CN112823166B

CN112823166B - Conditionally active chimeric antigen receptors for modified T cells

Info

Publication number: CN112823166B
Application number: CN201980058347.2A
Authority: CN
Inventors: 杰·M·肖特
Original assignee: Bioatla Inc
Current assignee: Bioatla Inc
Priority date: 2018-09-07
Filing date: 2019-08-23
Publication date: 2025-01-24
Anticipated expiration: 2039-08-23
Also published as: JP7432250B2; EP3847191A4; WO2020050993A2; CN112823166A; TWI835848B; AU2019334864A1; JP2021536256A; EP3847191A2; TW202024124A; WO2020050993A3; AU2019334864B2

Abstract

The present invention relates to chimeric antigen receptors for binding to tumor-specific target antigens. The chimeric antigen receptor comprises at least one antigen-specific targeting region evolved from a parent protein or fragment thereof, and the antigen-specific targeting region has the property that activity in an assay under normal physiological conditions is reduced compared to activity in an assay under aberrant conditions. Methods of producing the chimeric antigen receptor are also provided.

Description

Conditionally active chimeric antigen receptor for modified T cells

Data of related applications

The present application is a continuation-in-part of U.S. patent application Ser. No. 16/053,166, filed on even date 8 at 2018 (currently under examination), which U.S. patent application Ser. No. 16/053,166 is in turn a divisional application of U.S. patent application Ser. No. 15/052,487, which has been filed on even date 15/052,487, which is in turn a continuation-in-part of International application Ser. No. PCT/US15/47197, filed on even date 27 at 2015, which International application Ser. No. 15/47197 claims priority from U.S. provisional application Ser. 62/043,067, filed on even date 28 at 2014 (which has expired), all of which are incorporated herein by reference in their entirety.

Technical Field

The present invention relates to the field of protein evolution. In particular, the invention relates to methods of producing conditionally active chimeric antigen receptors from a parent or wild-type protein. Conditionally active chimeric antigen receptors are reversibly or irreversibly inactivated under wild-type normal physiological conditions, but active under abnormal conditions.

Background

There is a great deal of literature describing the potential to evolve various properties of proteins, particularly enzymes for example. For example, enzymes may be evolved to stabilize them for operation under different conditions (e.g., at elevated temperatures). In the case of an increase in activity at elevated temperatures, most of this increase is due to the higher kinetic activity generally described by the Q10 law, which is doubled for the enzyme, assessed for every 10 ℃ increase in temperature.

In addition, there are examples of natural mutations that destabilize proteins under normal operating conditions. Some mutants may have activity at lower temperatures but have reduced levels of activity compared to the parent or wild-type protein. This is also generally described as a decrease in activity governed by Q10 or similar rules.

It is desirable to obtain useful conditionally activated molecules. For example, it is desirable to obtain a molecule which is almost inactive under wild-type working conditions, but active under conditions other than wild-type working conditions, and whose level of activity is the same as or higher than that under wild-type working conditions, or which is activated or deactivated in a specific microenvironment, or which is activated or deactivated after a period of time. In addition to temperature, other conditions that enable protein evolution or optimization include pH, osmolality (osmolality), oxidative stress, and electrolyte concentration. Other target properties that can be optimized during evolution include chemical resistance and proteolytic resistance.

A number of strategies for evolving or engineering molecules have been published. However, engineering or evolution of a protein to be inactive or nearly inactive (less than 10% active, preferably less than 1% active) under wild-type working conditions, while maintaining activity comparable to or higher than its corresponding parent or wild-type protein under conditions other than wild-type working conditions, requires the coexistence of a destabilizing mutation (destabilizing mutations) with an activity enhancing mutation that does not combat this destabilizing effect (destabilizing effect). The activity of the protein with reduced destabilization is expected to be more effective than predicted by standard rules such as Q10, and thus, by evolving the ability to, for example, inactivate the corresponding parent or wild-type protein under normal operating conditions but operate effectively at low temperatures, an unexpected new class of proteins can be prepared.

Chimeric Antigen Receptors (CARs) have been used to treat cancer. US 2013/0280220 discloses methods and compositions providing 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 therapies. Although in particular embodiments such cell therapies are for cancer (including cancers involving solid tumors), the cell therapies may be applicable to any medical condition.

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

In the present application, various publications are referenced by author and date. To more fully describe the date of these publications known to those skilled in the art and the state of the art described in the claims that follow, these publications are incorporated herein by reference in their entirety.

Drawings

FIG. 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 expressing the conditionally active antibodies of example 1 as bivalent or monovalent antibodies does not significantly alter the selectivity of these antibodies above pH 6.0 and pH 7.4.

FIG. 4 is a spectrum of size exclusion chromatography showing that the conditionally active antibody of example 2 does not aggregate.

Fig. 5 shows the binding and dissociation rates of the conditionally active antibody of example 2 as measured by a Surface Plasmon Resonance (SPR) assay.

FIGS. 6A-6B show the selectivity of conditionally active antibodies as measured by SPR assay performed on example 2.

Fig. 7A shows that CAR-T cells have no effect on CHO cell populations that do not express target antigen X1 of CAR-T cells. The CAR molecule in the CAR-T cell of this example comprises an antibody against target antigen X1, although the antibody is not conditionally active (comparative example a).

Figure 7B shows that CAR-T cells reduced CHO-63 cell populations expressing target antigen X1 of CAR-T cells. These CAR-T cells were identical to the cells used to generate the data shown in fig. 7A (comparative example a).

Fig. 8A shows that CAR-T cells have no effect on CHO cell populations that do not express target antigen X1 of CAR-T cells. The CAR molecule in CAR-T cells of this example 3 comprises a conditionally active antibody against target antigen X1.

Figure 8B shows that CAR-T cells reduce CHO-63 cell populations expressing target antigen X1 of CAR-T cells tested in example 3. These CAR-T cells were identical to the cells used to generate the data shown in fig. 8A.

Figures 9A-9B show cytokine release induced by CAR-T cell binding to target antigen X1 as described in example 3.

Figure 10 shows a conditionally active antibody against target antigen X2.

Figure 11A shows the cytotoxic effect induced by CAR-T cells binding to Daudi cells expressing target antigen X2 and the cytotoxic effect induced by CAR-T cells on HEK293 cells not expressing target antigen X2 as described in example 4.

Figures 12A-12B show cytokine release induced by CAR-T cell binding to target antigen X1, as described in example 5.

Fig. 13A-13B show cytokine release induced by CAR-T cell binding to target antigen X2, as described in example 5.

Figure 14 shows a conditionally active antibody against target antigen CD22 suitable for construction of CAR-T cells.

Disclosure of Invention

In one aspect, the invention provides a Chimeric Antigen Receptor (CAR) for binding to a tumor-specific target antigen. The chimeric antigen receptor includes at least one antigen-specific targeting region that evolves from a parent protein or domain thereof. The CAR further comprises a transmembrane domain and an intracellular signaling domain. The at least one antigen-specific targeting region has the property that activity in an assay under normal physiological conditions is reduced compared to activity in an assay 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 the group consisting of lentiviral vectors, gamma retroviral vectors, foamy viral vectors, adeno-associated viral vectors, adenovirus vectors, poxviral vectors, herpesviral vectors, engineered hybrid viruses, and transposon mediated vectors.

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

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

In another aspect, the invention provides a method of producing a chimeric antigen receptor comprising at least one antigen-specific targeting region, a transmembrane domain, and an intracellular signaling domain. The method comprises the step of generating the at least one antigen-specific targeting region from a parent protein or domain thereof that specifically binds to a tumor-specific target antigen. These steps include (i) evolving DNA encoding a parent or wild-type protein or domain thereof using one or more evolution techniques to produce mutant DNA, (ii) expressing the mutant DNA to obtain a mutant polypeptide, (iii) performing an assay for the mutant polypeptide under normal physiological conditions and an assay under abnormal conditions, and (iv) selecting at least one antigen-specific targeting region from the mutant polypeptide expressed in step (ii) that has reduced activity in the assay under normal physiological conditions compared to activity in the assay under abnormal conditions.

Definition of the definition

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

The term "about" as used herein in connection with a measured quantity refers to the normal variation of the measured quantity as would be expected by one skilled in the art to make measurements and to carry out a level of attention (level ofcare) commensurate with the purpose of the measurement and the accuracy of the measuring instrument. Unless otherwise indicated, "about" means that the values provided vary by +/-10%.

The term "agent" as used herein refers to a compound, mixture of compounds, spatially localized compound series (e.g., VLSIPS peptide series, polynucleotide series, and/or combinatorial small molecule series), biological macromolecules, phage peptide display libraries, phage antibody (e.g., scFv) display libraries, polysaccharidopeptide display libraries, or extracts obtained from biological materials such as cells or tissues of bacteria, plants, fungi, or animals (particularly mammals). The potential enzymatic activity of the reagents was evaluated in the screening assays described below. The potential activity of the agent as a conditionally active biologic therapeutic enzyme is evaluated in a screening assay described below.

The term "amino acid" as used herein refers to any organic compound containing an amino group (-NH ₂) and a carboxyl group (-COOH), preferably as a free radical or as part of a peptide bond after condensation. "twenty α -amino acids forming a naturally encoded polypeptide" is understood in the art to mean alanine (ala or A), arginine (arg or R), asparagine (asn or N), aspartic acid (asp or D), cysteine (cys or C), glutamic acid (glu or E), glutamine (gin or Q), glycine (gly or G), histidine (his or H), isoleucine (ile or I), leucine (leu or L), lysine (lys or K), methionine (met or M), phenylalanine (phe or F), proline (pro or P), serine (ser or S), threonine (thr or T), tryptophan (tip or W), tyrosine (tyr or Y) and valine (val or V).

The term "amplification" as used herein refers to an increase in the copy number of a polynucleotide.

The term "antibody" as used herein refers to intact immunoglobulin molecules, as well as fragments of immunoglobulin molecules, such as Fab, fab ', (Fab') ₂, fv and SCA fragments, which are capable of binding to an epitope of an antigen. These antibody fragments retain some of the ability to selectively bind to the antigen (e.g., polypeptide antigen) of the antibody from which they were derived, and can be prepared using methods well known in the art (see, e.g., harlow and Lane, supra) and described further below. Antibodies can be used to isolate a prepared amount of antigen by immunoaffinity chromatography. Various other uses of such antibodies are in the diagnosis and/or staging of a disease (e.g., neoplasia) and therapeutic applications for treating a disease, such as neoplasia, autoimmune disease, AIDS, cardiovascular disease, infection, and the like. Chimeric, human-like, humanized or fully human antibodies are particularly useful for human patient administration.

Fab fragments consist of monovalent antigen binding fragments of antibody molecules and can be prepared by digesting an intact antibody molecule with papain to produce fragments consisting of an intact light chain and a portion of a heavy chain.

Fab' fragments of antibody molecules can be obtained by treating the whole antibody molecule with pepsin and then reducing to give a molecule consisting of the whole light chain and part of the heavy chain. Two Fab' fragments were obtained from each 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.

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

The term "antigen" or "Ag" as used herein is defined as a molecule that elicits an immune response. Such an immune response may be involved in antibody production or activation of specific immunocompetent cells or both. Those skilled in the art will appreciate that any macromolecule, including almost any protein or peptide, may be used as an antigen. Furthermore, the antigen may be derived from recombinant or genomic DNA. Those of skill in the art will understand that any DNA comprising a nucleotide sequence or portion of a nucleotide sequence encoding a protein that elicits an immune response, thus encodes the term "antigen" as used herein. Furthermore, one skilled in the art will appreciate that an antigen need not be encoded solely by the full length nucleotide sequence of a gene. It will be apparent that the 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 elicit the desired immune response. Furthermore, the skilled artisan will appreciate that antigens need not be encoded by a "gene" at all. It is apparent that the antigen may be produced, synthesized from, or may be derived from a biological sample. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

As used herein, an "antigen loss escape variant" refers to a cell that exhibits reduced or lost expression of a target antigen to which a CAR of the present invention targets.

The term "autoimmune disease" as used herein is defined as a condition caused by an autoimmune response. Autoimmune diseases are the result of inappropriate and excessive responses to self-antigens. Examples of autoimmune diseases include, but are not limited to, edison's disease, alopecia areata (alopecia greata), ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, crohn's disease, diabetes (type 1), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, graves ' disease, gillin-barre syndrome, hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, sjogren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxoedema, pernicious anemia, colitis, and the like.

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

The term "B cell related disorder" as used herein includes B cell immunodeficiency, autoimmune diseases and/or excessive/uncontrolled cell proliferation associated with B cells (including lymphomas and/or leukemias). Examples of diseases for which the bispecific CARs of the invention may be used in their 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, X-linked agaropectinemia, pre-B acute lymphoblastic leukemia, systemic lupus erythematosus, common variant immunodeficiency, chronic lymphoblastic leukemia, diseases associated with selective IgA deficiency and/or IgG subclass deficiency, B lineage lymphomas (hodgkin's lymphoma and/or non-hodgkin's lymphoma), immunodeficiency with thymoma, transient hypogammaglobulinemia and/or high IgM syndrome, and virus-mediated B cell diseases (such as EBV-mediated lymphoproliferative diseases) and chronic infections in which B cells participate in at a pathobiological point of view.

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 junctions within the brain capillary endothelial plasma membrane, creating a dense barrier that limits the transport of molecules, even very small molecules (e.g., urea (60 daltons)), to the brain. The blood-brain barrier within the brain, the blood-spinal cord barrier within the spinal cord, and the blood-retinal barrier within the retina are continuous capillary barriers within the Central Nervous System (CNS), and are collectively referred to herein as the "blood-brain barrier" or "BBB. The BBB also includes a blood-cerebrospinal fluid barrier (choroid plexus), where the barrier includes ependymal cells rather than capillary endothelial cells.

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

The term "chimeric antigen receptor" or "CAR" as used herein refers to an engineered receptor that specifically grafts an antigen onto cytotoxic cells such as T cells, NK cells, and macrophages. The CARs of the invention may comprise at least one antigen-specific targeting region (ASTR), an Extracellular Spacer Domain (ESD), a transmembrane domain (TM), one or more co-stimulatory domains (CSD), and an Intracellular Signaling Domain (ISD). In one embodiment, ESD and/or CSD are optional. In another embodiment, the CAR is a bispecific CAR that has specificity for two different antigens or epitopes. After ASTR specifically binds to the target antigen, ISD activates intracellular signaling. For example, ISD can redirect T cell specificity and reactivity to a selected target in a non-MHC-restricted manner, thereby exploiting the antigen binding properties of antibodies. non-MHC-restricted antigen recognition allows CAR-expressing T cells the ability to recognize antigens independent of antigen processing, bypassing the primary mechanism of tumor evasion. Furthermore, when expressed in T cells, the CAR advantageously does not dimerize with endogenous T Cell Receptor (TCR) alpha and beta 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 as a single polypeptide chain. For example, a CAR of the invention can be co-expressed with a therapeutic control (e.g., truncated epidermal growth factor (EGFRt)), where the CAR is encoded by a first polynucleotide strand and the therapeutic control is encoded by a second polynucleotide strand. In one embodiment, the first and second polynucleotide strands are linked by a nucleic acid sequence encoding a cleavable linker. Alternatively, the CAR and therapeutic control are encoded by two different polynucleotides that are not linked by a linker but are encoded by, for example, two different vectors.

The term "homologous (cognate)" as used herein refers to a genetic sequence that is evolutionarily and functionally related between species. For example, but not limited to, the human CD4 gene is a homologous gene to the mouse 3d4 gene in the human genome, since the sequence and structure of these two genes indicate that they have high homology, both genes encoding a protein that plays a role in transmitting T cell activation signals by limiting antigen recognition by mhc class ii.

The term "conditionally active biologic protein (conditionally active biologic protein)" refers to a variant or mutation of a parent or wild-type protein that is more or less active than the parent or wild-type protein under one or more normal physiological conditions. Such conditionally active proteins may also exhibit activity in selected areas of the body and/or exhibit increased or decreased activity under abnormal or allowable physiological conditions. The term "normal physiological condition" as used herein refers to one of temperature, pH, osmolality, oxidative stress, electrolyte concentration, concentration of small organic molecules such as glucose, lactic acid, pyruvic acid esters, nutritional components, other metabolites, and the like, concentration of other molecules such as oxygen, carbonates, phosphates, and carbon dioxide, and cell type and nutrient availability, which are considered to be within normal ranges at the site of administration or in tissues or organs at the site of action of an individual.

In one embodiment, the normal physiological condition is a normal physiological pH in the plasma of the mammalian subject in the range of greater than 7.0 to about 7.8, or about 7.2 to about 7.6, or about 7.3 to about 7.5. An abnormal condition is a 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 6.6.

The term "abnormal condition" as used herein refers to a condition that deviates from the normal acceptable range of the condition. In one aspect, a conditionally active biologic protein is almost inactive under normal physiological conditions, but active under abnormal conditions at a level equal to or better than the parent or wild-type protein from which it is derived. For example, in one aspect, the evolved conditionally active biologic protein is nearly inactive at body temperature, but active at lower temperatures. In another aspect, the conditionally active biologic 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 biologic protein is used as a drug or therapeutic. In another aspect, the protein is more or less active in high oxygenated blood, for example, after passage through the lung or in a lower pH environment found in the kidney.

"Conservative amino acid substitutions" refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains such as glycine, alanine, valine, leucine and isoleucine, a group of amino acids having aliphatic hydroxyl side chains such as serine and threonine, a group of amino acids having amide-containing side chains such as asparagine and glutamine, a group of amino acids having aromatic side chains such as phenylalanine, tyrosine and tryptophan, a group of amino acids having basic side chains such as lysine, arginine and histidine, and a group of amino acids having sulfur-containing side chains such as cysteine and methionine. Preferred conservative amino acid substitutions are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

The term "corresponding to" as used herein refers to polynucleotide sequences that are homologous (i.e., identical, non-strictly evolutionarily related) to all or part of a reference polynucleotide sequence, or polypeptide sequences that are identical to a reference polypeptide sequence. In contrast, the term "complementary" as used herein refers to the homology of its complementary sequence to all or part of the reference nucleotide sequence. For example, the nucleotide sequence "TATAC" corresponds to the reference sequence "TATAC", complementary to the reference sequence "GTATA".

The term "costimulatory ligand" as used herein includes molecules on antigen-presenting cells (e.g., dendritic cells, B cells, etc.) that specifically bind to cognate costimulatory molecules on T cells, thereby providing a signal that mediates T cell responses (including but not limited to proliferation, activation, differentiation, etc.) in addition to the first signal provided by the binding of, for example, a TCR/CD3 complex to a peptide-loaded MHC molecule. Co-stimulatory ligands may include, but are not limited to, CD7, B7-1 (CD 80), B7-2 (CD 86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible co-stimulatory ligands (ICOS-L), intercellular adhesion molecules (ICAM), CD30L, CD, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind Toll ligand receptors, and ligands that specifically bind B7-H3. Costimulatory ligands also include antibodies that specifically bind costimulatory 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, B-H3 and ligands that specifically bind to CD 83.

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

The term "costimulatory signal" as used herein refers to a signal that, in combination with a first signal (e.g., TCR/CD3 linkage), results in up-or down-regulation of T cell proliferation and/or a key molecule.

The term "cytotoxic cells" as used herein refers to cells that can damage or destroy invading microorganisms, tumor cells, or other diseased tissue cells. The term is intended to include Natural Killer (NK) cells, activated NK cells, neutrophils, T cells, eosinophils, basophils, B cells, macrophages and lymphokine-activated killer (LAK) cells, among other cell types. The cytotoxic cells are bound to the target cells by antibodies, receptors, ligands, or fragments/derivatives thereof to form stable complexes and stimulate the cytotoxic cells to destroy the target cells.

Cytotoxic cells may also include other immune cells with tumor lysis capabilities, 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,, volume 124, pages 2824-2833, 2014) and granulocytes. In addition, cytotoxic cells may include immune cells with phagocytic capacity, including but not limited to macrophages and granulocytes, cells with stem and/or progenitor characteristics, including but not limited to hematopoietic stem/progenitor cells (Zhen et al, "HIV-specific Immunity Derived From CHIMERIC ANTIGEN Receptor-ENGINEERED STEM CELLS," Mol ter., volume 23, pages 1358-1367, 2015), embryonic Stem Cells (ESCs), cord blood stem cells, and induced pluripotent stem cells (ipscs) (Themeli et al, "New cell sources forT CELL ENGINEERING AND adoptive immunotherapy," CellStem cell, volume 16, pages 357-366, 2015). In addition, cytotoxic cells include "synthetic cells" such as iPSC derived T cells (TiPSC) (Themeli et al, ,"Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy,"NatBiotechnol.,, vol.31, pages 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 as compared to a substrate that is not contacted with the enzyme.

The term "directed ligation" refers to ligation wherein the 5 'and 3' ends of the polynucleotide are not identical enough to determine the preferred ligation orientation. For example, untreated and undigested PCR products with two blunt ends, when ligated into cloning vectors that form blunt ends at digested multiple cloning sites, generally do not have a preferred ligation orientation, and thus, in these cases, generally do not occur. In contrast, directional ligation generally occurs when digested PCR products with an EcoR I treated 5 'end and a BamHI treated 3' end are ligated into a cloning vector with multiple cloning sites digested with EcoR I and BamHI.

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

The terms "genetically modified cell," "redirected cell," "genetically engineered cell," or "modified cell" as used herein refer to a cell expressing a CAR of the invention.

The term "DNA shuffling (shuffling)" as used herein refers to recombination between substantially homologous but not identical sequences, and in some embodiments, DNA shuffling may include crossover by non-homologous recombination (cross-over), such as by 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 produces a beneficial effect on the human or animal body when administered to the human or animal body. Preferably, the therapeutic agent includes a substance that can treat, cure, or alleviate one or more symptoms, diseases, or abnormalities in the human or animal body or a substance that can enhance the health of the human or animal body.

An "effective amount" refers to an amount of a conditionally active biologic protein or fragment that is administered to a living organism for a period of time effective to treat or prevent a disease in the living organism, e.g., to provide a therapeutic effect during a desired dosing interval.

The term "electrolyte" as used herein means a mineral (mineral) that is charged in blood or other body fluids. For example, in one aspect, normal physiological conditions and abnormal conditions may be conditions of "electrolyte concentration". In one aspect, the electrolyte concentration to be measured is selected from one or more of ionized calcium, sodium, potassium, magnesium, chlorine, bicarbonate, and phosphate concentrations. For example, in one aspect, the normal range for serum calcium is 8.5 to 10.2mg/dL. In this regard, abnormal serum calcium concentrations may be selected within a range above or below normal. In another example, in one aspect, the normal range of serum chlorine is 96-106 milliequivalents per liter (mEq/L). In this regard, the abnormal serum chlorine concentration may be selected within a range above or below normal. In another example, in one aspect, the normal range for serum magnesium is from 1.7-2.2mg/dL. In this regard, abnormal serum magnesium concentrations may be selected within a range above or below normal. In another example, in one aspect, the normal range for serum phosphorus is from 2.4mg/dL to 4.1mg/dL. In this regard, abnormal serum phosphorus concentrations may be selected within a range above or below normal. In another example, in one aspect, normal serum or blood sodium ranges from 135mEq/L to 145mEq/L. In this regard, the abnormal serum or blood sodium concentration may be selected to be above or below the normal range. In another example, in one aspect, normal serum or blood potassium ranges from 3.7mEq/L to 5.2mEq/L. In this regard, the abnormal serum or blood potassium concentration may be selected within a range above or below normal. In another aspect, the normal serum bicarbonate range is from 20mEq/L to 29mEq/L. In this regard, the concentration of bicarbonate in the abnormal serum or blood may be selected within a range above or below normal. In a different aspect, bicarbonate levels can be used to indicate a normal level of acidity (pH) in the blood. The term "electrolyte concentration" may also be used to define the condition of a particular electrolyte in a tissue or body fluid other than blood or plasma. In this case, the normal physiological condition is considered to be the clinically normal range of the tissue or fluid. In this regard, the electrolyte concentration of the abnormal tissue or body fluid may be selected within a range higher or lower than normal.

The term "epitope" as used herein refers to an epitope on an antigen, such as an enzyme polypeptide, to which an antibody paratope, such as an antibody specific for an enzyme, binds. An epitope is typically composed of a chemically active surface combination of molecules, such as amino acids or sugar side chains, and may have specific three-dimensional structural characteristics as well as specific charge characteristics. An "epitope" as used herein refers to that portion of an antigen or other macromolecule capable of forming an interaction (binding interaction) that interacts with the variable region conjugate of an antibody. Typically, such interactions are manifested as intermolecular contacts with one or more amino acid residues of the CDRs.

The term "evolution" or "evolution (evolving)" as used herein refers to the use of one or more mutagenesis methods to generate a new polynucleotide encoding a new polypeptide, which itself is an improved biomolecule and/or contributes to the production of another improved biomolecule. In one particular non-limiting aspect, the invention relates to the evolution of conditionally active biologic proteins from parent or wild-type proteins. For example, in one aspect, the evolution involves methods employing non-random polynucleotide chimerism (chimerization) and non-random site-directed mutagenesis as disclosed in U.S. patent application publication No. 2009/0130518. More specifically, the present invention provides methods of evolution of conditionally active biological enzymes that exhibit reduced activity under normal physiological conditions compared to the parent or wild-type enzyme parent molecule, and enhanced activity under one or more aberrant conditions compared to the antigen-specific targeting region of the parent or wild-type enzyme.

When the terms "fragment," "derivative," and "analog" are used in reference to a reference polypeptide, it is intended to include polypeptides that retain at least one biological function or activity that is at least substantially the same as the reference polypeptide. Furthermore, examples of the terms "fragment", "derivative" or "analogue" are "precursor forms" of molecules, such as low activity pro-proteins (proprotein), which can be modified by cleavage to yield mature enzymes with significantly higher activity.

The term "gene" as used herein refers to a DNA fragment involved in the production of a polypeptide chain, which includes regions preceding and following the coding region (leading and trailing regions) and the spacer sequences (introns) between the coding fragments (exons).

The term "heterologous" as used herein means that one single stranded nucleic acid sequence cannot hybridize to another single stranded nucleic acid sequence or its complement. Thus, a heterologous region refers to a polynucleotide region or region in the sequence of a polynucleotide that has a region or region that cannot hybridize to another nucleic acid or polynucleotide. Such a region or region is, for example, a mutation region.

As used herein, the term "homologous (homologous)" or "partially homologous (homeologous)" refers to a single-stranded nucleic acid sequence that hybridizes to the complement of the single-stranded nucleic acid sequence. The extent of hybridization may depend on a number 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 is greater than about 5bp, more preferably, the region of identity is greater than about 10bp.

The benefits of the present invention extend to "industrial applications" (or industrial processes), which term is used to include suitable applications in the commercial industry (or simply industry) as well as non-commercial industrial applications (e.g., in biomedical research in non-profit institutions). Related applications include those in the diagnostic, pharmaceutical, agricultural, manufacturing and academic fields.

The term "immune cell" as used herein 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 an immunity including, but not limited to, innate immunity, humoral immunity, cellular immunity, inflammatory response, acquired (adaptive) immunity, autoimmunity, and/or hyperimmunity.

The term "isolated" as used herein means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, naturally occurring nucleotides or enzymes present in living animals are not isolated, but the same nucleotides or enzymes isolated from some or all of the coexisting materials in the natural system are isolated. Such a polynucleotide may be part of a vector, and/or such a polynucleotide or enzyme may be part of a composition, which is still isolated since such a vector or composition is not part of its natural environment.

The term "isolated nucleic acid" as used herein is used to define a nucleic acid, such as a DNA or RNA molecule, that is not tightly linked to 5 'and 3' flanking sequences, but is typically tightly linked in the genome that is naturally occurring in the organism from which it is derived. Thus, the term describes, for example, nucleic acids integrated into a vector such as a plasmid or viral vector, nucleic acids integrated into the genome of a heterologous cell (or into a homologous cell genome but at a site different from that at which it naturally occurs), and nucleic acids present as separate molecules, such as DNA fragments produced by PCR amplification or restriction cleavage, or RNA molecules produced by in vitro transcription. The term also describes recombinant nucleotides that form part of a hybrid gene encoding other polypeptide sequences that may be used, for example, to produce fusion proteins.

The term "lentivirus" as used herein refers to a genus of the family retrovirus. Lentiviruses are unique among retroviruses, which are capable of infecting non-dividing cells, and they can deliver large amounts of genetic information into the DNA of host cells, and therefore they are one of the most effective ways to deliver gene delivery vectors. HIV, SIV and FIV are all examples of lentiviruses. Lentiviral-derived vectors 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 domain sequence, that is recognized by a particular receptor. Those skilled in the art will appreciate that a molecule (or macromolecular complex) may be either a receptor or a ligand. In general, a conjugate having a smaller molecular weight refers to a ligand, and a conjugate having a larger molecular weight refers to a receptor.

As used herein, the term "ligation" refers to the process of forming phosphodiester bonds between two double-stranded nucleic acid fragments (Sambrook et al, (1982) molecular cloning, A handbook of molecular cloning, cold spring harbor laboratory (Molecular Cloning:A Laboratory Manual.Cold Spring Harbour Laboratory),Cold Spring Harbor,NY.,p.146;Sambrook et al, a handbook of molecular cloning, a second edition (Molecular Cloning: alaboratorymanual,2 ^nd Ed.), cold Spring HarborLaboratoryPress, 1989). Unless otherwise specified, ligation may be accomplished using known buffers and conditions, using 10 units of T4 DNA ligase ("ligase") to ligate about equimolar amounts of DNA fragments per 0.5 microgram.

The term "linker" or "spacer" as used herein refers to a molecule or combination of molecules that links two molecules, such as a DNA binding protein and a random peptide, such that the two molecules are in a preferred configuration, e.g., such that the random peptide binds to the receptor of the DNA binding protein with minimal steric hindrance. As used herein, "linker" (L) or "linker domain" or "linker region" refers to an oligopeptide or polypeptide region of about 1 to 100 amino acids in length that connects together any domain/region of the CAR of the invention. The linker may be composed of flexible residues such as glycine and serine, allowing adjacent protein domains to move freely relative to each other. Longer linkers can be used when it is necessary to ensure that two adjacent domains do not spatially interfere with each other. The linker may 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, the linker comprises a picornavirus 2A-like linker, CHYSEL sequences of porcine teschovirus (P2A), a echinacea armyworm beta tetrad virus (Thosea asignavirus, T2A), or a combination, variant, and functional equivalent thereof. Other linkers will be apparent to those skilled in the art and may be used in conjunction with alternative embodiments of the present invention.

The term "mammalian cell surface display" as used herein refers to a technique whereby a protein or antibody or a portion of an antibody is expressed and displayed on the surface of a mammalian host cell for the purpose of screening, e.g., screening for specific antigen binding by a combination of magnetic beads and fluorescence activated cell sorting. In one aspect, mammalian expression vectors are used to express immunoglobulins in both a secreted form and a cell surface bound form, as described in DuBridge et al, US 2009/0136550. In another aspect, the techniques are used to screen viral vectors encoding libraries of antibodies or antibody fragments that are displayed on the cell membrane when expressed in cells, as described in Gao et al, US 2007/011260. Whole IgG surface display on mammalian cells is known. For example, akamatsuu et al developed a mammalian cell surface display vector suitable for direct isolation of IgG molecules based on their antigen binding affinity and biological activity. Antibody libraries were displayed as intact IgG molecules on the cell surface using Epstein-Barr virus-derived episomal vectors and screened for specific antigen binding by a combination of magnetic beads and fluorescence activated cell sorting. Plasmids encoding antibodies with the desired binding characteristics are recovered from the sorted cells and converted to a form suitable for the production of soluble IgG. See Akamatsuu et al J.Immunol. Methods, volume 327, pages 40-52, 2007.Ho et al used widely for cell surface display of single chain Fv antibodies for affinity maturation of human embryonic kidney 293T cells for transient protein expression. Cells expressing rare mutant antibodies with higher affinity were enriched 240-fold by single pass cell sorting from a large excess of cells expressing WT antibodies with slightly lower affinity. Furthermore, after randomization of the intrinsic antibody hotspots by a single selection of the combinatorial library, highly enriched mutants with enhanced binding affinity to CD22 were obtained. See Ho et al, "Isolation of anti-CD22 FV WITH HIGH AFFINITY by FV DISPLAY on human cells (isolation of anti-CD22 Fv with high affinity by Fv display on human cells)" ProcNatl AcadSci USA, vol 103, pages 9637-9642, 2006.

B cells specific for antigen may also be used. Such B cells can be isolated directly from Peripheral Blood Mononuclear Cells (PBMCs) of a human donor. Recombinant antigen-specific single chain Fv (scFv) libraries were generated from this B cell pool and screened by mammalian cell surface display using Sindbis virus expression systems. The Variable Regions (VR) of the Heavy (HC) and Light (LC) chains are isolated from positive clones and recombinant fully human antibodies produced as whole IgG or Fab fragments. In this way, several hypermutated high affinity antibodies, model viral antigens, and antibodies specific for nicotine that bind to qβ virus-like particles (VLPs) were isolated. See Beerli et al, "Isolation of human monoclonal antibodies by MAMMALIAN CELL DISPLAY (isolation of human monoclonal antibodies by mammalian cell display)", procNatlAcadSci USA, volume 105, pages 14336-14341, 2008.

Yeast cell surface display can also be used in the present invention, see for example, kondo and Ueda, "YEAST CELL-surface display-applications ofmolecular display (molecular display yeast cell surface display application)", appl. Microbiol. Biotechnol., vol.64, pages 28-40, 2004, which describes a cell surface engineering system using, for example, saccharomyces cerevisiae. Several representative display systems for expression in Saccharomyces cerevisiae are described in Lee et al, "Microbial cell-surface display," TRENDSinBitechnol., vol.21, pp.45-52, 2003, and Boder and Wittrup, "Yeast surface display for screening combinatorial polypeptide libraries (yeast cell display for screening combinatorial polypeptide libraries)," Nature Biotechnol., vol.15, pp.553, 1997.

The term "producing" as used herein refers to producing a protein in an amount sufficient to allow at least phase I clinical determination of a therapeutic protein or sufficient for regulatory approval of a diagnostic protein.

The term "microenvironment" as used herein refers to any portion or area of a tissue or body that has a sustained or temporary, physiological or chemical differential from other areas of the tissue or area of the body.

The term "molecular property to be evolved" as used herein includes molecules comprising polynucleotide sequences, molecules comprising polypeptide sequences, molecules comprising partial polynucleotides and partial polypeptide sequences. Particularly relevant to the molecular properties being evolved, but in no way limited to, examples include the activity of the protein under specific conditions, such as those related to temperature, salinity, osmotic pressure, pH, oxidative stress, and the concentration of glycerol, dimethyl sulfoxide, detergents, and/or any other molecular species that are contacted in the reaction environment. Other molecular properties that are particularly relevant to the evolved molecular properties, but are in no way limited to examples including stability, such as residual molecular properties that are exhibited after exposure to a particular environment for a period of time, such as would be encountered during storage.

The term "mutation (mutation)" as used herein refers to a change in sequence in a parent or wild-type nucleic acid sequence or a change in sequence in a peptide sequence. Such mutations may be point mutations, such as transitions or transversions. The mutation may be a deletion, insertion or repetition.

The term "multispecific antibody" as used herein is an antibody that has binding affinity for at least two different epitopes. Multispecific antibodies may be prepared as full length antibodies or antibody fragments (e.g., F (ab') ₂ bispecific antibodies). The engineered antibodies may bind to two, three or more (e.g. four) antigens (see e.g. US 2002/0004587 A1). One conditionally active antibody may be engineered to be a multispecific antibody, or two antibodies may be engineered to comprise a heterodimer that binds two antigens. Multispecific antibodies may also be multifunctional.

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

The term "naturally occurring" as used herein applies to objects where the fact is that it may be found in nature. For example, polypeptide or polynucleotide sequences present in organisms (including viruses) are naturally occurring, which can be isolated from natural sources and have not been intentionally altered by man in the laboratory. In general, the term naturally occurring refers to the presence of a subject in an individual that is not pathological (not diseased), which is common in species.

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

The term "nucleic acid molecule" as used herein consists of at least one base or one base pair, depending on whether it is single-stranded or double-stranded, respectively. Furthermore, the nucleic acid molecule may belong exclusively (exclusively) or chimeric (CHIMERICALLY) to any group of nucleoside containing molecules, as exemplified by, but not limited to, RNA, DNA, genomic nucleic acid, non-genomic nucleic acid, 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 RNAs, and nucleic acid molecules composed of a chimeric composition of one or more components that do not naturally occur with naturally occurring components.

In addition, a "nucleic acid molecule" may comprise in part one or more non-nucleoside based components, as exemplified by, but not limited to, amino acids and carbohydrates. Thus, by way of example, but not limitation, ribozymes are based in part on nucleosides and in part on proteins, and are considered "nucleic acid molecules".

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

The term "oligonucleotide" (or synonymously "oligonucleotide") refers to a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands that can be chemically synthesized. Such synthetic oligonucleotides may or may not have a 5' phosphate. Those oligonucleotides without 5' phosphate will not ligate to another oligonucleotide in the presence of kinase without addition of phosphate and ATP. The synthesized oligonucleotides will be ligated to fragments that are not dephosphorylated.

The term "operably linked" as used herein refers to a linkage between 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 coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.

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

The term "parent polynucleotide combination" as used herein is a combination consisting of one or more different nucleotides. Generally the term is used in relation to a combination of progeny polynucleotides, preferably obtained by mutagenizing a combination of parents, in which case the terms "parent", "starter" and "template" are used interchangeably.

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

The term "physiological condition" as used herein refers to biochemical parameters such as temperature, pH, osmotic pressure, ionic strength, viscosity, etc., which are compatible with living organisms and/or are typically present in cells of a living yeast culture cell or mammalian cell. For example, the intracellular conditions of yeast cells grown under typical laboratory culture conditions are physiological conditions. Suitable in vitro reaction conditions for in vitro transcription mixtures (cocktails) are generally physiological conditions. Typically, in vitro physiological conditions include 50-200mM NaCl or KCl, pH6.5-8.5,20-45℃and 0.001-10mM divalent cations (e.g., mg ⁺⁺、Ca⁺⁺), preferably about 150mM NaCl or KCl, pH 7.2-7.6,5mM divalent cations, and often 0.01-1.0% of non-specific proteins (e.g., bovine Serum Albumin (BSA)). Nonionic detergents (Tween, NP-40, triton X-100) are often present, typically about 0.001-2%, typically 0.05-0.2% (v/v). The particular aqueous conditions are selected by the physician in the routine manner. For general guidance, buffered aqueous conditions of 10-250mM NaCl,5-50mM Tris HCl,pH5-8, optionally with the addition of divalent cations and/or metal chelators and/or nonionic detergents and/or membrane components and/or defoamers and/or scintillation materials (scintillant) may be used. In some embodiments, the buffer may contain at least one of BSA, carbonate, bicarbonate, chloride salts, and the like. Normal physiological conditions refer to temperature, pH, osmolality, oxidative stress, and electrolyte concentrations at the site of administration or site of action in a patient or individual, which are considered to be within the normal range within the patient.

The sequence of a double stranded polynucleotide is described herein using standard convention (Standard convention) (5 'to 3').

The term "population" as used herein refers to, for example, a polynucleotide, a portion of a polynucleotide, or a collection of polynucleotide or protein components. "mixed population" refers to a collection of components that belong to the same nucleic acid or protein family (i.e., are related) but differ in sequence (i.e., are different sources) and thus differ in biological activity.

A molecule having a "precursor form" refers to a molecule that has undergone any combination of one or more covalent and non-covalent chemical modifications (e.g., glycosylation, proteolytic cleavage, dimerization or oligomerization, temperature-induced or pH-induced conformational change, attachment of cofactors, etc.), thereby obtaining a more mature, differently (e.g., increased activity) molecular form than the reference precursor molecule. When two or more chemical modifications (e.g., two proteolytic cleavage or one proteolytic cleavage and one deglycosylation) can be distinguished to yield a mature molecule, the reference precursor molecule may be referred to as a "pre-pro-form" molecule.

The term "receptor" as used herein refers to a molecule having an affinity for a given ligand. The receptor may be a naturally occurring or synthetic molecule. Receptors may be used in their unaltered state or to form aggregates with other species. Receptors may be linked to the binding unit either directly or through specific binding substances, covalently or noncovalently. Examples of receptors include, but are not limited to, antibodies, including monoclonal antibodies, and capable of undergoing an antisera reaction with a particular antigenic determinant (e.g., located on a virus, cell, or other material), cell membrane receptors, complexes of carbohydrates and glycoproteins, enzymes, and hormone receptors.

The term "reductive recombination (Reductive reassortment)" as used herein refers to an increase in molecular diversity by deletion (and/or insertion) events mediated by repeated sequences.

The term "restriction site" as used herein refers to a recognition sequence, including catalytic cleavage sites, necessary to demonstrate the action of a restriction enzyme. It will be appreciated that the cleavage site may or may not be included in a portion of the restriction site that contains the low ambiguity sequence (low ambiguity sequence), i.e., the sequence that contains the primary determinant of the frequency of occurrence of the restriction site. When referring to an enzyme (e.g., a restriction enzyme) to "cleave" a polynucleotide, it is understood that the restriction enzyme catalyzes or facilitates cleavage of the polynucleotide.

The term "single chain antibody" as used herein refers to a polypeptide comprising a VH domain and a VL domain linked by a polypeptide, typically linked by a spacer peptide, and which may comprise other amino acid sequences at the amino-and/or carboxy-terminus. For example, a single chain antibody may comprise a tether segment for linking to a coding polynucleotide. As an example, scFv is a single chain antibody. Single chain antibodies are typically proteins composed of one or more polypeptide segments of at least 10 contiguous amino acids that are substantially encoded by genes of the immunoglobulin superfamily (see, e.g., the Immunoglobulin Gene Superfamily (immunoglobulin gene superfamily ),A.F.Williams andA.N.Barclay,in Immunoglobulin Genes,T.Honjo,F.W.Alt,and THE.Rabbits,eds.,(1989)Academic press:San Diego,Calif.,pp.361-368), most often encoded by rodent, non-human primate, avian, porcine, bovine, ovine, caprine, or human heavy or light chain gene sequences).

If they bind to each other with a higher affinity than other non-specific molecules, then the members of the pair of molecules (e.g., antibody-antigen pair and ligand-receptor pair) are described as "specifically binding" to each other. For example, an antibody against an antigen binds more efficiently to the antigen than a non-specific protein, and may be described as an antibody specifically binding to the antigen.

The term "stimulation" as used herein refers to the induction of a primary response by binding of a stimulatory molecule (e.g., a TCR/CD3 complex) to its cognate ligand, thereby mediating a signaling event, such as, but not limited to, signaling through the TCR/CD3 complex. Stimulation may mediate altered expression of certain molecules, such as, for example, down regulation of TGF- β and/or recombination of cytoskeletal structures, etc.

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

The term "stimulatory ligand" as used herein refers to a ligand that, when present on an antigen presenting cell (e.g., dendritic cell, B cell, etc.), can specifically bind to a cognate binding partner (referred to herein as a "stimulatory molecule") on a T cell, thereby mediating a primary response of the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, etc. Stimulating ligands are well known in the art and include, inter alia, peptide-loaded MHC class I molecules, anti-CD 3 antibodies, super-agonist anti-CD 28 antibodies, and super-agonist anti-CD 2 antibodies.

The term "target cell" as used herein refers to a cell that is involved in a disease and that can be targeted by genetically modified cytotoxic cells of the invention (including but not limited to genetically modified T cells, NK cells and macrophages). 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 cell" and "T lymphocyte" are interchangeable and are used synonymously herein. Examples include, but are not limited to, naive T cells, central memory T cells, effector memory T cells, and combinations thereof.

The term "transduction" as used herein refers to the introduction of an exogenous nucleic acid into a cell using a viral vector. "transfection" as used herein refers to the use of recombinant DNA techniques to introduce exogenous nucleic acid into a cell. The term "transformation" refers to the introduction of an "exogenous" (i.e., external or extracellular) gene, DNA or RNA sequence into a host cell such that the host cell expresses the introduced gene or sequence to produce a desired substance, such as a protein or enzyme encoded by the introduced gene or sequence. The introduced gene or sequence may also be referred to as a "cloned" or "exogenous" gene or sequence, and may include regulatory or control sequences, such as initiation sequences, termination sequences, promoter sequences, signal sequences, secretion sequences, or other sequences used by the cytogenetic machinery. The gene or sequence may include nonfunctional sequences or sequences without known functions. Host cells that receive and express the introduced DNA or RNA are those that have been "transformed" and are "transformants" or "clones". The DNA or RNA introduced into the host cell may be 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 "treating" includes (1) preventing or delaying the appearance of a state, disease or condition that occurs in an animal that may have or is susceptible to the state, disease or condition but has not experienced or exhibited clinical or subclinical symptoms of the state, disease or condition, (2) inhibiting the state, disease or condition (i.e., preventing, reducing or delaying the progression of the disease, or maintaining the recurrence of the disease in the treated condition, or at least one clinical or subclinical symptom), and/or (3) alleviating the condition (i.e., causing regression of the state, disease or condition or causing regression of at least one clinical or subclinical symptom thereof). The benefit to the patient to be treated is statistically significant, or at least perceptible to the patient or physician.

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

The term "tumor microenvironment" as used herein refers to any and all elements of a tumor environment, including elements that create a structural and/or functional environment for a malignant process to survive and/or expand and/or spread.

The term "variable segment" as used herein refers to a portion of a nascent peptide that includes a random, pseudo-random or defined core sequence. "variable segment" refers to a portion of a nascent peptide that includes a random, pseudo-random, or defined core sequence. The variable segment may comprise variable or non-variable residue positions, and the degree of variability of the residues at the variable residue positions may be limited, with both options being prescribed by the physician. Typically, the variable segment is about 5 to 20 amino acid residues (e.g., 8 to 10) in length, although the variable segment may be longer and may include an antibody moiety or a receptor protein, such as an antibody fragment, a nucleic acid binding protein, a receptor protein, or the like

"Vector," "cloning vector," and "expression vector" as used herein refer to a polynucleotide sequence (e.g., a foreign gene) that can be introduced into a host cell in order to transform the host and facilitate expression (e.g., transcription and translation) of the introduced sequence. Vectors include plasmids, phages, viruses, and the like.

The term "wild-type" as used herein refers to a polynucleotide that does not include any mutations. "wild-type protein (wildtypeprotein)", "wild-type protein (wild-typeprotein)", "wild-type biological protein (wild-type biologic protein)" or "wild-type biological protein (wild type biologic protein)" refer to a protein that can be isolated from nature, has a level of activity under natural conditions and comprises an amino acid sequence found under natural conditions. The terms "parent molecule" and "protein of interest" also refer to wild-type proteins. The "wild-type protein" preferably has some desirable properties (e.g., higher binding affinity and/or enzymatic activity) that can be obtained by screening a library of proteins for desirable properties, including better stability, improved selectivity and/or solubility at different temperatures and/or pH.

The term "work" as in "work sample", for example, simply refers to one sample for work. Likewise, "working molecule" refers, for example, to a molecule that is used for working.

Detailed Description

For purposes of illustration, the principles of the present invention are described by reference to various exemplary embodiments. Although certain embodiments of the invention have been specifically described herein, those of ordinary skill in the art will readily recognize that the same principles are equally applicable and can be used with other systems and methods. Before explaining the disclosed embodiments of the invention in detail, it is to be understood that the invention is not limited in its application to the details of any particular embodiment shown. In addition, the terminology used herein is for the purpose of description and not of limitation. Furthermore, although certain methods are described with reference to steps presented herein in a particular order, in many cases, the steps may be performed in any order as will be appreciated by one of skill in the art. Thus, the novel method is not limited to the specific arrangement of steps disclosed herein.

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

The present invention relates to a Chimeric Antigen Receptor (CAR) for binding to a target antigen comprising at least one antigen specific targeting region evolving from a parent or wild-type protein or domain thereof and having the property that the activity in an assay under normal physiological conditions is reduced compared to the activity in an assay under abnormal conditions, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the chimeric antigen receptor further comprises an extracellular spacer domain or at least one co-stimulatory domain. The target antigen may be a tumor specific antigen, which may be Ax1, ROR2 or CD22

The CARs of the invention may have at least one of (1) their affinity for the target antigen is reversibly or irreversibly reduced under normal physiological conditions compared to the same CAR without the conditionally active antigen-specific targeting region, and (2) their affinity is enhanced under aberrant conditions compared to the same CAR without the conditionally active antigen-specific targeting region. These CARs can direct cytotoxic cells to the diseased site where abnormal conditions exist, such as tumor microenvironment or synovial fluid. As a result of these properties, because of the low affinity of CARs for normal tissue, CARs can preferentially direct cytotoxic cells to the diseased site. Such CARs can significantly reduce side effects and allow for the use of higher doses of therapeutic agents to enhance therapeutic efficacy. CARs are particularly valuable for developing new therapeutic agents that are required to stay in the individual for a short or limited period of time. Examples of beneficial applications include systemic treatment at high doses and local treatment at high concentrations.

Chimeric antigen receptors can include antigen-specific targeting regions that have reduced binding affinity for a target antigen as compared to the antigen-specific targeting regions of the parent or wild-type protein or domain thereof under normal physiological conditions.

Chimeric antigen receptors may include antigen-specific targeting regions that have increased activity in assays under aberrant conditions as compared to antigen-specific targeting regions of a parent or wild-type protein or domain thereof, and reduced binding affinity for a target antigen under normal physiological conditions as compared to antigen-specific targeting regions of a parent or wild-type protein or domain thereof.

In any of the chimeric antigen receptors described above, the antigen-specific targeting region has increased selectivity in an assay under aberrant conditions as compared to the antigen-specific targeting region of the parent or wild-type protein or domain thereof.

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

The CAR molecule includes a linker that connects the two antigen-specific targeting regions (fig. 1). The linker orients the two antigen-specific targeting regions in such a way that the two antigen-specific targeting regions on the CAR-T cells exhibit improved or optimized activity in binding to the target antigen (Jensen et al ,"Design and implementation of adoptive therapy with chimeric antigen receptor-modifiedT cells,",ImmunolRev.,, volume 257, pages 127-144, 2014). Thus, the linker is preferably capable of adopting a specific conformation that is capable of improving or optimizing the binding of the two antigen-specific targeting regions to the target antigen, thereby increasing the effectiveness of the CAR-T cell.

In some embodiments, the linker may be a Gly-Ser tandem repeat sequence (Grada,"TanCAR:ANovel Bispecific ChimericAntigen Receptor for CancerImmunotherapy,"Molecular TherapyNucleicAcids,, volume 2, e105,2013, 18-25 amino acids in length. The flexible linker can adopt a number of different conformations for improved or optimized presentation of two antigen-specific targeting regions for binding to a target antigen.

In some embodiments, the linker is capable of adopting different conformations under normal physiological conditions and abnormal conditions. In particular, the linker has a first conformation under aberrant conditions that is improved or optimized for presentation of two antigen-specific targeting regions for binding to a target antigen, while the same linker has a second conformation under normal physiological conditions that is less effective for presentation of two antigen-specific targeting regions for binding to a target antigen than the first conformation of the linker under aberrant conditions. Such linkers may be referred to as "conditional linkers" that allow two antigen-specific targeting regions to bind to a target antigen under aberrant conditions with higher binding activity than under normal physiological conditions. Thus, CAR-T cells comprising such conditional linkers are more active under abnormal conditions than the same CAR-T cells under normal physiological conditions.

Proteins that change conformation at different pH's have been described previously, for example in Di Russo et al, volume ("pH-Dependent conformational changes in proteins and their effect on experimental pK(a)s:the case of Nitrophorin 4,"PLoS Comput Biol.,, e1002761,2012. In addition, proteins having different conformations at different temperatures are described in Caldwell, volume "Temperature-induced protein conformational changes in barley root plasma membrane-enriched microsomes,"PlantPhysiol.,, page 924-929, 1989. The conformation of antibodies affected by pH and/or temperature is discussed in Gandhi, "Effect ofpH andtemperature on conformational changes of a humanized monoclonal antibody," from the master graduation paper at the university of rocgdeli, usa.

The choice of conditional linkers for the CAR molecule falls within the scope of the invention. The conditional linker may adopt a first conformation under aberrant conditions that is improved or optimized for presentation of the two antigen-specific targeting regions for binding to the target antigen, and a second conformation under normal physiological conditions that is suboptimal for presentation of the two antigen-specific targeting regions for binding to the target antigen. In some embodiments, the binding activity of the CAR molecule produced in the suboptimal conformation of the linker to the target antigen under normal physiological conditions 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 the binding activity of the CAR molecule in the modified or optimized conformation having the linker under aberrant conditions.

The conditional linker may be generated from a starter linker selected from the group consisting of a 2A linker, a 2A-like linker, a picornaviral 2A-like linker, the 2A peptide (P2A) of porcine teschovirus and the 2A peptide (T2A) of the b tetrad of echinococcosis minor, and variants and functional equivalents thereof. The initial linker is evolved to produce the mutein, which is then assayed under normal physiological conditions and under abnormal conditions. Selecting a protein from the muteins that has a conditional linker based on the selected protein exhibiting (a) a conditional linker having a first conformation under aberrant conditions that is improved or optimized for presentation of two antigen-specific targeting regions for binding to a target antigen, and (b) a second conformation of the conditional linker under normal physiological conditions that is suboptimal for presentation of two antigen-specific targeting regions for binding to a target antigen.

The CAR molecule also includes an extracellular spacer domain that links the two antigen-specific targeting regions with a transmembrane domain that in turn links the co-stimulatory domain and the intracellular signaling domain within the T cell (fig. 1). The extracellular spacer domain is preferably capable of supporting an antigen-specific targeting region to recognize and bind to a target antigen on a 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, thus allowing the antigen-specific targeting region to have a structure that optimally recognizes the specific structure and density of a target antigen on a cell, such as a tumor cell (Hudecek et al, ,"The non-signaling extracellular spacer domain ofchimeric antigen receptors is decisive for in vivo antitumor activity,"CancerImmunolRes.,, volume 3, pages 125-135, 2015). The flexibility of the extracellular spacer domain allows the extracellular spacer domain to adopt a number of different conformations.

In some embodiments, the extracellular spacer domain is capable of adopting different conformations under normal physiological conditions and abnormal conditions. In particular, the extracellular spacer domain has a first conformation under aberrant conditions that is improved or optimized for presentation of two antigen-specific targeting regions for binding to a target antigen, while the same extracellular spacer domain has a second conformation under normal physiological conditions that is suboptimal for presentation of two antigen-specific targeting regions for binding to a target antigen. Such an extracellular spacer domain may be referred to as a "conditional extracellular spacer domain" because it allows two antigen-specific targeting regions to bind a target antigen with a higher binding activity under aberrant conditions than under normal physiological conditions. Thus, with a conditional extracellular spacer domain, the activity of a CAR-T cell is higher under aberrant conditions than the same CAR-T cell under normal physiological conditions.

The selection of conditional extracellular spacer domains for CAR molecules falls within the scope of the invention. In some embodiments, the suboptimal conformation of the extracellular spacer domain under normal physiological conditions results in a CAR molecule that has 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 the binding activity to the target antigen of a CAR molecule having the optimal conformation of the same extracellular spacer domain under aberrant conditions.

It has been found that for regions comprising extracellular spacer domains and transmembrane domains, the anti-ubiquitinated forms 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 oft cells that is resistantto ubiquitylation,"Human Gene Therapy,, volume 24, pages 27-37, 2013). Within this region, the extracellular spacer domain is outside the CAR-T cell, and is therefore exposed to different conditions, and may become conditionally anti-ubiquitinated.

The extracellular spacer domain is conditionally anti-ubiquitinated, which is also within the scope of the invention. In particular, the extracellular spacer domain of the CAR molecule has a higher resistance to ubiquitination under aberrant conditions than under normal physiological conditions. Thus, CAR-T cells having a conditional anti-ubiquitinated extracellular spacer domain will have enhanced cytotoxicity under aberrant conditions relative to their cytotoxicity under normal physiological conditions.

The conditional anti-ubiquitination extracellular spacer domain may be selected to have a higher ubiquitination resistance at an aberrant pH or an aberrant temperature and a lower ubiquitination resistance at a normal physiological pH or a normal physiological temperature. In one embodiment, the conditional anti-ubiquitination extracellular spacer domain has a higher resistance to ubiquitination at the pH of the tumor microenvironment and a lower resistance to ubiquitination at normal physiological pH, e.g., at pH in human plasma at pH 7.2-7.6.

To generate the conditional extracellular spacer domain, a starting protein fragment selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, and a CH3 region of an antibody is evolved to generate a mutein. Muteins are assayed under normal physiological conditions and under abnormal conditions. The conditional extracellular spacer domain is selected from a mutein that exhibits (a) a conditional extracellular spacer domain having a first conformation under abnormal conditions for binding the antigen-specific targeting region to the target antigen with a higher binding activity and a second conformation under normal physiological conditions for binding the antigen-specific targeting region to the target antigen with a lower binding activity than under abnormal conditions, or a protein that has a higher ubiquitination resistance under abnormal conditions than under normal physiological conditions.

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

In an alternative embodiment, the invention provides a Chimeric Antigen Receptor (CAR) for binding to a target antigen, comprising at least one antigen-specific targeting region that evolves from a parent or wild-type protein or domain thereof and that has increased selectivity in an assay under aberrant conditions compared to the antigen-specific targeting region of the parent or wild-type protein or domain thereof, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the chimeric antigen receptor further comprises an extracellular spacer domain or at least one co-stimulatory domain.

The invention also relates to a method of evolving a parent or wild-type protein or domain thereof to produce a conditionally active protein having at least one of (a) a decreased activity compared to an antigen-specific targeting region of the parent or wild-type protein or domain thereof in an assay under normal physiological conditions, and (b) an increased activity compared to an antigen-specific targeting region of the parent or wild-type protein or domain thereof in an assay under aberrant conditions. The conditionally active protein may be engineered into a CAR.

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

The chimeric antigen receptor produced by the method can include an antigen-specific targeting region that has increased activity in an assay under aberrant conditions as 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 as compared to the antigen-specific targeting region of the parent or wild-type protein or domain thereof.

In any of the above chimeric antigen receptors produced by the method, the antigen-specific targeting region has increased selectivity in an assay under aberrant conditions as compared to the antigen-specific targeting region of the parent or wild-type protein or domain thereof.

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

In an alternative embodiment of the method, a Chimeric Antigen Receptor (CAR) for binding to a target antigen produced by the method comprises at least one antigen-specific targeting region that evolves from a parent or wild-type protein or domain thereof and has increased selectivity in an assay under aberrant conditions as compared to the antigen-specific targeting region of the parent or wild-type protein or domain thereof, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the chimeric antigen receptor further comprises an extracellular spacer domain or at least one co-stimulatory domain.

Chimeric antigen receptor

The immune system of mammals, particularly humans, has cytotoxic cells for targeting and destroying diseased tissues and/or pathogens. The use of these cytotoxic cells to remove unwanted tissue such as tumors (i.e., target tissue) is a promising therapeutic approach. Other tissues that can be targeted for removal include hyperplasia of glands (e.g., the prostate), warts, and unwanted adipose tissue. However, this relatively new treatment has to date met with limited success. For example, because cancer cells can adapt to new therapies by reducing expression of surface antigens to reduce the effectiveness of the therapies, the long-term benefits of using T cells to target and destroy tumors are relatively low. Cancer cells may even dedifferentiate to evade detection in response to tumor-specific T cells. See Maher,"Immunotherapy of Malignant Disease Using Chimeric Antigen Receptor Engrafted T Cells,"ISRN Oncology,, volume 2012, ARTICLE ID 278093,2012.

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

The chimeric antigen receptor of the 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, the CAR may further comprise an Extracellular Spacer Domain (ESD) and/or a co-stimulatory domain (CSD). See fig. 1.

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

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

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

In one embodiment, if V _H domain alone is sufficient to confer antigen specificity ("single domain antibody"), astm comprises a full length IgG heavy chain specific for the target antigen and having V _H domain, CH1 domain, hinge domain, and CH2 and CH3 (Fc) Ig domains. if both the V _H domain and the V _L domain are required to produce fully active ASTR, then the CAR containing V _H and the full-length lambda light chain (IgL) are introduced into the same cytotoxic cell to produce active ASTR. in another embodiment, each astm of the CAR comprises at least two single chain antibody variable fragments (scFv), each specific for a different target antigen. The C-terminal end of one of the variable domains (V _H or V _L) was linked to the scFv at the N-terminal end of the other variable domain (V _L or V _H, respectively) by a polypeptide linker without significantly disrupting antigen binding or specificity of binding (Chaudhary et al ,"A recombinant single-chain immunotoxin composed of anti-Tac variable regions and atruncated diphtheriatoxin( recombinant single chain immunotoxins consisting of an anti-Tac variable region and a truncated diphtheria toxin), "proc.Natl.Acad.Sci., volume 87, page 9491, 1990; immunological and structural characterization of the high affinity anti-fluorescein single chain antibody by bedzyk et al ,""Immunological and structural characterization ofa high affinity anti-fluorescein single-chain antibody()," j.biol.chem., volume 265, pages 18615, 1990). These scFv lack constant regions (fcs) present in the heavy and light chains of natural antibodies. scFv specific for at least two different antigens are arranged in tandem. In one embodiment, the extracellular spacer domain may be connected between the ASTR and the transmembrane domain.

In another embodiment, the scFv fragment may be fused to all or a portion of the constant domain of the heavy chain. In another embodiment, the ASTR of the CAR comprises a bivalent single-chain variable fragment (di-scFv, bi-scFv). In CARs comprising di-scFV, two scFV, each specific for antigen, were linked together to form a single peptide chain with two V _H regions and two V _L regions (Xiong et al ,"Development of tumor targeting anti-MUC-1multimer:effects of di-scFv unpaired cysteine location onPEGylation and tumorbinding( tumor targeting anti-MUC-1 multimer development: di-scFV was not paired with the effect of cysteine positions on pegylation and tumor binding), "ProteinEngineeringDesign andSelection, volume 19, 359-367, 2006; kufer et al," A revival ofbispecific antibodies (reactivation of bispecific antibodies), "Trends inBiotechnology, volume 22, pages 238-244, 2004).

In another embodiment, the ASTR comprises a diabody. In diabodies, scFv is produced using a linker peptide that is too short for the two variable regions to fold together, allowing the scFv to dimerize. Shorter linkers (one or two amino acids) lead to the formation of trimers, so-called tri-antibodies. Four antibodies may also be used for ASTR.

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

The CAR-targeted antigen is present on the cell surface or inside in the tissue targeted for removal (e.g., tumor, gland (e.g., prostate) hyperplasia, warts, and unwanted adipose tissue). Although the ASTR of the CAR recognizes and binds more efficiently to surface antigens, the CAR may also target intracellular antigens. In some embodiments, the target antigen is preferably specific for cancer, inflammatory disease, neuronal disease, diabetes, cardiovascular disease, or infectious disease. 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, autoimmune, and/or inflammatory diseases.

ASTR-targeted cancer specific antigens include 4-IBB, 5T4, adenocarcinoma antigen, alpha fetoprotein, axl, BAFF, B-lymphoma cells, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor )、CD28、CD30(TNFRSF8)、CD33、CD4、CD40、CD44 v6、CD51、CD52、CD56、CD74、CD80、CEA、CNT0888、CTLA-4、DR5、EGFR、EpCAM、CD3、FAP、 fibronectin ectodomain B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, igG1, LI-CM, IL-13 IL-6, insulin-like growth factor I receptor, integrin α5β1, integrin αvβ3, MORAb-009, MS4A1, MUC1, mucin Canag, N-glycolylneuraminic acid, NPC-1C, PDGF-R a, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, ROR1, ROR2, SCH 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF- β2, TGF- β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA.88, VEGF-A, VEGFR-1, VEGFR2, or vimentin.

ASTR-targeted inflammatory disease-specific antigens include one or more of AOC3 (VAP-1), CAM-3001, CCL11 (eosinophil chemokine-1), CD125, CD147 (basic immunoglobulin), CD154 (CD 40L), CD2, CD20, CD23 (IgE receptor), CD25 (chain of IL-2 receptor), CD3, CD4, CD5, IFN- α, IFN- γ, igE Fc region, IL-1, IL-12, IL-23, IL-13, IL-17A, IL-22, IL-4, IL-5, IL-6 receptor, integrin α4, integrin α4β7, llamA (LAMA GLAMA), LFA-1 (CD 11A), MEDI-528, myostatin, OX-40, rhuMAb β7, conchA sclerostin (scleroscin), SOST, TGF β1, TNF- α, or VEGF-A.

The ASTR-targeted neuronal disease specific antigens of the invention comprise one or more of beta amyloid or MABT 5102A. The ASTR-targeted diabetes-specific antigens of the invention include one or more of L-I beta or CD 3. The cardiovascular disease specific antigens targeted by the ASTR of the present invention include one or more of C5, cardiac myoglobin, CD41 (integrin alpha-lib), fibrin II, beta chain, ITGB2 (CD 18) and sphingosine-1-phosphate.

The infectious disease specific antigen targeted by ASTR of the invention comprises one or more of anthrax toxin, CCR5, CD4, aggregation factor A, 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-alpha.

Other examples of target antigens include specific or amplified forms of surface proteins found on cancer cells, e.g., IL-14 receptor, CD19, CD20 and CD40 of B cell lymphoma, lewis and CEA antigens of various cancers, tag72 antigen of breast and colorectal cancers, EGF-R of lung cancer, folate binding proteins and HER-2 proteins amplified generally in human breast and ovarian cancers, or viral proteins such as gp120 and gp41 envelope proteins of HIV, envelope proteins from hepatitis B and C viruses, glycoprotein B and other envelope glycoproteins from human cytomegalovirus, and envelope proteins from cancer viruses such as Kaposi's sarcoma-associated herpesvirus. Other potential target antigens include CD4, where the ligand is the HIVgp120 envelope glycoprotein, and other viral receptors such as ICAM, which are receptors for human rhinoviruses, and related receptor molecules for polioviruses.

In another embodiment, the CAR can target an antigen that binds (engage) to a cancer treatment cell (e.g., NK cells and other cells mentioned herein) to activate the cancer treatment cell by acting as an immune effector cell. One example of this is a CAR that targets the CD16A antigen to combat NK cells with CD30 expressing malignancies. Bispecific, tetravalent AFM13 antibodies are examples of antibodies that can deliver this effect. Further details of this type of embodiment can be found, for example, in the following documents, rothe, a., et al, ,"A phase 1study of the bispecific anti-CD30/CD16A antibody construct AFM13 in patients with relapsed or refractoryHodgkin lymphoma,"Blood,2015, 25, volume 125, 26, pages 4024-4031.

In one embodiment, the ASTR targets a tumor specific antigen selected from Ax1, ROR2 and CD 22.

In some embodiments, ASTR is a single chain antibody that targets the cancer antigen Axl, which may have a nucleotide sequence selected from SEQ ID NOS 2-5 or an amino acid sequence selected from SEQ ID NOS 9-12. These single chain antibodies targeting the cancer antigen Axl comprise 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. These single chain antibodies targeting the cancer antigen Axl have increased binding activity to Axl at pH 6.0 compared to the same binding activity to Axl at pH 7.4.

In another embodiment, 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. The binding activity of this single chain antibody targeting the cancer antigen ROR2 to ROR2 at pH 6.0 is increased compared to the homogeneous binding activity to ROR2 at pH 7.4.

Single chain antibodies targeting Ax1 or ROR2 are suitable for linking to the transmembrane domain and intracellular signaling domain to generate CAR structures.

The extracellular spacer domain of CAR is a hydrophilic region located between the ASTR and transmembrane domain. In some embodiments, the domain facilitates proper protein folding of the CAR. The extracellular spacer domain is an optional component of the CAR. The extracellular spacer domain may comprise 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, a CH3 region of an antibody, an artificial spacer sequence, or a combination thereof. Examples of extracellular spacer domains include CD8a hinges, artificial spacers consisting of polypeptides that can be as small as three glycine (Gly), and CH1 and CH3 domains of IgG (e.g., human IgG 4).

The transmembrane domain of a CAR is a region that is capable of crossing the plasma membrane of a cytotoxic cell. The transmembrane domain is selected from a transmembrane region of a transmembrane protein, such as a type I transmembrane protein, an artificial hydrophobic sequence or a combination thereof. Examples of transmembrane domains include the transmembrane region of the alpha, beta or zeta chain of a T cell receptor, CD8, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD 154. The synthetic transmembrane domain may comprise a triplet of phenylalanine, tryptophan and valine. Optionally, a short oligopeptide or polypeptide linker (preferably 2 to 10 amino acids in length) can form a link between the transmembrane domain and the intracellular signaling domain of the CAR. Glycine-serine diabodies provide particularly suitable linkers between transmembrane domains and intracellular signaling domains.

The CARs of the invention also comprise an intracellular signaling domain. The intracellular signaling domain transduces effector function signals and directs the cytotoxic cells to perform their specialized function, i.e., damage and/or destruction of the target cell. Examples of intracellular signaling domains include zeta chains of the T cell receptor complex or any homologue thereof, e.g. eta chain, fcsRly and beta chain, MB1 (Iga) chain, B29 (Ig) chain etc., human CD3 zeta chain, CD3 polypeptides (delta, delta and epsilon), syk family tyrosine kinases (Syk, ZAP 70 etc.), src family tyrosine kinases (Lck, fyn, lyn etc.), and other molecules involved in T cell transduction, e.g. CD2, CD5 and CD28. In particular, the intracellular signaling domain may be the human cd3ζ chain, the cytoplasmic tail of FcyRIII, fcsRI, fc receptors, cytoplasmic receptors carrying an immunoreceptor tyrosine-based activation motif (ITAM), and combinations thereof.

The intracellular signaling domains used in CARs can include several types of intracellular signaling domains of various other immune signaling receptors, including but not limited to first, second, and third generation T cell signaling proteins, including CD3 receptors, B7 family co-stimulatory receptors, and Tumor Necrosis Factor Receptor (TNFR) superfamily receptors (Park et al, "ARE ALL CHIMERIC ANTIGEN receptors created equal. In addition, intracellular signaling domains include those used by NK and NKT cells (Hermanson, et al, "Utilizing CHIMERIC ANTIGEN receptors to direct natural KILLER CELL ACTIVITY," Front immunol., volume 6, page 195, 2015), such as the signaling domains of NKp30 (B7-H6) (Zhang et al, volume ,"An NKp30-based chimeric antigen receptor promotes T cell effector functions and antitumor efficacy in vivo,"JImmunol.,, pages 2290-2299, 2012) and DAP12 (Topfer et al, "DAP12-based ACTIVATING CHIMERIC ANTIGEN receptor for NK cell tumor immunotherapy," JImmunol., volume 194, pages 3201-3212, 2015), NKG2D, NKp, NKp46, DAP10, and CD3 z. In addition, intracellular signaling domains also include signaling domains of human immunoglobulin receptors that comprise an immunoreceptor tyrosine-based activation motif (ITAM), such as FCGAMMARI, FCGAMMARIIA, FCGAMMARIIC, FCGAMMARIIIA, FCRL (Gillis et al, ,"Contribution of Human FcγRs to Disease with Evidence from Human Polymorphisms and Transgenic Animal Studies,"FrontImmunol.,, volume 5, page 254, 2014).

In some embodiments, the intracellular signaling domain comprises a cytoplasmic signaling domain of TCR ζ, fcrγ, fcrβ, cd3γ, cd3δ, cd3ε, CD5, CD22, CD79a, CD79b, or CD66 d. It is particularly preferred that the intracellular signaling domain in the CAR comprises the cytoplasmic signaling domain of human cd3ζ.

The CARs of the invention may comprise a co-stimulatory domain that has the function of enhancing cell proliferation, cell survival, and memory cell formation against the cytotoxic cells expressing the CAR. The CARs of the invention may comprise one or more co-stimulatory domains selected from the group consisting of co-stimulatory domains in protein 、CD28、CD137(4-IBB)、CD134(OX40)、Dap1O、CD27、CD2、CD7、CD5、ICAM-1、LFA-1(CD11a/CD18)、Lck、TNFR-I、PD-1、TNFR-II、Fas、CD30、CD40、ICOS LIGHT、NKG2C、B7-H3 of the TNFR superfamily or a combination thereof. If the CAR comprises more than one co-stimulatory domain, these domains may be arranged in tandem, optionally separated by a linker. The co-stimulatory domain is an intracellular domain that can be located between the transmembrane domain and the intracellular signaling domain in the CAR.

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

Antigen-specific targeting regions with conditional activity

CARs are chimeric proteins produced by fusing all of the different domains described above together to form a fusion protein. CARs are typically produced from expression vectors comprising polynucleotide sequences encoding different domains of the CAR. ASTRs of the invention that are capable of recognizing and binding to antigens on target cells have conditional activity. Specifically, the ASTRs of the invention have less or no activity to bind to a target antigen under normal physiological conditions and have activity to bind to a target antigen under abnormal conditions, as compared to the ASTRs of the corresponding parent or wild-type proteins. The present invention provides methods of producing conditionally active astm s from a parent or wild-type protein or binding domain thereof (parent or wild-type astm s).

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

In some embodiments, the wild-type protein is an antibody, and the wild-type antibody can be found by generating and screening a library of antibodies. The antibody library may be a polyclonal antibody library or a monoclonal antibody library. A polyclonal antibody library against a target antigen may be generated by injecting the antigen directly 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 an antigen. For the preparation of monoclonal antibody libraries, any technique that provides antibodies produced by continuous cell line culture may be used. Examples include hybridoma technology, triple-source hybridoma technology, human B-cell hybridoma technology, and EBV hybridoma technology (see, e.g., cole (1985) in Monoclonal Antibodies AND CANCER THERAPY, alan r.list, inc., pp.77-96). The techniques for producing single chain antibodies (see, e.g., U.S. Pat. No. 4,946,778) may be adapted to produce single chain antibody libraries.

Other methods exist for generating and screening antibody libraries for the discovery of wild-type antibodies. For example, a fully human antibody display library may be utilized. Such libraries are populations of antibodies displayed on the surface of host cells. Preferably, the antibody library represents human antibody repertoires that have a broad ability to bind a broad range of antigens. Because antibodies are displayed on the cell surface, the effective affinity (caused by the avidity of the antibody) of each antibody in the library is increased. Unlike other popular library types (e.g., phage display libraries, where antibody antigenicity is less desirable for screening and identification purposes), it is desirable in the present invention to provide excellent antibody antigenicity by cell surface display. Cell surface display libraries enable the identification of antibodies with low, medium and high binding affinities, as well as the identification of non-immunogenic weak epitopes in a screening or selection step.

Production of evolved molecules from parent molecules

The parent or wild-type protein or binding domain thereof (parent or wild-type astm) is subjected to a mutagenesis procedure to produce a population of mutant polypeptides, which are then screened to identify mutant astm s having the property of having an increased binding affinity to the target antigen compared to the parent or wild-type astm s under aberrant conditions and, optionally, substantially the same or reduced binding affinity to the target antigen compared to the parent or wild-type astm s under normal physiological conditions.

Any chemical synthesis method or recombinant mutagenesis method can be used to produce the mutant polypeptide population. Unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology, which are within the skill of the art, can be used in the practice of the invention. Such techniques are well explained in the literature. see, e.g., molecular Cloning A Laboratory Manual (molecular clone A laboratory Manual), 2nd Ed., main code Sambrook, fritsch and Maniatis (Cold Spring Harbor Laboratory Press:1989); DNA Cloning (DNA Cloning), volumes I and II (D.N.Glover ed., 1985); oligonucleotide Synthesis (oligonucleotide synthesis) (m.j. Gait ed., 1984); mullis et al U.S. Pat. No. 4,683,195;NucleicAcid Hybridization (nucleic acid hybridization) (B.D.Hames & S.J.Higgins eds.1984), transcriptionAndTranslation (transcription and translation) (B.D.Hames & S.J.Higgins eds.1984), culture OfAnimal Cells (animal cell culture) (R.I.Fresnel, alan R.List, inc., 1987), immobilized CellsAnd Enzymes (fixed cells and enzymes) (IRL Press, 1986), B.Perbal, A Practical Guide To Molecular Cloning (practice guidelines for molecular Cloning) (1984), THE TREATISE, methods In Enzymology (methods in monozymol) (ACADEMIC PRESS, inc., N.Y.), geneTransferVectors For MAMMALIAN CELLS (gene transfer vectors for mammalian cells) (J.H.Miller and M.P.Caeds., 1987,Cold Spring Harbor Laboratory), methods In Enzymology (methods in zymol), volumes 154 and 155 (methods in Wu et al), immunochemical Methods IN CELLAND Molecular Biology (immunochemical methods in cell and molecular biology) (MAYER AND WALKER, eds, ACADEMIC PRESS, 1987), and (methods in fluid-phase, 1988), and (methods in fluid-phase, J.J.Mildl., 35, 1988), and (methods in fluid-phase, 1988), n.y., 1986).

The present invention provides a method of producing a nucleic acid mutant encoding a conditionally active mutant polypeptide, the method comprising modifying a nucleic acid by (i) replacing one or more nucleotides with a different nucleotide, wherein the nucleotides comprise a natural or non-natural nucleotide, (ii) deleting one or more nucleotides, (iii) adding one or more nucleotides, or (iv) any combination thereof. In one aspect, the unnatural nucleotide comprises inosine. In another aspect, the method further comprises detecting altered enzymatic activity of a polypeptide encoded by the modified nucleic acid to determine the modified nucleic acid encoding the polypeptide having altered enzymatic activity. In one aspect, the modified step (a) consists of PCR, error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, cycle-by-cycle mutagenesis, exponential-by-whole mutagenesis, site-specific mutagenesis, gene reassembly, gene site saturation mutagenesis, ligase chain reaction, in vitro mutagenesis, ligase chain reaction, oligonucleotide synthesis, any DNA generating technique, and any combination thereof. In another aspect, the method further comprises repeating at least one modification step.

The invention further provides a method of generating a polynucleotide from two or more nucleic acids, the method comprising (a) determining identical and different portions between two or more nucleic acids, wherein at least one nucleic acid comprises a nucleic acid of the invention, (b) providing a set of oligonucleotides associated with at least two sequences of the two or more nucleic acids, and (c) extending the oligonucleotides with a polymerase, thereby generating a polynucleotide.

Any mutagenesis technique may be used in various embodiments of the invention. Random (Stochastic) or random (random) mutagenesis exemplifies the case of mutation (modified or altered) of a parent molecule, resulting in a set of offspring molecules with predetermined mutations. Thus, in vitro random mutagenesis reactions, for example, there is no specific predetermined product in the intended middle yield, and the exact nature of the mutation obtained, and also the product produced, is uncertain and therefore random. Random mutagenesis occurs, for example, in error-prone PCR and random shuffling methods, where the mutations obtained are random and predetermined. Variant forms may be generated by error-prone transcription, such as error-prone PCR, or using a polymerase lacking error correction (see Liao (1990) Gene 88:107-111), the first variant form, or by replicating the first variant form in a mutagenized strain (mutator strain) (the mutagenized host cells are discussed in further detail below, which are generally well known). The mutagenic strain may include any mutation in any organism with impaired mismatch repair function. These mutations include the mutated gene products of mutS, mutT, mutH, mutL, ovrD, dcm, vsr, umuC, umuD, sbcB, recJ, et al. Lesions are obtained by addition of agents such as small compounds or expressed antisense RNA or other techniques to produce gene mutations, allele replacement, selective inhibition. The damage may be to a gene, or to any organism's homologous gene.

Other mutagenesis methods include oligonucleotide-directed mutagenesis techniques, error-prone polymerase chain reaction (error-prone PCR) and cassette mutagenesis, in which specific regions of the parent polynucleotide are replaced by synthetically mutagenized oligonucleotides. In these cases, a number of mutation sites are created around some of the sites in the parent sequence.

In oligonucleotide-directed mutagenesis, short sequences are replaced by synthetic mutagenic oligonucleotides. In oligonucleotide-directed mutagenesis, a short sequence of a polynucleotide is removed from the polynucleotide using restriction enzyme digestion and replaced by a synthetic polynucleotide in which multiple bases are changed relative to the starting sequence. The polynucleotide sequence may also be altered by chemical mutagenesis. Chemical mutagens include, for example, sodium bisulfite, nitrous acid, hydroxylamine, hydrazine, or formic acid. Other reagents for analogs of nucleotide precursors include nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine. Generally, these reagents are added to the PCR reaction to replace nucleotide precursors, thereby mutating the sequence. Intercalating agents such as proflavine, acriflavine, quinacrine and the like may also be used. Random mutations in the polynucleotide sequence may also be obtained by X-ray or ultraviolet radiation. In general, the plasmid polynucleotide thus mutagenized is introduced into E.coli and propagated as a pool (pool) or library of hybrid plasmids.

Error-prone PCR uses low-precision (low-fidelity) polymerization conditions to introduce low levels of random point mutations on long sequences. In a mixture of fragments of unknown sequence, the mixture can be mutagenized using error-prone PCR.

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

Alternatively, any non-random mutagenesis technique may be used in various embodiments of the invention. An example of a non-random mutagenesis is, for example, a parent molecule that has been mutated (modified or altered) to produce a progeny molecule having one or more predetermined mutations. It will be appreciated that it is realistic that in many reactions where molecular processing occurs, there is an amount of background product whose presence does not affect the non-random nature of the mutagenesis method with the intended product. Site-directed saturation mutagenesis and synthetic ligation recombination are examples of mutagenesis techniques in which the exact chemical structure of the target product is predetermined.

One method of site-directed saturation mutagenesis is disclosed in U.S. patent application publication No. 2009/0130518. The method provides a set of degenerate primers corresponding to codons of a template polynucleotide, and polymerase extension is performed to generate polynucleotide offspring comprising sequences corresponding to the degenerate primers. The polynucleotide progeny may be expressed and screened for directed evolution. Specifically, this is a method of producing a set of progeny polynucleotides comprising the steps of (a) providing copies of a template polynucleotide, each comprising a plurality of codons encoding a sequence of the template polypeptide, and (b) subjecting each codon of the template polynucleotide to the steps of (1) providing a set of degenerate primers, each primer comprising a degenerate codon corresponding to a codon of the template polynucleotide and at least one contiguous sequence homologous to a contiguous sequence of codons of the template polynucleotide, (2) providing conditions to anneal the primers to the copies of the template polynucleotide, and (3) performing a polymerase extension reaction along the template from the primers, thereby producing polynucleotide progeny, each polynucleotide progeny comprising a sequence corresponding to the degenerate codon of the annealed primer, thereby producing a set of polynucleotide progeny. Site-directed saturation mutagenesis involves the directed evolution of nucleic acids and screening clones containing the evolved nucleic acids to obtain target binding activity.

Site-directed saturation mutagenesis generally involves the method 1) of preparing molecular offspring (including molecules consisting of polynucleotide sequences, molecules consisting of polypeptide sequences, and molecules consisting of portions of polynucleotide sequences and portions of polypeptide sequences), mutagenizing from one or more ancestral or parental templates to obtain at least one point mutation, addition, deletion, and/or chimeric; 2) screening the offspring molecules-preferably using a high throughput method-to obtain the desired binding affinity for the target antigen; 3) optionally obtaining and/or cataloging structures and/or functional information relating to the parent and/or offspring molecules; and 4) optionally repeating any of steps 1) -3).

In site-directed saturation mutagenesis, polynucleotide offspring are generated (e.g., from a parent polynucleotide template) that are termed "codon site-directed saturation mutagenesis," each containing a set of at least up to three consecutive point mutations (i.e., containing different bases from the new codon), such that each codon (or each degenerate codon family encoding the same amino acid) is represented at each codon position. Corresponding to and encoded by the polynucleotide progeny, a set of polypeptide progeny, each having at least one separate amino acid point mutation, is also produced. In a preferred aspect, what is known as "site-directed saturation mutagenesis of amino acids" is produced, a mutant polypeptide of 19 naturally encoded polypeptides each, along which an alpha-amino acid substitution is made at each amino acid position. If other amino acids are used in place of or in addition to the 20 naturally encoded amino acids, this results in a total of 20 different polypeptide offspring (at each amino acid position along the parent polypeptide), including the original amino acid or potentially ≡21 different polypeptide offspring.

Other mutagenesis techniques may also be employed, including methods of recombination and more particularly preparation of polynucleotides encoding polypeptides by in vivo rearrangement of polynucleotide sequences containing partial homology regions, recombining the polynucleotides to generate at least one polynucleotide, and screening the polynucleotides to produce polypeptides having useful properties.

In another aspect, mutagenesis techniques exploit the natural properties of cells to recombine molecules and/or mediate reducing processes that reduce sequence complexity and repeat or continue sequence ranges with homologous regions.

Various mutagenesis techniques, either alone or in combination, provide methods for preparing hybrid polynucleotides encoding biologically active hybrid polypeptides. In achieving these and other objects, according to one aspect of the invention, there is provided a method of introducing a polynucleotide into a suitable host cell and growing the host cell under conditions for the production of a hybrid polypeptide.

Chimeric genes were generated by ligating 2 polynucleotide fragments using compatible cohesive ends generated by restriction enzymes, wherein each fragment was from a separate ancestral (or parent) molecule. Another example is the mutagenesis (i.e., obtaining codon substitutions, additions or deletions) of a single codon position of a parent polynucleotide to produce a single polynucleotide progeny encoding a single site-mutated polypeptide.

In addition, in vivo site-specific recombination systems have been employed to produce gene hybrids, as well as random methods in vivo recombination, and recombination between homologous but truncated genes in plasmids. Mutagenesis by overlap extension and PCR has also been reported.

Non-random methods have been used to obtain a large number of point mutations and/or chimerisms (chimerization), e.g., comprehensive or exhaustive methods have been used to generate all molecules in a particular set of mutations, the function being attributed to a particular group of structures (e.g., a particular single amino acid position or a sequence of two or more amino acid positions) in a template molecule, to classify and compare a particular set of mutations.

Any of these or other evolutionary methods may be used in the present invention to generate a novel population (library) of mutant polypeptides from a parent or wild-type protein.

Expression of evolved molecules

The mutant polynucleotides resulting from the evolution step may or may not be size fractionated on agarose gel according to the disclosed protocol, inserted into an expression vector, and transfected into a suitable host cell to produce the mutant polypeptide (expression). The expression may be performed using conventional molecular biology techniques. Thus, the expression step may use various known methods.

For example, briefly, the mutant polynucleotide produced in the evolution step is then digested and ligated into an expression vector, such as plasmid DNA, using standard molecular biology techniques. The vector is then transformed into bacteria or other cells using standard methods (protocols). High throughput expression and screening can be performed in single wells of a multi-well plate, such as a 96-well plate. The method was repeated to obtain each mutant polynucleotide.

The screened and isolated polynucleotides are introduced into a suitable host cell. Suitable host cells are any cells capable of promoting recombination and/or reducing recombination. The polynucleotides thus screened are preferably already present in a vector which comprises the appropriate regulatory sequences. The host cell may be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or preferably the host cell may be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell may be calcium phosphate transfection, DEAE-dextran mediated transfection or electroporation (e.g.Ecker and Davis,1986, expression of the antisense RNA inhibitor gene in plant cells (Inhibition of gene expression inplant cells by expression ofantisense RNA),Proc NatlAcad Sci USA,83:5372-5376).

As representative examples, expression vectors which may be used, mention may be made of viral particles, baculoviruses, phages, plasmids, phagemids, cosmids, phagemids (fosmids), bacterial artificial chromosomes, viral DNA (e.g.vaccinia virus, adenovirus, fowl pox virus, pseudorabies and derivatives of SV 40), P1-based artificial chromosomes, yeast plasmids, yeast artificial chromosomes and any other specific host of interest (e.g.Bacillus, aspergillus niger and yeast). Thus, for example, the DNA may be included in any expression vector for expressing the polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences. A number of suitable carriers are well known in the art and are commercially available. The following vectors are provided by way of example, bacteria, pQE vector (Qiagen), pBluescript plasmid, pNH vector, lambda-ZAP vector (Stratagene), ptrc99a, pKK223-3, pDR540, pRIT2T (Pharmacia), eukaryotic, pXTl, pSG5 (Stratagene), pSVK3, pBPV, PMSG, pSVLSV (Pharmacia). However, any other plasmid or other vector may be used as long as it is replicable and viable in the host. Low copy or high copy number vectors may be used in the present invention.

The mutant polynucleotide sequence in the expression vector is operably linked to appropriate expression control sequences (promoters) to direct the synthesis of RNA. Specific named bacterial promoters include lacI, lacZ, T, T7, gpt, λPR, PL and trp. Eukaryotic promoters include CMV early (IMMEDIATE EARLY), HSV thymidine kinase, early and late SV40, LTR from retrovirus, and mouse metallothionein-1. The selection of suitable vectors and promoters is 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 also includes suitable sequences for amplifying the expression. The promoter region can be selected from any gene of interest using chloramphenicol transferase (CAT) vectors or other vectors with selectable markers. In addition, it is preferred that the expression vector contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or tetracycline or ampicillin resistance as in E.coli.

Eukaryotic DNA transcription may be increased by inserting enhancer sequences into expression vectors. Enhancers are cis-acting sequences of 10-300bp that enhance transcription by promoters. Enhancers can be effective in enhancing transcription when in the 5 'or 3' direction of the transcriptional unit. Enhancers are also useful if they are located within introns or within the coding sequence itself. Viral enhancers are commonly used, including the SV40 enhancer, cytomegalovirus enhancer, polyoma enhancers, and adenovirus enhancers. Enhancer sequences from mammalian systems are also commonly used, such as mouse immunoglobulin heavy chain enhancers.

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 that confers drug resistance. The first two marker genes preferably use mutant cell lines that lack the ability to grow without adding thymidine to the growth medium. Transformed cells can then be identified by their ability to grow on non-supplemented media. Examples of prokaryotic drug resistance genes used as markers include genes conferring resistance to G418, mycophenolic acid and hygromycin.

Depending on the type of cell production host, the expression vector containing the DNA segment of interest may be transferred into the host cell by well known methods. For example, calcium chloride transfection is commonly used for prokaryotic host cells, whereas calcium phosphate treatment, lipofection, or electroporation may 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).

Once the expression vector has been introduced into a suitable host, the host is maintained under conditions suitable for high level expression of the introduced mutant polynucleotide sequence to produce the mutant polypeptide. These expression vectors are generally replicable in the host organism as episomes or as part of the host chromosomal DNA. Typically, the expression vector will contain a selectable marker, such as tetracycline or neomycin, to allow detection of those cells transformed with the desired DNA sequence (see, e.g., U.S. Pat. No. 4,704,362).

Thus, in another aspect of the invention, mutant polynucleotides can be produced by means of reductive recombination. The method involves preparing constructs containing contiguous sequences (original coding sequences), inserting them into a suitable vector, and then introducing them into a suitable host cell. The rearrangement of individual molecular recognition (molecularidentity) is produced by a combinatorial approach between consecutive sequences in constructs with homologous regions or between quasi-repeat units. The rearrangement process recombines and/or reduces the complexity and size of the repeated sequences and creates new molecules. Various treatments may be employed to enhance recombination rates. These may include the use of ultraviolet light or chemicals that disrupt DNA and/or the use of host cells that exhibit higher levels of "genetic instability. Thus, the recombination method may include homologous recombination or the natural property of quasi-repeated sequences that direct self-evolution.

The cells are then propagated and subjected to "reductive recombination". If desired, the rate of reductive recombination can be stimulated by introducing DNA damage. In vivo recombination is focused on "intermolecular" methods collectively referred to as "recombination", which is generally regarded as a "RecA-dependent" phenomenon in bacteria. The present invention relies on recombination of host cells to recombine and rearrange sequences, or on the ability of the cells to mediate a reduction process to reduce the complexity of quasi-repeated sequences in the cells by deletion. This "reductive recombination" process occurs by an "intramolecular" RecA-independent process. The end result is a recombination of the molecules into all possible combinations.

In one aspect, the host organism or cell comprises a gram negative bacterium, a gram positive bacterium, or a eukaryotic organism. In another aspect of the invention, the gram negative bacteria include E.coli or Pseudomonas fluorescens. In another aspect of the invention, the gram positive bacteria include Streptomyces diversa, lactobacillus gasseri (Lactobacillus gasseri), lactococcus lactis (Lactococcus lactis), streptococcus cremoris (Lactococcus cremoris) or bacillus subtilis. In another aspect of the invention, the eukaryotic organism comprises Saccharomyces cerevisiae, schizosaccharomyces, pichia pastoris, kluyveromyces lactis, hansenula or Aspergillus niger (Aspergillus niger). As representative examples of suitable hosts, mention may be made of bacterial cells such as E.coli, streptomyces, salmonella typhimurium, fungal cells such as yeast, insect cells such as S2 and Sf9, animal cells such as CHO, COS or human melanoma, adenoviruses and plant cells. The selection of an appropriate host is considered to be within the teachings of those skilled in the art.

In addition to eukaryotic microorganisms such as yeast, mammalian tissue cell cultures may also be used to express the mutant polypeptides of the invention (see Winnacker, "From Genes to Clones (from gene to clone)," VCH Publishers, n.y., n.y. (1987)). Eukaryotic cells are preferred because many suitable host cell lines capable of secreting intact immunoglobulins have been developed in the art, including CHO cell lines, various COS cell lines, heLa cells, myeloma cell lines, B cells or hybridomas. Expression vectors for these cells may include expression control sequences such as origins of replication, promoters, enhancers (Queen et al, immunol. Rev., vol. 89, page 49, 1986) and necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites and transcription terminator sequences. Preferred expression 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、MiaPACA-2、ACHN、Jurkat、MM1、Ovcar 3、HT 1080、Panc-1、U266、769P、BT-474、Caco-2、HCC 1954、MDA-MB-468、LnCAP、NRK-49F and SP2/0 cell lines, and mouse spleen cells and rabbit PBMC. In one aspect, the mammalian host cell is selected from CHO or HEK293 cell lines. In a specific aspect, the mammalian host cell is a CHO-S cell line. In another specific aspect, the mammalian system is a HEK293 cell line. In another embodiment, the eukaryotic host is a yeast cell system. In one aspect, the eukaryotic host is selected from a Saccharomyces cerevisiae cell or a Pichia cell.

In another embodiment, mammalian host cells may be created commercially by contractual research or custom manufacturing organizations. For example, for recombinant antibodies or other proteins, lonza (Long Shaji groups of limited, bassell, switzerland) can use GS gene expression system ^TM technology to produce vectors expressing these products using CHOK1SV or NS0 cell production hosts. Host cells containing the polynucleotide of interest may be cultured in conventional nutrient media modified as appropriate for activating promoters, screening transformants, or amplifying genes. Culture conditions, such as temperature, pH, etc., are previously used to screen for expression of host cells, as will be apparent to those skilled in the art.

As described above, the optimization of expression of conditionally active ASTR may be achieved by optimization of the vector used (vector components, e.g. promoter, splice site, 5 'and 3' terminal and flanking sequences), genetic modification of the host cell to reduce gene deletions and rearrangements, evolution of host cell gene activity by methods of evolving the relevant gene in vivo or in vitro, optimization of host glycosylase by evolution of the relevant gene, and/or selection of cells with enhanced expression capacity by host cell mutagenesis and selection strategies at the chromosomal level.

Protein expression can be induced by a variety of known methods, and a number of genetic systems have been disclosed for inducing protein expression. For example, using a suitable system, the addition of an inducer may induce the protein expression. The cells were then pelleted by centrifugation and the supernatant discarded. Cells were incubated with dnase, rnase and lysozyme to enrich for periplasmic proteins. After centrifugation, the supernatant containing the new protein was transferred to a new multiwell plate and stored prior to detection.

Cells are typically collected by centrifugation, disrupted by physical or chemical means, and the resulting crude extract is used for further purification. Microbial cells used to express the protein may be disrupted by any convenient means, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysates. Such methods are well known to those skilled in the art. Expressed polypeptides or fragments thereof may be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. If necessary, a protein renaturation step may be used to complete the configuration of the polypeptide. High Performance Liquid Chromatography (HPLC) can be employed as the final purification step if desired. Screening for conditionally active astm can be aided by using convenient high throughput screening or selection methods. Cell surface display expression and screening techniques (e.g., as defined above) can be used to screen mutant proteins to obtain conditionally active ASTR.

Screening to identify reversible or irreversible mutants

Identification of the target molecule is accomplished directly, mainly by measuring the protein activity under permissive and wild-type conditions. Mutants with the greatest activity ratio (permissive/wild-type) can then be screened and the arrangement of point mutations generated by a combination of individual mutations (permutation) using standard methods. The combinatorial aligned protein library is then screened to give proteins that show the greatest difference in activity between permissive and wild-type conditions.

The supernatants can be screened for activity by a variety of methods, such as high throughput activity assays, e.g., fluorescent assays, to identify protein mutants that are sensitive to the desired characteristics (temperature, pH, etc.). For example, temperature sensitive mutants are screened and the enzyme activity or antibody activity of each mutant is determined using commercially available substrates at a lower temperature (e.g., 25 ℃) and at a temperature at which the original protein functions (e.g., 37 ℃). Screening can be performed in a variety of media, such as serum and BSA, among others. The reaction may initially be in the form of a multi-well assay, such as a 96-well assay, validated using a different format, such as a 14ml tube format.

In one aspect, the method further comprises modifying at least one nucleic acid or polypeptide prior to assaying the candidate polypeptide for conditional biological activity. In another aspect, the assaying step (c) further comprises detecting increased expression of the polypeptide in the host cell or host organism. In another aspect, the determining step (c) further comprises detecting the enzymatic activity over a pH range of about pH3 to about pH 12. In another aspect, the determining step (c) further comprises detecting the enzymatic activity over a pH range of about pH5 to about pH 10. In another aspect, the determining step (c) further comprises detecting the enzymatic activity over a pH range of about pH6 to about pH 8. In another aspect, the determining step (c) further comprises detecting the enzyme activity at pH6.7 and pH 7.5. In another aspect, the determining step (C) further comprises detecting the enzymatic activity over a temperature range of about 4 ℃ to about 55 ℃. In another aspect, the determining step (C) further comprises detecting the enzymatic activity over a temperature range of about 15 ℃ to about 47 ℃. In another aspect, the determining step (C) further comprises detecting the enzymatic activity over a temperature range of about 20 ℃ to about 40 ℃. In another aspect, the determining step (C) further comprises detecting the enzymatic activity over a temperature range of about 25 ℃ to about 37 ℃. In another aspect, the determining step (c) further comprises detecting the enzymatic activity under conditions of normal osmotic pressure and abnormal (positive or negative) osmotic pressure. In another aspect, the determining step (c) further comprises detecting the enzyme activity under conditions of normal electrolyte concentration and abnormal (positive or negative) electrolyte concentration. The test electrolyte concentration is selected from one of calcium, sodium, potassium, magnesium, chlorine, bicarbonate and phosphate concentrations, and in another aspect, the determining step (c) further comprises detecting an enzymatic activity that forms a stable reaction product.

In another aspect, the invention provides a purified antibody that specifically binds to a polypeptide of the invention having enzymatic activity or a fragment thereof. In one aspect, the invention provides antibody fragments that specifically bind to polypeptides having enzymatic activity.

Antibodies and antibody-based screening methods

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

The antibodies can be used in immunoprecipitation, staining, immunoaffinity columns, and the like. If desired, the nucleic acid sequence encoding a particular antigen can be generated by immunization followed by isolation, amplification or cloning of the polypeptide or nucleic acid and immobilization of the polypeptide to an array of the invention. Alternatively, the methods of the invention may be used to modify the structure of an antibody produced by a cell to be modified, e.g., the affinity of the antibody may be increased or decreased. Moreover, the ability to prepare or modify antibodies can be engineered into the phenotype of a cell by the methods of the invention.

Methods of immunization, methods of preparing and isolating ANTIBODIES (polyclonal and monoclonal) are known to those skilled in the art and are described in the scientific and patent literature, see for example Coligan, immunoepidemic handbook (CURRENT PROTOCOLS IN IMMUNOLOGY), wiley/Greene, N.Y. (1991); stites (eds.)) basic and clinical immunology (7 th edition )(BASIC AND CLINICAL IMMUNOLOGY)(7th ed.)Lange Medical Publications,Los Altos,Calif.("Stites");Goding, monoclonal antibody: principle and protocols (2 nd edition )(MONOCLONAL ANTIBODIES:PRINCIPLES AND PRACTICE)(2ded.)Academic Press,NewYork,N.Y.(1986);Kohler(1975)". Sequential culture (Continuous cultures of fused cells secreting antibody ofpredefined specificity)",Nature 256:495;Harlow(1988) antibody of fusion cells secreting specific ANTIBODIES, laboratory handbook (ANTBIES, A LABORATORY MANUAL)), cold Spring HarborPublications, newYork., in addition to traditional methods in animals, ANTIBODIES can also be produced in vitro, e.g., using recombinant antibody binding sites to express phage display libraries see for example, hoogenboom (1997)' design and optimization (Designing and optimizing library selection strategies for generating high-affinity antibodies)",Trends Biotechnol.15:62-70;and Katz(1997)" of library screening strategies for producing high antibody affinity, construction and mechanical determination of phage affinity and specificity of ligand found or engineered by display technology (Structural and mechanistic determinants ofaffinity and specificity ofligands discovered or engineered by phage display)",Annu.Rev.Biophys.Biomol.Struct.26:27-45.

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

In immunoaffinity methods, the antibody is attached to a solid support, such as a bead or other columnar matrix. The protein reagent is contacted with the antibody under conditions whereby the antibody specifically binds to a polypeptide of the invention. Washing is followed by removal of non-specifically bound proteins and elution of specifically bound polypeptides.

The ability of a protein in a biological sample to bind to an antibody can be determined using any method familiar to those skilled in the art. For example, binding may be determined by labeling the antibody with a detectable label, such as a fluorescent agent, an enzyme label, or a radioisotope. Alternatively, the ability of the antibody to bind to the sample may be determined using a secondary antibody with a detectable label. Specific assays include ELISA assays, diabody sandwich methods, radioimmunoassays and Western blots.

The polyclonal antibodies raised against the polypeptides of the invention may be obtained by direct injection of the polypeptides into an animal or by administration of the polypeptides to a non-human animal. The antibody thus obtained then binds to the polypeptide itself. In this manner, even sequences encoding only polypeptide fragments can be used to generate antibodies that bind to the entire native polypeptide. The antibodies can then be used to isolate the polypeptide from cells expressing the polypeptide.

For the preparation of monoclonal antibodies, any technique for producing antibodies from continuous cell line cultures may be employed. Examples include hybridoma technology, monoclonal antibody preparation, any technology that provides antibodies produced by continuous cell line culture may be used. Examples include hybridoma technology, triple-hybridoma technology, human B-cell hybridoma technology, and epstein barr virus hybridoma technology (see, e.g., cole (1985), monoclonal antibodies, and cancer therapies (monoclone antibodies AND CANCER THERAPY), alan r.liss, inc., pp.77-96).

The techniques described for producing single chain antibodies (see, e.g., U.S. Pat. No. 4946778) may be adapted for use in producing single chain antibodies that act on the polypeptides of the invention. Alternatively, transgenic mice can be used to express humanized antibodies that act on these polypeptides or fragments thereof. Antibodies raised against the polypeptides of the invention may be used to screen similar polypeptides (e.g., enzymes) from other organisms and samples. In these techniques, polypeptides from an organism are contacted with an antibody and those polypeptides that specifically bind to the antibody are detected. Any of the methods described above may be used to detect antibody binding.

Screening method and "on-line" detection device

In the operation of the methods of the invention, a variety of instruments and methods can be used for the polypeptides and nucleic acids of the invention, e.g., for screening polypeptides having enzymatic activity, for screening compounds that are potential modulators, e.g., activators or inhibitors of enzymatic activity, for screening antibodies that bind to the polypeptides of the invention, for screening nucleic acids that hybridize to the nucleic acids of the invention, for screening cells expressing the polypeptides of the invention, and the like.

Arrays or "biochips"

The nucleic acids or polypeptides of the invention may be immobilized on or applied to an array. Arrays can be used to screen or monitor libraries of compositions (e.g., small molecules, antibodies, nucleic acids, etc.), for their ability to bind or modulate the activity of a nucleic acid or polypeptide of the invention. For example, in one aspect of the invention, the parameter monitored is transcriptional expression of an enzyme gene. One or more or all of the transcripts of a cell may be determined by hybridization to a sample containing the cell transcript, or a nucleic acid representative of the cell transcript or its complement, by hybridization to a nucleic acid immobilized on an array or "biochip". By using a nucleic acid "array" on a microchip, some or all of the cellular transcripts can be quantified simultaneously. Alternatively, arrays containing genomic nucleic acid may be used to determine the genotype of cell lines newly engineered by the methods of the invention. An "array" of polypeptides may also be used to simultaneously quantify multiple proteins. The invention may be practiced using any known "array", also known as a "microarray" or "nucleic acid array" or "polypeptide array" or "antibody array" or "biochip" or variant thereof. Typically, an array is a large number of "spots" or "target elements", each target element comprising a defined amount of one or more biomolecules, e.g., oligonucleotides, immobilized to a specific region of the substrate surface for specific binding of a sample molecule, e.g., an mRNA transcript.

In the operation of the methods of the invention, any known array and/or method of making and using an array or variant thereof may be incorporated herein, in whole or in part, and is described, for example, in U.S. Pat. No. 6277628、6277489、6261776、6258606、6054270、6048695、6045996、6022963、6013440、5965452、5959098、5856174、5830645、5770456、5632957、5556752、5143854、5807522、5800992、5744305、5700637、5556752、5434049;, see also, for example, WO 99/51773, WO 99/09217, WO97/46313, WO 96/17958, see also, for example, johnston (1998) "Gene chip: desired array for understanding Gene regulation (GENE CHIPS: array ofhope for understanding gene regulation)", curr.biol.8: R171-R174; schummer (1997) ", direct hybridization (Direct hybridization oflarge-insert genomic clones on high-density gridded cDNA filter arrays)",Biotechniques 23:120-124;Solinas-Toldo(1997)" for genomic large clone insertion in high density grid cDNA filtration assays, matrix-based comparative genomic hybridization (Matrix-Based Comparative Genomic Hybridization:Biochips to Screen for Genomic Imbalances)",Genes,Chromosomes&Cancer20:399-407;Bowtell(1999)" by microarray for selection (Options Available--From Start to Finish--for Obtaining Expression DatabyMicroarray)",Nature Genetics Supp.21:25-32. for obtaining expression data from initiation to termination, see also published U.S. patent application Ser. Nos. 20010018642, 20010019827, 20010016322, 20010014449, 20010014448, 20010012537, 20010008765.

Capillary array

Capillary arrays, such as GIGAMATRIX ^TM Diversa Corporation, san Diego, calif., can be used in the methods of the invention. Nucleic acids or polypeptides of the invention may be immobilized on or applied to an array, including capillary arrays. Arrays can be used to screen or monitor libraries of compositions (e.g., small molecules, antibodies, nucleic acids, etc.), for their ability to bind or modulate the activity of a nucleic acid or polypeptide of the invention. The capillary array provides another system for capturing (holding) and screening samples. For example, the sample screening device may include a plurality of capillaries forming an adjacent array of capillaries, wherein each capillary includes at least one wall defining a cavity for retaining a sample. The device may further comprise a matrix material disposed between adjacent capillaries of the array, and one or more reference marks formed in the matrix material. A capillary for screening a sample, wherein the capillary is adapted to be incorporated in a capillary array, may include a first wall defining a cavity for retaining the sample, and a second wall formed of a filter material that filters excitation energy provided to the cavity to excite the sample. A polypeptide or nucleic acid, e.g., a ligand, may be introduced into the first component into at least a portion of one capillary of the capillary array. Each capillary of the capillary array may comprise at least one wall defining a cavity for retaining the first component. The bubbles may be introduced in a capillary tube behind the first component. The second component may be introduced into the capillary tube, wherein the second component is separated from the first component by the air bubble. The target sample may be introduced as a first liquid labeled with a detectable particle into capillaries of a capillary array, wherein each capillary of the capillary array comprises at least one wall defining a cavity for retaining the first liquid and the detectable particle, wherein at least one wall is coated with a binding substance for binding the detectable particle to the at least one wall. The method may further comprise removing the first liquid from the capillary, wherein the bound detectable particles remain within the capillary, and introducing a second liquid into the capillary. The capillary array may comprise a plurality of individual capillaries having at least one outer wall defining a cavity. The capillary outer wall may be one or more fused walls. Likewise, the walls may define a cavity that is cylindrical, square, hexagonal, or any other geometric shape, so long as the walls form a cavity that retains a liquid or sample. The capillaries of the capillary array may be brought together in a close proximity to form a planar structure. Capillaries may be bonded together by fusion (e.g., where the capillaries are made of glass), adhesion, bonding, or sidewall-sidewall clamping. The capillary array may be comprised of any number of individual capillaries, for example, capillaries ranging from 100 to 4,000,000. The capillary array may be formed from about 100,000 or more individual capillaries bonded together to form a microtiter plate.

Engineered conditionally active antibodies

The conditionally active antibody may be engineered to produce a multispecific conditionally active antibody. The multispecific antibody may be an antibody having polyepitopic specificity, as described in WO 2013/170168. Multispecific antibodies include, but are not limited to, antibodies comprising a heavy chain variable domain (V _H) and a light chain variable domain (V _L), wherein the V _HV_L unit has a multi-epitope specificity, antibodies having two or more V _L and V _H domains, wherein each V _HV_L unit binds a different epitope, antibodies having two or more single variable domains, wherein each single variable domain binds a different epitope, antibodies comprising one or more antibody fragments, and antibodies comprising covalently or non-covalently linked antibody fragments.

To construct multispecific antibodies, including bispecific antibodies, antibody fragments having at least one free thiol group are obtained. Antibody fragments may be obtained from full length conditionally active antibodies. The conditionally active antibody may be enzymatically digested to produce an antibody fragment. 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 ', F (ab') ₂, fv, diabody (Db), tandem diabody (taDb), linear antibodies (see U.S. Pat. No. 5,641,870; zapata et al, protein Eng., vol. 8, pages 1057-1062, (1995)), single arm antibodies, single variable domain antibodies, minibodies (Olafsen et al (2004) Protein Eng. Design & sel., vol. 17, pages 315-323), single chain antibody molecules, fragments produced by Fab expression libraries, anti-idiotype (anti-Id) antibodies, complementarity Determining Regions (CDRs), and epitope binding fragments. Antibody fragments may also be produced using DNA recombination techniques. The DNA encoding the antibody fragment may be cloned into a plasmid expression vector or a phagemid vector and expressed directly in E.coli. Methods of abzyme digestion, DNA cloning and recombinant protein expression are well known to those skilled in the art.

Antibody fragments may be purified using conventional techniques and may be reduced to produce free sulfhydryl groups. Antibody fragments having free thiol groups can be reacted with a cross-linking agent such as bismaleimide. Such cross-linked antibody fragments are purified and then reacted with a second antibody fragment having free thiol groups. The end products of the cross-linking of the two antibody fragments were purified. In certain embodiments, each antibody fragment is a Fab, and the end product in which two fabs are linked by a bismaleimide is referred to herein as a bismaleimide- (thio-Fab) ₂ or a bis-Fab. Such multispecific antibodies and antibody analogs (including di-Fab) can be utilized to rapidly synthesize a large number of antibody fragment combinations or structural variants of natural antibodies or specific antibody/fragment combinations.

Multispecific antibodies may be synthesized with modified crosslinkers such that other functional moieties may be attached to the multispecific antibody. The modifying cross-linker allows the attachment of any thiol-reactive moiety. In one embodiment, N-succinimidyl-S-acetylthioacetate (SATA) is attached to bismaleimide to form bismaleimide-acetylthioacetate (BMata). After deprotection of the masked thiol groups, any functional group with a thiol-reactive moiety may be attached to the multispecific antibody.

Exemplary thiol-reactive reagents include multifunctional linker reagents, capture reagents (i.e., affinity labeling reagents such as biotin-linker reagents), detection labels (e.g., fluorophore reagents), solid phase immobilization reagents (e.g., SEPHAROSE ^TM, polystyrene, or glass), or drug-linker intermediates. One example of a thiol-reactive reagent is N-ethylmaleimide (NEM). Such multispecific antibodies or antibody analogs with modified cross-linking agents may be further reacted with pharmaceutical moiety reagents or other labels. Reaction of the multispecific antibody or antibody analog with the drug-linker intermediate provides a multispecific antibody-drug conjugate or antibody analog-drug conjugate, respectively.

Many other techniques for preparing multispecific antibodies may also be used in the present invention. References describing these techniques include (1) MILSTEIN AND Cuello, nature, volume 305, page 537 (1983)), WO 93/08829, and Traunecker et al, emboj, volume 10, page 3655 (1991), regarding recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities; (2) U.S. Pat. No. 5,731,168, directed to pestle engineering, (3) WO 2009/089004A1, directed to engineered electrostatic steering effects for the preparation of antibody Fc heterodimer molecules, (4) U.S. Pat. No. 4,676,980, and Brennan et al, science, volume 229, page 81 (1985), directed to cross-linking two or more antibodies or fragments, (5) Kostelny et al, J.Immunol, volume 148, pages 1547-1553 (1992), directed to the use of leucine zippers to produce bispecific antibodies, (6) Hollinger et al, proc. Natl. Acad. Sci. USA, volume 90, pages 6444-6448 (1993), directed to the preparation of bispecific antibody fragments using the "diabody" technique, (7) Gruber et al, J.mu.nol, page 152 (1994), directed to the use of single chain Fv (sFv) dimer (sFv) and (Bv) for the preparation of antibodies, and more specifically directed to the antibody (Bv) of the three domains (U.S. 5, J.1290, whereby the antibodies can be more specifically directed to the antibodies (U.S. 5, J.1291 and more specifically directed to the antibodies (U.S. Pat. No. 5).

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

In one embodiment, conditionally active antibodies for crossing the Blood Brain Barrier (BBB) are engineered to produce multispecific antibodies (e.g., bispecific antibodies). The multispecific antibody comprises a first antigen-binding site that binds a BBB-R and a second antigen-binding site that binds a brain antigen. At least the first antigen binding site of the BBB-R is conditionally active. Brain antigens are antigens expressed in the brain, which can be targeted by antibodies or small molecules. Examples of such antigens include, but are not limited to, beta-secretase 1 (BACE 1), amyloid beta (Abeta), epidermal Growth Factor Receptor (EGFR), human epidermal growth factor receptor 2 (HER 2), tau, apolipoprotein E4 (ApoE 4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK 2), parkinson's protein, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR 6), amyloid Precursor Protein (APP), p75 neurotrophic factor receptor (p 75 NTR), and caspase 6. In one embodiment, the antigen is BACE1.

The BBB has an endogenous transport system mediated by the BBB receptor (BBB-R), a specific receptor that allows macromolecules to transport across the BBB. For example, antibodies that bind to the BBB-R can be transported across the BBB using an endogenous transport system. Such antibodies can be used as vectors for transporting drugs or other agents across the BBB by using an endogenous BBB receptor-mediated transport system across the BBB. Such antibodies need not have a high affinity for the BBB-R. Antibodies that are not conditionally active antibodies but have low affinity for the BBB-R have been described as more efficient at crossing the BBB than higher affinity antibodies, as described in US 2012/0171120.

Another approach for engineering antibodies into the brain is to engineer the antibodies so that they are delivered to the brain through the lymphatic vessels of the central nervous system. Thus, antibodies can be engineered to bind or mimic immune cells, such as T cells or synovial fluid or cerebrospinal fluid transported to the central nervous system by lymphatic vessels. Details of 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.337-341,2015, 7, 16, and articles cited therein and publicly available prior to the filing date 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 in the brain. The conditionally active antibody may have a high affinity for the BBB-R on the blood side of the BBB, while having little or no affinity for the brain side of the BBB. A drug (e.g., a drug conjugate) can be coupled to the conditionally active antibody to cross the BBB with the antibody and be transported into the brain.

BBB-R is a transmembrane receptor protein expressed on brain endothelial cells that is capable of transporting molecules across the blood brain barrier. Examples of BBB-R include transferrin receptor (TfR), insulin receptor, insulin-like growth factor receptor (IGF-R), low density lipoprotein receptor (including but not limited to low density lipoprotein receptor-related protein 1 (LRP 1) and low density lipoprotein receptor-related protein 8 (LRP 8)), and heparin-binding epidermal growth factor-like growth factor (HB-EGF). Exemplary BBB-R herein is transferrin receptor (TfR). TfR is a transmembrane glycoprotein (molecular weight of about 180,000) consisting of two subunits linked by disulfide bonds (each apparent molecular weight of about 90,000) that are involved in iron uptake in vertebrates.

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

Plasma is a body fluid that is very different from the extracellular fluid of the brain (ECF). As described in Somjen ("Ions in the Brain: normal Function, seizures, and Stroke," Oxford University Press,2004,16, and 33 pages) and volume Redzic("Molecular biology of the blood-brain and the blood-cerebrospinal fluid barriers:similarities and differences,"Fluids and Barriers ofthe CNS,8:3, 2011), brain extracellular fluid has significantly less K ⁺, more Mg ²⁺, and H ⁺ than plasma. The difference in ion concentration between plasma and brain ECF results in a significant difference in osmolality and osmolality between the two fluids. Table 1 shows the millimolar concentrations of common ions in plasma and brain ECF.

TABLE 1 common ions in plasma (arterial plasma) and brain extracellular fluid (CSF)

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).

Thus, there are several physiological conditions that differ between the two sides of the BBB, such as pH, concentration of various substances (e.g. lactose, glucose, K ⁺、Mg²⁺), osmolality and osmolality. 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 concentration of K ⁺ than human plasma. For physiological conditions of Mg ²⁺ concentration, human brain ECF has significantly more Mg ²⁺ than human plasma. For physiological conditions of osmotic pressure, human brain ECF has an osmotic pressure different from human plasma. In some embodiments, the physiological condition of the brain ECF may be the composition, pH, osmolality, and osmolality of the brain ECF of the patient with the particular neurological disorder, which may be different from the physiological condition of the brain ECF of the general population.

Accordingly, the present invention provides a method for evolving DNA encoding a template antibody against BBB-R to produce 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 the BBB-R under at least one plasma physiological condition and have a lower affinity or no affinity for the BBB-R than the template antibody under at least one brain physiological condition in the brain ECF. Thus, the mutant antibodies selected have low or high affinity for the BBB-R on the plasma side and low or no affinity for the BBB-R on the brain ECF side. The selected mutant antibodies are useful as conditionally active antibodies for transport across the BBB.

Such conditionally active antibodies are beneficial across the BBB and remain in brain ECF. The low affinity for the brain-side BBB-R reduces the rate at which conditionally active antibodies are transported across the BBB to the outside of the brain and back into the blood (or removed) relative to the template antibody.

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

Such conditionally active antibodies are advantageous in crossing the BBB and retaining in brain ECF. After binding to the BBB-R on the plasma side, the conditionally active antibody is transported across the BBB, with little or no affinity for the BBB-R on the brain ECF side meaning that the conditionally active antibody is 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 (IC 50), which is a measure of how much antibody is needed to inhibit 50% of the binding of a known BBB-R ligand to the BBB-R. A common method is to perform a competitive binding assay, such as a competitive ELISA assay. An exemplary competitive ELISA assay to measure IC50 of TfR (a BBB-R) is one in which increasing concentrations of anti-TfR antibodies compete with biotinylated TfR ^A for binding to TfR. anti-TfR antibody competition ELISA can be performed overnight at 4 ℃ in Maxisorp plates (neptene, n.j.) coated with 2.5 μg/ml of purified murine TfR extracellular domain in PBS. Plates were washed with PBS/0.05% tween 20 and blocked with Superblock blocking buffer (Thermo Scientific, hudson, new hampshire) in PBS. Titration of each individual anti-TfR antibody (1:3 serial dilution) was combined with biotinylated anti-TfR ^A (0.5 nM final concentration) and added to the plate for 1 hour at room temperature. Plates were washed with PBS/0.05% Tween 20, HRP-streptavidin (SouthernBiotech, bermingham) 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-TfR ^A bound to the plates was detected using TMB substrate (BioFX Laboratories, omega).

High IC50 means that more conditionally active antibody is required to inhibit binding of a known ligand of the BBB-R, and therefore the affinity of the antibody for that BBB-R is relatively low. Conversely, a low IC50 indicates that fewer conditionally active antibodies are required to inhibit binding of a known ligand, and thus the affinity of the antibody for the BBB-R is relatively high.

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

Conditionally active biological proteins for synovial fluid

Joint diseases are a major cause of disability and early retirement in industrialized countries. Joint diseases often result in injury at joints that are difficult to repair. Synovial fluid is a body fluid found in the synovial cavity of a joint (e.g., knee, hip, shoulder) of a human or animal body between cartilage and the synovium facing the joint forming surface. Synovial fluid provides nutrition to cartilage and also serves as a lubricant for joints. The cells of the cartilage and synovium secrete fluids that act as lubricants between the joint-forming surfaces. Human synovial fluid contains about 85% water. It is derived from the dialysate of plasma, itself composed of water, dissolved proteins, glucose, clotting 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 joint areas. Some other proteins are also found in human synovial fluid, including glycoproteins such as alpha-1-Acid Glycoprotein (AGP), alpha-1-antitrypsin (A1 AT) and lubricin.

Synovial fluid has a very different composition than other parts of the body. Thus, synovial fluid has different physiological conditions than other parts of the body (e.g. plasma). For example, synovial fluid has a glucose of less than about 10mg/dL, whereas the average normal glucose level in human plasma is about 100mg/dL, fluctuating in the range between 70-100mg/dL in a day. In addition, the total protein level in synovial fluid is about one third of the plasma protein level, because macromolecules such as proteins do not readily pass through the synovial membrane into the synovial fluid. Human synovial fluid has also been found to have a pH higher than that in human plasma (Jebens et al, "On the viscosity andpH ofsynovial fluid and the pH ofblood," The Journal ofBone and Joint Surgery, volume 41B, pages 388-400, 1959; farr et al, volume ,"Significance of the hydrogen ion concentration in synovial fluid in Rheumatoid Arthritis,"Clinical and Experimental Rheumatology,, pages 3, 99-104, 1985).

Thus, synovial fluid has several physiological conditions that differ from those of other parts of the body (e.g. those in plasma). Synovial fluid has a higher pH than other parts of the body, in particular plasma. Synovial fluid has a lower glucose concentration than other parts of the body (e.g. plasma). Synovial fluid also has a lower protein concentration than other parts of the body (e.g. plasma).

Several antibodies have been used to treat joint diseases by introducing the antibodies into synovial fluid. For example, synovial fluid in injured joints is known to contain many factors that have an effect on the progression of osteoarthritis (see, e.g., fernandes, et al, "The Role of Cytokines in Osteoarthritis Pathophysiology", biorheology, volume 39, pages 237-246, 2002). Cytokines produced by activated synovial cells, such as interleukin-1 (IL-I) and tumor necrosis factor-alpha (TNF-alpha), are known to up-regulate Matrix Metalloproteinase (MMP) gene expression. Upregulation of MMPs leads to degradation of matrix and non-matrix proteins in the joint. Antibodies that neutralize cytokines may prevent the progression of osteoarthritis.

The use of antibodies as drugs is a promising strategy for the treatment of joint diseases. For example, antibodies (e.g., anti-aggrecan or aggrecanase antibodies) have been developed to treat osteoarthritis, which has so far been the most prevalent in joint diseases (WO 1993/022429 A1). Antibodies to acetylated high mobility group box 1 (HMGB 1) have been developed for the diagnosis or treatment of joint diseases, which are inflammatory, autoimmune, neurodegenerative or malignant diseases/disorders, such as arthritis. The antibodies can be used to detect acetylated forms of HMGB1 in synovial fluid (WO 2011/157905 A1). Another antibody (CD 20 antibody) has also been developed for the treatment of connective tissue and cartilage injuries of joints.

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

Such conditionally active biologic protein may be a conditionally active antibody. In some embodiments, the invention also provides conditionally active biologic proteins that are proteins other than antibodies. For example, conditionally active immunomodulators may be developed by the present invention for preferential modulation of immune responses in synovial fluid, which may have little or no effect on the immune response of other parts of the body.

The conditionally active biologic protein may be a conditionally active cytokine signaling inhibitor (SOCS). Many of these SOCSs are involved in inhibiting JAK-STAT signaling pathways. Conditionally active cytokine signaling inhibitors may preferentially inhibit cytokine signaling in synovial fluid, but not or to a lesser extent, other parts of the body.

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

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

Conditionally active biological proteins for tumors

Cancer cells in solid tumors are able to form a tumor microenvironment around them to support growth and metastasis of the cancer cells. Tumor microenvironments are the cellular environment in which the tumor is present, including peripheral blood vessels, immune cells, fibroblasts, other cells, soluble factors, signaling molecules, extracellular matrix, and mechanical factors that can promote tumor transformation, support tumor growth and invasion, protect the tumor from host immunity, culture treatment resistance, and provide microenvironment for dormant metastasis thriving (MECHANICAL CUE). The tumor and its surrounding microenvironment are closely related and constantly interacting. Tumors can affect their microenvironment by releasing extracellular signals, promoting tumor angiogenesis, and inducing peripheral immune tolerance, while immune cells in the microenvironment can affect the growth and evolution of cancer cells. See warts et al, "Tumor Microenvironment Complexity: emerging Roles IN CANCER THERAPY," CANCERRES, volume 72, pages 2473-2480, 2012.

The tumor microenvironment is typically hypoxic. As the tumor mass increases, tumor ingrowth moves away from the existing blood supply, which makes it difficult to completely supply oxygen to the tumor microenvironment. In more than 50% of locally advanced solid tumors, the partial pressure of oxygen in the tumor environment is below 5mmHg compared to the partial pressure of oxygen in plasma of about 40 mmHg. In contrast, other parts of the body are not hypoxic. The hypoxic environment causes genetic instability, which is associated with cancer progression by downregulating the pathways of nucleotide excision repair and mismatch repair. Hypoxia also causes upregulation of hypoxia-inducible factor 1 alpha (HIF 1-alpha), which induces angiogenesis and is associated with a poor prognosis and activation of genes associated with metastasis. See Weber et al, "The tumor microenvironment," Surgical Oncology, volume 21, pages 172-177, 2012and Blagosklonny, "Antiangiogenic THERAPY AND tumorprogression," CANCERCELL, volume 5, pages 13-17, 2004.

Furthermore, tumor cells often rely on energy produced by lactic acid fermentation that does not require oxygen. Thus, tumor cells are unlikely to use normal aerobic respiration that requires oxygen. The result of fermentation using lactic acid is that the tumor microenvironment is acidic (pH 6.5-6.9), and other parts of the body are typically neutral or slightly alkaline compared to other parts of the body. For example, the pH of human plasma is about 7.4. See Estrella et al, "ACIDITY GENERATED by the Tumor Microenvironment Drives Local Invasion," cancer research, vol.73, pages 1524-1535, 2013. Because of the relatively high nutritional requirements of proliferative cancer cells compared to cells located elsewhere in the body, the availability of nutrients in the tumor microenvironment is also low.

In addition, tumor microenvironments also contain many different cell types that are not normally present in other parts of the body. These cell types include endothelial cells and precursors thereof, pericytes, smooth muscle cells, fibroblasts, cancer-related fibroblasts, myofibroblasts, neutrophils, eosinophils, basophils, mast cells, T lymphocytes and B lymphocytes, natural killer cells and Antigen Presenting Cells (APCs) (e.g., macrophages and dendritic cells) (Lorusso et al ,"The tumor microenvironment and its contribution to tumor evolution toward metastasis,"Histochem Cell Biol,, volume 130, pages 1091-1103, 2008).

Thus, the tumor microenvironment has at least several physiological conditions that are different from the physiological conditions of other parts of the body (e.g. physiological conditions in plasma). The pH (acidity) of the tumor microenvironment is lower than other parts of the body, in particular plasma (pH 7.4). The tumor microenvironment has a lower oxygen concentration than other parts of the body (e.g. plasma). In addition, the tumor microenvironment has a lower nutritional availability than other parts of the body (in particular plasma). Tumor microenvironments also have several different cell types that are not common in other parts of the body (especially plasma).

Some cancer drugs include antibodies that can penetrate into the tumor microenvironment and act on cancer cells therein. Antibody-based cancer therapies have matured and have become one of the most successful and important strategies for treating patients with hematological malignancies and solid tumors. There is a range of cell surface antigens expressed by human cancer cells in which the cell surface antigens are over-expressed, mutated or selectively expressed as compared to in normal tissue. These cell surface antigens are excellent targets for antibody cancer treatment.

Cancer cell surface antigens that can be targeted by antibodies fall into several different categories. Hematopoietic differentiation antigens are glycoproteins typically associated with Cluster of Differentiation (CD) groupings and include CD20, CD30, CD33, and CD52. Cell surface differentiation antigens are distinct groups of glycoproteins and carbohydrates found on the surface of normal and tumor cells. Antigens involved in growth and differentiation signaling are typically growth factors and growth factor receptors. Growth factors that are antibody targets in cancer patients include CEA2, epidermal growth factor receptor (EGFR; also known as ERBB 1) 12, ERBB2 (also known as HER 2) 13, ERBB3 (ref.18), MET (also known as HGFR) 19, insulin-like growth factor 1 receptor (IGF 1R) 20, ephrin receptor A3 (EPHA 3) 21, tumor Necrosis Factor (TNF) -related apoptosis-inducing ligand receptor 1 (TRAILR 1; also known as TNFRSF 10A), TRAILR2 (also known as TNFRSF 10B), and receptor activator of nuclear factor κb ligand (RANKL; also known as TNFSF 11) 22. Antigens involved in angiogenesis are typically proteins or growth factors that support the formation of the new microvasculature, including Vascular Endothelial Growth Factor (VEGF), VEGF receptor (VEGFR), integrin αvβ3 and integrin α5β1 (REF.10). Tumor stroma and extracellular matrix are support structures indispensable to tumors. Matrix antigens and extracellular matrix antigens that are therapeutic targets include Fibroblast Activation Protein (FAP) and tenascin. See Scott et al, "Antibody therapy of cancer," Nature REVIEWS CANCER, volume 12, pages 278-287, 2012.

In addition to antibodies, other biological proteins are also expected to be useful in cancer therapy. Examples include tumor inhibitors such as retinoblastoma protein (pRb), p53, pVHL, APC, CD, ST5, YPEL3, ST7, and ST14. Some proteins that induce apoptosis in cancer cells can also be introduced into tumors to reduce their size. There are at least two mechanisms by which tumor apoptosis can be induced, tumor necrosis factor-induced and Fas-Fas ligand-mediated mechanisms. At least some proteins involved in either of the two apoptotic mechanisms may be introduced into the tumor for treatment.

Cancer stem cells are cancer cells capable of producing all cell types found in a particular cancer sample, and are therefore neoplastic cells. They can produce tumors by a stem cell process that self-renews and differentiates into multiple cell types. Cancer stem cells are thought to persist in tumors as a distinct population and cause recurrence and metastasis by producing new tumors. Developing specific therapies that target cancer stem cells can improve survival and improve quality of life for cancer patients, particularly for patients with metastatic disease.

These drugs used to treat tumors often interfere with normal physiological functions of other parts of the body besides the tumor. For example, proteins that induce apoptosis in tumors can also induce apoptosis in certain other parts of the body, thereby causing side effects. In embodiments in which antibodies are used to treat tumors, the antigen of the antibody may also be expressed in other parts of the body where it performs normal physiological functions. For example, the monoclonal antibody bevacizumab (targeting vascular endothelial growth factor) prevents tumor vascular growth. The antibodies may also prevent vascular growth or repair in other parts of the body, resulting in bleeding, poor wound healing, blood clots, and kidney damage. More effective tumor treatment is highly desirable to develop conditionally active biologic proteins that target tumors either primarily or alone.

In some embodiments, the invention provides conditionally active biologic proteins produced by a parent or wild-type biologic protein, which may be candidates for tumor treatment. Conditionally active biologic proteins have lower activity than the parent or wild-type biologic proteins under at least one physiological condition in a body part other than the tumor microenvironment (e.g., plasma), and have higher activity than the parent or wild-type biologic proteins under at least one physiological condition in the tumor microenvironment. Such conditionally active biologic proteins can preferentially act on cancer cells in the tumor microenvironment to treat the tumor, thus being less likely to cause side effects. In embodiments where the biological protein is an antibody to an antigen on the surface of a tumor cell and the antigen is exposed to a tumor microenvironment in the tumor cell, the conditionally active antibody has a lower affinity for the antigen than the parent or wild-type antibody in other parts of the body (e.g., the 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 the antigen in other parts of the body, but bind better or strongly tightly to the antigen in the tumor microenvironment.

In some embodiments, the conditionally active antibody is an antibody to an immune checkpoint protein, resulting in inhibition of an immune checkpoint. Such conditionally active antibodies have at least one of (1) an increased binding affinity for an immune checkpoint protein in a tumor microenvironment as compared to the parent or wild-type antibody from which the conditionally active antibody is derived, and (2) a decreased binding affinity for an immune checkpoint protein in a non-tumor microenvironment as compared to the parent or wild-type antibody from which the conditionally active antibody is derived.

Immune checkpoints serve as an endogenous inhibitory pathway of the immune system to maintain self-tolerance and regulate the duration and extent of immune responses to antigen stimuli (i.e., exogenous molecules, cells, and tissues). See Pardoll, nature REVIEWS CANCER, volume 12, pages 252-264, 2012. Inhibition of an immune checkpoint by inhibition of one or more checkpoint proteins may cause hyperactivation of the immune system (particularly T cells), thereby inducing the immune system to attack the tumor. Checkpoint proteins suitable for use in the present invention include CTLA4 and its ligands CD80 and CD86, PD1 and its ligands PDL1 and PDL2, T cell immunoglobulin and mucin-3 (TIM 3) and its ligands GAL9, B and T Lymphocyte Attenuator (BTLA) and its ligand HVEM (herpes virus entry medium), receptors such as killer cell immunoglobulin-like receptor (KIR), lymphocyte activating gene-3 (LAG 3) and adenosine A2a receptor (A2 aR), and ligands B7-H3 and B7-H4. Other suitable immune checkpoint proteins are described in Pardoll, nature REVIEWS CANCER, volume 12, pages 252-264, 2012 and Nirschl & Drake, CLIN CANCERRES, volume 19, pages 4917-4924, 2013.

CTLA-4 and PD1 are the two best known immune checkpoint proteins. CTLA-4 can down-regulate the pathway of T cell activation (Fong et al, cancer Res.69 (2): 609-615,2009; and Weber, cancer immunol. 58:823-830,2009). Blocking CTLA-4 has been shown to enhance T cell activation and proliferation. Inhibitors of CTLA-4 include anti-CTLA-4 antibodies. anti-CTLA-4 antibodies bind to CTLA-4 and block the interaction of CTLA-4 with its ligand CD80 or CD86, thereby blocking down-regulation of the immune response elicited by the interaction of CTLA-4 with its ligand.

Checkpoint protein PD1 is known to inhibit T cell activity in peripheral tissues and limit autoimmunity when producing an inflammatory response to infection. In vitro PD1 blockade can enhance T cell proliferation and cytokine production in response to stimulation of specific antigen targets or allogeneic cells in mixed lymphocyte responses. A strong correlation between PD1 expression and reduced immune response has been shown to be caused by the inhibitory function of PD1, i.e., by induction of immune checkpoints (Pardoll, nature REVIEWS CANCER,12:252-264,2012). PD1 blocking can be achieved by a variety of mechanisms, including antibodies that bind to PD1 or its ligand PDL1 or PDL 2.

Past studies have found antibodies against several checkpoint proteins (CTLA 4, PD1, PD-L1). These antibodies are useful for treating tumors (hyperactivating immune system) by inhibiting immune checkpoints, thereby hyperactivating the immune system (particularly T cells) for attacking tumors (Pardoll, nature REVIEWS CANCER, volume 12, pages 252-264, 2012). However, super-activated T cells may also attack host cells and/or tissues, resulting in collateral damage to the patient's body. Thus, therapies based on the use of these known antibodies to inhibit immune checkpoints are difficult to control and risk to patients is also a serious problem. For example, FDA approved antibodies against CTLA-4 have black box warnings due to their high toxicity.

The present invention addresses the problem of collateral damage to super-activated T cells by providing conditionally active antibodies to immune checkpoint proteins. These conditionally active antibodies preferentially activate immune checkpoints in the tumor microenvironment. At the same time, the conditionally active antibody does not inhibit or less inhibits immune checkpoints in a non-tumor microenvironment (e.g., normal body tissue), such that the likelihood of collateral damage to the body is reduced in a non-tumor microenvironment. This objective is achieved by engineering conditionally active antibodies to be more active in the tumor microenvironment than in the non-tumor microenvironment.

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

Highly immunogenic tumors (e.g., malignant melanoma) are most vulnerable to the hyperactivation of the immune system obtained by manipulation of the immune system. Thus, conditionally active antibodies against immune checkpoint proteins are particularly effective for treating such hyperimmune tumors. However, other types of tumors are also vulnerable to hyperactivation of the immune system.

In some embodiments, conditionally active antibodies to immune checkpoint proteins may be used in combination therapies. For example, combination therapies may include conditionally active antibodies against tumor cell surface molecules (tumor specific antigens) and conditionally active antibodies against immune checkpoint proteins. In one embodiment, the binding activity of the conditionally active antibody to the tumor cell surface molecule and the binding activity of the conditionally active antibody to the immune checkpoint protein may be present in a single protein, i.e. the bispecific conditionally active antibody disclosed herein. In some further embodiments, the 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., the multispecific antibodies disclosed herein.

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

In some embodiments, the conditionally active antibody to the checkpoint protein may be in a prodrug form. For example, a conditionally active antibody may be a prodrug without the desired pharmaceutical activity before being cleaved and brought into a pharmaceutical form. Prodrugs may be preferentially cleaved in the tumor microenvironment either because the enzyme that catalyzes such cleavage is preferentially present in the tumor microenvironment or because the conditionally active antibody makes the cleavage site more accessible in the tumor microenvironment than in the non-tumor microenvironment.

Engineered conditionally active biologic proteins for stem cell niches including tumor stem cells

Stem cells are present in an environment called a stem cell niche in the body, which constitutes a fundamental unit of tissue physiology, integrating signals that mediate the response of stem cells to the needs of an organism. However, the niche can also induce pathology by imposing abnormal functions on stem cells or other targets. The interaction between stem cells and their niches creates a dynamic system required to maintain tissue and The final design of stem cell therapies (Scadden, "The stem-CELL NICHE AS AN ENTITY of action," Nature, volume 441, pages 1075-1079, 2006). Common stem cell niches in vertebrates include germ line stem cell niches, hematopoietic stem cell niches, hair follicle stem cell niches, intestinal stem cell niches, and cardiovascular stem cell niches.

Stem Cell niches are specialized environments (Drummond-Barbosa, "STEM CELLS, THEIR NICHES AND THE SYSTEMIC environmental: an working Network," Genetics, volume 180, pages 1787-1797, 2008; fuchs, "Socializing with the Neighbors: STEM CELLS AND THEIR NICHE," Cell, volume 116, pages 769-77, 8,2004) that are different from other parts of the body (e.g., plasma). The stem cell niche is anoxic in that oxidative DNA damage is reduced. Direct measurements of Oxygen levels have shown that bone marrow is generally quite hypoxic (.about.1% -2%O ₂) compared to plasma (Keith et al, "Hypoxia-Inducible Factors, STEM CELLS, AND CANCER," Cell, volume 129, pages 465-472, 2007; mohyeldin et al, "Oxygen IN STEM CELL Biology: A CRITICAL Component of THE STEM CELL NICHE," CELL STEM CELL, volume 7, pages 150-161, 2010). In addition, the stem cell niche needs to have several other factors to regulate the stem cell characteristics within the niche, factors of the extracellular matrix components, growth factors, cytokines and physicochemical properties of the environment, including pH, ionic strength (e.g., ca ²⁺ concentration) and metabolites.

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

Stem cell therapy is an interventional strategy to introduce new adult stem cells into damaged tissue in order to treat disease or injury. This strategy depends on the ability of stem cells to self-renew and subsequently produce offspring with varying degrees of differentiation. Stem cell therapy offers great potential for tissue regeneration, where regenerated tissue can potentially replace diseased and damaged areas in the body with minimal risk of rejection and side effects. Thus, the delivery of drugs (biological proteins (e.g., antibodies) or chemical compounds) to the stem cell niche to affect stem cell turnover and differentiation is an important part of stem cell therapies.

There are several examples of how the stem cell niche affects the renewal and/or differentiation of stem cells in mammals. The first is in the skin, where β -1 integrin is known to be differentially expressed on primary cells and to be involved in the constrained localization of stem cell populations by interaction with matrix glycoprotein ligands. Second, in the nervous system, the lack of tenascin-C alters the number and function of neural stem cells in the subventricular zone. tenascin-C appears to regulate the sensitivity of stem cells to fibroblast growth factor 2 (FGF 2) and bone morphogenic protein 4 (BMP 4), resulting in an enhanced propensity of stem cells. Third, another matrix protein, arg-Gly-Asp-containing sialic acid protein (osteopontin (OPN)) has now been shown to contribute to hematopoietic stem cell regulation. OPN interacts with several receptors known on hematopoietic stem cells, CD44 and α4 and α5β1 integrins. The change in OPN production is significant, especially in the case of osteoblast activation. Because the lack of OPN results in the expansion of hyper-physiological stem cells under stimulated conditions, animals lacking OPN have an increased HS cell number. Therefore, OPN appears to be a constraint on the number of hematopoietic stem cells, limiting the number of stem cells under steady state conditions or conditions with stimulation. See Scadden, "The step-CELL NICHE AS AN ENTITY of action," Nature, volume 441, pages 1075-1079, 2006.

Xie et al "Autocrine signaling based selection of combinatorial antibodies that transdifferentiate human stem cells,"Proc Natl Acad Sci U S A,, volume 110, pages 8099-8104, 2013 disclose methods of using antibodies to affect stem cell differentiation. The antibodies are agonists of granulocyte colony stimulating factor receptor. Unlike the natural granulocyte colony-stimulating factor, which activates the differentiation of cells along a predetermined pathway, the isolated agonist antibody transdifferentiates human myeloid cd34+ bone marrow cells into neural progenitor cells. Melidoni et al ("Selecting antagonistic antibodies that control differentiation through inducible expression in embryonic stem cells,"Proc Natl Acad Sci U S A,, volume 110, pages 17802-17807, 2013) also disclose the use of antibodies to interfere with the interaction between FGF4 and its receptor FGFR1 beta, thus blocking the autopcritic FGF 4-mediated differentiation of embryonic stem cells.

Knowledge of ligand/receptor function in stem cell differentiation enables strategies to be implemented that employ biological proteins to interfere with these ligands/receptors to modulate or even direct stem cell differentiation. The ability to control the differentiation of non-genetically modified human stem cells by administering antibodies into the stem cell niche can provide a new ex vivo or in vivo approach to stem cell-based therapeutics. In some embodiments, the invention provides conditionally active biologic proteins produced by a parent or wild-type biologic protein, which are capable of entering a stem cell niche comprising cancer stem cells to regulate stem cell or tumor progression. Conditionally active biologic proteins have lower activity than the parent or wild-type biologic proteins under at least one physiological condition of the rest of the body, and have higher activity than the parent or wild-type biologic proteins under at least one physiological condition of the stem cell niche (e.g., cancer stem cell environment). Such conditionally active biologic proteins are unlikely to cause side effects and act preferentially on the stem cell niche to regulate stem cell turnover and differentiation. In some embodiments, the conditionally active biologic protein is an antibody. Such conditionally active antibodies may bind weakly or not at all to antigens in other parts of the body, but bind strongly and tightly to antigens in the stem cell niche.

The conditionally active proteins for use in synovial fluid, tumor microenvironment and stem cell niches of the present invention are generated by a method of evolving DNA encoding a parent or wild type biological protein to generate a library of mutated DNA. The mutant DNA library is then expressed to obtain the mutant protein. The muteins are screened for conditionally active biologic proteins having a higher activity than the parent or wild-type biologic protein under at least one physiological condition selected from the group consisting of synovial fluid, tumor microenvironment and stem cell niche in a first portion of the body and a lower activity than the parent or wild-type biologic protein under at least one physiological condition in a second portion of the body different from the first portion of the body. The second body part may be plasma. Such selected mutant biological proteins are conditionally active biological proteins, which have a high activity in a first part of the body and a low activity in a second part of the body.

Such conditionally active biologic proteins are advantageous in reducing side effects of the parent or wild-type protein, as the conditionally active biologic protein has lower activity in other parts of the body where it is not intended to act. For example, if a conditionally active biologic protein is purposefully introduced into a tumor microenvironment, the fact that the conditionally active biologic protein has low activity in body parts other than the tumor microenvironment means that such a conditionally active biologic protein is unlikely to interfere with normal physiological functions of body parts other than the tumor microenvironment. Meanwhile, the conditionally active biologic protein has high activity in the tumor microenvironment, so that the conditionally active biologic protein has higher curative effect in the aspect of treating tumors.

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

Conditionally active biologic proteins for functioning in one of the synovial fluid, tumor microenvironment or stem cell niches also enable the use of new drug targets. The use of conventional biological proteins as therapeutic agents may lead to unacceptable side effects. For example, inhibition of the Epidermal Growth Factor Receptor (EGFR) can be very effective in inhibiting tumor growth. However, drugs that inhibit EGFR also inhibit the growth of the skin and Gastrointestinal (GI) tract. Side effects make EGFR unsuitable as a tumor drug target. The use of conditionally active antibodies that bind EGFR with high affinity only in the tumor microenvironment, but do not have or have very low affinity in any other part of the body, would significantly reduce side effects while inhibiting tumor growth. In this case, EGFR can be an effective new tumor drug target by using conditionally active antibodies.

In another example, inhibiting a cytokine is generally beneficial in repairing joint damage. However, inhibition of cytokines in other parts of the body may also inhibit the immune response of the body, causing immunodeficiency. Thus, cytokines in synovial fluid are not ideal targets for the development of traditional antibody drugs for the treatment of joint injury. However, by using conditionally active antibodies that preferentially bind to cytokines in synovial fluid while not binding or only weakly binding to the same cytokines in other parts of the body, the side effects of immunodeficiency may be significantly reduced. Thus, cytokines in synovial fluid can be suitable targets for repairing joint damage through the use of conditionally active antibodies.

Conditionally active biologic protein for inflamed organs/tissues

In some embodiments, the conditionally active biologic protein is designed to preferentially act in an organ or tissue that is prone to inflammation, such as lymph nodes, tonsils, adenoids, and sinuses. Other organs and tissues prone to inflammation can be found in anatomical textbooks, for example Gray's Anatomy, 41 st edition, published 2015 by Elsevier, HENRY GREY.

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

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

Conditionally active viral particles

Viral particles have long been used as delivery vehicles for transporting proteins, nucleic acid molecules, chemical compounds or radioisotopes to target cells or tissues. Viral particles commonly used as delivery vectors include retrovirus, adenovirus, lentivirus, herpesvirus, and adeno-associated virus. Typically in ligand-receptor binding systems, viral particles recognize their target cells via surface proteins that act as recognition proteins for specific binding to cellular proteins (Lentz,"The recognition event between virus and host cell receptor:a target for antiviral agents,"J.of Gen.Virol.,, volume 71, pages 751-765, 1990, which act as target proteins for the target cells. For example, the viral recognition protein may be a ligand for a receptor on the target cell. The specificity between the ligand and the receptor allows the viral particle to specifically recognize and deliver its contents to the target cell.

Techniques for developing artificial viral particles from wild-type viruses are well known to those skilled in the art. As delivery vectors, artificial viral particles are known to include retroviral particles based on viruses such as retrovirus (see, e.g., WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; U.S. Pat. No. 5,219,740; WO 93/11230; WO 93/10218; U.S. Pat. No.4,777,127; UK patent No. 2,200,651;EP 0 345 242; and WO 91/02805), alphaviruses (e.g., sindbis (Sindbis) viral vectors, semliki) forest viruses (ATCC VR-67; ATCC VR-1247), ross river viruses (ATCC VR-373; ATCC VR-1246), venezuelan equine encephalitis viruses (ATCC VR-923; ATCC VR-1250;ATCC VR 1249;ATCC VR-532)) and adeno-associated viruses (see, e.g., WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; 95/11984 and WO 95/00655).

Generally, artificial viral particles are constructed by inserting an exogenous recognition protein into the viral particle, which exogenous recognition protein replaces the natural recognition protein, typically by recombinant techniques. The exogenous recognition protein may be, for example, an antibody, receptor, ligand, or collagen binding domain. The present invention provides conditionally active recognition proteins that have no or low activity to bind to cells under normal physiological conditions and have high activity to bind to cells under abnormal conditions. The conditionally active recognition protein may thus preferentially bind to target cells at the diseased tissue and/or at the diseased site based on the presence of an abnormal condition at the diseased tissue and/or at the diseased site, and avoid or only minimally bind to cells of normal tissue in which normal physiological conditions are present. The conditionally active recognition protein may be expressed and displayed on the surface of the viral particle.

In some embodiments, the invention provides a method of evolving a parent or wild-type recognition protein and screening for a conditionally active recognition protein. Conditional activity recognition proteins have lower cell-binding activity than the parent or wild-type recognition protein under normal physiological conditions and higher cell-binding activity than the parent or wild-type recognition protein under abnormal conditions. Such conditionally active recognition proteins may be inserted into viral particles by well known recombinant techniques to produce conditionally active viral particles.

In another embodiment, the invention provides a conditionally active viral particle comprising a conditionally active recognition protein, the conditionally active recognition protein being such that the conditionally active viral particle recognizes and binds to a target cell of a diseased tissue or located at a diseased site, and does not recognize and bind to a normal tissue cell. Such conditionally active viral particles may preferentially deliver the therapeutic agent within the viral particle to diseased tissue or to the diseased site, while conditionally active viral particles deliver less or no therapeutic agent to cells of normal tissue.

In some embodiments, the target cells of the diseased site are within a region or microenvironment having an abnormal pH (e.g., pH 6.5) or abnormal temperature, as compared to the pH or temperature in other parts of the body that are healthy or not suffering from the particular disease or disease state. In this embodiment, the conditionally active recognition protein is less active 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 is more active 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 at sites that experience abnormal pH or temperature, thereby delivering the therapeutic agent to the affected site.

In one embodiment, the viral particles may comprise conditionally active antibodies of the invention, in particular the variable regions of antibodies (e.g., fab', fv). Such conditionally active antibodies may bind to target proteins (as antigens) of target cells with lower affinity than the parent or wild-type antibodies under normal physiological conditions that may be encountered at locations with normal tissue and with higher affinity than the parent or wild-type antibodies under abnormal physiological conditions that may be encountered at diseased sites or in diseased tissue. Conditionally active antibodies may be derived from parent or wild-type antibodies according to the methods of the invention.

In one embodiment, the target protein on the target cell comprises a tyrosine kinase growth factor receptor that is overexpressed on the cell surface, for example, in many tumors. Exemplary tyrosine kinase growth factors are VEGF receptors, FGF receptors, PDGF receptors, IGF receptors, EGF receptors, TGF-alpha receptors, TGF-beta receptors, HB-EGF receptors, erbB2 receptors, erbB3 receptors, and ErbB4 receptors.

Conditionally active DNA/RNA modified proteins

DNA/RNA modified proteins have been found as a form of new genome engineering tools, particularly one known as CRISPR, which can allow researchers to microsurgically manipulate genes, altering DNA sequences precisely and easily at precise locations on the chromosome (genome editing, mali et al, "Cas9 AS A VERSATILE tool for engineering biology," Nature Methods, volume 10, pages 957-963, 2013). For example, sickle cell anemia is caused by single base mutations, which can be corrected by using DNA/RNA modified proteins. This technique can even precisely delete or edit the position of a chromosome by changing a single base pair (Makarova et al, "Evolution and classification of THE CRISPR-CAS SYSTEMS," Nature Reviews Microbiology, volume 9, pages 467-477, 2011).

Genome editing with CRISPR enables rapid simultaneous polygenic changes to cells. Many human diseases, including heart disease, diabetes, and neurological diseases, are affected by multiple genetic mutations. This CRISPR-based technique has the potential to reverse mutations that cause diseases and treat or at least reduce the severity of these diseases. Genome editing relies on CRISPR-associated (Cas) proteins (enzyme families) for cleavage of genomic DNA. Typically, cas proteins are directed to a target region in the genome by a small guide RNA, where the guide RNA matches the target region. Because Cas proteins have little or no sequence specificity, guide RNAs serve as pointers to Cas proteins to achieve precise genome editing. In one embodiment, one Cas protein may be used with multiple guide RNAs to correct multiple gene mutations simultaneously.

There are many Cas proteins. Examples include Cas1、Cas2、Cas3'、Cas3"、Cas4、Cas5、Cas6、Cas6e、Cas6f、Cas7、Cas8a1、Cas8a2、Cas8b、Cas8c、Cas9、Cas10、Cas10d、Csy1、Csy2、Csy3、Cse1、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, volume 9, pages 467-477, 2011).

In order to perform genome editing, cas protein must enter the target cell. Cells in an individual may have different intracellular pH inside the cell. Some cells in diseased tissue have abnormal intracellular pH. For example, some tumor cells tend to have an alkaline intracellular pH of about 7.12-7.65, while cells in normal tissue have a neutral intracellular pH in the range of 6.99-7.20 (see Cardone et al, "The role ofdisturbed 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 about 7.2-7.5, 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 ofPhysiology-Lung Cellular andMolecular Physiology,, vol 289, pp.L 867-L874, 2005). Furthermore, in ischemic cells, the intracellular pH is generally in the range of 6.55-6.65, which is lower than that of normal tissue, volume pH(Haqberg,"IntracellularpH during ischemia in skeletal muscle:relationship to membrane potential,extracellular pH,tissue lactic acid and ATP,"Pflugers Arch.,, pages 342-347, 1985). Further examples of abnormal intracellular pH in diseased tissue are discussed in Han et al, "Fluorescent Indicators forIntracellularpH," chemrev., volume 110, pages 2709-2728, 2010.

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

In some embodiments, conditionally active Cas proteins may be delivered to target cells using the conditionally active viral particles of the present invention. The conditionally active viral particle comprises a conditionally active Cas protein and at least one guide RNA for guiding the Cas protein to the location of the genomic DNA to be edited by the Cas protein.

The multispecific antibodies have high selectivity to preferentially target tissue containing all or most of the targets (antigens) to which the multispecific antibodies can bind. For example, a bispecific antibody may provide selectivity for target cells by exhibiting greater preference for target cells expressing two antigens recognized by the bispecific antibody than non-target cells that may express only one of the two antigens. Thus, due to the kinetics of the system, at equilibrium, more bispecific antibodies bind to target cells than to non-target cells.

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

Engineered cytotoxic cells

Once a conditionally active astm is identified by the screening step, the chimeric antigen receptor can be assembled by ligating the polynucleotide sequences encoding the respective domains to form a single polynucleotide sequence (CAR gene encoding a conditionally active CAR). Individual domains include conditionally active ASTR, TM, and ISD. In some embodiments, other domains may also be incorporated into the CAR, including ab ESD and CSD (fig. 1). If the conditionally active CAR is a bispecific CAR, the CAR gene may be in the N-terminal to C-terminal direction, e.g., of the configuration N-terminal signal sequence-ASTR 1-linker-ASTR 2-extracellular spacer domain-transmembrane domain-co-stimulatory domain-intracellular signaling domain. In one embodiment, such CAR genes can comprise two or more co-stimulatory domains.

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

The conditionally active CAR is introduced into the cytotoxic cell by an expression vector. 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 viral vectors, adeno-associated viral (AAV) vectors, adenovirus vectors, engineered hybrid viruses, naked DNA, including but not limited to transposon mediated vectors such as sleep Beauty, piggybak, integrase (e.g. Phi 31). Some other suitable expression vectors include Herpes Simplex Virus (HSV) expression vectors and retroviral expression vectors.

Adenovirus expression vectors are based on adenoviruses, which have low capacity to integrate into genomic DNA but have high efficiency in transfecting host cells. The adenovirus expression vector comprises an adenovirus sequence sufficient to (a) support packaging of the expression vector and (b) ultimately express the CAR gene in a host cell. The adenovirus genome is a 36kb linear double-stranded DNA into which an exogenous DNA sequence (e.g., CAR gene) can be inserted in place of the large pieces of adenovirus DNA to prepare the expression vector of the invention (Grunhaus and Horwitz, "Adenoviruses as cloning vectors," settings virol., volume 3, pages 237-252, 1992).

Another expression vector is based on adeno-associated virus (AAV), which utilizes an adenovirus-coupled system. Such AAV expression vectors have a high frequency of integration into the host genome. It may even infect non-dividing cells, thus making it useful for delivering genes into mammalian cells, for example in tissue culture or in vivo. AAV vectors have a broad host range of infectivity. Details regarding the production and use of AAV vectors are described in U.S. Pat. nos. 5,139,941 and 4,797,368.

Retroviral expression vectors are capable of integrating into the host genome, delivering large amounts of exogenous genetic material, infecting a wide range of species and cell types and packaging in specific cell lines. Retroviral vectors are constructed by inserting nucleic acids (e.g., nucleic acids encoding CARs) into the viral genome at certain locations to produce replication defective viruses. Although retroviral vectors are capable of infecting a variety of cell types, integration and stable expression of the CAR gene requires division of the host cell.

Lentiviral vectors are derived from lentiviruses, which are complex retroviruses that contain, in addition to the common retroviral genes gag, pol and env, other genes with regulatory or structural functions (U.S. Pat. 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 are generated by attenuating HIV virulence genes multiple times, e.g., deleting genes env, vif, vpr, vpu and nef to make the vector biologically safe. Lentiviral vectors are capable of infecting non-dividing cells, and are useful for in vivo and ex vivo gene transfer and expression of CAR genes (U.S. Pat. No. 5,994,136).

The expression vector comprising the conditionally active CAR gene may be introduced into a host cell by any method known to those of skill in the art. If desired, the expression vector may include viral sequences for transfection. Alternatively, the expression vector may be introduced by fusion, electroporation, gene gun (biolistic), transfection, lipofection, etc. The host cells may be grown and expanded in culture prior to introduction of the expression vector and then suitably treated to introduce and integrate the vector. Host cells are then amplified and screened by means of the markers present in the vector. Various markers that may be used include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, and the like. The terms "cell", "cell line" and "cell culture" as used herein are used interchangeably. In some embodiments, the host cell is a T cell, NK cell, or NKT cell.

In another aspect, the invention also provides a genetically engineered cytotoxic cell comprising and stably expressing a conditionally active CAR of the invention. 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, which may be a mixed population or a single clonal population of CD4 ⁺/CD8^- and CD4 ^-/CD8⁺ cells, CD4 ⁺ T cells of the invention may also produce IL-2, IFN-. Gamma., TNF-. Alpha.and other T cell effector cytokines when co-cultured in vitro with cells expressing a target antigen (e.g., CD20 ⁺ and/or CD19 ⁺ tumor cells), CD8 ⁺ T cells of the invention may in some embodiments be CD45RA ⁺CD62L⁺ primary cells, CD45 CD62I7 memory cells, CD62L 62 "or any combination thereof," HIV-specific for use in a combination of human tumor cells, and human tumor cells, 35.

Genetically engineered cytotoxic cells can be produced by stably transfecting host cells with an expression vector comprising a CAR gene of the invention. Other methods of 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, optical transformation, gene electrotransfer, and/or hydrodynamic delivery), and/or particle-based methods (e.g., using a gene gun and/or magnetic transfection to puncture infection (impalefection)). Transfected cells that present a single integrated unrearranged vector and express a conditionally active CAR can be demonstrated to be amplified ex vivo.

Physical methods for introducing the expression vector into the host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, e.g., sambrook et al (2001,Molecular Cloning:A Laboratory Manual (molecular cloning: A laboratory Manual), cold Spring HarborLaboratory, newYork). Chemical methods for introducing expression vectors into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles and liposomes.

After introducing an expression vector containing a CAR gene into a host cell, the CAR gene will be expressed, thereby producing a CAR molecule that can bind to the target antigen. The resulting CAR molecule becomes a transmembrane protein due to the transmembrane domain. The host cell is then converted to a CAR cell, e.g., a CAR-T cell. Methods for producing engineered cytotoxic cells, such as CAR-T cells, with CAR molecules have been described, for example, in CARTELLIERI et al, ,"Chimeric antigenreceptor-engineered T cells for immunotherapy ofcancer,"JournalofBiomedicine andBiotechnology,, volume 2010, article ID 956304,2010, and Ma et al, "" VERSATILE STRATEGY for control THE SPECIFICITY AND ACTIVITY of ENGINEERED T CELLS, "PNAS, volume 113, E450-E458,2016.

Whether before or after genetically modifying cytotoxic cells to express a desired conditionally active CAR, the cells can be activated and expanded using methods described, for example, in U.S. patent nos. 6,352,694;6,534,055;6,905,680;6,692,964;5,858,358;6,887,466;6,905,681;7,144,575;7,067,318;7,172,869;7,232,566;7,175,843;5,883,223;6,905,874;6,797,514;6,867,041; and US20060121005. For example, T cells of the invention may be expanded by surface contact with a ligand having an agent attached thereto that stimulates a signal associated with the CD3/TCR complex and a costimulatory molecule on the surface of the T cell. In particular, the T cell population may be stimulated by contact with an anti-CD 3 antibody or antigen binding fragment thereof immobilized on a surface or an au anti-CD 2 antibody or by contact with a protein kinase C activator (e.g. bryostatin) conjugated to a calcium ionophore. To co-stimulate the accessory molecules on the surface of the T cells, ligands that bind the accessory molecules are used. For example, T cells may be contacted with an anti-CD 3 antibody and an anti-CD 28 antibody under conditions suitable to stimulate T cell proliferation. To stimulate proliferation of CD4 ⁺ T cells or CD8 ⁺ T cells, anti-CD 3 antibodies and anti-CD 28 antibodies are used. Examples of anti-CD 28 antibodies include 9.3, B-T3 and XR-CD28 (Bei Sangsong Diaclone, france) and these may be used in the present invention, as well as other methods commonly known in the art (Berg et al, transplantProc.30 (8): 3975-3977,1998; haanen et al, J.exp. Med.190 (9): 13191328,1999; garland et al, J.Immunol. Meth.227 (1-2): 53-63,1999).

In various embodiments, the invention provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a therapeutically effective amount of a conditionally active CAR of the invention. The conditionally active CAR in the composition may be any one or more of a polynucleotide encoding the CAR, a protein comprising the conditionally active 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 in the pharmaceutical industry as salts of therapeutic proteins, including, for example, salts of sodium, potassium, calcium, and the like, as well as ammonium salts of procaine, dibenzylamine, ethylenediamine, ethanolamine, methylglucamine, taurine, and the like, as well as acid addition salts such as hydrochloride salts, basic amino acids, and the like.

Pharmaceutically acceptable excipients can include any excipient used in the preparation of pharmaceutical compositions, which is generally safe, non-toxic and desirable, and includes veterinary uses as well as pharmaceutically acceptable excipients for human use. Such excipients may be solid, liquid, semi-solid, or in the case of aerosol compositions, gaseous. One type of excipient includes a pharmaceutically acceptable carrier that 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, saline, alcohols, and water. Solid carriers include starch, lactose, calcium sulfate, dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar and gelatin. The carrier may also include a slow release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax.

The pharmaceutically acceptable carrier is determined in part by the particular composition being administered and the particular method used to administer the composition. Thus, the pharmaceutical compositions of the present invention have a variety of suitable formulations. A variety of aqueous carriers may be used, such as buffered saline and the like. These solutions are sterile and generally free of undesirable substances. These compositions may be sterilized by conventional well-known sterilization techniques. The composition may contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like. The CAR concentration in these formulations can vary widely and is selected primarily based on liquid volume, viscosity and body weight and according to the particular mode of administration selected and desired by the patient.

In various embodiments, the pharmaceutical compositions of the present invention may be formulated for delivery by 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, transdermal, parenteral, implantable pump, continuous infusion, topical application, capsule and/or injection.

The pharmaceutical compositions of the present invention may be encapsulated, tableted or prepared as emulsions or syrups for oral administration. The pharmaceutical compositions are prepared according to conventional techniques of pharmacy, including grinding, mixing, granulating and tabletting (when needed) for tablet forms, or grinding, mixing and filling for hard capsule forms. When a liquid carrier is used, the formulation may be in the form of a syrup, elixir, emulsion or aqueous or non-aqueous suspension. The liquid formulation may be administered orally directly or filled into soft gelatin capsules.

The pharmaceutical compositions may be formulated as (a) liquid solutions, e.g., an effective amount of the encapsulated nucleic acid suspended in a diluent (e.g., water, saline, or PEG 400), (b) capsules, sachets, or tablets, each containing a predetermined amount of the active ingredient, e.g., liquid, solid, particles, or gelatin, (c) suspensions in a suitable liquid, and (d) suitable emulsions. In particular, suitable dosage forms include, but are not limited to, tablets, pills, powders, dragees, capsules, liquids, lozenges, gels, syrups, slurries, suspensions and the like.

Solid formulations comprise suitable solid excipients, for example carbohydrate or protein fillers, including, for example, sugars such as lactose, sucrose, mannitol or sorbitol, starches from corn, wheat, rice, potato or other plants, celluloses such as methylcellulose, hydroxypropyl methylcellulose and sodium carboxymethylcellulose, and gums including acacia and tragacanth, and proteins such as gelatin and collagen. A disintegrant or solubilizing agent, such as cross-linked polyvinylpyrrolidone, agar, alginic acid or a salt thereof, such as sodium alginate, may be added. Tablet forms may include lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, potato starch, tragacanth, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid and other excipients, colorants, fillers, binders, diluents, buffers, wetting agents, preservatives, flavoring agents, dyes, disintegrants and pharmaceutically acceptable carriers.

The liquid suspension comprises the conditionally active CAR, mixed with excipients suitable for the preparation of an aqueous suspension. Such excipients include suspending agents, such as sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, sodium alginate, polyvinylpyrrolidone, tragacanth and gum acacia, and dispersing or wetting agents, such as naturally-occurring phosphatides (e.g., lecithin), condensation products of alkylene oxides with fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide with long chain fatty alcohols (e.g., heptadecaethyleneoxy cetyl alcohol (HEPTADECAETHYLENE OXYCETANOL)), condensation products of ethylene oxide with partial esters derived from fatty acids and hexitols (e.g., polyoxyethylene sorbitol monooleate), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene sorbitan monooleate). The liquid suspension may also contain one or more preservatives (e.g., ethyl or n-propyl parahydroxybenzoate), one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, aspartame, or saccharin. Osmolality of the formulation can be adjusted.

Lozenge forms may comprise the flavored active ingredient, typically sucrose and acacia or tragacanth, as well as lozenges comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like, in addition to the active ingredient, carriers known in the art. It is known that conditionally active CARs must be protected from digestion when administered orally. This is typically accomplished by mixing the conditionally active CAR with a composition that renders it resistant to hydrolysis by an acid or enzyme or by coating the conditionally active CAR in a suitable resistant carrier, such as a liposome. Methods for protecting proteins from digestion are well known in the art. The pharmaceutical composition may be encapsulated, for example, in liposomes, or in a formulation, to provide slow release of the active ingredient.

Pharmaceutical compositions may be formulated as aerosols (e.g., they may be "nebulized") for administration by inhalation. The aerosol may be placed in a pressurized acceptable propellant such as dichlorodifluoromethane, propane, nitrogen, and the like. Suitable rectal administration agents include, for example, suppositories that consist of a suppository base encapsulated nucleic acid. Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons. Furthermore, it is also possible to use gelatin rectal capsules consisting of a combination of nucleic acids encapsulated by a matrix comprising, for example, liquid triglycerides, polyethylene glycols and paraffin hydrocarbons.

Pharmaceutical compositions may be formulated for parenteral administration, for example, by intra-articular (intra-articular), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, including aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, thiobis-dichlorophenol and solutes that render the formulation isotonic with the accepted blood, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In the practice of the invention, the compositions may be administered, for example, by intravenous infusion, oral administration, topical administration, intraperitoneal administration, intravesical administration, or intrathecal administration. In one aspect, the parenteral mode of administration is a preferred method of administration for the composition comprising the CAR protein and the genetically engineered cytotoxic cell. The compositions may conveniently be administered in unit dosage form and may be prepared by any of the methods well known in the pharmaceutical arts, e.g., as described in "Remington pharmaceutical science (Remington's Pharmaceutical Sciences)", mackpubishing co. Intravenous formulations may contain pharmaceutically acceptable carriers such as sterile water or saline, polyalkylene glycols such as polyethylene glycol, vegetable oils, hydrogenated naphthalenes and the like.

The pharmaceutical composition may be administered by at least one mode selected from the group consisting of parenteral, subcutaneous, intramuscular, intravenous, intra-articular, intrabronchial, intraperitoneal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebral, intracerebroventricular, intracolic, intrathecal, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal. The method may optionally further comprise administering, prior to, concurrently with, or subsequent to the administration of the conditionally active CAR, at least one composition comprising an effective amount of at least one compound or protein selected from the group consisting of a detectable label or reporter, a TNF antagonist, an antirheumatic, a muscle relaxant, an anesthetic, a non-steroidal anti-inflammatory drug (NSAK), an analgesic, an anesthetic, a sedative, a local anesthetic, a neuromuscular blocker, an antibacterial, an antipsoriatic, a corticosteroid, an anabolic steroid, an erythropoietin, an immunization, an immunoglobulin, an immunosuppressant, a growth hormone, a hormone replacement drug, a radiopharmaceutical, an antidepressant, an antipsychotic, an agonist, an asthma drug, a beta agonist, an inhaled steroid, epinephrine or an analog thereof, a cytotoxic or other anticancer, antimetabolite, such as methotrexate or an antiproliferative.

Types of cancers treated with the genetically engineered cytotoxic cells or pharmaceutical compositions of the invention include carcinomas, blastomas and sarcomas, as well as certain leukemia or lymphoid malignancies, benign and malignant tumors (benign AND MALIGNANT tumors) and malignant tumors (MALIGNANCIES), such as sarcomas, carcinomas and melanomas. The cancer may be a non-solid tumor (e.g., hematological tumor) or a solid tumor. Adult tumors/cancers and pediatric tumors/cancers are also included.

Hematological cancer is a cancer of the blood or bone marrow. Examples of hematologic (or hematologic) cancers include leukemias including acute leukemia (e.g., acute lymphoblastic leukemia, acute myelogenous leukemia and myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia and erythroleukemia), chronic leukemia (e.g., chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphomas, hodgkin's disease, non-hodgkin's lymphomas (painless and higher forms), multiple myelomas, waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplastic disorders.

Solid tumors are abnormal masses of tissue that typically do not contain cysts or areas of fluid. Solid tumors may be benign or malignant. Different types of solid tumors (e.g., sarcomas, carcinomas, and lymphomas) are named by the type of cells that form the solid tumor. Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovial carcinoma, mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma and CNS tumors (e.g., glioma such as brain stem glioma and mixed glioma), glioblastoma (glioblastoma, also known as astrocytoma, CNS lymphoma, blastoma, glioblastoma multiforme), medulloblastoma, neuroblastoma, schwannoma, angioma, angioblastoma, neuroblastoma and mydrioma.

The 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 adapted to administer the at least one conditionally active CAR by at least one means selected from the group consisting of parenteral, subcutaneous, intramuscular, intravenous, intra-articular, intrabronchial, intra-abdominal, intracapsular, intracartilaginous, intracavity, intracerebellar, intracerebroventricular, colonic, intrauterine, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus (bolus), vaginal, rectal, buccal, sublingual, intranasal, or transdermal means.

In another aspect, the invention provides a kit comprising at least one CAR protein, a polynucleotide sequence encoding a CAR, or a host cell expressing a CAR in lyophilized form in a first container, and optionally a second container comprising sterile water, sterile buffered water, or at least one preservative selected from phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride, alkyl parabens, benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, or a mixture thereof in an aqueous diluent. In one aspect, the contents of the second container are used to restore (reconstituted) the concentration of the conditionally active CAR or specific portion or variant in the first container to a concentration of about 0.1mg/ml to about 500mg/ml in the kit. 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 invention provides a method of treating at least one wild-type protein mediated disorder comprising administering to a patient in need thereof a formulation provided in a kit and reconstituted prior to administration.

Also provided are articles of manufacture for human pharmaceutical or diagnostic use comprising packaging material and a container comprising a solution or lyophilized form of at least one CAR protein, a polynucleotide sequence encoding a CAR, or a host cell expressing a CAR. The article of manufacture may optionally include the container as a component of a parenteral, subcutaneous, intramuscular, intravenous, intra-articular, intrabronchial, intra-abdominal, intracapsular, intracartilaginous, intracavity, intracavitary, intracerebellar, intracerebroventricular, intracolic, endocervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrafetal intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal delivery device or system.

In some embodiments, the invention provides a method comprising obtaining a cytotoxic cell from an individual, engineering the cytotoxic cell by introducing a CAR gene of the invention into the cytotoxic cell gene, and administering the genetically engineered cytotoxic cell to the individual. In some embodiments, the cytotoxic cell is selected from the group consisting of a T cell, a naive T cell, a memory T cell, an effector T cell, a natural killer cell, and a macrophage. In one embodiment, the cytotoxic cell is a T cell.

In one embodiment, the T cells are obtained from an individual. T cells can be obtained from a number of sources including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the invention, any number of T cell lines available in the art may be used. In certain embodiments of the invention, T cells may be obtained from blood obtained from an individual using any number of techniques known to those skilled in the art (e.g., ficoll ^TM isolation).

In a preferred embodiment, the cells from the circulating blood of the individual are obtained by apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one embodiment, cells collected by apheresis may be washed to remove plasma fractions and placed in an appropriate buffer or medium for subsequent processing steps. In one embodiment of the invention, the cells are washed with Phosphate Buffered Saline (PBS). In alternative embodiments, the wash solution lacks calcium and may lack magnesium or may lack many, if not all, divalent cations. Furthermore, surprisingly, the initial activation step in the absence of calcium results in amplified activation. As one of ordinary skill in the art will readily appreciate, the washing step may be performed by methods known to those skilled in the art, for example, by using a semi-automated "flow-through" centrifuge (e.g., cobe 2991 cell processor, baxter CytoMate or blood cell reinfusion apparatus (Haemonetics CELL SAVER) 5) and following manufacturer's instructions. After washing, the cells may be resuspended in various biocompatible buffers, such as PBS without Ca ²⁺ and without Mg ²⁺, boehmite a, or another saline solution with or without a buffer. Alternatively, the undesirable components of the apheresis sample may be removed and the cells resuspended directly in culture medium.

In another embodiment, T cells are isolated from peripheral blood by lysing the erythrocytes and removing monocytes (e.g., by PERCOLL ^TM gradient centrifugation or by countercurrent elutriation centrifugation). Specific T cell subsets, such as CD3 ⁺、CD28⁺、CD4⁺、CD8⁺、CD45RA⁺ and CD45RO ⁺ T cells, may be further isolated by positive or negative selection techniques. For example, enrichment of T cell populations by negative selection can be accomplished with a combination of antibodies directed against surface markers specific for the cells that are negatively selected. One method is cell sorting and/or selection by negative magnetic immunoadhesion or flow cytometry using a mixture of monoclonal antibodies directed against cell surface markers present on negatively selected cells. To enrich for CD4 ⁺ cells by negative selection, monoclonal antibody mixtures typically include antibodies directed against CD14, CD20, CD11b, CD16, HLA-DR and CD 8. In certain embodiments, it may be desirable to enrich or positively select regulatory T cells that normally express CD4 ⁺、CD25⁺、CD62L^hi、GITR⁺ and FoxP3 ⁺.

For example, in one embodiment, the antibody is conjugated to the CD3 antibody/CD 28 antibody (i.e., 3X 28) by beads (e.g.M-450 CD3/CD 28T) for a period of time sufficient to positively select the desired T cells. In one embodiment, the period of time is about 30 minutes. In another embodiment, the period of time ranges from 30 minutes to 36 hours or more, and all integer values therebetween. In another embodiment, the period of time is at least 1,2, 3, 4, 5, or 6 hours. In another preferred embodiment, the period of time is 10 to 24 hours. In a preferred embodiment, the incubation period is 24 hours. To isolate T cells from patients with leukemia, using a longer incubation time (e.g., 24 hours) can increase cell yield. In any case where there are fewer T cells than other cell types, such as in isolating Tumor Infiltrating Lymphocytes (TILs) from tumor tissue or immunocompromised individuals, longer incubation times may be used to isolate T cells. In addition, the use of longer incubation times may increase the efficiency of capturing CD8 ⁺ T cells. Thus, by simply shortening or extending the time for T cells to bind to CD3/CD28 beads and/or by increasing or decreasing the ratio of beads to T cells (as further described herein), a subpopulation of T cells can be preferentially selected for at the beginning of the culture or at other points in the process. In addition, by increasing or decreasing the proportion of CD3 antibodies and/or CD28 antibodies on the beads or other surfaces, the T cell subpopulation targeted can be preferentially selected at the beginning of the culture or at other desired time points. The skilled person will appreciate that multiple rounds of selection may also be employed in the context of the present invention. In certain embodiments, it may be desirable to perform a selection process and use "unselected" cells in the activation and expansion process. The "unselected" cells may also be subjected to other rounds of selection.

The resulting cytotoxic cells are then genetically engineered as described herein. The polynucleotide encoding the conditionally active CAR (typically in an expression vector) is introduced into the cytotoxic cell such that the cytotoxic cell will express, preferably stably express, 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 merely transient retention of the introduced polynucleotide is sufficient. In this way, there may be a short term effect in which cytotoxic cells may be introduced into the host and then turned on after a predetermined time, e.g., after the cells have been able to migrate to a particular treatment site.

Genetically engineered cytotoxic cells can be introduced into an individual (e.g., a mammal) in a variety of ways, depending on the nature of the cytotoxic cells and the disease to be treated. Genetically engineered cytotoxic cells can be introduced at the tumor site. In one embodiment, the genetically engineered cytotoxic cells are directed to cancer or are modified to direct cancer. The number of genetically engineered cytotoxic cells used will depend on many factors, such as the environment, the purpose of introduction, the lifetime of the cells, the regimen used (e.g., number of administrations), the ability of the cells to proliferate, and the stability of the recombinant construct. The genetically engineered cytotoxic cells may be administered as a dispersion, injected at or near the site of interest. The cells may be in a physiologically acceptable medium.

It will be appreciated that the method of treatment is affected by a number of variables, such as the cellular response to the CAR, the efficiency with which the cytotoxic cells express the CAR and, as the case may be, the secretion level, the activity of the expressed CAR, the specific needs of the individual (which may vary over time and situation), the rate of loss of cellular activity caused by loss of expression activity of genetically engineered cytotoxic cells or cells of the individual, and the like. Thus, it is desirable to monitor each individual patient for the proper dose for the individual, even if there are universal cells that can be administered to the entire population, and the practice of such monitoring patients is conventional in the art.

The following examples are illustrative of the process of the present invention and are not intended to be limiting. Other suitable modifications and adaptations of the various conditions and parameters normally encountered in the art and obvious to those skilled in the art are within the scope of the invention.

Examples

Example 1 production of scFv conditionally active antibodies against Axl

Two conditionally active single chain antibodies (CAB-scFv-63.9-4 and CAB-scFv-63.9-6) against the drug target antigen Axl were expressed as homodimers with wild-type human IgG1 Fc, SEQ ID NO:13 or 14 (resulting in 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 FIGS. 2 and 3) and as heterodimers in a "pestle and mortar" system, resulting in monovalent scFv (resulting in the monovalent antibodies scFv-63.9-4-02, SEQ ID NO:11 and CAB-scFv-63.9-6-02, SEQ ID NO: 12) in FIGS. 2 and 3.

The binding affinity of these antibodies to the drug target antigen Axl was measured by ELISA assay at pH 6.0 and pH 7.4. As shown in fig. 2, scFv antibodies showed comparable affinity to the drug target antigen Axl at both pH 6.0 and pH 7.4 as full bivalent (full bivalent antibodies) antibodies. In addition, the selectivity of these scFv antibodies at pH 6.0 and pH 7.4 as shown in figure 3 was also comparable to full bivalent antibodies. This example demonstrates that conditionally active antibodies of the invention have comparable affinity and selectivity to scFv antibodies or full bivalent antibodies. Thus, the conditionally active antibodies of the invention may be inserted as a single DNA strand into a DNA molecule encoding a CAR in a CAR-T platform of the invention.

Example 2 scFv antibodies against target antigen Axl for construction of CAR-T cells

According to one embodiment of the invention, conditionally active antibodies against the drug target antigen Axl are generated by simultaneous screening for selectivity and affinity at pH 6.0 and pH 7.4 and expression levels. The FLAG tag was used for screening in serum, as human antibodies were present in serum that could lead to false positive screening. The screening buffer was a carbonate buffer (krebs buffer with Ringer-standard buffer, but different from PBS). The resulting conditionally active antibody was found to have a higher affinity for the drug target antigen Axl compared to the wild-type antibody at pH 6.0, but a lower affinity for the same drug target antigen Axl compared to the wild-type antibody at pH 7.4. Furthermore, these conditionally active antibodies all have high expression levels as shown in table 2 below, wherein the "clone" column shows antibodies and expression levels "mg/ml" are shown in the second column.

Clones of these antibodies and the required expression levels ("ordered amounts"), expected expression levels ") are sent to the service provider. However, the actual expression levels ("delivery amounts") of these antibodies are very high and exceed the expected expression levels.

TABLE 2 conditionally active antibodies with high expression levels

Taking BAP063.9-13-1 antibody as an example, the conditionally active antibody does not show aggregation in buffer, as shown in FIG. 4. The BAP063.9-13-1 antibody was analyzed by size exclusion chromatography. In fig. 4, only one peak was detected, indicating little or no aggregation of the antibody.

Surface Plasmon Resonance (SPR) assays were also used to measure their rate of binding and dissociation to the drug target antigen Axl. SPR assays are known for measuring the rate of binding and dissociation of conditionally active antibodies. SPR assay is performed in the presence of bicarbonate. The in vivo rate of binding and dissociation of conditionally active antibodies (in animals and humans) is a very important feature of conditionally active antibodies.

It was observed that the conditionally active antibody had a fast binding rate at pH 6.0 and a slower binding rate at pH 7.4 compared to the negative control (BAP 06310F10, which had similar binding rates at pH 6.0 and pH 7.4) (fig. 5). Furthermore, raising the temperature from room temperature to 60 ℃ did not significantly change the SPR assay results (fig. 5). SPR assays also showed that these conditionally active antibodies were highly selective at pH 6.0 compared to pH 7.4 (fig. 6A-6B show one antibody as an example).

Conditionally active biological antibodies are summarized in table 3. Two of the antibodies were expressed as scFv (BAP 063.9-13.3 and BAP 063.9-48.3) that were ready to be inserted into a CAR in the CAR-T platform. Incubation of the antibodies for 1 hour at 60 ℃ did not alter the affinity of most antibodies ("thermostability"). In two columns of report data (last two columns of Table 3) for binding activity measured using SPR at pH 6.0 and pH 7.4, comparisons were made with "BAP063.6-hum10F10-FLAG" (negative control, second row of Table 3). The selectivity of these antibodies can be determined by the difference between the data in the last two columns. Both scFv antibodies had very high selectivity (75% and 50% selectivity at pH 6 compared to 0% at pH 7.4).

Comparative example A CAR-T cells with unconditionally active antibodies to target antigen Axl

The use of unconditionally active scFv antibodies against target antigen Axl to construct CAR-T cells that bind to target antigen Axl or CHO cells (CHO-Axl) expressing target antigen Axl on the cell surface is shown in fig. 7A-7B. An unconditionally active antibody is used as an ASTR for the CAR molecule, which is inserted into T cells to construct CAR-T cells that can bind to the target antigen Axl.

By way of comparison, CHO cells that did not express target antigen Axl were treated with (1) T cells transduced with no CAR molecule, (2) T cells transduced with CAR molecules that did not bind to target antigen Axl, and (3) T cells transduced with CAR molecules having unconditionally active antibodies to target antigen Axl (fig. 7A). CHO cell populations are indicated by a cell index (Y-axis in fig. 7A), and a decrease in cell index indicates cytotoxicity (cell killing) caused by CAR-T cells.

Referring to fig. 7A, CHO cells showed growth prior to T cell addition. Upon addition of CAR-T cells that bind to the target antigen Axl, the cell index initially decreases, indicating non-specific cytotoxicity of T cells. Shortly thereafter, however, CHO cells resume growth. More importantly, the difference between these three treatments was insignificant, indicating that CAR-T cells with unconditionally active antibodies to the target antigen Axl were not significantly cytotoxic to CHO cells that did not express the target antigen Axl.

CHO cells expressing the target antigen Axl were then treated in the same manner as described above (1) T cells transduced with no CAR molecule, (2) T cells transduced with CAR molecules that did not bind to the target antigen Axl, and (3) T cells transduced with CAR molecules having unconditionally active antibodies to the target antigen Axl (fig. 7B). After T cell addition, the cell index was significantly reduced by treatment with CAR cells with non-conditionally active antibodies to target antigen Axl, whereas by the other two treatments the cell index was not reduced, indicating that CAR cells with non-conditionally active antibodies to target antigen Axl were cytotoxic to CHO-X1 cells expressing target antigen Axl.

Example 3 CAR-T cells with conditionally active scFv antibodies against target antigen Axl

A CAR molecule was constructed using a conditionally active scFv antibody against the target antigen Axl. Transducing T cells with a CAR molecule such that the T cells express the CAR molecule (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. Non-transduced T cells (without CAR molecules) were used as controls (fig. 8A-8B).

Referring to fig. 8A, CHO cells that do not express the target antigen Axl were treated with CAR-T cells and non-transduced T cells. There was no significant difference between the two treatments, indicating that CAR-T cells were not cytotoxic to CHO cells. Refer to fig. 8B. Wherein CHO cells expressing the target antigen Axl (CHO-63) are treated in a similar manner, the CAR-T cells with conditionally active antibodies against the target antigen Axl significantly reduce the CHO-63 cell population compared to non-transduced T cells. This suggests that CAR-T cells with conditionally active antibodies against the target antigen Axl are cytotoxic to CHO-63 cells.

Upon binding to the target antigen Axl, CAR-T cells induce cytotoxicity. This effect was confirmed by measuring the levels of the cytokines interferon gamma (INFg) and IL 2. Cytokine data are shown in figures 9A-9B. In fig. 9A, binding of CAR-T cells to target antigen Axl on CHO-63 cells elicited a significant release INFg as compared to non-transduced T cells, as shown by the increased cytokine levels observed. Similarly, in fig. 9B, binding of CAR-T cells to the target antigen Axl on CHO-63 cells triggered significant release of IL2 as compared to non-transduced T cells, as shown by the increased cytokine levels observed.

Example 4 CAR-T cells with conditionally active scFv antibodies against target antigen ROR2

A conditionally active scFv antibody was generated against the target antigen ROR 2. Their binding activity to the target antigen ROR2 was measured using ELISA assay (fig. 10).

One of the scFv antibodies shown in fig. 10, scFv-116101, was used to construct CAR molecules for the production of CAR-T cells (116101 CAR-T). The constructed CAR-T cells were used to target Daudi cells expressing the target antigen ROR 2. Negative controls were T cells transduced with no CAR molecule (non-transduced T cells) and CAR-T cells transduced with CAR molecules that were unable to bind target antigen ROR2 (non-ROR 2 scFv CAR-T). The results are shown in fig. 11A. The ratio of the number of T cells to the number of Daudi cells in these treatments was 10:1. CAR-T cells with scFv antibodies targeting the target antigen ROR2 on Daudi cells (116101 CAR-T) induced significant cell death of Daudi cells as shown by the higher dead cell/live cell ratio in fig. 11A.

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

Example 5 cytokine Release by CAR-T cells with antibodies to target antigens Axl and ROR2 in examples 1-4

Cytokine release induced by CAR-T cell binding to target antigen was measured in this example. FIGS. 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, both INFg and IL2 cytokine levels were significantly increased compared to the control in which the same CAR-T cells were used to treat CHO cells that did not express the target antigen Axl, indicating release of INFg and IL2 cytokines. Furthermore, T cells not transduced with CAR molecules and CAR-T cells not binding to the target antigen Axl do not result in significant release of INFg and IL2 cytokines.

Figures 13A-13B show INFg and IL cytokine levels after CAR-T cells comprising conditionally active scFv antibodies against target antigen ROR2 bind to Rajib cells and Daudi cells, both expressing target antigen ROR 2. After treatment of Rajib cells and Daudi cells with CAR-T cells for 24 hours, a significant increase in INFg and IL2 cytokine levels was observed compared to a control in which the same CAR-T cells were used to treat HEK293 cells that did not express the target antigen ROR 2. In addition, T cells not transduced with CAR molecules and CAR-T cells not binding to the target antigen ROR2 did not significantly increase cytokine levels, indicating failure to induce significant release of INFg and IL2 cytokines.

Example 6 conditionally active scFv antibodies against target antigen CD22

Five conditionally active scFv antibodies were selected against the target antigen CD 22. The conditionally active scFv antibody was selected to be more active at ph6.0 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 CD 22.

It is to be understood that even though numerous characteristics and advantages of the present invention have been set forth in the foregoing description, together with details of the structure and function of the invention, this disclosure is illustrative only, and changes may be made in detail, especially in matters of shape, size and arrangement of parts within the principles of the present invention to the full extent indicated by the broad general meaning of the terms in which the appended claims are expressed.

Sequence listing

<110> Biological protein Co., ltd (Bioatla, LLC)

<120> Conditionally active chimeric antigen receptor for modified T cells

<130> KHP212110206.5

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<170> PatentIn version 3.5

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agtgggtctg ggacagactt cactctcacc atcagcagac tggagcctga agattttgca 660

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cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 900

tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 960

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ccagggaagg ggctggagtg gattggtctt attaaacctt ccaatggtgg tactagttac 180

aaccagaagt tcaagggcag agtcaccatc tcagccgaca agtccatcag caccgcctac 240

ctgcagtgga gcagcctgaa ggcctcggac accgccatgt attactgtgc acatggtcac 300

tacgagagtt acgaggctat ggactactgg ggccagggaa cgctggtcac cgtcagctca 360

ggcggcggag gaagcggcgg tggatccgga ggaggaggct ctgacatcca gatgacccag 420

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ctcatctatt ggcaggatac ccggcacact ggagtcccat caaggttcag cggcagtgga 600

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tactgtcagg aacattttag cactccgctc acgttcggcc aagggaccaa ggtggaaatc 720

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cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 900

tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 960

aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1020

aaggagtaca agtgcaaggt cagcaacaaa gccctcccag cccccatcga gaaaaccatc 1080

tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 1140

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gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1320

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gaggtccagc tggtacagtc tggggctgag gtgaagaagc ctggggctac agtgaaaatc 60

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ccagggaagg ggctggagtg gattggtctt attaaacctt ccaatggtgg tactagttac 180

aaccagaagt tcaagggcag agtcaccatc tcagccgaca agtccatcag caccgcctac 240

ctgcagtgga gcagcctgaa ggcctcggac accgccatgt attactgtgc acatggtcac 300

tacgagagtt acgaggctat ggactactgg ggccagggaa cgctggtcac cgtcagctca 360

ggcggcggag gaagcggcgg tggatccgga ggaggaggct ctgacatcca gatgacccag 420

tctccatcct ccctgtctgc atctgtagga gacagagtca ccatcacttg caaggccagt 480

caggatgtgg tttctgctgt agcctggtac cagcagaaac ctggccaggc tcccaggctc 540

ctcatctatt ggcaggatac ccggcacact ggagtcccat caaggttcag cggcagtgga 600

tctgggacag aattcactct caccatcagc agcctgcagc ctgatgattt tgcaacttat 660

tactgtcagg aacattttag ccctccgctc acgttcggcc aagggaccaa ggtggaaatc 720

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cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 900

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ctcatctatt ggcaggatac ccggcacact ggagtcccat caaggttcag cggcagtgga 600

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aaacgtacga cggacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg 780

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cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 900

tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 960

aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1020

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gtgctggact ccgacggctc cttcttcctc accagcaagc tcaccgtgga caagagcagg 1320

tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1380

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ccagggaagg ggctggagtg gattggtctt attaaacctt ccaatggtgg tactagttac 180

aaccagaagt tcaagggcag agtcaccatc tcagccgaca agtccatcag caccgcctac 240

ctgcagtgga gcagcctgaa ggcctcggac accgccatgt attactgtgc acatggtcac 300

tacgagagtt acgaggctat ggactactgg ggccagggaa cgctggtcac cgtcagctca 360

ggcggcggag gaagcggcgg tggatccgga ggaggaggct ctgacatcca gatgacccag 420

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ctcatctatt ggcaggatac ccggcacact ggagtcccat caaggttcag cggcagtgga 600

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tactgtcagg aacattttag ccctccgctc acgttcggcc aagggaccaa ggtggaaatc 720

aaacgtacga cggacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg 780

ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 840

cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 900

tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 960

aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1020

aaggagtaca agtgcaaggt cagcaacaaa gccctcccag cccccatcga gaaaaccatc 1080

tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 1140

gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1200

atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1260

gtgctggact ccgacggctc cttcttcctc accagcaagc tcaccgtgga caagagcagg 1320

tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1380

acgcagaaga gcctctccct gtctccgggt aaagattaca aggatgacga cgataag 1437

<210> 6

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<220>

<223> Synthetic sequence (SYNTHETIC SEQUENCE)

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gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 60

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300

tgcaaggtca gcaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 420

aaccaggtca gcctgtactg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 600

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 660

ctctccctgt ctccgggtaa a 681

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<223> Synthetic sequence (SYNTHETIC SEQUENCE)

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gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 60

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300

tgcaaggtca gcaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 420

aaccaggtca gcctgtactg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 600

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 660

ctctccctgt ctccgggtaa a 681

<210> 8

<211> 479

<212> PRT

<213> Artificial (Artifical)

<220>

<223> Synthetic sequence (SYNTHETIC SEQUENCE)

<400> 8

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Val Phe Thr Ser Tyr

20 25 30

Trp Leu His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly

115 120 125

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

130 135 140

Pro Gly Glu Arg Ala Thr Leu Ser Cys Lys Ser Ser Gln Ser Val Leu

145 150 155 160

Tyr Ser Ala Val Glu Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro

165 170 175

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

180 185 190

Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

195 200 205

Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys

210 215 220

Lys Gln Tyr Leu Ser Ser Trp Thr Phe Gly Gln Gly Thr Lys Val Glu

225 230 235 240

Ile Lys Arg Thr Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

245 250 255

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

260 265 270

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

275 280 285

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

290 295 300

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

305 310 315 320

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

325 330 335

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

340 345 350

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

355 360 365

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

370 375 380

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

385 390 395 400

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

405 410 415

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

420 425 430

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

435 440 445

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

450 455 460

Leu Ser Leu Ser Pro Gly Lys Asp Tyr Lys Asp Asp Asp Asp Lys

465 470 475

<210> 9

<211> 479

<212> PRT

<213> Artificial (Artifical)

<220>

<223> Synthetic sequence (SYNTHETIC SEQUENCE)

<400> 9

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

1 5 10 15

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

20 25 30

Thr Met Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Leu Ile Lys Pro Ser Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala His Gly His Tyr Glu Ser Tyr Glu Ala Met Asp Tyr Trp Gly Gln

100 105 110

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

115 120 125

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

130 135 140

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser

145 150 155 160

Gln Asp Val Val Ser Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln

165 170 175

Ala Pro Arg Leu Leu Ile Tyr Trp Gln Asp Thr Arg His Thr Gly Val

180 185 190

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr

195 200 205

Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Glu

210 215 220

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

225 230 235 240

Lys Arg Thr Thr Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

245 250 255

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

260 265 270

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

275 280 285

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

290 295 300

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

305 310 315 320

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

325 330 335

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

340 345 350

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

355 360 365

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

370 375 380

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

385 390 395 400

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

405 410 415

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

420 425 430

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

435 440 445

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

450 455 460

Leu Ser Leu Ser Pro Gly Lys Asp Tyr Lys Asp Asp Asp Asp Lys

465 470 475

<210> 10

<211> 479

<212> PRT

<213> Artificial (Artifical)

<220>

<223> Synthetic sequence (SYNTHETIC SEQUENCE)

<400> 10

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

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Ser Phe Trp Gly Ala

20 25 30

Thr Met Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Leu Ile Lys Pro Ser Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala His Gly His Tyr Glu Ser Tyr Glu Ala Met Asp Tyr Trp Gly Gln

100 105 110

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

115 120 125

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

130 135 140

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser

145 150 155 160

Gln Asp Val Val Ser Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln

165 170 175

Ala Pro Arg Leu Leu Ile Tyr Trp Gln Asp Thr Arg His Thr Gly Val

180 185 190

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr

195 200 205

Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Glu

210 215 220

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

225 230 235 240

Lys Arg Thr Thr Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

245 250 255

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

260 265 270

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

275 280 285

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

290 295 300

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

305 310 315 320

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

325 330 335

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

340 345 350

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

355 360 365

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

370 375 380

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

385 390 395 400

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

405 410 415

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

420 425 430

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

435 440 445

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

450 455 460

Leu Ser Leu Ser Pro Gly Lys Asp Tyr Lys Asp Asp Asp Asp Lys

465 470 475

<210> 11

<211> 479

<212> PRT

<213> Artificial (Artifical)

<220>

<223> Synthetic sequence (SYNTHETIC SEQUENCE)

<400> 11

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

1 5 10 15

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

20 25 30

Thr Met Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Leu Ile Lys Pro Ser Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala His Gly His Tyr Glu Ser Tyr Glu Ala Met Asp Tyr Trp Gly Gln

100 105 110

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

115 120 125

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

130 135 140

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser

145 150 155 160

Gln Asp Val Val Ser Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln

165 170 175

Ala Pro Arg Leu Leu Ile Tyr Trp Gln Asp Thr Arg His Thr Gly Val

180 185 190

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr

195 200 205

Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Glu

210 215 220

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

225 230 235 240

Lys Arg Thr Thr Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

245 250 255

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

260 265 270

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

275 280 285

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

290 295 300

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

305 310 315 320

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

325 330 335

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

340 345 350

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

355 360 365

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

370 375 380

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

385 390 395 400

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

405 410 415

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

420 425 430

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

435 440 445

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

450 455 460

Leu Ser Leu Ser Pro Gly Lys Asp Tyr Lys Asp Asp Asp Asp Lys

465 470 475

<210> 12

<211> 479

<212> PRT

<213> Artificial (Artifical)

<220>

<223> Synthetic sequence (SYNTHETIC SEQUENCE)

<400> 12

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

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Ser Phe Trp Gly Ala

20 25 30

Thr Met Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Leu Ile Lys Pro Ser Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala His Gly His Tyr Glu Ser Tyr Glu Ala Met Asp Tyr Trp Gly Gln

100 105 110

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

115 120 125

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

130 135 140

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser

145 150 155 160

Gln Asp Val Val Ser Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln

165 170 175

Ala Pro Arg Leu Leu Ile Tyr Trp Gln Asp Thr Arg His Thr Gly Val

180 185 190

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr

195 200 205

Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Glu

210 215 220

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

225 230 235 240

Lys Arg Thr Thr Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

245 250 255

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

260 265 270

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

275 280 285

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

290 295 300

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

305 310 315 320

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

325 330 335

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

340 345 350

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

355 360 365

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

370 375 380

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

385 390 395 400

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

405 410 415

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

420 425 430

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

435 440 445

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

450 455 460

Leu Ser Leu Ser Pro Gly Lys Asp Tyr Lys Asp Asp Asp Asp Lys

465 470 475

<210> 13

<211> 227

<212> PRT

<213> Artificial (Artifical)

<220>

<223> Synthetic sequence (SYNTHETIC SEQUENCE)

<400> 13

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Tyr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys

225

<210> 14

<211> 227

<212> PRT

<213> Artificial (Artifical)

<220>

<223> Synthetic sequence (SYNTHETIC SEQUENCE)

<400> 14

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Tyr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys

225

<210> 15

<211> 481

<212> PRT

<213> Artificial (Artifical)

<220>

<223> Synthetic sequence (SYNTHETIC SEQUENCE)

<400> 15

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Thr Gly

20 25 30

Tyr Tyr Trp Asn Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp

35 40 45

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

50 55 60

Lys Asn Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Phe Glu Gly Val Trp Tyr Gly Leu Asp Tyr Trp Gly Gln Gly

100 105 110

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

115 120 125

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

130 135 140

Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu

145 150 155 160

Ser Val Asp Arg Tyr Gly Asn Ser Phe Ile His Trp Tyr Gln Gln Lys

165 170 175

Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Arg Thr Tyr Asn Leu Glu

180 185 190

Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe

195 200 205

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

210 215 220

Cys Gln Gln Thr Asn Glu Asp Pro Trp Thr Phe Gly Gln Gly Thr Lys

225 230 235 240

Val Glu Ile Lys Arg Thr Asp Lys Thr His Thr Cys Pro Pro Cys Pro

245 250 255

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

260 265 270

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

275 280 285

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

290 295 300

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

305 310 315 320

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

325 330 335

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

340 345 350

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

355 360 365

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

370 375 380

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

385 390 395 400

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

405 410 415

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

420 425 430

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

435 440 445

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

450 455 460

Lys Ser Leu Ser Leu Ser Pro Gly Lys Asp Tyr Lys Asp Asp Asp Asp

465 470 475 480

Lys

<210> 16

<211> 1446

<212> DNA

<213> Artificial (Artifical)

<220>

<223> Synthetic sequence (SYNTHETIC SEQUENCE)

<400> 16

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60

acctgcactg tctctggcta ctccatcacc actggttatt actggaactg ggtgcgacag 120

gctcgtggac aacgccttga gtggataggt tacataacct acgacggtag caagaactac 180

aacccatctc tcaagaatag agtcaccata tcagtagaca cgtccaagaa ccagttctcc 240

ctgaagctga gctctgtgac cgccgcggac acggctgtgt attactgttc gagatttgag 300

ggagtttggt atggtttgga ctactggggc cagggaacgc tggtcaccgt cagctcaggc 360

ggcggaggaa gcggcggtgg atccggagga ggaggctctg ccatccagtt gacccagtct 420

ccatcctccc tgtctgcatc tgtaggagac agagtcacca tcacttgcag agccagtgaa 480

agtgttgata gatatggcaa tagttttata cactggtatc agcagaaacc agggaaagct 540

cctaagctcc tgatctatcg tacatacaac ctagaatctg ggatcccagc caggttcagt 600

ggcagtgggt ctgggacaga gttcactctc accatcagca gcctgcagtc tgaagatttt 660

gcagtttatt actgtcagca aactaatgag gatccgtgga cgttcggcca agggaccaag 720

gtggaaatca aacgtacgga caaaactcac acatgcccac cgtgcccagc acctgaactc 780

ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 840

cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 900

ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 960

cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 1020

aatggcaagg agtacaagtg caaggtcagc aacaaagccc tcccagcccc catcgagaaa 1080

accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 1140

cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 1200

agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1260

cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1320

agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1380

cactacacgc agaagagcct ctccctgtct ccgggtaaag attacaagga tgacgacgat 1440

aagtga 1446

Claims

1. A chimeric antigen receptor for binding to a tumor-specific target antigen, comprising:

i. at least one antigen specific targeting region evolving from a parent or wild type protein or domain thereof and having the property that the activity of the antigen specific targeting region in an assay under normal physiological conditions is reduced compared to the activity in an assay under abnormal conditions deviating from said normal physiological conditions;

transmembrane domain, and

An intracellular signaling domain,

Wherein the tumor specific target antigen is Axl and the at least one antigen specific targeting region is a single chain antibody having an amino acid sequence selected from the group consisting of SEQ ID NOS 9-12, or

The tumor-specific target antigen is ROR2 and the at least one antigen-specific targeting region is a single chain antibody having the amino acid sequence of SEQ ID No. 15.

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

3. The chimeric antigen receptor according to claim 1, wherein the tumor-specific target antigen is ROR2 and the at least one antigen-specific targeting region is a single chain antibody having the amino acid sequence of SEQ ID No. 15.

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

5. The chimeric antigen receptor of claim 4, wherein the normal physiological pH is in the range of greater than 7.0 to 7.8.

6. The chimeric antigen receptor of claim 5, wherein the normal physiological pH is in the range of 7.2 to 7.6.

7. The chimeric antigen receptor of claim 4, wherein the aberrant pH is in the range of 6.0 to less than 7.0.

8. The chimeric antigen receptor of claim 7, wherein the aberrant pH is in the range of 6.0 to 6.8.

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

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

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

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

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

14. The chimeric antigen receptor according to claim 1, wherein the transmembrane domain is selected from the group consisting of artificial hydrophobic sequences, and transmembrane domains of type I transmembrane proteins, alpha, beta or zeta chains of T cell receptors, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD 154.

15. The chimeric antigen receptor according to claim 1, wherein the intracellular signaling domain is selected from the group consisting of human CD3 zeta chain, cytoplasmic tail of FcyRIII, fcsRI, fc receptor, cytoplasmic receptor with immune receptor tyrosine activation motif (ITAM), TCR zeta, fcrgamma, fcrbeta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d cytoplasmic signaling domain.

16. The chimeric antigen receptor of claim 1, further comprising a co-stimulatory domain selected from the group consisting of the co-stimulatory domains of proteins in the TNFR superfamily, CD28, CD137, CD134, dap1O, CD, CD2, CD5, ICAM-1, LFA-1, lck, TNFR-I, TNFR-II, fas, CD, CD40, ICOS LIGHT, NKG2C and B7-H3.

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

18. The expression vector of claim 17, wherein the expression vector is selected from the group consisting of lentiviral vectors, gamma retrovirus vectors, foamy virus vectors, adeno-associated virus vectors, adenovirus vectors, poxvirus vectors, herpesvirus vectors, engineered hybrid viruses, and transposon mediated vectors.

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