WO2015143224A1 - Chimeric antigen receptor-modified t-cells - Google Patents

Abstract

This invention relates to cancer-reactive antibodies with one or more non- naturally encoded amino acids, chimeric antigen receptor-modified t-cells comprising one or more non-naturally encoded amino acids, and methods of using both in the detection, treatment, and prevention of cancer.

Description

Chimeric Antigen Receptor-Modified T-Cells

FIELD OF THE INVENTION

[0001] The immune system of vertebrates consists of a number of organs and ceil types which have evolved to accurately recognize foreign antigens, specifically bind to, and eliminate/destroy such foreign antigens. Lymphocytes, among other cell types, are critical to the immune system. Lymphocytes are divided into three major sub-populations, T cells, NK cells, and B cells. Although inter-dependent, T cells_, and NK cells are largely responsible for cell-mediated immunity and B cells are largely responsible for antibody production (humoral immunity). In humans, each B cell can produce an enormous number of antibody molecules.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is an international PCT application of U.S, Provisional Application Serial No, 61/955,455 filed on March 18, 2014, the specification and contents of which are incorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

[0003] This invention relates to cancer-reactive antibodies with one or more non-naturally encoded amino acids, chimeric antigen receptor-modified t-cells comprising one or more non-naturally encoded amino acids, and methods of using both in the detection, treatment, and prevention of cancer.

[0004] The immune system of vertebrates consists of a number of organs and cell types which have evolved to accurately recognize foreign antigens, specifically bind to, and eliminate/destroy such foreign antigens. Lymphocytes, among other cell types, are critical to the immune system. Lymphocytes are divided into three major sub-populations, T cells, NK cells, and B cells, Although inter-dependent, T cells and NK cells are largely responsible for cell-mediated immunity and B cells are largely responsible for antibody production (humoral immunity), In humans, each B cell can produce an enormous number of antibody molecules.

[0005] Such antibody production typically ceases (or substantially decreases) when a foreign antigen has been neutralized. Occasionally, however, proliferation of a particular B cell will continue unabated and may result in a cancer known as a B cell leukemias and lymphomas. B-cell lymphomas, such as the B-cell subtype of NHL, are significant contributors to cancer mortality. The response of B-cell malignancies to various forms of treatment is mixed. For example, in cases in which adequate clinical staging of NHL is possible, field radiation therapy can provide satisfactory treatment. Still, about one-half of the patients die from the disease. (Devesa et al, J, Nat'l Cancer Inst, 79:701 (1987))

[0006] The majority of acute and chronic lymphocytic leukemias are of the B-cell lineage (Freedman, Hematol, Oncol. Clin. North Am. 4:405 (1990)). Chronic lymphocytic leukemias are the most common leukemia in the Western world (Goodman et al., Leukemia and Lymphoma 22: 1 (1 96)). Acute leukemias are aggressive in nature and difficult to cure. Due to the very low rate of cellular proliferation, chronic lymphocytic leukemia is resistant to cytotoxic drug treatment. Over 30,000 new cases of Non-Hodgkin's lymphoma are diagnosed each year in the United States alone. (Shipp et al, Cancer: Principles and Practice of Oncology, Lippincott- aven Publishers, Philadelphia, 1997, p2165).

[0007] Traditional methods of treating B-cell malignancies, including chemotherapy and radiotherapy, and hematopoietic stem-cell transplantation, have limited utility due to toxic side effects. While current therapies have produced significant complete response rates, a large percentage of patients remain at a significant risk for disease relapse (Glass et al,, Cancer 80:231 1, 1997). Immune-based strategies for targeting minimal residual disease are under development and may provide additional modalities for consolidating standard chemotherapy and radiotherapy regimens,

[0008] The large majority of patients having B-celt malignancies, including chronic lymphocytic leukemia (CLL), will die from their disease, One approach to treating these patients is to genetically modify T cells to target antigens expressed on tumor cells through the expression of chimeric antigen receptors (CARs), CARs are antigen receptors that are designed to recognize cell surface antigens in a human leukocyte antigen-independent manner. Attempts in using genetically modified cells expressing CARs to treat these types of patients have met with very limited success. See for example, Brentjens et al., 201.0, Molecular Therapy, 18:4, 666-668; Morgan et al,, 2010, Molecular Therapy, published online February 23, 2010, pages 1 -9; and, Till et al, 2008, Blood, 1 12:2261 -2271.

[0009] In most cancers, tumor-specific antigens are not yet well defined, but in B cell malignancies, CD 19 is an attractive tumor target. Expression of CD 19 is restricted to normal and malignant B cells (Uckun, et al. Blood, 1988, 71 : 13-29), so that CD 19 is a widely accepted target to safely test CARs, While CARs can trigger T-cell activation in a manner similar to an endogenous T-cell receptor, a major impediment to the clinical application of this technology to date has been limited in vivo expansion of CAR+ T cells, rapid disappearance of the cells after infusion, and disappointing clinical activity (Jena, et al., Blood;, 2010, 1 16: 1035- 1044; Uckun, et al, Blood, 1988, 71 : 13-29).

[0010] Antibody-based therapeutics have emerged as important components of therapies for an increasing number of human malignancies in such fields as oncology, inflammatory and infectious diseases. In most cases, the basis of the therapeutic function is the high degree of specificity and affinity the antibody-based drug has for its target antigen. Arming monoclonal antibodies with drugs, toxins, or radionuclides is yet another strategy by which mAbs may induce therapeutic effect. By combining the exquisite targeting specificity of antibody with the tumor killing power of toxic effector molecules, immunoconjugates permit sensitive discrimination between target and normal tissue thereby resulting in fewer side effects than most conventional chemotherapeutic drugs.

[0011] Given the physical properties of PSMA and its expression pattern in relation to prostate cancer progression PSMA is an excellent target in the development of antibody-drug conjugates for imaging, diagnostic and therapeutic uses, The first PSMA-specific MAb reported, 7ΕΠ , was subsequently developed and commercialized as a diagnostic agent for tumor imaging (ProstaScint, Cytogen, Princeton, N.J.). However, this antibody recognizes an intracellular epitope of PSMA which limits its usefulness as an imaging agent for the detection of PSMA. More recently, MAbs such as J591 that recognize the extracellular portion of PSMA have been identified, Anti-PSMA antibody-drug conjugates that can be utilized for imaging, diagnostic and/or therapeutic uses are therefore needed, The present invention provides such antibody-drug conjugates for use in prostate cancer,

SUMMARY OF THE INVENTION

[0012] Disclosed herein are cancer-reactive antibodies with one or more non-naturally encoded amino acids; methods for tagging cancer cells in a patient comprising treating the patient with one or more cancer-reactive antibodies; an anti-tag chimeric antigen receptor- expressing (AT-CAR) T cell system comprising a) tagging cancer cells in a patient and b) administering to the patient, either simultaneously or subsequently, chimeric antigen receptor-expressing T cells (CAR T cells), in some embodiments with one or more non- naturally encoded amino acids within the CAR-T cell, wherein the CAR-T cells bind the tagged cancer cells.

[0013] In some embodiments, the cancer-reactive antibody comprises one or more post- translational modifications. In some embodiments, the cancer-reactive antibody is linked to a linker, polymer, or biologically active molecule. In some embodiments, the cancer-reactive antibody is linked to a bifunctional polymer, bifunctional linker, or at least one additional cancer-reactive antibody. In some embodiments, the cancer-reactive antibody is linked to a tag, In some embodiments of the present invention, more than one cancer-reactive antibody is administered to a patient in need thereof. In other embodiments of the present invention, the tag of each formulation of cancer-reactive antibodies is the same or different and the tag is selected from the group consisting of fluorescein isothiocyanate (FITC), streptavidin, biotin, histidine, dinitrophenol, peridinin chlorophyll protein complex, green fluorescent protein, phycoerythrin (PE), horse radish peroxidase, palmitoylation, nitrosylation, alkalanine phosphatase, glucose oxidase, and maltose binding protein. In other embodiments of the present invention, the antibody of the cancer-reactive antibodies is an antibody or an antigen- binding fragment thereof. In some embodiments, the antibody or antigen -binding fragment thereof is cetuximab, nimotuzumab, panitumumab, retuximab, omalizumab, tositumomab, trastuzumab, gemtuzumab, alemtuzumab, bevacuzimab or an antigen-binding fragment of any one thereof,

[0014] In some embodiments, the cancer-reactive antibody comprises a substitution, addition or deletion that modulates affinity of the cancer- reactive antibody polypeptide for a cancer-reactive antibody polypeptide receptor or binding partner, including but not limited to, a protein, polypeptide, small molecule, or nucleic acid, In some embodiments, the cancer- reactive antibody polypeptide comprises a substitution, addition, or deletion that increases the stability of the cancer-reactive antibody polypeptide when compared with the stability of the corresponding cancer-reactive antibody without the substitution, addition, or deletion. In some embodiments, the cancer-reactive antibody polypeptide comprises a substitution, addition, or deletion that modulates the rmmunogenicity of the cancer-reactive antibody polypeptide when compared with the immunogenicity of the corresponding cancer-reactive antibody without the substitution, addition, or deletion. In some embodiments, the cancer- reactive antibody polypeptide comprises a substitution, addition, or deletion that modulates serum half-life or circulation time of the cancer-reactive antibody polypeptide when compared with the serum half-life or circulation time of the corresponding cancer-reactive antibody without the substitution, addition, or deletion.

[0015] In some embodiments, the cancer-reactive antibody polypeptide comprises a substitution, addition, or deletion that increases the aqueous solubility of the cancer-reactive antibody polypeptide when compared to aqueous solubility of the corresponding cancer- reactive antibody without the substitution, addition, or deletion. In some embodiments, the cancer-reactive antibody polypeptide comprises a substitution, addition, or deletion that increases the solubility of the cancer-reactive antibody polypeptide produced in a host cell when compared to the solubility of the corresponding cancer-reactive antibody without the substitution, addition, or deletion, In some embodiments, the cancer-reactive antibody polypeptide comprises a substitution, addition, or deletion that increases the expression of the cancer-reactive antibody polypeptide in a host cell or increases synthesis in vitro when compared to the expression or synthesis of the corresponding cancer-reactive antibody without the substitution, addition, or deletion. The cancer-reactive antibody polypeptide comprising this substitution retains agonist activity and retains or improves expression levels in a host cell. In some embodiments, the cancer-reactive antibody polypeptide comprises a substitution, addition, or deletion that increases protease resistance of the cancer-reactive antibody polypeptide when compared to the protease resistance of the corresponding cancer- reactive antibody without the substitution, addition, or deletion. U.S. Pat. No. 6,716,626 indicated that potential sites that may be substituted to alter protease cleavage include, but are not limited to, a monobasic site within 2 residues of a proline, In some embodiments, the cancer-reactive antibody polypeptide comprises a substitution, addition, or deletion that modulates signal transduction activity of the cancer-reactive antibody receptor when compared with the activity of the receptor upon interaction with the corresponding cancer- reactive antibody polypeptide without the substitution, addition, or deletion. In some embodiments, the cancer-reactive antibody polypeptide comprises a substitution, addition, or deletion that modulates its binding to another molecule such as a receptor when compared to the binding of the corresponding cancer-reactive antibody polypeptide without the substitution, addition, or deletion.

[0016] In some embodiments, the cancer-reactive antibody polypeptide comprises a substitution, addition, or deletion that increases compatibility of the cancer-reactive antibody polypeptide with pharmaceutical preservatives (e.g., m-cresol, phenol, benzyl alcohol) when compared to compatibility of the corresponding cancer-reactive antibody without the substitution, addition, or deletion. This increased compatibility would enable the preparation of a preserved pharmaceutical formulation that maintains the physiochemical properties and biological activity of the protein during storage,

[0017] In some embodiments, the chimeric antigen receptor (CAR) in the CAR T cell comprises one or more post-translational modifications. In some embodiments, the CAR is linked to a linker, polymer, or biologically active molecule. In some embodiments, the CAR is linked to a bifunctional polymer, bifunctional linker, or at least one additional CAR, In some embodiments, the CAR is specific for a cancer-reactive antibody tag, In some embodiments of the present invention, each T cell expresses one CAR. In some embodiments of the present invention, some T cells express more than one CAR. In other embodiments of the present invention, each T cell expresses two CARs. In still other embodiments of the present invention, each T cell expresses more than one CAR. In some embodiments of the present invention, CAR T cells are administered to a patient in need thereof. In other embodiments of the present invention, a formulation of CAR T cells is administered to a patient in need thereof that binds to a specific cancer-reactive antibody tag. In some embodiments of the present invention, two formulations of CAR T cells are administered to a patient in need thereof that bind specifically to two different cancer-reactive antibody tags. In some embodiments of the present invention, two formulations of CAR T cells are administered to a patient in need thereof that bind specifically to the same cancer- reactive antibody tags. In some embodiments of the present invention, multiple formulations of CAR T cells are administered to a patient in need thereof that bind to multiple cancer- reactive antibody tags. The tag may be selected from, but is not limited to, the group consisting of fluorescein isothiocyanate (FITC), streptavidin, biotin, histidine, dinitrophenol, peridinin chlorophyll protein complex, green fluorescent protein, phycoerythrin (PE), horse radish peroxidase, palmitoylation, nitrosylation, alkalanine phosphatase, glucose oxidase, and maltose binding protein,

[0018] In some embodiments, the CAR comprises a substitution, addition or deletion that modulates affinity of the CAR to a cancer-reactive antibody tag or binding partner, including but not limited to, a protein, polypeptide, small molecule, or nucleic acid. In some embodiments, the CAR polypeptide comprises a substitution, addition, or deletion that increases the stability of the CAR polypeptide when compared with the stability of the corresponding CAR without the substitution, addition, or deletion, In some embodiments, the CAR polypeptide comprises a substitution, addition, or deletion that modulates the immunogenicity of the CAR polypeptide when compared with the immunogenicity of the corresponding CAR without the substitution, addition, or deletion, In some embodiments, the CAR polypeptide comprises a substitution, addition, or deletion that modulates serum half- life or circulation time of the CAR polypeptide when compared with the serum half-life or circulation time of the corresponding CAR without the substitution, addition, or deletion,

[0019] In some embodiments, the CAR polypeptide comprises a substitution, addition, or deletion that increases the aqueous solubility of the CAR polypeptide when compared to aqueous solubility of the corresponding CAR without the substitution, addition, or deletion. In some embodiments, the CAR polypeptide comprises a substitution, addition, or deletion that increases the solubility of the CAR polypeptide produced in a host cell when compared to the solubility of the corresponding CAR without the substitution, addition, or deletion. In some embodiments, the CAR polypeptide comprises a substitution, addition, or deletion that increases the expression of the CAR polypeptide in a host cell or increases synthesis in vitro when compared to the expression or synthesis of the corresponding CAR without the substitution, addition, or deletion. The CAR polypeptide comprising this substitution retains agonist activity and retains or improves expression levels in a host cell. In some embodiments, the CAR polypeptide comprises a substitution, addition, or deletion that increases protease resistance of the CAR polypeptide when compared to the protease resistance of the corresponding CAR without the substitution, addition, or deletion. U.S. Pat, No. 6,716,626 indicated that potential sites that may be substituted to alter protease cleavage include, but are not limited to, a monobasic site within 2 residues of a proline. In some embodiments, the CAR polypeptide comprises a substitution, addition, or deletion that modulates signal transduction activity of the CAR receptor when compared with the activity of the receptor upon interaction with the corresponding CAR polypeptide without the substitution, addition, or deletion, In some embodiments, the CAR polypeptide comprises a substitution, addition, or deletion that modulates its binding to another molecule .such as a receptor when compared to the binding of the corresponding CAR polypeptide without the substitution, addition, or deletion,

[0020] In some embodiments, the CAR polypeptide comprises a substitution, addition, or deletion that increases compatibility of the CAR polypeptide with pharmaceutical preservatives (e.g., m-cresol, phenol, benzyl alcohol) when compared to compatibility of the corresponding CAR without the substitution, addition, or deletion, This increased compatibility would enable the preparation of a preserved pharmaceutical formulation that maintains the physiochemical properties and biological activity of the protein during storage,

[0021] The present invention provides an anti-tag chimeric antigen receptor-expressing (AT-CAR) T cell system, In one aspect of the present invention the system comprises genetically transformed immune cells capable of recognizing a cancer cell. In other aspects of the present invention the system comprises genetically transformed, immune cells capable of recognizing a tumor antigen. In other aspects of the present invention the system comprises genetically transformed immune cells capable of recognizing two tumor antigens. In other aspects of the present invention the system comprises genetically transformed immune cells capable of recognizing three tumor antigens. In other aspects of the present invention the system comprises genetically transformed immune cells capable of recognizing more than one tumor antigen, In other aspects of the present invention the system comprises CAR T ceils that bind to a tumor antigen. The AT-CAR system of the present invention confers singular or multiple tumor antigen selectivity to T cells of the invention, In some embodiments, the AT-CAR system comprises in vivo cancer cell tagging. In some embodiments, the AT-CAR system of the present invention confers singular or multiple tumor antigen selectivity to T cells of the invention,

[0022] For example, and as further described herein, aFITC-CAR-expressing human T cells of the present invention express CARs that specifically recognize various human cancer cells when those cells are bound by cancer-reactive FITC-labeled antibodies, The activation of aFITC-CAR-expressing T cells has been shown to induce efficient target lysis, T cell proliferation, and cytokine/chemokine production in vitro and ex vivo. In vivo, aFITC-CAR- expressing T cells plus FITC-cetuximab (Ctx) have been shown to delay colon cancer tumor establishment but lead to the selection of tumor-associated antigen (TAA)— negative cancer cells (U.S. Patent Published Application Number 20130287752, hereby incorporated in its entirety by reference), Using a pancreatic tumor model with uniform TAA expression, aFITC-CAR-expressing T cells were observed to eradicate an established tumor and prevent tumor growth.

[0023] In certain embodiments, the invention is drawn to a method of treating cancer in a subject, comprising: (a) administering a formulation of tagged proteins to a subject in need of treatment, wherein the tagged proteins bind a cancer cell in the subject, and (b) administering a therapeutically-effective population of anti-tag chimeric antigen receptor (AT-CAR)- expressing T cells to the subject, wherein the AT-CAR-expressing T cells bind the tagged proteins and wherein one or both of the tagged proteins and/or the AT-CAR-expressing T cells contains one or more non-naturally encoded amino acids.

[0024] In a related embodiment, the invention is drawn to a method of treating cancer in a subject, comprising: (a) administering two formulations of tagged proteins to a subject in need of treatment, wherein the tagged proteins bind a cancer cell in the subject, and (b) administering two populations of AT-CAR-expressing T cells to the subject, wherein the AT- CAR-expressing T cells bind the tagged proteins and wherein one or both of the tagged proteins and/or the AT-CAR-expressing T cells contains one or more non-naturally encoded amino acids.

[0025] In a related embodiment, the invention is drawn to a method of treating cancer in a subject, comprising: (a) administering two formulations of tagged proteins to a subject in need of treatment, wherein the tagged proteins bind a cancer cell in the subject, and (b) administering one or more therapeutically-effective populations of AT-CAR-expressing T cells to the subject, wherein the AT-CAR-expressing T cells bind the tagged proteins and wherein one or both of the tagged proteins and/or the AT-CAR-expressing T cells contains one or more non-naturally encoded amino acids.

[0026] In a related embodiment, the invention is drawn to a method of treating cancer in a subject, comprising: (a) administering one or more formulations of tagged proteins to a subject in need of treatment, wherein the tagged proteins bind a cancer cell in the subject, and (b) administering one or more therapeutically-effective populations of AT-CAR-expressing T cells to the subject, wherein the AT-CAR-expressing T cells bind the tagged proteins and wherein one or both of the tagged proteins and/or the AT-CAR-expressing T cells contains one or more non-naturally encoded amino acids.

[0027] In a further related embodiment, the invention is drawn to a method of treating cancer in a subject, comprising: (a) administering at least two formulations of tagged proteins to a subject in need of treatment, wherein the tagged proteins bind a cancer cell in the subject, and (b) administering at least two therapeutically-effective populations of AT-CAR- expressing T cells to the subject, wherein the AT-CAR-expressing T cells bind the tagged proteins and induce cancer cell death, thereby treating cancer in a subject.

[0028] In particular aspects of the embodiments of the invention, the AT-CAR of each population of AT-CAR-expressing T cells is the same or different and the AT-CAR comprises a tag-binding domain, a transmembrane domain, and an activation domain. In some aspects, the tag-binding domain is an antibody or an antigen-binding fragment thereof. In some aspects, the tag-binding domain specifically binds a tag selected from the group consisting of fluorescein isothiocyanate (FITC), streptavidin, biotin, histidine, dinitrophenol, peridinin chlorophyll protein complex, green fluorescent protein, phycoerythrin (PE), horse radish peroxidase, palmitoylation, nitrosylation, alkalanine phosphatase, glucose oxidase, and maltose binding protein, In some aspects, the tag-binding domain specifically binds a tag selected from the group consisting of FITC, biotin, PE, histidine or streptavidin. In some aspects where the tag-binding domain is antigen-binding fragment, the antigen-binding fragment is a single chain variable fragment (scFv), such as a scFv that specifically binds a tag selected from the group consisting of fluorescein isothiocyanate (FITC), streptavidin, biotin, histidine, dinitrophenol, peridinin chlorophyll protein complex, green fluorescent protein, phycoerythrin (PE), horse radish peroxidase, palmitoylation, nitrosylation, alkalanine phosphatase, glucose oxidase, and maltose binding protein. In other aspects of the present invention the transmembrane domain is the hinge and transmembrane regions of the human CD8a chain. In other aspects, the activation domain comprises one or more of the cytoplasmic region of CD28, the cytoplasmic region of CD137 (41BB), OX40, HVEM, CD3 and FcRe.

[0029] In some embodiments of the invention, the T cells of each population of AT-CAR- expressing T cells are the same or different and wherein the T cells are selected from the group consisting of T cells of any HLA-background from peripheral blood mononuclear cells (PBMC), T cells isolated from a tumor explant of the subject, and intratumoral T cells of the subject, In some embodiments of the invention, the T cells of each population of AT-CAR- _. expressing T cells consist of HLA-A2+ peripheral blood mononuclear cells (PBMC),

[0030] In some of the embodiments of the present invention, the formulation of tagged protein is administered to the subject prior to administration of the therapeutically-effective population of AT-CAR-expressing T cells. In some of the embodiments of the present invention, the formulations of tagged proteins are administered to the subject prior to administration of the therapeutically-effective population of AT-CAR-expressing T cells. In some of the embodiments of the present invention, the formulations of tagged proteins are administered to the subject prior to administration of the therapeutically-effective populations of AT-CAR-expressing T cells.

[0031] In some of the embodiments of the present invention, the formulation of tagged protein is administered to the subject concurrently with administration of the therapeutically- effective population of AT-CAR-expressing T cells. In some of the embodiments of the present invention, the formulations of tagged proteins are administered to the subject concurrently with administration of the therapeutically-effective population of AT-CAR- expressing T cells. In some of the embodiments of the present invention, the formulations of tagged proteins are administered to the subject concurrently with administration of the therapeutically-effective populations of AT-CAR-expressing T cells.

[0032] In some of the embodiments of the present invention, the formulation of tagged protein is administered to the . subject after administration of the therapeutically-effective population of AT-CAR-expressing T cells. In some of the embodiments of the present invention, the formulations of tagged proteins are administered to the subject after administration of the therapeutically-effective population of AT-CAR-expressing T cells. In some of the embodiments of the present invention, the formulations of tagged proteins are administered to the subject after administration of the therapeutically-effective populations of AT-CAR-expressing T cells.

Π [0033] In some of the embodiments of the present invention, the formulation of tagged protein and the formulation of the therapeutically-effective population of AT-CAR- expressing T cells are administered in any order. In some of the embodiments of the present invention, the formulations of tagged proteins and the therapeutically-effective population of AT-CAR-expressing T ce!ls are administered in any order. In some of the embodiments of the present invention, the formulations of tagged proteins are administered to the subject and the therapeutically-effective populations of AT-CAR-expressing T cells are administered in any order.

[0034] In particular aspects of the embodiments of the invention, AT-CAR-expressing T cell binding to the tagged proteins induces cytolytic activation of the T cells.

[0035] Disclosed in the present invention are methods and/or compositions for use in medicine, for example, for use in immunotherapy, including immunotherapy for infection or cancer, for example, including methods of use and compositions of T cells engineered to express an antigen-specific chimeric antigen receptor (CAR). In some embodiments, the engineered T cell has impaired expression of its endogenous alpha T-cell receptor (TCR). In some embodiments, the engineered T cell has impaired expression of its endogenous beta TCR. In some embodiments, the engineered T cell has impaired expression of its endogenous alpha/beta T-cell receptor TCR. In some embodiments, the engineered T cell has a disrupted endogenous alpha T-cell receptor (TCR). In some embodiments, the engineered T cell has a disrupted beta TCR, In some embodiments, the engineered T cell has a disrupted endogenous alpha beta T-cell receptor TCR, In some embodiments, there is disruption of the T-cell receptor α/β in CAR-expressing T cells using zinc finger nucleases (ZFNs) for generating universal T cells for immunotherapy, In embodiments of the invention, there is knocking out of the T-cell receptor αβ-chain in CAR-expressing T cells, for example using zinc finger nucleases. In some embodiments, the engineered T cell has a non-naturally encoded amino acid that disrupts the endogenous alpha/beta T-cell receptor TCR. In some embodiments, the engineered T cell has a non-naturally encoded amino acid that disrupts the endogenous alpha T-cell receptor TCR, In some embodiments, the engineered T cell has a non-naturally encoded amino acid that disrupts the endogenous beta T-cell receptor TCR, [0036] In some embodiments of the present invention, the CAR is only expressed in the T cells when a non-naturally encoded amino acid is provided concurrently. In some embodiments of the present invention, expression of the T cell's alpha/beta TCR is engineered to be dependent upon administration of a non-naturally encoded amino acid. [0037] In some embodiments, there are universal CAR-expressing T cells, such as from a healthy donor, that may be suitably stored, for example, in the freezer, and then infused into allogeneic individuals on demand, The T cell products of the present invention are engineered to be antigen-specific. In some embodiments, the invention provides engineered T cells from allogeneic healthy donors that can be administered to any patient without causing GVHD, In some embodiments, the invention provides autologous CAR⁺TCR^neg T cells.

[0038] The skilled artisan recognizes that in some methods there is simple depletion of T ceils that continue to express TCR by using, for example, clinical-grade CDS -specific monoclonal antibody such that a T-cell product can be generated in which at least the majority have lost expression of endogenous TCR, Other ways include the use of fluorescence-activated ceil sorting (FACS) and TCR- specific monoclonal antibodies or aptamers to reduce and possibly eliminate the presence of T cells that continue to express TCR. In addition, TCR-specific antibodies can be combined with other purification schemes, such as addition of complement, toxins, or resetting to reduce/eliminate T cells that continue to express TCR. In certain embodiments there are methods of treating an individual in need of treatment using compositions encompassed by the invention. The treatment includes employing particular T cells having a CAR and also having genetic modifications to exclude functional TCR, Although in specific embodiments the modifications of the cells exclude functional TCR by any suitable means, in certain aspects the modifications include knock out (or in alternative embodiments, knock down, such as by siRNA and TALENs, for example) of the a and/or β chains or the gamma and/or delta chains of the TCR,

[0039] Some embodiments of the present invention provide a method of making a cell expressing a CAR comprising introducing an expression cassette in to the cell, wherein the expression cassette encodes a polypeptide comprising a heterologous human extracellular antigen binding domain, a transmembrane domain, one or more an intracellular signaling domain(s), and a non-naturally encoded amino acid. In some embodiments, the methods further comprise stimulating the cells with antigen presenting cells, recombinant antigen, or an antibody to the receptor to cause the cells to proliferate, kill, and/or make cytokines, In specific embodiments, the method further comprises stimulating the cells with antigen presenting cells to cause the cells to proliferate.

[0040] In some embodiments, there are recombinant antigen-specific TCR^nes cells expressing and bearing on the cell surface membrane an antigen-specific CAR human polypeptide comprising an intracellular signaling domain, a transmembrane domain and an extracellular domain, the extracellular domain comprising a human anti-antigen monoclonal antibody or antigen binding fragment thereof with a non-naturally encoded amino acid. In some embodiments, there are recombinant antigen-specific TCR^neg cells expressing and bearing on the cell surface membrane an antigen-specific CAR human polypeptide comprising an intracellular signaling domain with a non-naturally encoded amino acid, a transmembrane domain and an extracellular domain, the extracellular domain comprising a human anti-antigen monoclonal antibody or antigen binding fragment thereof. In some embodiments, there are recombinant antigen-specific TCR^neg cells expressing and bearing on the cell surface membrane an antigen-specific CAR human polypeptide comprising a non- naturally encoded amino acid, an intracellular signaling domain, a transmembrane domain and an extracellular domain, the extracellular domain comprising a human anti-antigen monoclonal antibody or antigen binding fragment thereof, In some embodiments, there are recombinant antigen-specific TCR^neg cells expressing and bearing on the cell surface membrane an antigen -specific CAR human polypeptide comprising an intracellular signaling domain, a transmembrane domain with a non-naturally encoded amino acid and an extracellular domain, the extracellular domain comprising a human anti-antigen monoclonal antibody or antigen binding fragment thereof. In some embodiments, there are recombinant antigen-specific TCR^neg cells expressing and bearing on the cell surface membrane an antigen-specific CAR human polypeptide comprising an intracellular signaling domain, a transmembrane domain and an extracellular domain with a non-naturally encoded amino acid, the extracellular domain comprising a human anti-antigen monoclonal antibody or antigen binding fragment thereof. In some embodiments, there are recombinant antigen- specific TCR^neg cells expressing and bearing on the cell surface membrane an antigen- specific CAR human polypeptide comprising an intracellular signaling domain, a transmembrane domain and an extracellular domain, the extracellular domain comprising a human anti-antigen monoclonal antibody or antigen binding fragment thereof and wherein the polypeptide comprises a non-naturally encoded amino acid. In some embodiments, there are recombinant antigen-specific TCR^neg cells expressing and bearing on the cell surface membrane an antigen-specific CAR human polypeptide comprising an intracellular signaling- domain, a transmembrane domain and an extracellular domain, the extracellular domain comprising a human anti-antigen monoclonal antibody or antigen binding fragment thereof and wherein the polypeptide comprises two non-naturally encoded amino acids. In some embodiments, there are recombinant antigen-specific TCR^nee cells expressing and bearing on the cell surface membrane an antigen-specific CAR human polypeptide comprising an intracellular signaling domain, a transmembrane domain and an extracellular domain, the extracellular domain comprising a human anti-antigen monoclonal antibody or antigen binding fragment thereof and wherein the polypeptide comprises at least one non-naturally encoded amino acid. In some embodiments, there are recombinant antigen-specific TCR^neg cells expressing and bearing on the cell surface membrane an antigen-specific CAR human polypeptide comprising an intracellular signaling domain, a transmembrane domain and an extracellular domain, the extracellular domain comprising a human anti-antigen monoclonal antibody or antigen binding fragment thereof and wherein the polypeptide comprises more than one non-naturally encoded amino acids. In some embodiments, there are recombinant antigen-specific TCR^nes cells expressing and bearing on the cell surface membrane an antigen-specific CAR human polypeptide comprising an intracellular signaling domain, a transmembrane domain and an extracellular domain, the extracellular domain comprising a human anti-antigen monoclonal antibody or antigen binding fragment thereof and wherein the polypeptide comprises more than two non-naturally encoded amino acids.

[0041] In some embodiments, there is a method of treating a human disease condition associated with a cell expressing endogenous CD 19 comprising infusing a patient with an amount of a recombinant TCR^neg cell expressing a human antigen-specific CAR (by way of non-limiting example this could be a CD19-specific CAR) sufficient to treat the condition, wherein, for this specific example, the human antigen-specific CAR comprises a heterologous human CD 19 extracellular binding domain, a transmembrane domain, and an intracellular signaling domain comprising a non-naturally encoded amino acid. In some embodiments, there is a method of treating a human disease condition associated with a cell expressing endogenous CD 19 comprising infusing a patient with an amount of a recombinant TCR^neE cell expressing a human antigen-specific CAR (by way of non-limiting example this could be a CD19-specific CAR) sufficient to treat the condition, wherein, for this specific example, the human antigen-specific CAR comprises a heterologous human CD 19 extracellular binding domain, a transmembrane domain, and an intracellular signaling domain comprising more than one non-naturally encoded amino acids.

[0042] In some embodiments more than one pair of zinc finger nucleases can be used to modify a cell. For example, both zinc finger nucleases targeting the alpha chain and the beta chain can be used to eliminate T-cell receptor expression. In another instance, the zinc finger nucleases targeting the T-cell receptor can be used (for example, sequentially) with zinc finger nucleases to target one or more human leukocyte antigen(s) (HLA). Some embodiments of the present invention provide methods and compositions of engineered T- cells that have lost both T-cell receptor expression and HLA expression.

[0043] In one embodiment there is an isolated T-cell population wherein cells of the population comprise an endogenous T-cell receptor coding sequence that is either not expressed or which encodes a nonfunctional T-cell receptor; and a recombinant chimeric antigen receptor comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising an antigen binding region. In one embodiment there is an isolated T-cell population wherein cells of the population comprise an endogenous T-cell receptor coding sequence that is not expressed in the presence of one or more non-naturally encoded amino acids. In one embodiment there is an isolated T-cell population wherein cells of the population comprise an endogenous T-cell receptor coding sequence that is not expressed in the presence of a non-naturally encoded amino acids. In one embodiment there is an isolated T-cell population wherein cells of the population comprise an endogenous T- cell receptor coding sequence that is either not expressed or which encodes a nonfunctional T-cell receptor; and a recombinant chimeric antigen receptor comprising one or more non- naturally encoded amino acids, an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising an antigen binding region, In one embodiment there is an isolated T-cell population wherein cells of the population comprise an endogenous T- cell receptor coding sequence that is either not expressed or which encodes a nonfunctional T-cell receptor; and a recombinant chimeric antigen receptor comprising a non-naturally encoded amino acid, an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising an antigen binding region. In one embodiment there is an isolated T-cell population wherein cells of the population comprise an endogenous T-cell receptor coding sequence that is either not expressed or which encodes a nonfunctional T-cell receptor; and a recombinant chimeric antigen receptor comprising two non-naturally encoded amino acids, an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising an antigen binding region.

[0044] In some embodiments, the T-cell receptor is nonfunctional by virtue of one or more disruptions in the coding sequence of a chain, β chain, or both. In some embodiments, the T- cell receptor is nonfunctional by virtue of a polynucleotide encoding a non-naturally encoded amino acid in the coding sequence of a chain, β chain, or both. In some embodiments, the T- cell receptor is nonfunctional by virtue of a polynucleotide substitution encoding a non- naturally encoded amino acid in the coding sequence of a chain, β chain, or both. In some embodiments, the T-cell receptor is nonfunctional by virtue of more than one polynucleotide substitutions encoding a non-naturally encoded amino acid in the coding sequence of a chain, β chain, or both. In some embodiments, the T-cell receptor is nonfunctional by virtue of one or more disruptions in the coding sequence of a chain. In some embodiments, the T-cell receptor is nonfunctional by virtue of a polynucleotide encoding a non-naturally encoded amino acid in the coding sequence of a chain, In some embodiments, the T-cell receptor is nonfunctional by virtue of a polynucleotide substitution encoding a non-naturally encoded amino acid in the coding sequence of a chain. In some embodiments, the T-cell receptor is nonfunctional by virtue of more than one polynucleotide substitutions encoding a non- naturally encoded amino acid in the coding sequence of chain, In some embodiments, the T-cell receptor is nonfunctional by virtue of one or more disruptions in the coding sequence of β chain. In some embodiments, the T-cell receptor is nonfunctional by virtue of a polynucleotide encoding a non-naturally encoded amino acid in the coding sequence of β chain. In some embodiments, the T-cell receptor is nonfunctional by virtue of a polynucleotide substitution encoding a non-naturally encoded amino acid in the coding sequence of β chain. In some embodiments, the T-cell receptor is nonfunctional by virtue of more than one polynucleotide substitutions encoding a non-naturally encoded amino acid in the coding sequence of β chain. In some embodiments, the T-cell receptor is nonfunctional by virtue of one or more disruptions in the coding sequence of both the a chain and β chain. In some embodiments, the T-cell receptor is nonfunctional by virtue of a polynucleotide encoding a non-naturally encoded amino acid in the coding sequence of both the a chain and β chain. In some embodiments, the T-cell receptor is nonfunctional by virtue of a polynucleotide substitution encoding a non-naturally encoded amino acid in the coding sequence of both the a chain and β chain, In some embodiments, the T-cell receptor is nonfunctional by virtue of more than one polynucleotide substitutions encoding a non- naturally encoded amino acid in the coding sequence of both the a chain and β chain,

[0045] In some embodiments, the endogenous T-cell receptor is knocked out. In some embodiments, the antigen binding region is an F(ab')2, Fab', Fab, Fv, or scFv and/or the antigen binding region binds a tumor associated antigen, such as CD 19, CD20, ROR] , carcinoembryonic antigen, alphafetoprotein, CA- 125, MUC- 1 , epithelial tumor antigen, melanoma-associated antigen, mutated p53, mutated ras, HER2/Neu, ERBB2, FTER3, folate binding protein, F1TV- 1 envelope glycoprotein gpl20, HIV- 1 envelope glycoprotein gp41 , GD2, CD 123, CD23.CD30 , CD56, c-Mef mesothelin, GD3, HERV-K, IL- 1 IRalpha, IL- 13Ralpha2, kappa chain, or lambda chain, CSPG4 (also known as, high molecular weight melanoma associated antigen), EGFRvIII, and VEGFR2, In some embodiments, the antigen binding region binds a pathogen antigen, such as a fungal, viral, or bacterial antigen. In some cases, the fungal antigen is from Aspergillus or Candida. In certain cases, the viral antigen is from HSV, RSV, EBV, CMV, JC virus, BK virus, or Ebola.

[0046] In embodiments of the invention, there is an intracellular signaling domain that is a T-lyrnphocyte activation domain. In some embodiments, the intracellular signaling domain comprises CD3, CD28, OX40/CD134, 4- 1BB/CD137, FceRIy, ICOS/CD278, ILRB/CD 122, IL--2RG/CD 132, DAP molecules, CD70, cytokine receptor, CD40, or a combination thereof.

[0047] In certain embodiments, the transmembrane domain comprises IgG4Fc hinge, Fc regions, CD4 transmembrane domain, CD28 transmembrane domain, the CD3 transmembrane domain, cysteine mutated hitman€Β3ζ domain, CD 16 transmembrane domain, CDS transmembrane domain, or erythropoietin receptor transmembrane domain.

[0048] In some embodiments, there is a chimeric antigen receptor that comprises: a) an antigen binding domain from a variable region of an antigen-specific monoclonal antibody; b) a signaling domain of 033ζ; and c) CD28, CD 137, CD 134, or combinations thereof. In some embodiments, there is a chimeric antigen receptor that comprises: a) an antigen binding domain from a variable region of an antigen-specific monoclonal antibody; b) a signaling domain of 033ζ; c) CD28, CD 137, CD 134, or combinations thereof; and d) a non-naturally encoded amino acid. In some embodiments, there is a chimeric antigen receptor that comprises: a) an antigen binding domain from a variable region of an antigen-specific monoclonal antibody; b) a signaling domain of 033ζ; c) CD28, CD 137, CD 134, or combinations thereof; and d) two non-naturally encoded amino acids. In some embodiments, there is a chimeric antigen receptor that comprises: a) an antigen binding domain from a variable region of an antigen-specific monoclonal antibody; b) a signaling domain of 033ζ; c) CD28, CD 137, CD 134, or combinations thereof; and d) at least one non-naturally encoded amino acid. In some embodiments, there is a chimeric antigen receptor that comprises: a) an antigen binding domain from a variable region of an antigen-specific monoclonal antibody; b) a signaling domain of 033ζ; c) CD28, CD 137, CD 134, or combinations thereof; and d) more than one non-naturally encoded amino acid. In . some embodiments, there is a chimeric antigen receptor that comprises: a) an antigen binding domain from a variable region of an antigen-specific monoclonal antibody that comprises one or more non-naturally encoded amino acid(s); b) a signaling domain of 033ζ; and c) CD28, CD 137, CD 134, or combinations thereof. In some embodiments, there is a chimeric antigen receptor that comprises: a) an antigen binding domain from a variable region of an antigen-specific monoclonal antibody; b) a signaling domain of 033ζ comprising at least one non-naturally encoded amino acid; and c) CD28, CD 137, CD 134, or combinations thereof. In some embodiments, there is a chimeric antigen receptor that comprises: a) an antigen binding domain from a variable region of an antigen- specific monoclonal antibody; b) a signaling domain of 033ζ; and c) CD28, CD 137, CD 134, or combinations thereof with at least one non-naturally encoded amino acid. In some embodiments, there is a chimeric antigen receptor that comprises: a) an antigen binding domain from a variable region of an antigen- specific monoclonal antibody that comprises one non-naturally encoded amino acid; b) a signaling domain of 033ζ; and c) CD28, CD 137, CD 134, or combinations thereof, In some embodiments, there is a chimeric antigen receptor that comprises: a) an antigen binding domain from a variable region of an antigen-specific monoclonal antibody; b) a signaling domain of 033ζ comprising one non-naturally encoded, amino acid; and c) CD28, CD 137, CD 134, or combinations thereof. In some embodiments, there is a chimeric antigen receptor that comprises: a) an antigen binding domain from a variable region of an antigen-specific monoclonal antibody; b) a signaling domain of 033ζ; and c) CD28, CD 137, CD 134, or combinations thereof with one non-naturally encoded amino acid.

[0049] In some embodiments of the present invention, there are methods of generating the cells of the invention, comprising the steps of; a) providing one or more T cells; b) modifying the T cell(s) to express a recombinant chimeric antigen receptor comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising an antigen binding region; and c) modifying the T cell(s) to harbor an endogenous T-eell receptor coding sequence that is not expressed or encodes a nonfunctional T-cell receptor, In some embodiments, step b) occurs before step c) or step c) occurs before step b), In certain aspects, the T cell is provided from an umbilical cord blood bank, is provided from a peripheral blood bank, is an induced pluripotent stem cell, or is a human embryonic stem cell. In some embodiments, the T cell is allogeneic in reference to one or more intended recipients. In some cases, there are methods of generating the cells of the invention, comprising the steps of: a) providing one or more T cells; b) modifying the T cell(s) to express a recombinant chimeric antigen receptor comprising a non-naturally encoded amino acid, an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising an antigen binding region; and c) modifying the T cell(s) to harbor an endogenous T-eell receptor coding sequence that is not expressed or encodes a nonfunctional T-cell receptor. In some cases, there are methods of generating the cells of the invention, comprising the steps of: a) providing one or more T cells; b) modifying the T cell(s) to express a recombinant chimeric antigen receptor comprising at least one non-naturally encoded amino acid an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising an antigen binding region; and c) modifying the T celi(s) to harbor an endogenous T-cell receptor coding sequence that is not expressed or encodes a nonfunctional T-cell receptor. In some cases, there are methods of generating the cells of the invention, comprising the steps of: a) providing one or more T cells; b) modifying the T cell(s) to express a recombinant chimeric antigen receptor comprising more than one non-naturally encoded amino acids an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising an antigen binding region; and c) modifying the T cell(s) to harbor an endogenous T-cell receptor coding sequence that is not expressed or encodes a nonfunctional T-cell receptor. In some cases, there are methods of generating the cells of the invention, comprising the steps of: a) providing one or more T cells; b) modifying the T cell(s) to express a recombinant chimeric antigen receptor comprising a non-naturally encoded amino acid within the intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising an antigen binding region; and c) modifying the T cell(s) to harbor an endogenous T-cell receptor coding sequence that is not expressed or encodes a nonfunctional T-cell receptor. In some cases, there are methods of generating the cells of the invention, comprising the steps of: a) providing one or more T cells; b) modifying the T cell(s) to express a recombinant chimeric antigen receptor comprising an intracellular signaling domain, a transmembrane domain comprising a non-naturally encoded amino acid, and an extracellular domain comprising an antigen binding region; and c) modifying the T cell(s) to harbor an endogenous T-cell receptor coding sequence that is not expressed or encodes a nonfunctional T-cell receptor. In some cases, there are methods of generating the cells of the invention, comprising the steps of: a) providing one or more T cells; b) modifying the T cell(s) to express a recombinant chimeric antigen receptor comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising a non- naturally encoded amino acid and an antigen binding region; and c) modifying the T cell(s) to harbor an endogenous T-eell receptor coding sequence that is not expressed or encodes a nonfunctional T-cell receptor. In some cases, there are methods of generating the cells of the invention, comprising the steps of: a) providing one or more T cells; b) modifying the T cell(s) to express a recombinant chimeric antigen receptor comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising an antigen binding region comprising a non-naturally encoded amino acid; and c) modifying the T cell(s) to harbor an endogenous T-cell receptor coding sequence that is not expressed or encodes a nonfunctional T-cell receptor. Combinations of the above will be apparent to one skilled in the art.

[0050] In some embodiments, the chimeric antigen receptor is stably introduced into the cell, In other embodiments, the polynucleotide that encodes the chimeric antigen receptor is introduced into the cell by a transposon/transposase system or a viral-based gene transfer system, such as by recombinant retrovirus or lentivirus. In some embodiments, following modification of the T cell(s), they are propagated by exposing the T cells to artificial antigen presenting cells, by using OK'T'3 (or equivalent to cross-fink CDS) optionally with other co- stimulatory antibodies (e.g., anti-CD2S) on beads, or by using OKT3 (or equivalent to crosslink CD3) optionally with other co- stimulatory antibodies (e.g., anti~CD28) mixed with peripheral blood mononuclear cells, In some embodiments, a polynucleotide that encodes the recombinant chimeric antigen receptor is electroporated into the T cell, in some embodiments, a polynucleotide that encodes the chimeric antigen receptor is present on a plasmid or viral vector. In some embodiments, the polynucleotide that encodes the recombinant chimeric antigen receptor encodes a non-natural amino acid. In some embodiments, the polynucleotide that encodes the recombinant chimeric antigen receptor encodes more than one non-natural amino acid.

[0051] In some embodiments, the T cell can be genetically modified with zinc finger nuclease or TLAE nuclease to eliminate HLA expression, The T cell may express a CAR and/or have been modified to eliminate TCR expression. In some embodiments, the endogenous T-cell receptor and/or HLA is disrupted by nonhomologous end joining repair, such as is generated by zinc finger nuclease, TALE nuclease, introduced into the cell by physical means, electro-transfer of mRNA species, viral vector, or non-viral vector. In some embodiments, there are methods of treating an individual with a medical condition (such as autoimmune disease, cancer, or infection, including Aspergillus or Candida), comprising the step of providing an effective amount of cells from the population of cells described herein, including more than once in some aspects, such as at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, or more days apart. In some embodiments, the cancer is lymphoma, leukemia, non- Hodgkin's lymphoma, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, chronic lymphocytic leukemia, or B cell-associated autoimmune diseases.

[0052] It is to be understood that the methods and compositions described herein and incorporated by reference are not limited to the particular methodology, protocols, cell lines, constructs, and reagents described herein and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the methods and compositions described herein, which will be limited only by the appended claims,

DEFINITIONS

[0053] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly indicates otherwise,

[0054] Unless defined otherwise, ail technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the inventions described herein belong, Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the inventions described herein, the preferred methods, devices and materials are now described,

[0055] All publications and patents mentioned herein are incorporated herein by reference in their entirety for the purpose of describing and disclosing, for example, the constructs and methodologies that are described, in the publications, which might be used in connection with the presently described inventions, The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors described herein are not entitled to antedate such disclosure by virtue of prior invention or for any other reason,

[0056] The terms "aldol-based linkage" or "mixed aldol-based linkage" refers to the acid- or base -catalyzed condensation of one carbonyl compound with the enolate/enol of another carbonyl compound, which may or may not be the same, to generate a β-hydroxy carbonyl compound— an aldol.

[0057] The term "affinity label," as used herein, refers to a label which reversibly or irreversibly binds another molecule, either to modify it, destroy it, or form a compound with it. By way of example, affinity labels include enzymes and their substrates, or antibodies and their antigens,

[0058] The terms "alkoxy," "alkylamino" and "alkylthio" (or thioalkoxy) are used in their conventional sense, and refer to those alkyl groups linked to molecules via an oxygen atom, an amino group, or a sulfur atom, respectively.

[0059] The term "alkyl," by itself or as part of another molecule means, unless otherwise stated, a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals, having the number of carbon atoms designated (i. e. Ci-Cio means one to ten carbons). Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n- hexyi, n-heptyl, n-octyl, and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds, Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4- pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. The term "alkyl," unless otherwise noted, is also meant to include those derivatives of alkyl defined in more detail herein, such as "heteroalkyl", "haloalkyl" and "homoalkyl".

[0060] The term "alkylene" by itself or as part of another molecule means a divalent radical derived from an alkane, as exemplified, by (-CH2-)n, wherein n may be 1 to about 24. By way of example only, such groups include, but are not limited to, groups having 10 or fewer carbon atoms such as the structures -CH2CH2- and -CH2CH2CH2CH2-. A "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms. The term "alkylene," unless otherwise noted, is also meant to include those groups described herein as "heteroalkylene."

[0061] The term "amino acid" refers to naturally occurring and non-natural amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrolysine and selenocysteine, Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring . amino acid, by way of example only, an -carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group. Such analogs may have modified R groups (by way of example, norleucine) or may have₌modified peptide backbones while still retaining the same basic chemical structure as a naturally occurring amino acid, Non-limiting examples of amino acid analogs include homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium,

[0062] Amino acids may be referred to herein by either their name, their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-1UB Biochemical Nomenclature Commission, Additionally, nucleotides, may be referred to by their commonly accepted single-letter codes.

[0063] An "amino terminus modification group" refers to any molecule that can be attached to a terminal amine group. By way of example, such terminal amine groups may be at the end of polymeric molecules, wherein such polymeric molecules include, but are not limited to, polypeptides, polynucleotides, and polysaccharides. Terminus modification groups include but are not limited to_t various water soluble polymers, peptides or proteins. By way of example only, terminus modification groups include polyethylene glycol or serum albumin. Terminus modification groups may be used to modify therapeutic characteristics of the polymeric molecule, including but not limited to increasing the serum half-life of peptides.

[0064] By "antibody" herein is meant a protein consisting of one or more polypeptides substantially encoded by all or part of the antibody genes. The immunoglobulin genes include, but are not limited to, the kappa, lambda, alpha, gamma (IgGl, IgG2, IgG3, and IgG4), delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Antibody herein is meant to include full-length antibodies and antibody fragments, and include antibodies that exist naturally in any organism or are engineered (e.g. are variants).

[0065] The term "antibody" refers to intact antibody, monoclonal or polyclonal antibodies, The term "antibody" also encompasses, multispecific antibodies such as bispecific antibodies. Human antibodies are usually made of two light chains and two heavy chains each comprising variable regions and constant regions, The light chain variable region comprises 3 CDRs, identified herein as CDRL 1, CDRL2 and CDRL3 flanked by framework regions, The heavy chain variable region comprises 3 CDRs, identified herein as CDRH1 , CDRH2 and CDRH3 flanked by framework regions.

[0066] The term "antigen-binding fragment", as used herein, refers to one or more fragments of an antibody that retain the ability to bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of an intact antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments. linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, ( 1989) Nature 341 :544-546), which consists of a VH domain; (vi) an isolated complementarity determining region (CDR), e.g., VH CDR3 comprising or not additional sequence (linker, framework region(s) etc.) and (v) a combination of two to six isolated CDRs comprising or not additional sequence (linker, framework region(s) etc.). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single polypeptide chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci, USA 85 :5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. Furthermore, the antigen-binding fragments include binding-domain immunoglobulin fusion proteins comprising (i) a binding domain polypeptide (such as a heavy chain variable region, a light chain variable region, or a heavy chain variable region fused to a light chain variable region via a linker peptide) that is fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. The hinge region may be modified by replacing one or more cysteine residues with serine residues so as to prevent dimerization, Such binding-domain immunoglobulin fusion proteins are further disclosed in US 2003/0118592 and US 2003/0133939. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0067] A typical antigen binding site is comprised of the variable regions formed by the pairing of a light chain immunoglobulin and a heavy chain immunoglobulin. The structure of the antibody variable regions is very consistent and exhibits very similar structures. These variable regions are typically comprised of relatively homologous framework regions (FR) interspaced with three hypervariable regions termed Complementarity Determining Regions (CDRs). The overall binding activity of the antigen binding fragment is often dictated by the sequence of the CDRs. The FRs often play a role in the proper positioning and alignment in three dimensions of the CDRs for optimal antigen binding,

[0068] In fact, because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that shows the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L, et al., 1998, Nature 332:323-327; Jones, P. et al„ 1986, Nature 321 :522-525; and Queen, C, et al„ 1989, Proc. Natl. Acad, See, U.S.A. 86: 10029-10033). Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences. These germline sequences will differ from mature antibody gene sequences because they will not include completely assembled variable genes, which are formed by V(D)J joining during B cell maturation. Germline gene sequences will also differ from the sequences of a high affinity secondary repertoire antibody which contains mutations throughout the variable gene but typically clustered in the CDRs. For example, somatic mutations are relatively infrequent in the amino terminal portion of framework region 1 and in the carboxy-terminal portion of framework region 4. Furthermore, many somatic mutations do not significantly alter the binding properties of the antibody. For this reason, it is not necessary to obtain the entire DNA sequence of a particular antibody in order to recreate an intact recombinant antibody having binding properties similar to those of the original antibody. Partial heavy and light chain sequence spanning the CDR regions is typically sufficient for this purpose. The partial sequence is used to determine which germline variable and joining gene segments contributed to the recombined antibody variable genes, The germline sequence is then used to fill in missing portions of the variable regions. Heavy and light chain leader sequences are cleaved during protein maturation and do not contribute to the properties of the final antibody. To add missing sequences, cloned cDNA sequences can be combined with synthetic oligonucleotides by ligation or PCR amplification. Alternatively, the entire variable region can be synthesized to create an entirely synthetic variable region clone. This process has certain advantages such as eli ination or inclusion of particular restriction sites, or optimization of particular codons,

[0069] Of course, the totality or portions of the framework region of the antibody described herein may be used in conjunction with the CDRs in order to optimize the affinity, specificity or any other desired properties of the antibody. By "antibody" herein is meant a protein consisting of one or more polypeptides substantially encoded by all or part of the antibody genes. The immunoglobulin genes include, but are not limited to, the kappa, lambda, alpha, gamma (IgGl , IgG2, IgG3, and IgG4), delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Antibody herein is meant to include full-length antibodies and antibody fragments, and include antibodies that exist naturally in any organism or are engineered (e.g. are variants).

[0070] By "antibody fragment" is meant any form of an antibody other than the full-length form. Antibody fragments herein include antibodies that are smaller components that exist within full-length antibodies, and antibodies that have been engineered, Antibody fragments include but are not limited to Fv, Fc, Fab, and Fab')2, single chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDRl, CDR2, CDR3, combinations of CDR's, variable regions, framework regions, constant regions, heavy chains, light chains, and variable regions, and alternative scaffold non-antibody molecules, bispecific antibodies, and the like (Maynard & Georgiou, 2000, Annu. Rev. Biomed. Eng. 2:339-76; Hudson, 1998, Curr. Opin. Biotechnol. 9:395-402). Another functional substructure is a single chain Fv (scFv), comprised of the variable regions of the immunoglobulin heavy and light chain, covalently connected by a peptide linker (S-z Hu et al., 1996, Cancer Research, 56, 3055- 3061). These small (Mr 25,000) proteins generally retain specificity and affinity for antigen in a single polypeptide and can provide a convenient building block for larger, antigen- specific molecules. Unless specifically noted otherwise, statements and claims that use the term "antibody" or "antibodies" specifically includes "antibody fragment" and "antibody fragments,"

[0071] By "antibody-drug conjugate, or "ADC", as used herein, refers to an antibody molecule, or fragment thereof, that is covalently bonded to one or more biologically active molecule(s). The biologically active molecule may be conjugated to the antibody through a linker, polymer, or other covalent bond.

[0072] The term "aromatic" or "aryl", as used herein, refers to a closed ring structure which has at least one ring having a conjugated pi electron system and includes both carbocyclic aryl and heterocyclic aryl (or "heteroaryl" or "heteroaromatic") groups, The carbocyclic or heterocyclic aromatic group may contain from 5 to 20 ring atoms, The term includes monocyclic rings linked covalently or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups. An aromatic group can be unsubstituted or substituted, Non- limiting examples of "aromatic" or "aryl", groups include phenyl, 1-naphthyl, 2-naphthyl, 4- biphenyl, anthracenyl, and phenanthracenyl, Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described herein,

[0073] For brevity, the term "aromatic" or "aryl" when used in combination with other terms (including but not limited to, aryloxy, arylthioxy, aralkyl) includes both aryl and heteroaryl rings as defined above. Thus, the term "aralkyl" or "alkaryl" is meant to include those radicals in which an aryl group is attached to an alkyl group (including but not limited to, benzyl, phenethyl, pyridylmethyl and the like) including those alkyl groups in which a carbon atom (including but not limited to, a methylene group) has been replaced by a heteroatom, by way of example only, by an oxygen atom. Examples of such aryl groups include, but are not limited to, phenoxymethyl, 2-pyridyloxymethyl, 3-(l- naphthyloxy)propyl, and the like.

[0074] The term "arylene", as used herein, refers to a divalent aryl radical, Non-limiting examples of "arylene" include phenylene, pyridinylene, pyrimidinylene and thiophenylene. Substituents for arylene groups are selected from the group of acceptable substituents described herein,

[0075] A "bifunctional polymer", also referred to as a "bifunctional linker", refers to a polymer comprising two functional groups that are capable of reacting specifically with other moieties to form covalent or non-covalent linkages. Such moieties may include, but are not limited to, the side groups on natural or non-natural amino acids or peptides which contain such natural or non-natural amino acids. The other moieties that may be linked to the bifunctional linker or bifunctional polymer may be the same or different moieties, By way of example only, a bifunctional linker may have a functional group reactive with a group on a first peptide, and another functional group which is reactive with a group on a second peptide, whereby forming a conjugate that includes the first peptide, the bifunctional linker and the second peptide. Many procedures and linker molecules for attachment of various compounds to peptides are known. See, e.g., European Patent Application No. 188,256; U.S. Patent Nos. 4,671,958, 4,659,839, 4,414, 148, 4,699,784; 4,680,338; and 4,569,789 which are incorporated by reference herein in their entirety, A "multi-functional polymer" also referred to as a "multi-functional linker", refers to a polymer comprising two or more functional groups that are capable of reacting with other moieties, Such moieties may include, but are not limited to, the side groups on natural or non-natural amino acids or peptides which contain such natural or non-natural amino acids (including but not limited to, amino acid side groups) to form covalent or non-covalent linkages. A bi-functional polymer or multifunctional polymer may be any desired length or molecular weight, and may be selected to provide a particular desired spacing or conformation between one or more molecules linked to a compound and molecules it binds to or the compound.

[0076] The term "bioavailability," as used herein, refers to the rate and extent to which a substance or its active moiety is delivered from a pharmaceutical dosage form and becomes available at the site of action or in the general circulation. Increases in bioavailability refers to increasing the rate and extent a substance or its active moiety is delivered from a pharmaceutical dosage form and becomes available at the site of action or in the general circulation, By way of example, an increase in bioavailability may be indicated as an increase in concentration of the substance or its active moiety in the blood when compared to other substances or active moieties, A non-limiting example of a method to evaluate increases in bioavailability is given in examples 21-25, This method may be used for evaluating the bioavailability of any polypeptide. [0077] The term "biologically active molecule", "biologically active moiety" or "biologically active agent" when used herein means any substance which can affect any physical or biochemical properties of a biological system, pathway, molecule, or interaction relating to an organism, including but not limited to, viruses, bacteria, bacteriophage, transposon, prion, insects, fungi, plants, animals, and humans, In particular, as used herein, biologically active molecules include but are not limited to any substance intended for diagnosis, cure, mitigation, treatment, or prevention of disease in humans or other animals, or to otherwise enhance physical or mental well-being of humans or animals. Examples of biologically active molecules include, but are not limited to, peptides, proteins, enzymes, small molecule drugs, hard drugs, soft drugs, prodrugs, carbohydrates, inorganic atoms or molecules, dyes, lipids, nucleosides, radionuclides, oligonucleotides, toxins, cells, viruses, liposomes, microparticles and micelles. Classes of biologically active agents that are suitable for use with the methods and compositions described herein include, but are not limited to, drugs, prodrugs, radionuclides, imaging agents, polymers, antibiotics, fungicides, anti-viral agents, anti-inflammatory agents, anti-tumor agents, cardiovascular agents, anti -anxiety agents, hormones, growth factors, steroidal agents, microbially derived toxins, and the like.

[0078] By "modulating biological activity" is meant increasing or decreasing the reactivity of a polypeptide, altering the selectivity of the polypeptide, enhancing or decreasing the substrate selectivity of the polypeptide. Analysis of modified biological activity can be performed by comparing the biological activity of the non-natural polypeptide to that of the natural polypeptide,

[0079] The term "biomaterial," as used herein, refers to a biologically-derived material, including but not limited to material obtained from bioreactors and/or from recombinant methods and techniques,

[0080] The term "biophysical probe," as used herein, refers to probes which can detect or monitor structural changes in molecules, Such molecules include, but are not limited to, proteins and the "biophysical probe" may be used to detect or monitor interaction of proteins with other macromolecules. Examples of biophysical probes include, but are not limited to, spin-labels, a fluorophores, and photoactivatible groups.

[0081] The term "biosynthetically," as used herein, refers to any method utilizing a translation system (cellular or non-cellular), including use of at least one of the following components; a polynucleotide, a codon, a tRNA, and a ribosome. By way of example, non- natural amino acids may be "biosynthetically incorporated" into non-natural amino acid polypeptides using the methods and techniques described herein, "In vivo generation of polypeptides comprising non-natural amino acids", and in the non-limiting example 20. Additionally, the methods for the selection of useful non-natural amino acids which may be "biosynthetically incorporated" into non-natural amino acid polypeptides are described in the non-limiting examples 20.

[0082] The term "biotin analogue," or also referred to as "biotin mimic", as used herein, is any molecule, other than biotin, which bind with high affinity to avidin and/or streptavidin.

[0083] The term "carbonyl" as used herein refers to a group containing at a moiety selecting from the group consisting of -C(O)-, -S(O)-, -S(0)2~, and -C(S)-, including, but not limited to, groups containing a least one ketone group, and/or at least one aldehyde groups, and/or at least one ester group, and/or at least one carboxylic acid group, and/or at least one thioester group. Such carbonyl groups include ketones, aldehydes, carboxylic acids, esters, and thioesters. In addition, such groups may be part of linear, branched, or cyclic molecules.

[0084] The term "carboxy terminus modification group" refers to any molecule that can be attached to a terminal carboxy group. By way of example, such terminal carboxy groups may be at the end of polymeric molecules, wherein such polymeric molecules include, but are not limited to, polypeptides, polynucleotides, and polysaccharides. Terminus modification groups include but are not limited to, various water soluble polymers, peptides or proteins. By way of example only, terminus modification groups include polyethylene glycol or serum albumin. Terminus modification groups may be used to modify therapeutic characteristics of the polymeric molecule, including but not limited to increasing the serum half-life of peptides,

[0085] The term "chemically cleavable group," also referred to as "chemically labile", as used herein, refers to a group which breaks or cleaves upon exposure to acid, base, oxidizing agents, reducing agents, chemical inititiators, or radical initiators.

[0086] The term "chemiluminescent group," as used herein, refers to a group which emits light as a result of a chemical reaction without the addition of heat. By way of example only, luminol (5-amino-2,3-dihydro-l,4-phthalazinedione) reacts with oxidants like hydrogen peroxide (H202) in the presence of a base and a metal catalyst to produce an excited state product (3-aminophthalate, 3-APA).

[0087] The term "chromophore," as used herein, refers to a molecule which absorbs light of visible wavelengths, UV wavelengths or IR wavelengths.

[0088] The term "cofactor," as used herein, refers to an atom or molecule essential for the action of a large molecule, Cofactors include, but are not limited to, inorganic ions, coenzymes, proteins, or some other factor necessary for the activity of enzymes. Examples include, heme in hemoglobin, magnesium in chlorophyll, and metal ions for proteins.

[0089] "Cofolding," as used herein, refers to refolding processes, reactions, or methods which employ at least two molecules which interact with each other and result in the transformation of unfolded or improperly folded molecules to properly folded molecules. By way of example only, "cofolding," employ at least two polypeptides which interact with each other and result in the transformation of unfolded or improperly folded polypeptides to native, properly folded polypeptides. Such polypeptides may contain natural amino acids and/or at least one non-natural amino acid.

[0090] A "comparison window," as used herein, refers a segment of any one of contiguous positions used to compare a sequence to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Such contiguous positions include, but are not limited to a group consisting of from about 20 to about 600 sequential units, including about 50 to about 200 sequential units, and about 100 to about 150 sequential units. By way of example only, such sequences include polypeptides and polypeptides containing non-natural amino acids, with the sequential units include, but are not limited to natural and non-natural amino acids, In addition, by way of example only, such sequences include polynucleotides with nucleotides being the corresponding sequential units. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, including but not limited to, by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math, 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J, Mol. Biol. 48:443, by the search for similanty method of Pearson and Lipman (1988) Proc. Nat'L Acad. Sci. USA 85 :2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

[0091] By way of example, an algorithm which may be used to determine percent sequence^' identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1997) Nuc, Acids Res. 25 :3389-3402, and Altschul et al, (1990) J, Mol, Biol. 215:403-410, respectively, Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 1 1, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a ordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci, USA 89: 10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands, The BLAST algorithm is typically performed with the "low complexity" filter turned off.

[0092] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul ( 1993) Proc. Natl. Acad. Sci. USA 90:5873- 5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, or less than about 0.01, or less than about 0,001.

[0093] The term "conservatively modified variants" applies to both natural and non-natural amino acid and natural and non-natural nucleic acid sequences, and combinations thereof. With respect to particular nucleic acid sequences, "conservatively modified variants" refers to those natural and non-natural nucleic acids which encode identical or essentially identical natural and non-natural amino acid sequences, or where the natural and non-natural nucleic acid does not encode a natural and non-natural amino acid sequence, to essentially identical sequences. By way of example, because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Thus by way of example every natural or non-natural nucleic acid sequence herein which encodes a natural or non-natural polypeptide also describes every possible silent variation of the natural or non-natural nucleic acid. One of ordinary skill in the art will recognize that each codon in a natural or non-natural nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a natural and non-natural nucleic acid which encodes a natural and non- natural polypeptide is implicit in each described sequence. [0094] As to amino acid sequences, individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single natural and non-natural amino acid or a small percentage of natural and non-natural amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the deletion of an amino acid, addition of an amino acid, or substitution of a natural and non-natural amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar natural amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the methods and compositions described herein.

[0095] Conservative substitution tables providing functionally similar amino acids are known to those of ordinary skill in the art. The following eight groups each contain amino acids that are conservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) As ragine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins; Structures and Molecular Properties (W H Freeman & Co.; 2nd edition (December 1993)

[0096] The terms "cycloalkyl" and "heterocycloalkyl", by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of "alky!" and "hetero lkyl", respectively. Thus, a cycloalkyl or heterocycloalkyl include saturated, partially unsaturated and fully unsaturated ring linkages. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule, The heteroatom may include, but is not limited to, oxygen, nitrogen or sulfur. Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1- cyclohexenyl, 3-cyclohexenyl, cycloheptyl and the like. Examples of heterocycloalkyl include, but are not limited to, l-(l,2,5,6-tetrahydropyridyl), 1 -piperidinyl, 2-piperidinyl, 3- piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. Additionally, the term encompasses multicyclic structures, including but not limited to, bicyclic and tricyclic ring structures, Similarly, the term "heterocycloalkylene" by itself or as part of another molecule means a divalent radical derived from heterocycloalkyl, and the term "cycloalkylene" by itself or as part of another molecule means a divalent radical derived from cycloalkyl.

[0097] The term "cyclodextrin," as used herein, refers to cyclic carbohydrates consisting of at least six to eight glucose molecules in a ring formation. The outer part of the ring contains water soluble groups; at the center of the ring is a relatively nonpolar cavity able to accommodate small molecules,

[0098] The term "cytotoxic," as used herein, refers to a compound which harms cells,

[0099] "Denaturing agent" or "denaturant," as used herein, refers to any compound or material which will cause a reversible unfolding of a polymer. By way of example only, "denaturing agent" or "denaturants," may cause a reversible unfolding of a protein. The strength of a denaturing agent or denaturant will be determined both by the properties and the concentration of the particular denaturing agent or denaturant. By way of example, denaturing agents or denaturants include, but are not limited to, chaotropes, detergents, organic, water miscible solvents, phospholipids, or a combination thereof. Non-limiting examples of chaotropes include, but are not limited to, urea, guanidine, and sodium thiocyanate. Non-limiting examples of detergents may include, but are not limited to, strong detergents such as sodium dodecyl sulfate, or polyoxyethylene ethers (e.g. Tween or Triton detergents), Sarkosyl, mild non-ionic detergents (e.g., digitonin), mild cationic detergents such as N->2,3-(Dioleyoxy)-propyl-N,N,N-trimethylammonium, mild ionic detergents (e.g. sodium cholate or sodium deoxycholate) or zwitterionic detergents including, but not limited to, sulfobetaines (Zwittergent), 3-(3-chlolamidopropyl)dimethylammonio-l -propane sulfate (CHAPS), and 3-(3-chlolamidopropyl)dimethylammonio-2-hydroxy-l-propane sulfonate (CHAPSO). Non-limiting examples of organic, water miscible solvents include, but are not limited to, acetonitrile, lower alkanols (especially C2 - C4 alkanols such as ethanol or isopropanol), or lower alkandiols (C2 - C4 alkandiols such as ethylene-glycol) may be used as denaturants, Non-limiting examples of phospholipids include, but are not limited to, naturally occurring phospholipids such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol or synthetic phospholipid derivatives or variants such as dihexanoylphosphatidylcholine or diheptanoylphosphatidylcholine.

[00100] The term "desired functionality" as used herein refers to any group selected from a label; a dye; a polymer; a water-soluble polymer; a derivative of polyethylene glycol; a photocrosslinker; a cytotoxic compound; a drug; an affinity label; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; an antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin label; a fluorophore; a metal- containing moiety; a radioactive moiety; a novel functional group; a group that covalently or noncovalently interacts with other molecules; a photocaged moiety; an actinic radiation excitable moiety; a ligand; a photoisomerizable moiety; biotin; a biotin analogue; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an elongated side chain; a carbon-linked sugar; a redox-active agent; an amino thioacid; a toxic moiety; an isotopically labeled moiety; a biophysical probe; a phosphorescent group; a chemiluminescent group; an electron dense group; a magnetic group; an intercalating group; a chromophore; an energy transfer agent; a biologically active agent (in which case, the biologically active agent can include an agent with therapeutic activity and the non-natural amino acid polypeptide or modified non-natural amino acid can serve either as a co- therapeutic agent with the attached therapeutic agent or as a means for delivery the therapeutic agent to a desired site within an organism); a detectable label; a small molecule; an inhibitory ribonucleic acid; a radionucleotlde; a neutron-capture agent; a derivative of biotin; quantum dot(s); a nanotransmitter; a radiotransmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an ag!ycan, an allergan, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, a mimotope, a receptor, a reverse micelle, and any combination thereof.

[00101] The term "diamine", as used herein, refers to groups/molecules comprising at least two amine functional groups, including, but not limited to, a hydrazine group, an amidine group, an imine group, a 1, 1 -diamine group, a 1,2-diamine group, a 1,3-diamine group, and a 1 ,4-diamine group. In addition, such groups may be part of linear, branched, or cyclic molecules.

[00102] The term "detectable label," as used herein, refers to a label which may be observable using analytical techniques including, but not limited to, fluorescence, chemiluminescence, electron-spin resonance, ultraviolet/visible absorbance spectroscopy, mass spectrometry, nuclear magnetic resonance, magnetic resonance, and electrochemical methods.

[00103] The term "dicarbonyl" as used herein refers to a group containing at least two moieties selected from the group consisting of -C(O)-, -S(O)-, -S(0)2-, and -C(S)-, including, but not limited to, 1 ,2-dicarbonyl groups, a 1,3 -dicarbonyl groups, and 1,4- dicarbonyl groups, and groups containing a least one ketone group, and/or at least one aldehyde groups, and/or at least one ester group, and/or at least one carboxylic acid group, and/or at least one thioester group. Such dicarbonyl groups include diketones, ketoaldehydes, ketoacids, ketoesters, and ketothioesters. In addition, such groups may be part of linear, branched, or cyclic molecules. The two moieties in the dicarbonyl group may be the same or different, and may include substituents that would produce, by way of example only, an ester, a ketone, an aldehyde, a thioester, or an amide, at either of the two moieties.

[00104] The term "drug," as used herein, refers to any substance used in the prevention, diagnosis, alleviation, treatment, or cure of a disease or condition.

[00105] The term "dye," as used herein, refers to a soluble, coloring substance which contains a chromophore.

[00106] The term "effective amount," as used herein, refers to a sufficient amount of an agent or a compound being administered which will relieve to some extent one or more of the symptoms of the disease or condition being treated. The result can be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. By way of example, an agent or a compound being administered includes, but is not limited to, a natural amino acid polypeptide, non-natural amino acid polypeptide, modified natural amino acid polypeptide, or modified non-amino acid polypeptide, Compositions containing such natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, or modified non-natural amino acid polypeptides can be administered for prophylactic, enhancing, and/or therapeutic treatments. An appropriate "effective" amount in any individual case may be determined using techniques, such as a dose escalation study.

[00107] The term "electron dense group," as used herein, refers to a group which scatters electrons when irradiated with an electron beam. Such groups include, but are not limited to, ammonium molybdate, bismuth subnitrate cadmium iodide, 99%, carbohydrazide, ferric chloride hexahydrate, hexamethylene tetramine, 98.5%, indium trichloride anhydrous, lanthanum nitrate, lead acetate trihydrate, lead citrate trihydrate, lead nitrate, periodic acid, phosphomolybdic acid, phosphotungstic acid, potassium ferricyanide, potassium ferrocyanide, ruthenium red, silver nitrate, silver proteinate (Ag Assay: 8,0-8.5%) "Strong", silver tetraphenylporphin (S-TPPS), sodium chloroaurate, sodium tungstate, thallium nitrate, thiosemicarbazide (TSC), uranyl acetate, uranyl nitrate, and vanadyl sulfate.

[00108] The term "energy transfer agent," as used herein, refers to a molecule which can either donate or accept energy from another molecule. By way of example only, fluorescence resonance energy transfer (FRET) is a dipole-dipole coupling process by which the excited- state energy of a fluorescence donor molecule is non-radiatively transferred to an unexcited acceptor molecule which then fluorescently emits the donated energy at a longer wavelength, [00109] The terms "enhance" or "enhancing" means to increase or prolong either in potency or duration a desired effect, By way of example, "enhancing" the effect of therapeutic agents refers to the ability to increase or prolong, either in potency or duration, the effect of therapeutic agents on during treatment of a disease, disorder or condition. An "enhancing- effective amount," as used herein, refers to an amount adequate to enhance the effect of a therapeutic agent in the treatment of a disease, disorder or condition, When used in a patient, amounts effective for this use will depend on the severity and course of the disease, disorder or condition, previous therapy, the patient's health status and response to the drugs, and the judgment of the treating physician.

[00110] As used herein, the term "eukaryote" refers to organisms belonging to the phylogenetic domain Eucarya, including but not limited to animals (including but not limited to, mammals, insects, reptiles, birds, etc), ciliates, plants (including but not limited to, monocots, dicots, and algae), fungi, yeasts, flagellates, microsporidia, and protists,

[00111] The term "fatty acid," as used herein, refers to carboxylic acids with about C6 or longer hydrocarbon side chain.

[00112] The term "fluorophore," as used herein, refers to a molecule which upon excitation emits photons and is thereby fluorescent.

[00113] The terms "functional group", "active moiety", "activating group", "leaving group", "reactive site", "chemically reactive group" and "chemically reactive moiety," as used herein, refer to portions or units of a molecule at which chemical reactions occur. The terms are somewhat synonymous in the chemical arts and are used herein to indicate the portions of molecules that perform some function or activity and are reactive with other molecules, [00114] The term "halogen" includes fluorine, chlorine, iodine, and bromine,

[00115] The term "haloacyl," as used herein, refers to acyl groups which contain halogen moieties, including, but not limited to, -C(0)CH3, -C(0)CF3, -C(0)CH20CH3, and the like. [00116] The term "haloalkyl," as used herein, refers to alkyl groups which contain halogen moieties, including, but not limited to, -CF3 and -CH2CF3 and the like.

[00117] The term "heteroalkyl," as used herein, refers to straight or branched chain, or cyclic hydrocarbon radicals, or combinations thereof, consisting of an alkyl group and at least one heteroatom selected from the group consisting of O, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N and S and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH2-CH2-0-CH3, - CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3_} -CH2-S-CH2-CH3, -CH2-CH2,-S(0)-CH3, - CH2-CH2-S(0)2-CH3, -CH=CH-0-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, and -CH=CH- N(CH3)-CH3. In addition, up to two heteroatoms may be consecutive, such as, by way of example, -CH2-NH-OCH3 and -CH2-0-Si(CH3)3.

[00118] The terms "heterocyclic-based linkage" or "heterocycle linkage" refers to a moiety formed from the reaction of a dicarbonyl group with a diamine group. The resulting reaction product is a heterocycle, including a heteroaryl group or a heterocycloalkyl group. The resulting heterocycle group serves as a chemical link between a non-natural amino acid or non-natural amino acid polypeptide and another functional group. In one embodiment, the heterocycle linkage includes a nitrogen-containing heterocycle linkage, including by way of example only a pyrazole linkage, a pyrrole linkage, an indole linkage, a benzodiazepine linkage, and a pyrazalone linkage.

[00119] Similarly, the term "heteroalkylene" refers to a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH2-CH2-S-CH2 CH2- and -CH2-S-CH2- CH2-NH-CH2-, For heteroalkylene groups, the same or different heteroatoms can also occupy either or both of the chain termini (including but not limited to, alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, aminooxyalkylene, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. By way of example, the formula -C(0)2R' represents both -C(0)2R' and -R'C(0)2 .

[00120] The term "heteroaryl" or "heteroaromatic," as used herein, refers to aryl groups which contain at least one heteroatom selected from N, O, and S; wherein the nitrogen and sulfur atoms may be optionally oxidized, and the nitrogen atom(s) may be optionally quaternized. Heteroaryl groups may be substituted or unsubstituted. A heteroaryl group may be attached to the remainder of the molecule through a heteroatom. Non-limiting examples of heteroaryl groups include 1 -pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyI, 4- unidazolyl, pyrazinyl, 2-oxazoIy!, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3- thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3- quinolyl, and 6-quinolyl.

[00121] The term "homoallcyl," as used herein refers to alky] groups which are hydrocarbon groups,

[00122] The term "identical," as used herein, refers to two or more sequences or subsequences which are the same. In addition, the term "substantially identical," as used herein, refers to two or more sequences which have a percentage of sequential units which are the same when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using comparison algorithms or by manual alignment and visual inspection, By way of example only, two or more sequences may be "substantially identical" if the sequential units are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a specified region. Such percentages to describe the "percent identity" of two or more sequences. The identity of a sequence can exist over a region that is at least about 75-100 sequential units in length, over a region that is about 50 sequential units in length, or, where not specified, across the entire sequence, This definition also refers to the complement of a test sequence. By way of example only, two or more polypeptide sequences are identical when the amino acid residues are the same, while two or more polypeptide sequences are "substantially identical" if the amino acid residues are about 60% identical, about 65% identical, about 70% identical, about 75%> identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a specified region. The identity can exist over a region that is at least about 75 to about 100 amino acids in length, over a region that is about 50 amino acids in length, or, where not specified, across the entire sequence of a polypeptide sequence. In addition, by way of example only, two or more polynucleotide sequences are identical when the nucleic acid residues are the same, while two or more polynucleotide sequences are "substantially identical" if the nucleic acid residues are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a specified region, The identity can exist over a region that is at least about 75 to about 100 nucleic acids in length, over a region that is about 50 nucleic acids in length, or, where not specified, across the entire sequence of a polynucleotide sequence.

[00123] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters,

[00124] The term "immunogenicity," as used herein, refers to an antibody response to administration of a therapeutic drug, The immunogenicity toward therapeutic non-natural amino acid polypeptides can be obtained using quantitative and qualitative assays for detection of anti -non -natural amino acid polypeptides antibodies in biological fluids. Such assays include, but are not limited to, Radioimmunoassay (RIA), Enzyme-linked immunosorbent assay (ELISA), luminescent immunoassay (LIA), and fluorescent immunoassay (FIA). Analysis of immunogenicity toward therapeutic non-natural amino acid polypeptides involves comparing the antibody response upon administration of therapeutic non-natural amino acid polypeptides to the antibody response upon administration of therapeutic natural amino acid polypeptides,

[00125] The term "intercalating agent," also referred to as "intercalating group," as used herein, refers to a chemical that can insert into the intramolecular space of a molecule or the intermolecular space between molecules. By way of example oniy an intercalating agent or group may be a molecule which inserts into the stacked bases of the DNA double helix.

[00126] The term "isolated," as used herein, refers to separating and removing a component of interest from components not of interest, Isolated substances can be in either a dry or semi- dry state, or in solution, including but not limited to an aqueous solution. The isolated component can be in a homogeneous state or the isolated component can be a part of a pharmaceutical composition that comprises additional pharmaceutically acceptable carriers and/or excipients. Purity and homogeneity may be determined using analytical chemistry techniques including, but not limited to, polyacrylamide gel electrophoresis or high performance liquid chromatography. In addition, when a component of interest is isolated and is the predominant species present in a preparation, the component is described herein as substantially purified. The term "purified," as used herein, may refer to a component of interest which is at least 85% pure, at least 90% pure, at least 95% pure, at least 99% or greater pure. By way of example only, nucleic acids or proteins are "isolated" when such nucleic acids or proteins are free of at least some of the cellular components with which it is associated in the natural state, or that the nucleic acid or protein has been concentrated to a level greater than the concentration of its in vivo or in vitro production, Also, by way of example, a gene is isolated when separated from open reading frames which flank the gene and encode a protein other than the gene of interest.

[00127] The term "label," as used herein, refers to a substance which is incorporated into a compound and is readily detected, whereby its physical distribution may be detected and/or monitored.

[00128] The term "linkage," as used herein to refer to bonds or chemical moiety formed from a chemical reaction between the functional group of a linker and another molecule. Such bonds may include, but are not limited to, covalent linkages and non-covalent bonds, while such chemical moieties may include, but are not limited to, esters, carbonates, imines phosphate esters, hydrazones, acetals, orthoesters, peptide linkages, and oligonucleotide linkages. Hydrolytica!ly stable linkages means that the linkages are substantially stable in water and do not react with water at useful pH values, including but not limited to, under physiological conditions for an extended period of time, perhaps even indefinitely. Hydrolytically unstable or degradable linkages means that the linkages are degradable in water or in aqueous solutions, including for example, blood. Enzymatically unstable or degradable linkages means that the linkage can be degraded by one or more enzymes. By way of example only, PEG and related polymers may include degradable linkages in the polymer backbone or in the linker group between the polymer backbone and one or more of the terminal functional groups of the polymer molecule. Such degradable linkages include, but are not limited to, ester linkages formed by the reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on a biologically active agent, wherein such ester groups generally hydrolyze under physiological conditions to release the biologically active agent. Other hydrolytically degradable linkages include but are not limited to carbonate linkages; imine linkages resulted from reaction of an amine and an aldehyde; phosphate ester linkages formed by reacting an alcohol with a phosphate group; hydrazone linkages which are reaction product of a hydrazide and an aldehyde; acetal linkages that are the reaction product of an aldehyde and an alcohol; orthoester linkages that are the reaction product of a formate and an alcohol; peptide linkages formed by an amine group, including but not limited to, at an end of a polymer such as PEG, and a carboxyl group of a peptide; and oligonucleotide linkages formed by a phosphoramidite group, including but not limited to, at the end of a polymer, and a 5' hydroxyl group of an oligonucleotide.

[00129] The terms "medium" or "media," as used herein, refer to any culture medium used to grow and harvest cells and/or products expressed and/or secreted by such cells. Such "medium" or "media" include, but are not limited to, solution, solid, semi-solid, or rigid supports that may support or contain any host cell, including, by way of example, bacterial host cells, yeast host cells, insect host cells, plant host cells, eukaryotic host cells, mammalian host cells, CHO cells, prokaryotic host cells, E. coli, or Pseudomonas host cells, and cell contents. Such "medium" or "media" includes, but is not limited to, medium or media in which the host cell has been grown into which a polypeptide has been secreted, including medium either before or after a proliferation step, Such "medium" or "media" also includes, but is not limited to, buffers or reagents that contain host cell lysates, by way of example a polypeptide produced intracellularly and the host cells are lysed or disrupted to release the polypeptide.

[00130] The term "metabolite," as used herein, refers to a derivative of a compound, by way of example natural amino acid polypeptide, a non-natural amino acid polypeptide, a modified natural amino acid polypeptide, or a modified non-natural amino acid polypeptide, that is formed when the compound, by way of example natural amino acid polypeptide, non-natural amino acid polypeptide, modified natural amino acid polypeptide, or modified non-natural amino acid polypeptide, is metabolized. The term "pharmaceutically active metabolite" or "active metabolite" refers to a biologically active derivative of a compound, by way of example natural amino acid polypeptide, a non-natural amino acid polypeptide, a modified natural amino acid polypeptide, or a modified non-natural amino acid polypeptide, that is formed when such a compound, by way of example a natural amino acid polypeptide, non- natural amino acid polypeptide, modified natural amino acid polypeptide, or modified non- natural amino acid polypeptide, is metabolized.

[00131] The term "metabolized," as used herein, refers to the sum of the processes by which a particular substance is changed by an organism. Such processes include, but are not limited to, hydrolysis reactions and reactions catalyzed by enzymes. Further information on metabolism may be obtained from The Pharmacological Basis of Therapeutics, 9th Edition, McGraw-Hill ( 1996). By way of example only, metabolites of natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, or modified non-natural amino acid polypeptides may be identified either by administration of the natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, or modified non-natural amino acid polypeptides to a host and analysis of tissue samples from the host, or by incubation of natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, or modified non-natural amino acid polypeptides with hepatic cells in vitro and analysis of the resulting compounds.

[00132] The term "metal chelator," as used herein, refers to a molecule which forms a metal complex with metal ions. By way of example, such molecules may form two or more coordination bonds with a central metal ion and may form ring structures.

[00133] The term "metal-containing moiety," as used herein, refers to a group which contains a metal ion, atom or particle, Such moieties include, but are not limited to, cisplatin, chelated metals ions (such as nickel, iron, and platinum), and metal nanoparticles (such as nickel, iron, and platinum).

[00134] The term "moiety incorporating a heavy atom," as used herein, refers to a group which incorporates an ion of atom which is usually heavier than carbon. Such ions or atoms include, but are not limited to, silicon, tungsten, gold, lead, and uranium.

[00135] The term "modified," as used herein refers to the presence of a change to a natural amino acid, a non-natural amino acid, a natural amino acid polypeptide or a non-natural amino acid polypeptide. Such changes, or modifications, may be obtained by post synthesis modifications of natural amino acids, non-natural amino acids, natural amino acid polypeptides or non-natural amino acid polypeptides, or by co-translational, or by post- translational modification of natural amino acids, non-natural amino acids, natural amino acid polypeptides or non-natural amino acid polypeptides. The form "modified or unmodified" means that the natural amino acid, non-natural amino acid, natural amino acid polypeptide or non-natura! amino acid polypeptide being discussed are optionally modified, that is, he natural amino acid, non-natural amino acid, natural amino acid polypeptide or non-natural amino acid polypeptide under discussion can be modified or unmodified.

[00136] As used herein, the term "modulated serum half-life" refers to positive or negative changes in the circulating half-life of a modified biologically active molecule relative to its non-modified form. By way of example, the modified biologically active molecules include, but are not limited to, natural amino acid, non-natural amino acid, natural amino acid polypeptide or non-natural amino acid polypeptide, By way of example, serum half-life is measured by taking blood samples at various time points after administration of the biologically active molecule or modified biologically active molecule, and determining the concentration of that molecule in each sample. Correlation of the serum concentration with time allows calculation of the serum half-life. By way of example, modulated serum half-life may be an increased in serum half-life, which may enable an improved dosing regimens or avoid toxic effects. Such increases in serum may be at least about two fold, at least about three-fold, at least about five-fold, or at least about ten-fold. Methods for evaluating serum half-life are known in the art and may be used for evaluating the serum half-life of antibodies and antibody drug conjugates of the present invention.

[00137] The term "modulated therapeutic half-life," as used herein, refers to positive or negative change in the half-life of the therapeutically effective amount of a modified biologically active molecule, relative to its non-modified form. By way of example, the modified biologically active molecules include, but are not limited to, natural amino acid, non-natural amino acid, natural amino acid polypeptide or non-natural amino acid polypeptide. By way of example, therapeutic half-life is measured by measuring pharmacokinetic and/or pharmacodynamic properties of the molecule at various time points after administration. Increased therapeutic half-life may enable a particular beneficial dosing regimen, a particular beneficial total dose, or avoids an undesired effect. By way of example, the increased therapeutic half-life may result from increased potency, increased or decreased binding of the modified molecule to its target, an increase or decrease in another parameter or mechanism of action of the non-modified molecule, or an increased or decreased breakdown of the molecules by enzymes such as, by way of example only, proteases. Methods for evaluating therapeutic half-life are known in the art and may be used for evaluating the therapeutic half-life of antibodies and antibody drug conjugates of the present invention.

[0 138] The term "nanoparticle," as used herein, refers to a particle which has a particle size between about 500 nm to about 1 mil.

[00139] The term "near-stoichiometric," as used herein, refers to the ratio of the moles of compounds participating in a chemical reaction being about 0.75 to about 1.5.

[00140] As used herein, the term "non-eukaryote" refers to non-eukaryotic organisms, By way of example, a non-eukaryotic organism may belong to the Eubacteria, (which includes but is not limited to, Escherichia coli, Thermus thermophilus, or Bacillus stearothermophilus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas putida), phylogenetic domain, or the Archaea, which includes, but is not limited to, Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, or Halobacterium such as Haloferax volcanii and Halobacterium species NRC-1, or phylogenetic domain, [00141] A "non-natural amino acid" refers to an amino acid that is not one of the 20 common amino acids or pyrotysine or selenocysteine, Other terms that may be used synonymously with the term "non-natural amino acid" is "non-naturally encoded amino acid," "unnatural amino acid," "non-naturally-occurring amino acid," and variously hyphenated and non-hyphenated versions thereof, The term "non-natural amino acid" includes, but is not limited to, amino acids which occur naturally by modification of a naturally encoded amino acid (including but not limited to, the 20 common amino acids or pyrrolysine and selenocysteine) but are not themselves incorporated into a growing polypeptide chain by the translation complex. Examples of naturally-occurring amino acids that are not naturally-encoded include, but are not limited to, N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine. Additionally, the term "non- natural amino acid" includes, but is not limited to, amino acids which do not occur naturally and may be obtained synthetically or may be obtained by modification of non-natural amino acids.

[00142] The term "nucleic acid," as used herein, refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in either single- or double-stranded form. By way of example only, such nucleic acids and nucleic acid polymers include, but are not limited to, (i) analogues of natural nucleotides which have similar binding properties as a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides; (ii) oligonucleotide analogs including, but are not limited to, PNA (peptidonucleic acid), analogs of DNA used in antisense technology (phosphorothioates, phosphoroamidates, and the like); (iii) conservatively modified variants thereof (including but not limited to, degenerate codon substitutions) and complementary sequences and sequence explicitly indicated. By way of example, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al„ Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al„ J. Biol. Chera. 260:2605- 2608 (1985); and Rossolini et ai., Mol. Cell. Probes 8:91-98 (1994)).

[00143] The term "oxidizing agent," as used herein, refers to a compound or material which is capable of removing an electron from a compound being oxidized, By way of example oxidizing agents include, but are not limited to, oxidized glutathione, cystine, cystamine, oxidized dithiothreitol, oxidized erythreitol, and oxygen. A wide variety of oxidizing agents are suitable for use in the methods and compositions described herein. [00144] The term "pharmaceutically acceptable", as used herein, refers to a material, including but not limited, to a salt, carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively nontoxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[00145] The term "photoaffinity label," as used herein, refers to a label with a group, which, upon exposure to light, forms a linkage with a molecule for which the label has an affinity. By way of example only, such a linkage may be covalent or non-covalent,

[00146] The term "photocaged moiety," as used herein, refers to a group which, upon illum ination at certain wavelengths, covalently or non-covalently binds other ions or molecules.

[00147] The term "photocleavable group," as used herein, refers to a group which breaks upon exposure to light,

[00148] The term "photocrosslinker," as used herein, refers to a compound comprising two or more functional groups which, upon exposure to light, are reactive and form a covalent or non-covalent linkage with two or more monomeric or polymeric molecules.

[00149] The term "photoisomerizable moiety," as used herein, refers to a group wherein upon illumination with light changes from one isomeric form to another.

[00150] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, That is, a description directed to a polypeptide applies equally to a description of a peptide and a description of a protein, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-natural amino acid. Additionally, such "polypeptides," "peptides" and "proteins" include amino acid chains of any length, including full length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

[00151] The term "post-translationally modified" refers to any modification of a natural or non-natural amino acid which occurs after such an amino acid has been translationally incorporated into a polypeptide chain, Such modifications include, but are not limited to, co- translational in vivo modifications, co-translational in vitro modifications (such as in a cell- free translation system), post-translational in vivo modifications, and post-translational in vitro modifications,

[00152] The terms "prodrug" or "pharmaceutically acceptable prodrug," as used herein, refers to an agent that is converted into the parent drug in vivo or in vitro, wherein which does not abrogate the biological activity or properties of the drug, and is relatively nontoxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. Prodrugs are generally drug precursors that, following administration to a subject and subsequent absorption, are converted to an active, or a more active species via some process, such as conversion by a metabolic pathway, Some prodrugs have a chemical group present on the prodrug that renders it less active and/or confers solubility or some other property to the drug. Once the chemical group has been cleaved and/or modified from the prodrug the active drug is generated. Prodrugs are converted into active drug within the body through enzymatic or non-enzymatic reactions. Prodrugs may provide improved physiochemica! properties such as better solubility, enhanced delivery characteristics, such as specifically targeting a particular cell, tissue, organ or ligand, and improved therapeutic value of the drug. The benefits of such prodrugs include, but are not limited to, (i) ease of administration compared with the parent drug; (ii) the prodrug may be bioavailable by oral administration whereas the parent is not; and (iii) the prodrug may also have improved solubility in pharmaceutical compositions compared with the parent drug. A pro-drug includes a pharmacologically inactive, or reduced-activity, derivative of an active drug, Prodrugs may be designed to modulate the amount of a drug or biologically active molecule that reaches a desired site of action through the manipulation of the properties of a drug, such as physiochemical, biopharmaceutical, or pharmacokinetic properties. An example, without limitation, of a prodrug would be a non-natural amino acid polypeptide which is administered as an ester (the "prodrug") to facilitate transmittal across a cell membrane where water solubility is detrimental to mobility but which then is metabolically hydrolyzed to the carboxylic acid, the active entity, once inside the cell where water solubility is beneficial. Prodrugs may be designed as reversible drug derivatives, for use as modifiers to enhance drug transport to site-specific tissues.

[00153] The term "prophylactically effective amount," as used herein, refers that amount of a composition containing at least one non-natural amino acid polypeptide or at least one modified non-natural amino acid polypeptide prophylactically applied to a patient which will relieve to some extent one or more of the symptoms of a disease, condition or disorder being treated. In such prophylactic applications, such amounts may depend on the patient's state of health, weight, and the like. It is considered well within the skill of the art for one to determine such prophylactically effective amounts by routine experimentation, including, but not limited to, a dose escalation clinical trial. [00154] The term "protected," as used herein, refers to the presence of a "protecting group" or moiety that prevents reaction of the chemically reactive functional group under certain reaction conditions. The protecting group will vary depending on the type of chemically reactive group being protected. By way of example only, (i) if the chemically reactive group is an amine or a hydrazide, the protecting group may be selected from tert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc); (ii) if the chemically reactive group is a thiol, the protecting group may be orthopyridyldisulfide; and (iii) if the chemically reactive group is a carboxylic acid, such as butanoic or propionic acid, or a hydroxyl group, the protecting group may be benzyl or an alkyl group such as methyl, ethyl, or tert-butyl.

[00155] By way of example only, blocking/protecting groups may be selected from:

ally! Bn Cbz alloc Me

[00156] Additionally, protecting groups include, but are not limited to, including photolabile groups such as Nvoc and MeNvoc and other protecting groups known in the art, Other protecting groups are described in Greene and Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, NY, 1999, which is incorporated herein by reference in its entirety.

[00157] The term "radioactive moiety," as used herein, refers to a group whose nuclei spontaneously give off nuclear radiation, such as alpha, beta, or gamma particles; wherein, alpha particles are helium nuclei, beta particles are electrons, and gamma particles are high energy photons.

[00158] The term "reactive compound," as used herein, refers to a compound which under appropriate conditions is reactive toward another atom, molecule or compound. [00159] The term "recombinant host cell," also referred to as "host cell," refers to a cell which includes an exogenous polynucleotide, wherein the methods used to insert the exogenous polynucleotide into a cell include, but are not limited to, direct uptake, transduction, f-mating, or other methods known in the art to create recombinant host cells. By way of example only, such exogenous polynucleotide may be a nonintegrated vector, including but not limited to a plasmid, or may be integrated into the host genome,

[00160] The term "redox-active agent," as used herein, refers to a molecule which oxidizes or reduces another molecule, whereby the redox active agent becomes reduced or oxidized. Examples of redox active agent include, but are not limited to, ferrocene, quinones, Ru2+/3+ complexes, Co2+/3+ complexes, and Os2+/3+ complexes.

[00161] The term "reducing agent," as used herein, refers to a compound or material which is capable of adding an electron to a compound being reduced. By way of example reducing agents include, but are not limited to, dithiothreitol (DTT), 2-mercaptoethanol, dithioerythritol, cysteine, cysteamine (2-aminoethanethiol), and reduced glutathione. Such reducing agents may be used, by way of example only, to maintain sulfhydryl groups in the reduced state and to reduce intra- or intermolecular disulfide bonds,

[00162] "Refolding," as used herein describes any process, reaction or method which transforms an improperly folded or unfolded state to a native or properly folded conformation. By way of example only, refolding transforms disulfide bond containing polypeptides from an improperly folded or unfolded state to a native or properly folded conformation with respect to disulfide bonds. Such disulfide bond containing polypeptides may be natural amino acid polypeptides or non-natural amino acid polypeptides,

[00163] The term "resin," as used herein, refers to high molecular weight, insoluble polymer beads. By way of example only, such beads may be used as supports for solid phase peptide synthesis, or sites for attachment of molecules prior to purification.

[00164] The term "saccharide," as used herein, refers to a series of carbohydrates including but not limited to sugars, monosaccharides, oligosaccharides, and polysaccharides,

[00165] The term "safety" or "safety profile," as used herein, refers to side effects that might be related to administration of a drug relative to the number of times the drug has been administered. By way of example, a drug which has been administered many times and produced only mild or no side effects is said to have an excellent safety profile. A non- limiting example of a method to evaluate the safety profile is given in example 26. This method may be used for evaluating the safety profile of any polypeptide. [00166] The phrase "selectively hybridizes to" or "specifically hybridizes to," as used herein, refers to the binding, duplexing, or hybridizing of a molecule to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture including but not limited to, total cellular or library DNA or RNA.

[00167] The term "spin label," as used herein, refers to molecules which contain an atom or a group of atoms exhibiting an unpaired electron spin (i.e. a stable paramagnetic group) that can be detected by electron spin resonance spectroscopy and can be attached to another molecule. Such spin-label molecules include, but are not limited to, nitryl radicals and nitroxides, and may be single spin-labels or double spin-labels.

[00168] The term "stoichiometric," as used herein, refers to the ratio of the moles of compounds participating in a chemical reaction being about 0.9 to about 1.1.

[00169] The term "stoichiometric-like," as used herein, refers to a chemical reaction which becomes stoichiometric or near-stoichiometric upon changes in reaction conditions or in the presence of additives. Such changes in reaction conditions include, but are not limited to, an increase in temperature or change in pH. Such additives include, but are not limited to, accelerants.

[00170] The phrase "stringent hybridization conditions" refers to hybridization of sequences of DNA, RNA, PNA or other nucleic acid mimics, or combinations thereof, under conditions of low ionic strength and high temperature, By way of example, under stringent conditions a probe will hybridize to its target subsequence in a complex mixture of nucleic acid (including but not limited to, total cellular or library DNA or RNA) but does not hybridize to other sequences in the complex mixture, Stringent conditions are sequence-dependent and will be different in different circumstances. By way of example, longer sequences hybridize specifically at higher temperatures. Stringent hybridization conditions include, but are not limited to, (i) about 5-10 oC lower than the thermal melting point (Tm) for the specific sequence at a defined, ionic strength and pH; (ii) the salt concentration is about 0.01 to about 1 ,0 M at about p^'H 7.0 to about pH 8.3 and the temperature is at least about 30 oC for short probes (including but not limited to, about 10 to about 50 nucleotides) and at least about 60 oC for long probes (including but not limited to, greater than 50 nucleotides); (iii) the addition of destabilizing agents including, but not limited to, formamide, (iv) 50% formamide, 5X SSC, and 1 % SDS, incubating at 42 oC, or 5X SSC, about 1% SDS, incubating at 65 oC, with wash in 0.2X SSC, and about 0.1% SDS at 65 oC for between about 5 minutes to about 120 minutes. By way of example only, detection of selective or specific hybridization, includes, but is not limited to, a positive signal at least two times background. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, 'Overview of principles of hybridization and the strategy of nucleic acid assays" (1993).

[00171] The term "subject" as used herein, refers to an animal which is the object of treatment, observation or experiment. By way of example only, a subject may be, but is not limited to, a mammal including, but not limited to, a human.

[00172] The term "substantially purified," as used herein, refers to a component of interest that may be substantially or essentially free of other components which normally accompany or interact with the component of interest prior to purification. By way of example only, a component of interest may be "substantially purified" when the preparation of the component of interest contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating components. Thus, a "substantially purified" component of interest may have a purity level of about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%o, about 99% or greater. By way of example only, a natural amino acid polypeptide or a non-natural amino acid polypeptide may be purified from a native cell, or host cell in the case of recombinantly produced natural amino acid polypeptides or non-natural amino acid polypeptides. By way of example a preparation of a natural amino acid polypeptide or a non- natural amino acid polypeptide may be "substantially purified" when the preparation contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%), less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about !% (by dry weight) of contaminating material. By way of example when a natural amino acid polypeptide or a non-natural amino acid polypeptide is recombinantly produced by host cells, the natural amino acid polypeptide or non-natural amino acid polypeptide may be present at about 30%, about 25%), about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1%> or less of the dry weight of the cells. By way of example when a natural amino acid polypeptide or a non-natural amino acid polypeptide is recombinantly produced by host cells, the natural amino acid polypeptide or non-natural amino acid polypeptide may be present in the culture medium at about 5g/L, about 4g/L, about 3g/L, about 2g/L, about l g/L, about 750mg/L, about 500mg L, about 250mg/L, about lOOmg/L, about 50mg/L, about 1.0m g/L, or about lmg/L or less of the dry weight of the cells. By way of example, "substantially purified" natural amino acid polypeptides or non-natural amino acid polypeptides may have a purity level of about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater as determined by appropriate methods, including, but not limited to, SDS/PAGE analysis, RP- HPLC, SEC, and capillary electrophoresis.

[00173] The term "substituents" also referred to as "non-interfering substituents" "refers to groups which may be used to replace another group on a molecule. Such groups include, but are not limited to, halo, C1-C10 alkyl, C2-C10 alkenyl, C2-C 10 alkynyl, C1-C10 alkoxy, C5- C12 aralkyl, C3-C12 cycloalkyl, C4-C12 cycloaikenyl, phenyl, substituted phenyl, toluolyl, xylenyi, biphenyl, C2-C12 alkoxyalkyl, C5-C12 alkoxyaryl, C5-C12 aryloxyalkyl, C7-C12 oxyaryl, C1-C6 alkyl sulfinyl, C1-C10 alkylsulfonyl, -(CH2)m-O-(Cl-C 10 alkyl) wherein m is from 1 to 8, aryl, substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclic radical, substituted heterocyclic radical, nitroalkyl, -N02, -CN, -NRC(O)-(Cl -C10 alkyl), -C(0)-(C 1 - C10 alkyl), C2-C10 alkthioalkyl, -C(O)O-(Cl-C 10 alkyl), -OH, -S02, =S, -COOH, -NR2, carbonyl, -C(O)-(C l-C10 alkyl)-CF3, -C(0)-CF3, -C(0)NR2, -(C 1-C10 aryl)-S-(C6-C10 aryl), -C(O)-(C6-C 10 aryl), -(CH2)m-O-(CH2)m-O-(C l-C 10 alkyl) wherein each m is from 1 to 8, -C(0)NR2, -C(S)N 2, -S02NR2, -NRC(0)NR2, -NRC(S)NR2, salts thereof, and the like, Each R group in the preceding list includes, but is not limited to, H, alkyl or substituted alkyl, aryl or substituted aryl, or alkary!, Where substituent groups are specified by their conventional chemical formulas, written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left; for example, -CH20- is equivalent to -OCH2-.

[00174] By way of example only, substituents for alkyl and heteroalkyl radicals (including those groups referred to as alkyl en e, alkenyl, heteroalkylene, hetero alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloaikenyl, and heterocycloalkenyl) includes, but is not limited to: -OR, =0, =NR, =N-OR, -NR2, -SR, -halogen, -SiR3, -OC(0)R, -C(0)R, -C02R, - CONR2, -OC(0)NR2, -NRC(0)R, ~NRC(0)NR2, -NR(0)2R, -NR-C(NR2)= R, -S(0)R, - S(0)2R, -S(0)2NR2, -NRS02R, -CN and -N02. Each R group in the preceding list includes, but is not limited to, hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, including but not limited to, aryl substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or aralkyl groups, When two R groups are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR2 is meant to include, but not be limited to, 1 - pyrrolidinyl and 4-morpholinyl. [00175] By way of example, substituents for aryl and heteroaryl groups include, but are not limited to, -OR, =0, =NR_; =N-OR₅ -NR2, -SR, -halogen, -SiR3, -OC(0)R, -C(0)R, -C02R, - CONR2, -OC(0)NR2, -NRC(0)R, -NRC(0)NR2, -NR(0)2R, -NR-C(NR2)=NR, -S(0)R, - S(0)2R, -S(0)2NR2, -NRS02R, -CN, -N02, -R₃ -N3, -CH(Ph)2, fluoro(Cl-C4)alkoxy, and fluoro(C l -C4)alkyl, in a number ranging from zero to the total number of open valences on the aromatic ring system; and where each R group in the preceding list includes, but is not limited to, hydrogen, alky], heteroalkyl, aryl and heteroaryl,

[00176] The term "therapeutically effective amount," as used herein, refers to the amount of a composition containing at least one non-natural amino acid polypeptide and/or at least one modified non-natural amino acid polypeptide administered to a patient already suffering from a disease, condition or disorder, sufficient to cure or at least partially arrest, or relieve to some extent one or more of the symptoms of the disease, disorder or condition being treated. The effectiveness of such compositions depend conditions including, but not limited to, the severity and course of the disease, disorder or condition, previous therapy, the patient's health status and response to the drugs, and the judgment of the treating physician. By way of example only, therapeutically effective amounts may be determined by routine experimentation, including but not limited to a dose escalation clinical trial.

[00177] The term "thioalkoxy," as used herein, refers to sulfur containing alkyl groups linked to molecules via an oxygen atom.

[00178] The term "thermal melting point" or Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of probes complementary to a target hybridize to the target sequence at equilibrium.

[00179] The term "toxic moiety" or "toxic group" as used herein, refers to a. compound which can cause harm, disturbances, or death. Toxic moieties include, but are not limited to, NCAl , auristatin, DNA minor groove binding agent, DNA minor groove alkylating agent, enediyne, lexitropsin, duocarmycm, taxane, puromycin, dolastatin, maytansinoid, vinca alkaloid, AFP, MMAF, MMAE, AEB, AEVB, auristatin E, paclitaxel, docetaxel, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, dolastatin-10, echinomycin, combretatstatin, chalicheamicin, maytansine, DM-1, netropsin, podophyllotoxin (e.g. etoposide, teniposide, etc.), baccatin and its derivatives, anti-tubuUn agents, cryptophysin, combretastatin, auristatin E, vincristine, vinblastine, vindesine, vinorelbine, VP-16, camptothecin, epothilone A, epothilone B, nocodazole, colchicines, colcimid, estramustine, cemadotin, discodermolide, maytansine, eleutherobin, mechlorethamine, cyclophosphamide, melphalan, carmustine, lomustine, semustine, streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozo!omide, ytarabine, cytosine arabinoside, fluorouracil, floxuridine, 6-thioguanine, 6- mercaptopurine, pentostatin, 5-fluorouracil, methotrexate, 10-propargyl-5,8-dideazafolate, 5,8-dideazatetrahydrofolic acid, leucovorin, fludarabine phosphate, pentostatine, gemcitabine, Ara-C, paclitaxel, docetaxel, deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine, brequinar, antibiotics (e.g., anthracyclme, gentamicin, cefalotin, vancomycin, telavancin, daptomycin, azithromycin, erythromycin, rocithromycin, furazolidone, amoxicillin, ampicillin, carbenicillin, flucloxacillin, methicillin, penicillin, ciprofloxacin, moxifloxacin, ofloxacin, doxycycline, minocycline, oxytetracycline, tetracycline, streptomycin, rifabutin, ethambutol, rifaximin, etc.), antiviral drugs (e.g., abacavir, acyclovir, ampligen, cidofovir, delavirdine, didanosine, efavirenz, entecavir, fosfonet, ganciclovir, ibacitabine, imunovir, idoxuridine, inosine, lopinavir, methisazone, nexavir, nevirapine, oseltamivir, penciclovir, stavudine, trifluridine, truvada, valaciclovir, zanamivir, etc), daunorubicin hydrochloride, daunomycin, rubidomycin, cerubidine, idarubicin, doxorubicin, epirubicin and morpholino derivatives, phenoxizone biscyclopeptides (e.g., dactinomycin), basic glycopeptides (e.g., bleomycin), anthr¾quinone glycosides (e.g., plicamycin, mithramycin), anthracenediones (e.g., mitoxantrone), azirinopyrrolo indolediones (e.g., mitomycin), macrocyclic immunosuppressants (e.g., cyclosporine, FK-506, tacrolimus, prograf, rapamycin etc.), navelbene, CPT-1 1, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, droloxafine, allocolchicine, Halichondrin B, colchicine, colchicine derivatives , maytansine, rhizoxin, paclitaxel, paclitaxel derivatives, docetaxel, thiocolchicine, trityl cysterin, vinblastine sulfate, vincristine sulfate, cisplatin, carboplatin, hydroxyurea, N- methylhydrazine, epidophyllotoxin, procarbazine, mitoxantrone, leucovorin, and tegafur, "Taxanes" include paclitaxel, as well as any active taxane derivative or pro-drug, Chemotherapeutic agents such as erlotinib (TARCEVA.RTM., Genentech/OSI Pharm.), bortezomib (VELCADE.RTM., Millenium Pharm.), fulvestrant (FASLODEX.RTM., AstraZeneca), sutent (SU11248, Pfizer), letrozole (FEMARA.RTM., Novartis), imatinib mesylate (GLEEVEC.RTM, Novartis), PTK787/ZK 222584 (Novartis), oxaliplatin (Eloxatin.RTM,, Sanofi), 5-FU (5-fluorouracil), leucovorin, Rapamycin (Sirolimus, RAPAMUNE.RTM., Wyeth), Iapatinib (TYKERB.RTM,, GSK572016, GlaxoSmithKline), lonafarnib (SCH 66336), sorafenib (BAY43-9006, Bayer Labs,), and gefitinib (IRESSA.RTM,, AstraZeneca), AG1478, AG1571 (SU 5271 ; Sugen), alkylating agents such as thiotepa and CYTOXAN. RTM, cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; antifolate antineoplastic such as pemetrexed (ALIMTA.RTM. Eli Lilly), aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methy!amelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB 1-TM1); eleutherobin; pan crati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics, calicheamicin, calicheamicin gammall and calicheamicin omegall ; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomysins, actinomycin, anthramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN.RTM. doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxombicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puTomycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptop rine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK.RTM. polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel (TAXOL.RTM., Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE.TM, Cremophor-free, albumin, nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, 111.), and TAXOTERE.RTM. doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR.RTM. gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; NAVELBINE.RTM. vinorelbine; novantrone; teniposide; edatrexate; daunomycm; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above,

[00180] The terms "treat," "treating" or "treatment", as used herein, include alleviating, abating or ameliorating a disease or condition symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition. The terms "treat," "treating" or "treatment", include, but are not limited to, prophylactic and/or therapeutic treatments.

[00181] As used herein, the term "water soluble polymer" refers to any polymer that is soluble in aqueous solvents, Such water soluble polymers include, but are not limited to, polyethylene glycol, polyethylene glycol propionaldehyde, mono CI -CIO alkoxy or aryloxy derivatives thereof (described in U.S. Patent No, 5,252,714 which is incorporated by reference herein), monomethoxy-polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, polyamino acids, divinylether maleic anhydride, N-(2-Hydroxypropyl)- methacrylamide, dextran, dextran derivatives including dextran sulfate, polypropylene glycol, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyol, heparin, heparin fragments, polysaccharides, oligosaccharides, glycans, cellulose and cellulose derivatives, including but not limited to methylcellulose and carboxymethyl cellulose, serum albumin, starch and starch derivatives, polypeptides, polyalkylene glycol and derivatives thereof, copolymers of polyalkylene glycols and derivatives thereof, polyvinyl ethyl ethers, and alpha-beta-poly[(2~hydroxyethy!)-DL-aspartamide, and the like, or mixtures thereof. By way of example only, coupling of such water soluble polymers to natural amino acid polypeptides or non-natural polypeptides may result in changes including, but not limited to, increased water solubility, increased or modulated serum half-life, increased or modulated therapeutic half-life relative to the unmodified form, increased bioavailability, modulated biological activity, extended circulation time, modulated immunogenicity, modulated physical association characteristics including, but not limited to, aggregation and multimer formation, altered receptor binding, altered binding to one or more binding partners, and altered receptor dhnerization or multimerization. In addition, such water soluble polymers may or may not have their own biological activity,

[00182] Unless otherwise indicated, conventional methods of mass spectroscopy, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art are employed.

[00183] Compounds, (including, but not limited to non-natural amino acids, non-natural amino acid polypeptides, modified non-natural amino acid polypeptides, and reagents for producing the aforementioned compounds) presented herein include isotopically-labeled compounds, which are identical to those recited in the various formulas and structures presented herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the present compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, such as 2H, 3H, 13C, 14C, 15N, I SO, 170, 35S, 18F, 36C1, respectively. Certain isotopically-labeled compounds described herein, for example those into which radioactive isotopes such as 3H and 14C are incorporated, are useful in drug and/or substrate tissue distribution assays, Further, substitution with isotopes such as deuterium, i.e., 2H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements.

[00184] Some of the compounds herein (including, but not limited to non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides, and reagents for producing the aforementioned compounds) have asymmetric carbon atoms and can therefore exist as enantiomers or diastereomers. Diasteromeric mixtures can be separated into their individual diastereomers on the basis of their physical chemical differences by methods known, for example, by chromatography and/or fractional crystallization. Enantiomers can be separated by converting the enantiomeric mixture into a diastereomeric mixture by reaction with an appropriate optically active compound (e.g., alcohol), separating the diastereomers and converting (e.g., hydrolyzing) the individual diastereomers to the corresponding pure enantiomers. All such isomers, including diastereomers, enantiomers, and mixtures thereof are considered as part of the compositions described herein.

[00185] In additional or further embodiments, the compounds described herein (including, but not limited to non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides, and reagents for producing the aforementioned compounds) are used in the form of pro-drugs. In additional or further embodiments, the compounds described herein (including, but not limited to non-natural amino acids, non- natural amino acid polypeptides and modified non-natural amino acid polypeptides, and reagents for producing the aforementioned compounds) are metabolized upon administration to an organism in need to produce a metabolite that is then used to produce a desired effect, including a desired therapeutic effect. In further or additional embodiments are active metabolites of non-natural amino acids and "modified or unmodified" non-natural amino acid polypeptides.

[00186] The methods and formulations described herein include the use of M-oxides, crystalline forms (also known as polymorphs), or pharmaceutically acceptable salts of non- natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides. In certain embodiments, non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides may exist as tautomers. All tautomers are included within the scope of the non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides presented herein. In addition, the non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides described herein can exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like, The solvated forms of the non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides presented herein are also considered to be disclosed herein.

[00187] Some of the compounds herein (including, but not limited to non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides and reagents for producing the aforementioned compounds) may exist in several tautomeric forms. All such tautomeric forms are considered as part of the compositions described herein. Also, for example all enol-keto forms of any compounds (including, but not limited to non-natural amino acids, non-natural amino acid polypeptides and modified non- natural amino acid polypeptides and reagents for producing the aforementioned compounds) herein are considered as part of the compositions described herein.

[00188] Some of the compounds herein (including, but not limited to non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides and reagents for producing either of the aforementioned compounds) are acidic and may form a salt with a pharmaceutically acceptable cation. Some of the compounds herein (including, but not limited to non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides and reagents for producing the aforementioned compounds) can be basic and accordingly, may form a salt with a pharmaceutically acceptable anion. All such salts, including di-salts are within the scope of the compositions described herein and they can be prepared by conventional methods. For example, salts can be prepared by contacting the acidic and basic entities, in either an aqueous, non-aqueous or partially aqueous medium. The salts are recovered by using at least one of the following techniques: filtration, precipitation with a non-solvent followed by filtration, evaporation of the solvent, or, in the case of aqueous solutions, lyophilization.

[00189] Pharmaceutically acceptable salts of the non-natural amino acid polypeptides disclosed herein may be formed when an acidic proton present in the parent non-natural amino acid polypeptides either is replaced by a metal ion, by way of example an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base. In addition, the salt forms of the disclosed non-natural amino acid polypeptides can be prepared using salts of the starting materials or intermediates. The non-natural amino acid polypeptides described herein may be prepared as a pharmaceutically acceptable acid addition salt (which is a type of a pharmaceutically acceptable salt) by reacting the free base form of non-natural amino acid polypeptides described herein with a pharmaceutically acceptable inorganic or organic acid, Alternatively, the non-natural amino acid polypeptides described herein may be prepared as pharmaceutically acceptable base addition salts (which are a type of a pharmaceutically acceptable salt) by reacting the free acid form of non-natural amino acid polypeptides described herein with a pharmaceutically acceptable inorganic or organic base.

[00190] The type of pharmaceutical acceptable salts, include, but are not limited to: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2- hydroxyethanesulfonic acid, benzenesulfonic acid, 2 -naphthalene sulfonic acid, 4- methylbicyclo-[2.2.2]oct-2-ene-l-carboxylic acid, glucoheptonic acid, 4,4'-methylenebis-(3- hydroxy-2-ene-l -carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N- methylglucamine, and the like. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like.

[00191] The corresponding counterions of the non-natural amino acid polypeptide pharmaceutical acceptable salts may be analyzed and identified using various methods including, but not limited to, ion exchange chromatography, ion chromatography, capillary electrophoresis, inductively coupled plasma, atomic absorption spectroscopy, mass spectrometry, or any combination thereof. In addition, the therapeutic activity of such non- natural amino acid polypeptide pharmaceutical acceptable salts may be tested using the techniques and methods described in examples 87-91.

[00192] It should be understood that a reference to a salt includes the solvent addition forms or crystal forms thereof, particularly solvates or polymorphs. Solvates contain either stoichiometric or non-stoichiometric amounts of a solvent, and are often formed during the process of crystallization with pharmaceutically acceptable solvents such as water, ethanol, and the like. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol. Polymorphs include the different crystal packing arrangements of the same elemental composition of a compound. Polymorphs usually have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optical and electrical properties, stability, and solubility. Various factors such as the recrystallization solvent, rate of crystallization, and storage temperature may cause a single crystal form to dominate.

[00193] The screening and characterization of non-natural amino acid polypeptide pharmaceutical acceptable salts polymorphs and/or solvates may be accomplished using a variety of techniques including, but not limited to, thermal analysis, x-ray diffraction, spectroscopy, vapor sorption, and microscopy. Thermal analysis methods address thermo chemical degradation or thermo physical processes including, but not limited to, polymorphic transitions, and such methods are used to analyze the relationships between polymorphic forms, determine weight loss, to find the glass transition temperature, or for excipient compatibility studies. Such methods include, but are not limited to, Differential scanning calorimetry (DSC), Modulated Differential Scanning Calorimetry (MDCS), Thermogravimetric analysis (TGA), and Thermogravi-metric and Infrared analysis (TG/IR). X-ray diffraction methods include, but are not limited to, single crystal and powder diffractometers and synchrotron sources. The various spectroscopic techniques used include, but are not limited to, Raman, FTIR, UVIS, and NMR (liquid and solid state), The various microscopy techniques include, but are not limited to, polarized light microscopy, Scanning Electron Microscopy (SEM) with Energy Dispersive X-Ray Analysis (EDX), Environmental Scanning Electron Microscopy with EDX (in gas or water vapor atmosphere), IR microscopy, and Raman microscopy,

INCORPORATION BY REFERENCE

[00194] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

DETAILED DESCRIPTION OF THE INVENTION

[00195] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby,

I, Introduction

[00196] Recently, an entirely new technology in the protein sciences has been reported, which promises to overcome many of the limitations associated with site-specific modifications of proteins. Specifically, new components have been added to the protein biosynthetic machinery of the prokaryote Escherichia coli (E. coli) (e.g., L. Wang, et al., (2001 ), Science 292:498-500) and the eukaryote Sacchromyces cerevisiae (S. cerevisiae) (e.g., J. Chin et al, Science 301 :964-7 (2003)), which has enabled the incorporation of non- natural amino acids to proteins in vivo, A number of new amino acids with novel chemical, physical or biological properties, including photoaffmity labels and photoisomerizable amino acids, keto amino acids, and glycosylated amino acids have been incorporated efficiently and with high fidelity into proteins in E, coli and in yeast in response to the amber codon, TAG, using this methodology. See, e.g., J. W. Chin et al,, (2002), Journal of the American Chemical Society 124:9026-9027 (incorporated by reference in its entirety); J. W. Chin, & P. G, Schultz, (2002), ChemBioChem 3(1 1): 1 135-1137 (incorporated by reference in its entirety); J. W. Chin, et al., (2002), PNAS United States of America 99(17): 11020-1 1024 (incorporated by reference in its entirety); and, L. Wang, & P. G. Schultz, (2002), Chem. Comm., 1 -1 1 (incorporated by reference in its entirety), These studies have demonstrated that it is possible to selectively and routinely introduce chemical functional groups that are not found in proteins, that are chemically inert to all of the functional groups found in the 20 common, genetically-encoded amino acids and that may be used to react efficiently and selectively to form stable covalent linkages.

II. Overview

[00197] Disclosed herein are cancer-reactive antibodies with one or more non-naturally encoded amino acids; methods for tagging cancer cells in a patient comprising treating the patient with one or more cancer-reactive antibodies; an anti-tag chimeric antigen receptor- expressing (AT-CAR) T cell system comprising a) tagging cancer cells in a patient and b) administering to the patient, either simultaneously or subsequently, chimeric antigen receptor-expressing T cells (CAR T cells), in some embodiments with one or more non- naturally encoded amino acids within the CAR-T cell, wherein the CAR-T cells bind the tagged cancer cells.

[00198] In some embodiments, the cancer-reactive antibody comprises one or more post- translational modifications, In some embodiments, the cancer-reactive antibody is linked to a linker, polymer, or biologically active molecule. In some embodiments, the cancer-reactive antibody is linked to a bifunctional polymer, Afunctional linker, or at least one additional cancer-reactive antibody. In some embodiments, the cancer-reactive antibody is linked to a tag, In some embodiments of the present invention, more than one cancer-reactive antibody is administered to a patient in need thereof. In other embodiments of the present invention, the tag of each formulation of c ncer-reactive antibodies is the same or different and the tag is selected from the group consisting of fluorescein isothiocyanate (FITC), streptavidin, biotin, histidine, dinitrophenol, peridinin chlorophyll protein complex, green fluorescent protein, phycoerythrin (PE), horse radish peroxidase, palmitoylation, nitrosylation, alkalanine phosphatase, glucose oxidase, and maltose binding protein. In other embodiments of the present invention, the antibody of the cancer-reactive antibodies is an antibody or an antigen- binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof is cetuximab, nimotuzumab, panitumumab, retuximab, omalizumab, tositumomab, trastuzumab, gemtuzumab, alemtuzumab, bevacuzimab or an antigen-binding fragment of any one thereof.

[00199] In some embodiments, the cancer-reactive antibody comprises a substitution, addition or deletion that modulates affinity of the cancer-reactive antibody polypeptide for a cancer-reactive antibody polypeptide receptor or binding partner, including but not limited to, a protein, polypeptide, small molecule, or nucleic acid. In some embodiments, the cancer- reactive antibody polypeptide comprises a substitution, addition, or deletion that increases the stability of the cancer-reactive antibody polypeptide when compared with the stability of the corresponding cancer-reactive antibody without the substitution, addition, or deletion. In some embodiments, the cancer-reactive antibody polypeptide comprises a substitution, addition, or deletion that modulates the immunogenicity of the cancer-reactive antibody polypeptide when compared with the immunogenicity of the corresponding cancer-reactive antibody without the substitution, addition, or deletion. In some embodiments, the cancer- reactive antibody polypeptide comprises a substitution, addition, or deletion that modulates serum half-life or circulation time of the cancer-reactive antibody polypeptide when compared with the serum half-life or circulation time of the corresponding cancer-reactive antibody without the substitution, addition, or deletion,

[00200] In some embodiments, the cancer-reactive antibody polypeptide comprises a substitution, addition, or deletion that increases the aqueous solubility of the cancer-reactive antibody polypeptide when compared to aqueous solubility of the corresponding cancer- reactive antibody without the substitution, addition, or deletion. In some embodiments, the cancer-reactive antibody polypeptide comprises a substitution, addition, or deletion that increases the solubility of the cancer-reactive antibody polypeptide produced in a host cell when compared to the solubility of the corresponding cancer-reactive antibody without the substitution, addition, or deletion, In some embodiments, the cancer-reactive antibody polypeptide comprises a substitution, addition, or deletion that increases the expression of the cancer-reactive antibody polypeptide in a host cell or increases synthesis in vitro when compared to the expression or synthesis of the corresponding cancer-reactive antibody without the substitution, addition, or deletion. The cancer-reactive antibody polypeptide comprising this substitution retains agonist activity and retains or improves expression levels in a host cell, In some embodiments, the cancer-reactive antibody polypeptide comprises a substitution, addition, or deletion that increases protease resistance of the cancer-reactive antibody polypeptide when compared to the protease resistance of the corresponding cancer- reactive antibody without the substitution, addition, or deletion. U.S. Pat. No. 6,716,626 indicated that potential sites that may be substituted to alter protease cleavage include, but are not limited to, a monobasic site within 2 residues of a proline, In some embodiments, the cancer-reactive antibody polypeptide comprises a substitution, addition, or deletion that modulates signal transduction activity of the cancer-reactive antibody receptor when compared with the activity of the receptor upon interaction with the corresponding cancer- reactive antibody polypeptide without the substitution, addition, or deletion, In some embodiments, the cancer-reactive antibody polypeptide comprises a substitution, addition, or deletion that modulates its binding to another molecule such as a receptor when compared to the binding of the corresponding cancer-reactive antibody polypeptide without the substitution, addition, or deletion.

[00201] In some embodiments, the cancer-reactive antibody polypeptide comprises a substitution, addition, or deletion that increases compatibility of the cancer-reactive antibody polypeptide with pharmaceutical preservatives (e.g., m-cresol, phenol, benzyl alcohol) when compared to compatibility of the corresponding cancer-reactive antibody without the substitution, addition, or deletion. This increased compatibility would enable the preparation of a preserved pharmaceutical formulation that maintains the physiochemical properties and biological activity of the protein during storage,

[00202] In some embodiments, the chimeric antigen receptor (CAR) in the CAR T cell comprises one or more post-translational modifications. In some embodiments, the CAR is linked to a linker, polymer, or biologically active molecule. In some embodiments, the CAR is linked to a bifunctional polymer, bifunctional linker, or at least one additional CAR. In some embodiments, the CAR is specific for a cancer-reactive antibody tag. In some embodiments of the present invention, each T cell expresses one CAR. In some embodiments of the present invention, some T cells express more than one CAR, In other embodiments of the present invention, each T cell expresses two CARs. In still other embodiments of the present invention, each T cell expresses more than one CAR, In some embodiments of the present invention, CAR T cells are administered to a patient in need thereof. In other embodiments of the present invention, a formulation of CAR T cells is administered to a patient in need thereof that binds to a specific cancer-reactive antibody tag. In some embodiments of the present invention, two formulations of CAR T cells are administered to a patient in need thereof that bind specifically to two different cancer-reactive antibody tags, In some embodiments of the present invention, two formulations of CAR T cells are administered to a patient in need thereof that bind specifically to the same cancer- reactive antibody tags, In some embodiments of the present invention, multiple formulations of CAR T cells are administered to a patient in need thereof that bind to multiple cancer- reactive antibody tags. The tag may be selected from, but is not limited to, the group consisting of fluorescein isothiocyanate (FITC), streptavidin, biotin, histidine, dinitrophenol, peridinin chlorophyll protein complex, green fluorescent protein, phycoerythrin (PE)₅ horse radish peroxidase, palmitoylation, nitrosylation, alkalanine phosphatase, glucose oxidase, and maltose binding protein.

[00203] In some embodiments, the CAR comprises a substitution, addition or deletion that modulates affinity of the CAR to a cancer-reactive antibody tag or binding partner, including but not limited to, a protein, polypeptide, small molecule, or nucleic acid. In some embodiments, the CAR polypeptide comprises a substitution, addition, or deletion that increases the stability of the CAR polypeptide when compared with the stability of the corresponding CAR without the substitution, addition, or deletion. In some embodiments, the CAR polypeptide comprises a substitution, addition, or deletion that modulates the immunogenicity of the CAR polypeptide when compared with the immuno genie ity of the corresponding CAR without the substitution, addition, or deletion. In some embodiments, the CAR polypeptide comprises a substitution, addition, or deletion that modulates serum half- life or circulation time of the CAR polypeptide when compared with the serum half-life or circulation time of the corresponding CAR without the substitution, addition, or deletion.

[00204] In some embodiments, the CAR polypeptide comprises a substitution, addition, or deletion that increases the aqueous solubility of the CAR polypeptide when compared to aqueous solubility of the corresponding CAR without the substitution, addition, or deletion. In some embodiments, the CAR polypeptide comprises a substitution, addition, or deletion that increases the solubility of the CAR polypeptide produced in a host cell when compared to the solubility of the corresponding CAR without the substitution, addition, or deletion, In some embodiments, the CAR polypeptide comprises a substitution, addition, or deletion that increases the expression of the CAR polypeptide in a host cell or increases synthesis in vitro when compared to the expression or synthesis of the corresponding CAR without the substitution, addition, or deletion, The CAR polypeptide comprising this substitution retains agonist activity and retains or improves expression levels in a host cell. In some embodiments, the CAR polypeptide comprises a substitution, addition, or deletion that increases protease resistance of the CAR polypeptide when compared to the protease resistance of the corresponding CAR without the substitution, addition, or deletion. U.S. Pat. No, 6,716,626 indicated that potential sites that may be substituted to alter protease cleavage include, but are not limited to, a monobasic site within 2 residues of a proline. In some embodiments, the CAR polypeptide comprises a substitution, addition, or deletion that modulates signal transduction activity of the CAR receptor when compared with the activity of the receptor upon interaction with the corresponding CAR polypeptide without the substitution, addition, or deletion. In some embodiments, the CAR polypeptide comprises a substitution, addition, or deletion that modulates its binding to another molecule such as a receptor when compared to the binding of the corresponding CAR polypeptide without the substitution, addition, or deletion.

[00205] In some embodiments, the CAR polypeptide comprises a substitution, addition, or deletion that increases compatibility of the CAR polypeptide with pharmaceutical preservatives (e.g., m-cresol, phenol, benzyl alcohol) when compared to compatibility of the corresponding CAR without the substitution, addition, or deletion. This increased compatibility would enable the preparation of a preserved pharmaceutical formulation that maintains the physiochemical properties and biological activity of the protein during storage.

[00206] The present invention provides an anti-tag chimeric antigen receptor- expressing (AT-CAR) T cell system. In one aspect of the present invention the system comprises genetically transformed immune cells capable of recognizing a cancer cell. In other aspects of the present invention the system comprises genetically transformed immune cells capable of recognizing a tumor antigen. In other aspects of the present invention the system comprises genetically transformed immune cells capable of recognizing two tumor antigens. In other aspects of the present invention the system comprises genetically transformed immune cells capable of recognizing three tumor antigens. In other aspects of the present invention the system comprises genetically transformed immune cells capable of recognizing more than one tumor antigen. In other aspects of the present invention the system comprises CAR T ceils that bind to a tumor antigen. The AT-CAR system of the present invention confers singular or multiple tumor antigen selectivity to T cells of the invention. In some embodiments, the AT-CAR system comprises in vivo cancer cell tagging. In some embodiments, the AT-CAR system of the present invention confers singular or multiple tumor antigen selectivity to T cells of the invention.

[00207] For example, and as further described herein, aFITC-CAR-expressing human T cells of the present invention express CARs that specifically recognize various human cancer cells when those cells are bound by cancer-reactive FITC-labeled antibodies. The activation of aFITC-CAR-expressing T cells has been shown to induce efficient target lysis, T cell proliferation, and cytokine/chemokine production in vitro and ex vivo. In vivo, aFITC-CAR- expressing T cells plus FITC-cetuximab (Ctx) have been shown to delay colon cancer tumor establishment but lead to the selection of tumor-associated antigen (TAA)— negative cancer cells (U.S. Patent Published Application Number 20130287752, hereby incorporated in its entirety by reference). Using a pancreatic tumor model with uniform TAA expression, aFITC-CAR-expressing T cells were observed to eradicate an established tumor and prevent tumor growth,

[00208] In certain embodiments, the invention is drawn to a method of treating cancer in a subject, comprising: (a) administering a formulation of tagged proteins to a subject in need of treatment, wherein the tagged proteins bind a cancer cell in the subject, and (b) administering a therapeutically-effective population of anti-tag chimeric antigen receptor (AT-CAR)- expressing T cells to the subject, wherein the AT-CAR-expressing T cells bind the tagged proteins and wherein one or both of the tagged proteins and/or the AT-CAR-expressing T cells contains one or more non-naturally encoded amino acids.

[00209] In a related embodiment, the invention is drawn to a method of treating cancer in a subject, comprising: (a) administering two formulations of tagged proteins to a subject in need of treatment, wherein the tagged proteins bind a cancer cell in the subject, and (b) administering two populations of AT-CAR-expressing T cells to the subject, wherein the AT- CAR-expressing T cells bind the tagged proteins and wherein one or both of the tagged proteins and/or the AT-CAR-expressing T cells contains one or more non-naturally encoded amino acids.

[00210] In a related embodiment, the invention is drawn to a method of treating cancer in a subject, comprising: (a) administering two formulations of tagged proteins to a subject in need of treatment, wherein the tagged proteins bind a cancer cell in the subject, and (b) administering one or more therapeutically-effective populations of AT-CAR-expressing T cells to the subject, wherein the AT-CAR-expressing T cells bind the tagged proteins and wherein one or both of the tagged proteins and/or the AT-CAR-expressing T cells contains one or more non-naturally encoded amino acids. [00211] In a related embodiment, the invention is drawn to a method of treating cancer in a subject, comprising: (a) administering one or more formulations of tagged proteins to a subject in need of treatment, wherein the tagged proteins bind a cancer cell in the subject, and (b) administering one or more therapeutically-effective populations of AT-CAR-expressing T cells to the subject, wherein the AT-CAR-expressing T cells bind the tagged proteins and wherein one or both of the tagged proteins and/or the AT-CAR-expressing T cells contains one or more non -naturally encoded amino acids.

[00212] In a further related embodiment, the invention is drawn to a method of treating cancer in a subject, comprising: (a) administering at least two formulations of tagged proteins to a subject in need of treatment, wherein the tagged proteins bind a cancer cell in the subject, and (b) administering at least two therapeutically-effective populations of AT-CAR- expressing T cells to the subject, wherein the AT-CAR-expressing T cells bind the tagged proteins and induce cancer cell death, thereby treating cancer in a subject.

[00213] In particular aspects of the embodiments of the invention, the AT-CAR of each population of AT-CAR-expressing T cells is the same or different and the AT-CAR comprises a tag-binding domain, a transmembrane domain, and an activation domain, In some aspects, the tag-binding domain is an antibody or an antigen-binding fragment thereof. In some aspects, the tag-binding domain specifically binds a tag selected from the group consisting of fluorescein isothiocyanate (FITC), streptavidin, biotin, histidine, dinitrophenol, peridinin chlorophyll protein complex, green fluorescent protein, phycoerythrin (PE), horse radish peroxidase, palmitoylation, nitrosylation, alkalanine phosphatase, glucose oxidase, and maltose binding protein. In some aspects, the tag-binding domain specifically binds a tag selected from the group consisting of FITC, biotin, PE, histidine or. streptavidin. In some aspects where the tag-binding domain is antigen-binding fragment, the antigen-binding fragment is a single chain variable fragment (scFv), such as a scFv that specifically binds a tag selected from the group consisting of fluorescein isothiocyanate (FITC), streptavidin, biotin, histidine, dinitrophenol, peridinin chlorophyll protein complex, green fluorescent protein, phycoerythrin (PE), horse radish peroxidase, palmitoylation, nitrosylation, alkalanine phosphatase, glucose oxidase, and maltose binding protein. In other aspects of the present invention the transmembrane domain is the hinge and transmembrane regions of the human CD8a chain. In other aspects, the activation domain comprises one or more of the cytoplasmic region of CD28, the cytoplasmic region of CD137 (41BB), OX40, HVEM, CD3 and. FcRs. [00214] In some embodiments of the invention, the T cells of each population of AT-CAR- expressing T cells are the same or different and wherein the T cells are selected from the group consisting of T cells of any HLA-background from peripheral blood mononuclear cells (PBMC), T cells isolated from a tumor explant of the subject, and intratumoral T cells of the subject, In some embodiments of the invention, the T cells of each population of AT-CAR- expressing T cells consist of HLA-A2+ peripheral blood mononuclear cells (PBMC),

[00215] In some of the embodiments of the present invention, the formulation of tagged protein is administered to the subject prior to administration of the therapeutically-effective population of AT-CAR-expressing T cells, In some of the embodiments of the present invention, the formulations of tagged proteins are administered to the subject prior to administration of the therapeutically-effective population of AT-CAR-expressing T cells. In some of the embodiments of the present invention, the formulations of tagged proteins are administered to the subject prior to administration of the therapeutically-effective populations of AT-CAR-expressing T cells.

[00216] In some of the embodiments of the present invention, the formulation of tagged protein is administered to the subject concurrently with administration of the therapeutically- effective population of AT-CAR-expressing T cells, In some of the embodiments of the present invention, the formulations of tagged proteins are administered to the subject concurrently with administration of the therapeutically-effective population of AT-CAR- expressing T cells, In some of the embodiments of the present invention, the formulations of tagged proteins are administered to the subject concurrently with administration of the therapeutically-effective populations of AT-CAR-expressing T cells,

[0O217] In some of the embodiments of the present invention, the formulation of tagged protein is administered to the subject after administration of the therapeutically-effective population of AT-CAR-expressing T cells, In some of the embodiments of the present invention, the formulations of tagged proteins are administered to the subject after administration of the therapeutically-effective population of AT-CAR-expressing T cells. In some of the embodiments of the present invention, the formulations of tagged proteins are administered to the subject after administration of the therapeutically-effective populations of AT-CAR-expressing T cells.

[00218] In some of the embodiments of the present invention, the formulation of tagged protein and the formulation of the therapeutically-effective population of AT-CAR- expressing T cells are administered in any order. In some of the embodiments of the present invention, the formulations of tagged proteins and the therapeutically-effective population of AT-CAR-expressing T cells are administered in any order. In some of the embodiments of the present invention, the formulations of tagged proteins are administered to the subject and the therapeutically-effective populations of AT-CAR-expressing T cells are administered in any order.

[00219] In particular aspects of the embodiments of the invention, AT-CAR-expressing T cell binding to the tagged proteins induces cytolytic activation of the T cells.

[00220] Disclosed in the present invention are methods and/or compositions for use in medicine, for example, for use in immunotherapy, including immunotherapy for infection or cancer, for example, including methods of use and compositions of T cells engineered to express an antigen-specific chimeric antigen receptor (CAR), In some embodiments, the engineered T cell has impaired expression of its endogenous alpha T-cell receptor (TCR). In some embodiments, the engineered T cell has impaired expression of its endogenous beta TCR. In some embodiments, the engineered T cell has impaired expression of its endogenous alpha beta T-cell receptor TCR. In some embodiments, the engineered T cell has a disrupted endogenous alpha T-cell receptor (TCR), In some embodiments, the engineered T cell has a disrupted beta TCR, In some embodiments, the engineered T cell has a disrupted endogenous alpha beta T-cell receptor TCR, In some embodiments, there is disruption of the T-cell receptor α/β in CAR-expressing T cells using zinc finger nucleases (ZFNs) for generating universal T cells for immunotherapy. In embodiments of the invention, there is knocking out of the T-cell receptor αβ-chain in CAR-expressing T cells, for example using zinc finger nucleases. In some embodiments, the engineered T cell has a non-naturally encoded amino acid that disrupts the endogenous alpha/beta T-cell receptor TCR. In some embodiments, the engineered T cell has a non-naturally encoded amino acid that disrupts the endogenous alpha T-cell receptor TCR. In some embodiments, the engineered T cell has a non-naturally encoded amino acid that disrupts the endogenous beta T-cell receptor TCR, [00221] In some embodiments of the present invention, the CAR is only expressed in the T cells when a non-naturally encoded amino acid is provided concurrently. In some embodiments of the present invention, expression of the T cell's alpha/beta TCR is engineered to be dependent upon administration of a non-naturally encoded amino acid.

[00222] In some embodiments, there are universal CAR-expressing T cells, such as from a healthy donor, that may be suitably stored, for example, in the freezer, and then infused into allogeneic individuals on demand.. The T cell products of the present invention are engineered to be antigen-specific. In some embodiments, the invention provides engineered T cells from allogeneic healthy donors that can be administered to any patient without causing GVHD, In some embodiments, the invention provides autologous CAR⁺TCR^neg T cells,

[00223] The skilled artisan recognizes that in some methods there is simple depletion of T ceils that continue to express TCR by using, for example, clinical-grade CDS -specific monoclonal antibody such that a T-cell product can be generated in which at least the majority have lost expression of endogenous TCR. Other ways include the use of fluorescence-activated ceil sorting (FACS) and TCR- specific monoclonal antibodies or aptamers to reduce and possibly eliminate the presence of T cells that continue to express TCR. In addition, TCR-specific antibodies can be combined with other purification schemes, such as addition of complement, toxins, or resetting to reduce/eliminate T cells that continue to express TCR. In certain embodiments there are methods of treating an individual in need of treatment using compositions encompassed by the invention. The treatment includes employing particular T cells having a CAR and also having genetic modifications to exclude functional TCR, Although in specific embodiments the modifications of the cells exclude functional TCR by any suitable means, in certain aspects the modifications include knock out (or in alternative embodiments, knock down, such as by siRNA and TALENs, for example) of the a and/or β chains or the gamma and/or delta chains of the TCR.

[00224] Some embodiments of the present invention provide a method of making a cell expressing a CAR comprising introducing an expression cassette in to the cell, wherein the expression cassette encodes a polypeptide comprising a heterologous human extracellular antigen binding domain, a transmembrane domain, one or more an intracellular signaling domain(s), and a non-naturally encoded amino acid, In some embodiments, the methods further comprise stimulating the cells with antigen presenting cells, recombinant antigen, or an antibody to the receptor to cause the cells to proliferate, kill, and/or make cytokines. In specific embodiments, the method further comprises stimulating the cells with antigen presenting cells to cause the cells to proliferate,

[00225] In some embodiments, there are recombinant antigen-specific TCR^neg cells expressing and bearing on the cell surface membrane an antigen-specific CAR human polypeptide comprising an intracellular signaling domain, a transmembrane domain and an extracellular domain, the extracellular domain comprising a human anti-antigen monoclonal antibody or antigen binding fragment thereof with a non-naturally encoded amino acid. In some embodiments, there are recombinant antigen-specific TCR^lieg cells expressing and bearing on the cell surface membrane an antigen-specific CAR human polypeptide comprising an intracellular signaling domain with a non-naturally encoded amino acid, a transmembrane domain and an extracellular domain, the extracellular domain comprising a human anti-antigen monoclonal antibody or antigen binding fragment thereof. In some embodiments, there are recombinant antigen-specific TCR^neg cells expressing and bearing on the cell surface membrane an antigen-specific CAR human polypeptide comprising a non- naturally encoded amino acid, an intracellular signaling domain, a transmembrane domain and an extracellular domain, the extracellular domain comprising a human anti-antigen monoclonal antibody or antigen binding fragment thereof. In some embodiments, there are recombinant antigen-specific TCR^neg cells expressing and bearing on the cell surface membrane an antigen-specific CAR human polypeptide comprising an intracellular signaling domain, a transmembrane domain with a non-naturally encoded amino acid and an extracellular domain, the extracellular domain comprising a human anti-antigen monoclonal antibody or antigen binding fragment thereof. In some embodiments, there are recombinant antigen-specific TCR^neg cells expressing and bearing on the cell surface membrane an antigen-specific CAR human polypeptide comprising an intracellular signaling domain, a transmembrane domain and an extracellular domain with a non-naturally encoded amino acid, the extracellular domain comprising a human anti-antigen monoclonal antibody or antigen binding fragment thereof. In some embodiments, there are recombinant antigen- specific TCR^neg cells expressing and bearing on the cell surface membrane an antigen- specific CAR human polypeptide comprismg an intracellular signaling domain, a transmembrane domain and an extracellular domain, the extracellular domain comprising a human anti-antigen monoclonal antibody or antigen binding fragment thereof and wherein the polypeptide comprises a non-naturally encoded amino acid. In some embodiments, there are recombinant antigen-specific TCR^neg cells expressing and bearing on the cell surface membrane an antigen-specific CAR human polypeptide comprising an intracellular signaling domain, a transmembrane domain and an extracellular domain, the extracellular domain comprising a human anti-antigen monoclonal antibody or antigen binding fragment thereof and wherein the polypeptide comprises two non-naturally encoded amino acids. In some embodiments, there are recombinant antigen-specific TCR^neg cells expressing and bearing on the cell surface membrane an antigen-specific CAR human polypeptide comprising an intracellular signaling domain, a transmembrane domain and an extracellular domain, the extracellular domain comprising a human anti-antigen monoclonal antibody or antigen binding fragment thereof and wherein the polypeptide comprises at least one non-naturally encoded amino acid. In some embodiments, there are recombinant antigen-specific TCR^nQg cells expressing and bearing on the cell surface membrane an antigen-specific CAR human polypeptide comprising an intracellular signaling domain, a transmembrane domain and an extracellular domain, the extracellular domain comprising a human anti-antigen monoclonal antibody or antigen binding fragment thereof and wherein the polypeptide comprises more than one non-naturally encoded amino acids. In some embodiments, there are recombinant antigen-specific TCR^neg cells expressing and bearing on the cell surface membrane an antigen-specific CAR human polypeptide comprising an intracellular signaling domain, a transmembrane domain and an extracellular domain, the extracellular domain comprising a human anti-antigen monoclonal antibody or antigen binding fragment thereof and wherein the polypeptide comprises more than two non-naturally encoded amino acids,

[00226] In some embodiments, there is a method of treating a human disease condition associated with a cell expressing endogenous CD 19 comprising infusing a patient with an amount of a recombinant TCR^neg cell expressing a human antigen-specific CAR (by way of non-limiting example this could be a CD1 -specific CAR) sufficient to treat the condition, wherein, for this specific example, the human antigen-specific CAR comprises a heterologous human CD 19 extracellular binding domain, a transmembrane domain, and an intracellular signaling domain comprising a non-naturally encoded amino acid. In some embodiments, there is a method of treating a human disease condition associated with a cell expressing endogenous CD 19 comprising infusing a patient with an amount of a recombinant TCR^neg cell expressing a human antigen-specific CAR (by way of non-limiting example this could be a CD1 -specific CAR) sufficient to treat the condition, wherein, for this specific example, the human antigen-specific CAR comprises a heterologous human CD 19 extracellular binding domain, a transmembrane domain, and an intracellular signaling domain comprising more than one non-naturally encoded amino acids.

[00227] In some embodiments more than one pair of zinc finger nucleases can be used to modify a cell. For example, both zinc finger nucleases targeting the alpha chain and the beta chain can be used to eliminate T-cell receptor expression. In another instance, the zinc finger nucleases targeting the T-cell receptor can be used (for example, sequentially) with zinc finger nucleases to target one or more human leukocyte antigen(s) (HLA), Some embodiments of the present invention provide methods and compositions of engineered T- cells that have lost both T-cell receptor expression and HLA expression.

[00228] In one embodiment there is an isolated T-cell population wherein cells of the population comprise an endogenous T-cell receptor coding sequence that is either not expressed or which encodes a nonfunctional T-cell receptor; and a recombinant chimeric antigen receptor comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising an antigen binding region, In one embodiment there is an isolated T-cell population wherein cells of the population comprise an endogenous T-cell receptor coding sequence that is not expressed in the presence of one or more non-naturally encoded amino acids. In one embodiment there is an isolated T-cel! population wherein cells of the population comprise an endogenous T-cell receptor coding sequence that is not expressed in the presence of a non-naturally encoded amino acids. In one embodiment there is an isolated T-cell population wherein cells of the population comprise an endogenous T- cell receptor coding sequence that is either not expressed or which encodes a nonfunctional T-cell receptor; and a recombinant chimeric antigen receptor comprising one or more non- naturally encoded amino acids, an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising an antigen binding region. In one embodiment there is an isolated T-cell population wherein cells of the population comprise an endogenous T- cell receptor coding sequence that is either not expressed or which encodes a nonfunctional T-cell receptor; and a recombinant chimeric antigen receptor comprising a non-naturally encoded amino acid, an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising an antigen binding region. In one embodiment there is an isolated T-cell population wherein cells of the population comprise an endogenous T-cell receptor coding sequence that is either not expressed or which encodes a nonfunctional T-cell receptor; and a recombinant chimeric antigen receptor comprising two non-naturally encoded amino acids, an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising an antigen binding region.

[00229] In some embodiments, the T-cell receptor is nonfunctional by virtue of one or more disruptions in the coding sequence of a chain, β chain, or both. In some embodiments, the T- cell receptor is nonfunctional by virtue of a polynucleotide encoding a non-naturally encoded amino acid in the coding sequence of a chain, β chain, or both. In some embodiments, the T- cell receptor ^' is nonfunctional by virtue of a polynucleotide substitution encoding a non- naturally encoded amino acid in the coding sequence of a chain, β chain, or both. In some embodiments, the T-cell receptor is nonfunctional by virtue of more than one polynucleotide substitutions encoding a non-naturally encoded amino acid in the coding sequence of a chain, β chain, or both. In some embodiments, the T-cell receptor is nonfunctional by virtue of one or more disruptions in the coding sequence of chain. In some embodiments, the T-cell receptor is nonfunctional by virtue of a polynucleotide encoding a non-naturally encoded amino acid in the coding sequence of a chain. In some embodiments, the T-cell receptor is nonfunctional by virtue of a polynucleotide substitution encoding a non-naturally encoded amino acid in the coding sequence of a chain. In some embodiments, the T-cell receptor is nonfunctional by virtue of more than one polynucleotide substitutions encoding a non- naturally encoded amino acid in the coding sequence of a chain. In some embodiments, the T-cell receptor is nonfunctional by virtue of one or more disruptions in the coding sequence of β chain. In some embodiments, the T-cell receptor is nonfunctional by virtue of a polynucleotide encoding a non-naturally encoded amino acid in the coding sequence of β chain. In some embodiments, the T-cell receptor is nonfunctional by virtue of a polynucleotide substitution encoding a non-naturally encoded amino acid in the coding sequence of β chain. In some embodiments, the T-cell receptor is nonfunctional by virtue of more than one polynucleotide substitutions encoding a non-naturally encoded amino acid in the coding sequence of β chain. In some embodiments, the T-cell receptor is nonfunctional by virtue of one or more disruptions in the coding sequence of both the a chain and β chain. In some embodiments, the T-cell receptor is nonfunctional by virtue of a polynucleotide encoding a non-naturally encoded amino acid in the coding sequence of both the a chain and β chain. In some embodiments, the T-cell receptor is nonfunctional by virtue of a polynucleotide substitution encoding a non-naturally encoded amino acid in the coding sequence of both the a chain and β chain, In some embodiments, the T-cell receptor is nonfunctional by virtue of more than one polynucleotide substitutions encoding a non- naturally encoded amino acid in the coding sequence of both the a chain and β chain,

[00230] In some embodiments, the endogenous T-cell receptor is knocked out, In some embodiments, the antigen binding region is an F(ab')2, Fab', Fab, Fv, or scFv and/or the antigen binding region binds a tumor associated antigen, such as CD 19, CD20, ROR] , carcinoembryonic antigen, alphafetoprotein, CA- 125, MUC- 1 , epithelial tumor antigen, melanoma-associated antigen, mutated p53, mutated ras, HER2/Neu, ERBB2, FTER3, folate binding protein, F1TV- 1 envelope glycoprotein gpl20, HIV- 1 envelope glycoprotein gp41 , GD2, CD 123, CD23.CD30 , CD56, c-Mef mesothelin, GD3, HERV-K, IL- 1 IRalpha, IL- 13Ralpha2, kappa chain, or lambda chain, CSPG4 (also known as, high molecular weight melanoma associated antigen), EGFRvIII, and VEGFR2, In some embodiments, the antigen binding region binds a pathogen antigen, such as a fungal, viral, or bacterial antigen. In some cases, the fungal antigen is from Aspergillus or Candida. In certain cases, the viral antigen is from HSV, RSV, EBV, CMV, JC virus, BK virus, or Ebola.

[00231] In embodiments of the invention, there is an intracellular signaling domain that is a T-lymphocyte activation domain. In some embodiments, the intracellular signaling domain comprises CD3, CD28, OX40/CD134, 4- 1BB/CD 137, FceRIy, ICOS/CD278, ILRB/CD 122, IL--2RG/CD 132, DAP molecules, CD70, cytokine receptor, CD40, or a combination thereof.

[00232] In certain embodiments, the transmembrane domain comprises IgG4Fc hinge, Fc regions, CD4 transmembrane domain, CD28 transmembrane domain, the CD3 transmembrane domain, cysteine mutated hitman €Β3ζ domain, CD 16 transmembrane domain, CDS transmembrane domain, or erythropoietin receptor transmembrane domain.

[00233] In some embodiments, there is a chimeric antigen receptor that comprises: a) an antigen binding domain from a variable region of an antigen-specific monoclonal antibody; b) a signaling domain of 033ζ; and c) CD28, CD 137, CD 134, or combinations thereof. In some embodiments, there is a chimeric antigen receptor that comprises: a) an antigen binding domain from a variable region of an antigen-specific monoclonal antibody; b) a signaling domain of 033ζ; c) CD28, CD 137, CD 134, or combinations thereof; and d) a non-naturally encoded amino acid. In some embodiments, there is a chimeric antigen receptor that comprises: a) an antigen binding domain from a variable region of an antigen-specific monoclonal antibody; b) a signaling domain of 033ζ; c) CD28, CD 137, CD 134, or combinations thereof; and d) two non-naturally encoded amino acids, In some embodiments, there is a chimeric antigen receptor that comprises: a) an antigen binding domain from a variable region of an antigen-specific monoclonal antibody; b) a signaling domain of 033ζ; c) CD28, CD 137, CD 134, or combinations thereof; and d) at least one non-naturally encoded amino acid. In some embodiments, there is a chimeric antigen receptor that comprises: a) an antigen binding domain from a variable region of an antigen-specific monoclonal antibody; b) a signaling domain of 033ζ; c) CD28, CD 137, CD 134, or combinations thereof; and d) more than one non-naturally encoded amino acid. In some embodiments, there is a chimeric antigen receptor that comprises: a) an antigen binding domain from a variable region of an antigen-specific monoclonal antibody that comprises one or more non-naturally encoded amino acid(s); b) a signaling domain of 033ζ; and c) CD28, CD 137, CD 134, or combinations thereof. In some embodiments, there is a chimeric antigen receptor that comprises: a) an antigen binding domain from a variable region of an antigen-specific monoclonal antibody; b) a signaling domain of 033ζ comprising at least one non-naturally encoded amino acid; and c) CD28, CD 137, CD 134, or combinations thereof. In some embodiments, there is a chimeric antigen receptor that comprises: a) an antigen binding domain from a variable region of an antigen-specific monoclonal antibody; b) a signaling domain of 033ζ; and c) CD28, CD 137, CD 134, or combinations thereof with at least one non-naturally encoded amino acid, In some embodiments, there is a chimeric antigen receptor that comprises; a) an antigen binding domain from a variable region of an antigen- specific monoclonal antibody that comprises one non-naturally encoded amino acid; b) a signaling domain of 033ζ; and c) CD28, CD 137, CD 134, or combinations thereof. In some embodiments, there is a chimeric antigen receptor that comprises; a) an antigen binding domain from a variable region of an antigen-specific monoclonal antibody; b) a signaling domain of 033ζ comprising one non-naturally encoded amino acid; and c) CD28, CD 137, CD 134, or combinations thereof. In some embodiments, there is a chimeric antigen receptor that comprises: a) an antigen binding domain from a variable region of an antigen-specific monoclonal antibody; b) a signaling domain of 033ζ; and c) CD28, CD 137, CD 134, or combinations thereof with one non-naturally encoded amino acid.

[00234] In some embodiments of the present invention, there are methods of generating the cells of the invention, comprising the steps of: a) providing one or more T cells; b) modifying the T cell(s) to express a recombinant chimeric antigen receptor comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising an antigen binding region; and c) modifying the T cell(s) to harbor an endogenous T-eell receptor coding sequence that is not expressed or encodes a nonfunctional T-cell receptor. In some embodiments, step b) occurs before step c) or step c) occurs before step b). in certain aspects, the T cell is provided from an umbilical cord blood bank, is provided from a peripheral blood bank, is an induced pluripotent stem cell, or is a human embryonic stem cell. In some embodiments, the T cell is allogeneic in reference to one or more intended recipients, In some cases, there are methods of generating the cells^' of the invention, comprising the steps of: a) providing one or more T cells; b) modifying the T cell(s) to express a recombinant chimeric antigen receptor comprising a non-naturally encoded amino acid, an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising an antigen binding region; and c) modifying the T cell(s) to harbor an endogenous T-cell receptor coding sequence that is not expressed or encodes a nonfunctional T-cell receptor. In some cases, there are methods of generating the cells of the invention, comprising the steps of: a) providing one or more T cells; b) modifying the T cell(s) to express a recombinant chimeric antigen receptor comprising at least one non-naturally encoded amino acid an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising an antigen binding region; and c) modifying the T cell(s) to harbor an endogenous T-eell receptor coding sequence that is not expressed or encodes a nonfunctional T-cell receptor. In some cases, there are methods of generating the cells of the invention, comprising the steps of: a) providing one or more T cells; b) modifying the T cell(s) to express a recombinant chimeric antigen receptor comprising more than one non-naturally encoded amino acids an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising an antigen binding region; and c) modifying the T cell(s) to harbor an endogenous T-cell receptor coding sequence that is not expressed or encodes a nonfunctional T-cell receptor, In some cases, there are methods of generating the cells of the invention, comprising the steps of; a) providing one or more T cells; b) modifying the T cell(s) to express a recombinant chimeric antigen receptor comprising a non-naturally encoded amino acid within the intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising an antigen binding region; and c) modifying the T cell(s) to harbor an endogenous T-cell receptor coding sequence that is not expressed or encodes a nonfunctional T-cell receptor. In some cases, there are methods of generating the cells of the invention, comprising the steps of: a) providing one or more T cells; b) modifying the T cell(s) to express a recombinant chimeric antigen receptor comprising an intracellular signaling domain, a transmembrane domain comprising a non-naturally encoded amino acid, and an extracellular domain comprising an antigen binding region; and c) modifying the T cell(s) to harbor an endogenous T-cell receptor coding sequence that is not expressed or encodes a nonfunctional T-ceil receptor. In some cases, there are methods of generating the cells of the invention, comprising the steps of: a) providing one or more T cells; b) modifying the T cell(s) to express a recombinant chimeric antigen receptor comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising a non- naturally encoded amino acid and an antigen binding region; and c) modifying the T cell(s) to harbor an endogenous T-eell receptor coding sequence that is not expressed or encodes a nonfunctional T-cell receptor. In some cases, there are methods of generating the cells of the invention, comprising the steps of; a) providing one or more T cells; b) modifying the T cell(s) to express a recombinant chimeric antigen receptor comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising an antigen binding region comprising a non-naturally encoded amino acid; and c) modifying the T cell(s) to harbor an endogenous T-cell receptor coding sequence that is not expressed or encodes a nonfunctional T-cell receptor. Combinations of the above will be apparent to one skilled in the art.

[00235] In some embodiments, the chimeric antigen receptor is stably introduced into the cell. In other embodiments, the polynucleotide that encodes the chimeric antigen receptor is introduced into the cell by a transposon/transposase system or a viral-based gene transfer system, such as by recombinant retrovirus or lentivirus. ■ In some embodiments, following modification of the T cell(s), they are propagated by exposing the T cells to artificial antigen presenting cells, by using O 'T'3 (or equivalent to cross-fink CDS) optionally with other co- stimulatory antibodies (e.g., anti-CD2S) on beads, or by using OKT3 (or equivalent to crosslink CD3) optionally with other co-stimulatory antibodies (e.g., anti-CD28) mixed with peripheral blood mononuclear cells, In some embodiments, a polynucleotide that encodes the recombinant chimeric antigen receptor is electroporated into the T cell, in some embodiments, a polynucleotide that encodes the chimeric antigen receptor is present on a plasmid or viral vector. In some embodiments, the polynucleotide that encodes the recombinant chimeric antigen receptor encodes a non-natural amino acid. In some embodiments, the polynucleotide that encodes the recombinant chimeric antigen receptor encodes more than one non-natural amino acid.

[00236] In some embodiments, the T cell can be genetically modified with zinc finger nuclease or TLAE nuclease to eliminate HLA expression. The T cell may express a CAR and/or have been modified to eliminate TCR expression. In some embodiments, the endogenous T-cell receptor and/or HLA is disrupted by nonhomologous end joining repair, such as is generated by zinc finger nuclease, TALE nuclease, introduced into the cell by physical means, electro-transfer of mRNA species, viral vector, or non-viral vector, In some embodiments, there are methods of treating an individual with a medical condition (such as autoimmune disease, cancer, or infection, including Aspergillus or Candida), comprising the step of providing an effective amount of cells from the population of cells described herein, including more than once in some aspects, such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, or more days apart, In some embodiments, the cancer is lymphoma, leukemia, non- Hodgkin's lymphoma, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, chronic lymphocytic leukemia, or B cell-associated autoimmune diseases.

III. Non-Natural Amino Acid Derivatives

[00237] The non-natural amino acids used in the methods and compositions described herein have at least one of the following four properties: (1) at least one functional group on the sidechain of the non-natural amino acid has at least one characteristics and/or activity and/or reactivity orthogonal to the chemical reactivity of the 20 common, genetically- encoded amino acids (i,e,, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), or at least orthogonal to the chemical reactivity of the naturally occurring amino acids present in the polypeptide that includes the non-natural amino acid; (2) the introduced non-natural amino acids are substantially chemically inert toward the 20 common, genetically-encoded amino acids; (3) the non-natural amino acid can be stably incorporated into a polypeptide, preferably with the stability commensurate with the naturally- occurring amino acids or under typical physiological conditions, and further preferably such incorporation can occur via an in vivo system; and (4) the non-natural amino acid includes an oxime functional group or a functional group that can be transformed into an oxime group by reacting with a reagent, preferably under conditions that do not destroy the biological properties of the polypeptide that includes the non-natural amino acid (unless of course such a destruction of biological properties is the purpose of the modification/transformation), or where the transformation can occur under aqueous conditions at a pH between about 4 and about 8, or where the reactive site on the non-natural amino acid is an electrophilic site. Any number of non-natural amino acids can be introduced into the polypeptide. Non-natural amino acids may also include protected or masked oximes or protected or masked groups that can be transformed into an oxime group after deprotection of the protected group or unmasking of the masked group. Non-natural amino acids may also include protected or masked carbonyl or dicarbonyl groups, which can be transformed into a carbonyl or dicarbonyl group after deprotection of the protected group or unmasking of the masked group and thereby are available to react with hydroxylamines or oximes to form oxime groups.

[00238] Non-natural amino acids that may be used in the methods and compositions described herein include, but are not limited to, amino acids comprising a amino acids with novel functional groups, amino acids that covalently or noncovalently interact with other molecules, glycosylated amino acids such as a sugar substituted serine, other carbohydrate modified amino acids, keto-containing amino acids, aldehyde- containing amino acids, amino acids comprising polyethylene glycol or other polyethers, heavy atom substituted amino acids, chemically cleayable and/or photocleavable amino acids, amino acids with an elongated side chains as compared to natural amino acids, including but not limited to, polyethers or long chain hydrocarbons, including but not limited to, greater than about 5 or greater than about 10 carbons, carbon-linked sugar-containing amino acids, redox-active amino acids, amino thioacid containing amino acids, and amino acids comprising one or more toxic moiety.

[00239] In some embodiments, non-natural amino acids comprise a saccharide moiety. Examples of such amino acids include N-acetyl-L-glucosaminyl-L-serine, N-acetyl-L- galactosaminyl-L-serine, N-acetyl-L-glucosaminyl-L-threonine, N-acetyl-L-glucosaminyl-L- asparagine and Omannosaminyl-L-serine. Examples of such amino acids also include examples where the naturally- occurring N- or O- linkage between the amino acid and the saccharide is replaced by a covalent linkage not commonly found in nature - including but not limited to, an alkene, an oxime, a thioether, an amide and the like. Examples of such amino acids also include saccharides that are not commonly found in naturally-occurring proteins such as 2-deoxy-glucose, 2-deoxygalactose and the like.

[00240] The chemical moieties incorporated into polypeptides via incorporation of non- natural amino acids into such polypeptides offer a variety of advantages and manipulations of polypeptides, For example, the unique reactivity of a carbonyl or dicarbonyl functional group (including a 'keto- or aldehyde- functional group) allows selective modification of proteins with any of a number of hydrazine- or hydroxylamine-containing reagents in vivo and in vitro. A heavy atom non-natural amino acid, for example, can be useful for phasing x-ray structure data, The site-specific introduction of heavy atoms using non-natural amino acids also provides selectivity and flexibility in choosing positions for heavy atoms. Photoreactive non-natural amino acids (including but not limited to, amino acids with benzophenone and arylazides (including but not limited to, phenylazide) side chains), for example, allow for efficient in vivo and in vitro photocrosslinking of polypeptides, Examples of photoreactive non-natural amino acids include, but are not limited to, p-azido-phenylalanine and p-benzoyl- phenylalanine. The polypeptide with the photoreactive non-natural amino acids may then be crosslinked at will by excitation of the photoreactive group-providing temporal control. In a non-limiting example, the methyl group of a non-natural amino can be substituted with an isotopically labeled, including but not limited to, with a methyl group, as a probe of local structure and dynamics, including but not limited to, with the use of nuclear magnetic resonance and vibrational spectroscopy.

A. Structure and Synthesis of Non-Natural Amino Acid Derivatives: Carbonyl, Carbonyl like, Masked Carbonyl, and Protected Carbonyl Groups

[00241] Amino acids with an electrophilic reactive group allow for a variety of reactions to link molecules via various chemical reactions, including, but not limited to, nucleophilic addition reactions. Such electrophilic reactive groups include a carbonyl- or dicarbonyl-group (including a keto- or aldehyde group), a carbonyl-like- or dicarbonyl-like- group (which has reactivity similar to a carbonyl- or dicarbonyl-group and is structurally similar to a carbonyl- or dicarbonyl-group), a masked carbonyl- or masked dicarbonyl-group (which can be readily converted into a carbonyl- or dicarbonyl-group), or a protected carbonyl- or protected dicarbonyl-group (which has reactivity similar to a carbonyl- or dicarbonyl-group upon deprotection), Such amino acids include amino acids having the structure of Formula (XXXVII);

(XXXVII)

wherein:

A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -0-, -0-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(0)k- where k is 1, 2, or 3, - S(0)_k(alkylene or substituted alkylene)-, -C(0)-, -NS(0)₂-, -0S(0)₂-, -C(0)-(alkylene or substituted alkylene)-, -C(S)-_} -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'- (alkylene or substituted alkylene)-, -C(0)N(R , -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(0)0-, -S(0)_kN(R , -N(R')C(0)N(R , -N(R')C(S)N(R')-₅ -N(R')S(0)_kN(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, - C(R⁵)=N-N=, -C(R')₂-N=N-, and -C(R>N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl;

R is H, alkyl, substituted alkyl, cycloalky!, or substituted cycloalkyl;

each R" is independently H, alkyl, substituted alkyl, or a protecting group, or when more than one R" group is present, two R" optionally form a heterocycloaikyl; Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

R₂ is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide;

each of R3 and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl;

or the -A-B-K-R groups together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl comprising at least one carbonyl group, including a dicarbonyl group, protected carbonyl group, including a protected dicarbonyl group, or masked carbonyl group, including a masked dicarbonyl group;

or the -K-R group together forms a monocyclic or bicyclic cycloalkyl or heterocycloalkyl comprising at least one carbonyl group, including a dicarbonyl group, protected carbonyl group, including a protected dicarbonyl group, or masked carbonyl group, including a masked dicarbonyl group;

with a proviso that when A is phenylene and each R3 is H, B is present; and that when A is - (CHz)4- and each R₃ is Ii_} B is not -NHC(0)(CH₂CH₂)-; and that when A and B are absent and each 3 is H, R is not methyl. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[00242] In certain embodiments, compounds of Formula (XXXVII) are stable in aqueous solution for at least 1 month under mildly acidic conditions. In certain embodiments, compounds of Formula (XXXVII) are stable for at least 2 weeks under mildly acidic conditions. In certain embodiments, compound of Formula (XXXVII) are stable for at least 5 days under mildly acidic conditions. In certain embodiments, such acidic conditions are pH 2 to 8.

[00243] In certain embodiments of compounds of Formula (XXXVII), B is lower alkylene, substituted lower alkylene, -0-(alkyIene or substituted alkylene)-, -C(R')=N-N(R')-, - N(R')CO-, -C(O)-, -C(R')=N~, -C(0)-(alkylene or substituted alkylene)-, -CON(R')-(alkylene or substituted alkylene)-, -Sfalkylene or substituted alkylene)-, - S(0)(alkylene or substituted alkylene)-, or -S(0)₂(alkylene or substituted alkylene)-. In certain embodiments of compounds of Formula (XXXVII), B is -0(CH₂)-, -CH=N-, -CH=N-NH-, -NHCH2-, -NHCO-, -C(0 , -C(0)-(CH₂)-, -CONH-(CH₂)-, -SCH₂-, - S(=0)CH₂-, or -S(0)₂CH2-. In certain embodiments of compounds of Formula (XXXVII), R is Ci-6 alkyl or cycloalkyl. In certain embodiments of compounds of Formula (XXXVII) R is -CH3, -CH(CH3)₂, or cyclopropyl. In certain embodiments of compounds of Formula (XXXVII), i is H, tert-butyloxycarbonyl (Boc), 9- Fluorenylmethoxycarbonyi (Fmoc), N- acetyl, tetrafluoroacetyl (TFA), or benzyloxycarbonyl (Cbz). In certain embodiments of compounds of Formula (XXXVII), Ri is a resin, amino acid, polypeptide, antibody, or polynucleotide. In certain embodiments of compounds of Formula (XXXVII), R2 is OH, O- methyl, O-ethyl, or O-i-butyl. In certain embodiments of compounds of Formula (XXXVII), R-2 is a resin, amino acid, polypeptide, antibody, or polynucleotide, In certain embodiments of compounds of Formula (XXXVII), R2 is a polynucleotide. In certain embodiments of compounds of Formula (XXXVII), R2 is ribonucleic acid (RNA).

[00244] In certain embodiments of compounds of Formula (XXXVII), ¾ ⁰ is selected from the group consisting of:

(i) A is substituted lower alkylene, C^arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

B is optional, and when present is a divalent linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, -0-, -0-(alkylene or substituted alkylene)-, -S-, - S(O)-, -S(0)₂-, -NS(0)₂-, -OS(0)₂-, -C(O)-, -C(0)-(alkylene or substituted alkylene)-, -C(S>, -N(R⁵)-, -C(0)N(R , -CON(R')~(alkylene or substituted alkylene)-, -CSN(R')-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(0)0-, -N(R')C(S)-, -S(0)N(R')_s -S(0)₂N(R'), -N(R')C(0)N(R')-, -N(R')C(S)N(R')-, -N(R')S(0)N(R')-, -N(R')S(0)₂N(R')-, -N(R')-N=, - C(R')=N-N(R , -C(R')=N-N=, -C(R')₂-N=N-, and -C(R')2-N(R')-N(R')-;

(ii) A is optional, and when present is substituted lower alkylene, ⁽LVarylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

B is a divalent linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, - 0-, -0-(alkylene or substituted alkylene)-, -S-, -S(O)-, -S(0)₂-, -NS(0)₂-, - OS(0)₂-₅ -C(O)-, -C(0)-(alkylene or substituted alkylene)-, -C(S)-, -N(R')-, -C(0)N(R')-, -CON(R^!)-(alkylene or substituted alkylene)-, -CSN(R')-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(0)0-, -N(R')C(S)-, -S(0)N(R'), -S(0)₂N(R'), -N(R')C(0)N(R')-, -N(R')C(S)N(R')-, -N(R')S(0)N(R')-_! -N(R')S(0)₂N(R')-, -N(R^!)-N=, -C(R')=N~N(R')~, - C(R')=N-N=, -C(R')₂~N-N-, and -C(R')₂-N(R')-N(R')-;

(iii) A is lower alkylene;

B is optional, and when present is a divalent linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, -0-, -0-(alkylene or substituted alkylene)-, -S-, - S(0)-, -S(0)₂-, -NS(0)₂-, -0S(0)₂-, -C(0)-, -C(0)-(alkylene or substituted ' alkylene)-, -C(S)-, -N(R')-, -C(0)N(R')-, -CSN(R')-, -CON(R')-(alkylene or substituted alkylene)-, -N(R')C(0)0-, -N(R')C(S)-, -S(0)N(R'), -S(0)₂N(R'), -N(R')C(0)N(R')-, -N(R')C(S)N(R')-, -N(R')S(0)N(R')-, -N(R')S(0)₂N(R')- , -N(R')-N=, -C(R')=N-N(R')-₃ -C(R')=N-N=, -C(R')₂-N=N-, and -C(R')₂-N(R')-N(R ; and

(iv) A is phenylene;

B is a divalent linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, - 0-, -O-Calkylene or substituted alkylene)-, -S-, -S(0 , -S(0)₂-, -NS(0)₂-, - 0S(0)₂-, -C(0)-, -C(0)-(alkylene or substituted alkylene)-, -C(S)-, -N(R')-, -C(0)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(0)0-, -N(R')C(S)-, -S(0)N(R'), -S(0)₂N(R'), -N(R')C(0)N(R')-, -N(R')C(S)N(R')-, -N(R')S(0)N(R')-, -N(R')S(0)₂N(R')-₃ -N(R')-N= -C(R')=N-N(R')-, - C(R')=N-N= -C(R')2-N=N-_} and -C(R')₂-N(R')-N(R')-;

each R' is independently H, alkyl, or substituted a!kyl; Ri is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; and each R3 and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[00245] In addition, amino acids having the structure of Formula (XXXVIII) are included:

(XXXVIII), wherein:

A is optional, and when present is lower alkylene, substituted lower alkylene, lower cyclo alkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted hetero alkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -0-, -0-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(0)_k- where k is 1 , 2, or 3, - S(0)k(alkylene or substituted alkylene)-, -C(O)-, -NS(0)₂-, -OS(0)₂-, -C(0)-(alkylene or substituted alkylene)-, -C(S , -C(S)-(alkylene or substituted alkylene)-, -N(R , -NR'- (alkylene or substituted alkylene)-, -C(0)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(0)0-, -S(0 N(R')-, -N(R')C(0)N(R')-, -N(R')C(S)N(R , -N(R')S(0),_tN(R')-, -N(R')-N=, -C(R^!)=N-, -C(R')=N-N(R')-, - C(R')=N-N=, -C(R')₂-N=N-, and -C(R')2-N( ')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

Ri is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; and

R2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; with a proviso that when A is phenylene, B is present; and that when A is -(CH.)4-, B is not - NHC(0)(CH2CH2) and that when A and B are absent, R is not methyl. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[00246] In addition, amino acids having the structure of Formula (XXXIX) are included:

(XXXIX),

wherein:

B is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower hetero alkylene, -0-, -0~(alkylene or substituted alkylene)-, -S-, ~S-(alkylene or substituted alkylene)-, ~S(Q)k~ where k is 1, 2, or 3, -S(0)k(alkylene or substituted alkylene)-, -C(O)-, -NS(0)₂-, -OS(0)₂-, -C(0)-(alkylenc or substituted alkylene)-, -C(S)-, -C(S)-(allcylene or substituted alkylene)-, -N(R')-, -NR' -(alkylene or substituted alkylene)-, -C(0)N(R' , -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(0)0-₃ -S(0)_kN(R')-, -N(R')C(0)N(R')-, -N(R')C(S)N(R')-, -N(R')S(0)_kN(R')-, -N(R')-N= -C(R')=N-_} -C(R')=N-N(R')-, -C(R')=N-N=, -C(R N=N-, and -C(R')₂-N(R')-N(R')-, where each R^! is independently H, alkyl, or substituted alkyl;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R] is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each Rg is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R')₂, -C(0)_kR' where k is 1, 2, or 3, -C(0)N(R')₂, -OR', and -S(0)kR', where each R' is independently H, alkyl, or substituted alkyl. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[00247] In addition, the following amino acids are included:

Such non-natural amino acids may be are optionally amino protected group, carboxyl protected and/or in the form of a salt, or may be incorporated into a non-natura! amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[00248] In addition, the following amino acids having the structure of Formula (XXXX) are included:

(XXXX)

wherein ~NS(0)2", -OS(0)2-, optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -0-, -0-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(0)k- where k is 1, 2, or 3, -S(0)k(alkylene or substituted alkylene)-, -C(0)-, -C(0)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, - R' -(alkylene or substituted alkylene)-, -C(0)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, - CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(0)0-, -S(0)kN(R , -N(R')C(0)N(R')-, -N(R')C(S)N(R')-, -N(R')S(0)_kN(R')-, -N(R')-N= -C(R')=N-₅ -C(R')=N-N(R')-, - C(R')=N-N=, -C(R')2-N=N-, and -C(R')₂-NCR^,)-N(R')-, where each R' is independently H, alkyl, or substituted alkyl;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each R_a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R')_2} -C(0),_cR' where k is 1, 2, or 3, -C(0)N(R')2, -OR', and -S(0)_kR\ where each R' is independently H, alkyl, or substituted alkyl; and n is 0 to 8; with a proviso that when A is -(Cl b)^, B is not -NHC(0)(CH2CH₂)-. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[00249] In addition, the following amino acids are included;

wherein such compounds are optionally amino protected, optionally carboxyl protected, optionally amino protected and carboxyl protected, or a salt thereof, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[00250] In addition, the following amino acids having the structure of Formula (XXXXI) are included:

(XXXXI),

wherein,

A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkyny!ene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -0-, -0-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(0)k- where k is 1, 2, or 3, - S(0)k(alkylene or substituted alkylene)-, -C(0)-, -NS(0)₂-, -0S(0)₂-, -C(0)-(alkylene or substituted alkylene)-, -C(S)-_} -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'- (alkylene or substituted alkylene)-, -C(0)N(R')-, -CON(R')-(aIkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(0)0-, -S(0)_kN(R')-, -N(R')C(0)N(R , -N(R')C(S)N(R^! , -N(R')S(0)_kN(R , -N(R')~N=, -C(R')=N-, -C(R')=N-N(R')-, - C(R')=N-N= -C(R')₂-N=N-, and -C(R' )2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl;

Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

R₂ is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non- natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translational!y modified.

[00251] In addition, the following amino acids having the structure of Formula (ΧΧΧΧΠ) are included;

XXXXil),

wherein,

B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -0-, -0-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(0)_k- where k is 1, 2, or 3, - S(0)_k(alkylene or substituted alkylene)-, -C(O)-, -NS(0)₂-, -OS(0)₂-, -C(0)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'- (alkylene or substituted alkylene)-, -C(0)N(R , -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R*)C(0)0-, -S(0)i_iN(R')-₃ -N(R')C(0)N(R')-, -N(R')C(S)N(R , -N(R')S(0)kN(R^s)-, -N(R^!)-N=, -C(R')=N-₃ -C(R')=N-N(R'h - C(R')=N-N=, -C(R')z-N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; wherein each R_¾ is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R')₂, -C(0)_kR' where k is 1, 2, or 3, -C(0)N(R')2, -OR', and -S(0)kR', where each R' is independently H, alkyl, or substituted alkyl.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non- natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified,

[00252] In addition, the following amino acids are included:

wherein such compounds are optionally amino protected, optionally carboxyl protected, optionally amino protected and carboxyl protected, or a salt thereof, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[00253] In addition, the following amino acids having the structure of Formula (XXXXIV) are included:

(XXXXIV), wherein,

B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkyl ene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -0-, -0-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(0)_k- where k is 1, 2, or 3, - S(0)_k(alkylene or substituted alkylene)-, -C(O)-, -NS(0)₂-, -OS(0)₂-, -C(0)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted, alkylene)-, -N(R')-, -NR'- (alkylene or substituted alkylene)-, -C(0)N(R')-_} -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R (alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(0)0-, -S(0)_kN(R')-, -N(R')C(0)N(R')-_s -N(R')C(S)N(R')-, -N(R')S(0)_kN(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R , - C(R')=N-N=, -C(R')₂-N=N-, and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each R_a is independently selected from the group consisting of H_; halogen, alkyl, substituted alkyl, -N(R')₂, -C(0)_kR' where k is 1, 2, or 3, ~C(0)N(R')₂, -OR', and -S(0)_kR\ where each R' is independently H, alkyl, or substituted alkyl; and n is 0 to 8,

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non- natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[00254] In addition, the following amino acids are included:

wherein such compounds are optionally amino protected, optionally carboxyl protected, optionally amino protected and carboxyl protected, or a salt thereof, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[00255] In addition to monocarbonyl structures, the non-natural amino acids described herein may include groups such as dicarbonyl, dicarbonyl like, masked dicarbonyl and protected dicarbonyl groups.

For example, the following amino acids having the structure of Formula (XXXXV) are included:

(XXXXV), wherein,

A is optional, and when present is lower alkylene, substituted lower afkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, allcynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -0-, -0-(aikylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(0)k- where k is 1, 2, or 3, - S(0)_k(alkylene or substituted alkylene)-, -C(O)-, -NS(0)₂-, -OS(0)₂-, -C(0)-(alkylene or substituted alkylene)-, -C(S , -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'- (alkylene or substituted alkylene)-, -C(0)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R (alky!ene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(0)0-, -S(0)_kN(R')-, -N(R')C(0)N(R⁵)-, -N(R')C(S)N(R')-_} -N(R')S(0)kN(R , -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, - C(R')=N-N= -C(R')₂-N=N-, and -C(R^!)2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non- natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[00256] In addition, the following amino acids having the structure of Formula (XXXXVI) are included:

(XXXXVI),

wherein,

B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -0-, -0-(aIkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(0)i_<- where k is 1, 2, or 3, - S(0)k(alkylene or substituted alkylene)-, -C(O)-, -NS(0)₂-, -OS(0)₂-, -C(0)~(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R , -NR'- (alkylene or substituted alkylene)-, -C(0)N(R')-_; -CON(R (alkylene ₀r substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(0)0-, -S(0)_kN(R , -N(R')C(0)N(R' , -N(R')C(S)N(R')-, -N(R')S(0)kN(R')-, -N(R')~N= -C(R')=N-, -C(R')=N-N(R , - C(R')=N-N=, -C(R')₂-N=N-, and -C(R')₂-N(R')-N(R⁵)-, where each R' is independently H, alkyl, or substituted alkyl;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and. R.2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; wherein each R_a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R')2, -C(0)_kR' where k is 1, 2, or 3, -C(0)N(R¹)_2; -OR', and -S(0)kR'_} where each R' is independently H, alkyl, or substituted alkyl,

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non- naturai amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[00257] In addition, the following amino acids are included;

wherein such compounds are optionally amino protected and carboxyl protected, or a salt thereof. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[00258] In addition, the following amino acids having the structure of Formula (XXXXVII) are included:

(XXXXVII), wherein,

B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -0-, -0-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(0)k- where k is 1, 2, or 3, - S(0)_k(alkylene or substituted alkylene)-, -C(0 , -NS(0)₂-, -OS(0)₂-, -C(0)-(alkylcne or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'- (alkylene or substituted alkylene)-, -C(0)N(R')-_} -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(0)0-, -S(0)_kN(R⁵)-, -N(R')C(0)N(R')-, -N(R')C(S)N(R')-, -N(R')S(0)_kN(R , -N(R')-N=, -C(R')=N-, -CfR'^ -NiR')-, - C(R')=N-N=₎ -C(R^!)₂-N=N-, and -C(R')₂-N(R')-N(R')-_; where each R' is independently H, alkyl, or substituted alkyl;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each R_a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R')₂₅ -C(0)_kR' where k is 1, 2, or 3, -C(0)N(R')2, -OR', and - S(0)kR', where each R' is independently H, alkyl, or substituted alkyl; and n is 0 to 8.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non- natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[00259] In addition, the following amino acids are included:

wherein such compounds are optionally amino protected and carboxyl protected, or a salt thereof, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified,

[00260] In addition, the following amino acids having the structure of Formula (XXXXVHI) are included;

(XXXXVIII); wherein:

A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower hetero lkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocyclo lkylene, arylene, substituted arylene, heteroarylene, substituted hetero arylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide;

Xi is C, S, or S(O); and L is alkylene, substituted alkylene, N(R')(alkylene) or N(R')(substituted alkylene), where R' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non- natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified,

[00261] In addition, the following amino acids having the structure of Formula (XXXXIX) are included:

(XXXXIX) wherein:

A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkyl ene, substituted lower eye lo alkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted hetero alkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted, alkarylene, aralkylene, or substituted aralkylene;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide;

L is alkylene, substituted alkylene, N(R')(alkylene) or N(R')(substituted alkylene), where R' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non- natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[00262] In addition, the following amino acids having the structure of Formula (XXXXX) are included;

(XXXXX) wherein:

A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloa!kylene, substituted lower heterocyclo lkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

R₂ is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide;

L is alkylene, substituted alkylene, N(R')(alkylene) or N(R')(substituted alkylene), where R' is H, aikyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non- natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified,

[00263] In addition, the following amino acids having the structure of Formula (XXXXXI) are included;

(XXXXXI); wherein:

A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide;

Xi is C, S, or S(O); and n is 0, 1, 2, 3, 4, or 5; and each R^s and R⁹ on each CR⁸R⁹ group is independently selected from the group consisting of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R⁸ and R⁹ can together form =0 or a cycloalkyl, or any to adjacent R⁸ groups can together form a cycloalkyl.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non- natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[00264] In addition, the following amino acids having the structure of Formula (XXXXXII) are included:

(XXXXXII) wherein:

A is optional, and when present is lower alkylene, substituted lower alkylene, lower eye lo alky lene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

Ri is H, an amino protectmg group, resin, amino acid, polypeptide, or polynucleotide; and R.2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; n is 0, 1, 2, 3, 4, or 5; and each R⁸ and R⁹ on each CR⁸R⁹ group is independently selected from the group consisting of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R^s and R⁹ can together form =0 or a cycloalkyl, or any to adjacent R⁸ groups can together form a cycloalkyl,

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non- natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified,

[00265] In addition, the following amino acids having the structure of Formula (XXXXXIII) are included:

(XXXXXIII)

wherein:

A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocyclo lkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralky!ene, or substituted aralkyiene;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; n is 0, 1, 2, 3, 4, or 5 ; and each R⁸ and R⁹ on each CR⁸R⁹ group is independently selected from the group consisting of H, alkoxy, alkylamine, halogen, alley!, aryl, or any R^s and R⁹ can together form =0 or a cycloalkyl, or any to adj cent R⁸ groups can together form a cycloalkyl.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non- natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[00266] In addition, the following amino acids having the structure of Formula (XXXXXIV) are included :

(XXXXXIV); wherein:

A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

R] is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide;

Xi is C, S, or S(O); and L is alkylene, substituted alkylene, N(R')(alkylene) or N(R') (substituted alkylene), where R' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non- natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified, [00267] In addition, the following amino acids having the structure of Formula (XXXXXV) are included:

(XXXXXV) wherein:

A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted hetero alkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

R₂ is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide;

L is alkylene, substituted alkylene, N(R')(alkylene) or N(R')(substituted alkylene), where R' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non- natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[00268] In addition, the following amino acids having the structure of Formula (XXXXXVI) are included:

(XXXXXVI) wherein:

A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloaikylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloaikylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; ] is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide;

L is alkylene, substituted alkylene, N(R')(alkylene) or N(R')(substituted alkylene), where R' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non- natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[00269] In addition, amino acids having the structure of Formula (XXXXXVII) are included:

7

(XXXXXVII), wherein:

A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycioalkylene, lower alkenylene, substituted lower alkenylene, alkynyiene, lower heteroalkylene, substituted hetero alkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, hetero arylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

where (a) indicates bonding to the A group and (b) indicates bonding to respective carbonyl groups, R3 and R4 are independently chosen from H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, or R₃ and R4 or two R3 groups or two R4 groups optionally form a cycloalkyl or a heterocycloalkyl;

R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

T3 is a bond, C(R)(R), O, or S_s and R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R.2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non- natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[00270] In addition, amino acids having the structure of Formula (XXXXXVIII) are included:

(XXXXXVIII), wherein:

, where (a) indicates bonding to the A group and (b) indicates bonding to respective carbonyl groups, R3 and R4 are independently chosen from H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, or R3 and R₄ or two R3 groups or two R groups optionally form a cycloalkyl or a heterocycloalkyl;

R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

T3 is a bond, C(R)(R), O, or S, and R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R.2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each R_a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R')2, -C(0)_kR' where k is 1, 2, or 3, -C(0)N(R -OR', and - S(0)kR^!, where each R' is independently H, alkyl, or substituted alkyl.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non- natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[00271] In addition, amino acids having the structure of Formula (XXXXXIX) are included:

(XXXXXIX), wherein;

R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; and

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non- natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[00272] In addition, amino acids having the structure of Formula (XXXXXX) are included:

(XXXXXX), wherein:

R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl,

[00273] In addition, the following amino acids having structures of Formula (XXXXXX) are included:

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non- natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[00274] The carbonyl or dicarbonyl functionality can be reacted selectively with a hydroxylamine-containing reagent under mild conditions in aqueous solution to form the corresponding oxime linkage that is stable under physiological conditions. See, e.g., Jencks, W. P., J, Am. Chem. Soc, 81, 475-481 (1959); Shao, J. and Tam, J. P., J. Am. Chem. Soc. 1 17( 14):3893-3899 (1995), Moreover, the unique reactivity of the carbonyl or dicarbonyl group allows for selective modification in the presence of the other amino acid side chains, See, e.g., Cornish, V. W., et al., J. Am. Chem. Soc. 118:8150-8151 (1996); Geoghegan, K. F, & Stroh, J, G„ Bioconjug. Chem. 3 : 1 38-146 (1992); Mahal, L, K„ et al., Science 276: 1 125- 1 128 (1997).

[00275] The synthesis of p-acetyl-(+/-)-phenylalanine and m-acetyl-(+/-)-phenylalanine is described in Zhang, Z., et al, Biochemistry 42: 6735-6746 (2003), incorporated by reference. Other carbonyl- or dicarbonyl-containing amino acids can be similarly prepared. [00276] In some embodiments, a polypeptide comprising a non-natural amino acid is chemically modified to generate a reactive carbonyl or dicarbonyl functional group. For instance, an aldehyde functionality useful for conjugation reactions can be generated from a functionality having adjacent amino and hydroxyl groups. Where the biologically active molecule is a polypeptide, for example, an N-terminal serine or threonine (which may be normally present or may be exposed via chemical or enzymatic digestion) can be used to generate an aldehyde functionality under mild oxidative cleavage conditions using periodate, See, e.g., Gaertner, et. al., Bioconjug. Chem. 3 : 262-268 (1992); Geoghegan, K. & Stroh, J., Bioconjug. Chem. 3 : 138-146 (1992); Gaertner et al., J. Biol. Chem. 269:7224-7230 (1994). However, methods known in the art are restricted to the amino acid at the N-terminus of the peptide or protein.

[00277] Additionally, by way of example a non-natural amino acid bearing adjacent hydroxyl and amino groups can be incorporated into a polypeptide as a "masked" aldehyde functionality. For example, 5 -hydroxy lysine bears a hydroxyl group adjacent to the epsilon amine. Reaction conditions for generating the aldehyde typically involve addition of molar excess of sodium metaperiodate under mild conditions to avoid oxidation at other sites within the polypeptide. The pH of the oxidation reaction is typically about 7,0. A typical reaction involves the addition of about 1.5 molar excess of sodium meta periodate to a buffered solution of the polypeptide, followed by incubation for about 10 minutes in the dark, See, e.g. U.S. Patent No. 6,423,685.

B. Structure and Synthesis of Non-Natural Amino Acids: Dicarbonyl, Dicarbonyl-like, Masked Dicarbonyl, and Protected Dicarbonyl Groups

[00278] Amino acids with an electrophilic reactive group allow for a variety of reactions to link molecules via nucleophilic addition reactions among others. Such electrophilic reactive groups include a dicarbonyl group (including a diketone group, a ketoaldehyde group, a ketoacid group, a ketoester group, and a ketothioester group), a dicarbonyl-like group (which has reactivity similar to a dicarbonyl group and is structurally similar to a dicarbonyl group), a masked dicarbonyl group (which can be readily converted into a dicarbonyl group), or a protected dicarbonyl group (which has reactivity similar to a dicarbonyl group upon deprotection). Such amino acids include amino acids having the structure of Formula (XXXVII):

(XXXVII), wherein;

A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower hetero alkylene, substituted hetero alkylene, lower heterocycloalkylene, substituted lower heterocycloaikylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

B is optional, and when present is a linker linked at one end. to a diamine containing moiety, the linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -0-(alkylene or substituted alkylene)-, -S-(alkylene or substituted alkylene)-, -C(0)R"-, -S(0)k(alkylene or substituted alkylene)-, where k is 1, 2, or 3, -C(0)-(alkylene or substituted alkylene)-, -C(S)-(alkylene or substituted alkylene)-, -NR"-(alkylene or substituted alkylene)-, -CON(R")-(alkylene or substituted alkylene)-, -CSN(R")-(alkylene or substituted alkylene)-, and -N(R")CO (alkylene or substituted alkylene)-, where each R" is independently H, alkyl, or substituted alkyl;

where, Ti is a bond, optionally substituted C1 -C4 alkylene, optionally substituted C1 -C4 alkenylene, or optionally substituted heteroalkyl; wherein each optional substituents is independently selected from lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroaryiene, a!karylene, substituted alkarylene, aralkylene, or substituted aralkylene;

T2, is selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -0-, -0-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(0)k- where k is 1, 2, or 3, -S(0)k(alkylene or substituted alkylene)-, -C(0 , -C(0)-(alky1ene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(0)N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')- (alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)-, -N(R')C(0)0-, -S(0)_kN(R')-, -N(R')C(0)N(R')-, -N(R')C(S)N(R , -N(R')S(0)_kN(R , -N(R')-N= -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=

and -C(R')2-N(R')-N(R')-, where each R' is independently H, alkyl, or substituted alkyl;

where each i is independently selected from the group consisting of -0-, -S-, -N(H)-, - N(R)-₃ -N(Ac)-, and -N(OMe)-; X₂ is -OR, -OAc, -SR, -N(R)z, -N(R)(Ac), - N(R)(OMe), or N₃, and where each R' is independently H, alkyl, or substituted alkyl;

R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and 2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; or the -A-B-K-R groups together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl comprising at least one carbonyl group, including a dicarbonyl group, protected carbonyl group, including a protected dicarbonyl group, or masked carbonyl group, including a masked dicarbonyl group; or the -K-R group together forms a monocyclic or bicyclic cycloalkyl or heterocycloalkyl comprising at least one carbonyl group, including a dicarbonyl group, protected carbonyl group, including a protected dicarbonyl group, or masked carbonyl group, including a masked dicarbonyl group.

[00279] Non-limiting example of dicarbonyl amino acids having the structure of Formula (XXXVII) include:

[00280] The following amino acids having structures of Formula (XXXVII) are also included:

ο , and Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non- natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

C, Structure and Synthesis of Non-Natural Amino Acids: Ketoalkyne, Ketoalkyne -like, Masked Ketoalkyne, Protected Ketoalkyne Group, Alkyne, and Cycloalkyne Groups

[00281] Amino acids containing reactive groups with dicarbonyl-like reactivity allow for the linking of molecules via nucleophilic addition reactions. Such e!ectrophilic reactive groups include a ketoalkyne group, a ketoalkyne-like group (which has reactivity similar to a ketoalkyne group and is structurally similar to a ketoalkyne group), a masked ketoalkyne group (which can be readily converted into a ketoalkyne group), or a protected ketoalkyne group (which has reactivity similar to a ketoalkyne group upon deprotection). In some embodiments, amino acids containing reactive groups with a terminal alkyne, internal alkyne or cycloalkyne allow for Unking of molecules via cycloaddition reactions (e.g., 1,3-dipolar cycloadditions, azide-alkyne Huisgen cycloaddition, etc.) Such amino acids include amino acids hav :

(XXXXXXI-B), wherein:

A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted hetero alkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

B is optional, and when present is a linker linked at one end to a diamine containing moiety, the linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -0-(alkylene or substituted alkylene)-, -S-(alkylene or substituted alkylene)-, -C(0) "-₅ -S(0)k(alkylene or substituted alkylene)-, where k is 1 , 2, or 3, -C(0)-(alkylene or substituted alkylene)-, -C(S)-(alkylene or substituted alkylene)-, -NR"- (alkylene or substituted alkylene)-, -CON(R")-(alkylene or substituted alkylene)-, -CSN(R")-(alkylene or substituted alkylene)-, and -N(R")CO- (alkylene or substituted alkylene)-, where each R" is independently H, alkyl, or substituted alkyl;

G is optional, and when present is

T is a carbonyl protecting group including, but not limited to,

, where each Xi is independently selected from the group consisting of -0-, -S-, -N(H)-, -N(R)-, -N(Ac)-, and -N(OMe)-; X₂ is -OR, - OAc, -SR, -N(R)₂, -N(R)(Ac), -N(R)(OMe), or N₃, and where each R' is independently H, alkyl, or substituted alkyl;

R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

R] is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide;

R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each of R3 and R is independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl; each R| 9 is independently selected from, the group consisting of Ci-Ce alkyl, Cj-Ce alkoxy, ester, ether, thioether, aminoa!kyl, halogen, alkyl ester, aryl ester, amide, aryl amide, alkyl halide, alkyl amine, alkyl sulfonic acid, alkyl nitro, thioester, sulfonyl ester, halosuffonyl, nitrile, alkyl nitrile, and nitro; and q is 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 1 0 or 1 1 ,

D. Structure and Synthesis of Non-Natural Amino Acids: Ketoamine, Ketoamine-like, Masked Ketoamine, and Protected Ketoamine Groups

[00282] Amino acids containing reactive groups with dicarbonyl-like reactivity allow for the linking of molecules via nucleophilic addition reactions. Such reactive groups include a ketoamine group, a ketoamine-like group (which has reactivity similar to a ketoamine group and is structurally similar to a ketoamine group), a masked ketoamine group (which can be readily converted into a ketoamine group), or a protected ketoamine group (which has reactivity similar to a ketoamine group upon deprotection), Such amino acids include amino acids having the structure of Formula (XXXXXXII):

(XXXXXXII) wherein:

A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower hetero cycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

B is optional, and when present is a linker linked at one end to a diamine containing moiety, the linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -0-(alkylene or substituted alkylene)-, -S-(alkylene or substituted alkylene)-, -C(0)R"-, -S(0)k(alkyIene or substituted alkylene)-, where k is 1 , 2, or 3, -C(0)-(alkylene or substituted alkylene)-, -C(S)-(alkylene or substituted alkylene)-, -NR"- (alkylene or substituted alkylene)-, -CON(R")-(alkylene or substituted alkylene)-, -CSN(R" (alkylene or substituted alkylene)-, and -N(R")CO- (alkylene or substituted alkylene)-, where each R" is independently H, alkyl, or substituted alkyl;

Ti is an optionally substituted C1-C4 alkylene, an optionally substituted C1 -C4 alkenylene, or an optionally substituted heteroalkyl;

T4 is a carbony! protecting group including, but not limited to, ^R'° ^0R' ,

where each Xj is independently selected from the group consisting of -0-, -S-, -N(H)-, - N(R')-, -N(Ac)-, and -N(OMe)-; X₂ is -OR, -OAc, -S ', -N(R')a, -N(R')(Ac), - N(R')(OMe), or N3, and where each R' is independently H, alkyl, or substituted alkyl;

R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each of Rj and R is independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and 4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl.

[00283] Amino acids having the structure of Formula (ΧΧΧΧΧΧΠ) include amino acids hav Il) and Formula (XXXXXXIV):

( XXXXXIV)

wherein each R_a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R')i, -C(0)_kR' where k is 1, 2, or 3, -C(0)N(R')₂, -OR', and - S(0)kR', where each R' is independently Ft, alkyl, or substituted alkyl.

E. Structure and Synthesis of Non-Natural Amino Acids: Diamine, Diamine-like, Masked Diamine, Protected Amines andAzides

[00284] Amino acids with a nucleophilic reactive group allow for a variety of reactions to link molecules via electrophilic addition reactions among others. Such nucleophilic reactive groups include a diamine group (including a hydrazine group, an amidine group, an imine group, a 1 , 1 -diamine group, a 1,2-diamine group, a 1,3-diamine group, and a 1,4-diamine group), a diamine-like group (which has reactivity similar to a diamine group and is structurally similar to a diamine group), a masked diamine group (which can be readily converted into a diamine group), or a protected diamine group (which has reactivity similar to a diamine group upon deprotection). In some embodiments, amino acids containing reactive groups with azides allow for linking of molecules via cycloaddition reactions (e.g., 1,3- dipolar cycloadditions, azide-alkyne Huisgen cycloaddition, etc.).

[00285] In another aspect are methods for the chemical synthesis of hydrazine-substituted molecules for the derivatization of carbonyl-substituted dolastatin derivatives. In one embodiment, the hydrazine-substituted molecule can dolastatin linked derivatives. In one embodiment are methods for the preparation of hydrazine-substituted molecules suitable for the derivatization of carbonyl-containing non-natural amino acid polypeptides, including by way of example only, ketone-, or aldehyde-containing non-natural amino acid polypeptides. In a further or additional embodiment, the non-natural amino acids are incorporated site- specifically during the in vivo translation of proteins. In a further or additional embodiment, the hydrazine-substituted dolastatin derivatives allow for the site-specific derivatization of carbonyl-containing non-natural amino acids via nucleophilic attack of each carbonyl group to form a heterocycle-derivatized polypeptide, including a nitrogen-containing heterocycle- derivatized polypeptide in a site-specific fashion. In a further or additional embodiment, the method for the preparation of hydrazine-substituted dolastatin derivatives provides access to a wide variety of site-specifically derivatized polypeptides. In a further or additional embodiment are methods for synthesizing hydrazine-functionalized polyethyleneglycol (PEG) linked dolastatin derivatives.

[00286] Such amino acids include amino acids having the structure of Formula (XXXVII- A) or (XXXVII-B):

(XXXVII), (XXXVII-B),

wherein:

A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower hetero alkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

B is optional, and when present is a linker linked at one end to a diamine containing moiety, the linker selected from the group consisting of lower alkylene, substituted lower allcylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -0-(alkylene or substituted alkylene)-, -S-(a!kylene or substituted alkylene)-, -C(0)R"-, -C(0)R"-, -S(0)_k(alkylene or substituted alkylene)-, where k is 1, 2, or 3, -C(0)-(alkylene or substituted alkylene)-, -C(S)- (alkylene or substituted alkylene)-, -NR"-(alkylene or substituted alkylene)-, -CON(R")-(alkylene or substituted alkylene)-, -CSN(R")-(alkylene or substituted alkylene)-, and -N(R")CO-(alkylene or substituted alkylene)-, where each R" is independently H, alkyl, or substituted alkyl;

where:

Rs and R9 are independently selected from H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, or amine protecting group;

Tj is a bond, optionally substituted C 1 -C4 alkylene, optionally substituted C1-C4 alkenylene, or optionally substituted heteroalkyl;

T2 is optionally substituted C1 -C4 alkylene, optionally substituted C 1 -C4 alkenylene, optionally substituted heteroalkyl, optionally substituted aryl, or optionally substituted heteroaryl; wherein each optional substituents is independently selected from lower alkyl, substituted lower alkyl, lower cycloalkyl, substituted lower cycloalkyl, lower alkenyl, substituted lower alkenyl, alkynyl, lower heteroalkyl, substituted heteroalkyl, lower heterocycloalkyl, substituted lower heterocycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each of R3 and R is independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R or two R₃ groups optionally form a cycloalkyl or a heterocycloalkyl; or the -A-B-K-R groups together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl comprising at least one diamine group, protected diamine group or masked diamine group; or the -B-K-R groups together form a bicyclic or tricyclic cycloalkyl or cycloaryl or heterocycloalkyl comprising at least one diamine group, protected diamine group or masked diamine group; or the -K-R group together forms a monocyclic or bicyclic cycloalkyl or hetero eye lo alley 1 comprising at least one diamine group, protected diamine group or masked diamine group; wherein at least one amine group on -A-B-K-R is optionally a protected amine,

[00287] In one aspect are compounds comprising the structures 1 or 2:

1 2

wherein:

A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

B is optional, and when present is a linker linked at one end to a diamine containing moiety, the linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -0-(alkylene or substituted alkylene)-, -S-(alkylene or substituted alkylene)-, -C(0)R"-,- S(0)k(alkylene or substituted alkylene)-, where k is 1, 2, or 3, -C(0)-(alkylene or substituted alkylene)-, -C(S)-(alkylene or substituted alkylene)-, -NR"- (alkylene or substituted, alkylene)-, -CON(R")-(alkylene or substituted alkylene)-, -CSN(R")-(alkylene or substituted alkylene)-, and -N(R")CO- (alkylene or substituted alkylene)-, where each R" is independently H, alkyl, or substituted alkyl;

T] is a bond or CEh; and T2 is CH; wherein each optional substituents is independently selected from lower alkyl, substituted lower alkyl, lower cycloalkyl, substituted lower cycloalkyl, lower alkenyl, substituted lower alkenyl, alkynyl, lower heteroalkyl, substituted heteroalkyl, lower heterocycloalkyl, substituted lower heterocycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl;

Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and

R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each of R3 and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R or two 3 groups optionally form a cycloalkyl or a heterocycloalkyl; or the A-B-diamine containing moiety together form a bicyclic cycloalkyl or heterocycloalkyl comprising at least one diamine group, protected diamine group or masked diamine group; or the -B-diamine containing moiety groups together form a bicyclic or tricyclic cycloalkyl or cycloaryl or heterocycloalkyl comprising at least one diamine group, protected diamine group or masked diamine group; wherein at least one amine group on -A-B -diamine containing moiety is optionally a protected amine; or an active metabolite, salt, or a pharmaceutically acceptable prodrug or solvate thereof,

[00288] The following non-limiting examples of amino acids having the structure of Formula (XXXVII) are included:

Such non-natural amino acids may also be in the form of a salt or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and/or optionally post translationally modified. [00289] In certain embodiments, compounds of Formula (XXXVII) are stable in aqueous solution for at least 1 month under mildly acidic conditions. In certain embodiments, compounds of Formula (XXXVII) are stable for at least 2 weeks under mildly acidic conditions. In certain embodiments, compound of Formula (XXXVII) are stable for at least 5 days under mildly acidic conditions. In certain embodiments, such acidic conditions are pH about 2 to about 8.

[00290] In certain embodiments of compounds of Formula (XXXVII), B is lower alkylene, substituted lower alkylene, 0-(alkylene or substituted alkylene)-, C(R')=NN(R')-, - N(R')CO-₃ C(0)~, -C(R')=N-, C(0)-(alkylene or substituted alkylene)-, CON(R')(alkylene or substituted alkylene)-, -S(alkylene or substituted alkylene)-, -S(O) (alkylene or substituted alkylene)-, or -S(0)₂(alkylene or substituted alkylene)-. In certain embodiments of compounds of Formula (XXXVII), B is -0(CH₂)-, -CH=N-, CH= H-, -NHCH2-, -NHCO-, C(O)-, C(0)(CH₂)-, CONH(CH₂)-, -SCH₂-_: -S(=0)CH₂-, or -S(0)₂CH₂-. In certain embodiments of compounds of Formula (XXXVII), R is O-6 alkyl or cycloalkyl. In certain embodiments of compounds of Formula (XXXVII) R is -CH3, -CH(CH3)₂, or cyclopropyl. In certain embodiments of compounds of Formula (XXXVII), Ri is H_; tert-butyloxycarbonyl (Boc), 9- Fluorenylmethoxycarbonyl (Fmoc), N-acetyl, tetrafluoroacetyl (TFA), or benzyloxycarbonyl (Cbz). In certain embodiments of compounds of Formula (XXXVII), Ri is a resin, amino acid, polypeptide, or polynucleotide, In certain embodiments of compounds of Formula (XXXVII), i is an antibody, antibody fragment or monoclonal antibody, In certain embodiments of compounds of Formula (XXXVII), R₂ is OH, Omethyl, O-ethyl, or O-t-butyl, In certain embodiments of compounds of Formula (XXXVII), R₂ is a resin, at least one amino acid, polypeptide, or polynucleotide, In certain embodiments of compounds of Formula (XXXVII), R2 is an antibody, antibody fragment or monoclonal antibody.

[00291] The following non-limiting examples of amino acids having the structure of Formula (XXXVII) are also included:

F. Structure and Synthesis of Non-Natural Amino Acids: Aromatic Amines

[00293] Non-natural amino acids with nucleophilic reactive groups, such as, by way of example only, an aromatic amine group (including secondary and tertiary amine groups), a masked aromatic amine group (which can be readily converted into a aromatic amine group), or a protected aromatic amine group (which has reactivity similar to an aromatic amine group upon deprotection) allow for a variety of reactions to link molecules via various reactions, including but not limited to, reductive alkylation reactions with aldehyde containing dolastatin linker derivatives. Such aromatic amine containing non-natural amino acids include amino acids having the structure of Formula (XXXXXXV):

(XXXXXXV)

wherein:

is selected from the group consisting of a monocyclic aryl ring, a bicyclic aryl ring, a multicyclic aryl ring, a monocyclic heteroaryl ring, a bicyclic heteroaryl ring, and a multicyclic heteroaryl ring;

A is independently CR_a, or N;

B is independently CR_a, N, O, or S; each Ra is independently selected from the group consisting of H, halogen, alkyl, -NC , -CN, substituted alkyl, -N(R')₂, -C(0)_kR' , -C(0)N(R')₂, -OR', and -S(0)_kR', where k is 1, 2, or 3 ; and n is 0, 1, 2, 3, 4, 5, or 6;

R] is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; and

R2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; each of R3 and R4 is independently H, halogen, lower alkyl, or substituted Sower alkyl, or R3 and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl;

M is H or -CH2R5 ; or the M-N-C(Rs) moiety may form a 4 to 7 membered ring structure;

R5 is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyi, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -C(0)R", -C(0)OR", -C(0)N(R")2, -C(0)NHCH(R")2, -(alkylene or substituted alkylene)-N(R")₂, -(alkenylene or substituted alkenylene)-N(R")2, -(alkylene or substituted alkylene)-(aryl or substituted aryl), -(alkenylene or substituted alkenylene)-(aryl or substituted aryl), -(alkylene or substituted alkylene)-ON(R")2, -(alkylene or substituted alkylene)-C(0)SR", -(alkylene or substituted alkylene)-S-S-(aryl or substituted aryl), wherein each R" is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or -C(0)OR'; or two R5 groups optionally form a cycloalkyl or a heterocycloalkyl; or R5 and any R_a optionally form a cycloalkyl or a heterocycloalkyl; and each R' is independently H, alkyl, or substituted alkyl.

Such non-natural amino acids may also be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally reductively alkylated.

The structure

(as presented in all examples herein) does not present the relative orientations of "A," "B," "NH-M" and "R_a"; rather these four features of this structure may be oriented in any chemically-sound manner (along with other features of this structure), as illustrated by example herein.

[00294] Non-natural amino acids containing an aromatic amine moiety having the structure of Formula (A) include non-natural amino acids having the structures:

, and

M

wherein, each A' is independently selected from CR_S, N, or C—NH , and up to two A'

M

I

may be ^c NH with the remaining A' selected from CR_a, or N,

Such non-natural amino acids may also be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally reductively alkylated.

[00295] Non-limiting examples of non-natural amino acids containing an aromatic amine moiety having the structure of Formula (XXXXXXV) include non-natural amino acids having Formula (XXXXXXVII),

(XXXXXXVI I),

wherein; G is an amine protecting group, including, but not limited to,

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non- natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally reductively alkylated.

[00296] Non-natural amino acids containing an aromatic amine moiety have the following structures:

wherein each R_a is independently selected from the group consisting of H, halogen, alkyi, - N0₂₎ -CN, substituted alkyi, -N(R')2, -C(0)_kR', -C(0)N(R% -OR', and -S(0)_kR\ where k is 1, 2, or 3;

M is H or -CH2R5; or the M-N-C(Rs) moiety may form a 4 to 7 membered ring structure;

Ri is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide;

R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide;

Rs is alkyi, substituted alkyi, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -C(0)R", -C(0)OR", -C(0)N(R")₂, -C(0)NHCH(R")2, -(alkylene or substituted alkylene)-N(R")₂, -(alkenylene or substituted alkenylene)-N(R")2, -(alkylene or substituted alkylene) -(aryl or substituted aryl), -(alkenylene or substituted alkenylene)-(aryl or substituted aryl), -(alkylene or substituted alkylene)-ON(R")₂, -(alkylene or substituted alkylene)-C(0)SR", -(alkylene or substituted alkylene)-S-S-(aryl or substituted aryl), wherein each R" is independently hydrogen, alkyi, substituted alkyi, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or -C(0)0R'; or Rs and any R_a optionally form a cycloalkyl or a hetero cycloalkyl; and each R' is independently H, alkyl, or substituted alkyl. Such non-natural amino acids may also be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide.

[00297] Such non-natural amino acids of Formula (XXXXXXV) may be formed by reduction of protected or masked amine moieties on the aromatic moiety of a non-natural amino acid. Such protected or masked amine moieties include, but are not limited to, imines, hydrazines, nitro, or azide substituents. The reducing agents used to reduce such protected or masked amine moieties include, but are not limited to, TCEP, Na2S, Na2S2C>4, L1AIH4, NaBH₄ or NaBCNHs.

IV Location of non-natural amino acids in dolastatin linker derivatives

[00298] The methods and compositions described herein include incorporation of one or more non-natura! amino acids into a chimeric antigen receptor (CAR) and/or a cancer- reactive antibody, and/or a tag on the cancer-reactive antibody One or more non-natural amino acids may be incorporated at one or more particular positions which, in some embodiments, do not disrupt activity. In other embodiments, including as for previous examples such as producing a population of T cells with non-functioning alpha and/or beta chains in the TCR, These can be achieved by making "conservative" substitutions, including but not limited to, substituting hydrophobic amino acids with non-natural or natural hydrophobic amino acids, bulky amino acids with non-natural or natural bulky amino acids, hydrophilic amino acids with non-natural or natural hydrophilic amino acids) and/or inserting the non-natural amino acid in a location that is not required for activity.

[00299] A variety of biochemical and structural approaches can be employed to select the desired sites for substitution with a non-natural amino acid. In some embodiments, the non- natural amino acid is linked at the C-terminus of the antibody or CAR. In other embodiments, the non-natural amino acid is linked at the N-terminus of the antibody or CAR. Any position of the of the antibody or CAR is suitable for selection to incorporate a non- natural amino acid, and selection may be based on rational design or by random selection for any or no particular desired purpose. Selection of desired sites may be based on producing a non-natural amino acid polypeptide (which may be further modified or remain unmodified) having any desired property or activity, including but not limited to a receptor binding modulators, receptor activity modulators, modulators of binding to binder partners, binding partner activity modulators, binding partner conformation modulators, dimer or multimer formation, no change to activity or property compared to the native molecule, or manipulating any physical or chemical property of the polypeptide such as solubility, aggregation, or stability, Alternatively, the sites identified as critical to biological activity may also be good candidates for substitution with a non-natural amino acid, again depending on the desired activity sought for the polypeptide. Another alternative would be to simply make serial substitutions in each position on the polypeptide chain with a non-natural amino acid and observe the effect on the activities of the polypeptide. Any means, technique, or method for selecting a position for substitution with a non-natural amino acid into any polypeptide is suitable for use in the methods, techniques and compositions described herein.

[00300] The structure and activity of naturally-occurring mutants of a polypeptide that contain deletions can also be examined to determine regions of the protein that are likely to be tolerant of substitution with a non-natural amino acid, Once residues that are likely to be intolerant to substitution with non-natural amino acids have been eliminated, the impact of proposed substitutions at each of the remaining positions can be examined using methods including, but not limited to, the three-dimensional structure of the relevant polypeptide, and any associated ligands or binding proteins, X-ray crystallographic and NMR structures of many polypeptides are available in the Protein Data Bank, a centralized database containing three-dimensional structural data of large molecules of proteins and nucleic acids, one can be used to identify amino acid positions that can be substituted with non-natural amino acids, . In addition, models may be made investigating the secondary and tertiary structure of polypeptides, if three-dimensional structural data is not available, Thus, the identity of amino acid positions that can be substituted with non-natural amino acids can be readily obtained,

[00301] Exemplary sites of incorporation of a non-natural amino acid include, but are not limited to, those that are excluded from potential receptor binding regions, or regions for binding to binding proteins or ligands may be fully or partially solvent exposed, have minimal or no hydrogen-bonding interactions with nearby residues, may be minimally exposed, to nearby reactive residues, and/or may be in regions that are highly flexible as predicted by the three-dimensional crystal structure of a particular polypeptide with its associated receptor, ligand or binding proteins,

[00302] A wide variety of non-natural amino acids can be substituted for, or incorporated into, a given position in a polypeptide, By way of example, a particular non-natural amino acid may be selected for incorporation based on an examination of the three dimensional crystal structure of a polypeptide with its associated ligand, receptor and/or binding proteins, a preference for conservative substitutions

[00303] In one embodiment, the methods described herein include incorporating into the antibody or CAR, where the of the antibody or CAR comprises a first reactive group; and contacting the first reactive group with a molecule (including but not limited to a second protein or polypeptide or polypeptide analog; an antibody or antibody fragment; and any combination thereof) that comprises a second reactive group, In certain embodiments, the first reactive group is a hydroxylamine moiety and the second reactive group is a carbonyl or dicarbonyl moiety, whereby an oxime linkage is formed. In certain embodiments, the first reactive group is a carbonyl or dicarbonyl moiety and the second reactive group is a hydroxylamine moiety, whereby an oxime linkage is formed. In certain embodiments, the first reactive group is a carbonyl or dicarbonyl moiety and the second reactive group is an oxime moiety, whereby an oxime exchange reaction occurs. In certain embodiments, the first reactive group is an oxime moiety and the second reactive group is carbonyl or dicarbonyl moiety, whereby an oxime exchange reaction occurs.

[00304] In some cases, the antibody and/or CAR incorporation(s) will be combined with other additions, substitutions, or deletions within the polypeptide to affect other chemical, physical, pharmacologic and/or biological traits. In some cases, the other additions, substitutions or deletions may increase the stability (including but not limited to, resistance to proteolytic degradation) of the polypeptide or increase affinity of the polypeptide for its appropriate receptor, ligand and/or binding proteins. In some cases, the other additions, substitutions or deletions may increase the solubility (including but not limited to, when expressed in K coli or other host cells) of the polypeptide, In some embodiments sites are selected for substitution with a naturally encoded or non-natural amino acid in addition to another site for incorporation of a non-natural amino acid for the purpose of increasing the polypeptide solubility following expression in K coli, or other recombinant host cells. In some embodiments, the polypeptides comprise another addition, substitution, or deletion that modulates affinity for the associated ligand, binding proteins, and/or receptor, modulates (including but not limited to, increases or decreases) receptor dimerization, stabilizes receptor dimers, modulates circulating half-life, modulates release or bio-availability, facilitates purification, or improves or alters a particular route of administration. Similarly, the non- natural amino acid polypeptide can comprise chemical or enzyme cleavage sequences, protease cleavage sequences, reactive groups, antibody-binding domains (including but not limited to, FLAG or poly-His) or other affinity based sequences (including but not limited to, FLAG, poly-His, GST, etc) or linked molecules (including but not limited to, biotin) that improve detection (including but not limited to, GFP), purification, transport thru tissues or cell membranes, prodrug release or activation, size reduction, or other traits of the polypeptide,

Claims

WHAT IS CLAIMED IS:

1. A method of treating cancer in a subject, comprising: (a) administering a formulation of tagged proteins to a subject in need of treatment, wherein the tagged proteins bind a cancer cell in the subject, and (b) administering a therapeutically-effective population of anti-tag chimeric antigen receptor (AT-CAR)-expressing T cells to the subject, wherein the AT-CAR-expressing T cells bind the tagged proteins and induce cancer cell death, thereby treating cancer in a subject,

2. A method of treating cancer in a subject, comprising: (a) administering one or more formulations of tagged proteins to a subject in need of treatment, wherein the tagged proteins bind a cancer cell in the subject, and (b) administering one or more therapeutically- effective populations of AT-CAR-expressing T cells to the subject, wherein the AT-CAR- expressing T cells bind the tagged proteins and induce cancer cell death, thereby treating cancer in a subject.

3. A method of treating cancer in a subject, comprising: (a) administering at least two formulations of tagged proteins to a subject in need of treatment, wherein the tagged proteins bind a cancer cell in the subject, and (b) administering at least two therapeutically- effective populations of AT-CAR-expressing T cells to the subject, wherein the AT-CAR- expressing T cells bind the tagged proteins and induce cancer cell death, thereby treating cancer in a subject,

4. The method of claim 1, wherein the tag of each formulation of tagged proteins is the same or different and the tag is selected from the group consisting of fluorescein isothiocyanate (FITC), streptavidin, biotin, histidine, dinitrophenol, peridinin chlorophyll protein complex, green fluorescent protein, phycoerythrin (PE), horse radish peroxidase, palmitoylation, nitrosylation, alkalanine phosphatase, glucose oxidase, and maltose binding protein.

5. The method of claim 1, wherein the protein of each formulation of tagged proteins is the same or different and the protein is an antibody or an antigen-binding fragment thereof.

6. The method of claim 5, wherein the antibody or antigen-binding fragment thereof is cetuximab, nimotuzumab, panitumumab, retuximab, omalizumab, tositumomab, trastuzumab, geratuzumab, alemtuzumab, bevacuzimab or an antigen-binding fragment of any one thereof.

7. The method of claim 1, wherein the AT-CAR of each population of AT-CAR- expressing T cells is the same or different and the AT-CAR comprises a tag-binding domain, a transmembrane domain, and an activation domain.

8. The method of claim 7, wherein the tag-binding domain is an antibody or an antigen -binding fragment thereof,

9. The method of claim 7, wherein one of the tag-binding domain, transmembrane domain, and activation domain comprises one or more non-naturally encoded amino acid(s).

10. The method of claim 1, wherein the tagged protein comprises one or more non-naturally encoded amino acids.

1 1. The method of claim 7, wherein the tag-binding domain specifically binds FITC, biotin, PE, histidine or streptavidin.

12. The method of claim 8, wherein the antigen-binding fragment is a single chain variable fragment (scFv).

13. The method of claim 8, wherein the antigen-binding fragment is a single chain variable fragment (scFv) that specifically binds FITC, biotin, PE, histidine or streptavidin,

14. The method of claim 7, wherein the transmembrane domain is the hinge and transmembrane regions of the human CD8 a-chain,

15. The method of claim 7, wherein the activation domain comprises one or more of the cytoplasmic region of CD28, the cytoplasmic region of CD137 (41BB), OX40, HVEM, D% and FcRs.

16. The method of claim 1, wherein the T cells of each population of AT-CAR- expressing T cells are the same or different and wherein the T cells are selected from the group consisting of T cells of any HLA-background from peripheral blood mononuclear cells (PBMC), T cells isolated from a tumor explant of the subject, and intratumoral T cells of the subject,

17. The method of claim 1, wherein the T cells of each population of AT-CAR- expressing T cells consist of HLA-A2+ peripheral blood mononuclear cells (PBMC).

18. The method of claim 1 , wherein the formulation(s) of tagged proteins are administered to the subject prior to administration of the therapeutically-effective population(s) of AT-CAR-expressing T cells.

19. The method of claim 1. wherein the formulation(s) of tagged proteins are administered to the subject concurrently with administration of the therapeutically-effective population(s) of AT-CAR-expressing T cells.

20. The method of claim 1, wherein the formulation(s) of tagged proteins are administered to the subject after administration of the therapeutically-effective population(s) of AT-CAR-expressing T cells.

21. The method of claim 1 , wherein the formulation(s) of tagged proteins and the therapeutically-effective population(s) of AT-CAR-expressing T cells are administered to the subject in any order.

22. The method of claim 1 , wherein AT-CAR-expressing T cell binding to the tagged proteins induces cytolytic activation of the T cells.

23. The method of claim 1 , wherein the subject is a human.