such as Phi31. Some other suitable expression vectors include herpes simplex virus (HSV) and retroviral expression vectors.

Adenovirus expression vectors are based on adenovirus, which has a low ability to integrate into genomic DNA, but a high transfection efficiency of host cells. The adenoviral expression vector contains sufficient adenoviral sequences to (a) assist in packaging the expression vector, and (b) ultimately express the CAR gene in the host cell. The adenovirus genome is a 36 kb linear double-stranded DNA in which foreign DNA sequences (such as the CAR gene) can be inserted to replace large fragments of the adenovirus DNA to create the expression vectors of the invention. (Grunhaus and Horwitz, “Adenoviruses as cloning vectors,” Seminars Virol., vol. 3, pages 237-252, 1992).

Another expression vector is based on adeno-associated viruses, which utilizes the adenovirus coupling system. This AAV expression vector has a high frequency of integration into the host genome. It is capable of infecting even non-dividing cells, thus making it useful for gene delivery to mammalian cells, eg in tissue culture or in vivo. AAV vectors have a wide host range in terms of infectivity. Details regarding the construction and use of AAV vectors are described in US Pat. No. 5,139,941 and US Pat. No. 4,797,368.

Retroviral expression vectors have the ability to integrate into the host genome, deliver large amounts of foreign genetic material, infect a wide range of species and cell types, and be packaged into specific cell lines. Retroviral vectors are constructed by inserting a nucleic acid (eg, encoding a CAR) into a specific location in the viral genome to create a replication-defective virus. Although retroviral vectors can infect a wide variety of cell types, integration and stable expression of the CAR gene requires host cell division.

Lentiviral vectors are derived from lentiviruses, which are complex retroviruses that contain the common retroviral genes gag, pol, and env, as well as other genes with regulatory or structural functions ( (U.S. Pat. No. 6,013,516 and U.S. Pat. No. 5,994,136). Some examples of lentiviruses include human immunodeficiency virus (HIV-1, HIV-2) and simian immunodeficiency virus (SIV). Lentiviral vectors have been created by multiple attenuation of HIV virulence genes, eg, by deletion of the genes env, vif, vpr, vpu and nef, making the vector biologically safe. Lentiviral vectors have the ability to infect non-dividing cells and can be used for gene transfer and expression of CAR genes both in vivo and ex vivo (US Pat. No. 5,994,136).

Expression vectors containing conditionally active CAR genes can be introduced into host cells by any means known to those skilled in the art. The expression vector may contain viral sequences for transfection, if necessary. Alternatively, expression vectors may be introduced by fusion, electroporation, particle bombardment, transfection, lipofection, and the like. The host cells may be grown and expanded in culture prior to introduction of the expression vector, followed by appropriate treatments for introduction and integration of the vector. The host cells are then expanded and screened by markers present on the vector. A variety of 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 and stably expressing a conditionally active CAR of the invention. In one embodiment, the genetically engineered cells include T lymphocytes (T cells), naive T cells (T _N ), memory T cells (e.g., central memory T cells) that have the ability to generate therapeutically relevant progeny. (T _CM ), effector memory cells (T _EM )), natural killer cells, and macrophages. In another embodiment, the genetically engineered cells are autologous cells. Examples of suitable T cells include CD4 ⁺ /CD8 ^- , CD4 ^- /CD8 ⁺ , CD4 ^- /CD8 ^- or CD4 ⁺ /CD8 ⁺ T cells. T cells may be a mixed population of CD4 ⁺ /CD8 ⁻ and CD4 ⁻ /CD8 ⁺ cells or a single clonal population. CD4 ⁺ T cells of the invention also exhibit IL-2, IFN-γ, TNF-α, and other T cells when co-cultured in vitro with cells expressing target antigens (e.g., CD20 ⁺ and/or CD19 ⁺ tumor cells). Cellular effector cytokines may also be produced. CD8 ⁺ T cells of the invention are capable of lysing cells expressing target antigens. In some embodiments, the T cells can be any one or more of CD45RA ⁺ CD62L ⁺ naive cells, CD45RO CD62I7 central memory cells, CD62L ^" effector memory cells, or combinations thereof (Berger et al. ., “Adaptive transfer of virus-specific and tumor-specific T cell immunity,” Curr. Opin. Immunol., vol. 21, pages 224-232, 2009).

Genetically engineered cytotoxic cells can be generated by stably transfecting host cells with expression vectors containing the CAR genes of the invention. Additional methods of genetically engineering cytotoxic cells using expression vectors include chemical transformation methods (e.g., use of calcium phosphate, dendrimers, liposomes and/or cationic polymers), non-chemical transformation methods (e.g. , electroporation, optical transformation, gene electrotransfer and/or hydrodynamic delivery) and/or particle-based methods (e.g. impalefection, use of gene guns and/or magnetofection). It will be done. Transfected cells exhibiting the presence of a single unrearranged integrated vector and expressing conditionally active CAR can be expanded ex vivo.

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

After introducing the expression vector containing the CAR gene into a host cell, the CAR gene will be expressed, thus creating a CAR molecule capable of binding the target antigen. The produced CAR molecule has a transmembrane domain and thus becomes a transmembrane protein. The host cells will then be converted into CAR cells, such as CAR-T cells. For methods of generating engineered cytotoxic cells, such as CAR-T cells, containing CAR molecules, see, for example (Cartellieri et al., "Chimeric Antigen Receptor Engineered T Cells for Cancer Immunotherapy"). "Chimeric antigen receptor-engineered T cells for immunotherapy of cancer", Journal of Biomedicine and Biotechnology, vol. 201 0, Article ID 956304, 2010; and Ma et al., “Controlling the specificity and activity of engineered T cells. ``Versatile strategy for controlling the specificity and activity of engineered T cells'', PNAS, vol. 113, E450-E458, 2016) It is listed.

Whether before or after genetic modification of cytotoxic cells to express the desired conditionally active CAR, e.g., U.S. Pat. No. 6,352,694; Specification No. 055; Specification No. 6,905,680; Specification No. 6,692,964; Specification No. 5,858,358; Specification No. 6,887,466; Specification No. 6,905,681; Specification No. 7,144,575; Specification No. 7,067,318; Specification No. 7,172,869; Specification No. 7,232,566 Specification of No. 7,175,843; Specification of No. 5,883,223; Specification of No. 6,905,874; Specification of No. 6,797,514; Cells can be activated and expanded in number using methods such as those described in US Pat. No. 6,867,041; and US Patent Application Publication No. 20060121005. For example, T cells of the invention can be expanded by contacting a surface that has bound an agent that stimulates CD3/TCR complex associated signals and a ligand that stimulates co-stimulatory molecules on the surface of the T cell. In particular, T cell populations are isolated by contacting surface-immobilized anti-CD3 antibodies, or antigen-binding fragments thereof, or anti-CD2 antibodies, or by contacting protein kinase C activators (e.g., bryostatin) together with calcium ionophores. ) can be stimulated by contact with Co-stimulation of accessory molecules on the surface of T cells involves the use of ligands that bind to the accessory molecules. 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 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 are used in this book as well as other methods commonly known in the art. (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 pharmaceutical compositions comprising a pharmaceutically acceptable excipient and a therapeutically effective amount of a conditionally active CAR of the invention. The conditionally active CAR in the composition can be any one or more of a polynucleotide encoding a CAR, a protein comprising a CAR, or a genetically modified cell that expresses a CAR protein. CAR proteins may be in the form of pharmaceutically acceptable salts. Pharmaceutically acceptable salts include, for example, salts such as sodium, potassium, and calcium, and amine salts such as procaine, dibenzylamine, ethylenediamine, ethanolamine, methylglucamine, taurine, and acid addition salts such as hydrochloride. Refers to salts that can be used as salts of therapeutic proteins in the pharmaceutical industry, including salts, basic amino acids, and the like.

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

Pharmaceutically acceptable carriers will depend, in part, on the particular composition being administered, as well as on the particular method used to administer the composition. Accordingly, suitable formulations of the pharmaceutical compositions of the present invention vary widely. Various aqueous carriers can be used, such as buffered saline. These solutions are sterile and generally free of undesirable substances. These compositions may be sterilized by conventional, well-known sterilization techniques. The composition may contain appropriate pharmaceutically acceptable auxiliary substances to approximate physiological conditions, such as pH adjusting agents and buffering agents, toxicity adjusting agents, etc., for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. May contain. The concentration of CAR in these formulations can vary widely and will be selected according to the particular administration method chosen and the needs of the patient, primarily based on fluid volume, viscosity, and body weight.

In various embodiments, pharmaceutical compositions of the present invention may be formulated for delivery by any suitable route of administration. "Route of Administration" includes, but is not limited to, aerosol, nasal, oral, intravenous, intramuscular, intraperitoneal, inhalation, transmucosal, transdermal, parenteral, implantable pump, continuous infusion, topical application, capsule and It may refer to any route of administration known in the art, including/or injection.

Pharmaceutical compositions according to the invention may be encapsulated, tableted, or prepared in emulsions or syrups for oral administration. The pharmaceutical compositions are made according to conventional pharmaceutical techniques involving crushing, mixing, granulating, and compressing (if necessary) for tablet forms; or crushing, mixing, and filling for hard gelatin capsule forms. If a liquid carrier is used, the formulation may be in the form of a syrup, elixir, emulsion or aqueous or non-aqueous suspension. Such liquid formulations may be administered directly orally or filled into soft gelatin capsules.

The pharmaceutical composition comprises (a) an effective amount of the packaged nucleic acid suspended in a liquid solution, e.g., water, saline, or a diluent such as PEG 400; (b) each of a liquid, solid, granular or capsules, sachets or tablets containing a predetermined amount of the 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, dragees, capsules, solutions, 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, potato, or other plants; methylcellulose, hydroxypropyhnethyl cellulose, and sodium carboxymethylcellulose. Suitable solid excipients include carbohydrate or protein fillers, including cellulose; and gums, including arabic and tragacanth; and proteins, such as gelatin and collagen. Disintegrants or solubilizers may be added, such as cross-linked polyvinylpyrrolidone, agar, alginic acid, or its salts such as sodium alginate. The tablet form contains lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, potato starch, tragacanth, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscannellose sodium, talc, magnesium stearate, of stearic acid and other excipients, colorants, fillers, binders, diluents, buffers, wetting agents, preservatives, flavoring agents, dyes, disintegrants, and pharmaceutically acceptable carriers. may include one or more.

Liquid suspensions have the conditionally active CAR in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum arabic, and naturally occurring phosphatides (e.g. lecithin), alkylene oxides, etc. condensation products of ethylene oxide with fatty acids (e.g. polyoxyethylene stearate), condensation products of ethylene oxide with long-chain aliphatic alcohols (e.g. heptadecaethylene oxycetanol), partial esters derived from ethylene oxide and fatty acids and hexitols. Nucleic acid agents or wetting agents such as condensation products (e.g. polyethylene sorbitol monooleate) or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydride (e.g. polyoxyethylene sorbitan monooleate) may be included. I can do it. Liquid suspensions may also contain one or more preservatives, such as ethyl or n-propyl-p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and sucrose, aspartame or saccharin. may contain one or more sweeteners. The formulation may be adjusted for osmotic pressure.

Lozenges usually have the active ingredient in a flavoring such as sucrose and acacia or tragacanth, and pastilles have the active ingredient in an inert base such as gelatin and glycerin or a sucrose and acacia emulsion, a gel. In addition to these active ingredients, they can have carriers known in the art. It is understood that conditionally active CARs must be protected from digestion when administered orally. This is generally done by conjugating the conditional CAR with a composition that confers resistance to acidic and enzymatic hydrolysis, or by conjugating the conditionally active CAR to a suitable CAR, such as a liposome. This is achieved by packaging it in a resistant carrier. Methods of protecting proteins from digestion are known in the art. Pharmaceutical compositions can be encapsulated, for example, in liposomes or in formulations that provide for sustained release of the active ingredient.

The pharmaceutical compositions may be formulated as aerosol formulations (eg, they can be "nebulized") for administration by inhalation. Aerosol formulations can be placed in a pressurized acceptable propellant, such as dichlorodifluoromethane, propane, nitrogen, and the like. Formulations suitable for rectal administration include, for example, suppositories consisting of the packaging nucleic acid with a suppository base. Suitable suppository bases include natural or synthetic triglycerides or paraffinic hydrocarbons, in addition to gelatin, which consists of combinations of packaging nucleic acids with bases containing, for example, liquid triglycerides, polyethylene glycols, and paraffinic hydrocarbons. It is also possible to use rectal capsules.

The pharmaceutical compositions may be formulated for parenteral administration, such as by intra-articular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes. aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostatic agents, and solutes to render the formulation isotonic with the blood of the intended recipient; In the practice of this invention, the compositions may include sterile aqueous and non-aqueous suspensions that may contain clouding agents, solubilizing agents, thickening agents, stabilizers, and preservatives; for example, by intravenous infusion. It can be administered orally, topically, intraperitoneally, intravesically or intrathecally. In one aspect, parenteral administration is a preferred method of administration for compositions comprising CAR proteins or genetically engineered cytotoxic cells. The compositions may conveniently be administered in unit dosage form, such as those available from Remington's Pharmaceutical Sciences, Mack Publishing Co. Easton Pa. , ^18th Ed. , 1990, by any method well known in the pharmaceutical art. Formulations for intravenous administration may contain pharmaceutically acceptable carriers such as sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes, and the like.

The pharmaceutical composition may be administered parenterally, subcutaneously, intramuscularly, intravenously, intraarticularly, intrabronchially, intraperitoneally, intracapsularly, intrachondrally, intrasinally, intracavitally, intracerebellarly, intracerebroventricularly, colonically. Internal, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, synovial fluid. Administration may be by at least one method selected from intracapsular, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal. The method optionally includes detectable labels or reporters, TNF antagonists, antirheumatic drugs, muscle relaxants, narcotics, non-steroidal anti-inflammatory drugs (NSAK), before, simultaneously with, or after conditionally activated CAR. )), analgesics, anesthetics, sedatives, local anesthetics, neuromuscular blockers, antibacterial agents, antipsoriatics, corticosteroids, anabolic steroids, erythropoietin, immunizations, immunoglobulins, immunosuppressants, Metabolism of growth hormone, hormone replacement drugs, radiopharmaceuticals, antidepressants, antipsychotics, stimulants, asthma drugs, beta agonists, inhaled corticosteroids, epinephrine or its analogues, cytotoxic drugs or other anticancer drugs, methotrexate, etc. The method may further include 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 an anti-proliferative agent.

Types of cancers treated with the genetically engineered cytotoxic cells or pharmaceutical compositions of the invention include carcinomas, blastomas, and sarcomas, and certain leukemias or lymphoid malignancies, benign and malignant tumors, and Malignant diseases include sarcomas, carcinomas, and melanomas. The cancer may be a non-solid tumor (such as a hematologic tumor) or a solid tumor. Also included are adult tumors/cancers and pediatric tumors/cancers.

Hematological cancers are cancers of the blood or bone marrow. Examples of blood cancers (or hematogenous cancers) include leukemias, such as acute leukemias (acute lymphocytic leukemia, acute myelocytic leukemia, acute myelocytic leukemia as well as myeloblastic, promyelocytic, myelomonocytic) , monocytic and erythroleukemia), chronic leukemia (including chronic myelocytic (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high-grade forms), multiple myeloma, Waldenström's macroglobulinemia, heavy chain disease, myelodysplastic syndromes, hairy cell leukemia, and myelodysplasia.

Solid tumors are abnormal tissue masses that usually do not contain cysts or areas of fluid. 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 sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synoviomas, mesothelioma, Ewing's tumor, leiomyosarcoma, striated muscle cancer, colon cancer, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, brown Cytoma, sebaceous carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, Wilms tumor, cervical cancer, testicular tumor, seminoma, bladder cancer , melanoma, and CNS tumors (such as gliomas (such as brainstem gliomas and mixed gliomas), glioblastomas (also known as glioblastoma multiforme), astrocytomas, CNS lymphomas, embryonic Cytoma, medulloblastoma, schwannoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma and brain metastases).

The invention also provides medical devices comprising at least one CAR protein, a polynucleotide sequence encoding a CAR, or a host cell expressing a CAR, which devices can be administered parenterally, subcutaneously, intramuscularly, intravenously, intraarticularly. intraticular, intrabronchial, intraperitoneal, intrasaccular, intrachondral, intrasinus, intracavity, intracerebellar, intraventricular, intracolon, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, pelvic intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal It is suitable to administer the at least one conditionally active CAR by at least one method selected from lateral, sublingual, intranasal, or transdermal.

In a further aspect, the invention provides at least one CAR protein, a polynucleotide sequence encoding a CAR, or a host cell expressing a CAR in lyophilized form in a first container and sterile water or sterile buffered water. or phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride, alkylparaben, benzalkonium chloride in an aqueous diluent. an optional second container comprising at least one preservative selected from the group consisting of: , benzethonium chloride, sodium dehydroacetate and thimerosal, or mixtures thereof. In one embodiment, in the kit, the concentration of the conditionally active CAR or specific portion or variant in the first container is from about 0.1 mg/ml to about 500 mg/ml in the second container. reconstituted to a concentration of In another embodiment, the second container further includes an isotonic agent. In another embodiment, the second container further includes a physiologically acceptable buffer. In one aspect, the present disclosure provides a method of treating a condition mediated by at least one wild-type protein, comprising administering to a patient in need thereof a formulation provided in a kit and reconstituted prior to administration. A method is provided comprising steps.

Also provided is a product for human medical or diagnostic use comprising a container and packaging material containing at least one CAR protein, a polynucleotide sequence encoding a CAR, or a host cell expressing a CAR in solution or lyophilized form. . The product may optionally be administered parenterally, subcutaneously, intramuscularly, intravenously, intraarticularly, intrabronchially, intraperitoneally, intracapsularly, intrachondrally, intrasinally, intracavitally, intracerebellarly, intracerebroventricularly. , intracolon, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, pelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, This may include having the container as a component of an intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal delivery device or system.

In some embodiments, the invention provides the steps of: recovering cytotoxic cells from a subject; genetically manipulating the cytotoxic cells by introducing a CAR gene of the invention into the cytotoxic cells; administering the engineered cytotoxic cells to a subject. 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, the 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, umbilical cord blood, thymus tissue, tissue at the site of infection, ascites, pleural fluid, spleen tissue, and tumors. In certain embodiments of the invention, any T cell line available in the art may 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 skilled in the art, such as Ficoll™ separation.

In one preferred embodiment, cells from an individual's circulating blood are obtained by apheresis. Apheresis products typically contain lymphocytes such as T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells collected by apheresis can be washed to remove the plasma fraction and place the cells 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 alternative embodiments, the cleaning 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 one of ordinary skill in the art will readily understand, washing steps can include the use of a semi-automatic "flow-through" centrifuge (e.g., Cobe 2991 Cell Processor, Baxter CytoMate, or Haemonetics Cell Saver 5) according to the manufacturer's instructions. This can be achieved by methods known to those skilled in the art. After washing, cells can be resuspended in a variety of biocompatible buffers, such as Ca ²⁺ -free, Mg ²⁺ -free PBS, PlasmaLyte A, or another saline solution with or without buffer. . Alternatively, undesired components of the apheresis sample may be removed and cells resuspended directly in culture medium.

In another embodiment, T cells are isolated from peripheral blood by lysing red blood cells and centrifuging, for example in a PERCOLL™ gradient, or depleting monocytes by countercurrent centrifugal elutriation. Certain 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 directed against surface markers unique to the cells being negatively selected. One method is cell sorting and/or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on the cells to be negatively selected. To enrich CD4 ⁺ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In certain embodiments, it may be desirable to enrich or positively select regulatory T cells that typically express CD4 ⁺ , CD25 ⁺ , CD62L ^hi , GITR ⁺ , and FoxP3 ⁺ .

For example, in one embodiment, T cells are combined with anti-CD3/anti-CD28 (i.e., 3x28) conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T, for positive selection of desired T cells. Isolated by incubation for a sufficient period of time. In one embodiment, this time is about 30 minutes. In further embodiments, the time ranges from 30 minutes to 36 hours or more, and all integer values therebetween. In further embodiments, this time is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, this time is 10-24 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 yield. Longer incubation times are useful for isolating T cells in any situation where T cells are scarce when compared to other cell types, such as isolation of tumor infiltrating lymphocytes (TILs) from tumor tissue or immunocompromised individuals. Can be used. Furthermore, using longer incubation times may increase the efficiency of capturing CD8+ T cells. Therefore, by simply shortening or prolonging the time that T cells are allowed to bind to CD3/CD28 beads and/or increasing or decreasing the bead to T cell ratio (as further described herein). ), preferential positive or negative selection of some T cell populations can be performed at the beginning of culture or at other points 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 positive activation of some T cell populations at the beginning of culture or other points during the process can be achieved. Or you can make a negative selection. 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 perform a selection procedure and use "unselected" cells for the activation and expansion process. "Unselected" cells can also be subjected to further rounds of selection.

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

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

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

The following examples are illustrative of the methods of this disclosure and are not limiting. Other suitable modifications and adaptations of the various conditions and parameters commonly encountered in the field, which will be apparent to those skilled in the art, are within the scope of this disclosure.

Example 1: Generation of scFv conditionally activated antibodies against Axl Two conditionally activated single chain antibodies (CAB-scFv-63.9-4 and CAB-scFv-63.9-6) against drug target antigen Axl was expressed as a homodimer with wild-type human IgG1 Fc, SEQ ID NO: 13 or 14 (bivalent antibody CAB-scFv-63.9-4-01, SEQ ID NO: 9, and CAB -scFv-63.9-6-01, SEQ ID NO: 10), and expressed as a heterodimer in a knob-in-hole system to obtain a monovalent scFv (Fig. Monovalent antibodies 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 to drug target antigen Axl at pH 6.0 and pH 7.4 was measured by ELISA analysis. As shown in Figure 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 fully bivalent antibodies. Furthermore, as shown in Figure 3, the selectivity of these scFv antibodies at pH 6.0 compared to pH 7.4 was also comparable to fully bivalent antibodies. This example demonstrated that the conditionally activated antibodies of the invention have comparable affinity and selectivity as either scFv antibodies or fully bivalent antibodies. Thus, the conditionally active antibodies of the invention may be inserted as a single DNA strand into a DNA molecule encoding a CAR in the CAR-T platform of the invention.

Example 2: scFv antibodies against target antigen Axl for constructing CAR-T cells In one embodiment of the invention, simultaneously screened for selectivity and affinity and expression levels at both pH 6.0 and pH 7.4. By doing so, a conditionally activated antibody against the drug target antigen Axl was generated. Screening was performed using the FLAG tag in serum, as there were human antibodies in serum that could produce false positives for screening. The screening buffer was carbonate buffer (Krebs buffer containing Ringer's standard buffer but different from PBS). The conditionally activated antibodies generated both have higher affinity for the drug target antigen Axl at pH 6.0, but at pH 7.4 for the same drug target antigen Axl, both in comparison to the wild type antibody. It was found that the affinity was lower. Furthermore, these conditionally active antibodies all have high expression levels, as shown in Table 2 below (column "Clone" indicates the antibody and expression level "mg/ml" is indicated in the second column). has.

These antibody clones were sent to the service provider along with the requested expression level ("order quantity", expected expression level). However, the actual expression levels ("delivery") of these antibodies were extremely high and exceeded the expected expression levels.

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

The conditionally activated antibody was also analyzed using surface plasmon resonance (SPR) to measure its on and off kinetics towards the drug target antigen Axl. SPR analysis is known to measure the on and off rates of conditionally active antibodies. SPR analysis was performed in the presence of bicarbonate. The on and off kinetics of conditionally active antibodies in vivo (in animals and humans) is a critically important characteristic for conditionally active antibodies.

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

Conditionally 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), which were ready for immediate insertion into CARs on the CAR-T platform. Ta. Antibodies were incubated at 60°C for 1 hour, but the affinity of many of the antibodies did not change ("thermostability"). In the two columns reporting binding activity measurement data at pH 6.0 and pH 7.4 using SPR (last two columns of Table 3), "BAP063.6-hum10F10-FLAG" (negative control, Table 3 (second column). The selectivity of these antibodies can be determined by the difference between the two final column data. The two scFv antibodies had extremely high selectivity (75% and 50% at pH 6 versus 0% at pH 7.4).

Comparative Example A: CAR-T cells with non-conditionally activated antibodies against target antigen Axl A non-conditionally activated scFv antibody against target antigen Axl is used to express target antigen Axl or target antigen Axl on the cell surface. CAR-T cells that bind to CHO cells (CHO-Axl) were constructed, FIGS. 7A to 7B. A non-conditionally activated antibody was used as the ASTR of the CAR molecule, and this 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 were treated with (1) T cells not transduced with a CAR molecule, (2) T cells transduced with a CAR molecule that does not bind to the target antigen Axl, and (3) the target. were treated with T cells transduced with CAR molecules bearing non-conditionally activated antibodies 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 before adding T cells. After adding CAR-T cells that bind to the target antigen Axl, the cell index initially decreased, suggesting non-specific cytotoxicity of T cells. However, CHO cells resumed growth shortly thereafter. More importantly, the differences between the three treatments were not significant, with CAR-T cells bearing non-conditionally activated antibodies against the target antigen Axl having significant cytotoxicity against CHO cells not expressing the target antigen Axl. It was shown that there is no

Next, CHO cells expressing the target antigen Axl were treated in the same manner as above: (1) T cells not transduced with a CAR molecule, (2) T cells transduced with a CAR molecule that does not bind to the target antigen Axl, and (3) treated with T cells transduced with CAR molecules carrying non-conditionally activated antibodies against the target antigen Axl (FIG. 7B). After addition of T cells, the cell index was significantly reduced by treatment with CAR-T cells carrying non-conditionally activated antibodies against the target antigen Axl, but not by the other two treatments, Cytotoxicity of CAR-T cells with non-conditionally active antibodies against Axl against CHO-Axl cells expressing the target antigen Axl is shown.

Example 3: CAR-T cells with conditionally active scFv antibodies against target antigen Axl CAR molecules were constructed using conditionally active scFv antibodies against target antigen Axl. T cells were transduced with this CAR molecule so that they 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 molecules) were used as a control (Figures 8A-8B).

Referring to FIG. 8A, CHO cells that do not express the target antigen Axl were treated with CAR-T cells and non-transduced T cells. There was no significant difference between these two treatments, indicating no cytotoxicity of CAR-T cells towards CHO cells. Referring to Figure 8B, in which CHO cells expressing the target antigen Axl (CHO-63) were similarly treated, CAR-T cells with conditionally activated antibodies against the target antigen Axl were shown to be CHO-63 compared to untransduced T cells. -63 cell population was significantly reduced. This demonstrated that CAR-T cells with conditionally activated antibodies 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 Figures 9A-9B. In Figure 9A, binding of CAR-T cells bearing the target antigen Axl to CHO-63 cells resulted in a significant increase in INFg as indicated by the observed increase in cytokine levels compared to non-transduced T cells. caused the release. Similarly, in FIG. 9B, binding of CAR-T cells bearing the target antigen Axl to CHO-63 cells was shown to increase IL2 as indicated by the increased cytokine levels observed compared to non-transduced T cells. caused significant release of

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

One of the scFv antibodies shown in FIG. 10, scFv-116101, was used to construct a CAR molecule and 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 are not transduced with CAR molecules (non-transduced T cells) and CAR-T cells that are transduced with CAR molecules that do not have the ability to bind to the target antigen ROR2 (non-ROR2 scFv CAR-T). Met. The results are shown in Figure 11A. The ratio between the number of T cells and 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 Figure 11A. was induced.

HEK293 cells were treated with the same T cells used to treat Daudi cells. The results are shown in Figure 11B. Since HEK293 cells do not express target antigen ROR2 on the cell surface, CAR-T cells with scFv antibody targeting target antigen ROR2 (116101 CAR-T) showed significant cell death in HEK293 cells when compared to negative control. (Fig. 11B).

Example 5: Cytokine release of CAR-T cells bearing antibodies against target antigens Axl and ROR2 of Examples 1-4 In this example, cytokine release induced by binding of CAR-T cells to target antigens was measured. Figures 12A-12B show INFg and IL2 release after CAR-T cells containing conditionally active scFv antibodies against target antigen Axl bind to CHO-63 cells expressing target antigen Axl. After treating CAR-T cells with these CHO-63 cells for 24 hours, there was a significant increase in both INFg and IL2 cytokine levels, compared to CHO cells that do not express the target antigen Axl using the same CAR-T cells. The release of both INFg and IL2 cytokines compared to the control treated with was demonstrated. Furthermore, T cells that were not transduced with CAR molecules and CAR-T cells that did not bind the target antigen Axl did not result in significant release of INFg and IL2 cytokines.

Figures 13A-13B show INFg and IL cytokine levels after CAR-T cells containing conditionally active scFv antibodies against target antigen ROR2 bind to Large B cells and Daudi cells, both expressing target antigen ROR2. shows. After treating Raji B cells and Daudi cells with CAR-T cells for 24 hours, there was a significant increase in INFg and IL2 cytokine levels compared to a control in which the same CAR-T cells were used to treat HEK293 cells, which do not express the target antigen ROR2. 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 in CAR-T cells that did not bind the target antigen ROR2, thereby failing to induce significant release of INFg and IL2 cytokines. It was shown that

Example 6: Conditionally activated scFv antibodies against target antigen CD22 Five conditionally active scFv antibodies against target antigen CD22 were selected. The selected conditionally active scFv antibodies are 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, although the foregoing description indicates many features and advantages of the invention, as well as details of the structure and function of the invention, this disclosure is intended to be illustrative only, and the details, particularly the shape, size of the elements, etc. It is to be understood that changes may be made within the principles of the invention in terms of structure and arrangement to the fullest extent indicated by the broad general meaning of the terms expressing the appended claims. be.

Claims (20)

a chimeric antigen receptor for binding a tumor-specific target antigen, comprising:
i. at least one antigen-specific target region evolved from a parent or wild-type protein or domain thereof, wherein the activity of the antigen in assays under normal physiological conditions is compared to that in assays in abnormal conditions deviating from normal physiological conditions; at least one antigen-specific target region in which the activity of the specific target region is reduced;
ii. a transmembrane domain; and iii. contains an intracellular signaling domain;
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, or
A chimeric antigen receptor, 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 chimeric antigen receptor according to claim 1, wherein the tumor-specific target antigen is Axl and the at least one antigen-specific target region is a single chain antibody having an amino acid sequence selected from SEQ ID NOs: 9-12. body. 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. A claim in which the normal physiological conditions and abnormal conditions are the same conditions selected from temperature, pH, osmotic pressure, osmolarity, oxidative stress, electrolyte concentration, small organic molecule concentration, inorganic molecule concentration, cell type, and nutrient availability. Item 1. The chimeric antigen receptor according to item 1. 2. The chimeric antigen receptor according to claim 1, wherein the normal physiological condition is the normal physiological pH in the plasma of a mammalian subject, and the abnormal condition is the pH in the tumor microenvironment. 6. The chimeric antigen receptor according to claim 5 , wherein the normal physiological pH ranges from greater than 7.0 to 7.8. The chimeric antigen receptor according to claim 6 , wherein the normal physiological pH is in the range of 7.2 to 7.6. The chimeric antigen receptor according to claim 5 , wherein the abnormal pH ranges from 6.0 to less than 7.0. The chimeric antigen receptor according to claim 8 , wherein the abnormal pH is in the range of 6.0 to 6.8. 2. The chimeric antigen receptor of claim 1, further comprising an extracellular spacer domain or at least one costimulatory domain. The chimeric antigen of claim 10 , 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 combinations thereof. receptor. 2. The chimeric antigen receptor of claim 1, wherein at least one antigen-specific target region has a ratio of activity under abnormal conditions to the same activity under normal physiological conditions of at least 2. 2. The chimeric antigen receptor of claim 1, wherein the at least one antigen-specific target region comprises two antigen-specific target regions connected by a linker. 14. The chimeric antigen receptor of claim 13 , wherein the two antigen-specific target regions each bind different target antigens or different epitopes of the same target antigen. The transmembrane domain is an artificial hydrophobic sequence and a transmembrane domain of a type I transmembrane protein, an alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, 2. The chimeric antigen receptor of claim 1 selected from the group consisting of CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. Intracellular signaling domain includes the cytoplasmic signaling domain of human CD3 zeta chain, FcyRIII, FcsRI, cytoplasmic tail of Fc receptor, cytoplasmic receptor with immunoreceptor tyrosine-based activation motif (ITAM), TCR zeta, FcR 2. The chimeric antigen receptor of claim 1, 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, DaplO, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS LIGHT, NKG2C, and The chimeric antigen receptor of claim 1, further comprising a costimulatory domain selected from the group consisting of the costimulatory domains of B7-H3. An expression vector comprising a polynucleotide sequence encoding the chimeric antigen receptor of claim 1. The expression vector is derived from lentiviral vectors, gammaretroviral vectors, foamy viral vectors, adeno-associated viral vectors, adenoviral vectors, poxvirus vectors, herpesvirus vectors, genetically engineered hybrid viruses, and transposon-mediated vectors. 19. The expression vector according to claim 18 , selected from the group consisting of: A genetically engineered cytotoxic cell comprising a polynucleotide sequence encoding a chimeric antigen receptor according to claim 1.