TWI835848B - Conditionally active chimeric antigen receptors for modified t-cells - Google Patents

Abstract

This disclosure relates to a chimeric antigen receptor for binding with a tumor specific target antigen. The chimeric antigen receptor comprises at least one antigen specific targeting region evolved from a parent protein or a fragment thereof and having a decrease in activity in the assay at the normal physiological condition compared to the activity in the assay under the aberrant condition. A method for producing the chimeric antigen receptor is also provided.

Description

Conditionally active chimeric antigen receptors in engineered T cells

The present invention is related to the field of protein evolution. In particular, the present invention relates to a method of generating conditionally active chimeric antigen receptors from parental or wild-type proteins. Conditionally active chimeric antigen receptors are reversibly or irreversibly inactive under wild-type normal physiological conditions but are active under abnormal conditions.

A large literature describes the possibility of evolving proteins, especially enzymes, for a variety of properties. For example, enzymes can be evolved to operate stably under different conditions, such as at high temperatures. In cases where there is an increase in activity at high temperatures, much of the increase can be attributed to higher kinetic activity, which is often described by the Q10 rule, where it is estimated that in the case of enzymes, turnover doubles for every 10°C increase.

In addition, there are examples of natural mutations that destabilize proteins under their normal operating conditions. Certain mutants may be active at lower temperatures, but at reduced levels compared to the parent or wild-type protein. This is also typically described by a reduction in activity as directed by Q10 or similar rules.

It is necessary to generate suitable molecules for conditional activation. For example, it is desirable to produce molecules that are nearly inactive under wild-type operating conditions, but are active under conditions other than wild-type operating conditions, at levels equal to or better than wild-type operating conditions, or that are activated or inactive in certain microenvironments. active or deactivated over time. In addition to temperature, other conditions under which proteins can evolve or be 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.

A variety of strategies have been published for evolving or engineering molecules. However, a protein is engineered or evolved to be inactive or nearly inactive (less than 10% activity and preferably less than 1% activity) under wild-type operating conditions, while maintaining the same activity as its corresponding parent or wild-type protein in addition to the wild-type protein. Equivalent or better activity under conditions other than type operating conditions requires destabilization of coexisting mutations where the increased activity does not offset the destabilizing mutation. Destabilization is expected to reduce protein activity greater than that predicted by standard rules such as Q10. Thus, the ability to evolve proteins to function efficiently at lower temperatures, eg, while being inactive under normal operating conditions corresponding to the parent or wild-type protein, yields unexpectedly novel classes of proteins.

Chimeric antigen receptors (CARs) have been used to treat cancer. US 2013/0280220 discloses methods and compositions that provide improved cells encoding chimeric antigen receptors specific for two or more antigens, including tumor antigens. Cells expressing chimeric antigen receptors can be used in cell therapy. Such cell therapy may be applied to any medical condition, but in certain embodiments, the cell therapy is used for cancer, including cancer involving solid tumors.

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

Throughout this application, various publications are cited by author and date. The disclosures of these publications are hereby incorporated by reference in their entirety into this application in order to more fully describe the current state of the art known to those skilled in the art, including the disclosures described and claimed herein.

In one aspect, the invention provides a chimeric antigen receptor (CAR) for binding to a tumor-specific target antigen. The chimeric antigen receptor contains at least one antigen-specific targeting 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 targeting region has reduced activity when assayed under normal physiological conditions 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 vector is selected from lentiviral vectors, gamma retroviral vectors, foamy virus vectors, adeno-associated virus vectors, adenovirus vectors, poxvirus vectors, herpes virus vectors, engineered hybrid viruses and transposon-mediated vectors.

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

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

In yet another aspect, the present invention provides a method for generating a chimeric antigen receptor comprising at least one antigen-specific targeting region, a transmembrane domain, and an intracellular signaling domain. The method includes the step of generating at least one antigen-specific targeting region from a parent protein or domain thereof that specifically binds to a tumor-specific target antigen. Such steps include (i) using one or more evolution techniques to evolve DNA encoding the parent or wild-type protein or domains thereof to generate mutant DNA; (ii) expressing the mutant DNA to obtain a mutant polypeptide; (iii) expressing the mutant polypeptide Subject to analysis under normal physiological conditions and analysis under abnormal conditions; and (iv) select at least one antigen-specific targeting region from the mutant polypeptide expressed in step (iii) that exhibits a greater expression compared to analysis under abnormal conditions. The activity in the assay was reduced under normal physiological conditions.

Related application information

This application is a continuation-in-part of U.S. Patent Application No. 16/053,166 filed on August 2, 2018, currently pending, which is a divisional of now abandoned U.S. Patent Application No. 15/052,487, which is a continuation-in-part of International Application No. PCT/US15/47197 filed on August 27, 2015 and designating the United States, which claims the benefit of U.S. Provisional Application No. 62/043,067 filed on August 28, 2014, now expired, all of which are hereby incorporated by reference in their entirety. Definitions

To facilitate understanding of the examples provided herein, certain frequently occurring methods and/or terms will be defined.

As used herein, with respect to a quantity measured, the term "approximately" means that a person skilled in the art would expect that the measurement and operation would be consistent with the purpose of the measurement and the accuracy of the measuring equipment used for the quantity concerned. Match the normal variation in quantity measurements. Unless otherwise specified, "approximately" means a variation of +/-10% of the value provided.

The term "agent" is used herein to refer to chemical compounds, mixtures of chemical compounds, arrays of spatially localized compounds (e.g., VLSIPS peptide arrays, polynucleotide arrays, and/or combinatorial small molecule arrays), biological macromolecules, phage peptides Presentation libraries, phage antibody (eg scFv) presentation libraries, polyribosomal peptide presentation libraries or extracts made from biological material such as bacterial, plant, fungal or animal (certain mammalian) cells or tissues. The potential enzymatic activity of the agents is assessed by inclusion in the screening assays described herein below. Agents are evaluated for their potential activity as conditionally active biotherapeutic enzymes by inclusion in the screening assays described herein below.

As used herein, the term "amino acid" refers to any organic compound containing an amine group ( _--NH2 ) and a carboxyl group (--COOH); preferably in the form of a free radical or as part of a peptide bond after condensation part. The "twenty alpha amino acids that form naturally encoded polypeptides" are understood in the art and refer to: alanine (ala or A), arginine (arg or R), asparagine ( asn or N), aspartic acid (asp or D), cysteine (cys or C), glutamic acid (glu or E), glutamine (gin or Q), glycine (gly or G), histamine (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).

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

As used herein, the term "antibody" refers to intact immunoglobulin molecules as well as fragments of immunoglobulin molecules capable of binding to epitopes of an antigen, such as Fab, Fab', (Fab')2, Fv, and SCA fragments. Such antibody fragments may be prepared using methods well known in the art (see, e.g., Harlow and Lane, supra) and are further described below, retaining certain antigen (e.g., polypeptide antigen) selection from the antibody from which they are derived. The capacity for sexual union. Antibodies can be used to isolate prepared amounts of antigen by immunoaffinity chromatography. Various other uses of such antibodies are therapeutic applications for diagnosing and/or staging diseases, such as neoplasia, and for treating diseases such as neoplasia, autoimmune diseases, AIDS, cardiovascular disease , infections and similar diseases. Chimeric, human-like, humanized or fully human antibodies are particularly suitable for administration to human patients.

Fab fragments consist of the monovalent antigen-binding fragment of an antibody molecule and can be produced by digesting whole antibody molecules with the enzyme papain, yielding a fragment consisting of the entire light chain and a portion of the heavy chain.

Fab' fragments of antibody molecules can be obtained by treating the whole antibody molecule with pepsin and then reducing it, resulting in a molecule consisting of an entire light chain and a portion of a heavy chain. Two Fab' fragments are obtained per antibody molecule treated in this way.

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

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

The term "antigen" or "Ag" as used herein is defined as a molecule that causes an immune response. This immune response may involve antibody production or activation of specific immunocompetent cells or both. Those skilled in the art will understand that any macromolecule, including almost all proteins or peptides, can serve as an antigen. In addition, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA including a nucleotide sequence or a partial nucleotide sequence encoding a protein that causes an immune response thereby encodes the term "antigen" as used herein. In addition, those skilled in the art will understand that an antigen need not be encoded by the full length of the nucleotide sequence of a gene. Obviously, the present invention includes (but is not limited to) the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to encode polypeptides that cause the desired immune response. In addition, those skilled in the art will appreciate that antigens need not be "genetically" encoded at all. Obviously, antigens can be produced, synthesized, or derived from biological samples. Such biological samples may include (but are not limited to) tissue samples, tumor samples, cells, or biological fluids.

As used herein, "antigen-deficient escape mutants" refer to cells that display reduced or absent expression of target antigens that are targeted by the CAR of the present invention.

As used herein, the term "autoimmune disease" is defined as a condition resulting from an autoimmune response. Autoimmune diseases are the result of inappropriate and excessive responses to self-antigens. Examples of autoimmune diseases include, inter alia, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune mumps, and Crohn's disease. , diabetes mellitus (type 1), dystrophic epidermolysis bullosa, adhesions, glomerulonephritis, Graves' disease, Guillain-Barr syndrome, Hashimoto's disease ), hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sugarhren's syndrome (Sjogren's syndrome), spondyloarthropathy, thyroiditis, vasculitis, vitiligo, mucinous adenoma, pernicious anemia, ulcerative colitis.

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

As used herein, the term "B cell-related disease" includes B cell immunodeficiencies, autoimmune diseases, and/or excessive/uncontrolled cell proliferation related to B cells (including lymphoma and/or leukemia). Examples of such diseases for which the bispecific CARs of the invention may be used in methods of treatment 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, agammaglobulinemia in X-linked blood, pre-B Acute lymphoblastic leukemia, systemic lupus erythematosus, common variant immunodeficiency disorder, chronic lymphocytic leukemia, diseases associated with selective IgA deficiency and/or IgG subclass deficiency, B-lineage lymphoma (Host Hodgkin's lymphoma and/or non-Hodgkin's lymphoma), immunodeficiency with thymoma, transient hypogammaglobulinemia and/or hyperIgM syndrome, and viral mediators Caused B cell diseases, such as EBV-mediated lymphoproliferative diseases and chronic infections in which B cells are involved in the pathophysiology.

The term "blood-brain barrier" or "BBB" refers to the physiological barrier between the peripheral circulation and the brain and spinal cord, which is formed by tight binding within the endothelial membrane of brain capillaries that produces limiting molecules, even very small molecules such as urea (60 daltons)) to the brain. The blood-brain barrier in the brain, the blood-spinal cord barrier in the spinal cord, and the blood-retina 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 encompasses the blood-cerebrospinal fluid barrier (choroid plexus) when the barrier includes ependymal cells (rather than capillary endothelial cells).

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

As used herein, the term "chimeric antigen receptor" or "CAR" or "CARs" refers to engineered receptors that specifically graft antigens onto cytotoxic cells, such as T cells, NK cells, and macrophages. body. The CAR of the present invention may include at least one antigen-specific targeting region (ASTR), extracellular spacer domain (ESD), transmembrane domain (TM), one or more costimulatory domains (CSD) and intracellular signaling domain (ISD). In one embodiment, ESD and/or CSD are optional. In another embodiment, the CAR is a bispecific CAR specific for two different antigens or epitopes. After ASTR specifically binds to the target antigen, ISD activates intracellular signaling. For example, ISD can exploit the antigen-binding properties of antibodies to redirect T cell specificity and reactivity toward a selected target in a non-MHC-restricted manner. Non-MHC-restricted antigen recognition confers the ability of CAR-expressing T cells to recognize antigens independently of antigen processing, thus bypassing major tumor escape mechanisms. In addition, when expressed in T cells, CAR should not dimerize with endogenous T cell receptor (TCR) α and β chains.

The term "co-expression" as used herein refers to the simultaneous expression of two or more genes. A gene may be a nucleic acid encoding, for example, a single protein or a chimeric protein in the form of a single polypeptide chain. For example, a CAR of the invention can be co-expressed with a therapeutic control, such as epidermal growth factor truncated (EGFRt), wherein the CAR is encoded by a first polynucleotide strand and the therapeutic control is encoded by a second polynucleotide strand. Encoding. In one embodiment, 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 connected via a linker but are actually encoded by, for example, two different vectors.

As used herein, the term "homologous" refers to gene sequences that are functionally related between evolutionary and species. For example, but not limited to, in the human genome, the human CD4 gene is a homologous gene of the mouse 3d4 gene, because the sequence and structure of these two genes indicate that they are highly homologous and both genes encode proteins that play a role in signaling T cell activation through MHC class II 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. Such conditionally active proteins also exhibit activity in selected areas of the body and/or exhibit increased or decreased activity under abnormal (or permissive) physiological conditions. As used herein, the term "normal physiological conditions" refers to temperature; pH; osmotic pressure; weight molar osmotic concentration; oxidative pressure; electrolyte concentration; concentrations of small organic molecules, such as glucose, lactate, pyruvate, nutrient components, other metabolites and the like; concentrations of other molecules, such as oxygen, carbonate, phosphate and carbon dioxide; and cell type; and one of nutrient availability, which would be considered within the normal range for a tissue or organ at the site of administration to a subject or at the site of action.

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

The term "abnormal conditions" as used herein refers to conditions that deviate from the normal acceptable range for those conditions. In one aspect, a conditionally active biological protein is nearly inactive under normal physiological conditions but is active under abnormal conditions at a level equal to or better than the parent or wild-type protein from which it is derived. For example, in one aspect, a conditionally active biological protein evolved to be nearly inactive at body temperature but active at lower temperatures. In another aspect, the conditionally active biological protein is reversibly or irreversibly inactivated under normal physiological conditions. In another aspect, the parent or wild-type protein is a therapeutic protein. In another aspect, the conditionally active biological protein is used as a drug or therapeutic agent. In yet another aspect, the protein is more or less active in highly oxygenated blood, such as the lower pH environment seen after passage through the lungs or in the kidneys.

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

The term "corresponds to" is used herein to mean that a polynucleotide sequence is homologous (i.e., identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to that of a reference polynucleotide sequence. The peptide sequences are identical. In contrast, the term "complementary to" is used to mean that the complementary sequence is homologous to all or a portion of the reference polynucleotide sequence. For purposes of illustration, the nucleotide sequence "TATAC" corresponds to the reference "TATAC" and is complementary to the reference sequence "GTATA".

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

As used herein, the term "co-stimulatory molecule" refers to a molecule that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response of T cells, such as (but not limited to) a cognate binding partner on a proliferating T cell. Co-stimulatory molecules include (but are not limited to) class 1 MHC molecules, BTLA, and Toll ligand receptors.

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

As used herein, the term "cytotoxic cell" means a cell that can damage or destroy invading microorganisms, tumor cells, or other diseased tissue cells. This term is intended to include natural killer (NK) cells, activated NK cells, neutrophils, T cells, eosinophils, eosinophils, B cells, macrophages, and lymphokine-activated killer (LAK) cells, as well as other cell types. Cytotoxic cells bind to target cells via antibodies, receptors, ligands, or fragments/derivatives thereof to form a stable complex, and stimulate the cytotoxic cells to destroy the target cells.

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

The term "degradation effective" amount refers to the amount of enzyme required to process at least 50% of the substrate compared to the substrate without contact with the enzyme.

The term "directional joining" refers to joining in which the 5' and 3' ends of a polynucleotide are sufficiently different to indicate a preferred joining orientation. For example, an otherwise untreated and undigested PCR product with two blunt ends will typically not have a preferred joining orientation when joined to a digested cloning vector to generate blunt ends in its multiple cloning sites; therefore, directional joining will typically not be exhibited in these cases. In contrast, when a PCR product with a 5' EcoR I-treated end and a 3' BamH I digestion is joined to a cloning vector with multiple cloning sites digested with EcoR I and BamH I, directional joining will typically be exhibited.

As used herein, the term "disease targeted by genetically modified cytotoxic cells" encompasses genetically modified cells of the present invention targeting any relevant cells in any disease in any manner, regardless of whether the genetically modified cells target diseased cells or healthy cells to achieve a therapeutically beneficial result. Genetically modified cells include (but are not limited to) genetically modified T cells, NK cells, and macrophages. Genetically modified cells express the CAR of the present invention, which can target any of the antigens expressed on the surface of the target cells. Examples of targetable antigens include, but are not limited to, antigens expressed on B cells; antigens expressed on carcinomas, sarcomas, lymphomas, leukemias, germ cell tumors, and blastomas; antigens expressed on various immune cells; and antigens expressed on cells associated with various hematological diseases, autoimmune diseases, and/or inflammatory diseases. Other targetable antigens will be apparent to those skilled in the art, and alternative embodiments thereof may be targeted by the CAR of the present invention.

As used herein, the terms "genetically modified cells," "redirected cells," "genetically engineered cells," or "modified cells" refer to cells that express the CARs of the invention.

The term "DNA shuffling" is used herein to indicate the recombination of substantially homologous but non-identical sequences. In some embodiments, DNA shuffling may involve exchange via non-homologous recombination, such as via cer/lox and/or flp/frt systems and the like. DNA shuffling may be random or non-random.

The term "drug" or "drug molecule" refers to a therapeutic agent that includes a substance that has a beneficial effect on the human or animal body when administered to the human or animal body. Preferably, therapeutic agents include substances that treat, cure or alleviate one or more symptoms, diseases or abnormal conditions of the human or animal body or enhance the health of the human or animal body.

An "effective amount" is an amount of a conditionally active biological protein or fragment that is effective in treating or preventing a condition in a living organism to which it is administered over a certain period of time, for example, an amount that provides a therapeutic effect during the desired dosing time interval.

The term "electrolyte" as used herein defines an electrically charged mineral in blood or other body fluids. For example, in one aspect, normal physiological conditions and abnormal conditions may be "electrolyte concentration" conditions. In one aspect, the electrolyte concentration to be tested is selected from one or more of ionized calcium, sodium, potassium, magnesium, chloride ions, bicarbonate and phosphate concentrations. For example, in one condition, the normal range for serum calcium is 8.5 to 10.2 mg/dL. In this aspect, the abnormal serum calcium concentration can be selected from above or below the normal range. In another example, in one aspect, the normal range for serum chloride is 96-106 milliequivalents per liter (mEq/ L). In this aspect, the abnormal serum chloride ion concentration can be selected from above or below the normal range. In another example, in one aspect, the normal range of serum magnesium is 1.7-2.2 mg/dL. In this aspect, the abnormal serum magnesium concentration may be selected from above or below the normal range. In another example, in one aspect, the normal range for serum phosphorus is 2.4 to 4.1 mg/dL. In this aspect, the abnormal serum phosphorus 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 to 145 mEq/L. In this aspect, 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 of serum or blood potassium is 3.7 to 5.2 mEq/L. In this aspect, the abnormal serum or blood potassium concentration may be selected from above or below the normal range. In another aspect, the normal range of serum bicarbonate is 20 to 29 mEq/L. In this aspect, the abnormal serum or blood bicarbonate concentration may be selected from above or below the normal range. In various aspects, bicarbonate levels can be used to indicate normal levels of acidity (pH) in the blood. The term "electrolyte concentration" may also be used to define the condition of a specific electrolyte in tissues or body fluids other than blood or plasma. In this case, normal physiological conditions are considered to be the clinically normal range of that tissue or fluid. In this aspect, abnormal tissue or fluid electrolyte concentrations may be selected from above or below normal ranges.

The term "epitope" as used herein refers to an epitope on an antigen, such as an enzyme polypeptide, to which the complement of an antibody, such as an enzyme-specific antibody, binds. Antigenic determinants typically consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, and may have specific three-dimensional structural properties as well as charge-to-mass ratio properties. As used herein, "epitope" refers to a portion of an antigen or other macromolecule capable of forming a binding interaction with a variable region binding partner of an antibody. Typically, such binding interactions manifest as intermolecular contacts with one or more amino acid residues of the CDR.

As used herein, the term "evolution" or "evolving" refers to the use of one or more mutagenesis methods to generate novel polynucleotides encoding novel polypeptides that are themselves improved biological molecules and/or or help generate other improved biological molecules. In certain non-limiting aspects, the invention relates to the evolution of conditionally active biological proteins from parental or wild-type proteins. In one aspect, for example, evolution relates to a method of performing non-random polynucleotide chimerism and non-random site-directed mutagenesis disclosed in US Patent Application Publication 2009/0130718. More specifically, the present invention provides methods for evolving conditionally active biological enzymes that exhibit reduced activity under normal physiological conditions compared to a parent or wild-type enzyme precursor molecule, but that exhibit less activity than the parent or wild-type enzyme parent molecule. The antigen-specific targeting region has enhanced activity under one or more abnormal conditions.

When referring to a reference polypeptide, the terms "fragment," "derivative" and "analogue" include polypeptides that retain at least one biological function or activity that is at least substantially the same as the reference polypeptide. Furthermore, the terms "fragment," "derivative," or "analog" are exemplified by a "pro-form" molecule, such as a less active proprotein that can be engineered by cleavage to produce a mature enzyme with significantly higher activity.

The term "gene" as used herein means a DNA segment involved in the production of a polypeptide chain; it includes the regions preceding and following the coding region (leader and tail sequences) as well as the regions between individual coding segments (exons). Intervening sequences (introns).

As used herein, the term "heterologous" means that a single stranded nucleic acid sequence cannot be mixed into other single stranded nucleic acid sequences or their complementary sequences. Thus, a heterologous region means that a polynucleotide or a region of a polynucleotide has a region or area within its sequence that cannot be mixed into other nucleic acids or polynucleotides. Such a region or area is, for example, a mutant region.

The term "homologous" or "homeologous" as used herein means that a single-stranded nucleic acid sequence can be intermingled with a complementary single-stranded nucleic acid sequence. The degree of hybridization can depend on a variety of factors, including the amount of identity between sequences and hybridization conditions, such as temperature and salt concentration, as discussed later. Preferably, the region of identity exceeds about 5 bp, more preferably, the region of identity exceeds 10 bp.

The benefits of the invention extend to "industrial applications" (or industrial processes), which term is used to include applications in commercial industry (or simply industry) as well as non-commercial industrial applications (such as biomedical research by non-profit organizations). Relevant applications include those in the fields of diagnosis, medicine, agriculture, manufacturing and academia.

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

The term "immune response" as used herein refers to immunity, including (but not limited to) innate immunity, humoral immunity, cellular immunity, immunity, inflammatory response, acquired (resilient) immunity, autoimmunity and/or hypersensitivity Activating immunity.

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

As used herein, the term "isolated nucleic acid" defines a nucleic acid, such as a DNA or RNA molecule, that is not immediately continuous with 5' and 3' flanking sequences (which are normally immediately continuous when present in the genome of the naturally occurring organism from which it is derived). The term thus describes, for example, nucleic acids incorporated into a vector, such as a plasmid or viral vector; incorporated into the genome of a heterologous cell (or the genome of a homologous cell, but at a site different from that in which it occurs in nature); and nucleic acids that exist as separate molecules, such as DNA fragments generated by PCR amplification or restriction enzyme digestion or RNA molecules generated by in vitro transcription. The term also describes recombinant nucleic acids that form part of a hybrid gene encoding an additional polypeptide sequence that can be used, for example, to produce a fusion protein.

As used herein, the term "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses that are capable of infecting non-dividing cells; they can deliver large amounts of genetic information into host cell DNA, making them one of the most efficient methods of delivering gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses provide a means to achieve significant 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 specific receptor. Those skilled in the art will recognize that a molecule (or macromolecular complex) can be both a receptor and a ligand. Generally speaking, the binding partner with the smaller molecular weight is called the ligand and the binding partner with the larger molecular weight is called the receptor.

As used herein, the term "ligation" refers to a process for forming phosphodiester bonds between two double-stranded nucleic acid fragments (Sambrook et al., (1982). Molecular Cloning: A Laboratory Manual. Cold Spring Harbour Laboratory, Cold Spring Harbor, NY., p. 146; Sambrook et al., Molecular Cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989). Unless otherwise provided, ligation can be achieved using known buffers and conditions, using 10 units of T4 DNA ligase ("ligase") per 0.5 micrograms of approximately equivalent molar amounts of the DNA fragments to be joined.

As used herein, the term "linker" or "spacer" refers to a molecule or a group of molecules that connect two molecules, such as a DNA binding protein and a random peptide, and is used to place the two molecules in a preferred structure, for example so that the random peptide can bind to the receptor with minimal steric hindrance from the DNA binding protein. As used herein, "linker" (L) or "linker domain" or "linker region" refers to an oligo or polypeptide region of about 1 to 100 amino acids in length that connects any of the domains/regions of the CAR of the present invention together. Linkers can be composed of flexible residues, such as glycine and serine, so that adjacent protein domains can move freely relative to each other. Longer linkers can be used when it is necessary to ensure that two adjacent domains do not interfere with each other in space. Linkers can be cleavable or non-cleavable. Examples of cleavable linkers include 2A linkers (e.g., T2A), 2A-like linkers, or functional equivalents thereof, and combinations thereof. In some embodiments, linkers include: picornavirus 2A-like linkers; CHYSEL sequences of Teschovirus suis (P2A), Thosea asigna virus (T2A), or combinations, variants, and functional equivalents thereof. 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 presentation" as used herein refers to a technique whereby a protein or antibody of interest or a portion of an antibody is expressed and presented on the surface of a mammalian host cell in screening; for example, by magnetic beads and fluorescent Combination screening of light-activated cell sorting for specific antigen binding. In one aspect, mammalian expression vectors are used to express immunoglobulins simultaneously in both secreted and cell surface-bound forms, as in DuBridge et al., US 2009/0136950. In another aspect, these techniques are used to screen viral vectors encoding libraries of antibodies or antibody fragments that exhibit membrane expression when expressed in cells, as in Gao et al., US 2007/0111260. Intact IgG surface presentation on mammalian cells is known. For example, Akamatsuu et al. developed mammalian cell surface presentation vectors suitable for direct isolation of IgG molecules based on their antigen-binding affinity and biological activity. Using Epstein-Barr virus-derived episomal vectors, the antibody library is presented as intact IgG molecules on the cell surface and screened for specific antigen binding by a combination of magnetic beads and fluorescence-activated cell sorting. Plasmids encoding antibodies with desired binding properties are recovered from the sorted cells and converted into a form suitable for the production of soluble IgG. See Akamatsuu et al . J. Immunol. Methods , Vol. 327, pp. 40-52, 2007. Ho et al. used human embryonic kidney 293T cells, which are widely used for transient protein expression, to display affinity matured single-chain Fv antibodies on the cell surface. Cells expressing the rare mutant antibody with higher affinity were enriched 240-fold by a single pass cell sorting from a large number of cells expressing the WT antibody with slightly lower affinity. Furthermore, highly enriched mutants with increased binding affinity to CD22 were obtained after a single selection of a combinatorial library that randomized native antibody hotspots. See Ho et al., "Isolation of anti-CD22 Fv with high affinity by Fv display on human cells", Proc Natl Acad Sci USA , vol. 103, pp. 9637-9642, 2006.

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 library of recombinant antigen-specific single-chain Fv (scFv) is generated from this B cell pool and screened by mammalian cell surface presentation using the Sindbis virus expression system. The variable regions (VR) of the heavy chain (HC) and light chain (LC) can be isolated from positive clones and recombinant fully human antibodies are generated as intact IgG or Fab fragments. In this way, several hypermutated high-affinity antibodies that bind to Qβ virus-like particles (VLPs), model viral antigens, and antibodies specific for nicotine can be isolated. See Beerli et al., “Isolation of human monoclonal antibodies by mammalian cell display”, Proc Natl Acad Sci USA , Vol. 105, pp. 14336-14341, 2008.

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

The term "manufacture" as used herein refers to the production of a protein in sufficient quantities to permit at least Phase I clinical testing of a therapeutic protein or regulatory approval of a diagnostic protein.

As used herein, the term "microenvironment" means any part or region of a tissue or body that has constant or transient physical or chemical differences compared to other regions of the tissue or other regions of the body.

As used herein, the term "molecular properties to be evolved" includes reference to molecules comprising a polynucleotide sequence, molecules comprising a polypeptide sequence, and molecules included in a polynucleotide sequence portion and in a polypeptide sequence portion. Particularly relevant (but by no means limiting) examples of molecular properties to be evolved include protein activity under specified conditions, such as temperature; salinity; osmotic pressure; pH; oxidative stress, and the concentration of glycerol, DMSO, detergents, and/or any other molecular species encountered in the reaction environment. Additional particular (but by no means limiting) examples of molecular properties to be evolved include stability, such as the amount of residual molecular properties present after a specified exposure time in a specified environment, such as might be encountered during storage.

As used herein, the term "mutation" means a change in a parental sequence or wild-type nucleic acid sequence or a change in a peptide sequence. Such mutations may be point mutations, such as transitions or substitutions. Mutations may 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 can bind to two, three, or more than three (eg, four) antigens (see, eg, US 2002/0004587 A1). One conditionally active antibody can be engineered to be multispecific, or two antibodies can be engineered to include heterodimers that bind to two antigens. Multispecific antibodies can also be multifunctional.

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

The term "naturally occurring" as used herein, when applied to an object, refers to the fact that the object can be found in nature. For example, naturally occurring polypeptide or polynucleotide sequences are present in organisms, including viruses, that can be isolated from sources in nature and have not been intentionally engineered by humans in the laboratory. Generally speaking, the term naturally occurring refers to an object present in non-pathological (non-diseased) individuals, such as would be typical for substances.

As used herein, "normal physiological conditions" or "wild-type operating conditions" are those conditions of temperature, pH, osmolality, osmolality, oxidative stress, and electrolyte concentration that would be considered at the site of administration in an individual. The point or site of action is within the normal range.

As used herein, the term "nucleic acid molecule" includes at least one base or one base pair, depending on whether it is single-stranded or double-stranded, respectively. In addition, nucleic acid molecules can belong exclusively or chimerically to any group of molecules containing nucleotides, as exemplified by (but not limited to) the following nucleic acid molecule groups: RNA, DNA, genomic nucleic acids, non-genomic nucleic acids, naturally occurring and non-naturally occurring nucleic acids, and synthetic nucleic acids. By way of non-limiting example, this includes nucleic acids associated with any organelle, such as mitochondria, ribosomal RNA, and nucleic acid molecules chimerically including one or more components that do not naturally occur together with naturally occurring components.

In addition, a "nucleic acid molecule" may contain in part one or more non-nucleotide components, such as, but not limited to, amino acids and sugars. Thus, by way of example (but not limitation), partially nucleotide-based and partially protein-based ribonucleases are considered "nucleic acid molecules".

As used herein, the term "nucleic acid sequence encoding..." or "DNA coding sequence of..." or "nucleotide sequence encoding..." refers to a DNA sequence that is transcribed and translated into an enzyme when under the control of an appropriate regulatory sequence, such as a promoter. A "promoter" is a DNA regulatory region that is capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. A promoter is a portion of a DNA sequence. This sequence region has a start codon at its 3' end. The promoter sequence does include a minimum number of bases in which the elements necessary to initiate transcription are detectable above background levels. However, after RNA polymerase binds to the sequence and transcription begins at the start codon (with the 3' end of the promoter), transcription proceeds downstream in the 3' direction. Within the promoter sequence will be found the transcription start site responsible for RNA polymerase binding (suitably defined by nuclease S1 mapping) as well as the protein binding domain (consensus sequence).

The term "oligonucleotide" (or synonymously "oligo") refers to a single strand of polydeoxynucleotide or two complementary polydeoxynucleotide strands that can be chemically synthesized. Such synthetic oligonucleotides may or may not have a 5' phosphate. Without the addition of phosphate and ATP in the presence of kinase, those without a 5' phosphate will not ligate to other oligonucleotides. Synthetic oligonucleotides will be ligated to fragments that have not yet been dephosphorylated.

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

When RNA polymerase will transcribe two coding sequences into a single mRNA, the coding sequence is "operably linked" to the other coding sequence, which is subsequently translated into a single polypeptide with amino acids derived from both coding sequences. The coding sequences need not be contiguous with each other, as long as the expressed sequence is ultimately processed to produce the desired protein.

As used herein, the term "parent polynucleotide set" is a set that includes one or more different polynucleotide species. Typically this term is used to refer to a progeny set of polynucleotides preferably obtained by mutagenesis of a parent set, in which case the terms "parent", "starting" and "template" are used interchangeably.

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

As used herein, the term "physiological conditions" refers to temperature, pH, osmotic pressure, ionic strength, viscosity and similar biochemical parameters that are compatible with viable organisms and/or that typically exist intracellularly in viable cultured yeast cells or mammalian cells. For example, intracellular conditions in yeast cells grown under typical laboratory culture conditions are physiological conditions. Suitable in vitro reaction conditions for in vitro transcription mixtures are general physiological conditions. Generally, in vitro physiological conditions include 50-200 mM NaCl or KCl, pH 6.5-8.5, 20-45°C and 0.001-10 mM divalent cations (e.g., Mg ^++" , Ca ⁺⁺ ); preferably about 150 mM NaCl or KCl, pH 7.2-7.6, 5 mM divalent cations, and usually include 0.01-1.0% non-specific protein (e.g., bovine serum albumin (BSA)). Non-ionic detergents (Tween, NP-40, Triton X-100) may often be present, usually at about 0.001 to 2%, typically 0.05-0.2%. (v/v). The physician may select specific aqueous conditions according to known methods. For general guidance, the following buffered aqueous conditions may be applicable: 10-250 mM NaCl, 5-50 mM Tris HCl, pH 5-8, with the addition of divalent cations and/or metal chelators and/or non-ionic detergents and/or membrane solubilizers and/or defoamers and/or scintillation agents as appropriate. Normal physiological conditions refer to conditions of temperature, pH, osmotic pressure, weight molar osmotic concentration, oxidative pressure and electrolyte concentration in a patient or individual in vivo at the site of administration or site of action, which will be considered to be within the normal range for the patient.

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

The term "population" as used herein means a collection of components, such as polynucleotides, moieties or polynucleotides or proteins. "Mixed population" means a collection of components that belong to the same nucleic acid or protein family (i.e., are related), but that differ in their sequence (i.e., are not identical) and therefore in their biological activity.

A molecule having a "proform" refers to a molecule that undergoes any combination of one or more covalent and non-covalent chemical modifications (e.g., glycosylation, proteolytic cleavage, dimerization or oligomerization, temperature-induced or pH-induced conformational changes, association with cofactors, etc.) en route to a more mature molecular form having a property difference (e.g., increased activity) compared to the reference proform molecule. When two or more chemical modifications (e.g., two proteolytic cleavages or proteolytic cleavage and deglycosylation) can be distinguished en route to produce a mature molecule, the reference proprotein molecule can be referred to as a "pre-proform" molecule.

As used herein, the term "receptor" refers to a molecule that has an affinity for a given ligand. Receptors may be naturally occurring or synthetic molecules. Receptors may be employed in an unchanged state or in aggregate with other substances. Receptors may be covalently or non-covalently attached to binding members directly or via specific binding substances. Examples of receptors include, but are not limited to, antibodies, including monoclonal antibodies and antisera that react with specific antigenic determinants (e.g., on viruses, cells or other materials), cell membrane receptors, complex carbohydrates and glycoproteins, enzyme and hormone receptors.

As used herein, the term "reduced rearrangement" refers to the increase in molecular diversity generated by repeated sequence-mediated deletion (and/or insertion) events.

The term "restriction site" as used herein refers to the recognition sequence necessary to manifest the action of a restriction enzyme, and includes catalytic cleavage sites. It will be appreciated that the cleavage site may or may not be within a portion of the restriction site that includes a low-ambiguity sequence (ie, a sequence that contains a major determinant of the frequency of occurrence of the restriction site). When an enzyme (eg, a restriction enzyme) is referred to as "cleaving" a polynucleotide, it is understood to mean that the restriction enzyme catalyzes or promotes cleavage of the polynucleotide.

As used herein, the term "single chain antibody" refers to a polypeptide that includes a VH domain and a VL domain in a polypeptide bond typically linked via a spacer peptide, and which may include additional amino acids at the amine and/or carboxyl terminus sequence. For example, a single chain antibody may include a tether segment for attachment to an encoding polynucleotide. As an example, scFv is a single chain antibody. Single-chain antibodies are generally proteins composed of one or more polypeptide segments of at least 10 consecutive amine groups essentially encoded by immunoglobulin superfamily genes (see, for example, The Immunoglobulin Gene Superfamily, A. F. Williams and A. N. Barclay, in Immunoglobulin Genes, T. Honjo, F. W. Alt, and THE. Rabbits, eds. (1989) Academic press: San Diego, Calif., pp. 361 to 368, most commonly found in rodents, nonhuman primates, avian species, A porcine, bovine, ovine, goat, or human gene sequence encoding a heavy or light chain. Functional single-chain antibodies generally contain a sufficient portion of the immunoglobulin superfamily gene product to retain binding to a specific target molecule The specific target molecule is usually a receptor or an antigen (epitope).

Members of a molecular pair (such as antibody-antigen pairs and ligand-receptor pairs) are said to "specifically bind" to each other if they bind to each other with greater affinity than other non-specific molecules. For example, an elevated antibody that binds an antigen more efficiently than a nonspecific protein may be described as specifically binding to the antigen.

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

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

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

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

The terms "T cells" and "T lymphocytes" are used interchangeably and synonymously herein. Examples include, but are not limited to, naive T cells, central memory T cells, effector memory T cells, and combinations thereof.

As used herein, the term "transduction" refers to the introduction of exogenous nucleic acid into a cell using a viral vector. As used herein, "transfection" refers to the introduction of exogenous nucleic acid into a cell using recombinant DNA technology. The term "transformation" means the introduction of an "exogenous" (i.e., foreign or extracellular) gene, DNA or RNA sequence into a host cell so that the host cell will express the introduced gene or sequence to produce a desired substance, such as a protein or enzyme encoded by the introduced gene or sequence. The introduced gene or sequence may also be referred to as a "selected" or "exogenous" gene or sequence, and may include regulatory or control sequences, such as start, stop, promoter, signal, secretory or other sequences used by the cell's genetic machinery. The gene or sequence may include a non-functional sequence or a sequence with no known function. A host cell that receives and expresses the introduced DNA or RNA has been "transformed" and is a "transformant" or "clone." The DNA or RNA introduced into a host cell can come 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 "treatment" includes: (1) preventing or delaying the occurrence of clinical or subclinical symptoms of a symptom, disorder or condition in an animal (especially a mammal and The occurrence of clinical symptoms of a disease, disorder or condition, especially in humans); (2) Suppression of the disease, disorder or condition (e.g., arresting, reducing or delaying the progression of the disease or its recurrence (during maintenance treatment) (in the case of) or at least one of its clinical or subclinical symptoms); and/or (3) alleviating the condition (i.e. causing the resolution of the condition, disease or condition or at least one of its clinical or subclinical symptoms). The benefit to the patient being treated is statistically significant or at least detectable by the patient or by the physician.

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

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

As used herein, the term "variable segment" refers to a portion of a nascent peptide that includes a random, pseudo-random, or defined core sequence. "Variable segment" means a portion of a nascent peptide that includes a random, pseudo-random or defined core sequence. Variable segments may include both variant and invariant residue positions, and the degree of residue change at variant residue positions may be limited: both options are left to the discretion of the physician. Typically, the variable segments are about 5 to 20 amino acid residues long (eg, 8 to 10), but the variable segments can be longer and can include antibody portions or receptor proteins, such as antibody fragments, nucleic acid binding proteins, receptor proteins and their analogs.

As used herein, "vector", "cloning vector" and "expression vector" refer to a medium by which a polynucleotide sequence (e.g., a foreign gene) can be introduced into a host cell in order to transform the host and promote the expression (e.g., transcription and translation) of the introduced sequence. Vectors include plasmids, bacteriophages, viruses, etc.

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

For example, the term "processed" as in "processed sample" simply means a sample by processing. Likewise, for example, a "processed molecule" is a molecule that has been processed.

For purposes of illustration, the principles of the present invention are described by reference to various exemplary embodiments. Although certain embodiments of the present invention are specifically described herein, those generally skilled in the art will readily recognize that the same principles are equally applicable and can be used in other systems and methods. Before explaining the embodiments disclosed by the present invention in detail, it should be understood that the present invention is not limited in its application to the details of any particular embodiment shown. In addition, the derivatives used herein are for descriptive and not limiting purposes. In addition, although certain methods are described with reference to steps presented herein in a certain order, in many cases, such steps can be performed in any order that can be understood by those skilled in the art; the novel methods are therefore not limited to the specific step arrangements disclosed herein.

It must be noted that, as used herein and in the appended claims, the singular forms "a/an" and "the" include plural references unless the context clearly dictates otherwise. Furthermore, the terms "a or an", "one or more" and "at least one" are used interchangeably herein. The terms "includes," "includes," "has," and "constructs from" are also used interchangeably.

The present invention relates to a chimeric antigen receptor (CAR) for binding to a target antigen, comprising at least one evolved from a parental or wild-type protein or domain thereof and compared to activity in assays under abnormal conditions. Antigen-specific targeting regions with reduced activity analyzed under normal physiological conditions; transmembrane domains; and intracellular signaling domains. In some embodiments, the chimeric antigen receptor further includes an extracellular spacer domain or at least one costimulatory domain. The target antigen can be a tumor-specific antigen, which can be Axl, ROR2 or CD22.

The CAR of the present invention may have at least one of the following: (1) its affinity for the target antigen is reversibly or irreversibly reduced under normal physiological conditions, and (2) increased affinity compared to the same CAR without a conditionally active antigen-specific targeting region. These CARs can guide cytotoxic cells to disease sites where abnormal conditions exist, such as tumor microenvironments or synovial fluid. Due to these characteristics, the CAR can preferentially guide cytotoxic cells to disease sites due to its low affinity for normal tissues. This type of CAR can significantly reduce side effects and allow higher doses of therapeutic agents to be used to improve therapeutic efficacy. CARs are particularly valuable for the development of novel therapeutic agents that are required to last for a short or limited period of time in an individual. Examples of advantageous applications include high-dose systemic treatment and high-concentration local treatment.

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

Most chimeric antigen receptors include antigen-specific targeting regions with increased activity in assays under abnormal conditions compared to the parent or wild-type protein or domains thereof, and antigens with increased activity compared to the parent or wild-type protein or domains thereof. The specific targeting region is an antigen-specific targeting region that has reduced binding affinity to the target antigen under normal physiological conditions.

In any of the aforementioned chimeric antigen receptors, the selectivity of the antigen-specific targeting region in an assay under abnormal conditions may also be improved compared to the antigen-specific targeting region of the parent or wild-type protein or domain thereof.

In some embodiments, the antigen-specific targeting region can have 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 The activity in abnormal conditions of 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 is the same as in normal physiological conditions activity ratio.

The CAR molecule includes a linker connecting two antigen-specific targeting regions (Figure 1). The linker directs the two antigen-specific targeting regions on the CAR-T cell in a manner that enables the two antigen-specific targeting regions on the CAR-T cell to exhibit improved or optimal activity in binding to the target antigen (Jensen et al., "Design and implementation of adoptive therapy with chimeric antigen receptor-modified T cells", Immunol Rev. , Vol. 257, pp. 127-144, 2014). The linker is therefore more capable of adopting a specific configuration that achieves improved or optimal binding of the two antigen-specific targeting regions to the target antigen, thereby improving the effect of the CAR-T cell.

In some embodiments, the linker can be a Gly-Ser tandem repeat sequence with a length of 18-25 amino acids (Grada, "TanCAR: A Novel Bispecific Chimeric Antigen Receptor for Cancer Immunotherapy", Molecular Therapy Nucleic Acids , Volume 2, e105, 2013). This flexible linker can adopt a variety of different configurations to facilitate improved or optimal performance of the two antigen-specific targeting regions that bind to the target antigen.

In some embodiments, the linker is capable of adopting different configurations under normal physiological conditions and abnormal conditions. Specifically, the linker has a first configuration under abnormal conditions that is improved or optimal for presenting two antigen-specific targeting regions to bind to the target antigen, while the same linker has a second configuration under normal physiological conditions that is less effective in presenting two antigen-specific targeting regions to bind to the target antigen than the first configuration of the linker under abnormal conditions. Such linkers may be referred to as "conditional linkers," which allow the two antigen-specific targeting regions to bind to the target antigen with higher binding activity under abnormal conditions than under normal physiological conditions. Therefore, CAR-T cells including such conditional linkers are more active under abnormal conditions compared to the same CAR-T cells under normal physiological conditions.

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

The selection of conditional linkers for use in CAR molecules is within the scope of the invention. The conditional linker may adopt a first configuration under abnormal conditions that is improved or optimal for presenting two antigen-specific targeting regions for binding to the target antigen, and adopt a second configuration under normal physiological conditions , which is suboptimal for presenting two antigen-specific targeting regions bound to the target antigen. In some embodiments, a suboptimal configuration of the linker under normal physiological conditions results in a binding activity for the target antigen that is less than about 90% of the binding activity of a CAR molecule with a modified or optimal linker configuration under abnormal conditions. %, or about 80%, or about 70%, or about 60%, or about 50%, or about 40%, or about 30%, or about 20%, or about 10%, or about 5% of the CAR molecules.

Conditional linkers can be generated from a starting linker selected from a 2A linker, a 2A-like linker, a picornavirus 2A-like linker, a 2A peptide of the genus Teschovirus (P2A) of the family Suidae, and a 2A peptide of the genus Trametes virus (T2A), as well as variants and functional equivalents thereof. The starting linker is evolved to generate a mutant protein; the mutant protein is then subjected to analysis under normal physiological conditions and analysis under abnormal conditions. The protein having a conditional linker is selected from mutant proteins based on the following: the selected protein exhibits (a) a conditional linker having a first conformation under abnormal conditions that is improved or optimal for presenting two antigen-specific targeting regions for binding to the target antigen, and (b) a conditional linker having a second conformation under normal physiological conditions that is suboptimal for presenting two antigen-specific targeting regions for binding to the target antigen.

CAR molecules also include an extracellular spacer domain linking two antigen-specific targeting regions to a transmembrane domain, which in turn is linked to a costimulatory domain and an intracellular signaling domain inside the T cell (Figure 1). The extracellular spacer domain is preferably capable of supporting the antigen-specific targeting region to recognize and bind to the target antigen on the target cell (Hudecek et al., "The non-signaling extracellular spacer domain of chimeric antigen receptors is decisive for in vivo antitumor activity" , Cancer Immunol Res ., Volume 3, Pages 125–135, 2015). In some embodiments, the extracellular spacer domain is a flexible domain, thereby allowing the antigen-specific targeting region to have a structure that optimally recognizes the specific structure and density of the target antigen on cells, such as tumor cells (Hudecek et al., "The non-signaling extracellular spacer domain of chimeric antigen receptors is decisive for in vivo antitumor activity", Cancer Immunol Res ., Vol. 3, pp. 125-135, 2015). The flexibility of the extracellular spacer domain allows the extracellular spacer domain to adopt a variety of different configurations.

In some embodiments, the extracellular spacer subdomain is capable of adopting different conformations under normal physiological conditions and abnormal conditions. Specifically, the extracellular spacer subdomain has a first conformation under abnormal conditions that is improved or optimal for presenting two antigen-specific targeting regions to bind to the target antigen, while the same extracellular spacer subdomain has a second conformation under normal physiological conditions that is suboptimal for presenting two antigen-specific targeting regions to bind to the target antigen. Such an extracellular spacer subdomain may be referred to as a "conditional extracellular spacer subdomain" because it enables two antigen-specific targeting regions to bind to the target antigen with higher binding activity under abnormal conditions than under normal physiological conditions. Therefore, in the case of a conditional extracellular spacer subdomain, the activity of CAR-T cells under abnormal conditions can be higher compared to the same CAR-T cells under normal physiological conditions.

The selection of conditional extracellular spacer subdomains for use in CAR molecules is within the scope of the present invention. In some embodiments, a suboptimal conformation of an extracellular spacer subdomain under normal physiological conditions produces a CAR molecule with a binding activity to a target antigen that is less than about 90%, or about 80%, or about 70%, or about 60%, or about 50%, or about 40%, or about 30%, or about 20%, or about 10%, or about 5% of that of a CAR molecule with an optimal conformation of the same extracellular spacer subdomain under abnormal conditions.

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

The extracellular spacer subdomain is conditionally ubiquitinated and resistant to ubiquitination. Specifically, the extracellular spacer subdomain of the CAR molecule is more resistant to ubiquitination under abnormal conditions than under normal physiological conditions. Therefore, a CAR-T cell having a conditionally ubiquitinated extracellular spacer subdomain will have enhanced cytotoxicity under abnormal conditions relative to its cytotoxicity under normal physiological conditions.

The conditional ubiquitination-resistant extracellular spacer subdomain can be selected to be more resistant to ubiquitination at abnormal pH or abnormal temperature and less resistant to ubiquitination at normal physiological pH or normal physiological temperature. In one embodiment, the conditional ubiquitination-resistant extracellular spacer subdomain is more resistant to ubiquitination at the pH of the tumor microenvironment and less resistant to ubiquitination at normal physiological pH, such as pH 7.2-7.6 in human plasma.

To generate a conditional extracellular spacer domain, a starting protein fragment selected from the group consisting of the Fc fragment of an antibody, the hinge region of an antibody, the CH2 region of an antibody, and the CH3 region of an antibody is evolved to produce a mutant protein. Mutated proteins are subjected to analysis under normal physiological conditions as well as analysis under abnormal conditions. The conditional extracellular spacer domain is selected from mutant proteins exhibiting: (a) a conditional extracellular spacer domain that under abnormal conditions has a first configuration such that the antigen-specific targeting region binds to it with higher binding activity The target antigen has a second configuration of the conditional extracellular binding domain under normal physiological conditions such that the antigen-specific targeting region binds to the target antigen with lower binding activity than under abnormal conditions; or (b) compared to For proteins that are more resistant to ubiquitination under normal physiological conditions and under abnormal conditions.

Any of the foregoing chimeric antigen receptors can be configured such that the protein containing the antigen receptor is expressed at an increased level compared to the parent or wild-type protein or domain thereof.

In an alternative embodiment, the present invention provides a chimeric antigen receptor (CAR) for binding to a target antigen, comprising at least one antigen-specific targeting region evolved from a parent or wild-type protein or a domain thereof and having improved selectivity in an assay under abnormal conditions compared to the antigen-specific targeting region of the parent or wild-type protein or a 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 synergistic stimulatory domain.

The invention also relates to methods of evolving a parent or wild-type protein, or a domain thereof, to produce a conditionally active protein that has at least one of the following: (a) an antigen-specific target compared to the parent or wild-type protein, or domain thereof; (b) the antigen-specific targeting region of the parent or wild-type protein or domain thereof has increased activity in the assay under abnormal conditions compared to the antigen-specific targeting region of the parent or wild-type protein or domain thereof. Conditionally active proteins can be engineered into CARs.

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

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

In any of the aforementioned chimeric antigen receptors produced by the method, the selectivity of the antigen-specific targeting region in an assay under abnormal conditions can also be improved compared to the antigen-specific targeting region of the parent or wild-type protein or domain thereof.

Any of the chimeric antigen receptors described above produced by this method can be configured such that the protein containing the antigen receptor is expressed at an increased level compared to the parent or wild-type protein or domain thereof.

In an alternative embodiment of the method, a chimeric antigen receptor (CAR) generated by the method for binding to a target antigen includes at least one evolved from a parental or wild-type protein or domain thereof and compared to Antigen-specific targeting regions of the parent or wild-type protein or domains thereof Antigen-specific targeting regions with improved selectivity in assays under abnormal conditions; transmembrane domains; and intracellular signaling domains. In some embodiments, the chimeric antigen receptor further includes an extracellular spacer domain or at least one costimulatory domain. chimeric antigen receptor

The immune system of mammals, especially humans, has cytotoxic cells that target and destroy diseased tissues and/or pathogens. The use of these cytotoxic cells to remove unwanted tissues (i.e., target tissues) such as tumors is a promising therapeutic approach. Other tissues that can be targeted for removal include glandular hyperplasia (e.g., prostate), warts, and unwanted adipose tissue. However, this relatively new therapeutic approach has achieved only limited success to date. For example, the use of T cells to target and destroy tumors has relatively low long-term benefits because cancer cells can adapt to new therapies by reducing the expression of surface antigens, thereby reducing the effectiveness of such therapies. Cancer cells can even dedifferentiate to evade detection by tumor-specific T cells. See 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 significantly enhance the specificity and sensitivity of these cytotoxic cells. For example, CAR-expressing T cells (CAR-T cells) can use the CAR to direct the T cells to target tumor cells that express cell surface antigens that specifically bind to the CAR. Such CAR-T cells can more selectively deliver cytotoxic agents to tumor cells. CAR-T cells recognize target molecules directly and are therefore typically not restricted by polymorphic presentation elements such as human leukocyte antigen (HLA). The advantages of this CAR targeting strategy are threefold. First, because CAR-T cell function does not depend on HLA status, the same CAR-based approach can in principle be used for all tumor patients expressing the same target surface antigen. Second, impairment of antigen processing and presentation mechanisms is a common property of tumor cells and can promote immune evasion. However, this does not provide protection to CAR-T cells. Third, this system can be used to target a range of macromolecules, including proteins, carbohydrates, and glycolipids.

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

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

ASTRs may also include other protein functional domains to recognize and bind to target antigens. Because the target antigen may have other biological functions, such as acting as a receptor or ligand, the ASTR may alternatively include functional domains for specific binding to the antigen. Some examples of proteins with functional domains include binding interleukins (which enable recognition of cells bearing interleukin receptors), affibodies, ligand binding domains from naturally occurring receptors, for receptors, For example, soluble protein/peptide ligands on tumor cells. Virtually any molecule capable of binding with high affinity to a given antigen can be used in an ASTR, as those skilled in the art will appreciate.

In one embodiment, the CAR of the invention includes at least two ASTRs targeting at least two different antigens or two epitopes on the same antigen. In one embodiment, a CAR includes three or more ASTRs targeting at least three or more different antigens or epitopes. When multiple ASTRs are present in a CAR, the ASTRs can be configured in tandem and separated by linking peptides (Figure 1).

In one embodiment, the ASTR comprises a full-length IgG heavy chain specific for a target antigen and has _VH , CH1, hinge, and CH2 and CH3 (Fc) Ig domains, if the _VH domain alone is sufficient to confer antigen specificity ("single domain antibody"). If both, the _VH and _VL domains are necessary to produce a fully active ASTR, a CAR containing a _VH and a full-length lambda light chain (IgL) are introduced into the same cytotoxic cell to produce an active ASTR. In another embodiment, each ASTR of the CAR comprises at least two single-chain antibody variable fragments (scFv), each specific for a different target antigen. scFvs have been developed in which the C-terminus of one variable domain ( _VH or _VL ) is tethered to the N-terminus of the other variable domain ( _VL or _VH , respectively) via a polypeptide linker without significantly disrupting antigen binding or binding specificity (Chaudhary et al., "A recombinant single-chain immunotoxin composed of anti-Tac variable regions and a truncated diphtheria toxin", Proc. Natl. Acad. Sci ., Vol. 87, p. 9491, 1990; Bedzyk et al., "Immunological and structural characterization of a high affinity anti-fluorescein single-chain antibody" , J. Biol. Chem ., Vol. 265, p. 18615, 1990). These scFvs do not have the constant region (Fc) present in the heavy and light chains of natural antibodies. scFvs specific for at least two different antigens are configured in tandem. In one embodiment, the extracellular spacer subdomain can be linked between the ASTR and the transmembrane domain.

In another embodiment, the scFv fragment can be fused to all or part of the heavy chain constant domain. In another embodiment, the ASTR of the CAR includes a bivalent (or bivalent) single chain variable fragment (di-scFv, bi-scFv). In a CAR including a di-scFV, two scFvs each having specificity for an antigen are linked together to form a single peptide chain having two _VH and two _VL 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, pp. 359-367, 2006; Kufer et al., "A revival of bispecific antibodies", Trends in Biotechnology , Vol. 22, pp. 238-244, 2004).

In another embodiment, the ASTR includes bifunctional antibodies. In bifunctional antibodies, scFvs are produced with linker peptides that are too short for the two variable regions to fold together, driving the scFv to dimerize. Shorter linkers (one or two amino acids) allow trimers to form, so-called trifunctional antibodies (triabodies/tribodies). Tetrafunctional antibodies can also be used in ASTRs.

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

The antigens targeted by CAR exist on the surface or inside cells in the tissue, and they target the removal of tumors, glandular hyperplasia (such as prostate), warts, and unwanted adipose tissue. Although the ASTR of CAR more effectively recognizes and binds to surface antigens, intracellular antigens can also be targeted by CAR. In some embodiments, the target antigen is preferably specific to cancer, inflammatory diseases, neurological diseases, diabetes, cardiovascular diseases, or infectious diseases. Examples of target antigens include antigens expressed by various immune cells, carcinomas, sarcomas, lymphomas, leukemias, germ cell tumors, blastomas, and cells associated with various hematological diseases, autoimmune diseases, and/or inflammatory diseases.

Antigens specific to cancer that can be targeted by ASTR include one or more of the following: 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 disperser 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-hydroxyacetylceraminic acid, NPC-1C, PDGF-R a, PDL192, phospholipid Serine, 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.

Antigens specific for inflammatory diseases that can be targeted by ASTR include one or more of the following: AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (basal Immunoglobulin), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (IL-2 receptor chain), CD3, CD4, CD5, IFN-a, IFN-γ, IgE, IgE Fc Area, IL-1, IL-12, IL-23, IL-13, IL-17, IL-17A, IL-22, IL-4, IL-5, IL-5, IL-6, IL-6 body, integrin a4, integrin α4β7, llama, LFA-1 (CD1 la), MEDI-528, myostatin, OX-40, rhuMAb β7, sclerostin, 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. Antigens specific for diabetes that can be targeted by the ASTRs of the invention include one or more of L-Ιβ or CD3. Antigens specific for cardiovascular disease that can be targeted by the ASTRs of the present invention include C5, cardiac myosin, CD41 (integrin α-lib), fibrin II, β chain, ITGB2 (CD 18), and nerve sheaths One or more of the aminoalcohol-1-phosphates.

Antigens specific for infectious diseases that can be targeted by the ASTR of the present invention include one or more of the following: 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.

Other examples of target antigens include surface proteins found on cancer cells in a specific or amplified manner, such as the IL-14 receptor, CD19, CD20 and CD40 (for B-cell lymphomas), Lewis Y and CEA antigens ( for various cancers), Tag72 antigen (for breast and colorectal cancer), EGF-R (for lung cancer), folate-binding protein and HER-2 protein (which is often amplified in human breast and ovarian cancer tumors) or viral proteins , such as HIV gpl20 and gp41 envelope proteins, envelope proteins from hepatitis B and C viruses, glycoprotein B and other envelope glycoproteins from human cytomegalovirus, and from tumor viruses, such as Kaposi's sarcoma-related Herpes virus envelope protein. Other potential target antigens include CD4, for which the ligand is the HIV gpl20 envelope glycoprotein, and other viral receptors, such as ICAM, which is a receptor for human rhinovirus and a related receptor molecule for poliovirus.

In another embodiment, CAR can target and engage cancer therapeutic cells, such as antigens of NK cells and other cells mentioned herein to activate cancer therapeutic cells by acting as immune effector cells. This example is a CAR that targets the CD16A antigen to engage NK cells against malignant diseases expressing CD30. The bispecific tetravalent AFM13 antibody is an example of an antibody that can deliver this effect. Other details of this type of embodiment can be found in, for example, Rothe, A. et al., "A phase 1 study of the bispecific anti-CD30/CD16A antibody construct AFM13 in patients with relapsed or refractory Hodgkin lymphoma", Blood , June 25, 2015, Vl. 125, Issue 26, Pages 4024 to 4031.

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

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

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

Single chain antibodies targeting Axl or ROR2 are suitable for linking to transmembrane domains and intracellular signaling domains to generate CAR structures.

The extracellular spacer domain of CAR is a hydrophilic region located between the ASTR and the transmembrane domain. In some embodiments, this domain promotes proper protein folding of the CAR. The extracellular spacer domain is an optional component of the CAR. The extracellular spacer domain may include a domain selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, an antibody CH3 region, an artificial spacer sequence, or a combination thereof. Examples of extracellular spacer domains include the CD8a hinge, artificial spacers made from polypeptides that can be as small as three glycines (Gly), and the CH1 and CH3 regions of IgG (such as human IgG4).

The transmembrane domain of CAR is a region that is able to cross the plasma membrane of cytotoxic cells. The transmembrane domain is selected from transmembrane proteins, such as type I transmembrane proteins, artificial hydrophobic sequences, or transmembrane regions of a combination thereof. Examples of transmembrane domains include transmembrane regions of the α, β, or ζ chains of T cell receptors, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Synthetic transmembrane domains may include a triad of phenylalanine, tryptophan, and valine. As appropriate, a short oligo or polypeptide linker with a preferred length of between 2 and 10 amino acids may form a bond between the transmembrane domain of CAR and the intracellular signaling domain. The glycine-serine dyad provides a particularly suitable linker between the transmembrane domain and the intracellular signaling domain.

The CAR of the present invention also includes an intracellular signaling domain. Intracellular signaling domains transduce effector function signals and direct cytotoxic cells to perform their specialized functions, that is, to injure and/or destroy target cells. Examples of intracellular signaling domains include the zeta chain of any one of the T cell receptor complex or homologs thereof, such as n chain, FcsRly and β chain, MB 1 (Iga) chain, 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 those involved in T cell transduction Other molecules such as CD2, CD5 and CD28. Specifically, the intracellular signaling domain may be the human CD3 zeta chain, FcyRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor with an immunoreceptor tyrosine-based activation motif (ITAM), and combinations thereof.

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

In some embodiments, the intracellular signaling domain includes the cytoplasmic signaling domain of TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, or CD66d. The intracellular signaling domain in the CAR particularly preferably includes the cytoplasmic signaling domain of human CD3ζ.

The CAR of the present invention may include a synergistic stimulatory domain, which has the function of enhancing cell proliferation, cell survival and development of memory cells for the cytotoxic cells expressing CAR. The CAR of the present invention may include one or more synergistic stimulatory domains selected from the synergistic stimulatory domains of proteins in the TNFR superfamily, CD28, CD137 (4-1BB), CD134 (OX40), Dap10, CD27, CD2, CD7, CD5, ICAM-1, LFA-1 (CD11a/CD18), Lck, TNFR-I, PD-1, TNFR-II, Fas, CD30, CD40, ICOS LIGHT, NKG2C, B7-H3 or a combination thereof. If the CAR includes more than one synergistic stimulatory domain, these domains can be configured in tandem, separated by a linker as appropriate. The co-stimulatory domain is an intracellular domain that can be located between the transmembrane domain and the intracellular signaling domain in the CAR.

In some embodiments, two or more components of the CARs of the invention are separated by one or more linkers. For example, in a CAR including at least two ASTRs, the two ASTRs can be separated by a linker. The linker is an oligo or polypeptide region of about 1 to 100 amino acids in length. 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 can be composed of flexible residues such as glycine and serine, allowing adjacent protein domains to move freely relative to each other. Longer linkers, such as those longer than 100 amino acids, may be used in conjunction with alternative embodiments of the invention, and may be selected, for example, to ensure that two adjacent domains do not sterically interfere with each other. Examples of linkers useful 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 region

CAR is a chimeric protein produced by fusing all the different domains discussed above together to form a fusion protein. CAR is typically produced by an expression vector comprising a polynucleotide sequence encoding the different domains of CAR. The ASTR of the present invention used to recognize and bind to an antigen on a target cell is conditionally active. Specifically, compared to the ASTR corresponding to the parent or wild-type protein, the ASTR is less active or inactivated under normal physiological conditions and is active under abnormal conditions for binding to the target antigen. The present invention provides a method for producing a conditionally active ASTR from a parent or wild-type protein or its binding domain (parent or wild-type ASTR).

Wild-type proteins suitable for use in whole or in part with at least their binding domain for a target antigen as an ASTR in the present invention can be found by generating a protein library and screening the library for proteins with the desired binding affinity for the target antigen. Wild-type protein can be found by screening cDNA libraries. A cDNA library is a collection of selected cDNA (complementary DNA) fragments inserted into a collection of host cells, which together constitute portions of an organism's transcriptome. cDNA is produced from fully transcribed mRNA and therefore contains coding sequences for the expression of proteins in an organism. The information in cDNA libraries is a powerful and applicable tool for discovering proteins with desired properties by screening protein libraries with required binding affinities for target antigens.

In some embodiments, when the wild-type protein is an antibody, the wild-type antibody can be found by generating and screening an antibody library. The antibody library can be a polyclonal antibody library or a monoclonal antibody library. A polyclonal antibody library 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 polyclonal antibody library that binds to the antigen. For the preparation of a monoclonal antibody library, any technology that provides antibodies produced by continuous cell line culture can be used. Examples include fusion tumor technology, tri-source fusion tumor technology, human B cell fusion tumor technology, and EBV fusion tumor technology (see, e.g., Cole (1985) in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pages 77 to 96). Techniques describing the generation of single chain antibodies (see, e.g., U.S. Patent No. 4,946,778) can be adapted to generate libraries of single chain antibodies.

There are other methods for generating and screening antibody libraries for wild-type antibodies. For example, fully human antibody display libraries can be used. This type of library is a group of antibodies presented on the surface of host cells. Preferably, the antibody library represents a human antibody library of antibodies because of its ability to bind to a wide range of antigens. Because the antibodies are presented on the surface of the cell, the effective affinity (attributed to avidity) of each antibody in the library is increased. Unlike other commonly used library types, such as phage display libraries, in the case where the affinity of the antibody is not much needed for screening and identification purposes, the superaffinity provided by cell surface presentation is desirable in the present invention. Cell surface display libraries enable identification of low, medium and high binding affinity antibodies, as well as identification of non-immunogenic and weak antigenic determinants in screening or selection steps.

Methods in which a parent or wild-type protein or binding domain thereof (parental or wild-type ASTR) is subjected to a mutagenesis to generate a population of mutant polypeptides that can subsequently be screened to identify proteins that, compared to the parent or wild-type ASTR, behave under abnormal conditions A mutant ASTR with enhanced binding affinity for the target antigen under normal physiological conditions, and as appropriate, substantially the same or reduced binding affinity for the target antigen under normal physiological conditions.

Any chemical synthesis or recombinant mutation method can be used to generate mutant polypeptide populations. Unless otherwise indicated, the present invention can be practiced using the knowledge of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology, which are within the skill of the art. Such techniques are fully explained in the literature. See, e.g., Molecular Cloning A Laboratory Manual, 2nd ed., Sambrook, Fritsch, and Maniatis, eds. (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Vol. I and Vol. 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 and S. J. Higgins, eds., 1984); Transcription And Translation (B. D. Hames and 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 Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Cabs, eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vol. 154 and Vol. 155 (Wu et al., eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Vol. 1-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

The invention provides a method for generating a nucleic acid mutant encoding a conditionally active mutant polypeptide, the method comprising modifying the nucleic acid by the following steps: (i) one or more nucleotides are substituted for different nucleotides, wherein the nucleotide Includes natural or non-natural nucleotides; (ii) deletion of one or more nucleotides, (iii) addition of one or more nucleotides, or (iv) any combination thereof. In one aspect, the non-natural nucleotide includes inosine. In another aspect, the method further includes analyzing the polypeptide encoded by the modified nucleic acid with altered enzymatic activity, thereby identifying the modified nucleic acid encoding the polypeptide with altered enzymatic activity. In one aspect, step (a) is modified by PCR, error-prone PCR, shuffling, oligonucleotide-guided mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursion Collective mutation induction, exponential collective mutation induction, site-specific mutation induction, gene recombination, gene site saturation mutation induction, ligase chain reaction, in vitro mutation induction, ligase chain reaction, oligonucleotide synthesis, any DNA production technology and any combination thereof. In another aspect, the method further includes at least one iteration of the modifying step.

The invention further provides a method for preparing polynucleotides from two or more nucleic acids, the method comprising: (a) identifying regions of identity and diversity between two or more nucleic acids a region, wherein at least one of the nucleic acids includes a nucleic acid of the invention; (b) providing a set of oligonucleotides with sequences corresponding to at least two of two or more nucleic acids; and (c) using a polymerase Polynucleotides are made by elongating oligonucleotides.

Any mutation induction technique may be employed in the various embodiments of the present invention. Random or random mutation induction is exemplified by mutating (modifying or altering) a parent molecule to obtain a set of progeny molecules with unpredicted mutations. Thus, in an in vitro random mutation induction reaction, for example, there is no specific predetermined product that is expected to be produced; rather, there is uncertainty (hence randomness) about the precise nature of the mutations obtained and therefore also about the products produced. Random mutation induction is embodied in processes such as error-prone PCR and random shuffling, in which the mutations obtained are random or not predetermined. Variant forms may be generated by error-prone transcription, such as error-prone PCR or use of a polymerase without proofreading activity (see Liao (1990) Gene 88: 107-111), a first variant form, or by replication of the first form in a mutant strain (mutating host cells is discussed in further detail below and is generally well known). Mutant strains may include any mutant in any organism in which mismatch repair function is impaired. These include mutant gene products of mutS, mutT, mutH, mutL, ovrD, dcm, vsr, umuC, umuD, sbcB, recJ, etc. Attenuation is achieved by gene mutation, allele replacement, by addition of reagents, such as small compounds or selective inhibition expressing antisense RNA, or other techniques. The genes mentioned or homologous genes in any organism may be attenuated.

Other mutagenesis methods include oligonucleotide-guided mutagenesis, error-prone polymerase chain reaction (error-prone PCR), and cassette mutagenesis, in which specific regions of the parent polynucleotide are synthetically mutagenized oligos. Nucleotide substitution. In these cases, multiple mutation sites are created around certain positions in the parental sequence.

In oligonucleotide-guided mutagenesis, short sequences are replaced by synthetically mutagenic oligonucleotides. In oligonucleotide-guided mutagenesis, short sequences of polynucleotides are removed from the polynucleotide using restriction enzyme digestion and replaced by synthetic polynucleotides in which different bases have been changed relative to the original sequence. Polynucleotide sequences can also be altered through chemical mutagenesis. Chemical mutagens include, for example, sodium bisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid. Other agents that are nucleotide precursor analogs include nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine. Generally, these agents are added to the PCR reaction rather than to the nucleotide precursor, thereby mutating the sequence. Intercalating agents such as proflavine, acriflavine, quinacrine and the like may also be used. Random mutation induction of polynucleotide sequences can also be achieved by irradiation with X-rays or ultraviolet light. Generally, the so mutagenized plasmid polynucleotides are introduced into E. coli and propagated into a pool or library of promiscuous plasmids.

Error-prone PCR uses low-fidelity polymerization conditions to randomly introduce low-level point mutations within long sequences. In a mixture of fragments of unknown sequence, error-prone PCR can be used to induce the mixture.

In cassette mutagenesis, sequence blocks of a single template are typically subjected to (partially) random combinatorial sequence replacement. Reidhaar-Olson JF and Sauer RT: Combinatorial cassette mutagenesis as a probe of the informational content of protein sequences. Science 241(4861):53-57, 1988.

Alternatively, any technique of non-random or non-random mutation induction may be employed in various embodiments of the invention. Non-random mutagenesis is exemplified by mutating (engineering or altering) a parent molecule to obtain a progeny molecule having one or more predetermined mutations. It will be appreciated that the presence of a certain amount of background products is a reality in many reactions involving molecular manipulations, and that the presence of such background products does not detract from the non-random nature of mutagenesis methods with predetermined products. Site saturation mutagenesis and synthetic conjugation recombination into the precise chemical structure of the expected product are examples of predetermined mutagenesis techniques.

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

Site saturation mutagenesis induction is generally related to the following methods: 1) preparing progeny molecules, including: molecules comprising polynucleotide sequences, molecules comprising polypeptide sequences, and molecules comprising both polynucleotide sequences and polypeptide sequences, i.e., molecules induced to achieve at least one point mutation, addition, deletion and/or chimerism relative to one or more previous or parental templates; 2) screening the progeny molecules for the desired binding affinity to the target antigen (preferably using high-throughput methods); 3) obtaining and/or cataloging structural and/or functional information about the parental and/or progeny molecules, as appropriate; and 4) repeating any of steps 1) to 3), as appropriate.

In site saturation mutation induction, progeny polynucleotides are generated (e.g., from a parental polynucleotide template) in a process called "codon site saturation mutation induction," each having at least one set of up to three consecutive site mutations (i.e., different bases including new codons) such that each codon (or each family of degenerate codons encoding the same amino acid) is represented at each codon position. Corresponding to (and encoded by) these progeny polynucleotides, a set of progeny polypeptides are also generated, each having at least one single amino acid site mutation. In a preferred aspect, one such mutant polypeptide for each of the 19 naturally encoded polypeptides is generated in a process called "amino acid site saturation mutation induction," resulting in an α-amino acid substitution at each amino acid position along the polypeptide. This results in a total of 20 different progeny polypeptides (for each amino acid position along the parent polypeptide) that include the original amino acid, or potentially more than 21 different progeny polypeptides if additional amino acids are used instead of or in addition to the 20 naturally encoded amino acids.

Other mutation induction techniques may also be employed that involve recombination and more particularly a method of preparing polynucleotides encoding polypeptides by in vivo reassortment of polynucleotide sequences containing regions of partial homology, assembling polynucleotides to form at least one polynucleotide and screening polynucleotides for the production of polypeptides having useful properties.

In another aspect, mutagenesis techniques take advantage of the natural properties of cells in combination with molecules and/or mediate reductive processing that reduces sequence complexity and the extent of repetitive or contiguous sequences with regions of homology.

Various mutagenesis techniques can be used alone or in combination to provide methods for generating hybrid polynucleotides encoding biologically active hybrid polypeptides. In achieving these and other objects, there has been provided, in accordance with an aspect of the invention, a method for introducing a polynucleotide into a suitable host cell and growing the host cell under conditions that produce a hybrid polypeptide.

Chimeric genes have been made by joining two polynucleotide fragments, each derived from a separate progenitor (or parental) molecule, using compatible sticky ends generated by restriction enzymes. Another example is mutagenesis of a single codon position in a parent polynucleotide (ie, to effect a codon substitution, addition or deletion) to produce a single progeny polynucleotide encoding a single site mutagenesis polypeptide.

In addition, in vivo site-specific recombination systems have been used to generate gene hybrids as well as random methods of in vivo recombination and recombination between homologous but plastidically truncated genes. Mutation induction has also been reported by overlapping extension and PCR.

Non-random methods have been used to achieve larger numbers of point mutations and/or chimeras, for example, comprehensive or exhaustive approaches have been used to generate all molecular species within a specific set of mutations, in order to attribute function to specific structural groups in the template molecule (e.g., specific single amino acid positions or sequences including two or more amino acid positions), and to classify and compare specific sets of mutations.

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

The mutant polynucleotides resulting from the evolution step may or may not be size fractionated on an agarose gel according to published protocols, inserted into an expression vector, and transfected into an appropriate host cell to produce the mutant polypeptide (expression). Performance can be performed using conventional molecular biology techniques. Therefore, the rendering step can use various known methods.

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

The polynucleotide selected and isolated as described is introduced into a suitable host cell. Suitable host cells are any cells that can promote recombination and/or reduce rearrangement. The selected polynucleotide is preferably already in a vector including appropriate control sequences. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or preferably, the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into host cells can be achieved by calcium phosphate transfection, DEAE-polydextrose mediated transfection or electroporation (e.g., Ecker and Davis, 1986, Inhibition of gene expression in plant cells by expression of antisense RNA, Proc Natl Acad Sci USA , 83:5372-5376).

As representative examples of expression vectors that can be used, mention may be made of virions, baculoviruses, phages, plasmids, phagemids, myxosomes, fosmids, bacterial artificial chromosomes, viral DNA (e.g. vaccinia , adenovirus, varicella virus, pseudorabies and SV40 derivatives), P1-based artificial chromosomes, yeast plastids, yeast artificial chromosomes and any other specific host (such as Bacillus, Aspergillus and yeast) carrier. Thus, for example, DNA can be included in any of a variety of expression vectors used to express polypeptides. Such vectors include chromosomal, non-chromosomal and synthetic DNA sequences. A large number of suitable vectors are known to those skilled in the art and are commercially available. The following vectors are provided by way of examples; bacterial: pQE vector (Qiagen), pBluescript plasmid, pNH vector, (λ-ZAP vector (Stratagene); ptrc99a, ρKK223-3, pDR540, pRIT2T (Pharmacia); eukaryotic: pXTl, ρSG5 (Stratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia). However, any other plasmid or other vector can be used as long as it is replicable and viable in the host. The present invention can use low copy number or High copy count vector.

The mutant polynucleotide sequence in the expression vector is operably linked to an appropriate expression control sequence (promoter) to direct RNA synthesis. Bacterial promoters of particular mention include lad, lacZ, T3, T7, gpt, λPR, PL and trp. Eukaryotic promoters include pre-early CMV, HSV thymidine kinase, pre- and post-SV40, LTRs from retroviruses, and mouse metallothionein-1. The selection of appropriate vectors and promoters is well within the level of ordinary skill in the art. The expression vector also contains ribosome binding sites for translation initiation and transcription termination. The vector may also include appropriate sequences for amplifying expression. The promoter region may be selected from any desired gene using a chloramphenicol transferase (CAT) vector or other vectors with selectable markers. In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait to facilitate selection of transformed host cells, such as dihydrofolate reductase or neomycin resistance (for eukaryotic cell culture) or in E. coli such as tetracycline or ampicillin resistance.

Eukaryotic DNA transcription can be increased by inserting an enhancer sequence into the expression vector. An enhancer is a cis-acting sequence between 10 and 300 bp that increases transcription by the promoter. Enhancers are effective in increasing transcription when 5' or 3' to the transcription unit. They are also effective if located within an intron or within the coding sequence itself. Typically, viral enhancers are used, including the SV40 enhancer, the cytomegalovirus enhancer, the polyoma enhancer, and the 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 selectable marker genes. Examples of suitable markers include the dihydrofolate reductase gene (DHFR), the thymidine kinase gene (TK) or a prokaryotic gene conferring drug resistance. The first two marker genes prefer the use of mutant cell lines that are incapable of growing without the addition of thymidine to the growth medium. Transformed cells can subsequently be identified by their ability to grow on unsupplemented medium. Examples of prokaryotic resistance genes used as markers include genes conferring resistance to G418, mycophenolic acid, and hygromycin.

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

After the expression vector has been introduced into an appropriate host, the host is maintained under conditions suitable for high-level expression of the introduced mutant polynucleotide sequence to produce the mutant polypeptide. Expression vectors typically replicate in the host organism as episomes or as integral portions of the host's chromosomal DNA. Typically, the expression vector will contain a selectable marker, such as tetracycline or neomycin, to allow detection of transformed cells with the desired DNA sequence (see, eg, US Pat. No. 4,704,362).

Therefore, in another aspect of the invention, mutant polynucleotides can be generated by a reduction rearrangement method. The method involves the generation of a construct containing a continuous sequence (the original coding sequence), its insertion into an appropriate vector, and its subsequent introduction into an appropriate host cell. The rearrangement of the individual molecular identities is performed by a combinatorial method between continuous sequences or between semi-repeated units in the construct having regions of homology. The rearrangement method recombines and/or reduces the complexity and extent of the repetitive sequences and results in the generation of novel molecular species. Various treatments can be applied to enhance the rearrangement rate. These may include treatment with ultraviolet light or DNA damaging chemicals and/or the use of host cell strains that exhibit enhanced levels of "genetic instability". Thus, shuffling methods may involve homologous recombination or the natural property of half-repeated sequences to direct their own evolution.

The cells then multiply and achieve "reduced rearrangement." The rate of rearrangement methods can be reduced by introducing DNA damaging stimuli when necessary. In vivo rearrangements focus on "intermolecular" processes, collectively known as "recombination" in bacteria, and are generally regarded as "RecA-dependent" phenomena. The present invention may rely on the ability of host cells to recombine and rearrange sequences, or cell-mediated reduction processes to reduce the complexity of half-repeated sequences in the cell by deletions. This "reduced rearrangement" approach is performed via an "intramolecular", RecA-independent approach. The end result is the rearrangement of molecules into all possible combinations.

In one aspect, the host organism or cell includes a Gram-negative bacterium, a Gram-positive bacterium, or a eukaryotic organism. In another aspect of the invention, the Gram-negative bacteria include E. coli or Pseudomonas fluorescens. In another aspect of the invention, the Gram-positive bacteria include Streptomyces diversa, Lactobacillus gasseri, Lactococcus lactis, Lactococcus cremoris Or Bacillus subtilis. In another aspect of the invention, the eukaryotic organisms include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Kluyveromyces lactis Kluyveromyces lactis), Hansenula plymorpha or Aspergillus niger. As representative examples of suitable hosts may be mentioned: 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; adenovirus; and plant cells. Selection of an appropriate host is deemed to be within the realm of those skilled in the art based on the teachings herein.

In addition to eukaryotic microorganisms such as yeast, mammalian tissue cell cultures can also be used to express the mutant polypeptides of the invention (see Winnacker, "From Genes to Clones", VCH Publishers, NY, NY (1987)). Eukaryotic cells are preferred because a variety of suitable host cell lines that can secrete complete immunoglobulins have been developed in this technology, including CHO cell lines, various COS cell lines, HeLa cells, myeloma cell lines, and B cells. or fusion tumor. Expression vectors for these cells may include expression control sequences such as origins of replication, promoters, enhancers (Queen et al., Immunol. Rev. , vol. 89, p. 49, 1986), and expression control sequences such as ribosome binding sites, Essential processing information sites for RNA splicing sites, polyadenylation sites, and transcription terminator sequences. Better performing control sequences are promoters derived from immunoglobulin genes, cytomegalovirus, SV40, adenovirus, bovine papilloma virus and the like.

In one embodiment, the eukaryotic host cell is selected from CHO, HEK293, IM9, DS-1, THP-1, Hep G2, COS, NIH 3T3, C33a, A549, A375, SK-MEL-28, DU 145, PC-3, HCT 116, 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 spleen cells and rabbit PBMC. In one aspect, the mammalian host cell is selected from CHO or HEK293 cell lines. In one specific aspect, the mammalian host cell is a CHO-S cell strain. In another specific aspect, the mammalian system is a HEK293 cell strain. In another embodiment, the eukaryotic host is a yeast cell system. In one aspect, the eukaryotic host is selected from brewer's yeast cells or Pichia pastoris cells.

In another embodiment, mammalian host cells can be commercially produced by contract research or custom manufacturing organizations. For example, for recombinant antibodies or other proteins, Lonza (Lonza Group Ltd, Basel, Switzerland) can use the GS Gene Expression System™ technology to produce vectors expressing such products using CHOK1SV or NS0 cell production hosts. Host cells containing the relevant polynucleotides can be cultured in an engineered nutrient medium suitable for activating promoters, selecting transformants, or amplifying genes. The culture conditions (such as temperature, pH, and the like) are those previously used for the host cells selected for expression and will be apparent to those of ordinary skill in the art.

As discussed above, optimization of the performance of conditionally active ASTRs can be achieved by optimization of the vector used (vector components such as promoters, splice sites, 5' and 3' ends and flanking sequences), gene Modification of host cells to reduce gene deletions and rearrangements, evolution of host cell gene activity by in vivo or in vitro methods of evolving relevant genes, host glycosylation by evolution of relevant genes and/or induction of host cell mutations by chromosome wide This is achieved through enzyme optimization and selection strategies to select cells with enhanced expressive capabilities.

Protein expression can be induced by a variety of known methods, and a variety of genetic systems for inducing protein expression have been published. For example, with an appropriate system, the addition of an inducer will induce protein expression. Cells were then pelleted by centrifugation and the supernatant was removed. Periplasmic proteins can be enriched by incubating cells with DNase, RNase, and lysozyme. After centrifugation, the supernatant containing new proteins was transferred to a new multiwell plate and stored prior to analysis.

Cells are typically harvested by centrifugation, disrupted by physical or chemical means and the resulting crude extract retained for further purification. Microbial cells employed in protein expression may be disrupted by any suitable method, including freeze-thaw cycles, sonication, mechanical disruption or use of cell lysing agents. Such methods are well known to those skilled in the art. The expressed polypeptide or fragment thereof may be recovered and purified from recombinant cell cultures by methods including precipitation with ammonium sulfate or ethanol, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography. Optionally, a protein refolding step may be used to complete the polypeptide structure. If necessary, a final purification step may be performed using high performance liquid chromatography (HPLC). Screening for conditionally active ASTRs may be aided by the availability of appropriate high throughput screening or selection methods. Cell surface display expression and screening techniques (e.g., as defined above) may be used to screen mutant proteins for conditionally active ASTRs. Screening to Identify Reversible or Irreversible Mutants

Identification of the desired molecule is most directly accomplished by measuring protein activity under permissive and wild-type conditions. Mutants with the greatest activity ratio (permissive/wild type) can then be selected and point mutation substitutions generated by combining individual mutations using standard methods. The combinatorial permutation protein library is then screened for those proteins that exhibit the greatest differential activity between permissive and wild-type conditions.

Supernatants can be screened for activity using a variety of methods, such as using high-throughput activity assays, such as fluorescence assays, to identify protein mutants that are sensitive to any desired property (temperature, pH, etc.). For example, to screen for time-sensitive mutants, the enzymatic activity of each individual mutant is determined using commercially available substrates at a lower temperature (such as 25 degrees Celsius) and at a temperature at which the original protein is functional (such as 37 degrees Celsius). or antibody activity. Screening can be performed in a variety of media, such as serum and BSA, among others. Reactions can be performed initially in a multi-well assay format, such as a 96-well assay, and validated using a different format, such as a 14 ml tube format.

In one aspect, the method further comprises modifying at least one of the nucleic acid or polypeptide prior to testing the candidate for conditional biological activity, in another aspect, the testing of step (c) further comprises testing the polypeptide for improved expression in a host cell or host organism, in another aspect, the testing of step (c) further comprises testing for enzyme activity at a pH in the range of about pH 3 to about pH 12. In another aspect, the testing of step (c) further comprises testing for enzyme activity at a pH in the range of about pH 5 to about pH 10. In another aspect, the testing of step (c) further comprises testing for enzyme activity at a pH in the range of about pH 6 to about pH 8. In another aspect, the testing of step (c) further comprises testing for enzyme activity at pH 6.7 and pH 7.5. In another aspect, the testing of step (c) further comprises testing the enzyme activity at a temperature in the range of about 4°C to about 55°C. In another aspect, the testing of step (c) further comprises testing the enzyme activity at a temperature in the range of about 15°C to about 47°C. In another aspect, the testing of step (c) further comprises testing the enzyme activity at a temperature in the range of about 20°C to about 40°C. In another aspect, the testing of step (c) further comprises testing the enzyme activity at a temperature of 25°C and 37°C. In another embodiment, the test of step (c) further comprises testing the enzyme activity under normal osmotic pressure and abnormal (positive or negative) osmotic pressure, in another embodiment, the test of step (c) further comprises testing the enzyme activity under normal electrolyte concentration and abnormal (positive or negative) electrolyte concentration. The electrolyte concentration to be tested is selected from one of calcium, sodium, potassium, magnesium, chloride ion, bicarbonate and phosphate concentration, in another embodiment, the test of step (c) further comprises testing the enzyme activity that leads to the stabilization reaction product.

In another aspect, the present invention provides a purified antibody having enzymatic activity that specifically binds to the polypeptide of the present invention or a fragment thereof. In one aspect, the present invention provides a fragment of an antibody having enzymatic activity that specifically binds to a polypeptide. Antibodies and antibody-based screening methods

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

Antibodies can be used in immunoprecipitation, staining, immunoaffinity columns and the like. If necessary, nucleic acid sequences encoding specific antigens can be generated by immunization, followed by isolation of polypeptides or nucleic acids, amplification or cloning of polypeptides and immobilization on arrays of the invention. Alternatively, the methods of the invention can be used to alter the structure of antibodies produced by cells to be modified, for example, to increase or decrease the affinity of the antibodies. In addition, the ability to make or alter antibodies can be a phenotype engineered into cells by the methods of the invention.

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

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

In immunoaffinity procedures, antibodies are attached to solid supports such as beads or other column substrates. The protein preparation is contacted with the antibody under conditions such that the antibody specifically binds to one of the polypeptides of the invention. After washing to remove unbound proteins, the specifically bound polypeptides are eluted.

The ability of proteins in a biological sample to bind to antibodies can be determined using any of a variety of procedures familiar to those skilled in the art. For example, binding can be determined by labeling the antibody with a detectable label such as a fluorescent agent, an enzyme label, or a radioactive isotope. Alternatively, antibody binding to the sample can be detected using a secondary antibody having such a detectable label thereon. Specific assays include ELISA assays, sandwich assays, radioimmunoassays, and Western blotting.

Polyclonal antibodies raised against the polypeptides of the invention can be obtained by injecting the polypeptide directly into the animal or by administering the polypeptide to a non-human animal. The antibody thus obtained will then bind the polypeptide itself. In this manner, even sequences encoding only fragments of a polypeptide can be used to generate antibodies that bind to the intact native polypeptide. Such antibodies can subsequently be used to isolate the polypeptide from cells expressing the polypeptide.

For the preparation of monoclonal antibodies, any technique that provides antibodies produced by continuous cell line culture may be used. Examples include the hybridoma technique, the tri-focal hybridoma technique, the human B cell hybridoma technique, and the EBV hybridoma technique (see, e.g., Cole (1985) in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

Techniques described for generating single chain antibodies (see, eg, US Pat. No. 4,946,778) can be adapted to generate single chain antibodies to the polypeptides of the invention. Alternatively, transgenic mice can be used to express humanized antibodies expressing such polypeptides or fragments thereof. Antibodies raised against polypeptides of the invention can be used to screen for similar polypeptides (eg, enzymes) from other organisms and samples. In such techniques, polypeptides from an organism are contacted with antibodies and those polypeptides that specifically bind to the antibodies are detected. Any of the procedures described above can be used to detect antibody binding. Screening methods and "online" monitoring devices

In practicing the methods of the present invention, a variety of apparatus and methods can be used in conjunction with the polypeptides and nucleic acids of the present invention, for example, to screen for enzymatic activity of polypeptides, to screen for compounds that are potential modulators of enzyme activity, such as activators or inhibitors, to screen for antibodies that bind to polypeptides of the present invention, to screen for nucleic acids that are mixed into nucleic acids of the present invention, to screen for cells expressing polypeptides of the present invention, and the like. Array or "biochip"

Nucleic acids or polypeptides of the invention can be immobilized or applied to arrays. Arrays can be used to screen or monitor a library of compositions (eg, small molecules, antibodies, nucleic acids, etc.) for their ability to bind to or modulate the activity of a nucleic acid or polypeptide of the invention. For example, in one aspect of the invention, the monitored parameter is the transcriptional performance of an enzyme gene. One or more or all transcripts of a cell can be quantified by hybridization of a sample including the cellular transcript or nucleic acids representative of or complementary to the cellular transcript, by hybridization to immobilized nucleic acids on an array or "biochip" Test. By using "arrays" of nucleic acids on microchips, some or all of the cellular transcripts can be quantified simultaneously. Alternatively, arrays including genomic nucleic acids may be used to determine the genotype of new engineered strains produced by the methods of the invention. "Peptide arrays" can also be used to quantify multiple proteins simultaneously. The present invention can be implemented with any known "array", which is also called "microarray" or "nucleic acid array" or "polypeptide array" or "antibody array" or "biochip" or variations thereof. An array is generally a plurality of "spots" or "target elements", each target element including a defined amount of one or more biomolecules, such as oligonucleotides immobilized on a defined area of the substrate surface for specific binding to sample molecules, e.g. mRNA transcripts.

In practicing the methods of the present invention, any known array and/or method of making and using an array may be incorporated in whole or in part or in variations thereof, such as, for example, U.S. Patent Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606; 6,054,270; 6,048,695; 6,045,996; 6,022,963; 6,013,440; 5,965,45 No. 2; No. 5,959,098; No. 5,856,174; No. 5,830,645; No. 5,770,456; No. 5,632,957; No. 5,556,752; No. 5,143,854; No. 5,807,522; No. 5,800,992; No. 5,744,305; No. 5,700,637; No. 5,556,752; No. 5,434,049; see also, for example, WO 99/51773; WO 99/09217; WO 97/46313; WO 96/17958; see also, for example, Johnston (1998) "Gene chips: Array of hope for understanding gene regulation", Curr. Biol . 8:R171-R174; Schummer (1997) "Inexpensive Handheld Device for the Construction of High-Density Nucleic Acid Arrays", Biotechniques 23:1087-1092; Kern (1997) "Direct hybridization of large-insert genomic clones on high-density gridded cDNA filter arrays", Biotechniques 23:120-124; Solinas-Toldo (1997) "Matrix-Based Comparative Genomic Hybridization: Biochips to Screen for Genomic Imbalances", Genes, Chromosomes & Cancer 20:399-407; Bowtell (1999) "Options Available-From Start to Finish~for Obtaining Expression Data by Microarray", Nature Genetics Supp . 21:25-32. See also published U.S. patent applications Nos. 20010018642; 20010019827; 20010016322; 20010014449; 20010014448; 20010012537; 20010008765. Capillary arrays

Capillary arrays, GIGAMATRIX™ Diversa Corporation, San Diego, Calif., can be used in the methods of the present invention. Nucleic acids or polypeptides of the present invention can be immobilized or applied to arrays, including in capillary arrays. Arrays can be used to screen or monitor the ability of a library of compositions (e.g., small molecules, antibodies, nucleic acids, etc.) to bind to or modulate the activity of nucleic acids or polypeptides of the present invention. Capillary arrays provide additional systems for holding and screening samples. For example, a sample screening apparatus can include a plurality of capillaries formed into an array of adjacent capillaries, wherein each capillary includes at least one wall defining an inner cavity for holding a sample. The apparatus may further include an interstitial material disposed between adjacent capillaries in the array and one or more reference markers formed in the interstitial material. Capillaries for screening samples, wherein the capillaries are adapted to be incorporated into a capillary array, may include: a first wall defining an inner cavity for retaining the sample; and a second wall formed of a filtering material for filtering excitation energy provided to the inner cavity to excite the sample. Polypeptides or nucleic acids, such as ligands, may be introduced into the first component as at least a portion of the capillaries of the capillary array. Each capillary of the capillary array may include at least one wall defining an inner cavity for retaining the first component. Air bubbles may be introduced into the capillaries after the first component. A second component may be introduced into the capillary, wherein the second component is separated from the first component by an air bubble. A related sample may be introduced into a capillary of a capillary array in the form of a first liquid labeled with a detectable particle, wherein each capillary of the capillary array includes at least one wall defining an inner cavity for retaining the first liquid and the detectable particle, and wherein at least one wall is coated with a binding material for binding the detectable particle to at least one wall. The method may further include removing the first liquid from the capillary, wherein the bound detectable particle is maintained in the capillary, and introducing the second liquid into the capillary. The capillary array may include a plurality of individual capillaries, which include at least one outer wall defining an inner cavity. The outer wall of the capillary may be one or more walls fused together. Similarly, the walls may define an interior cavity that is cylindrical, square, hexagonal, or any other geometric shape, so long as the walls form an interior cavity for retaining a liquid or sample. The capillaries of a capillary array may be held together in close proximity to form a planar structure. The capillaries may be joined together by fusion (e.g., where the capillaries are made of glass), gluing, bonding, or clamping them side by side. Capillary arrays may be formed from any number of individual capillaries, e.g., in the range of 100 to 4,000,000 capillaries. Capillary arrays may form a microtiter plate having about 100,000 or more individual capillaries joined together. Engineering conditionally active antibodies

Conditionally active antibodies can be engineered to generate multispecific conditionally active antibodies. Multispecific antibodies may be antibodies with multiple antigenic determinant specificities, as described in WO 2013/170168. Multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable domain ( _VH ) and a light chain variable domain ( _VL ), wherein the _{VH VL} unit has multiple antigenic determinant specificities; antibodies having two or more _VL and _VH domains, wherein each _{VH VL} unit binds to a different antigenic determinant; antibodies having two or more single variable domains, wherein each single variable domain binds to a different antigenic determinant; and antibodies comprising one or more antibody fragments; and antibodies comprising antibody fragments that have been covalently or non-covalently linked.

To construct multispecific antibodies, including bispecific antibodies, antibody fragments having at least one free sulfhydryl group are obtained. Antibody fragments can be obtained from full-length conditionally active antibodies. Conditionally active antibodies can be enzymatically digested to produce antibody fragments. Exemplary 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, diabody (Db); tandem diabody (taDb), linear antibody (see U.S. Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng ., Vol. 8, pp. 1057-1062 (1995)); single-arm antibodies, single variable domain antibodies, minibodies (Olafsen et al. (2004) Protein Eng. Design & Sel ., Vol. 17, pp. 315-323), single-chain antibody molecules, fragments generated from Fab expression libraries, anti-idiotypic (anti-Id) antibodies, complementarity determining regions (CDRs) and epitope binding fragment. Antibody fragments can also be produced using recombinant DNA techniques. DNA encoding antibody fragments can be cloned into plastid expression vectors or phagemid vectors and expressed directly in E. coli. Antibody enzymatic digestion methods, DNA cloning and recombinant protein expression methods are well known to those familiar with this technology.

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

Multispecific antibodies can be synthesized with engineered cross-linkers to allow for the attachment of additional functional moieties to the multispecific antibodies. The modified cross-linkers allow for the attachment of any sulfhydryl-reactive moiety. In one embodiment, N-succinimidyl-S-acetylthioacetate (SATA) is attached to bis-cis-butylenediimide to form bis-cis-butylenediimide-acetylthioacetate (BMata). After deprotection of the masked thiol group, any functional group with a sulfhydryl-reactive (or thiol-reactive) moiety can be attached to the multispecific antibody.

Exemplary thiol-reactive reagents include multifunctional linker reagents, acquisition (i.e., affinity) labeling reagents (eg, biotin-linker reagents), detection tags (eg, fluorophore reagents), solid phase immobilization reagents (e.g. SEPHAROSE™, polystyrene or glass) or drug-linker intermediates. One example of a thiol-reactive reagent is N-ethylmaleimide (NEM). Such multispecific antibodies or antibody analogs with engineered cross-linkers can be further reacted with drug moiety reagents or other labels. Reaction of a multispecific antibody or antibody analog with a drug-linker intermediate provides a multispecific antibody-drug conjugate or antibody analog-drug conjugate, respectively.

Other techniques for making multispecific antibodies may also be used in the present invention. References describing these techniques include: (1) Milstein and Cuello, Nature, Vol. 305, p. 537 (1983)); WO 93/08829; and Traunecker et al., EMBO J., Vol. 10, p. 3655 Page (1991) on the recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities; (2) US Patent No. 5,731,168 on "pestle-mortar"engineering; (3) WO 2009/089004A1 On engineered electrostatic guidance for making antibody Fc-heterodimeric molecules; (4) U.S. Patent No. 4,676,980; and Brennan et al., Science, Vol. 229, p. 81 (1985) on cross-linking two or more than two antibodies or fragments; (5) Kostelny et al., J. Immunol., Vol. 148, pp. 1547-1553 (1992) on the use of leucine zippers to generate bispecific antibodies; (6) Hollinger et al., Proc. Natl. Acad. Sci. USA, Volume 90, Pages 6444 to 6448 (1993) on the use of "bifunctional antibody" technology to prepare bispecific antibody fragments; (7) Gruber et al. Human, J. Immunol., vol. 152, p. 5368 (1994) on the use of single-chain Fv (sFv) dimers; (8) Tutt et al. J. Immunol. 147: 60 (1991) on the preparation of trispecific Antibodies; and (9) US 2006/0025576A1 and Wu et al. Nature Biotechnology , Volume 25, Pages 1290 to 1297 (2007) on engineered proteins with three or more functional antigen binding sites Antibodies, including "octopus antibodies" or "dual variable domain immunoglobulins" (DVD).

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

In one embodiment, conditionally active antibodies are engineered to cross the blood-brain barrier (BBB) to make multispecific antibodies (eg, bispecific antibodies). The multispecific antibody includes a first antigen-binding site that binds BBB-R and a second antigen-binding site that binds a brain antigen. At least the first antigen binding site of BBB-R is conditionally active. Brain antigens are antigens expressed in the brain that can be targeted with antibodies or small molecules. Examples of such antigens include (but are not limited to): beta-secretase 1 (BACE1), amyloid beta (Aβ), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau, Apolipoprotein E4 (ApoE4), 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 apoptotic protease 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 macromolecules to be transported throughout the BBB. For example, antibodies that can bind to the BBB-R can be transported throughout the BBB using the endogenous transport system. Such antibodies can serve as a medium for transporting drugs or other agents across the BBB using an endogenous BBB receptor-mediated transport system that passes through the BBB. Such antibodies do not need to have a high affinity for the BBB-R. Antibodies that are not conditionally active antibodies with low affinity for the BBB-R have been described as more effectively crossing the BBB than high-affinity antibodies, as described in US 2012/0171120.

Another method for engineering antibodies to enter the brain is to engineer antibodies to be delivered to the brain via the central nervous system lymphatic vessels. Thus, antibodies can be engineered to bind to or mimic immune cells, such as T cells, or to travel to the synovium or cerebrospinal fluid of the central nervous system via lymphatic vessels. The details of the lymphatic vessels of the central nervous system are described, for example, in Louveau, A. et al., "Structural and functional features of central nervous system lymphatic vessels", Nature 523, pp. 337-341, July 16, 2015, and the article cited this article as publicly available at the time of the present application.

Unlike traditional antibodies, conditionally active antibodies do not need to have low affinity for the BBB-R to cross the BBB and remain inside the brain. Conditionally active antibodies can have high affinity for the BBB-R on the blood side of the BBB and little or no affinity for the brain side of the BBB. Drugs, such as drug conjugates, can be coupled to conditionally active antibodies to be delivered with the antibodies throughout the BBB into the brain.

BBB-Rs are transmembrane receptor proteins expressed on brain endothelial cells that are capable of transporting molecules across the blood-brain barrier. Examples of BBBRs include: transferrin receptor (TfR); insulin receptor; insulin-like growth factor receptor (IGF-R); low-density lipoprotein receptors, including but not limited to low-density lipoprotein receptor-related protein 1 (LRP1) and low-density lipoprotein receptor-related protein 8 (LRP8); and heparin-binding epidermal growth factor-like growth factor (HB-EGF). An exemplary BBB-R herein is transferrin receptor (TfR). TfR is a transmembrane glycoprotein (molecular weight of approximately 180,000) that includes two disulfide-bonded subunits (each with an apparent molecular weight of approximately 90,000) that are involved in the uptake of iron in vertebrates.

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

Plasma is a very different body fluid than the brain's extracellular fluid (ECF). For example, Somjen ("Ions in the Brain: Normal Function, Seizures, and Stroke", Oxford University Press, 2004, pp. 16 and 33) and Redzic ("Molecular biology of the blood-brain and the blood-cerebrospinal fluid barriers"": similarities and differences", Fluids and Barriers of the CNS , Vol. 8:3, 2011), brain extracellular fluid has significantly less K ⁺ and more Mg ²⁺ and H ⁺ than plasma. The difference in ion concentration between plasma and brain ECF results in significant differences in osmolality and molar osmolarity between the two fluids. Table 1 shows the concentrations of common ions in millimoles for two plasma and brain ECF. Table 1. Common ions in plasma ( arterial plasma ) and brain extracellular fluid (CSF) arterial plasma CSF human rat human rat Na ⁺ 150 148 147 152 K ⁺ 4.6 5.3 2.9 3.4 Ca , total 2.4 3.1 1.14 1.1 Ca ²⁺ , free 1.4 1.5 1.0 1.0 pCa Mg , total 0.86 0.8 1.15 1.3 Mg ²⁺ , free 0.47 0.44 0.7 0.88 H ⁺ 0.000039 0.000032 0.000047 0.00005 pH 7.41 7.5 7.3 7.3 Cl ^- 99 119 _HCO3- ^​ 26.8 31 23.3 28

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

Therefore, several physiological conditions are different between the two sides of the BBB, such as pH, concentrations of various substances (such as lactose, glucose, K+, Mg2+), osmotic pressure, and weight molar osmotic concentration. For physiological conditions of pH, human plasma has a higher pH than human brain ECF. For physiological conditions of K+ concentration, brain ECF has a lower K+ concentration than human plasma. For physiological conditions of Mg2+ concentration, human brain ECF has significantly more Mg2+ than human plasma. For physiological conditions of osmotic pressure, human brain ECF has a different osmotic pressure than human plasma. In some embodiments, the physiological condition of brain ECFs may be the composition, pH, osmotic pressure, and weight molar osmotic concentration of brain ECFs of patients with a particular neurological disorder, which may be different from the physiological condition of brain ECFs of the general population.

The present invention therefore provides a method for evolving DNA encoding template antibodies against BBB-R to generate a mutant DNA library. The mutant DNA library is then expressed to obtain mutant antibodies. Screening mutant antibodies for conditionally active antibodies that bind to the BBB-R under at least one plasma physiological condition and have low affinity or no binding to the BBB-R under at least one brain physiological condition in brain ECF compared to the template antibody. Have affinity. Therefore, the selected mutant 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. This selected mutant antibody serves as a conditionally active antibody delivered throughout the BBB.

Such conditionally active antibodies favor crossing the BBB and remaining in the brain ECF. Low affinity to the BBB-R on the brain side reduces the rate (or removal) of conditionally active antibodies that are transported across the BBB from the brain and back into the blood relative to template antibodies.

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

Such conditionally active antibodies favor crossing the BBB and remaining in the brain ECF. After binding to the BBB-R on the plasma side, the conditionally active antibodies are transported across the BBB, and the little affinity for the BBB-R on the brain ECF side means that conditionally active antibodies are unlikely to be transported out of the brain.

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

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

In some embodiments, the IC50 of a conditionally active antibody to BBB-R in plasma can be from about 1 nM to about 100 μM, or from about 5 nM to about 100 μM, or from about 50 nM to about 100 μM, or 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. Synovial fluid conditionally active biological protein

Joint disease is a leading cause of disability and early retirement in industrialized countries. Joint disease often leads to damage to the joints that is difficult to repair. Synovial fluid is the body fluid found in the synovial cavity of joints of the human or animal body (eg, knees, hips, shoulders) between the cartilage and synovium facing the articular surfaces. Synovial fluid provides nutrients to cartilage and also serves as a lubricant for joints. Cartilage and synovial cells secrete fluid that acts as a lubricant between joint surfaces. Human synovial fluid consists of approximately 85% water. It is derived from plasma permeate, which itself is composed of water, dissolved proteins, glucose, coagulation factors, mineral ions, hormones, etc. Proteins such as albumin and globulin are present in synovial fluid and are believed to play an important role in lubricating the joint area. 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 than the rest of the body. Therefore, synovial fluid has different physiological conditions than other parts of the body, such as plasma. For example, synovial fluid has less than about 10 mg/dL of glucose, while the average normal glucose level in human plasma is about 100 mg/dL, fluctuating between 70 and 100 mg/dL throughout the day. In addition, the total protein content in synovial fluid is about one-third of the protein content of plasma because large molecules such as proteins are not easily transported through the synovium into the synovial fluid. It has also been found that the pH of human synovial fluid is higher than the pH in human plasma (Jebens et al., "On the viscosity and pH of synovial fluid and the pH of blood", The Journal of Bone and Joint Surgery, Vol. 41 B, pp. 388-400, 1959; Farr et al., "Significance of the hydrogen ion concentration in synovial fluid in Rheumatoid Arthritis", Clinical and Experimental Rheumatology, Vol. 3, pp. 99-104, 1985).

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

Several antibodies have been used to treat joint diseases by introducing them into the synovial fluid. For example, synovial fluid in injured joints is known to contain a variety of factors that affect the progression of osteoarthritis (see, e.g., Fernandes et al., "The Role of Cytokines in Osteoarthritis Pathophysiology", Biorheology , Vol. 39, pp. 237-246, 2002). Cytokines such as interleukin-1 (IL-I) and tumor necrosis factor-α (TNF-α), produced by activated synovial cells, are known to upregulate matrix metalloproteinase (MMP) gene expression. MMP upregulation leads to a decrease in matrix and non-matrix proteins in the joint. Antibodies that neutralize interleukins can prevent the progression of osteoarthritis.

Using antibodies as drugs is a promising strategy for treating joint diseases. For example, antibodies (such as those directed against aggrecan or aggrecanase) have been developed to treat osteoarthritis, which has by far the greatest incidence among joint diseases (WO1993/022429A1). 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/conditions, such as arthritis. This antibody can be used to detect the acetylated form of HMGB1 in synovial fluid (WO 2011/157905A1). Other antibodies (CD20 antibodies) have also been developed to treat damage to connective tissue and cartilage in joints.

However, the antigens of these antibodies are often expressed in other parts of the body with important physiological functions. Antibodies directed against these antigens (but effective in treating joint diseases) can also significantly interfere with the normal physiological functions of these antigens in other parts of the body. As a result, patients may experience severe side effects. Therefore, there is a need to develop therapeutic agents, such as antibodies directed against cytokines or other antigens that can preferentially bind to their antigens (proteins or other macromolecules) with higher affinity in synovial fluid, while not binding or only weakly binding to the same antigens in other parts of the body in order to reduce side effects.

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

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

In some embodiments, the present invention provides conditionally active biological proteins derived from parental or wild-type biological proteins. A conditionally active biological protein is less active in some part of the body, such as in plasma, under at least one physiological condition than a parent or wild-type biological protein, and is less active than a parent or wild-type biological protein. Higher activity in synovial fluid under at least one physiological condition. Such conditionally active biological proteins may act preferentially in synovial fluid but not or to a lesser extent in other parts of the body. Therefore, such conditionally active biological proteins may have reduced side effects.

In some embodiments, the conditionally active biological protein is an antibody directed against an antigen in or exposed to the synovial fluid. Such antigens can be any protein involved in the immune response/inflammation in joint diseases, but the antigen is often an interleukin. The conditionally active antibody has a lower affinity for the antigen in other parts of the body (such as plasma) under at least one physiological condition than the parent or wild-type antibody to the same antigen, but has a higher affinity for the antigen under at least one physiological condition in the synovial fluid than the parent or wild-type antibody. Such conditionally active antibodies may bind weakly or not at all to antigens in other parts of the body, but bind, for example, strongly and tightly or more strongly to antigens in the synovial fluid. Conditionally Active Biological Proteins for Tumors

Cancer cells in a solid tumor can form a tumor microenvironment in its environment to support the growth and metastasis of cancer cells. The tumor microenvironment is the cellular environment in which the tumor exists, including surrounding blood vessels, immune cells, fibroblasts, other cells, soluble factors, signaling molecules, extracellular matrix, and mechanical lines that can promote mesenchymal transformation, support tumor growth and invasion, protect tumors from host immunity, promote resistance to therapeutic agents, and provide an ecological niche for the thriving growth of potential metastatic tumors. Tumors and their surrounding microenvironment are closely related and constantly interact with each other. Tumors can affect their microenvironment by releasing extracellular signals, promoting tumor angiogenesis, and inducing peripheral immune tolerance, and immune cells in the microenvironment can affect the growth and evolution of cancer cells. See Swarts et al., “Tumor Microenvironment Complexity: Emerging Roles in Cancer Therapy”, Cancer Res, Vol. 72, pp. 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, which leads to difficulties in fully supplying oxygen to the tumor microenvironment. In more than 50% of locally advanced solid tumors, the oxygen partial pressure in the tumor environment is less than 5 mm Hg, compared to an oxygen partial pressure of approximately 40 mm Hg in plasma. In contrast, the rest of the body is not hypoxic. The hypoxic environment causes genetic instability, which is associated with cancer progression through downregulation of nucleotide excision repair and mismatch repair pathways. Hypoxia also causes upregulation of hypoxia-inducing factor 1 alpha (HIF1-α), which induces angiogenesis and is associated with a poorer prognosis and activation of genes associated with cancer metastasis. See Weber et al., “The tumor microenvironment”, Surgical Oncology , Vol. 21, pp. 172-177, 2012 and Blagosklonny, “Antiangiogenic therapy and tumor progression”, Cancer Cell , Vol. 5, pp. 13-17, 2004.

Additionally, tumor cells tend to rely on energy generated by lactic acid fermentation, which does not require oxygen. Thus, tumor cells are unlikely to use normal aerobic respiration, which does require oxygen. As a result of using lactic acid fermentation, the tumor microenvironment is acidic (pH 6.5-6.9), in contrast to the rest of the body, which is typically neutral or slightly alkaline. For example, human plasma has a pH of about 7.4. See Estrella et al., “Acidity Generated by the Tumor Microenvironment Drives Local Invasion”, Cancer Research , Vol. 73, pp. 1524-1535, 2013. The availability of nutrients in the tumor microenvironment is due to the relatively high nutrient requirements of proliferating cancer cells compared to cells located in other parts of the body.

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

The tumor microenvironment therefore has at least some physiological conditions that differ from those in other parts of the body, such as those in plasma. The tumor microenvironment has a lower pH (acidic) than the rest of the body, especially plasma (pH 7.4). The tumor microenvironment has lower concentrations of oxygen than other parts of the body, such as plasma. Furthermore, the tumor microenvironment has lower nutrient availability compared to other parts of the body, especially plasma. The tumor microenvironment also contains different cell types not commonly found in other parts of the body, especially in plasma.

Some cancer drugs include antibodies that penetrate into the tumor microenvironment and act on the cancer cells within it. Antibody-based therapies for cancer are long established and have become one of the most successful and important strategies for treating patients with hematological malignancies and solid tumors. There is a broader array of cell surface antigens expressed by human cancer cells that are overrepresented in normal tissue, mutated, or selectively expressed in cancer cells. These cell surface antigens are excellent targets for antibody cancer therapies.

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

In addition to antibodies, other biological proteins have shown promise in treating 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: the tumor necrosis factor-induced mechanism and the Fas-Fas ligand-mediated mechanism. At least some of the proteins involved in either of the two apoptotic mechanisms can be introduced into tumors for treatment.

Cancer stem cells are cancer cells that have the ability to generate all the cell types found in a particular cancer sample, and therefore form tumors. They can generate tumors through self-renewing stem cell processes and differentiation into a variety of cell types. It is believed that cancer stem cells remain in tumors in different groups and cause recurrence and metastasis by generating new tumors. The development of specific therapies targeting cancer stem cells may improve the survival and quality of life of cancer patients, especially for patients with metastatic disease.

Such drugs used to treat tumors often interfere with normal physiological functions in other parts of the body besides the tumor. For example, a protein that induces apoptosis of tumor cells may also induce apoptosis of some other parts of the body, thus causing side effects. In embodiments where antibodies are used to treat tumors, the antigen of the antibody may also be expressed in other parts of the body that perform normal physiological functions. For example, the monoclonal antibody bevacizumab (targeting vascular endothelial growth factor) blocks tumor blood vessel growth. This antibody may also block blood vessel growth or repair in other parts of the body, thus causing bleeding, poor wound healing, blood clots, and kidney damage. The development of conditionally active biological proteins that focus primarily or solely on targeting tumors is particularly needed for more effective tumor therapy.

In some embodiments, the present invention provides a conditionally active biological protein produced from a parent or wild-type biological protein that can be a candidate for tumor treatment. The conditionally active biological protein has lower activity in body parts other than the tumor microenvironment, such as plasma, under at least one physiological condition compared to the parent or wild-type biological protein, and has higher activity in the tumor microenvironment under at least one physiological condition compared to the parent or wild-type biological protein. Such conditionally active biological proteins can preferentially act on cancer cells in the tumor microenvironment in order to treat tumors, and therefore will be less likely to cause side effects. In embodiments where the biological protein is an antibody directed against an antigen on the surface of a tumor cell, wherein the antigen is exposed in the tumor microenvironment, the conditionally active antibody has a lower affinity for the antigen than the parent or wild-type antibody in other parts of the body, such as in a non-tumor microenvironment, and a higher affinity for the antigen than the parent or wild-type antibody in the tumor microenvironment. Such conditionally active antibodies may bind weakly or not at all to antigens in other parts of the body, but bind more strongly or strongly and tightly to antigens in the tumor microenvironment.

In some embodiments, the conditionally active antibody is an antibody directed against an immune checkpoint protein such that the immune checkpoint is inhibited. Such conditionally active antibodies have at least one of the following: (1) increased binding affinity for immune checkpoint proteins in the tumor microenvironment compared to the parent or wild-type antibody from which the conditionally active antibody originated; and (2) with Conditionally active antibodies originate from parental or wild-type antibodies that have reduced binding affinity for immune checkpoint proteins in the non-tumor microenvironment.

Immune checkpoints act as endogenous inhibitory pathways of the immune system to maintain self-tolerance and regulate the duration and extent of immune responses to antigenic stimuli, i.e., foreign molecules, cells, and tissues, see Pardoll, Nature Reviews Cancer , Vol. 12, pp. 252-264, 2012. Inhibition of immune checkpoints by inhibiting one or more checkpoint proteins may lead to hyperactivation of the immune system, especially T cells, thereby inducing the immune system to attack tumors. Checkpoint proteins suitable for the present invention include CTLA4 and its ligands CD80 and CD86, PD1 and its ligands PDL1 and PDL2, T cell immunoglobulin and mucin-3 (TIM3) and its ligand GAL9, B and T lymphocyte attenuation factor (BTLA) and its ligand HVEM (herpes virus entry mediator), receptors such as killer cell immunoglobulin-like receptor (KIR), lymphocyte activation gene-3 (LAG3) and adenosine A2A receptor (A2aR), and ligands B7-H3 and B7-H4. Additional suitable immune checkpoint proteins are described in Pardoll, Nature Reviews Cancer , Vol. 12, pp. 252-264, 2012 and Nirschl and Drake, Clin Cancer Res , Vol. 19, pp. 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 enhance T cell activation and proliferation. CTLA-4 inhibitors include anti-CTLA-4 antibodies. Anti-CTLA-4 antibodies bind to CTLA-4 and block the interaction between CTLA-4 and its ligand CD80 or CD86, thereby blocking the downregulation of the immune response induced by the interaction between CTLA-4 and its ligand.

The checkpoint protein PD1 is known to inhibit the activity of T cells in peripheral tissues and limit autoimmunity during the inflammatory response to infection. In vitro PD1 blockade enhances T cell proliferation and interleukin production in response to stimulation of specific antigen targets or allogeneic cells in mixed lymphocyte reactions. The strong correlation between PD1 expression and reduced immune response was shown to result from the inhibitory function of PD1, that is, by inducing immune checkpoints (Pardoll, Nature Reviews Cancer , 12: 252-264, 2012). PD1 blockade can be achieved by a variety of mechanisms, including antibodies PDL1 or PDL2 that bind PD1 or its ligands.

Past studies have identified antibodies against several checkpoint proteins (CTLA4, PD1, PD-L1). These antibodies are effective in treating tumors by inhibiting immune checkpoints, thereby hyperactivating the immune system, especially T cells, to attack tumors (Pardoll, Nature Reviews Cancer , Vol. 12, pp. 252-264, 2012) . However, hyperactivated T cells can also attack host cells and/or tissues, causing collateral damage to the patient's body. Therefore, therapies based on immune checkpoint inhibition using these known antibodies are difficult to treat and patient risk is a serious issue. For example, FDA-approved antibodies against CTLA-4 carry a black box warning due to their high toxicity.

The present invention solves the problem of collateral damage by providing conditionally active antibodies against immune checkpoint proteins through superactivated T cells. These conditionally active antibodies preferentially activate immune checkpoints in the tumor microenvironment. At the same time, immune checkpoints in non-tumor microenvironments, such as normal body tissues, are not inhibited by conditionally active antibodies or are inhibited less so that the possibility of collateral damage to the body in the non-tumor microenvironment is reduced. This purpose is achieved by engineering conditionally active antibodies to be more active in the tumor microenvironment than in the non-tumor microenvironment.

In some embodiments, a conditionally active antibody directed against an immune checkpoint protein can have 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 immune checkpoint proteins in the tumor microenvironment The ratio of binding activity to that of the same immune checkpoint protein in the non-tumor microenvironment. A typical assay used to measure the binding activity of an antibody is an ELISA assay.

Highly immunogenic tumors such as malignant melanoma are most susceptible to attack by a hyperactivated immune system through immune system manipulation. Therefore, conditionally active antibodies against immune checkpoint proteins may be particularly effective in treating these highly immunogenic tumors. However, other types of tumors are also susceptible to attack by a hyperactivated 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 the conditionally active antibody against a tumor cell surface molecule and the binding activity of the conditionally active antibody against an immune checkpoint protein can be present in a single protein, that is, a bispecific conditionally active antibody as disclosed herein. In some other embodiments, a combination therapy may include 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. In one embodiment, all of these binding activities may be present in a single protein, i.e., a multispecific antibody as disclosed herein.

Because conditionally active antibodies are more active in the tumor microenvironment than the activity of the parental or wild-type antibody directed against the same tumor cell surface molecule or checkpoint protein from which the conditionally active antibody originated, these combination therapies can provide both enhanced efficacy and significantly reduced toxicity. The reduced toxicity of these conditionally active antibodies, especially antibodies directed against immune checkpoint proteins, can allow the safe use of potent antibodies, such as ADC antibodies as described herein, as well as higher doses of antibodies.

In some embodiments, conditionally active antibodies directed against checkpoint proteins can be in prodrug form. For example, a conditionally active antibody may be a prodrug that does not possess the desired pharmaceutical activity until cleaved and changed to the pharmaceutical form. Prodrugs may be preferentially cleaved in the tumor microenvironment because enzymes that catalyze such cleavage are preferentially present in the tumor microenvironment or because of the accessibility of the cleavage site in the tumor microenvironment compared to the non-tumor microenvironment. Cleavage sites in the environment are more accessible. Conditionally active biological proteins targeting stem cell niches, including cancer stem cells

Stem cells exist in an environment called the stem cell niche in the body, which constitutes the basic unit of tissue physiology and integrates signals that mediate stem cell responses to the needs of the organism. However, the niche can also induce pathology by exerting abnormal functions on stem cells or other targets. The interaction between stem cells and their niche creates a dynamic system necessary for the maintenance of tissues and the ultimate design of stem cell therapeutics (Scadden, "The stem-cell niche as an entity of action", Nature, Vol. 441, pp. 1075-1079, 2006). Common stem cell niches in vertebrates include germ line stem cell niche, hematopoietic stem cell niche, hair follicle stem cell niche, intestinal stem cell niche and cardiovascular stem cell niche.

Stem cells' niche is a specialized environment that is distinct from the rest of the body (e.g., plasma) (Drummond-Barbosa, "Stem Cells, Their Niches and the Systemic Environment: An Aging Network," Genetics, vol. 180, pp. 1787-1797 Page, 2008; Fuchs, “Socializing with the Neighbors: Stem Cells and Their Niche,” Cell, vol. 116, pp. 769–778, 2004). The stem cell niche is hypoxic, where oxidative DNA damage is reduced. Direct measurement of oxygen content has revealed that bone marrow is generally completely hypoxic (approximately 1%-2% O2) compared with plasma (Keith et al., "Hypoxia-Inducible Factors, Stem Cells, and Cancer," Cell, Vol. 129, pp. 465-472, 2007; Mohyeldin et al., “Oxygen in Stem Cell Biology: A Critical Component of the Stem Cell Niche,” Cell Stem Cell, vol. 7, pp. 150-161, 2010). Additionally, a stem cell niche requires several other factors that regulate the properties of the stem cells within the niche: extracellular matrix components, growth factors, interleukins, and metabolites including pH, ionic strength (e.g., ^Ca2+ concentration) Factors of the physiological and chemical properties of the environment.

Therefore, the stem cell habitat has at least some physiological conditions that are different from those in other parts of the body, such as the physiological conditions in plasma. The stem cell habitat has a lower oxygen concentration (1-2%) than other parts of the body, especially plasma. Other physiological conditions of the stem cell habitat, including pH and ionic strength, may also be different from other parts of the body.

Stem cell therapy is an interventional strategy that introduces new adult stem cells into damaged tissue in order to treat disease or injury. This strategy relies on the ability of stem cells to self-renew and produce subsequent progeny with varying degrees of differentiation capacity. Stem cell therapy offers the significant possibility of tissue renewal that can potentially replace diseased and damaged areas in the body with minimal risk of rejection and side effects. Therefore, delivery of drugs (biological proteins (e.g., antibodies) or chemical compounds) to the stem cell niche in order to affect stem cell renewal and differentiation is an important part of stem cell therapy.

There are several examples of how stem cell niche affects stem cell renewal and/or differentiation in mammals. First in the skin, where β-1 integrin is known to be differentially expressed on primitive cells and involved in the restricted localization of stem cell populations via interactions with matrix glycoprotein ligands. Second, in the nervous system, the absence of tenascin C alters the number and function of neural stem cells in the subventricular zone. Tenascin C appears to regulate stem cell sensitivity to fibroblast growth factor 2 (FGF2) and bone morphogenetic protein 4 (BMP4), making stem cells more prone to stem cells. Third, other matrix proteins, the Arg-Gly-Asp-containing salivary protein osteopontin (OPN), have been shown to promote hematopoietic stem cell regulation. OPN interacts with several receptors known to be on hematopoietic stem cells, CD44 and α4 and α5β1 integrins. OPN production can change significantly, especially in the presence of osteoblast activation. OPN-deficient animals have increased HS cell numbers because the absence of OPN results in supraphysiological stem cell expansion under stimulatory conditions. Therefore, OPN appears to act as a constraint on hematopoietic stem cell numbers, limiting stem cell numbers under homeostatic conditions or stimulation. See Scadden, “The stem-cell niche as an entity of action”, Nature, vol. 441, pp. 1075-1079, 2006.

Xie et al. "Autocrine signaling based selection of combinatorial antibodies that transdifferentiate human stem cells", Proc Natl Acad Sci USA , Volume 110, Pages 8099 to 8104, 2013) reveal a method of using antibodies to influence stem cell differentiation. Antibodies are agonists of granulocyte colony-stimulating factor receptors. Unlike natural granulocyte colony-stimulating factors that activate cells to differentiate along predetermined pathways, isolated agonist antibodies transdifferentiate human myeloid lineage CD34+ myeloid cells into neural pioneer-like cells. Melidoni et al. ("Selecting antagonistic antibodies that control differentiation through inducible expression in embryonic stem cells", Proc Natl Acad Sci USA , Volume 110, Pages 17802 to 17807, 2013) also revealed the use of antibodies to interfere with FGF4 and its receptor FGFR1β interaction between them, thereby blocking autocrine FGF4-mediated differentiation of embryonic stem cells.

Knowledge of the function of ligands/receptors in stem cell differentiation has enabled strategies to apply biological proteins to interfere with these ligands/receptors for the purpose of regulating or even guiding stem cell differentiation. The ability to control the differentiation of non-genetically modified human stem cells by administering antibodies into the stem cell niche may provide new ex vivo or in vivo avenues for stem cell-based therapeutics. In some embodiments, the present invention provides conditionally active biological proteins produced from parental or wild-type biological proteins that are capable of entering stem cell niches, including cancer stem cells, to modulate stem cell or tumor development. A conditionally active biological protein has a lower activity in other parts of the body under at least one physiological condition than a parent or wild-type biological protein that is less active in other parts of the body under at least one physiological condition than a parent or wild-type biological protein. Stem cell niches such as cancer stem cells have higher activity in the environment. Such conditionally active biological proteins will be less likely to cause side effects and preferentially act on stem cell niches to regulate stem cell renewal and differentiation. In some embodiments, the conditionally active biological protein is an antibody. Such conditionally active antibodies may bind weakly or not at all to their antigens in other parts of the body, but bind strongly and tightly to their antigens in the stem cell niche.

The conditionally active biological proteins of the present invention that target synovial fluid, tumor microenvironment, and stem cell niches are produced by evolving DNA encoding parental or wild-type biological proteins to generate mutant DNA libraries. The mutant DNA library is then expressed to obtain mutant proteins. Screening for mutated proteins for conditionally active biological proteins that are more potent than parental or wild-type biological proteins under at least one physiological condition in a first part of the body selected from the group consisting of synovial fluid, tumor microenvironment, and stem cell niche. High activity and less activity under at least one physiological condition in a second part of the body that is different from the first part of the body compared to the parent or wild-type biological protein. The second part of the body can 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.

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

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

Conditionally active biological proteins acting in one of the synovial fluid, tumor microenvironment, or stem cell niches may also enable the use of new drug targets. The use of conventional bioproteins as therapeutic agents may lead to unacceptable side effects. For example, inhibiting the epidermal growth factor receptor (EGFR) is highly effective in inhibiting tumor growth. However, drugs that inhibit EGFR will also inhibit growth in the skin and gastrointestinal tract (GI). Side effects make EGFR unsuitable as a tumor drug target. Using conditionally active antibodies that bind to EGFR with high affinity only in the tumor microenvironment and have no or very low affinity in any other part of the body will significantly reduce side effects while inhibiting tumor growth. In this context, EGFR could become a potent new oncology drug target through the use of conditionally active antibodies.

In another example, inhibiting cytokines is often beneficial in repairing joint damage. However, suppressing interleukins in other parts of the body can also suppress the body's immune response, causing immune deficiency. Therefore, interleukins in synovial fluid are not ideal targets for the development of traditional antibody drugs for the treatment of joint injuries. However, the side effects of immune deficiency can be significantly reduced by using conditionally active antibodies that preferentially bind to interleukins in synovial fluid and do not bind or only weakly bind to the same interleukins in other parts of the body. Therefore, by using conditionally active antibodies, interleukins in synovial fluid may be suitable targets for repair of joint damage. Conditionally active biological protein for organs / tissues prone to inflammation

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

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

In some embodiments, conditionally active biological proteins can be produced by the present invention using abnormal conditions selected from one or more abnormal conditions encountered in an inflamed area and normal physiological conditions in human plasma. Such conditionally active biological proteins will therefore have higher activity in organs/tissues under inflamed conditions compared to the activity of the parent or wild-type biological protein and lower activity in human plasma compared to the activity of the parent or wild-type biological protein. Such conditionally active biological proteins may act preferentially in inflamed areas of the body, but will have little or no activity in non-inflamed areas of the body. Conditionally Active Virus Particles

Viruses have long been used as delivery vehicles for delivering 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. Virions often recognize their target cells in a ligand-receptor binding system via surface proteins that serve as recognition proteins that specifically bind to cellular proteins that serve as target proteins for the target cell (Lentz, "The recognition event between virus and "host cell receptor: a target for antiviral agents", J. of Gen. Virol ., vol. 71, pp. 751-765, 1990). For example, a viral recognition protein can be a ligand for a receptor on a target cell. The specificity between ligand and receptor allows viral particles to specifically recognize and deliver their contents to target cells.

Techniques for developing artificial virions from wild-type viruses are well known to those skilled in the art. Known artificial virions as delivery vehicles include those based on retroviruses (see, e.g., WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; US Patent No. 5,219,740; WO 93 /11230; WO 93/10218; US Patent No. 4,777,127; GB Patent No. 2,200,651; EP 0 345 242; and WO 91/02805), alpha viruses (such as Sindbis virus vectors, Semliki forest virus 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 (see, for example, WO 94/12649; WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95 /00655).

Generally speaking, artificial virus particles are constructed by inserting foreign recognition proteins into the virus particles and often replacing the natural recognition proteins through recombinant technology. The foreign recognition protein may be, for example, an antibody, a receptor, a ligand or a collagen binding domain. The present invention provides conditionally active recognition proteins that are inactivated or have reduced activity when bound to cells under normal physiological conditions and are active or have increased activity when bound to cells under abnormal conditions. Based on the presence of abnormal conditions at the site, the conditionally active recognition protein can thereby preferentially bind to diseased tissue and/or target cells at the disease site and avoid or only minimally bind to normal tissue cells where normal physiological conditions exist. The conditionally active recognition protein can be expressed and presented on the surface of the virion.

In some embodiments, the invention provides a method of evolving a parental or wild-type recognition protein and screening for conditional activity of the recognition protein. A conditionally active recognition protein is less active than the parental or wild-type recognition protein in binding to cells under normal physiological conditions, and is more active than the parental or wild-type recognition protein in binding to cells under abnormal conditions. Such conditionally active recognition proteins can be inserted into virions by well-known recombinant techniques to produce conditionally active virions.

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

In some embodiments, the target cells at the disease site are within a region or microenvironment with an abnormal pH (e.g., pH 6.5) or abnormal temperature compared to the pH or temperature in other parts of the body that are healthy or not suffering from a specific disease or disease condition. In this embodiment, the conditionally active recognition protein has a lower activity than the parent or wild-type recognition protein in binding to the target protein of the target cell at normal physiological pH or temperature, and has a higher activity than the parent or wild-type recognition protein in binding to the target protein of the target cell at abnormal pH or temperature. In this way, the recognition protein will preferentially bind to the site where the abnormal pH or temperature is encountered, thereby delivering the treatment to the disease site.

In one embodiment, the viral particle may include a conditionally active antibody of the present invention, and in particular a variable region (e.g., Fab, Fab', Fv) of the antibody. Such conditionally active antibodies may bind to a target protein (in the form of an antigen) of a target cell with lower affinity than the parent or wild-type antibody under normal physiological conditions that may be encountered at a normal tissue location and with higher affinity than the parent or wild-type antibody under abnormal conditions that may be encountered at a disease site or diseased tissue. According to the method of the present invention, the conditionally active antibody may be derived from a parent or wild-type antibody.

In one embodiment, the target protein on the target cell includes a tyrosine kinase growth factor receptor, which is overexpressed on the cell surface, for example, in various tumors. Exemplary tyrosine kinase growth factors are VEGF receptor, FGF receptor, PDGF receptor, IGF receptor, EGF receptor, TGF-α receptor, TGF-β receptor, HB-EGF receptor, ErbB2 receptor, ErbB3 receptor, and ErbB4 receptor. Conditionally Active DNA/RNA Modifying Proteins

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

Genome editing using CRISPR has the ability to rapidly and simultaneously produce multiple genetic changes in cells. A variety of human diseases, including heart disease, diabetes and neurological diseases, are affected by mutations in a variety of genes. This CRISPR-based technology has the potential to reverse disease-causing mutations and cure these diseases or at least reduce their severity. Genome editing relies on related CRISPR (Cas) proteins (a family of enzymes) in order to cut genomic DNA. Typically, Cas proteins are guided to targeted regions in the genome by a small guide RNA that matches the target region. Because Cas proteins have little or no sequence specificity, guide RNAs are used as pointers to Cas proteins to achieve precise genome editing. In one embodiment, a Cas protein can be used with multiple guide RNAs to correct multiple genetic mutations simultaneously.

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

In order to perform genome editing, the Cas protein must enter the target cell. Cells within an individual may have different intracellular pHs within the cells. Some cells in diseased tissue have abnormal intracellular pH. For example, some tumor cells tend to have an alkaline intracellular pH of approximately 7.12-7.65, whereas cells in normal tissue have a neutral intracellular pH in the range of 6.99-7.20. See Cardone et al., "The role of disturbed pH dynamics and the Na(+)/H(+) exchanger in metastasis," Nat. Rev. Cancer , vol. 5, pp. 786-795, 2005. In chronic hypoxia, cells in diseased tissue have an intracellular pH of approximately 7.2-7.5, which is also higher than that 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 Molecular Physiology , Volume 289, Pages L867 to L874, 2005). Additionally, in ischemic cells, the intracellular pH is typically in the range of 6.55 to 6.65, which is lower than the intracellular pH of normal tissue (Haqberg, "Intracellular pH during ischemia in skeletal muscle: relationship to membrane potential, extracellular pH, tissue lactic acid and ATP", Pflugers Arch ., vol. 404, pp. 342-347, 1985). More examples of abnormal intracellular pH in diseased tissue are discussed in Han et al., "Fluorescent Indicators for Intracellular pH," Chem Rev. , Vol. 110, pp. 2709-2728, 2010.

The present invention provides a method for producing conditionally active Cas protein from parent or wild-type Cas protein, wherein the conditionally active Cas protein has at least one of the following: (1) relative to the activity of parent or wild-type Cas protein in normal cells Enzyme activity that is reduced under normal physiological conditions within, and (2) activity that is increased under abnormal conditions within a target cell, such as one of the diseased cells discussed above, relative to the activity of the parental or wild-type Cas protein Enzyme activity. In some embodiments, normal physiological conditions are approximately neutral intracellular pH and abnormal conditions are varying intracellular pH above or below neutral. In one embodiment, the abnormal condition is an intracellular pH of 7.2 to 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 present invention. The conditionally active viral particles include conditionally active Cas proteins and at least one guide RNA for guiding the Cas protein to the location where the Cas protein will edit genomic DNA.

Multispecific antibodies are highly selective at preferentially targeted tissues that contain all or most of the targets (antigens) to which the multispecific antibody can bind. For example, bispecific antibodies provide selectivity for target cells by presenting a greater preference to target cells that express both of the antigens recognized by the bispecific antibody, compared to non-target cells that may express only one of the antigens. Thus, due to the dynamics of the system, there are more bispecific antibodies bound to target cells than to non-target cells at equilibrium.

The engineered multispecific antibodies or antigen-recognizing fragments thereof herein can be used as ASTRs in the chimeric antigen receptors of the present invention. Engineered Cytotoxic Cells

After the conditionally active ASTR is identified by the screening step, the chimeric antigen receptor can be assembled by joining the polynucleotide sequences encoding the individual domains to form a single polynucleotide sequence (CAR gene, which encodes the conditionally active CAR). The individual domains include conditionally active ASTR, TM and ISD. In some embodiments, other domains can also be introduced into the CAR, including ab ESD and CSD (Figure 1). If the conditionally active CAR is a bispecific CAR, the CAR gene can be, for example, in the following structure in the N-terminal to C-terminal direction: N-terminal signal sequence-ASTR 1-linker-ASTR 2-extracellular spacer domain-transmembrane domain-co-stimulatory domain-intracellular signaling domain. In one embodiment, such a CAR gene may include two or more co-stimulatory domains.

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

Conditionally active CAR is introduced into cytotoxic cells via expression vectors. Also provided herein are expression vectors comprising polynucleotide sequences encoding the conditionally active CARs of the invention. Suitable expression vectors include lentiviral vectors, gamma retroviral vectors, foamy virus vectors, adeno-associated virus (AAV) vectors, adenoviral vectors, engineered hybrid viruses, naked DNA, including (but not limited to) transposons 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.

Adenoviral expression vectors are based on adenovirus, which has a low capacity to integrate into genomic DNA, but a high efficiency to transfect host cells. Adenoviral expression vectors contain adenoviral sequences sufficient to achieve the following: (a) support expression vector packaging and (b) ultimately express the CAR gene in host cells. The adenoviral genome is a 36 kb linear double-stranded DNA into which foreign DNA sequences (such as the CAR gene) can be inserted to replace large fragments of adenoviral DNA, thereby making the expression vector of the present invention (Grunhaus and Horwitz, "Adenoviruses as cloning vectors", Seminars Virol ., Vol. 3, pp. 237-252, 1992).

Other expression vector systems are based on adeno-associated viruses, which utilize an adenovirus coupling system. This AAV expression vector has a high frequency of integration into the host genome. It can infect even non-dividing cells, thus making it suitable for gene delivery into mammalian cells, for example in tissue culture or in vivo. AAV vectors have a wide host range of infectivity. Details regarding the generation and use of AAV vectors are described in U.S. Patent Nos. 5,139,941 and 4,797,368.

Retroviral expression vectors are able 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 in specific cell lines. Retroviral vectors are constructed by inserting nucleic acids (e.g., encoding one of the CARs) into the viral genome at certain locations to produce replication-defective viruses. Although retroviral vectors are able to infect a wide variety of cell types, integration and stable expression of the CAR gene requires dividing host cells.

Lentiviral vectors are derived from lentiviruses, which are complex retroviruses that contain other genes with regulatory or structural functions in addition to the common retroviral genes gag, pol, and env (U.S. Patent Nos. 6,013,516 and 5,994,136). Some examples of lentiviruses include human immunodeficiency virus (HIV-1, HIV-2) and simian immunodeficiency virus (SIV). Lentiviral vectors have been generated by multiple reductions in HIV toxicity genes, such as deletion of genes env, vif, vpr, vpu, and nef to make the vector biosafe. Lentiviral vectors are able to infect non-dividing cells and can be used for both in vivo and ex vivo gene transfer and CAR gene expression (U.S. Patent No. 5,994,136).

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

In another aspect, the present invention also provides genetically engineered cytotoxic cells that include and stably express the conditionally active CAR of the present invention. In one embodiment, the genetically engineered cells include T lymphocytes (T cells), naive T cells ( _TN ), memory T cells (e.g., central memory T cells ( _TCM ), effector memory cells ( _TEM )), natural killer cells, and macrophages capable of producing therapeutically relevant progeny. In another embodiment, the genetically engineered cells are autologous cells. Examples of suitable T cells include CD4 ⁺ /CD8 ^- , CD4 ^- /CD8 ⁺ , CD4 ^- /CD8 ^- , or CD4 ⁺ /CD8 ⁺ T cells. T cells can be a mixed population of CD4 ⁺ /CD8 ^- and CD4 ^- /CD8 ⁺ cells or a single clone population. When co-cultured with cells expressing target antigens (e.g., CD20 ⁺ and/or CD 19 ⁺ tumor cells) in vitro, the CD4 ⁺ T cells of the present invention can also produce IL-2, IFN-γ, TNF-α and other T cell effector cytokines. The CD8 ⁺ T cells of the present invention can lyse cells expressing target antigens. In some embodiments, the T cells may be any one or more of CD45RA ⁺ CD62L ⁺ naive cells, CD45RO CD62I7 central memory cells, CD62L ^" effector memory cells, or a combination thereof (Berger et al., "Adoptive transfer of virus-specific and tumor-specific T cell immunity", Curr. Opin. Immunol ., Vol. 21, pp. 224-232, 2009).

Genetically engineered cytotoxic cells can be generated by stably transfecting host cells with an expression vector comprising a CAR gene of the present invention. Additional methods for genetically engineering cytotoxic cells using expression vectors include chemical transformation methods (e.g., using calcium phosphate, dendrimers, liposomes, and/or cationic polymers), non-chemical transformation methods (e.g., electroporation, phototransformation, gene electrotransfer, and/or hydrodynamic delivery), and/or particle-based methods (e.g., puncture infection, using a gene gun, and/or magnetofection). Transfected cells demonstrate the presence of a single integrated, unreconfigured vector and can amplify the expression of conditionally active CARs in vitro.

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

After the expression vector containing the CAR gene is introduced into the host cell, the CAR gene will be expressed, thus producing a CAR molecule that can bind to the target antigen. The resulting CAR molecules become transmembrane proteins by virtue of having a transmembrane domain. The host cells will then be converted into CAR cells, such as CAR-T cells. Methods for generating engineered cytotoxic cells, such as CAR-T cells, with CAR molecules have been described, for example (Cartellieri et al., "Chimeric antigen receptor-engineered T cells for immunotherapy of cancer", Journal of Biomedicine and Biotechnology , Volume 2010, Article ID 956304, 2010; and Ma et al., “Versatile strategy for controlling the specificity and activity of engineered T cells,” PNAS, Volume 113, E450-E458, 2016).

Whether before or after genetic modification of the cytotoxic cells to express the desired conditionally active CAR, methods such as those described in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Pat. The method described in 20060121005 activates and expands the number of cells. For example, the T cells of the present invention can be expanded by contacting with a surface to which is attached an agent that stimulates CD3/TCR complex-related signals and a ligand that stimulates a co-stimulatory molecule on the surface of the T cells. Specifically, the T cell population can be stimulated by contacting with an anti-CD3 antibody or an antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contacting with a protein kinase C activator (e.g., lichen planifolia) bound to a calcium ion carrier. In order to co-stimulate an accessory molecule on the surface of the T cells, a ligand that binds to the accessory molecule is used. For example, T cells can be contacted with anti-CD3 antibodies and anti-CD28 antibodies under conditions suitable for stimulating T cell proliferation. To stimulate CD4 ⁺ T cells or CD8 ⁺ T cells to proliferate, anti-CD3 antibodies and anti-CD28 antibodies are used. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besançon, France), which can be used as generally 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 including the CAR, or a genetically modified cell expressing the CAR protein. The CAR protein may be in the form of a pharmaceutically acceptable salt. Pharmaceutically acceptable salts refer to salts that can be used as therapeutic protein salts in the pharmaceutical industry, including, for example, sodium salts, potassium salts, calcium salts and similar salts; and procaine, diphenylmethylamine, ethanol Amine salts of diamines, ethanolamine, methylglucamine, taurine and the like; and acid addition salts such as hydrochlorides and basic amino acids and the like.

Pharmaceutically acceptable excipients may include excipients suitable for the preparation of pharmaceutical compositions that are generally safe, nontoxic, and acceptable, and include excipients that are acceptable for veterinary as well as human pharmaceutical use. Such excipients may be solid, liquid, semi-solid, or, in the case of aerosol compositions, gaseous. One type of excipient includes pharmaceutically acceptable carriers, which may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Liquid carriers include syrup, peanut oil, olive oil, glycerin, physiological saline, alcohols and water. Solid carriers include starch, lactose, calcium sulfate, dihydrate, clay, magnesium stearate or stearic acid, talc, pectin, acacia, agar and gelatin. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax.

The pharmaceutically acceptable carrier is determined in part by the specific composition administered and by the specific method used to administer the composition. Therefore, there are a variety of suitable formulations of the pharmaceutical compositions of the present invention. A variety of aqueous carriers can be used, such as buffered saline and the like. These solutions are sterile and generally do not contain undesirable substances. These compositions can be sterilized by known sterilization techniques. The composition may contain pharmaceutically acceptable auxiliary substances, such as pH adjusters and buffers, toxicity regulators and the like, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like, as required by general physiological conditions. The concentration of CAR in these formulations can vary widely and will be selected primarily based on fluid volume, viscosity, and weight, in accordance with the particular mode of administration chosen and the patient's needs.

In various embodiments, pharmaceutical compositions according to the invention may be formulated for delivery via any suitable route of administration. "Route of administration" may refer to any route of administration known in the art, including (but not limited to) aerosol, nasal, oral, intravenous, intramuscular, intraperitoneal, inhalation, transmucosal, trans. Dermal, parenteral, implantable pump, continuous infusion, topical application, capsule and/or injection.

The pharmaceutical compositions according to the present invention may be encapsulated, tableted or prepared in emulsions or syrups for oral administration. The pharmaceutical compositions are prepared following known pharmaceutical techniques involving grinding, mixing, granulation and, if necessary, compression for tablet form, or grinding, mixing and filling for hard gelatin capsule form. When a liquid carrier is used, the preparation 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.

Pharmaceutical compositions may be formulated as: (a) liquid solutions, such as an effective amount of encapsulated nucleic acid suspended in a diluent, such as water, physiological saline, or PEG 400; (b) capsules, sachets, or lozenges, each containing a predetermined amount The active ingredient in liquid, solid, granular or gelatin form; (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, liquids, buccal tablets, gels, syrups, slurries, suspensions, and the like.

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

Liquid suspensions include 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, tragacanth, and gum arabic; and dispersing agents or humectants, such as naturally occurring phospholipids (e.g., lecithin), condensation products of alkylene oxides and fatty acids (e.g., polyoxyethylene stearate), ethylene oxide and long-chain aliphatic alcohols (e.g., heptadecanethylene Condensation products of ethylene oxide and partial esters derived from fatty acids and hexitols (such as polyoxyethylene sorbitol monooleate), or condensation products of ethylene oxide and partial esters derived from fatty acids and hexitols Condensation products of partial esters of fatty acids and hexitol anhydrides (such as polyoxyethylene sorbitan monooleate). Liquid suspensions may also contain one or more preservatives, such as ethyl paraben or n-propyl paraben; one or more coloring agents; one or more flavoring agents; and one or more sweetening agents, such as sucrose. , aspartame or saccharin. The formulations can be adjusted for molar osmolarity.

Lozenge forms for oral administration may include flavorings, usually sucrose and the active ingredients of acacia or tragacanth, as well as tablets, in an inert base such as gelatin and glycerol, or sucrose and acacia, emulsions, gels and the like. Containing active ingredients, the inert matrix contains, in addition to the active ingredients, carriers known in the art. It is recognized that conditionally active CAR must be protected from digestion when administered orally. This is typically accomplished by complexing the conditionally active CAR with a composition to render it resistant to acidic and enzymatic hydrolysis or by encapsulating the conditionally active CAR in an appropriate resistant carrier, such as liposomes. Means of protecting proteins from digestion are well known in the art. Pharmaceutical compositions may be encapsulated, for example, in liposomes, or in formulations that provide slow release of the active ingredient.

Pharmaceutical compositions can be formulated as an aerosol formulation (eg, they can be "nebulized") for administration via inhalation. Aerosol formulations may be placed in acceptable pressurized propellants such as dichlorodifluoromethane, propane, nitrogen, and the like. Suitable formulations for rectal administration include, for example, suppositories, which consist of encapsulated nucleic acid and a suppository base. Suitable suppository bases include natural or synthetic triglycerides or paraffins. Alternatively, it is possible to use gelatin rectal consisting of a combination of encapsulated nucleic acids and a base including, for example, liquid triglycerides, polyethylene glycol, and paraffins. Capsules.

Pharmaceutical compositions can be formulated for parenteral administration, such as by intra-articular (in the joint), intravenous, intramuscular, intradermal, intraperitoneal and subcutaneous routes, including aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats and solutes that render the formulation isotonic to the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions, which may include suspending agents, solubilizers, thickening agents, stabilizers and preservatives. In the practice of the present invention, the composition may be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically or intrathecally. In one embodiment, parenteral administration is a preferred method for administering a composition comprising a CAR protein or a genetically engineered cytotoxic cell. The composition may be suitably administered in a unit dosage form and may be prepared by any of the methods well known in the pharmaceutical art, such as described in Remington's Pharmaceutical Sciences, Mack Publishing Co. Easton Pa., 18th edition, 1990. Formulations for intravenous administration may contain a pharmaceutically acceptable carrier such as sterile water or saline, polyalkylene glycols such as polyethylene glycol, vegetable oils, hydrogenated naphthalenes and the like.

The pharmaceutical composition can be administered by at least one mode selected from the group consisting of parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracavitary, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginally, rectally, buccally, sublingually, intranasally, and transdermally. 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 the following: a detectable marker or reporter, a TNF antagonist, an anti-rheumatic agent, a muscle relaxant, an anesthetic, a non-steroidal anti-inflammatory drug (NSAK), an analgesic, an anesthetic, a sedative, a local anesthetic, a myoneural blockade, or a combination thereof before, simultaneously, or after the conditionally active CAR. diuretics, antibacterial agents, antipsoriatics, corticosteroids, anabolic steroids, erythropoietin, immunizations, immunoglobulins, immunosuppression, growth hormones, hormone replacement drugs, radiopharmaceuticals, antidepressants, antipsychotics, stimulants, asthma medications, beta agonists, inhaled steroids, adrenaline or its analogs, cytotoxic or other anticancer agents, antimetabolites (such as methotrexate), or antiproliferative agents.

Types of cancer to be 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 malignancies, such as sarcomas, carcinomas and melanomas. Cancers can be non-solid tumors (such as hematological tumors) or solid tumors. Adult tumors/cancers and pediatric tumors/cancers are also included.

Hematological cancer is a cancer of the blood or bone marrow. Examples of hematological (or blood-related) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemias), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (refractory and advanced forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

Solid tumors are abnormal masses of tissue that usually do not contain cysts or areas of fluid. Solid tumors can be benign or malignant. Different types of solid tumors are named according to 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, synovialoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma Sarcoma, colon carcinoma, 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 cancer, pheochromocytoma sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, Cervical cancer, testicular tumors, seminoma, air sac cancer, melanoma, and CNS tumors such as gliomas (such as brainstem gliomas and mixed gliomas), glioblastomas (also known as multiple Glioblastoma) Astrocytoma, CNS lymphoma, blastoma, medulloblastoma, schwannoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, auditory nerve tumors, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma and brain metastases).

The present invention also provides a medical device comprising at least one CAR protein, a polynucleotide sequence encoding a CAR, or a host cell expressing a CAR, wherein the device is suitable for administering at least one conditionally active CAR by at least one mode selected from the following: parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracavitary, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginally, rectally, buccally, sublingually, intranasally, or transdermally.

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

Also provided is an article of manufacture for human pharmaceutical or diagnostic use, which includes an encapsulating material and a container including at least one CAR protein in solution or lyophilized form, a polynucleotide sequence encoding the CAR, or a host cell expressing the CAR. The article may optionally include having a container as parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraabdominal, intracystic, intrachondral, intracavity, intracoelomic, intracerebellar, intracerebroventricular, intracolonic, Intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intravertebral, intrasynovial, intrathoracic A component of an intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal or transdermal delivery device or system.

In some embodiments, the present invention provides a method comprising retrieving cytotoxic cells from an individual, genetically engineering the cytotoxic cells by introducing the CAR gene of the present invention into the cytotoxic cells, and administering the genetically engineered cytotoxic cells to the individual. 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 cells are T cells.

In one embodiment, T cells are obtained from an individual. T cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present invention, a variety of T cell strains available in the art can be used. In certain embodiments of the present invention, T cells can be obtained from blood collected from an individual using any number of techniques known to those skilled in the art, such as Ficoll™ separation.

In a preferred embodiment, cells from the individual's circulating blood are obtained by hemocytosis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells collected by hemocytosis can be washed to remove the plasma fraction and the cells placed in an appropriate buffer or culture medium for subsequent processing steps. In one embodiment of the present invention, cells are washed with phosphate buffered saline (PBS). In alternative embodiments, the wash solution is deficient in calcium and may be deficient in magnesium or may be deficient in many, if not all, divalent cations. Again, unexpectedly, the initial activation step in the absence of calcium resulted in amplified activation. As will be readily understood by those skilled in the art, the washing step may be accomplished by methods known to those skilled in the art, such as by using semi-automatic "flow-through" centrifugation according to the manufacturer's instructions (e.g. Cobe 2991 Cell Processor, Baxter CytoMate or Haemonetics Cell Saver 5). After washing, the cells can be resuspended in a variety of biocompatible buffers, such as Ca ²⁺ -free, Mg ²⁺ -free PBS, PlasmaLyte A, or other buffers with or without physiological saline solution. Alternatively, undesired components of the apheresis sample can be removed and the cells resuspended directly in culture medium.

In another embodiment, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, such as by PERCOLL ^™ gradient centrifugation or by countercurrent centrifugation. Specific subsets of T cells (such as CD3 ⁺ , CD28 ⁺ , CD4 ⁺ , CD8 ⁺ , CD45RA ⁺ and CD45RO ⁺ T cells) can be further isolated by positive or negative selection techniques. For example, enrichment of T cell populations by negative selection can be accomplished using a combination of antibodies directed against surface markers unique to the cells being negatively selected. One method is cell sorting and/or selection via negative magnetic immunoadhesion or flow cytometry using a mixture of monoclonal antibodies directed against cell surface markers present on negatively selected cells. To enrich CD4+ cells by negative selection, monoclonal antibody cocktails typically include antibodies to CD 14, CD20, CD11b, CD 16, HLA-DR and CD8. In certain embodiments, it may be desirable to enrich or positively select regulatory T cells, which typically express CD4 ⁺ , CD25 ⁺ , CD62L ^hi , GITR ⁺ and FoxP3 ⁺ .

For example, in one embodiment, T required for positive selection are obtained by incubating T with anti-CD3/anti-CD28 (i.e., 3×28)-conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T cells for a sufficient period of time to isolate T cells. In one embodiment, the time period is about 30 minutes. In another embodiment, the time period ranges from 30 minutes to 36 hours or more and all integer values therebetween. In another embodiment, the time period is at least 1, 2, 3, 4, 5 or 6 hours. In another preferred embodiment, the time period is 10 to 24 hours. In a preferred embodiment, the incubation period is 24 hours. To isolate T cells from leukemia patients, using longer incubation times (such as 24 hours) may increase cell yield. Longer incubation times can be used to isolate T cells in any situation where T cells are rare compared to other cell types, such as isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or from immunocompromised individuals. In addition, using a longer incubation time can increase the efficiency of capturing CD8+ T cells. Thus, simply shortening or lengthening the time required for T cells to bind to CD3/CD28 beads, and/or by increasing or decreasing the bead to T cell ratio (as further described herein), at the beginning of the culture or during the method At other time points during the period, T cell subsets are preferentially selected accordingly. Additionally, by increasing or decreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on beads or other surfaces, T cell subsets can be prioritized accordingly at the beginning of culture or at other desired time points. Those skilled in the art will understand that multiple selection rounds may also be used in the context of the present invention. In some embodiments, it may be necessary to perform a selection procedure and use "unselected" cells during activation and expansion. "Unselected" cells can also undergo additional rounds of selection.

The resulting cytotoxic cells are then genetically engineered as described herein. A CAR-encoding polynucleotide, typically located in an expression vector, is introduced into the cytotoxic cells such that the cytotoxic cells will express (preferably stably) the CAR. The polynucleotide encoding the CAR is typically integrated into the cytotoxic cell host genome. In some embodiments, polynucleotide introduction need not result in integration, but rather merely the temporary maintenance of the introduced polynucleotide may be sufficient. In this way, one can have a short-term effect, where cytotoxic cells can be introduced into the host and subsequently turned on after a predetermined time, for example after the cells have been able to migrate to a specific treatment site.

Depending on the nature of the cytotoxic cells and the disease to be treated, genetically engineered cytotoxic cells can be introduced into an individual, such as a mammal, by a wide variety of methods. Genetically engineered cytotoxic cells can be introduced at the tumor site. In one embodiment, genetically engineered cytotoxic cells navigate to cancer or are engineered to navigate to cancer. The number of genetically engineered cytotoxic cells used will depend on a variety of factors, such as the situation, purpose of introduction, cell life cycle, and protocol for use. For example, the number of administrations, cell doubling capacity and stability of the recombinant construct. Genetically engineered cytotoxic cells can be administered as a dispersion injected at or near the site of interest. The cells can be in a physiologically acceptable medium.

It is understood that treatment methods are subject to a variety of variables, such as the cellular response of the CAR, the efficacy and, if appropriate, the level of secretion of the CAR expressed by the cytotoxic cells, the activity of the expressed CAR, the specific needs of the individual, which may vary over time and circumstances, the rate of loss of cellular activity due to loss of genetically engineered cytotoxic cells or the expression activity of individual cells, and the like. Therefore, it is expected that for each individual patient, even if there are universal cells that can be administered to the general population, each patient will be monitored for an individual's appropriate dose, and such routine monitoring of patients is routine in the art.

The following examples are for illustrative but not limiting purposes of the method of the present invention. Other suitable modifications and adaptations of various conditions and parameters commonly encountered in the art and apparent to those skilled in the art are within the scope of the invention. Examples Example 1 : Generation of scFv conditionally active antibodies against Axl

Two conditionally active single chain antibodies (CAB-scFv-63.9-4 and CAB-scFv-63.9-6) directed against the drug target antigen Axl were expressed with wild-type human IgG1 Fc, SEQ ID NO: 13 or 14 (generating Figure 2 Homodimers of the bivalent antibodies CAB-scFv-63.9-4-01, SEQ ID NO: 9 and CAB-scFv-63.9-6-01, SEQ ID NO: 10) in -3 and the production of monovalent scFv (production The two different pestle-mortar systems of monovalent antibody scFv CAB-scFv-63.9-4-02, SEQ ID NO: 11 and CAB-scFv-63.9-6-02, SEQ ID NO: 12) in Figure 2-3 aggregate.

The binding affinity of these antibodies to the drug target antigen Axl at pH 6.0 and pH 7.4 was measured by ELISA analysis. As shown in Figure 2, the scFv antibody exhibited affinity for the drug target antigen Axl at both pH 6.0 and pH 7.4, which was comparable to all bivalent antibodies. In addition, as shown in Figure 3, the selectivity of these scFv antibodies at pH 6.0 over pH 7.4 was also comparable to all bivalent antibodies. This example demonstrates that the conditionally active antibodies of the present invention have affinity and selectivity comparable to scFv antibodies or all bivalent antibodies. Therefore, in the CAR-T platform of the present invention, the conditionally active antibodies of the present invention can be inserted as a single DNA chain into the DNA molecule encoding the CAR. Example 2 : scFv antibodies against the target antigen Axl for constructing CAR-T cells

According to one embodiment of the present invention, conditionally active antibodies for the drug target antigen Axl are generated by simultaneously screening for selectivity and affinity as well as expression at pH 6.0 and pH 7.4. Screening is performed using a FLAG tag in serum because human antibodies are present in serum that can cause false positives in screening. The screening buffer is a carbonate buffer (Krebs buffer and Ringer's standard buffer, but different from PBS). It was found that the conditionally active antibodies generated had a higher affinity for the drug target antigen Axl at pH 6.0, but a lower affinity for the same drug target antigen Axl at pH 7.4, both compared to the wild-type antibody. In addition, these conditionally active antibodies all had high expression levels, as shown in the following Table 2, where the row "Strain" displays the antibody and the expression level "mg/ml" is shown in the second row.

These antibody clones were delivered to the service provider at the required expression level ("ordered level", expected expression level). However, the actual expression level of these antibodies ("delivered level") was extremely high and exceeded the expected expression level. Table 2. Conditionally active antibodies with high expression levels Clonal strain mg/ml Target volume Amount BAP063.6-huml0F10-FLAG 7 150 294 BAP063.6-HC-H100Y-FLAG 6.6 150 238 BAP063.8-LC046HC04-FLAG 7 200 332.5 BAP063.8-LC062HC02-FLAG 5.8 200 220. 4 BAP063.9-13-1-FLAG 5.3 50 123 BAP063.9-29-2-FLAG 4.9 50 102 BAP063.9-45-2-FLAG 5.4 50 129 BAP063.9-13-3-FLAG 5.9 50 130 BAP063.9-21-3-FLAG 5.3 50 117 BAP063.9-21-4-FLAG 7 50 176 BAP063.9-29-4-FLAG 8.2 50 196 BAP063.9-48-3-FLAG 7 50 125 BAP063.9-49-4-FLAG 5.3 50 126 BAP063.9-61-1-FLAG 5.1 50 97 BAP063.9-61-2-FLAG 5 50 92

Using the BAP063.9-13-1 antibody as an example, the conditionally active antibody did not exhibit aggregation in buffer, as demonstrated in Figure 4. BAP063.9-13-1 antibody was analyzed by size exclusion chromatography. In Figure 4, only one peak was detected, indicating little or no aggregation of the antibody.

Conditionally active antibodies were also analyzed using surface plasmon resonance (SPR) to measure their on and off rates for the drug's target antigen Axl. SPR analysis is known to measure the rate at which conditionally active antibodies turn on and off. SPR analysis was performed in the presence of bicarbonate. The rate at which conditionally active antibodies are turned on and off in vivo (in animals and humans) is an extremely important characteristic of conditionally active antibodies.

It was observed that the conditionally active antibody had a fast turn-on rate at pH 6.0 and a slower turn-on rate at pH 7.4 compared to the negative control (BAP063 10F10, which had similar turn-on rates at pH 6.0 and pH 7.4) (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 more selective at pH 6.0 compared to pH 7.4 (Figures 6A-6B show one antibody as an example).

Conditionally active biological antibodies are summarized in Table 3. Two antibodies are represented as scFv (BAP063.9-13.3 and BAP063.9-48.3), which are intended to be inserted into the CAR in the CAR-T platform. Incubating antibodies at 60°C for one hour does not change the affinity of most antibodies ("thermal stability"). In the two rows of report data (the last two rows of Table 3) using SPR to measure the binding activity at pH 6.0 and pH 7.4, it is the same as "BAP063.6-hum10F10-FLAG" (negative control, the second row in Table 3). column) for comparison. The selectivity of these antibodies can be determined by the difference between the last two rows of data. Both scFv antibodies were extremely selective (75% and 50% at pH 6 and over 0% at pH 7.4). Table 3 - Overview of Conditionally Active Antibodies clone CABscFv mg/ml Booking volume Delivery volume Aggregation (PBS , pH 7.4) Thermal stability (60 ℃ 1 hour ) Improved binding at pH 7.4 after heat treatment Ka [Ms] Kd[s ^-1] KD[M] pH6.0 pH 6.0 SPR activity SPR active pH 7.4 BAF063 .6-hum10F10-FLAG 7 150 294 no 100% no 5.14E+06 8.38E-04 1.63E-10 100% 100% BAF063.6-HC-H100Y-FLAG 6.6 150 238 ND 2.41E+06 5.12E-03 2.12E-09 80% 40% BAF063.9-13-1-FLAG 5.3 50 12 3 no 100% yes 1.98E+06 2.88E-03 1.46E-09 100% 75% BAF063.9-29-2-FLAG 4.9 50 102 no 100% yes 1.19E+06 2.14E-03 1.79E-09 90% 50% BAF063.9-45-2-FLAG 5.4 50 12 9 no reduce yes 1.53E+06 2.31E-03 1.51E-09 75% 25% BAF063.9-13-3-FLAG yes 5.9 50 130 no 100% yes 1.42E+06 1.82E-03 1.28E-09 75% 0% BAF063.9-21-3-FLAG 5.3 50 117 no 100% no 1.53E+06 4.13E-03 2.69E-09 50% 25% BAF063.9-21-4-FLAG 7 50 176 no 100% no 1.03E+06 3.26E-03 3.16E-09 50% 0% BAF063.9-29-4-FLAG 8.2 50 196 no 100% (yes) 1.40E+06 2.21E-03 1.58E-09 75% 0% BAF063.9-43-3-FLAG yes 7 50 12 5 <5% reduce no 8.92E+05 2.33E-03 2.61E-09 50% 0% Comparative Example A : CAR-T cells with non-conditionally active antibodies against target antigen Axl

The non-conditionally active scFv antibody against the target antigen Axl is used to construct CAR-T cells that bind to the target antigen Axl or CHO cells (CHO-Axl) that express the target antigen Axl on the cell surface, Figures 7A-7B. The non-conditionally active antibody is used as an ASTR inserted into T cells to construct the CAR molecule of the CAR-T cell that can 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) T cells transduced with CAR molecules with non-conditionally active antibodies against the target antigen Axl (Fig. 7A). The CHO cell population is indicated by cell index (Y-axis in Figure 7A), where a decrease in cell index indicates cytotoxicity (cell killing) of CAR-T cells.

Referring to Figure 7A, CHO cells demonstrate growth prior to addition of T cells. After the addition of CAR-T cells binding to the target antigen Axl, the cell index initially decreased, indicating non-specific cytotoxicity of the T cells. However, the CHO cells resumed growth shortly thereafter. More importantly, the differences among the three treatments were not significant, indicating that CHO cells that do not exhibit significant cytotoxicity for the target antigen Axl of CAR-T cells with non-conditionally active antibodies against the target antigen Axl.

The CHO cells expressing the target antigen Axl were then treated in the same manner as above with: (1) T cells not transduced with a CAR molecule, (2) T cells transduced with a CAR molecule that does not bind to the target antigen Axl, and (3) T cells transduced with a CAR molecule having an unconditionally active antibody against the target antigen Axl ( FIG. 7B ). After the addition of T cells, the cell index was significantly reduced by treating the CAR-T cells with an unconditionally active antibody against the target antigen Axl, but not by the other two treatments, indicating cytotoxicity of CHO-X1 cells expressing the target antigen Axl by CAR-T cells having an unconditionally active antibody against the target antigen Axl. Example 3 : CAR-T cells having a conditionally active scFv antibody against the target antigen Axl

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

Referring to Figure 8A, CHO cells that do not express the target antigen Axl are treated with CAR-T cells and non-transduced T cells. There was no significant difference between the two treatments, indicating that CAR-T cells were not cytotoxic to CHO cells. Referring to Figure 8B, CHO cells (CHO-63) expressing target antigen Axl were similarly treated. Compared with non-transduced T cells, CAR-T cells with conditionally active antibodies against target antigen Axl significantly reduced CHO cells. -63 cell population. This indicates that CAR-T cells with conditionally active antibodies against the target antigen Axl are cytotoxic to CHO-63 cells.

After binding to the target antigen Axl, CAR-T cells induce cytotoxicity. This effect was confirmed by measuring the levels of interferon gamma (INFg) and IL2. The interleukin data are shown in Figures 9A-9B. In Figure 9A, the binding of CAR-T cells to the target antigen Axl on CHO-63 cells triggers a significant release of INFg compared to untransduced T cells, as shown by the observed increased interleukin levels. Similarly, in Figure 9B, the binding of CAR-T cells to the target antigen Axl on CHO-63 cells triggers a significant release of IL2 compared to untransduced T cells, as shown by the observed increased interleukin levels. Example 4 : CAR-T cells with conditionally active scFv antibodies against target antigen ROR2

Conditionally active scFv antibodies against the target antigen ROR2 were generated. The binding activity of the scFv antibodies against the target antigen ROR2 was measured using ELISA analysis ( FIG. 10 ).

One of the scFv antibodies shown in FIG. 10 , scFv-116101, was used to construct a CAR molecule (116101 CAR-T) for generating CAR-T cells. The constructed CAR-T cells were used to target Dowdy cells expressing the target antigen ROR2. Negative controls were T cells that were not transduced with the CAR molecule (untransduced T cells) and CAR-T cells that were transduced with a CAR molecule that could not bind to the target antigen ROR2 (non-ROR2 scFv CAR-T). The results are shown in FIG. 11A . The ratio of the number of T cells to the number of Dowdy cells in these treatments was 10:1. CAR-T cells (116101 CAR-T) with scFv antibodies targeting ROR2, a target antigen on Daudi cells, induced significant cell death against Daudi cells, as shown by the higher dead/survival cell ratio in FIG11A .

HEK293 cells were treated with the same T cells as those used to treat Dowdy cells. The results are shown in Figure 11B. Because HEK293 cells do not express the target antigen ROR2 on the cell surface, CAR-T cells with scFv antibodies targeting the target antigen ROR2 (116101 CAR-T) do not induce significant cell death of HEK293 cells compared to negative controls (Figure 11B). Example 5 : Cytokine release of CAR -T cells with antibodies against target antigens Axl and ROR2 in Examples 1 to 4

In this example, interleukin release induced by binding of CAR-T cells to target antigens was measured. Figures 12A-12B show INFg and IL2 release after binding of CAR-T cells containing conditionally active scFv antibodies against target antigen Axl to CHO-63 cells expressing target antigen Axl. After 24 hours of treatment of CAR-T cells with these CHO-63 cells, INFg and IL2 interleukin levels were significantly increased compared to controls in which the same CAR-T cells were used to treat CHO cells that do not express target antigen Axl, indicating INFg and IL2 interleukin release. In addition, T cells transduced with CAR molecules and CAR-T cells that did not bind to the target antigen Axl did not lead to significant release of INFg and IL2 cytokines.

Figures 13A-13B show the INFg and IL interleukin content after CAR-T cells containing conditionally active scFv antibodies against the target antigen ROR2 are combined with Rajib cells and Dowdy cells, both of which express the target antigen ROR2. After 24 hours of treatment of Rajib and Daudi cells with CAR-T cells, INFg and IL2 interleukin were observed compared to a control where the same CAR-T cells were used to treat HEK293 cells that do not express the target antigen ROR2. content significantly increased. In addition, T cells transduced with CAR molecules and CAR-T cells that did not bind to the target antigen ROR2 did not significantly increase interleukin levels, indicating that significant release of INFg and IL2 interleukins was not induced. Example 6 : Conditionally active scFv antibody against target antigen CD22

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

It should be understood, however, that although many features and advantages of the invention have been described in the foregoing description together with details of its structure and function, the invention is illustrative only and may be varied in detail, especially with respect to the shape, size and arrangement of parts within the principles of the invention, to the greatest extent indicated by the broad general meanings of the terms, within which the scope of the appended patent applications is expressed.

Figure 1 depicts a schematic representation of a chimeric antigen receptor according to one embodiment of the 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.

Figures 2 and 3 show that the conditionally active antibodies of Example 1 behave as bivalent or monovalent antibodies without significantly altering the selectivity of these antibodies at pH 6.0 and above pH 7.4.

FIG. 4 is a curve of size exclusion analysis indicating that the conditionally active antibody of Example 2 does not aggregate.

Figure 5 shows the on and off rates of the conditionally active antibody of Example 2 as measured by surface plasmon resonance (SPR) analysis.

Figures 6A-6B show the selectivity of conditionally active antibodies as measured by SPR analysis of Example 2.

Figure 7A shows that CAR-T cells have no effect on a CHO cell population that does not express the target antigen X1 of the CAR-T cells. The CAR molecule in the CAR-T cells of this example includes an antibody against the target antigen X1, but this antibody is not conditionally active (compare to Example A).

Figure 7B shows that CAR-T cells reduce the CHO-63 cell population expressing the 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).

Figure 8A shows that CAR-T cells have no effect on a CHO cell population that does not express the target antigen X1 of the CAR-T cells. The CAR molecule in the CAR-T cells of this Example 3 includes a conditionally active antibody against the target antigen X1.

Figure 8B shows CAR-T cell reduction in a CHO-63 cell population expressing target antigen X1 of CAR-T cells as tested in Example 3. These CAR-T cells were the same cells used to generate the data shown in Figure 8A.

9A-9B show the release of interleukins induced by the binding of CAR-T cells to the target antigen X1 as described in Example 3.

Figure 10 shows conditionally active antibodies against target antigen X2.

11A-11B show the cytotoxicity induced by CAR-T cells bound to Daudi cells expressing target antigen X2 (A) and the cytotoxicity induced by CAR-T cells on HEK293 cells that do not express target antigen X2 (B), as described in Example 4.

12A-12B show the release of interleukins induced by the binding of CAR-T cells to the target antigen X1 as described in Example 5.

Figures 13A-13B show interleukin release induced by binding of CAR-T cells to target antigen X2 as described in Example 5.

Figure 14 shows conditionally active antibodies against target antigen X3 suitable for constructing CAR-T cells.

Claims (17)

A chimeric antigen receptor that binds to the Axl antigen, comprising: i. at least one antigen-specific targeting region, which is a single-chain antibody having an amino acid sequence selected from SEQ ID NO: 9-12, and is similar to Having reduced activity in assays at normal physiological pH compared to the activity of the antigen-specific targeting region in assays at abnormal pH that deviates from normal physiological pH; ii. A transmembrane domain selected from artificial hydrophobic sequences And the transmembrane domain of type I transmembrane protein, the α, β or ζ chain of T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86 , CD134, CD137 and CD154; and iii. intracellular signaling domain, which is selected from the cytoplasmic signaling domain of the human CD3ζ chain, FcyRIII, FcsRI, the cytoplasmic tail region of the Fc receptor, with an immunoreceptor tyrosine-based Cytoplasmic receptors of activation motifs (ITAM), TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b and CD66d. A chimeric antigen receptor that binds to a ROR2 antigen, comprising: i. at least one antigen-specific targeting region, which is a single-chain antibody having an amino acid sequence selected from SEQ ID NO: 15, and having reduced activity in an assay at normal physiological pH compared to the activity of the antigen-specific targeting region in an assay at an abnormal pH that deviates from normal physiological pH; ii. a transmembrane domain selected from an artificial hydrophobic sequence and a transmembrane domain of a type I transmembrane protein, an α, β or ζ chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD 80, CD86, CD134, CD137 and CD154; and iii. an intracellular signaling domain selected from the cytoplasmic signaling domain of the human CD3ζ chain, FcyRIII, FcsRI, the cytoplasmic tail of the Fc receptor, a cytoplasmic receptor with an immunoreceptor tyrosine-based activation motif (ITAM), TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b and CD66d. The chimeric antigen receptor of any one of claims 1 to 2, wherein the normal physiological pH is the normal physiological pH in the plasma of a mammalian individual and the abnormal pH is the pH in the tumor microenvironment. The chimeric antigen receptor of claim 3, wherein the normal physiological pH is in the range of greater than 7.0 to about 7.8. The chimeric antigen receptor of claim 4, wherein the normal physiological pH is in the range of about 7.2 to about 7.6. The chimeric antigen receptor of claim 3, wherein the abnormal pH is in the range of 6.0 to less than 7.0. The chimeric antigen receptor of claim 6, wherein the abnormal pH is in the range of 6.0 to about 6.8. The chimeric antigen receptor of any one of claims 1 to 2 further comprises an extracellular spacer domain or at least one synergistic stimulatory domain. The chimeric antigen receptor of claim 8, wherein the extracellular spacer domain is selected from the following Group: Fc fragment of antibody, hinge region of antibody, CH2 region of antibody, CH3 region of antibody, artificial spacer sequence and combinations thereof. A chimeric antigen receptor as claimed in any one of claims 1 to 2, wherein the at least one antigen-specific targeting region has a ratio of at least about 2 of the activity under the abnormal condition to the same activity under the normal physiological condition. The chimeric antigen receptor as claimed in any one of claims 1 to 2, wherein the at least one antigen-specific targeting region includes two antigen-specific targeting regions connected to a linker. Such as the chimeric antigen receptor of claim 11, wherein the two antigen-specific targeting regions each bind to different target antigens or different epitopes of the same target antigen. The chimeric antigen receptor of any one of claims 1 to 2 further comprises a synergistic stimulatory domain selected from the group consisting of: synergistic stimulatory domains of proteins in the TNFR superfamily, CD28, CD137, CD134, DaplO, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, LIGHT, NKG2C and B7-H3. An expression vector comprising a polynucleotide sequence encoding a chimeric antigen receptor as described in any one of claims 1 to 2. The expression vector of claim 14, wherein the expression vector is selected from the group consisting of lentiviral vectors, gamma reverse transcription Viral vectors, foamy virus vectors, adeno-associated virus vectors, adenovirus vectors, poxvirus vectors, herpes virus vectors, engineered hybrid viruses and transposon-mediated vectors. A genetically engineered cytotoxic cell comprising a polynucleotide sequence encoding a chimeric antigen receptor as claimed in any one of claims 1 to 2. A method for generating a chimeric antigen receptor comprising at least one antigen-specific targeting region, a transmembrane domain selected from the group consisting of an artificial hydrophobic sequence and a transmembrane domain of a type I transmembrane protein, and an intracellular signaling domain. Membrane domain, α, β or ζ chain of T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, the The intracellular signaling domain is selected from the cytoplasmic signaling domain of the human CD3ζ chain, FcyRIII, FcsRI, the cytoplasmic tail region of Fc receptors, cytoplasmic receptors with immunoreceptor tyrosine-based activation motifs (ITAM), TCRζ , FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b and CD66d, the method includes the following steps: from a parent or wild-type protein that specifically binds to a tumor-specific target antigen selected from Axl and ROR2 or a domain thereof generates the at least one antigen-specific targeting region by: i. evolving DNA encoding the parent or wild-type protein or domain thereof using one or more evolutionary techniques to generate mutant DNA; ii. Expression of the mutant DNA to obtain mutant polypeptides; iii. Subjecting the mutant polypeptides to analysis at normal physiological pH and analysis at abnormal pH deviating from the normal physiological pH; and iv. Expression from step (ii) Mutated polypeptides selected to specifically target the at least one antigen A zone exhibiting reduced activity in the assay at the normal physiological pH compared to activity in the assay at the abnormal pH.