Adaptors for adoptive cell therapy and compounds for preparing same

Abstract

The present disclosure provides adaptor molecules for use in adoptive cell therapies of Formula I, chimeric antigen receptor (CAR) systems based on the same, and methods of treatment using the same. Further provided are compounds of Formula II useful in preparing adaptor molecules, such as those of Formula III.

Description

[0001] ADAPTORS FOR ADOPTIVE CELL THERAPY AND COMPOUNDS FOR PREPARING SAME CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to United States Provisional Application No. 63 / 541,954 filed October 2, 2023, the disclosure of which is incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under Grant No. GM142007 awarded by the National Institutes of Health. The Government has certain rights in the invention. BACKGROUND The ability to make a universal yet versatile system to generate T cells that can recognize various types of cancers has important clinical implications for the use of T cell- based therapies. One current strategy incorporates the use of genetic engineering to express a chimeric antigen receptor (CAR) on T cells. The extracellular domain of a typical CAR consists of VH and VL domains – single chain fragment variable (scFv) – from the antigen binding sites of a monoclonal antibody. The scFv is linked to a flexible transmembrane domain followed by a tyrosine-based activation motif such as that from CD3ζ. Second and third-generation CARs include additional activation domains from co-stimulatory molecules such as CD28 and CD137 (41BB), which serve to enhance T cell survival and proliferation. CAR T cells offer the opportunity to seek out and destroy cancer cells by recognizing tumor-associated antigens (TAA) expressed on their surface. As such, the recognition of tumor cells occurs via an MHC- independent mechanism. Various preclinical and early-phase clinical trials highlight the efficacy of CAR T cells in treating cancer patients with solid tumors and hematopoietic malignancies. Despite the promise that CAR T cells might have in treating cancer patients, there are several limitations to the generalized clinical application of CAR T cells. First, since no single tumor antigen is universally expressed by all cancer types, scFv in CAR needs to be constructed for each tumor antigen to be targeted. Second, the financial cost and labor-intensive tasks associated with identifying and engineering scFvs against a variety of tumor antigens pose a major challenge. Third, tumor antigens targeted by CAR could be downregulated or mutated in response to treatment, resulting in tumor evasion. Since current CAR T cells recognize only one target antigen, such changes in tumors negate the therapeutic effects. Therefore, the generation of CAR T cells capable of recognizing multiple tumor antigens is highly desired. Finally, CAR T cells react with the target antigen weakly expressed on non-tumor cells, potentially causing severe adverse effects. Use of scFvs with higher specificity to tumor antigen is required to avoid such “on-target off-tumor” reactions. Although ongoing studies are focused on generating methods to shut off CAR T cells in vivo, this system has yet to be developed and might pose additional inherent challenges.

[0002] Modifications to existing CAR T systems that address and overcome the hurdles currently preventing further development into effective means of in vivo treatment are therefore needed.

[0003] SUMMARY

[0004] The present disclosure provides adaptor molecules for use in adoptive cell therapies, chimeric antigen receptor (CAR) systems based on the same, and methods of treatment using the same. In one aspect, the adaptor molecules comprise a compound of Formula I:

[0005] T-L-A (I) or a pharmaceutically acceptable salt thereof. In some aspects, T in Formula I comprises a tag ligand. In some aspects, L in Formula I comprises a linker moiety. In some aspects, A in Formula I comprises a moiety capable of binding a cell surface protein selected from a folate receptor, a carbonic anhydrase, prostate-specific antigen, and programmed death-ligand 1 (PD- Ll). Pharmaceutical compositions comprising the adaptor molecules described herein and a pharmaceutically acceptable carrier or excipient are also provided.

[0006] In another aspect, a compound of Formula II is provided:

[0007] E-LA-TA(II)

[0008] In some aspects, E in Formula II comprises an active ester moiety. In some aspects, LAin Formula II comprises a linker moiety. In some aspects, TAin Formula II comprises a ligand for Halo-tag.

[0009] In another aspect, a method of synthesizing an adaptor molecule of Formula III is provided: AA-(C=O)-LA-TA(III) wherein AAis selected from an antibody or antigen recognizing fragment thereof. In some aspects, the method comprises reacting a compound of Formula II with an antibody or an antigen recognizing fragment thereof to form the adaptor molecule of Formula III. In another aspect, an adaptor molecule of Formula III is provided synthesized by the method provided herein.

[0010] In another aspect, A chimeric antigen receptor (CAR) system is also provided. In some aspects, the CAR system comprises an adaptor molecule described herein. In some aspects, the CAR system comprises a CAR comprising a receptor that is capable of binding to T on the adaptor molecule, a hinge domain, and a signaling domain. In another aspect, a cell is provided comprising the CAR system described herein.

[0011] In another aspect, a method of treating a medical disorder in a subject in need thereof is provided. In some aspects, the method comprises administering to the subject a cell described herein. In another aspect, the method comprises: a) providing an adaptor molecule described herein; b) administering to the subject a cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises a receptor that is capable of binding to T on the adaptor molecule, a hinge domain, and a signaling domain; and c) administering the adaptor molecule to the subject.

[0012] In some aspects, the medical disorder is a cancer.

[0013] The details of one or more aspects of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the disclosure will be apparent from the description and drawings, and from the claims.

[0014] DESCRIPTION OF DRAWINGS

[0015] FIG. 1 depicts the concept of universal SNAP-CAR targeting by adaptor molecules described herein.

[0016] FIG. 2 depicts a representative therapeutic administration strategy for the administration of the adaptor molecules described herein. SNAP-CAR T cells are administered via a one-time fusion. Antigen targeting by the SNAP-CAR T cells is controlled by repeated oral administration of one or more adaptor molecules described herein targeting tumor cells and / or immunosuppressive cells in the tumor site.

[0017] FIGs. 3A-3C provide representative small molecule adaptors targeting cancer cellsurface markers CAIX, PMSA, and the folic acid receptor.

[0018] FIG. 4 shows the adaptor dose-responsive activation of SNAP-CAR T cells coincubated with the indicated acetazolamide (AZ)-benzylguanine (BG) small molecule adaptors and concentrations as indicated by up-regulation of CD 107a and CD69 T cell activation markers measured by antibody staining and flow cytometry.

[0019] FIG. 5 shows the adaptor dose-responsive lysis of tumor cells mediated by SNAP-CAR T cells co-incubated with the indicated acetazolamide (Az)-benzylguanine (BG) small molecule adaptors and concentrations.

[0020] FIG. 6 depicts pre-loading of SNAP-CAR T cells with small molecule adaptors and data showing tumor cell lysis mediated by pre-assembled small molecular adaptor (Az-PEGIO- BG) / SNAP-CAR T cells.

[0021] FIG. 7 depicts conditional control of T cell activation and lysis by universal SNAPCAR targeting by small molecule adaptors.

[0022] FIG. 8 is a scheme of a representative synthesis of an exemplary intermediate used in the synthesis of compounds described herein.

[0023] FIG. 9 provides an exemplary synthetic scheme of a representative adaptor of Formula I targeting PD-L1.

[0024] FIG. 10 provides an exemplary synthetic scheme of a representative adaptor of Formula I targeting PD-L1.

[0025] FIG. 11 provides an exemplary synthetic scheme for representative adaptors of Formula I targeting the folate receptor.

[0026] FIG. 12 provides an exemplary synthetic scheme for a representative adaptor of Formula I targeting prostate specific antigen.

[0027] FIG. 13 provides an exemplary synthetic scheme for a representative adaptor of Formula I targeting carbonic anhydrase.

[0028] FIG. 14 provides exemplary syntheses of compounds of Formula II for conjugation of antibodies. The top comprises an NHS-activated ester and the bottom comprises a pentafluorophenyl-activated ester, the latter which may offer lower reactivity and increased hydrolytic stability providing higher antibody ratios for the resulting adaptors.

[0029] FIG. 15 provides a representative schematic of the concept of Halo-CAR T cells with A) an antibody adaptor and B) with a small molecule adaptor. The chloroalkane tag forms a covalent bond with Halo-CAR and directs antigen recognition on the target cell.

[0030] FIG. 16 shows that antibody adaptors make using the compounds of Formula II mediate antigen-specific T cell activation of Halo-CAR T cells. Ratios indicate the molar ratio of chloroalkane NHS ester to antibody amount used to generate the resulting adaptors. Halo-CAR T cells were coincubated with CD20+ and HER2+ K562 target cells and antibody adaptors targeting CD20 or HER2 for 24 hours. The cells were then analyzed by flow cytometry for CD69 T cell activation marker expression.

[0031] FIGs. 17A-17E depict how in 24hr co-incubation experiments with primary human SNAP-CAR T cells and the indicated cell line and adaptor, Az-PEG6-BG and Az-PEGIO-BG demonstrate CAIX-specific specific lysis of target cells and activation of SNAP-CAR T cells with no activity on Nalm6 CD20 (CAIX-) cells by flow cytometry. n=3 replicates, error bars indicate S.D. The chemical structures of adaptors tested are provided in FIG. 17E.

[0032] FIGs. 18A-18D depict how preloading SNAP-CAR with Az-PEGIO-BG adaptor demonstrate robust specific lysis of U87-MG cells and SNAP-CAR T cells activation and compares to activation by free-floating adaptor. n=3 replicates, error bars indicate S.D.

[0033] FIGs. 19A-19E are data that, in 24hr co-incubation experiments with primary human SNAP-CAR T cells and the indicated cell line and folate-PEG12-BG adaptor, demonstrate F0LR1 and F0LR2 specific specific target cell lysis and CAR T activation and no activity on K562 WT cells by flow cytometry. n=3 replicates, error bars indicate S.D. The chemical structure of the adaptor tested is provided in FIG. 19E.

[0034] FIGs. 20A-20E depict how, in 48hr co-incubation experiments with primary human SNAP-CAR T cells, the various indicated cell lines, and folate-PEG12-BG adaptor, significant specific target cell lysis is observed by flow cytometry. Mirvetuximab-BG (Mirv-BG) adaptor is included as a positive control for F0LR1 targeting. E:T signifies the effector ratio of CAR T cells (E) to target cells (T). n=3 replicates, error bars indicate S.D.

[0035] FIGs. 21A-21E depict how, in 24hr co-incubation experiments with primary human SNAP-CAR T cells and the indicated cell line and adaptor, PSMA-PEG12-BG demonstrated PSMA-specific specific lysis of target cells and activation of SNAP-CAR T cells with no activity on PC3 (PSMA-) cells by flow cytometry. n=3 replicates, error bars indicate S.D. The chemical structures of the adaptors tested are provided in FIG. 2 IE.

[0036] FIGs. 22A-22E depict how in 24hr co-incubation experiments with primary human Halo-CAR T cells and the U87-MG cell line and indicated adaptor concentrations, Az-PEG12- CA demonstrates CATX-specific specific lysis of target cells and activation of Halo-CAR T cells by flow cytometry. n=3 replicates, error bars indicate S.D. The chemical structure of the adaptor tested is provided in FIG. 22E.

[0037] FIGs. 23A-23E are data that, in 24hr co-incubation experiments with primary human Halo-CAR T cells and the indicated cell line and folate- PEGO-C A, folate-PEG6-CA and folate- PEG12-CA adaptors, demonstrate F0LR1 and FOLR2 specific specific target cell lysis and CAR T activation and no activity on K562 WT cells by flow cytometry. n=3 replicates, error bars indicate S.D. The chemical structures of the adaptors tested are provided in FIG. 23E.

[0038] FIGs. 24A-24E depict how, in 24hr co-incubation experiments with primary human mSA2-CAR T cells and the U87-MG cell line and indicated adaptor concentrations, Az-PEG5- biotin demonstrates specific lysis of target cells and activation of mSA2-CAR T cells by flow cytometry. n=3 replicates, error bars indicate S.D. The chemical structure of the adaptor tested is provided in FIG. 24E.

[0039] FIGs. 25A-25E depict how, in 24hr co-incubation experiments with primary human mSA2-CAR T cells and the K562+FOLR1 cell line and indicated adaptor concentrations, folate-PEG5-biotin demonstrates specific lysis of target cells and activation of mSA2-CAR T cells by flow cytometry. n=3 replicates, error bars indicate S.D. The chemical structure of the adaptor tested is provided in FIG. 25E.

[0040] Like reference symbols in the various drawings indicate like elements.

[0041] DETAILED DESCRIPTION

[0042] The following description of the disclosure is provided as an enabling teaching of the disclosure in its best, currently known aspects. Many modifications and other aspects disclosed herein will come to mind to one skilled in the art to which the disclosed compositions and methods pertain, benefiting from the teachings presented in the descriptions herein and the associated drawings. Therefore, it is understood that the disclosures are not limited to the specific aspects disclosed and that modifications and other aspects are intended to be included within the scope of the appended claims. The skilled artisan will recognize many variants and adaptations of the aspects described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein.

[0043] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0044] As apparent to those of skill in the art upon reading this disclosure, each of the individual aspects described and illustrated herein has discrete components and features that may be readily separated from or combined with the features of any of the other several aspects without departing from the scope or spirit of the present disclosure.

[0045] Any recited method can be carried out in the order of events recited or any other order that is logically possible. Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not explicitly state in the claims or descriptions that the steps are to be limited to a particular order, it is in no way intended that an order be inferred in any respect. This holds for any possible non-express basis for interpretation, including logic concerning arrangement of steps or operational flow, meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0046] All publications mentioned herein are incorporated by reference to disclose and describe the methods or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure before the filing date of the present application. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.

[0047] It is also to be understood that the terminology herein describes particular aspects only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methods belong. It can be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0048] Before describing the various aspects of the present disclosure, the following definitions are provided and should be used unless otherwise indicated. Additional terms may be defined elsewhere in the present disclosure. As used herein, “comprising” is interpreted as specifying the presence of the stated features, integers, steps, or components but does not preclude the presence or addition of one or more features, integers, steps, components, or groups thereof. Moreover, each of the terms “by,” “comprising,” “comprises,” “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of’ and “consisting of.” Similarly, “consisting essentially of’ is intended to include examples encompassed by the term “consisting of.”

[0049] As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context dictates otherwise. Thus, for example, reference to “an adaptor molecule,” “a ligand,” or “a cell” includes, but is not limited to, two or more such adaptor molecules, ligands, or cells, and the like.

[0050] Ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. Further, the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. There are many values disclosed herein, and each value is also disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value and to “about” another particular value. Similarly, when values are expressed as approximations, using the antecedent “about,” the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0051] When a range is expressed, a further aspect includes from the one particular value and to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g., the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’ . The range can also be expressed as an upper limit, e.g., ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x,’ ‘about y,’ and ‘about z’ as well as the ranges of Tess than x,’ Tess than y.’ and Tess than z.’ Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x,’ ‘about y,’ and ‘about z’ as well as the ranges of ‘greater than x,’ greater than y,’ and ‘greater than z.’ In addition, the phrase “about ‘x’ to ‘y’,” where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’.” Such a range format is used for convenience and brevity and, thus, should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5% but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0052] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact but may be approximate, larger or smaller, as desired, reflecting tolerances, conversion factors, rounding, measurement error, and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, as used herein, “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter, or other quantity or characteristic is “about,” “approximate,” or “at or about,” whether or not expressly stated to be such. Where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself unless expressly stated otherwise.

[0053] As used herein, the term “therapeutically effective amount” refers to an amount sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms but generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors, including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex, and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the particular compound employed and like factors within the knowledge and expertise of the health practitioner and which may be well known in the medical arts. In the case of treating a particular disease or condition, in some instances, the desired response can be inhibiting the progression of the disease or condition. This may involve only slowing the progression of the disease temporarily. However, in other instances, it may be desirable to permanently halt the progression of the disease. This can be monitored by routine diagnostic methods known to one of ordinary skill in the art for any particular disease. The desired response to treatment of the disease or condition can also be delaying the onset or even preventing the onset.

[0054] For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to increase the dosage gradually until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for administration. Consequently, single-dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The individual physician can adjust the dosage in the event of any contraindications. It is generally preferred that a maximum dose of the pharmacological agents of the disclosure (alone or in combination with other therapeutic agents) be used, that is, the highest safe dose according to sound medical judgment. However, a patient may insist on a lower or tolerable dose for medical reasons, psychological reasons, or virtually any other reason.

[0055] A response to a therapeutically effective dose of a disclosed compound or composition can be measured by determining the physiological effects of the treatment or medication, such as the decrease or lack of disease symptoms following the administration of the treatment or pharmacological agent. Other assays will be known to one of ordinary skill in the art and can be employed for measuring the level of the response. The amount of a treatment may be varied, for example, by increasing or decreasing the amount of a disclosed compound or pharmaceutical composition, changing the disclosed compound or pharmaceutical composition administered, changing the route of administration, changing the dosage timing, and so on. Dosage can vary and can be administered in one or more dose administrations daily for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.

[0056] As used herein, “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur. The description includes instances where said event or circumstance occurs and those where it does not.

[0057] As used interchangeably herein, “subject,” “individual,” or “patient” can refer to a vertebrate organism, such as a mammal (e.g., human). “Subject” can also refer to a cell, a population of cells, a tissue, an organ, or an organism, preferably to a human and constituents thereof.

[0058] As used herein, “treating” and “treatment” generally refer to obtaining a desired pharmacological or physiological effect. The effect can be but does not necessarily have to be prophylactic in preventing or partially preventing a disease, symptom, or condition such as a cancer. The effect can be therapeutic regarding a partial or complete cure of a disease, condition, symptom, or adverse effect attributed to the disease, disorder, or condition. The term “treatment” as used herein can include any treatment of a disorder in a subject, particularly a human. It can include any one or more of the following: (a) preventing the disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., mitigating or ameliorating the disease or its symptoms or conditions. The term “treatment,” as used herein, can refer to both therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment. Those in need of treatment (i.e., subjects in need thereof) can include those already with the disorder or those in which the disorder is to be prevented. As used herein, the term “treating” can include inhibiting the disease, disorder, or condition, e.g., impeding its progress, and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder, or condition. Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, e.g., such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain.

[0059] As used herein, “dose,” “unit dose,” or “dosage” can refer to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of a disclosed compound or a pharmaceutical composition thereof calculated to produce the desired response or responses in association with its administration.

[0060] As used herein, “therapeutic” can refer to treating, healing, or ameliorating a disease, disorder, condition, or side effect or decreasing the rate of advancement of a disease, disorder, condition, or side effect.

[0061] Compounds are described using standard nomenclature. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this disclosure belongs. The compounds described herein include enantiomers, mixtures of enantiomers, diastereomers, tautomers, racemates, and other isomers, such as rotamers, as if each is specifically described unless otherwise indicated or otherwise excluded by context. It is to be understood that the compounds provided herein may contain chiral centers. Such chiral centers may be of either the (R-) or (.S'-) configuration. The compounds provided herein may either be enantiomerically pure or diastereomeric or enantiomeric mixtures. It is to be understood that the chiral centers of the compounds provided herein may undergo epimerization in vivo. As such, one of skill in the art will recognize that the administration of a compound in its (7?-) form is equivalent, for compounds that undergo epimerization in vivo, to the administration of the compound in its (51-) form. Unless stated to the contrary, a formula with chemical bonds shown only as solid lines and not as wedges or dashed lines contemplates each possible isomer, e.g., each enantiomer, diastereomer, and meso compound, and a mixture of isomers, such as a racemic or scalemic mixture.

[0062] A dash that is not between two letters or symbols is used to indicate a point of attachment for a substituent. For example, -(C=0)NH2 is attached through the carbon of the keto (C=O) group.

[0063] The term “substituted,” as used herein, means that any one or more hydrogens on the designated atom or group are replaced with a moiety selected from the indicated group, provided that the designated atom’s normal valence is not exceeded and the resulting compound is stable. For example, when the substituent is oxo (i.e., =0), then two hydrogens on the atom are replaced. For example, a pyridyl group substituted by oxo is a pyridine. Combinations of substituents and / or variables are permissible only if such combinations result in stable compounds or useful synthetic intermediates. A stable active compound refers to a compound that can be isolated and can be formulated into a dosage form with a shelf life of at least one month. A stable manufacturing intermediate or precursor to an active compound is stable if it does not degrade within the period needed for reaction or other use. A stable moiety or substituent group is one that does not degrade, react or fall apart within the period necessary for use. Non-limiting examples of unstable moieties are those that combine heteroatoms in an unstable arrangement, as typically known and identifiable to those of skill in the art.

[0064] Any suitable group may be present on a “substituted” or “optionally substituted” position that forms a stable molecule and meets the desired purpose of the disclosure and includes, but is not limited to: alkyl, haloalkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocycle, aldehyde, amino, carboxylic acid, ester, ether, halo, hydroxy, keto, nitro, cyano, azido, oxo, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, sulfonylamino, or thiol.

[0065] “Alkyl” is a straight chain or branched saturated aliphatic hydrocarbon group. In certain aspects, the alkyl is C1-C2, C1-C3, or C1-C6 (i.e., the alkyl chain can be 1, 2, 3, 4, 5, or 6 carbons in length). The specified ranges as used herein indicate an alkyl group with length of each member of the range described as an independent species. For example, Ci-Cealkyl as used herein indicates an alkyl group having from 1, 2, 3, 4, 5, or 6 carbon atoms and is intended to mean that each of these is described as an independent species and Ci-C4alkyl as used herein indicates an alkyl group having from 1, 2, 3, or 4 carbon atoms and is intended to mean that each of these is described as an independent species. When Co-Cnalkyl is used herein in conjunction with another group, for example (C3-C7cycloalkyl)Co-C4alkyl, or -Co-C4(C3- C?cycloalkyl), the indicated group, in this case cycloalkyl, is either directly bound by a single covalent bond (Coalkyl), or attached by an alkyl chain, in this case 1, 2, 3, or 4 carbon atoms. Alkyls can also be attached via other groups such as heteroatoms, as in -0-Co-C4alkyl(C3- C?cycloalkyl). Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, isopentyl, tert-pentyl, neopentyl, n- hexyl, 2-methylpentane, 3-methylpentane, 2,2-dimethylbutane, and 2,3-dimethylbutane. In one aspect, the alkyl group is optionally substituted as described herein.

[0066] “Cycloalkyl” is a saturated mono- or multi-cyclic hydrocarbon ring system. When composed of two or more rings, the rings may be joined together in a fused or bridged fashion. Non-limiting examples of typical cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. In one aspect, the cycloalkyl group is optionally substituted as described herein.

[0067] “Alkenyl” is a straight or branched chain aliphatic hydrocarbon group having one or more carbon-carbon double bonds, each of which is independently either cis or trans, that may occur at a stable point along the chain. Non-limiting examples include C2-C4alkenyl and C2- Coalkenyl (i.e., having 2, 3, 4, 5, or 6 carbons). The specified ranges as used herein indicate an alkenyl group having each member of the range described as an independent species, as described above for the alkyl moiety. Examples of alkenyl include, but are not limited to, ethenyl and propenyl. In one aspect, the alkenyl group is optionally substituted as described herein. “Alkynyl” is a straight or branched chain aliphatic hydrocarbon group having one or more carbon-carbon triple bonds that may occur at any stable point along the chain, for example, C2-C4alkynyl or C2-C6alkynyl (i.e., having 2, 3, 4, 5, or 6 carbons). The specified ranges as used herein indicate an alkynyl group having each member of the range described as an independent species, as described above for the alkyl moiety. Examples of alkynyl include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1 -pentynyl, 2- pentynyl, 3-pentynyl, 4-pentynyl, 1 -hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, and 5-hexynyl. In one aspect, the alkynyl group is optionally substituted as described herein.

[0068] “Alkoxy” is an alkyl group as defined above covalently bound through an oxygen bridge (-O-). Examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, 2-butoxy, tert-butoxy, n-pentoxy, 2-pentoxy, 3-pentoxy, isopentoxy, neopentoxy, n-hexoxy, 2-hexoxy, 3-hexoxy, and 3 -methylpentoxy. Similarly, an “alkylthio” or “thioalkyl” group is an alkyl group as defined above with the indicated number of carbon atoms covalently bound through a sulfur bridge (-S-). Tn one aspect, the alkoxy group is optionally substituted as described herein.

[0069] “Alkanoyl” is an alkyl group as defined above covalently bound through a carbonyl (C=O) bridge. The carbonyl carbon is included in the number of carbons, for example C2alkanoyl is a CH3(C=0)- group. In one aspect, the alkanoyl group is optionally substituted as described herein.

[0070] “Halo” or “halogen” indicates, independently, any of fluoro, chloro, bromo or iodo.

[0071] “Aryl” indicates an aromatic group containing only carbon in the aromatic ring or rings. In one aspect, the aryl group contains 1 to 3 separate or fused rings and is 6 to 14 or 18 ring atoms, without heteroatoms as ring members. When indicated, such aryl groups may be further substituted with carbon or non-carbon atoms or groups. Such substitution may include fusion to a 4- to 7- or 5- to 7-membered saturated or partially unsaturated cyclic group that optionally contains 1, 2, or 3 heteroatoms independently selected from N, O, B, P, Si and S, to form, for example, a 3,4-methylenedioxyphenyl group. Aryl groups include, for example, phenyl and naphthyl, including 1 -naphthyl and 2-naphthyl. In one aspect, aryl groups are pendant. An example of a pendant ring is a phenyl group substituted with a phenyl group. In one aspect, the aryl group is optionally substituted as described herein.

[0072] The term “heterocycle” refers to saturated and partially saturated heteroatomcontaining ring radicals, where the heteroatoms may be selected from N, O, and S. The term heterocycle includes monocyclic 3-12 members rings, as well as bicyclic 5-16 membered ring systems (which can include fused, bridged, or spiro bicyclic ring systems). It does not include rings containing -O-O-, -O-S-, and -S-S- portions. Examples of saturated heterocycle groups including saturated 4- to 7-membered monocyclic groups containing 1 to 4 nitrogen atoms [e.g., pyrrolidinyl, imidazolidinyl, piperidinyl, pyrrolinyl, azetidinyl, piperazinyl, and pyrazolidinyl]; saturated 4- to 6-membered monocyclic groups containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms [e.g., morpholinyl]; and saturated 3- to 6-membered heteromonocyclic groups containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms [e.g., thiazolidinyl]. Examples of partially saturated heterocycle radicals include, but are not limited, dihydrothienyl, dihydropyranyl, dihydrofuryl, and dihydrothiazolyl. Examples of partially saturated and saturated heterocycle groups include, but are not limited to, pyrrolidinyl, imidazolidinyl, piperidinyl, pyrrolinyl, pyrazolidinyl, piperazinyl, morpholinyl, tetrahydropyranyl, thiazolidinyl, dihydrothienyl, 2,3-dihydro-benzo[l ,4]dioxanyl, indolinyl, isoindolinyl, dihydrobenzothienyl, dihydrobenzofuryl, isochromanyl, chromanyl, 1,2- dihydroquinolyl, 1,2,3,4-tetrahydro-isoquinolyl, 1,2,3,4-tetrahydro-quinolyl, 2, 3, 4, 4a, 9,9a- hexahydro-lH-3-aza-fluorenyl, 5,6,7-trihydro-l,2,4-triazolo|3,4-a|isoquinolyl, 3,4-dihydro- 2H-benzo[l,4]oxazinyl, benzofl, 4]dioxanyl, 2,3,-dihydro-lH-benzo[d]isothazol-6-yl, dihydropyranyl, dihydrofuryl, and dihydrothiazolyl. Bicyclic heterocycle includes groups wherein the heterocyclic radical is fused with an aryl radical wherein the point of attachment is the heterocycle ring. Bicyclic heterocycle also includes heterocyclic radicals that are fused with a carbocyclic radical. Representative examples include, but are not limited to, partially unsaturated condensed heterocyclic groups containing 1 to 5 nitrogen atoms, for example indoline and isoindoline, partially unsaturated condensed heterocyclic groups containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, partially unsaturated condensed heterocyclic groups containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms, and saturated condensed heterocyclic groups containing 1 to 2 oxygen or sulfur atoms.

[0073] “Heteroaryl” refers to a stable monocyclic, bicyclic, or multicyclic aromatic ring which contains from 1 to 4, or in some aspects 1, 2, or 3 heteroatoms selected from N, O, S, B, and P (and typically selected from N, O, and S) with remaining ring atoms being carbon, or a stable bicyclic or tricyclic system containing at least one 5, 6, or 7 membered aromatic ring which contains from 1 to 4, or in some aspects from 1 to 3 or from 1 to 2, heteroatoms selected from N, O, S, B, or P, with remaining ring atoms being carbon. In one aspects, the only heteroatom is nitrogen. In one aspect, the only heteroatom is oxygen. In one aspect, the only heteroatom is sulfur. Monocyclic heteroaryl groups typically have from 5 to 6 ring atoms. In some aspects, bicyclic heteroaryl groups are 8- to 10-membered heteroaryl groups, that is groups containing 8 or 10 ring atoms in which one 5-, 6-, or 7-membered aromatic ring which contains from 1 to 4 heteroatoms selected from N, O, S, B, or P is fused to a second aromatic or non-aromatic ring, wherein the point of attachment is an aromatic ring. When the total number of S and O atoms in the heteroaryl ring exceeds 1, these heteroatoms are not adjacent to one another within the ring. In one aspect, the total number of S and O atoms in the heteroaryl ring is not more than 2. In another aspect, the total number of S and O atoms in the heteroaryl ring is not more than 1. Examples of heteroaryl groups include, but are not limited to, pyridinyl, imidazolyl, imidazopyridinyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, furyl, thienyl, isoxazolyl, thiazolyl, oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, tetrahydroisoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, triazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl.

[0074] A “pharmaceutically acceptable salt” is a derivative of the disclosed compound in which the parent compound is modified by making inorganic and organic, pharmaceutically acceptable, acid or base addition salts thereof. The salts of the present compounds can be synthesized from a parent compound that contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting free acid forms of these compounds with a stoichiometric amount of the appropriate base (such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate, or the like) or by reacting free base forms of these compounds with a stoichiometric amount of the appropriate acid. Such reactions are typically carried out in water or in an organic solvent, or in a mixture of the two. Generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are typical, where practicable. Salts of the present compounds further include solvates of the compounds and of the compound salts. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include salts that are acceptable for human consumption and the quaternary ammonium salts of the parent compound formed, for example, from inorganic or organic salts. Examples of such salts include, but are not limited to, those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, mesylic, esylic, besylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, HOOC-(CH2)I-4-COOH, and the like, or using a different acid that produced the same counterion. Lists of additional suitable salts may be found, e.g., in Remington’s Pharmaceutical Sciences, 17thed., Mack Publishing Company, Easton, PA., p. 1418 (1985).

[0075] As used herein, substantially pure means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), nuclear magnetic resonance (NMR), gel electrophoresis, high- performance liquid chromatography (HPLC) and mass spectrometry (MS), gaschromatography mass spectrometry (GC-MS), and similar, used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Both traditional and modem methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound may, however, be a mixture of stereoisomers.

[0076] The present disclosure also includes compounds with at least one desired isotopic substitution of an atom at an amount above the natural abundance of the isotope, i.e., enriched.

[0077] Examples of isotopes that can be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, and chlorine, such as2H,3H,nC,13C,15N,170,18O,18F,31P, 32P,35s,36C1, and125I, respectively. In one aspect, isotopically labeled compounds can be used in metabolic studies (with14C), reaction kinetic studies (with, for example2H or3H), detection or imaging techniques, such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT) including drug and substrate tissue distribution assays, or in radioactive treatment of patients. In particular, an18F labeled compound may be particularly desirable for PET or SPECT studies. Isotopically labeled compounds of this disclosure and prodrugs thereof can generally be prepared by carrying out the procedures disclosed herein by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.

[0078] By way of general example and without limitation, isotopes of hydrogen, for example deuterium (2H) and tritium (3H) may optionally be used anywhere in described structures that achieve the desired result. Alternatively, or in addition, isotopes of carbon, e.g.,13C and14C, may be used. In one aspect, the isotopic substitution is replacing hydrogen with a deuterium at one or more locations on the molecule to improve the performance of the molecule as a drug, for example, the pharmacodynamics, pharmacokinetics, biodistribution, half-life, stability, AUC, Tmax, Cmax, etc. For example, the deuterium can be bound to carbon in allocation of bond breakage during metabolism (an alpha-deuterium kinetic isotope effect) or next to or near the site of bond breakage (a beta-deuterium kinetic isotope effect).

[0079] Isotopic substitutions, for example, deuterium substitutions, can be partial or complete. Partial deuterium substitution means that at least one hydrogen is substituted with deuterium. In certain aspects, the isotope is 80, 85, 90, 95, or 99% or more enriched in an isotope at any location of interest. In some aspects, deuterium is 80, 85, 90, 95, or 99% enriched at a desired location. Unless otherwise stated, the enrichment at any point is above natural abundance, and, in one aspect, is enough to alter a detectable property of the compounds as a drug in a human.

[0080] The compounds of the present disclosure may form a solvate with solvents (including water). Therefore, in one aspect, the disclosure includes a solvated form of the active compound. The term “solvate” refers to a molecular complex of a compound of the present disclosure (including a salt thereof) with one or more solvent molecules. Non-limiting examples of solvents are water, ethanol, dimethyl sulfoxide, acetone, and other common organic solvents. The term “hydrate” refers to a molecular complex comprising a disclosed compound and water. Pharmaceutically acceptable solvates in accordance with the disclosure include those wherein the solvent of crystallization may be isotopically substituted, e.g., DoO, de-acetone, or de-DMSO. A solvate can be in a liquid or solid form.

[0081] A “prodrug,” as used herein, means a compound that, when administered to a host in vivo, is converted into a parent drug. As used herein, the term “parent drug” means any of the presently described compounds herein. Prodrugs can be used to achieve any desired effect, including enhancing properties of the parent drug or improving the pharmaceutic or pharmacokinetic properties of the parent, including increasing the half-life of the drug in vivo. Prodrug strategies provide choices in modulating the conditions for in vivo generation of the parent drug. Non-limiting examples of prodrug strategies include covalent attachment of removable groups or removable portions of groups, for example, but not limited to, acylating, phosphorylation, phosphonylation, phosphoramidate derivatives, amidation, reduction, oxidation, esterification, alkylation, other carboxy derivatives, sulfoxy or sulfone derivatives, carbonylation, or anhydrides, among others. In certain aspects, the prodrug renders the parent compound more lipophilic. In certain aspects, a prodrug can be provided that has several prodrug moieties in a linear, branched, or cyclic manner. For example, non-limiting examples include the use of a divalent linker moiety such as a dicarboxylic acid, amino acid, diamine, hydroxycarboxylic acid, hydroxyamine, di-hydroxy compound, or other compounds that have at least two functional groups that can link the parent compound with another prodrug moiety and are typically biodegradable in vivo. In some aspects, 2, 3, 4, or 5 prodrug biodegradable moieties are covalently bound in a sequence, branched, or cyclic fashion to the parent compound. Non-limiting examples of prodrugs according to the present disclosure are formed with: a carboxylic acid on the parent drug and a hydroxylated prodrug moiety' to form an ester; a carboxylic acid on the parent drug and an amine prodrug to form an amide; an amino on the parent drug and a carboxylic acid prodrug moiety to form an amide; an amino on the parent drug and a sulfonic acid to form a sulfonamide; a sulfonic acid on the parent drug and an amino on the prodrug moiety' to form a sulfonamide; a hydroxyl group on the parent drug and a carboxylic acid on the prodrug moiety' to fonn an ester; a hy droxyl on the parent drug and a hydroxylated prodrug moiety to form an ester; a phosphonate on the parent drug and a hydroxylated prodrug moiety’ to form a phosphonate ester; a phosphoric acid on the parent drug and a hydroxylated prodrug moiety to form a phosphate ester; a hydroxyl on the parent drug and a phosphonate on the prodrug to form a phosphonate ester; a hydroxyl on the parent drug and a phosphoric acid prodrug moiety' to form a phosphate ester; a carboxylic acid on the parent drug and a prodrug of the structure HO-(CH2)2-O-(C2-24 alkyl) to form an ester; a carboxylic acid on the parent drug and a prodrug of the structure HO-(CH2)2-S-(C2-24 alkyl) to form a thioester; a hydroxyl on the parent drug and a prodrug of the structure HO-(CH2)2-O-(C2-24 alkyl) to form an ether; a hydroxyl on the parent drug and a prodrug of the structure HO-(CH2)2- O-(C2-24 alkyl) to fonn an thioether; and a carboxylic acid, oxime, hydrazide, hydrazine, amine or hydroxyl on the parent compound and a prodrug moiety that is a biodegradable polymer or oligomer including but not limited to polylactic acid, polylactide-co-glycolide, polyglycolide, polyethylene glycol, polyanhydride, polyester, polyamide, or a peptide.

[0082] In some aspects, a prodrug is provided by attaching a natural or non-natural amino acid to an appropriate functional moiety on the parent compound, for example, oxygen, nitrogen, or sulfur, and typically oxygen or nitrogen, usually in a manner such that the amino acid is cleaved in vivo to provide the parent drug. The amino acid can be used alone or covalently linked (straight, branched or cyclic) to one or more other prodrug moieties to modify the parent drug to achieve the desired performance, such as increased half-life, lipophilicity or other drug delivery or pharmacokinetic properties. The amino acid can be any compound with an amino group and a carboxylic acid, which includes an aliphatic amino acid, alkyl amino acid, aromatic amino acid, heteroaliphatic amino acid, heteroalkyl amino acid, heterocyclic amino acid, or heteroaryl amino acid.

[0083] Adaptor Molecules of Formula I

[0084] The present disclosure provides adaptor molecules for adoptive cell therapy. The disclosed adaptor molecules utilize small molecule ligands for a folate receptor, a carbonic anhydrase, prostate-specific antigen, and programmed death- ligand 1 (PD-L1) instead of adaptor molecules comprising antibodies tagged with tag ligand motifs. These small molecule adaptors complement previously described antibody adaptors due to their drug-like pharmacokinetics and pharmacodynamics. These adaptor molecules are easier to synthesize (especially at scale), have lower production costs, higher stability (including longer shelf-life and storage at room temperature), oral availability (allowing repeat application at home instead of a clinical setting), cell permeability, blood- brain barrier penetrance, and lack of immunogenicity. Further, covalent T cell modification with the adaptor molecules negates the often short circulation half-life otherwise found for small molecules.

[0085] In one aspect, an adaptor molecule for adoptive cell therapy of Formula I is provided:

[0086] T-L-A (I) or a pharmaceutically acceptable salt thereof; wherein:

[0087] T comprises a tag ligand;

[0088] L comprises a linker moiety; and

[0089] A comprises a moiety capable of binding a cell surface protein selected from a folate receptor, a carbonic anhydrase, prostate-specific antigen, and programmed death-ligand 1 (PD- Ll).

[0090] In adaptor molecules of Formula I, T comprises a tag ligand. In some aspects, T comprises a ligand for Isopep-tag, SpyTag, SnoopTag, DogTag, SdyTag, HaloTag, SNAP-tag, CLIP-tag, HUH-tag, or mSA2. In some particular aspects, T comprises a ligand for SNAP-tag, CLIP-tag, Halo-tag, SpyTag, SnoopTag, mSA2, or Isopep-tag. In some aspects, T comprises a benzylguanine, chloropyrimidine, benzylcytosine, biotin, or chloroalkane moiety. In some aspects, T comprises a ligand for Halo-tag. In some particular aspects, T comprises -(C1-C20 alkyl)-Cl. In some aspects, T comprises -(C 1 alkyl)-Cl. In some aspects, T comprises -(C2 alkyl)-Cl. In some aspects, T comprises -(C3 alkyl)-Cl. In some aspects, T comprises -(C4 alkyl)-Cl. In some aspects, T comprises -(Cs alkyl)-Cl. In some aspects, T comprises -(C6 alkyl)-Cl. In some aspects, T comprises -(C7 alkyl)-Cl. In some aspects, T comprises -(C8 alkyl) -Cl. In some aspects, T comprises -(C9 alkyl)-Cl. In some aspects, T comprises — (C10 alkyl)-Cl. In some aspects, T comprises -(Cn alkyl)-Cl. In some aspects, T comprises — (C12 alkyl)-Cl. In some aspects, T comprises -(C13 alkyl)-Cl. In some aspects, T comprises -(C14 alkyl)-Cl. In some aspects, T comprises -(C15 alkyl)-Cl. In some aspects, T comprises -(Ci6 alkyl)-Cl. In some aspects, T comprises -(C17 alkyl)-Cl. In some aspects, T comprises -(C18 alkyl)-Cl. In some aspects, T comprises -(C19 alkyl)-Cl. In some aspects, T comprises -(C20 alkyl)-Cl.

[0091] •Cl

[0092] In some aspects, T comprises

[0093] In adaptor molecules of Formula I, A comprises a moiety capable of binding a cell surface protein selected from a folate receptor, a carbonic anhydrase, prostate-specific antigen, and programmed death-ligand 1 (PD-L1).

[0094] In some aspects, A comprises a moiety capable of binding a folate receptor. In some aspects, the folate receptor comprises folate receptor 1 (adult) / FOLRl, folate receptor 2 (fetal) / FOLR2, or folate receptor gamma / F0LR3.

[0095] In some aspects, A comprises a moiety derived from a ligand selected from:

[0096] zz

[0097] H009

[0098] HOO5

[0099] , and wherein the point of attachment of L is provided by replacement of a hydrogen or other suitable moiety in the above structures with a covalent bond to L. For example, the point of attachment of L in the above ligands may be provided by replacement of a hydrogen, alkyl, alkoxy, alkynyl, amino, carboxylic acid, halo, or hydroxy group in the above structures with a covalent bond to L.

[0100] In some aspects, A comprises In some aspects, A comprises a moiety capable of binding a carbonic anhydrase. In some aspects, the carbonic anhydrase (CA) comprises a cytosolic CA isoform (such as, for example, CA-I, CA-II, CA-III, CA-VII, and CA-XIII), a mitochondrial CA isoform (such as, for example, CA-VA and CA-VB), a secreted CA isoform (such as, for example, CAVI), or a membrane-associated CA isoform (such as, for example, CA-IV, CA-IX, CA-XII, CA-XIV, and CA-XV).

[0101] In some aspects, A comprises a moiety derived from a ligand selected from:

[0102] wherein the point to attachment of L is provided by replacement of a hydrogen or other suitable moiety in the above structures with a covalent bond to L. For example, the point of attachment of L in the above ligands may be provided by replacement of a hydrogen, alkyl, haloalkyl, alkoxy, amino, ether, halo, hydroxy, or sulfonylamino group in the above structures with a covalent bond to L.

[0103] In some particular aspects, A comprises

[0104] In some aspects, A comprises a moiety capable of binding prostate-specific antigen.

[0105] In some aspects, A comprises a moiety derived from a ligand selected from: CO2H

[0106]

[0107] wherein the point of attachment to L is provided by replacement of a hydrogen or other suitable moiety in the above structures with a covalent bond to L. For example, the point of attachment of L in the above ligands may be provided by replacement of a hydrogen, alkyl, alkoxy, amino, carboxylic acid, halo, hydroxy, or thiol group in the above structures with a covalent bond to L.

[0108] In some aspects, A comprises In some aspects, A comprises a moiety capable of binding PD-L1.

[0109] In some aspects, A comprises a moiety derived from a ligand selected from:

[0110] CO2H

[0111] F

[0112]

[0113] wherein the point of attachment to L is provided by replacement of a hydrogen or other suitable moiety in the above structures with a covalent bond to L. For example, the point of attachment of L in the above ligands may be provided by replacement of a hydrogen, alkyl, haloalkyl, alkoxy, amino, carboxylic acid, halo, hydroxy, or cyano group in the above structures with a covalent bond to L.

[0114] In some aspects, A comprises In some aspects, A compris

[0115] A linker moiety L is included in the adaptor molecules of Formula I. A linker moiety is a chemically stable bivalent group that attaches T to A. A linker moiety L, as described herein, can be used in either direction, i.e., either the left end is linked to T and the right end to A, or the left end is linked to A and the right end to T.

[0116] In some aspects, the linker moiety L is a chain of 2 to 14, 15, 16, 17, 18, 19, or 20 or more carbon atoms, of which one or more carbons can be optionally replaced by a heteroatom such as O, N, S, or P.

[0117] In some aspects, the chain has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 19, or 20 contiguous atoms. For example, the chain may include 1 or more ethylene glycol units that can be contiguous, partially contiguous or non-contiguous (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, or 12 ethylene glycol units).

[0118] In some aspects, the chain has at least 1, 2, 3, 4, 5, 6, 7, or 8 contiguous units which can be branched and which can be independently alkyl, aryl, heteroaryl, alkenyl, or alkynyl, cycloalkyl, or heterocycloalkyl substituents.

[0119] In some aspects, the linker moiety L can include or be comprised of one or more ethylene glycol, propylene glycol, lactic and / or glycolic acid units. Block and random lactic acid-co-glycolic acid moieties, as well as ethylene glycol and propylene glycol, are known in the art and can be modified to obtain the desired half-life and hydrophilicity. In certain aspects, these units can be flanked or interspersed with other moieties, such as alkyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, etc., as desired to achieve the appropriate properties.

[0120] In some aspects, the linker moiety L is an optionally substituted (poly)ethylene glycol having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more, ethylene glycol units, or optionally substituted alkyl groups interspersed with optionally substituted O, N, S, P or Si atoms.

[0121] In some aspects, the linker moiety L is flanked, substituted, or interspersed with an alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group.

[0122] In some aspects, the linker moiety L may be asymmetric or symmetric. In some aspects, the linker moiety L can be a non-linear chain and can be, or include, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl cyclic moieties.

[0123] In some aspects, the linker moiety L is selected from LI: In some aspects, the linker moiety L is selected from the group consisting of a moiety of Formula LI, Formula L2, Formula L3, Formula L4, Formula L5, Formula L6, Formula L7,

[0124] Formula L8, Formula L9, or Formula LIO: wherein:

[0125] X101and X102are independently at each occurrence selected from a bond, aryl, heteroaryl, cycloalkyl, heterocycle, NR130, C(R130)2, O, C(O), and S; Rioo, R101, R102, R103, and R104are independently at each occurrence selected from the group consisting of a bond, alkyl, -C(O)-, -C(O)O-, -OC(O)-, -SO2-, -S(O)-, C(S)-, -C(O)NR130-, -NR130C(O)-, -O-, -S-, -NR130-, -C(R130R130)-, -P(O)(OR106))-, -R(O)(OR106)-, alkenyl, alkynyl, haloalkyl, alkoxy, aryl, heterocycloalkyl, cycloalkyl, heteroaryl, lactic acid, or glycolic acid, each of which may be optionally substituted with one or more (for example, 1, 2, 3, or 4) substituents independently selected from R140;

[0126] R106is independently at each occurrence selected from the group consisting of hydrogen, alkyl, arylalkyl, heteroarylalkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl;

[0127] R130is independently as each occurrence selected from the group consisting of hydrogen, alkyl, cycloalky], heterocycloalkyl, aryl, heteroaryl, -C(O)H, -C(O)OH, -C(O)alkyl, -C(O)Oalkyl, -C(O)(cycloalkyl, heterocycloalkyl, aryl, or heteroaryl), -C(O)O(cycloalkyl, heterocycloalkyl, aryl, or heteroaryl), alkenyl, or alkynyl; and

[0128] R140is independently at each occurrence selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, fluoro, bromo, chloro, hydroxyl, alkoxy, azide, amino cyano, -NH(alkyl, cycloalkyl, heterocyloalkyl, aryl, or heteroaryl), -N(independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl), -NHSCMalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl), -N(alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl)SO2alkyl, -NHSOhalkenyl, -N(alkyl)SO2alkenyl, -NI ISChalkynyl, -N(alkyl)SO2alkynyl, haloalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

[0129] In some aspects, R100, R101, R102, R103, and R104within the linker moiety L are selected in such manner that: no two -C(=O)- moieties are adjected to each other; no two -O- or -NH- moieties are adjacent to each other; and / or no moieties are otherwise selected in an order such that an unstable molecule results (as defined as producing a molecule that has a shelf life at ambient temperature of less than about six months, five months, or four months) due to decomposition caused by the selection and order of R100, R101, R102, R103, and R104.

[0130] The following are non-limiting examples of linker moieties and / or moieties which comprise the same in whole or in part that can be used in this disclosure. Based on this elaboration, those of skill in the art will understand how to use the full breadth of linker moieties that will accomplish the goal of the disclosure.

[0131] Non-limiting examples of moieties that may comprise the linker moiety L, either in whole or in part, include, but are not limited to: a bond; -C(=O)-; -C=C-; -NH-; -N / CH3)-; -0-; -CH2-; -(CH2)2-; -(CH2)3-; -(CH2)4-; -(CH2)5-; -(CH2)6-; -(CH2)7-; -(CH2)8-; -(CH2)9-;

[0132] -(CH2)IO-; -NH(C=O)-; -C(=O)NH-; -C(=O)CH2-; -C(=O)(CH2)2-; -C(=O)(CH2)3-;

[0133] -C(=O)(CH2)4-; -C(=O)(CH2)5-; -C(=O)(CH2)6-; -CH2C(=O)-; -(CH2)2C(=O)-;

[0134] -(CH2)3C(=O)-; -(CH2)4C(=O)-; -(CH2)5C(=O)-; -(CH2)6C(=O)-; -CH2NH-; -(CH2)2NH-; -(CH2)3NH-; -(CH2)4NH-; -(CH2)5NH-; -(CH2)6NH-; -NHCH2-; -NH(CH2)2-; -NH(CH2)3-;

[0135] -NH(CH2)4-; -NH(CH2)5-; -NH(CH2)6-; -CH2O-; -(CH2)2O-; -(CH2)3O-; -(CH2)4O-;

[0136] -(CH2)5O-; -(CH2)6O-; -OCH2-; -O(CH2)2-; -O(CH2)3-; -O(CH2)4-; -O(CH2)5-; -O(CH2)6-;

[0137] Further non-limiting examples of moieties which may comprise the linker moiety L, either in whole or in part, include, but are not limited to:

[0138] In some aspects, the linker moiety L may comprise, either in whole or in part, a moiety selected from: and O

[0139] In some aspects, the linker moiety L may comprise, either in whole or in part, a moiety selected from: and

[0140] In some aspects, the linker moiety L may comprise, either in whole or in part, a moiety

[0141] 0

[0142] O

[0143] In some aspects, the linker moiety L may comprise, either in whole or in part,

[0144] In some aspects, the linker moiety L may comprise, either in whole or in part,

[0145] In some aspects, the linker moiety L may comprise, either in whole or in part, a moiety selected from:

[0146] In some aspects, the linker moiety L may comprise, either in whole or in part, a moiety selected from:

[0147] In some aspects, the linker moiety L may comprise, either in whole or in part, a moiety selected from:

[0148] 0 o

[0149] In some aspects, the linker moiety L may comprise, either in whole or in part, a moiety selected from: o

[0150] In some aspects, the linker moiety L may comprise, either in whole or in part, a moiety selected from:

[0151] In some aspects, the linker moiety L may comprise, either in whole or in part, a moiety selected from: o o

[0152] In some aspects, the linker moiety L may comprise, either in whole or in part, a moiety selected from:

[0153] In some aspects, the linker moiety L may comprise, either in whole or in part, a moiety selected from: o o

[0154] In some aspects, the linker moiety L may comprise, either in whole or in part, a moiety selected from:

[0155] In some aspects, the linker moiety L may comprise, either in whole or in part, a moiety selected from:

[0156] wherein n is independently selected at each occurrence from 1, 2, 3, 4, 5, and 6; and all other variables are as defined herein. In some aspects, the linker moiety L may comprise, either in whole or in part, a moiety selected from:

[0157] In some aspects, the linker moiety L may comprise, either in whole or in part, a moiety selected from:

[0158] In some aspects, the linker moiety L may comprise, either in whole or in part, a moiety selected from:

[0159] In some aspects, the linker moiety L may comprise, either in whole or in part, a moiety selected from: o o In some aspects, the linker moiety L may comprise, either in whole or in part, a moiety selected from:

[0160] In some aspects, the linker moiety L may comprise, at least in part, a cleavable group.

[0161] In some aspects, the cleavable group may be cleaved under reductive conditions. In some aspects, the cleavable group may be cleaved under oxidative conditions. In some aspects, the cleavable group may be cleaved under acidic conditions. In some aspects, the cleavable group may be cleaved under alkaline conditions. In some aspects, the cleavable group may be cleaved photolytically. In some aspects, the cleavable group may be cleaved by a transition metal catalyst. In some aspects, the cleavable group may be cleaved enzymatically.

[0162] In some aspects, the cleavable group may comprise, at least in part, a disulfide (-S-S-) or an azo (-N=N-) moiety. In some aspects, the disulfide or azo moiety may be cleaved under reductive conditions. In some aspects, a disulfide or azo moiety may replace the groups represented by any one of R100, R101, R102, R103, and R104in the linker moieties of Formula LI .

[0163] In some aspects, the cleavable group may be cleaved by a protease. In some aspects, the cleavable moiety may comprise, at least in part, a moiety having the structure:

[0164]

[0165] In some aspects, the cleavable group is photocleavable. In some aspects, the cleavable moiety may comprise, at least in part, a moiety selected from: In some aspects, the above groups may replace the group represented by any one of

[0166] Rioo, R101, R102, R103, and R104in the linker moiety L of Formula LI .

[0167] Representative examples of adaptor molecules of Formula I include, but are not limited to:

[0168] or a pharmaceutically acceptable salt thereof, wherein n is an integer selected from 0 to 12 (for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12).

[0169] Further representative examples of adaptor molecules include, but are not limited to:

[0170] or a pharmaceutically acceptable salt thereof, wherein n is an integer selected from 0 to 12 (for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, , or 12).

[0171] Even further representative examples of adaptor molecules of Formula I include:

[0172]

[0173] Compounds of Formula II and Synthesis of Adaptor Molecules of Formula III

[0174] In further aspects, compounds are also provided which may be used in the synthesis and manufacture of adaptor molecules. In particular, such molecules may be used in the preparation of adaptor molecules other than those containing small molecule ligands, such as those containing antibody or antibody fragments. Thus, in one aspect, a compound is provided of Formula II

[0175] E-LA-TA(II) wherein:

[0176] E comprises an active ester moiety;

[0177] LAcomprises a linker moiety; and

[0178] TAcomprises a ligand for Halo-tag.

[0179] E may comprise any suitable active ester moiety as known in the art. As used herein, an “active ester” refers to an ester functional group that is highly susceptible toward nucleophilic attack. Activation may be impaired by modifications of the alkoxy component of the ester, for example by substituting electronegative substituents. In some aspects, E comprises an active ester moiety selected from an N-hydroxysuccinimidyl ester, a sulfosuccinimidyl ester, a N-hydroxyphthalimidyl ester, a N-hydroxysulfophthalimidyl ester, an o-nitrophenyl ester, a p-nitrophenyl ester, a 2,4-dinitrophenyl ester, a 3-sulfonyl-4- nitrophenyl ester, a 3-carboxy-4-nitrophenyl ester, a pentafluorophenyl ester, and a sulfonyltetrafluorophenyl ester.

[0180] In some aspects, E comprises an active ester moiety selected from

[0181] In some aspects, TAcomprises a chloroalkane moiety. In some particular aspects, TAcomprises -(C1-C20 alkyl)-Cl. In some aspects, TAcomprises -(Ci alkyl)-Cl. In some aspects, TAcomprises -(C2 alkyl)-Cl. In some aspects, TAcomprises -(C3 alkyl)-Cl. In some aspects, TAcomprises -(C4 alkyl)-Cl. In some aspects, TAcomprises -(C5 alkyl)-Cl. In some aspects, TAcomprises -(Cs alkyl)-Cl. In some aspects, TAcomprises -(C7 alkyl)-Cl. In some aspects, TAcomprises -(Cs alkyl)-Cl. In some aspects, TAcomprises -(C9 alkyl)-Cl. In some aspects, TAcomprises -(C10 alkyl)-Cl. In some aspects, TAcomprises — (Ci 1 alkyl)-Cl. In some aspects, TAcomprises -(C12 alkyl)-Cl. In some aspects, TAcomprises -(C13 alkyl)-Cl. In some aspects, TAcomprises -(C14 alkyl)-Cl. In some aspects, TAcomprises — (C15 alkyl)-Cl. In some aspects, TAcomprises -(Ci6 alkyl)-Cl. In some aspects, TAcomprises -(C17 alkyl)-Cl. In some aspects, TAcomprises -(Cis alkyl)-Cl. In some aspects, TAcomprises -(C19 alkyl)-Cl. In some aspects, TAcomprises -(C20 alkyl)-Cl. ■Cl

[0182] In some aspects, TAcomprises

[0183] A linker moiety LAis included in the compounds of Formula II. A linker moiety LAis a chemically stable bivalent group that attaches TAto E. A linker moiety, as described herein, can be used in either direction, i.e., either the left end is linked to TAand the right end to E, or the left end is linked to E and the right end to TA.

[0184] In some aspects, the linker moiety LAis a chain of 2 to 14, 15, 16, 17, 18, 19, or 20 or more carbon atoms, of which one or more carbons can be optionally replaced by a heteroatom such as O, N, S, or P.

[0185] In some aspects, the chain has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 19, or 20 contiguous atoms. For example, the chain may include 1 or more ethylene glycol units that can be contiguous, partially contiguous or non-contiguous (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 ethylene glycol units).

[0186] In some aspects, the chain has at least 1, 2, 3, 4, 5, 6, 7, or 8 contiguous units which can be branched and which can be independently alkyl, aryl, heteroaryl, alkenyl, or alkynyl, cycloalkyl, or heterocycloalkyl substituents.

[0187] In some aspects, the linker moiety LAcan include or be comprised of one or more ethylene glycol, propylene glycol, lactic and / or glycolic acid units. Block and random lactic acid-co-glycolic acid moieties, as well as ethylene glycol and propylene glycol, are known in the art and can be modified to obtain the desired half-life and hydrophilicity. In certain aspects, these units can be flanked or interspersed with other moieties, such as alkyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, etc., as desired to achieve the appropriate properties.

[0188] In some aspects, the linker moiety LAis an optionally substituted (poly)ethylene glycol having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more, ethylene glycol units, or optionally substituted alkyl groups interspersed with optionally substituted O, N, S, P or Si atoms.

[0189] In some aspects, the linker moiety LAis flanked, substituted, or interspersed with an alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group.

[0190] In some aspects, the linker moiety LAmay be asymmetric or symmetric. In some aspects, the linker moiety LAcan be a non-linear chain and can be, or include, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl cyclic moieties.

[0191] In some aspects, the linker moiety LAis selected from LI: In some aspects, the linker moiety LAis selected from the group consisting of a moiety of Formula LI, Formula L2, Formula L3, Formula L4, Formula L5, Formula L6, Formula L7,

[0192] Formula L8, Formula L9, or Formula LIO:

[0193] (L2)

[0194] (L3) heteroaryl

[0195] \Ri°3(L4) wherein:

[0196] X101and X102are independently at each occurrence selected from a bond, aryl, heteroaryl, cycloalkyl, heterocycle, NR130, C(R130)2, O, C(O), and S; Rioo, R101, R102, R103, and R104are independently at each occurrence selected from the group consisting of a bond, alkyl, -C(O)-, -C(O)O-, -OC(O)-, -SO2-, -S(O)-, C(S)-, -C(O)NR130-, -NR130C(O)-, -O-, -S-, -NR130-, -C(R130R130)-, -P(O)(OR106))-, -R(O)(OR106)-, alkenyl, alkynyl, haloalkyl, alkoxy, aryl, heterocycloalkyl, cycloalkyl, heteroaryl, lactic acid, or glycolic acid, each of which may be optionally substituted with one or more (for example, 1, 2, 3, or 4) substituents independently selected from R140;

[0197] R106is independently at each occurrence selected from the group consisting of hydrogen, alkyl, arylalkyl, heteroarylalkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl;

[0198] R130is independently as each occurrence selected from the group consisting of hydrogen, alkyl, cycloalky], heterocycloalkyl, aryl, heteroaryl, -C(O)H, -C(O)OH, -C(O)alkyl, -C(O)Oalkyl, -C(O)(cycloalkyl, heterocycloalkyl, aryl, or heteroaryl), -C(O)O(cycloalkyl, heterocycloalkyl, aryl, or heteroaryl), alkenyl, or alkynyl; and

[0199] R140is independently at each occurrence selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, fluoro, bromo, chloro, hydroxyl, alkoxy, azide, amino cyano, -NH(alkyl, cycloalkyl, heterocyloalkyl, aryl, or heteroaryl), -N(independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl), -NHSCMalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl), -N(alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl)SO2alkyl, -Nl lSChalkenyl, -N(alkyl)SO2alkenyl, -NI ISChalkynyl, -N(alkyl)SO2alkynyl, haloalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

[0200] In some aspects, R100, R101, R102, R103, and R104within the linker moiety are selected in such manner that: no two -C(=O)- moieties are adjected to each other; no two -O- or -NH- moieties are adjacent to each other; and / or no moieties are otherwise selected in an order such that an unstable molecule results (as defined as producing a molecule that has a shelf life at ambient temperature of less than about six months, five months, or four months) due to decomposition caused by the selection and order of R100, R101, R102, R103, and R104.

[0201] The following are non-limiting examples of linker moieties and / or moieties which comprise the same in whole or in part that can be used in this disclosure. Based on this elaboration, those of skill in the art will understand how to use the full breadth of linker moieties that will accomplish the goal of the disclosure.

[0202] Non-limiting examples of moieties that may comprise the linker moiety LA, either in whole or in part, include, but are not limited to: a bond; -C(=O)-; -C=C-; -NH-; -N / CHs)-; -O-; -CH2-; -(CH2)2-; -(CH2)3-; -(CH2)4-; -(CH2)5-; -(CH2)6-; -(CH2)7-; -(CH2)8-; -(CH2)9-;

[0203] -(CH2)IO-; -NH(C=O)-; -C(=O)NH-; -C(=O)CH2-; -C(=O)(CH2)2-; -C(=O)(CH2)3-;

[0204] -C(=O)(CH2)4-; -C(=O)(CH2)5-; -C(=O)(CH2)6-; -CH2C(=O)-; -(CH2)2C(=O)-;

[0205] -(CH2)3C(=O)-; -(CH2)4C(=O)-; -(CH2)5C(=O)-; -(CH2)6C(=O)-; -CH2NH-; -(CH2)2NH-; -(CH2)3NH-; -(CH2)4NH-; -(CH2)5NH-; -(CH2)6NH-; -NHCH2-; -NH(CH2)2-; -NH(CH2)3-;

[0206] -NH(CH2)4-; -NH(CH2)5-; -NH(CH2)6-; -CH2O-; -(CH2)2O-; -(CH2)3O-; -(CH2)4O-;

[0207] -(CH2)5O-; -(CH2)6O-; -OCH2-; -O(CH2)2-; -O(CH2)3-; -O(CH2)4-; -O(CH2)5-; -O(CH2)6-; o

[0208] Further non-limiting examples of moieties which may comprise the linker moiety LA, either in whole or in part, include, but are not limited to:

[0209] In some aspects, the linker moiety LAmay comprise, either in whole or in part, a moiety

[0210] O

[0211] In some aspects, the linker moiety LAmay comprise, either in whole or in part, a moiety selected from: and

[0212] In some aspects, the linker moiety LAmay comprise, either in whole or in part, a moiety

[0213] 0 0

[0214] O

[0215] In some aspects, the linker moiety LAmay comprise, either in whole or in part,

[0216] In some aspects, the linker moiety LAmay comprise, either in whole or in part,

[0217] In some aspects, the linker moiety LAmay comprise, either in whole or in part, a moiety selected from:

[0218] In some aspects, the linker moiety LAmay comprise, either in whole or in part, a moiety selected from:

[0219] In some aspects, the linker moiety LAmay comprise, either in whole or in part, a moiety selected from:

[0220] 0 o

[0221] In some aspects, the linker moiety LAmay comprise, either in whole or in part, a moiety selected from:

[0222] In some aspects, the linker moiety LAmay comprise, either in whole or in part, a moiety selected from:

[0223] In some aspects, the linker moiety LAmay comprise, either in whole or in part, a moiety selected from: o o selected from:

[0224] In some aspects, the linker moiety LAmay comprise, either in whole or in part, a moiety selected from: o o

[0225] In some aspects, the linker moiety LAmay comprise, either in whole or in part, a moiety selected from:

[0226] In some aspects, the linker moiety LAmay comprise, either in whole or in part, a moiety selected from:

[0227] wherein n is independently selected at each occurrence from 1, 2, 3, 4, 5, and 6; and all other variables are as defined herein. In some aspects, the linker moiety LAmay comprise, either in whole or in part, a moiety selected from:

[0228] H

[0229] In some aspects, the linker moiety LAmay comprise, either in whole or in part, a moiety selected from:

[0230] In some aspects, the linker moiety LAmay comprise, either in whole or in part, a moiety selected from:

[0231] In some aspects, the linker moiety LAmay comprise, either in whole or in part, a moiety selected from: o o In some aspects, the linker moiety LAmay comprise, either in whole or in part, a moiety selected from:

[0232] In some aspects, the linker moiety LAmay comprise, at least in part, a cleavable group.

[0233] In some aspects, the cleavable group may be cleaved under reductive conditions. In some aspects, the cleavable group may be cleaved under oxidative conditions. In some aspects, the cleavable group may be cleaved under acidic conditions. In some aspects, the cleavable group may be cleaved under alkaline conditions. In some aspects, the cleavable group may be cleaved photolytically. In some aspects, the cleavable group may be cleaved by a transition metal catalyst. In some aspects, the cleavable group may be cleaved enzymatically.

[0234] In some aspects, the cleavable group may comprise, at least in part, a disulfide (-S-S-) or an azo (-N=N-) moiety. In some aspects, the disulfide or azo moiety may be cleaved under reductive conditions. In some aspects, a disulfide or azo moiety may replace the groups represented by any one of R100, R101, R102, R103, and R104in the linker moiety LAof Formula LI.

[0235] In some aspects, the cleavable group may be cleaved by a protease. In some aspects, the cleavable moiety may comprise, at least in part, a moiety having the structure:

[0236] In some aspects, the cleavable group is photocleavable. In some aspects, the cleavable moiety may comprise, at least in part, a moiety selected from: In some aspects, the above groups may replace the group represented by any one of

[0237] Rioo, R101, R102, R103, and R104in the linker moiety LAof Formula LI .

[0238] Representative compounds of Formula II include:

[0239] In another aspect, a method is provided of synthesizing an adaptor molecule of Formula

[0240] III AA-(C=O)-LA-TA(III) wherein AAis selected from an antibody or antigen recognizing fragment thereof; and all other variables are as defined herein; the method comprising: reacting a compound of Formula II with an antibody or an antigen recognizing fragment thereof to form the adaptor molecule of Formula III.

[0241] In another aspect, an adaptor molecule of Formula III is provided prepared according to the methods described herein.

[0242] The adaptor molecules of Formula III contain a moiety comprising an antibody or antigen recognizing fragment thereof. As used herein, the term “antibody” is used in the broadest sense and specifically covers various aspects including, but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies formed from at least two intact antibodies), and antibody fragments such as diabodies so long as they exhibit a desired biological activity. Antibodies are primarily amino-acid based molecules but may also comprise one or more modifications such as with sugar moieties.

[0243] “Antibody fragments” comprise a portion of an intact antibody, preferably comprising an antigen binding region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site. Also produced is a residual “Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen. For the purposes herein, an “antibody” may comprise a heavy and light variable domain as well as an Fc region.

[0244] “Native antibodies” are usually heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.

[0245] As used herein, the term “variable domain” refers to specific antibody domains that differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. As used herein, the term “Fv” refers to antibody fragments which contain a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association.

[0246] Antibody “light chains” from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda based on amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies can be assigned to different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl , IgG2, IgG3, TgG4, IgA, and IgA2.

[0247] “Single-chain Fv” or “scFv,” as used herein, refers to a fusion protein of VH and VL antibody domains, wherein these domains are linked together into a single polypeptide chain. In some aspects, the Fv polypeptide linker enables the scFv to form the desired structure for antigen binding.

[0248] The term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain VH connected to a light chain variable domain VL in the same polypeptide chain. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93 / 11161 ; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993), the contents of each of which are incorporated herein by reference in their entirety.

[0249] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous cells (or clones), i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variants that may arise during production of the monoclonal antibody, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[0250] The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. The monoclonal antibodies herein include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies.

[0251] “Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the hypervariable region from an antibody of the recipient are replaced by residues from the hypervariable region from an antibody of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.

[0252] The term “hypervariable region” when used herein in reference to antibodies refers to regions within the antigen binding domain of an antibody comprising the amino acid residues that are responsible for antigen binding. The amino acids present within the hypervariable regions determine the structure of the complementarity determining region (CDR). As used herein, the “CDR” refers to the region of an antibody that comprises a structure that is complimentary to its target antigen or epitope.

[0253] In some aspects, the compositions of the present disclosure may include antibody mimetics. The term “antibody mimetic” refers to any molecule which mimics the function or effect of an antibody and which binds specifically and with high affinity to their molecular targets. As such, antibody mimics include nanobodies and the like.

[0254] In some aspects, antibody mimetics may be those known in the art including, but are not limited to affibody molecules, affilins, affitins, anticalins, avimers, DARPins, Fynomers and Kunitz and domain peptides. In other aspects, antibody mimetics may include one or more non-peptide region.

[0255] As used herein, the term “antibody variant” refers to a biomolecule resembling an antibody in structure and / or function comprising some differences in their amino acid sequence, composition or structure as compared to a native antibody.

[0256] The preparation of antibodies, whether monoclonal or polyclonal, is known in the art. Techniques for the production of antibodies are well known in the art and described, e.g. in Harlow and Lane “Antibodies, A Laboratory Manual”, Cold Spring Harbor Laboratory Press, 1988 and Harlow and Lane “Using Antibodies: A Laboratory Manual” Cold Spring Harbor Laboratory Press, 1999.

[0257] In one aspect, antibodies, antibody fragments, their variants or derivatives as described above are specifically immunoreactive with allergens. Antibodies or fragments of antibodies may also bind to target sites on allergens. Antibodies as used in the present disclosure may be characterized by their target molecule(s), by the antigens used to generate them, by their function (whether as agonists or antagonists) and / or by the cell niche in which they function.

[0258] Measures of antibody function may be made relative to a standard under normal physiologic conditions, in vitro or in vivo. Measurements may also be made relative to the presence or absence of the antibodies. Such methods of measuring include standard measurement in tissue or fluids such as serum or blood such as Western blot, enzyme-linked immunosorbent assay (ELISA), activity assays, reporter assays, luciferase assays, polymerase chain reaction (PCR) arrays, gene arrays, real time reverse transcriptase (RT) PCR and the like.

[0259] Antibodies may bind or interact with any number of locations on or along an antigen protein. Antigen antibody target sites contemplated include any and all possible sites for detecting said antigen.

[0260] Antibodies as used in the present disclosure exert their effects via binding (reversibly or irreversibly) to one or more antigen target sites. While not wishing to be bound by theory, target sites which represent a binding site for an antibody, are most often formed by proteins or protein domains or regions. However, target sites may also include biomolecules such as sugars, lipids, nucleic acid molecules or any other form of binding epitope.

[0261] Antibodies as used in the present disclosure, as well as antigens used to generate them, are primarily amino acid-based molecules. These molecules may be “peptides,” “polypeptides,” or “proteins.”

[0262] As used herein, the term “peptide” refers to an amino-acid based molecule having from 2 to 50 or more amino acids. Special designators apply to the smaller peptides with “dipeptide” referring to a two amino acid molecule and “tripeptide” referring to a three amino acid molecule. Amino acid based molecules having more than 50 contiguous amino acids are considered polypeptides or proteins.

[0263] The terms “amino acid” and “amino acids” refer to all naturally occurring L-alpha- amino acids as well as non-naturally occurring amino acids. Amino acids are identified by either the one-letter or three-letter designations as follows: aspartic acid (Asp: D), isoleucine (He: I), threonine (Thr: T), leucine (Leu: L), serine (Ser: S), tyrosine (Tyr: Y), glutamic acid (Glu: E), phenylalanine (Phe: F), proline (Pro: P), histidine (His: H), glycine (Gly: G), lysine (Lys: K), alanine (Ala: A), arginine (Arg: R), cysteine (Cys: C), tryptophan (Trp: W), valine (Vai: V), glutamine (Gin: Q) methionine (Met: M), and asparagine (Asn: N), where the amino acid is listed first followed parenthetically by the three and one letter codes, respectively. Antibodies of the present disclosure may be polyclonal or monoclonal or recombinant, as produced by methods known in the art.

[0264] In some aspects, the antibodies as used in the present disclosure may be labeled for purposes of detection with a detectable label known by one of skill in the art. The label can be a radioisotope, fluorescent compound, chemiluminescent compound, enzyme, or enzyme cofactor, or any other labels known in the art. In some aspects, the antibody that binds to a desired antigen is not labeled, but may be detected by binding of a labeled secondary antibody that specifically binds to the primary antibody.

[0265] Antibodies as used in the present disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, fragments produced by a Fab expression library, anti- idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the disclosure), intracellularly made antibodies (i.e., intrabodies), and epitope-binding fragments of any of the above. Antibodies as used in the present disclosure can be from any animal origin including birds and mammals. Preferably, such antibodies are of human, murine (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken origin. The antibodies as used in the present disclosure can be monospecific or multispecific (e.g., bispecific, trispecific, or of greater multispecificity). Multispecific antibodies can be specific for different epitopes of a peptide as described in the present disclosure, or can be specific for both a peptide as described in the present disclosure, and a heterologous epitope, such as a heterologous peptide or solid support material. (See, e.g., WO 93 / 17715; WO 92 / 08802; WO 91 / 00360; WO 92 / 05793; Tutt, A. et al., Trispecific F(ab)3 derivatives that use cooperative signaling via the TCR / CD3 complex and CD2 to activate and redirect resting cytotoxic T cells. J Immunol. 1991 Jul. 1 ; 147(l):60-9; U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; and Kostelny, S. A. et al., Formation of a bispecific antibody by the use of leucine zippers. J Immunol. 1992 Mar. 1; 148(5): 1547-53).

[0266] In some aspects, antibodies can be prepared from any region of an antigen. In the present disclosure, the peptides for generating antibodies preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, and, preferably, between about 5 to about 50 amino acids in length, more preferably between about 10 to about 30 amino acids in length, even more preferably between about 10 to about 20 amino acids in length. In certain aspects of the present disclosure, where larger polypeptides or proteins are used for generating antibodies, these preferably are at least 50, at least 55, at least 60, at least 70, at least 80, at least 90, or more amino acids in length.

[0267] Monoclonal antibodies as used in the present disclosure can be prepared using well- established methods known by those skilled in the art. In one aspect, the monoclonal antibodies are prepared using hybridoma technology (Kohler, G. et al., Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975 Aug. 7: 256 (5517): 495-7). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent (e.g., a peptide of the disclosure) to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, J. W., Monoclonal Antibodies: Principles and Practice. Academic Press. 1986; 59-1031 ). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, rabbit, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT -deficient cells.

[0268] Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif, and the American Type Culture Collection, Manassas, Va. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, D. et al., A human hybrid myeloma for production of human monoclonal antibodies. J Immunol. 1984 December; 133(6): 3001-5; Brodeur, B. et al., Monoclonal Antibody Production Techniques and Applications. Marcel Dekker, Inc., New York. 1987; 33:51-63).

[0269] The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies. Preferably, the binding specificity (i.e., specific immunoreactivity) of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (MA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known by those skilled in the art. The binding specificity of the monoclonal antibody can, for example, be determined by Scatchard analysis (Munson, P. J. et al., Ligand: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem. 1980 Sep. 1; 107(l):220-39).

[0270] After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium or RPML1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.

[0271] The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0272] In another aspects, the monoclonal antibodies as used in the present disclosure can also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567, which is hereby incorporated by reference in its entirety. DNA encoding the monoclonal antibodies of the disclosure can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the disclosure serve as a preferred source of DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the disclosure, or can be substituted for the variable domains of one antigen-combining site of an antibody of the disclosure to create a chimeric bivalent antibody. In another aspect, antibodies as used in the present disclosure can also be produced by various procedures known by those skilled in the art. For the production of polyclonal antibodies in vivo, host animals, such as rabbits, rats, mice, sheep, or goats, are immunized with either free or carrier-coupled peptides, for example, by intraperitoneal and / or intradermal injection. Injection material is typically an emulsion containing about 100 pg of peptide or carrier protein. Various adjuvants can also be used to increase the immunological response, depending on the host species. Adjuvants include, but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and other useful human adjuvants such as BCG (Bacillus Calmette- Guerin) and Corynebacterium parvum. Such adjuvants are also well known in the art. Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface. The titer of antibodies in serum from an immunized animal can be increased by selection of antibodies, e.g., by adsorption of the peptide onto a solid support and elution of the selected antibodies according to methods well known in the art.

[0273] Antibodies, variants and fragments thereof may be selected and produced using high throughput methods of discovery. In one aspect, synthetic antibodies, variants and fragments thereof are produced through the use of display libraries. The term “display” as used herein, refers to the expression or “display” of proteins or peptides on the surface of a given host. The term “library” as used herein, refers to a collection of unique cDNA sequences. A library may contain from as little as two unique cDNAs to hundreds of billions of unique cDNAs. In a preferred aspect, synthetic antibodies are produced using antibody display libraries or antibody fragment display libraries. The term “antibody fragment display library” as used herein, refers to a display library wherein each member encodes an antibody fragment containing at least one variable region of an antibody. Such antibody fragments are preferably Fab fragments, but other antibody fragments such as single-chain variable fragments (scFvs) are contemplated as well. In an Fab antibody fragment library, each Fab encoded may be identical except for the amino acid sequence contained within the variable loops of the complementarity determining regions (CDRs) of the Fab fragment. In an alternative or additional aspect, amino acid sequences within the individual VH and / or VL regions may differ as well. Display libraries may be expressed in a number of possible hosts including, but not limited to yeast, bacteriophage, bacteria and retroviruses. Additional display technologies that may be used include ribosome-display, microbead-display and protein-DNA linkage techniques. In a preferred aspect, Fab display libraries are expressed in yeast or in bacteriophages (also referred to herein as “phages” or “phage particles.” When expressed, the Fabs decorate the surface of the phage or yeast where they can interact with a given antigen. An antigen comprising an allergen or an antigen from a desired target site may be used to select phage particles or yeast cells expressing antibody fragments with the highest affinity for that antigen. The DNA sequence encoding the CDR of the bound antibody fragment can then be determined through sequencing using the bound particle or cell. In one aspect, positive selection is used in the development of antibodies. As used herein, the term “positive selection” refers to processes by which antibodies and / or fragments thereof are selected from display libraries based on affinity for antigens containing target sites. In some aspects, negative selection is utilized in the development of antibodies. As used herein, the term “negative selection” refers to processes by which antigens that lack target sites for antibody production are used to exclude antibodies and / or fragments thereof from a given display library during antibody development. In some aspects, both positive and negative selection processes are utilized during multiple rounds of selection in the development of antibodies using display libraries.

[0274] In yeast display, cDNA encoding different antibody fragments are introduced into yeast cells where they are expressed and the antibody fragments are “displayed” on the cell surface as described by Chao et al. (Chao, G. et al., Isolating and engineering human antibodies using yeast surface display. Nat Protoc. 2006; l(2):755-68). In yeast surface display, expressed antibody fragments contain an additional domain comprising the yeast agglutinin protein, Aga2p. This domain allows the antibody fragment fusion protein to attach to the outer surface of the yeast cell through the formation of disulfide bonds with surface-expressed Agalp. The result is a yeast cell, coated in a particular antibody fragment. Display libraries of cDNA encoding these antibody fragments are utilized initially in which the antibody fragments each have a unique sequence. These fusion proteins are expressed on the cell surface of millions of yeast cells where they can interact with a desired antigenic target peptide, incubated with the cells. Target peptides may be covalently or otherwise modified with a chemical or magnetic group to allow for efficient cell sorting after successful binding with a suitable antibody fragment takes place. Recovery may be by way of magnetic-activated cell sorting (MACS), fluorescence-activated cell sorting (FACS) or other cell sorting methods known in the art. Once a subpopulation of yeast cells is selected, the corresponding plasmids may be analyzed to determine the CDR sequence.

[0275] Bacteriophage display methods typically utilize filamentous phage including fd, Fl and Ml 3 virions. Such strains are non-lytic, allowing for continued propagation of the host and increased viral titres. Examples of phage display methods that can be used to make the antibodies of the present disclosure include those disclosed in Miersch et al. (Miersch, S. et al., Synthetic antibodies: Concepts, potential and practical considerations. Methods. 2012 August; 57(4): 486-98), Bradbury et al. (Bradbury, A. R. et al., Beyond natural antibodies: the power of in vitro display technologies. Nat Biotechnol. 2011 March; 29(3):245-54), Brinkman et al. (Brinkmann, U. et al., Phage display of disulfide-stabilized Fv fragments. J Immunol Methods. 1995 May 11; 182(l):41-50); Ames et al. (Ames, R. S. et al., Conversion of murine Fabs isolated from a combinatorial phage display library to full length immunoglobulins. J Immunol Methods. 1995 Aug. 18; 184(2): 177-86); Kettleborough et al. (Kettleborough, C. A. et al., Isolation of tumor cell-specific single-chain Fv from immunized mice using phage-antibody libraries and the re-construction of whole antibodies from these antibody fragments. Eur J Immunol. 1994 April; 24(4):952-8); Persic et al. (Persic, L. et al., An integrated vector system for the eukaryotic expression of antibodies or their fragments after selection from phage display libraries. Gene. 1997 Mar. 10; 187(1):9-18); PCT application No. PCT / GB91 / 01134; PCT publications WO 90 / 02809; WO 91 / 10737; WO 92 / 01047; WO 92 / 18619; WO 93 / 11236; WO 95 / 15982; WO 95 / 20401 ; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5, 969,108, each of which is incorporated herein by reference in its entirety.

[0276] Antibody fragment expression on bacteriophages may be carried out by inserting the cDNA encoding the fragment into the gene expressing a viral coat protein. The viral coat of filamentous bacteriophages is made up of five coat proteins, encoded by a single-stranded genome. Coat protein pill is the preferred protein for antibody fragment expression, typically at the N-terminus. If antibody fragment expression compromises the function of pill, viral function may be restored through coexpression of a wild type pill, although such expression will reduce the number of antibody fragments expressed on the viral coat, but may enhance access to the antibody fragment by the target antigen. Expression of viral as well as antibody fragment proteins may alternatively be encoded on multiple plasmids. This method may be used to reduce the overall size of infective plasmids and enhance the transformation efficiency. As described above, after selection of a host expressing a high affinity antibody or antibody fragment, the coding regions from the antibody or antibody fragment can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria.

[0277] The DNA sequence encoding a high affinity antibody can be mutated for additional rounds of selection in a process known as affinity maturation. The term “affinity maturation,” as used herein, refers to a method whereby antibodies are produced with increasing affinity for a given antigen through successive rounds of mutation and selection of antibody- or antibody fragment-encoding cDNA sequences. In a preferred aspect, this process is carried out in vitro. To accomplish this, amplification of CDR coding sequences may be carried out using error- prone PCR to produce millions of copies containing mutations including, but not limited to point mutations, regional mutations, insertional mutations and deletional mutations. As used herein, the term “point mutation” refers to a nucleic acid mutation in which one nucleotide within a nucleotide sequence is changed to a different nucleotide. As used herein, the term “regional mutation” refers to a nucleic acid mutation in which two or more consecutive nucleotides are changed to different nucleotides. As used herein, the term “insertional mutation” refers to a nucleic acid mutation in which one or more nucleotides are inserted into a nucleotide sequence. As used herein, the term “deletional mutation” refers to a nucleic acid mutation in which one or more nucleotides are removed from a nucleotide sequence. Insertional or deletional mutations may include the complete replacement of an entire codon or the change of one codon to another by altering one or two nucleotides of the starting codon.

[0278] Mutagenesis may be carried out on CDR-encoding cDNA sequences to create millions of mutants with singular mutations in CDR heavy and light chain regions. In another approach, random mutations are introduced only at CDR residues most likely to improve affinity. These newly generated mutagenic libraries can be used to repeat the process to screen for clones that encode antibody fragments with even higher affinity for the target peptide. Continued rounds of mutation and selection promote the synthesis of clones with greater and greater affinity (Chao, G. et al., Isolating and engineering human antibodies using yeast surface display. Nat. Protoc. 2006; l(2):755-68).

[0279] Examples of techniques that can be used to produce antibodies and antibody fragments, such as Fabs and scFvs, include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498;

[0280] Miersch et al. (Miersch, S. et al., Synthetic antibodies: Concepts, potential and practical considerations. Methods. 2012 August; 57(4):486-98), Chao et al. (Chao, G. et al., Isolating and engineering human antibodies using yeast surface display. Nat Protoc. 2006; 1(2):755- 68), Huston et al. (Huston, J. S. et al., Protein engineering of single-chain Fv analogs and fusion proteins. Methods Enzymol. 1991; 203:46-88); Shu et al. (Shu, L. et al., Secretion of a single-gene-encoded immunoglobulin from myeloma cells. Proc. Natl. Acad. Sci. U.S.A 1993 Sep. 1; 90(17):7995-9); and Skerra et al. (Skerra, A. et al., Assembly of a functional immunoglobulin Fv fragment in Escherichia coli. Science. 1988 May 20; 240(4855): 1038-41 ), each of which is incorporated herein by reference in its entirety.

[0281] For some uses, including the in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal immunoglobulin and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. (Morrison, S. L., Transfectomas provide novel chimeric antibodies. Science. 1985 Sep. 20; 229(4719): 1202-7; Gillies, S. D. et al., High-level expression of chimeric antibodies using adapted cDNA variable region cassettes. J Immunol Methods. 1989 Dec. 20; 125(1-2): 191-202; and U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporated herein by reference in their entirety).

[0282] Humanized antibodies are antibody molecules from non-human species that bind to the desired antigen and have one or more complementarity determining regions (CDRs) from the nonhuman species and framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions are substituted with corresponding residues from the CDR and framework regions of the donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding, and by sequence comparison to identify unusual framework residues at particular positions. (U.S. Pat. Nos. 5,693,762 and 5,585,089; Riechmann, L. et al., Reshaping human antibodies for therapy. Nature. 1988 Mar. 24; 332(6162):323-7, which are incorporated herein by reference in their entireties).

[0283] Antibodies can be humanized using a variety of techniques known in the art, including, for example, CDR-grafting (EP 239,400; PCT publication WO 91 / 09967; U.S. Pat. Nos. 5,225,539; 5,530,101 ; and 5,585,089); veneering or resurfacing (EP 592,106; EP 519,596; Padlan, E. A., A possible procedure for reducing the immunogenicity of antibody variable domains while preserving their ligand-binding properties. Mol Immunol. 1991 April-May; 28(4-5):489-98; Studnicka, G. M. et al., Human-engineered monoclonal antibodies retain full specific binding activity by preserving non-CDR complementarity-modulating residues. Protein Eng. 1994 June; 7(6):805-14; Roguska, M. A. et al., Humanization of murine monoclonal antibodies through variable domain resurfacing. Proc. Natl. Acad. Sci. U.S.A 1994 Feb. 1; 91(3):969-73); and chain shuffling (U.S. Pat. No. 5,565,332); each of which is incorporated herein by reference in their entirety.

[0284] Completely human antibodies are particularly desirable for therapeutic treatment of human patients, so as to avoid or alleviate immune reaction to foreign protein. Human antibodies can be made by a variety of methods known in the art, including the antibody display methods described above, using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111 ; and PCT publications WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735, and WO 91 / 10741 ; each of which is incorporated herein by reference in its entirety.

[0285] Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin polynucleotides. For example, the human heavy and light chain immunoglobulin polynucleotide complexes can be introduced randomly, or by homologous recombination, into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells, in addition to the human heavy and light chain polynucleotides. The mouse heavy and light chain immunoglobulin polynucleotides can be rendered nonfunctional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the disclosure.

[0286] Thus, using such a technique, it is possible to produce useful human IgG, IgA, IgM, IgD and IgE antibodies. For an overview of the technology for producing human antibodies, see Lonberg and Huszar (Eonberg, N. et al., Human antibodies from transgenic mice. Int. Rev. Immunol. 1995; 13(l):65-93). For a detailed discussion of the technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publications WO 98 / 24893; WO 92 / 01047; WO 96 / 34096; WO 96 / 33735; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771 ; 5,939,598; 6,075,181; and 6,114,598, each of which are incorporated by reference herein in their entirety. In addition, companies such as Abgenix, Inc. (Fremont, Calif.), Protein Design Labs, Inc. (Mountain View, Calif.) and Genpharm (San Jose, Calif.) can be engaged to provide human antibodies directed against a selected antigen using technology similar to the above described technologies.

[0287] Once an antibody molecule as would be used in the present disclosure has been produced by an animal, a cell line, chemically synthesized, or recombinantly expressed, it can be purified (i.e., isolated) by any method known in the art for the purification of an immunoglobulin or polypeptide molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen, Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Tn addition, the antibodies as used in the present disclosure or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.

[0288] In some aspects, the antibody may conjugated to a labeling agent, such as various organic small molecules, inorganic compounds, nanoparticles, enzymes or enzyme substrates, fluorescent materials, luminescent materials (e.g., luminol), bioluminescent materials (e.g., luciferase, luciferin, and aequorin), chemiluminescent materials, radioactive materials (e.g., 18F, 67Ga, 81mKr, 82Rb, 11 lln, 1231, 133Xe, 201T1, 1251, 35S, 14C, 3H, or 99mTc (e.g., as pertechnetate (technetate(VII), TcO4)), and contrast agents (e.g., gold (e.g., gold nanoparticles), gadolinium (e.g., chelated Gd), iron oxides (e.g., superparamagnetic iron oxide (SPIO), monocrystalline iron oxide nanoparticles (MIONs), and ultrasmall superparamagnetic iron oxide (USPIO)), manganese chelates (e.g., Mn-DPDP), barium sulfate, iodinated contrast media (iohexol), microbubbles, or perfluorocarbons). Such optically-detectable labels include for example, without limitation, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid; acridine and derivatives (e.g., acridine and acridine isothiocyanate); 5-(2'- aminoethyl)aminonaphthalene-l -sulfonic acid (EDANS); 4-amino-N-[3- vinylsulfonyl)phenyl]naphthalimide-3 ,5 disulfonate; N-(4-anilino- 1 -naphthyl)maleimide; anthranilamide; BODIPY; Brilliant Yellow; coumarin and derivatives (e.g., coumarin, 7- amino-4-methylcoumarin (AMC, Coumarin 120), and 7-amino-4-trifluoromethylcoumarin (Coumarin 151)); cyanine dyes; cyanosine; 4',6-diaminidino-2-phenylindole (DAPI); 5' 5"- dibromopyrogallol-sulfonaphthalein (bromopyrogallol Red); 7-diethylamino-3-(4'- isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'- diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'- disulfonic acid; 5- [dimethylamino] -naphthalene- 1 -sulfonyl chloride (DNS, dansylchloride); 4- dimethylaminophenylazophenyl-4’-isothiocyanate (DABITC); eosin and derivatives (e.g., eosin and eosin isothiocyanate); erythrosin and derivatives (e.g., erythrosin B and erythrosin isothiocyanate); ethidium; fluorescein and derivatives (e.g., 5 -carboxyfluorescein (FAM), dichlorotriazin-2-yl)aminofluorescein (DTAF), 2',7'-dimethoxy-4',5'-dichloro-6- carboxyfluorescein, fluorescein, fluorescein isothiocyanate, X-rhodamine-5-(and-6)- isothiocyanate (QFITC or XRITC), and fluorescamine); 2-[2-[3-[[l,3-dihydro-l,l-dimethyl-3- (3-sulfopropyl)-2H-benz[e]indol-2-ylidene]ethylidene]-2-[4-(ethoxycarbonyl)-l-piperazinyl]- 1-cyclopente n-l-yl]ethenyl]-l,l-dimethyl-3-(3-sulforpropyl)-lH-benz[e]indolium hydroxide, inner salt, compound with N,N-diethylethanamine(l : l) (IR144); 5-chloro-2-[2-[3-[(5-chloro- 3-ethyl-2(3H)-benzothiazol-ylidene)ethylidene]-2-(diphenylamino)-l-cyclopenten-l- yl]ethenyl]-3-ethyl benzothiazolium perchlorate (IR140); Malachite Green isothiocyanate; 4- methylumbelliferone orthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B- phycoerythrin; o-phthaldialdehyde; pyrene and derivatives (e.g., pyrene, pyrene butyrate, and succinimidyl 1-pyrene); butyrate quantum dots; Reactive Red 4 (CIBACRON™ Brilliant Red 3B-A); rhodamine and derivatives (e.g., 6-carboxy-X-rhodanine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA) tetramethyl rhodamine, and tetramethyl rhodamine isothiocyanate (TRITC)); riboflavin; rosolic acid; terbium chelate derivatives; Cyanine-3 (Cy3); Cyanine-5 (Cy5); cyanine-5.5 (Cy5.5), Cyanine-7 (Cy7); IRD 700; IRD 800; Alexa 647; La Jolta Blue; phthalo cyanine; and naphthalo cyanine. In particular aspects, the antibody is conjugated to a porphyrin.

[0289] Pharmaceutical Compositions

[0290] The compounds described herein can be administered by any suitable method and technique presently or prospectively known to those skilled in the art. For example, the active components described herein can be formulated in a physiologically or pharmaceutically acceptable form and administered by any suitable route known in the art including, for example, oral and parenteral routes of administering. As used herein, the term “parenteral” includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, and intrastemal administration, such as by injection. Administration of the active components of their compositions can be a single administration, or at continuous and distinct intervals as can be readily determined by a person skilled in the art.

[0291] Compositions, as described herein, comprising an active compound and a pharmaceutically acceptable carrier or excipient of some sort may he useful in a variety of medical and non- medical applications. For example, pharmaceutical compositions comprising an active compound and an excipient may be useful for the treatment or prevention of a cancer in a subject in need thereof.

[0292] "Pharmaceutically acceptable carrier" (sometimes referred to as a "carrier") means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic and includes a carrier that is acceptable for veterinary and / or human pharmaceutical or therapeutic use. The terms "carrier" or "pharmaceutically acceptable carrier" can include, but are not limited to, phosphate-buffered saline solution, water, emulsions (such as an oil / water or water / oil emulsion) and / or various types of wetting agents. As used herein, the term "carrier" encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well-known in the art for use in pharmaceutical formulations and as described further herein.

[0293] “Excipients” include any and all solvents, diluents or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, as suited to the particular dosage form desired. General considerations in formulation and / or manufacture can be found, for example, in Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980), and Remington: The Science and Practice of Pharmacy, 21st Edition (Lippincott Williams & Wilkins, 2005).

[0294] Exemplary excipients include, but are not limited to, any non-toxic, inert solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as excipients include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as com starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; com oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; detergents such as Tween 80; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. As would be appreciated by one of skill in this art, the excipients may be chosen based on what the composition is useful for. For example, with a pharmaceutical composition or cosmetic composition, the choice of the excipient will depend on the route of administration, the agent being delivered, the time course of delivery of the agent, etc., and can be administered to humans and / or to animals, orally, rectally, parenterally, intracisternally, intravaginally, intranasally, intraperitoneally, topically (as by powders, creams, ointments, or drops), buccally, or as an oral or nasal spray. In some aspects, the active compounds disclosed herein are administered topically.

[0295] Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and combinations thereof.

[0296] Exemplary granulating and / or dispersing agents include potato starch, com starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose, and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carbo xymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, etc., and combinations thereof.

[0297] Exemplary surface active agents and / or emulsifiers include natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and Veegum [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxy vinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate [Tween 20], polyoxyethylene sorbitan [Tween 60], polyoxyethylene sorbitan monooleate [Tween 80], sorbitan monopalmitate [Span 40], sorbitan monostearate [Span 60], sorbitan tristearate [Span 65], glyceryl monooleate, sorbitan monooleate [Span 80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate [Myrj 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. Cremophor), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether [Brij 30]), polyvinylpyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F 68, Poloxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and / or combinations thereof. Exemplary binding agents include starch (e.g., cornstarch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly (vinyl-pyrrolidone), magnesium aluminum silicate (Veegum), and larch arabogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, etc., and / or combinations thereof.

[0298] Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives.

[0299] Exemplary antioxidants include alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.

[0300] Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like), citric acid and salts and hydrates thereof (e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and salts and hydrates thereof, and tartaric acid and salts and hydrates thereof. Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.

[0301] Exemplary antifungal preservatives include butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.

[0302] Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.

[0303] Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, beta- carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid. Other preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus, Phenonip, methylparaben, Germall 115, Germaben II, NeoIone, Kathon, and Euxyl. In certain aspects, the preservative is an antioxidant. In other aspects, the preservative is a chelating agent.

[0304] Exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer’s solution, ethyl alcohol, etc., and combinations thereof. Exemplary lubricating agents include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and combinations thereof.

[0305] Exemplary natural oils include almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, chamomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary synthetic oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and combinations thereof.

[0306] Additionally, the composition may further comprise a polymer. Exemplary polymers contemplated herein include, but are not limited to, cellulosic polymers and copolymers, for example, cellulose ethers such as methylcellulose (MC), hydroxyethylcellulose (HEC), hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC), methylhydroxyethylcellulose (MHEC), methylhydroxypropylcellulose (MHPC), carboxymethyl cellulose (CMC) and its various salts, including, e.g., the sodium salt, hydroxyethylcarboxymethylcellulose (HECMC) and its various salts, carboxymethylhydroxyethylcellulose (CMHEC) and its various salts, other polysaccharides and polysaccharide derivatives such as starch, dextran, dextran derivatives, chitosan, and alginic acid and its various salts, carageenan, varoius gums, including xanthan gum, guar gum, gum arabic, gum karaya, gum ghatti, konjac and gum tragacanth, glycosaminoglycans and proteoglycans such as hyaluronic acid and its salts, proteins such as gelatin, collagen, albumin, and fibrin, other polymers, for example, polyhydroxyacids such as polylactide, polyglycolide, polyl(lactide-co-glycolide) and poly(.epsilon.-caprolactone-co-glycolide)-, carboxyvinyl polymers and their salts (e.g., carbomer), polyvinylpyrrolidone (PVP), polyacrylic acid and its salts, polyacrylamide, polyacrylic acid / acrylamide copolymer, polyalkylene oxides such as polyethylene oxide, polypropylene oxide, poly(ethylene oxide- propylene oxide), and a Pluronic polymer, polyoxy ethylene (polyethylene glycol), poly anhydrides, polyvinylalchol, polyethyleneamine and polypyrridine, polyethylene glycol (PEG) polymers, such as PEGylated lipids (e.g., PEG-stearate, l,2-Distearoyl-sn-glycero-3-Phosphoethanolamine-N- [Methoxy(Polyethylene glycol)-1000], 1 ,2-Distearoyl-sn-glycero-3-Phosphoethanolamine-N- [Methoxy(Polyethylene glycol)-2000], and l,2-Distearoyl-sn-glycero-3- Phosphoethanolamine-N- [Methoxy (Poly ethylene glycol)-5000]), copolymers and salts thereof.

[0307] Additionally, the composition may further comprise an emulsifying agent. Exemplary emulsifying agents include, but are not limited to, a polyethylene glycol (PEG), a polypropylene glycol, a polyvinyl alcohol, a poly-N-vinyl pyrrolidone and copolymers thereof, poloxamer nonionic surfactants, neutral water-soluble polysaccharides (e.g., dextran, Ficoll, celluloses), non-cationic poly(meth)acrylates, non-cationic poly acrylates, such as poly (meth) acrylic acid, and esters amide and hydroxy alkyl amides thereof, natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and Veegum [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxy vinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate [Tween 20], polyoxyethylene sorbitan [Tween 60], polyoxyethylene sorbitan monooleate [Tween 80], sorbitan monopalmitate [Span 40], sorbitan monostearate [Span 60], sorbitan tristearate [Span 65], glyceryl monooleate, sorbitan monooleate [Span 80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate [Myrj 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. Cremophor), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether [Brij 30]), polyvinylpyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F 68, Poloxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and / or combinations thereof. In certain aspects, the emulsifying agent is cholesterol.

[0308] Liquid compositions include emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compound, the liquid composition may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents, and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0309] Injectable compositions, for example, injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be an injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents for pharmaceutical or cosmetic compositions that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. Any bland fixed oil can be employed, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. In certain aspects, the particles are suspended in a carrier fluid comprising 1 % (w / v) sodium carboxymethyl cellulose and 0.1% (v / v) Tween 80. The injectable composition can be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions, which can be dissolved or dispersed in sterile water or other sterile injectable media prior to use.

[0310] Compositions for rectal or vaginal administration may be in the form of suppositories which can be prepared by mixing the particles with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol, or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the particles. Solid compositions include capsules, tablets, pills, powders, and granules. In such solid compositions, the particles are mixed with at least one excipient and / or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar- agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

[0311] Tablets, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

[0312] Compositions for topical or transdermal administration include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The active compound is admixed with an excipient and any needed preservatives or buffers as may be required.

[0313] The ointments, pastes, creams, and gels may contain, in addition to the active compound, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0314] Powders and sprays can contain, in addition to the active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates, polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons.

[0315] Transdermal patches have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the nanoparticles in a proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a ratecontrolling membrane or by dispersing the particles in a polymer matrix or gel.

[0316] The active ingredient may be administered in such amounts, time, and route deemed necessary in order to achieve the desired result. The exact amount of the active ingredient will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the medical disorder, the particular active ingredient, its mode of administration, its mode of activity, and the like. The active ingredient, whether the active compound itself or the active compound in combination with an agent, is preferably formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the active ingredient will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the active ingredient employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of the treatment; drugs used in combination or coincidental with the specific active ingredient employed; and like factors well known in the medical arts.

[0317] The active ingredient may be administered by any route. In some aspects, the active ingredient is administered via a variety of routes, including oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and / or drops), mucosal, nasal, bucal, enteral, sublingual; by intratracheal instillation, bronchial instillation, and / or inhalation; and / or as an oral spray, nasal spray, and / or aerosol. In general, the most appropriate route of administration will depend upon a variety of factors, including the nature of the active ingredient (e.g., its stability in the environment of the gastrointestinal tract), the condition of the subject (e.g., whether the subject is able to tolerate oral administration), etc. The exact amount of an active ingredient required to achieve a therapeutically or prophylactically effective amount will vary from subject to subject, depending on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular compound(s), mode of administration, and the like. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower, or the same as that administered to an adult.

[0318] Useful dosages of the active agents and pharmaceutical compositions disclosed herein can be determined by comparing their in vitro activity and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art.

[0319] The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms or disorder are affected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex, and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary and can be administered in one or more dose administrations daily for one or several days.

[0320] Chimeric Antigen Receptor (CAR) Systems

[0321] In another aspect, a chimeric antigen receptor (CAR) system is provided comprising i) an adaptor molecule described herein, and ii) a CAR comprising a receptor that is capable of binding toT on the adaptor molecule, a hinge domain, and a signaling domain.

[0322] In some aspects, the hinge domain is selected from a CD8a domain, a CD28 hinge, or modified IgG hinges with a deletion or modification of the CH2 and / or CH3 domain). In some aspects, the signaling domain is selected from a DAP10, NKG2D, NKG2C, NKp44, CD28, CD27, MeglO, CD32, CD16, 2B4, ZAP-70, LAT, SLP-76, or CD3 signaling domain.

[0323] In some aspects, the CAR further comprises one or more co- stimulation domains (such as for example, CD27, CD28, ICOS, 4- IBB, or 0X40).

[0324] In another aspect, a cell is provided comprising a CAR system described herein. In some aspects, the cell comprises an immune cell (including, but not limited to, T cells, NK cells, NK T cells, B cells, y6 T cells, and macrophages), a neuron, an epithelial cell, an endothelial cell, or a stem cell.

[0325] In some aspects, the disclosed CAR systems are made from and / or ultimately expressed on T cells, NK cells, NK T cells, B cells, y5 T cells, and / or macrophages. The cells used to make and express the CAR systems disclosed herein, as well as any cell comprising said receptors, may be from an autologous, syngeneic, or allogeneic source, with the selection dependent on the disease to be treated and the means available to do so. Suitable populations of effector cells that may be used in the methods include any immune cells with cytolytic activity, such as T cells. Exemplary sub-populations of T cells include but are not limited to, those expressing CD3+, including CD3+CD8+ T cells, CD3+CD4+ T cells, and NKT cells. In one aspect, the T cells are peripheral blood mononuclear cells (PBMC) of any HLA background from PBMCs and are utilized in autologous, syngeneic, or allogeneic systems. T cells may also be isolated from any source, including but not limited to a tumor explant of the subject being treated or intratumoral T cells of the subject being treated. For the sake of convenience, the effector cells are commonly referred to herein as T cells, but it should be understood that any reference to T cells unless otherwise indicated, is a reference to all effector cell types as defined herein.

[0326] It is understood that one way to define any known variants and derivatives, or those that might arise, of the disclosed genes and proteins herein is through defining the variants and derivatives in terms of homology to specific known sequences. Specifically disclosed are variants of these and other genes and proteins herein disclosed, which have at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent homology to the stated sequence. Those of skill in the art readily understand how to determine the homology of two proteins or nucleic acids, such as genes. For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest level.

[0327] Another way of calculating homology can be performed by published algorithms. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444 (1988), 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 inspection.

[0328] The same types of homology can be obtained for nucleic acids by, for example, the algorithms disclosed in Zuker, M. Science 244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger et al. Methods Enzymol. 183:281 -306, 1989, which are herein incorporated by reference for at least material related to nucleic acid alignment.

[0329] There are a number of compositions and methods which can be used to deliver nucleic acids to cells, either in vitro or in vivo. These methods and compositions can largely be broken down into two classes: viral-based delivery systems and non- viral-based delivery systems. For example, the nucleic acids can be delivered through a number of direct delivery systems such as electroporation, lipofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or via transfer of genetic material in cells or carriers such as cationic liposomes. Appropriate means for transfection, including viral vectors, chemical transfectants, or physico-mechanical methods such as electroporation and direct diffusion of DNA, are described by, for example, Wolff, J. A., et al., Science, 247, 1465- 1468, (1990); and Wolff, J. A. Nature, 352, 815-818, (1991). Such methods are well-known in the art and readily adaptable for use with the compositions and methods described herein. In certain cases, the methods can be modified to specifically function with large DNA molecules. Further, these methods can be used to target certain diseases and cell populations by using the targeting characteristics of the carrier.

[0330] Transfer vectors can be any nucleotide construction used to deliver genes into cells (e.g., a plasmid) or as part of a general strategy to deliver genes, e.g., as part of recombinant retrovirus or adenovirus (Ram et al. Cancer Res. 53:83-88, (1993)).

[0331] As used herein, plasmid or viral vectors are agents that transport the disclosed nucleic acids, such as a chimeric antigen receptor, into the cell without degradation and include a promoter yielding expression of the gene in the cells into which it is delivered. Viral vectors are, for example, Adenovirus, Adeno-associated virus, Herpes virus, Vaccinia virus, Polio virus, neuronal trophic virus, lentiviruses, Sindbis and other RNA viruses, including these viruses with the HIV backbone. Also preferred are any viral families which share the properties of these viruses which make them suitable for use as vectors. Retroviruses include Murine Maloney Leukemia virus, MMLV, and retroviruses that express the desirable properties of MMLV as a vector. Retroviral vectors are able to carry a larger genetic payload, i.e., a transgene or marker gene, than other viral vectors, and for this reason are a commonly used vector. However, they are not as useful in non-proliferating cells. Adenovirus vectors are relatively stable and easy to work with, have high titers, and can be delivered in aerosol formulation, and can transfect non-dividing cells. Pox viral vectors are large and have several sites for inserting genes, they are thermostable and can be stored at room temperature. A preferred aspect is a viral vector which has been engineered so as to suppress the immune response of the host organism, elicited by the viral antigens. Preferred vectors of this type can carry coding regions for Interleukin 8 or 10.

[0332] Viral vectors can have higher transaction (ability to introduce genes) abilities than chemical or physical methods to introduce genes into cells. Typically, viral vectors contain, nonstructural early genes, structural late genes, an RNA polymerase III transcript, inverted terminal repeats necessary for replication and encapsidation, and promoters to control the transcription and replication of the viral genome. When engineered as vectors, viruses typically have one or more of the early genes removed and a gene or gene / promotor cassette is inserted into the viral genome in place of the removed viral DNA. Constructs of this type can carry up to about 8 kb of foreign genetic material. The necessary functions of the removed early genes are typically supplied by cell lines which have been engineered to express the gene products of the early genes in trans.

[0333] A retrovirus is an animal virus belonging to the virus family of Retroviridae, including any types, subfamilies, genus, or tropisms, including, but not limited to lentiviruses (including HIV based lentiviral vectors and gammaretroviral vectors). Retroviral vectors, in general, are described by Verma, I.M., Retroviral vectors for gene transfer.

[0334] A retrovirus is essentially a package which has packed into it nucleic acid cargo. The nucleic acid cargo carries with it a packaging signal, which ensures that the replicated daughter molecules will be efficiently packaged within the package coat. In addition to the package signal, there are a number of molecules which are needed in cis, for the replication, and packaging of the replicated virus. Typically, a retroviral genome, contains the gag, pol, and env genes which are involved in the making of the protein coat. It is the gag, pol, and env genes which are typically replaced by the foreign DNA that it is to be transferred to the target cell. Retrovirus vectors typically contain a packaging signal for incorporation into the package coat, a sequence which signals the start of the gag transcription unit, elements necessary for reverse transcription, including a primer binding site to bind the tRNA primer of reverse transcription, terminal repeat sequences that guide the switch of RNA strands during DNA synthesis, a purine rich sequence 5' to the 3' LTR that serve as the priming site for the synthesis of the second strand of DNA synthesis, and specific sequences near the ends of the LTRs that enable the insertion of the DNA state of the retrovirus to insert into the host genome. The removal of the gag, pol, and env genes allows for about 8 kb of foreign sequence to be inserted into the viral genome, become reverse transcribed, and upon replication be packaged into a new retroviral particle. This amount of nucleic acid is sufficient for the delivery of a one to many genes depending on the size of each transcript. It is preferable to include either positive or negative selectable markers along with other genes in the insert.

[0335] Since the replication machinery and packaging proteins in most retroviral vectors have been removed (gag, pol, and env), the vectors are typically generated by placing them into a packaging cell line. A packaging cell line is a cell line which has been transfected or transformed with a retrovirus that contains the replication and packaging machinery, but lacks any packaging signal. When the vector carrying the DNA of choice is transfected into these cell lines, the vector containing the gene of interest is replicated and packaged into new retroviral particles, by the machinery provided in cis by the helper cell. The genomes for the machinery are not packaged because they lack the necessary signals.

[0336] The construction of replication-defective adenoviruses has been described (Berkner et al., J. Virology 61 :1213-1220 (1987); Massie et al., Mol. Cell. Biol. 6:2872-2883 (1986); Haj- Ahmad et al., J. Virology 57:267-274 (1986); Davidson et al., J. Virology 61 :1226-1239 (1987); Zhang "Generation and identification of recombinant adenovirus by liposome-mediated transfection and PCR analysis" BioTechniques 15:868-872 (1993)). The benefit of the use of these viruses as vectors is that they are limited in the extent to which they can spread to other cell types, since they can replicate within an initial infected cell, but are unable to form new infectious viral particles. Recombinant adenoviruses have been shown to achieve high efficiency gene transfer after direct, in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma and a number of other tissue sites (Morsy, J. Clin. Invest. 92: 1580-1586 (1993); Kirshenbaum, J. Clin. Invest. 92:381-387 (1993); Roessler, J. Clin. Invest. 92: 1085-1092 (1993); Moullier, Nature Genetics 4:154-159 (1993); La Salle, Science 259:988-990 (1993); Gomez-Foix, J. Biol. Chem. 267:25129-25134 (1992); Rich, Human Gene Therapy 4:461-476 (1993); Zabner, Nature Genetics 6:75-83 (1994); Guzman, Circulation Research 73:1201-1207 (1993); Bout, Human Gene Therapy 5:3-10 (1994); Zabner, Cell 75:207-216 (1993); Caillaud, Eur. J. Neuroscience 5: 1287-1291 (1993); and Ragot, J. Gen. Virology 74:501-507 (1993)). Recombinant adenoviruses achieve gene transduction by binding to specific cell surface receptors, after which the virus is internalized by receptor-mediated endocytosis, in the same manner as wild type or replication-defective adenovirus (Chardonnet and Dales, Virology 40:462-477 (1970); Brown and Burlingham, J. Virology 12:386-396 (1973); Svensson and Persson, J. Virology 55:442-449 (1985); Seth, et al., J. Virol. 51:650-655 (1984); Seth, et al., Mol. Cell. Biol. 4:1528-1533 (1984); Varga et al., J. Virology 65:6061-6070 (1991); Wickham et al., Cell 73:309-319 (1993)).

[0337] A viral vector can be one based on an adenovirus which has had the El gene removed and these virons are generated in a cell line such as the human 293 cell line. In another preferred aspect both the El and E3 genes are removed from the adenovirus genome.

[0338] Another type of viral vector is based on an adeno-associated virus (AAV). This defective parvovirus is a preferred vector because it can infect many cell types and is nonpathogenic to humans. AAV type vectors can transport about 4 to 5 kb and wild type AAV is known to stably insert into chromosome 19. Vectors which contain this site specific integration property are preferred. An especially preferred aspect of this type of vector is the P4.1 C vector produced by Avigen, San Francisco, CA, which can contain the herpes simplex virus thymidine kinase gene, HSV-tk, and / or a marker gene, such as the gene encoding the green fluorescent protein, GFP.

[0339] In another type of AAV virus, the AAV contains a pair of inverted terminal repeats (ITRs) which flank at least one cassette containing a promoter which directs cell-specific expression operably linked to a heterologous gene. Heterologous in this context refers to any nucleotide sequence or gene which is not native to the AAV or B19 parvovirus.

[0340] Typically, the AAV and B 19 coding regions have been deleted, resulting in a safe, noncytotoxic vector. The AAV ITRs, or modifications thereof, confer infectivity and sitespecific integration, but not cytotoxicity, and the promoter directs cell-specific expression. United states Patent No. 6,261,834 is herein incorporated by reference for material related to the AAV vector.

[0341] The disclosed vectors thus provide DNA molecules which are capable of integration into a mammalian chromosome without substantial toxicity.

[0342] The inserted genes in viral and retroviral usually contain promoters, and / or enhancers to help control the expression of the desired gene product. A promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard to the transcription start site. A promoter contains core elements required for basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and response elements.

[0343] Molecular genetic experiments with large human herpesviruses have provided a means whereby large heterologous DNA fragments can be cloned, propagated and established in cells permissive for infection with herpesviruses (Sun et al., Nature genetics 8: 33-41 , 1994; Cotter and Robertson, .Curr Opin Mol Ther 5: 633-644, 1999). These large DNA viruses (herpes simplex virus (HSV) and Epstein-Barr virus (EBV), have the potential to deliver fragments of human heterologous DNA > 150 kb to specific cells. EBV recombinants can maintain large pieces of DNA in the infected B-cells as episomal DNA. Individual clones carried human genomic inserts up to 330 kb appeared genetically stable The maintenance of these episomes requires a specific EBV nuclear protein, EBNA1, constitutively expressed during infection with EBV. Additionally, these vectors can be used for transfection, where large amounts of protein can be generated transiently in vitro. Herpesvirus amplicon systems are also being used to package pieces of DNA > 220 kb and to infect cells that can stably maintain DNA as episomes.

[0344] Other useful systems include, for example, replicating and host-restricted nonreplicating vaccinia virus vectors.

[0345] The disclosed compositions can be delivered to the target cells in a variety of ways. For example, the compositions can be delivered through electroporation, or through lipofection, or through calcium phosphate precipitation. The delivery mechanism chosen will depend in part on the type of cell targeted and whether the delivery is occurring for example in vivo or in vitro.

[0346] Thus, the compositions can comprise vectors for example, lipids such as liposomes, such as cationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionic liposomes. Liposomes can further comprise proteins to facilitate targeting a particular cell, if desired. Administration of a composition comprising a compound and a cationic liposome can be administered to the blood afferent to a target organ or inhaled into the respiratory tract to target cells of the respiratory tract. Regarding liposomes, see, e.g., Brigham et al. Am. J. Resp. Cell. Mol. Biol. 1:95-100 (1989); Feigner et al. Proc. Natl. Acad. Sci USA 84:7413-7417 (1987); U.S. Pat. No.4,897,355. Furthermore, the compound can be administered as a component of a microcapsule that can be targeted to specific cell types, such as macrophages, or where the diffusion of the compound or delivery of the compound from the microcapsule is designed for a specific rate or dosage.

[0347] In the methods described above which include the administration and uptake of exogenous DNA into the cells of a subject (i.e., gene transduction or transfection), delivery of the compositions to cells can be via a variety of mechanisms. As one example, delivery can be via a liposome, using commercially available liposome preparations such as LIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, MD), SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison, WI), as well as other liposomes developed according to procedures standard in the art. In addition, the disclosed nucleic acid or vector can be delivered in vivo by electroporation, the technology for which is available from Genetronics, Inc. (San Diego, CA) as well as by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, AZ).

[0348] The materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol, 42:2062-2065, (1991)). These techniques can be used for a variety of other specific cell types. Vehicles such as "stealth" and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

[0349] Nucleic acids that are delivered to cells which are to be integrated into the host cell genome, typically contain integration sequences. These sequences are often viral related sequences, particularly when viral based systems are used. These viral integration systems can also be incorporated into nucleic acids which are to be delivered using a non-nucleic acid based system of deliver, such as a liposome, so that the nucleic acid contained in the delivery system can be come integrated into the host genome.

[0350] Other general techniques for integration into the host genome include, for example, systems designed to promote homologous recombination with the host genome. These systems typically rely on sequence flanking the nucleic acid to be expressed that has enough homology with a target sequence within the host cell genome that recombination between the vector nucleic acid and the target nucleic acid takes place, causing the delivered nucleic acid to be integrated into the host genome. These systems and the methods necessary to promote homologous recombination are known to those of skill in the art.

[0351] As described above, the compositions can be administered in a pharmaceutically acceptable carrier and can be delivered to the subject=s cells in vivo and / or ex vivo by a variety of mechanisms well known in the art (e.g., uptake of naked DNA, liposome fusion, intramuscular injection of DNA via a gene gun, endocytosis and the like).

[0352] If ex vivo methods are employed, cells or tissues can be removed and maintained outside the body according to standard protocols well known in the art. The compositions can be introduced into the cells via any gene transfer mechanism, such as, for example, calcium phosphate mediated gene delivery, electroporation, microinjection or proteoliposomes. The transduced cells can then be infused (e.g., in a pharmaceutically acceptable carrier) or homotopically transplanted back into the subject per standard methods for the cell or tissue type. Standard methods are known for transplantation or infusion of various cells into a subject.

[0353] The nucleic acids that are delivered to cells typically contain expression controlling systems. For example, the inserted genes in viral and retroviral systems usually contain promoters, and / or enhancers to help control the expression of the desired gene product. A promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard to the transcription start site. A promoter contains core elements required for basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and response elements.

[0354] Preferred promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters, e.g. beta actin promoter. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication (Fiers et al., Nature, 273: 113 (1978)). The immediate early promoter of the human cytomegalovirus is conveniently obtained as a / fzndlll E restriction fragment (Greenway, P.J. et al., Gene 18: 355-360 (1982)). Of course, promoters from the host cell or related species also are useful herein.

[0355] Enhancer generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5' (Laimins, L. et al., Proc. Natl. Acad. Sci. 78: 993 (1981)) or 3' (Lusky, M.L., et al., Mol. Cell Bio. 3: 1108 (1983)) to the transcription unit. Furthermore, enhancers can be within an intron (Banerji, J.L. et al., Cell 33: 729 (1983)) as well as within the coding sequence itself (Osborne, T.F., et al., Mol. Cell Bio. 4: 1293 (1984)). They are usually between 10 and 300 bp in length, and they function in cis. Enhancers f unction to increase transcription from nearby promoters. Enhancers also often contain response elements that mediate the regulation of transcription. Promoters can also contain response elements that mediate the regulation of transcription. Enhancers often determine the regulation of expression of a gene. While many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, -fetoprotein and insulin), typically one will use an enhancer from a eukaryotic cell virus for general expression. Preferred examples are the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0356] The promotor and / or enhancer may be specifically activated either by light or specific chemical events which trigger their function. Systems can be regulated by reagents such as tetracycline and dexamethasone. There are also ways to enhance viral vector gene expression by exposure to irradiation, such as gamma irradiation, or alkylating chemotherapy drugs. In certain aspects the promoter and / or enhancer region can act as a constitutive promoter and / or enhancer to maximize expression of the region of the transcription unit to be transcribed. In certain constructs the promoter and / or enhancer region be active in all eukaryotic cell types, even if it is only expressed in a particular type of cell at a particular time. A preferred promoter of this type is the CMV promoter (650 bases). Other preferred promoters are SV40 promoters, cytomegalovirus (full length promoter), and retroviral vector LTR.

[0357] It has been shown that all specific regulatory elements can be cloned and used to construct expression vectors that are selectively expressed in specific cell types such as melanoma cells. The glial fibrillary acetic protein (GFAP) promoter has been used to selectively express genes in cells of glial origin.

[0358] Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human or nucleated cells) may also contain sequences necessary for the termination of transcription which may affect mRNA expression. These regions are transcribed as polyadenylated segments in the untranslated portion of the mRNA encoding tissue factor protein. The 3' untranslated regions also include transcription termination sites. It is preferred that the transcription unit also contains a polyadenylation region. One benefit of this region is that it increases the likelihood that the transcribed unit will be processed and transported like mRNA. The identification and use of polyadenylation signals in expression constructs is well established. It is preferred that homologous polyadenylation signals be used in the transgene constructs. In certain transcription units, the polyadenylation region is derived from the SV40 early polyadenylation signal and consists of about 400 bases. It is also preferred that the transcribed units contain other standard sequences alone or in combination with the above sequences improve expression from, or stability of, the construct.

[0359] The viral vectors can include nucleic acid sequence encoding a marker product. This marker product is used to determine if the gene has been delivered to the cell and once delivered is being expressed. Preferred marker genes are the E. Coli lacZ gene, which encodes B-galactosidase, and green fluorescent protein.

[0360] In some aspects the marker may be a selectable marker. Examples of suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hydromycin, and puromycin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure. There are two widely used distinct categories of selective regimes. The first category is based on a cell's metabolism and the use of a mutant cell line which lacks the ability to grow independent of a supplemented media. Two examples are: CHO DHFR- cells and mouse LTK- cells. These cells lack the ability to grow without the addition of such nutrients as thymidine or hypoxanthine. Because these cells lack certain genes necessary for a complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented media. An alternative to supplementing the media is to introduce an intact DHFR or TK gene into cells lacking the respective genes, thus altering their growth requirements. Individual cells which were not transformed with the DHFR or TK gene will not be capable of survival in non-supplemented media.

[0361] The second category is dominant selection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell. Those cells which have a novel gene would express a protein conveying drag resistance and would survive the selection. Examples of such dominant selection use the drugs neomycin, (Southern P. and Berg, P., J. Molec. Appl. Genet. 1: 327 (1982)), mycophenolic acid, (Mulligan, R.C. and Berg, P. Science 209: 1422 (1980)) or hygromycin, (Sugden, B. et al., Mol. Cell. Biol. 5: 410-413 (1985)). The three examples employ bacterial genes under eukaryotic control to convey resistance to the appropriate drag G418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively. Others include the neomycin analog G418 and puramycin.

[0362] Methods of Treatment

[0363] The present disclosure also provides methods for treating or preventing medical disorders in a subject, comprising administering to the subject a therapeutically effective amount of a composition disclosed herein. Representative examples of such medical disorders include, but are not limited to, cancers, autoimmune diseases, or viral infections. The methods can further comprise administering one or more additional therapeutic agents, for example anti- cancer agents or anti-inflammatory agents. Additionally, the method can further comprise administering a therapeutically effective amount of ionizing radiation to the subject.

[0364] Methods of killing a cell, for example a cancer or tumor cell, or an immune cell, are also provided comprising contacting the cell with an effective amount of a compound or composition as described herein. The methods can further include administering one or more additional therapeutic agents or administering an effective amount of ionizing radiation, for example, if the medical disorder comprises a cancer.

[0365] The disclosed methods can optionally include identifying a patient who is or can be in need of treatment of a medical disorder. The patient can be a human or other mammal, such as a primate (monkey, chimpanzee, ape, etc.), dog, cat, cow pig, or horse, or other animals having an oncological disorder. In some aspects, the subject can receive the therapeutic compositions prior to, during, or after surgical intervention, for example, to remove part or all of a tumor.

[0366] Compositions disclosed herein can be locally administered at one or more anatomical sites, such as sites of unwanted cell growth (such as a tumor site or benign skin growth, e.g., injected or topically applied to the tumor or skin growth), optionally in combination with a pharmaceutically acceptable carrier such as an inert diluent. Compounds and compositions disclosed herein can also be systemically administered, such as intravenously or orally, optionally in combination with a pharmaceutically acceptable carrier such as an inert diluent, or an assimilable edible carrier for oral delivery. In addition, the active compound can be incorporated into sustained release preparations and / or devices.

[0367] For the treatment of oncological disorder, compounds, agents, and compositions disclosed herein can be administered to a patient in need of treatment prior to, subsequent to, or in combination with other antitumor or anticancer agents or substances (e.g., chemotherapeutic agents, immunotherapeutic agents, radiotherapeutic agents, cytotoxic agents, etc.) and / or with radiation therapy and / or with surgical treatment to remove a tumor. For example, compounds, agents, and compositions disclosed herein can be used in methods of treating cancer wherein the patient is to be treated or is or has been treated with mitotic inhibitors such as taxol or vinblastine, alkylating agents such as cyclophosphamide or ifosfamide, antimetabolites such as 5-fluorouracil or hydroxyurea, DNA intercalators such as adriamycin or bleomycin, topoisomerase inhibitors such as etoposide or camptothecin, antiangiogenic agents such as angiostatin, antiestrogens such as tamoxifen, and / or other anti- cancer drugs or antibodies, such as, for example, imatinid or trastuzumab. These other substances or radiation treatments can be given at the same time as or at different times from the compounds disclosed herein. Examples of other suitable chemotherapeutic agents include, but are not limited to, altretamine, bleomycin, bortezomib, busulphan, calcium folinate, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gefitinib, gemcitabine, hydroxyurea, idarubicin, ifosfamide, imatinib, irinotecan, liposomal doxorubicin, lomustine, melphalan, mercaptopurine, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pentostatin, procarbazine, raltitrexed, streptozocin, tegafur-uraxil, temozolomide, thiotepa, tioguanine / thioguanine, topotexan, treosulfan, vinblastine, vincristine, vindesine, and vinorelbine. Examples of suitable immunotherapeutic agents include, but are not limited to, alemtuzumab, cetuximab, gemtuzumab, iodine 131 tositumomab, rituximab, and trastuzumab. Cytotoxic agents include, for example, radioactive isotopes and toxins of bacterial, fungal, plant, or animal origin. Also disclosed are methods of treating an oncological disorder comprising administering an effective amount of a compound described herein prior to, subsequent to, and / or in combination with administration of a chemotherapeutic agent, an immunotherapeutic agent, a radiotherapeutic agent, or radiotherapy.

[0368] In one aspect, a method of treating a medical disorder in a subject in need thereof is provided comprising administering to the subject a cell comprising a CAR system described herein. In some aspects, the medical disorder comprises a cancer.

[0369] In another aspect, a method of treating a medical disorder in a subject in need thereof is provided comprising: a) providing an adaptor molecule described herein; b) administering to the subject a cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises a receptor that target T on the adaptor molecule, a hinge domain, and a signaling domain; and c) administering the adaptor molecule to the subject. In some aspects, the medical disorder comprises a cancer.

[0370] In some aspects, the method further comprises administering a stimulus reactive blocking group that blocks the CAR from binding to T on the adaptor molecule. In some aspects, the stimulus reactive blocking group is reactive to a stimulus. Representative examples of such stimuli include, but are not limited to, a light (such as, for example, 365, 405, 544, or 780 nm), an enzyme (such as, for example, legumain, matrix metalloproteinase, pyridoxal kinase (PDXK), aldehyde dehydrogenase 7 family, member Al , (ALDH7A1), lipase C, hepatic type (LIPC), poly(ADP-ribose) polymerase 1 (PARP1), pyruvate kinase M2 (PKM2), phosphoglycerate kinase 1 (PGK1), ketohexokinase-A (KHK-A), hexokinases (HK), nucleoside diphosphate kinase (NDPK or NDK), and 6-phosphofructo-2-kinase / fructose-2,6- biphosphatase 4 (PFKFB4), mitochondrial a-ketoglutarate dehydrogenase (a-KGDH), lysine acetyltransferase 2A (KAT2A), acetyl-CoA synthetase short-chain family member 2 (ACSS2), ATP-citrate lyase (ACLY), pyruvate dehydrogenase complex (PDC), a-ketoglutarate dehydrogenase (a- KGDH), CD39, CD73, or fumarase), a small molecule (such as, for example, phosphine or tetrazine), pH, hypoxia, H2O2, and / or ROS. In some aspects, the stimulus reactive blocking group ceases to block the CAR from binding to T on the adaptor molecule upon reaction with the stimulus.

[0371] In some aspects, the adaptor molecule is provided as an oral dosage form and the cell is administered as a parenteral dosage form.

[0372] The term “neoplasia” or “cancer” is used throughout this disclosure to refer to the pathological process that results in the formation and growth of a cancerous or malignant neoplasm, i.e., abnormal tissue (solid) or cells (non-solid) that grow by cellular proliferation, often more rapidly than normal and continues to grow after the stimuli that initiated the new growth cease. Malignant neoplasms show partial or complete lack of structural organization and functional coordination with the normal tissue and most invade surrounding tissues, can metastasize to several sites, are likely to recur after attempted removal and may cause the death of the patient unless adequately treated. As used herein, the term neoplasia is used to describe all cancerous disease states and embraces or encompasses the pathological process associated with malignant, hematogenous, ascitic and solid tumors. The cancers which may be treated by the compositions disclosed herein may comprise carcinomas, sarcomas, lymphomas, leukemias, germ cell tumors, or blastomas.

[0373] Carcinomas which may be treated by the compositions of the present disclosure include, but are not limited to, acinar carcinoma, acinous carcinoma, alveolar adenocarcinoma, carcinoma adenomatosum, adenocarcinoma, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellular, basaloid carcinoma, basosquamous cell carcinoma, breast carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedocarcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epibulbar carcinoma, epidermoid carcinoma, carcinoma epitheliate adenoids, carcinoma exulcere, carcinoma fibrosum, gelatinform carcinoma, gelatinous carcinoma, giant cell carcinoma, gigantocellulare, glandular carcinoma, granulose cell carcinoma, hair matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher’s carcinoma, Kulchitzky-cell carcinoma, lentivular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma mastotoids, carcinoma medullare, medullary carcinoma, carcinoma melanodes, melanotonic carcinoma, mucinous carcinoma, carcinoma muciparum, carcinoma mucocullare, mucoepidermoid carcinoma, mucous carcinoma, carcinoma myxomatodes, masopharyngeal carcinoma, carcinoma nigrum, oat cell carcinoma, carcinoma ossificans, osteroid carcinoma, ovarian carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prostate carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, scheinderian carcinoma, scirrhous carcinoma, carcinoma scrota, signet-ring cell carcinoma, carcinoma simplex, small cell carcinoma, solandoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberrosum, tuberous carcinoma, verrucous carcinoma, and carcinoma vilosum.

[0374] Representative sarcomas which may be treated by the compositions of the present disclosure include, but are not limited to, liposarcomas (including myxoid liposarcomas and pleomorphic liposarcomas), leiomyosarcomas, rhabdomyosarcomas, neurofibrosarcomas, malignant peripheral nerve sheath tumors, Ewing's tumors (including Ewing's sarcoma of bone, extraskeletal or non-bone) and primitive neuroectodermal tumors (PNET), synovial sarcoma, hemangioendothelioma, fibrosarcoma, desmoids tumors, dermatofibrosarcoma protuberance (DFSP), malignant fibrous histiocytoma(MFH), hemangiopericytoma, malignant mesenchymoma, alveolar soft-part sarcoma, epithelioid sarcoma, clear cell sarcoma, desmoplastic small cell tumor, gastrointestinal stromal tumor (GIST) and osteosarcoma (also known as osteogenic sarcoma) skeletal and extra-skeletal, and chondrosarcoma.

[0375] The compositions of the present disclosure may be used in the treatment of a lymphoma. Lymphomas which may be treated include mature B cell neoplasms, mature T cell and natural killer (NK) cell neoplasms, precursor lymphoid neoplasms, Hodgkin lymphomas, and immunodeficiency-associated lymphoproliferative disorders. Representative mature B cell neoplasms include, but are not limited to, B-cell chronic lymphocytic leukemia / small cell lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma (such as Waldenstrom macroglobulinemia), splenic marginal zone lymphoma, hairy cell leukemia, plasma cell neoplasms (such as plasma cell myeloma / multiple myeloma, plasmacytoma, monoclonal immunoglobulin deposition diseases, and heavy chain diseases), extranodal marginal zone B cell lymphoma (MALT lymphoma), nodal marginal zone B cell lymphoma, follicular lymphoma, primary cutaneous follicular center lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, diffuse large B-cell lymphoma associated with chronic inflammation, Epstein-Barr virus-positive DLBCL of the elderly, lyphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK+ large B-cell lymphoma, plasmablastic lymphoma, primary effusion lymphoma, large B- cell lymphoma arising in HHV8- associated multicentric Castleman’s disease, and Burkitt lymphoma / leukemia. Representative mature T cell and NK cell neoplasms include, but are not limited to, T-cell prolymphocytic leukemia, T-cell large granular lymphocyte leukemia, aggressive NK cell leukemia, adult T-cell leukemia / lymphoma, extranodal NK / T-cell lymphoma, nasal type, enteropathy-associated T-cell lymphoma, hepatosplenic T-cell lymphoma, blastic NK cell lymphoma, lycosis fungoides / Sezary syndrome, primary cutaneous CD30-positive T cell lymphoproliferative disorders (such as primary cutaneous anaplastic large cell lymphoma and lymphomatoid papulosis), peripheral T-cell lymphoma not otherwise specified, angioimmunoblastic T cell lymphoma, and anaplastic large cell lymphoma. Representative precursor lymphoid neoplasms include B-lymphoblastic leukemia / lymphoma not otherwise specified, B-lymphoblastic leukemia / lymphoma with recurrent genetic abnormalities, or T-lymphoblastic leukemia / lymphoma. Representative Hodgkin lymphomas include classical Hodgkin lymphomas, mixed cellularity Hodgkin lymphoma, lymphocyte-rich Hodgkin lymphoma, and nodular lymphocyte-predominant Hodgkin lymphoma.

[0376] The compositions of the present disclosure may be used in the treatment of a Leukemia. Representative examples of leukemias include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), hairy cell leukemia (HCL), T-cell prolymphocytic leukemia, adult T-cell leukemia, clonal eosinophilias, and transient myeloproliferative disease.

[0377] The compositions of the present disclosure may be used in the treatment of a germ cell tumor, for example germinomatous (such as germinoma, dysgerminoma, and seminoma), non germinomatous (such as embryonal carcinoma, endodermal sinus tumor, choriocarcinoma, teratoma, polyembryoma, and gonadoblastoma) and mixed tumors.

[0378] The compositions of the present disclosure may be used in the treatment of blastomas, for example hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma, retinoblastoma, and glioblastoma multiforme. Representative cancers which may be treated include, but are not limited to: bone and muscle sarcomas such as chondrosarcoma, Ewing’s sarcoma, malignant fibrous histiocytoma of bone / osteosarcoma, osteosarcoma, rhabdomyosarcoma, and heart cancer; brain and nervous system cancers such as astrocytoma, brainstem glioma, pilocytic astrocytoma, ependymoma, primitive neuroectodermal tumor, cerebellar astrocytoma, cerebral astrocytoma, glioma, medulloblastoma, neuroblastoma, oligodendroglioma, pineal astrocytoma, pituitary adenoma, and visual pathway and hypothalamic glioma; breast cancers including invasive lobular carcinoma, tubular carcinoma, invasive cribriform carcinoma, medullary carcinoma, male breast cancer, Phyllodes tumor, and inflammatory breast cancer; endocrine system cancers such as adrenocortical carcinoma, islet cell carcinoma, multiple endocrine neoplasia syndrome, parathyroid cancer, phemochromocytoma, thyroid cancer, and Merkel cell carcinoma; eye cancers including uveal melanoma and retinoblastoma; gastrointestinal cancers such as anal cancer, appendix cancer, cholangiocarcinoma, gastrointestinal carcinoid tumors, colon cancer, extrahepatic bile duct cancer, gallbladder cancer, gastric cancer, gastrointestinal stromal tumor, hepatocellular cancer, pancreatic cancer, and rectal cancer; genitourinary and gynecologic cancers such as bladder cancer, cervical cancer, endometrial cancer, extragonadal germ cell tumor, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, penile cancer, renal cell carcinoma, renal pelvis and ureter transitional cell cancer, prostate cancer, testicular cancer, gestational trophoblastic tumor, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Wilms tumor; head and neck cancers such as esophageal cancer, head and neck cancer, nasopharyngeal carcinoma, oral cancer, oropharyngeal cancer, paranasal sinus and nasal cavity cancer, pharyngeal cancer, salivary gland cancer, and hypopharyngeal cancer; hematopoietic cancers such as acute biphenotypic leukemia, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid dendritic cell leukemia, AIDS-related lymphoma, anaplastic large cell lymphoma, angioimmunoblastic T-cell lymphoma, B-cell prolymphocytic leukemia, Burkitt’s lymphoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, cutaneous T-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, hepatosplenic T-cell lymphoma, Hodgkin’s lymphoma, hairy cell leukemia, intravascular large B-cell lymphoma, large granular lymphocytic leukemia, lymphoplasmacytic lymphoma, lymphomatoid granulomatosis, mantle cell lymphoma, marginal zone B-cell lymphoma, Mast cell leukemia, mediastinal large B cell lymphoma, multiple myeloma / plasma cell neoplasm, myelodysplastic syndroms, mucosa- associated lymphoid tissue lymphoma, mycosis fungoides, nodal marginal zone B cell lymphoma, non-Hodgkin lymphoma, precursor B lymphoblastic leukemia, primary central nervous system lymphoma, primary cutaneous follicular lymphoma, primary cutaneous immunocytoma, primary effusion lymphoma, plasmablastic lymphoma, Sezary syndrome, splenic marginal zone lymphoma, and T-cell prolymphocytic leukemia; skin cancers such as basal cell carcinoma, squamous cell carcinoma, skin adnexal tumors (such as sebaceous carcinoma), melanoma, Merkel cell carcinoma, sarcomas of primary cutaneous origin (such as dermatofibrosarcoma protuberans), and lymphomas of primary cutaneous origin (such as mycosis fungoides); thoracic and respiratory cancers such as bronchial adenomas / carcinoids, small cell lung cancer, mesothelioma, non-small cell lung cancer, pleuropulmonary blastoma, laryngeal cancer, and thymoma or thymic carcinoma; HIV / AIDs-related cancers such as Kaposi sarcoma; epithelioid hemangioendothelioma; desmoplastic small round cell tumor; and liposarcoma.

[0379] Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms of the disorder are affected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.

[0380] The following aspects of the present disclosure are also provided:

[0381] Aspect Al. An adaptor molecule for adoptive cell therapy of Formula I:

[0382] T-L-A (I) or a pharmaceutically acceptable salt thereof; wherein:

[0383] T comprises a tag ligand;

[0384] L comprises a linker moiety; and A comprises a moiety capable of binding a cell surface protein selected from a folate receptor, a carbonic anhydrase, prostate-specific antigen, and programmed death-ligand 1 (PD- Ll).

[0385] Aspect A2. The adaptor molecule of aspect Al, wherein T comprises a ligand for SNAP- tag, CLIP-tag, Halo-tag, SpyTag, SnoopTag, or Isopep-tag.

[0386] Aspect A3. The adaptor molecule of aspect Al or aspect A2, wherein T comprises a benzylguanine, chloropyrimidine, benzylcytosine, or chloroalkane moiety.

[0387] Aspect A4. The adaptor molecule of any one of aspects A1-A3, wherein A comprises a moiety capable of binding a folate receptor. Aspect A5. The adaptor molecule of aspect A4, wherein A comprises a moiety derived from a ligand selected from:

[0388] COOH

[0389] COOH

[0390] wherein the point of attachment of L is provided by replacement of a hydrogen or other suitable moiety in the above structures with a covalent bond to L.

[0391] Aspect A6. The adaptor molecule of aspect A4, wherein A comprises

[0392] Aspect A7. The adaptor molecule of any one of aspects A1-A3, wherein A comprises a moiety capable of binding a carbonic anhydrase.

[0393] Aspect A8. The adaptor molecule of aspect A7, wherein A comprises a moiety derived from a ligand selected from:

[0394] wherein the point to attachment of L is provided by replacement of a hydrogen or other suitable moiety in the above structures with a covalent bond to L. Aspect A9. The adaptor molecule of aspect A7, wherein A comprises

[0395] Aspect A10. The adaptor molecule of any one of aspects A1-A3, wherein A comprises a moiety capable of binding prostate- specific antigen.

[0396] Aspect Al l. The adaptor molecule of aspect A 10, wherein A comprises a moiety derived from a ligand selected from:

[0397] CO2H wherein the point of attachment to L is provided by replacement of a hydrogen or other suitable moiety in the above structures with a covalent bond to L.

[0398] Aspect A12. The adaptor molecule of aspect A10, wherein A comprises

[0399] Aspect A 13. The adaptor molecule of any one of aspects A1-A3, wherein A comprises a moiety capable of binding PD-L1. Aspect A 14. The adaptor molecule of aspect A 13, wherein A comprises a moiety derived from a ligand selected from:

[0400] CO2H

[0401] F

[0402] F

[0403]

[0404] wherein the point of attachment to L is provided by replacement of a hydrogen or other suitable moiety in the above structures with a covalent bond to L.

[0405] Aspect Al 5. The adaptor molecule of aspect A13, wherein

[0406] A comprises

[0407] Aspect A16. The adaptor molecule of any one of aspects A1-A15, wherein L is selected from

[0408] LI : wherein:

[0409] X101and X102are independently at each occurrence selected from a bond, aryl, heteroaryl, cycloalkyl, heterocycle, NR130, C(R130)2, O, C(O), and S;

[0410] Rioo, R101, R102, R103, and R104are independently at each occurrence selected from the group consisting of a bond, alkyl, -C(O)-, -C(O)O-, -OC(O)-, -SO2-, -S(O)-, C(S)-, -C(O)NR130-, -NR130C(O)-, -O-, -S-, -NR130-, -C(R130R130)-, -P(O)(OR106))-, -R(O)(OR106)-, alkenyl, alkynyl, haloalkyl, alkoxy, aryl, heterocycloalkyl, cycloalkyl, heteroaryl, lactic acid, or glycolic acid, each of which may be optionally substituted with one or more (for example, 1, 2, 3, or 4) substituents independently selected from R140;

[0411] R106is independently at each occurrence selected from the group consisting of hydrogen, alkyl, arylalkyl, heteroarylalkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl;

[0412] R130is independently as each occurrence selected from the group consisting of hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, -C(O)H, -C(O)OH, -C(O)alkyl, -C(O)Oalkyl, -C(O)(cycloalkyl, heterocycloalkyl, aryl, or heteroaryl), -C(O)O(cycloalkyl, heterocycloalkyl, aryl, or heteroaryl), alkenyl, or alkynyl; and R140is independently at each occurrence selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, fluoro, bromo, chloro, hydroxyl, alkoxy, azide, amino cyano, -NH(alkyl, cycloalkyl, heterocyloalkyl, aryl, or heteroaryl), -N(independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl), -NHSCMalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl), -N(alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl)SO2alkyl, -NHSOialkenyl, -N(alkyl)SO2alkenyl, -NHSChalkynyl, -N(alkyl)SO2alkynyl, haloalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

[0413] Aspect A17. The adaptor molecule of aspect Al selected from:

[0414] or a pharmaceutically acceptable salt thereof, wherein n is an integer selected from 0 to 12 (for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12).

[0415] Aspect Al 8. A pharmaceutical composition comprising an adaptor molecule of any one of aspects A1-A17, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient or carrier.

[0416] Aspect A19. A chimeric antigen receptor (CAR) system, comprising i) an adaptor molecule of any one of aspects A1-A17, and ii) a CAR comprising a receptor that is capable of binding to T on the adaptor molecule, a hinge domain, and a signaling domain. Aspect A20. A cell comprising the CAR system of aspect A19. Aspect A21. The cell of aspect A20, wherein the cell is an immune cell, a neuron, an epithelial cell, an endothelial cell, or a stem cell.

[0417] Aspect A22. The cell of aspect A20 or aspect A21, wherein the cell is a T cell.

[0418] Aspect A23. The cell of any one of aspects A20-A22, wherein the adaptor molecule is bound to the CAR.

[0419] Aspect A24. A method of treating a medical disorder in a subject in need thereof comprising administering to the subject a cell of any one of aspects A20-A23.

[0420] Aspect A25. A method of treating a medical disorder in a subject in need thereof comprising: a) providing an adaptor molecule of any one of aspects A1-A17; b) administering to the subject a cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises a receptor that target T on the adaptor molecule, a hinge domain, and a signaling domain; and c) administering the adaptor molecule to the subject.

[0421] Aspect A26. The method of aspect A24 or A25, wherein the medical disorder is a cancer, an autoimmune disorder, or a viral infection.

[0422] Aspect A27. The method of aspect A26, wherein the medical disorder is a cancer.

[0423] Aspect A28. The method of aspect KT1 , wherein the cancer is selected from the group consisting of lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin’s disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of head and neck, lung cancers, small cell lung cancer, nonsmall cell lung cancer, neuroblastoma, glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung, cervical carcinoma, breast cancer, epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, hematopoietic cancers, testicular cancer, colon cancer, rectal cancer, prostatic cancer, and pancreatic cancer.

[0424] Aspect A29. The method of any one of aspects A24-A28, further comprising administering a stimulus reactive blocking group that blocks the CAR from binding to T on the adaptor molecule. Aspect A30. The method of aspect A29, wherein the stimulus reactive blocking group is reactive to a stimulus selected from light, enzyme activity (e.g., from a protease), binding of a small molecule, pH, reactive oxygen species (ROS), and / or hypoxia.

[0425] Aspect A31. The method of aspect A30, wherein the stimulus reactive blocking group ceases to block the CAR from binding to T on the adaptor molecule upon reaction with the stimulus.

[0426] The following further alternative aspects of the present disclosure are also provided:

[0427] Aspect Bl. An adaptor molecule for adoptive cell therapy of Formula I:

[0428] T-L-A (I) or a pharmaceutically acceptable salt thereof; wherein:

[0429] T comprises a tag ligand;

[0430] L comprises a linker moiety; and

[0431] A comprises a moiety capable of binding a cell surface protein selected from a folate receptor, a carbonic anhydrase, prostate-specific antigen, and programmed death-ligand 1 (PD- Ll).

[0432] Aspect B2. The adaptor molecule of aspect Bl, wherein T comprises a ligand for SNAP- tag, CLIP-tag, Halo-tag, SpyTag, SnoopTag, mSA2, or Isopep-tag.

[0433] Aspect B3. The adaptor molecule of aspect Bl or aspect B2, wherein T comprises a benzylguanine, chloropyrimidine, benzylcytosine, biotin, or chloroalkane moiety.

[0434] Aspect B4. The adaptor molecule of any one of aspects B1-B3, wherein T comprises a chloroalkane moiety.

[0435] Aspect B5. The adaptor molecule of any one of aspects B1-B4, wherein T comprises — (Ci- Cio alkyl)-Cl.

[0436] Aspect B6. The adaptor molecule of any one of aspects B 1-B5, wherein T comprises

[0437] Aspect B7. The adaptor molecule of any one of aspects B1-B6, wherein A comprises a moiety capable of binding a folate receptor. Aspect B8. The adaptor molecule of aspect B7, wherein A comprises a moiety derived from a ligand selected from:

[0438] wherein the point of attachment of L is provided by replacement of a hydrogen or other suitable moiety in the above structures with a covalent bond to L.

[0439] Aspect B9. The adaptor molecule of aspect B7, wherein A comprises

[0440]

[0441] Aspect BIO. The adaptor molecule of any one of aspects B 1-B6, wherein A comprises a moiety capable of binding a carbonic anhydrase.

[0442] Aspect Bl 1. The adaptor molecule of aspect BIO, wherein A comprises a moiety derived from a ligand selected from: wherein the point to attachment of L is provided by replacement of a hydrogen or other suitable moiety in the above structures with a covalent bond to L.

[0443] Aspect B12. The adaptor molecule of aspect BIO, wherein A comprises

[0444] Aspect B 13. The adaptor molecule of any one of aspects B 1 -6, wherein A comprises a moiety capable of binding prostate-specific antigen.

[0445] Aspect Bl 4. The adaptor molecule of aspect B13, wherein A comprises a moiety derived from a ligand selected from: wherein the point of attachment to L is provided by replacement of a hydrogen or other suitable moiety in the above structures with a covalent bond to L.

[0446] Aspect Bl 5. The adaptor molecule of aspect Bl 3, wherein A comprises

[0447] Aspect Bl 6. The adaptor molecule of any one of aspects B 1-B6, wherein A comprises a moiety capable of binding PD-L1. Aspect Bl 7. The adaptor molecule of aspect B 16, wherein A comprises a moiety derived from a ligand selected from:

[0448] wherein the point of attachment to L is provided by replacement of a hydrogen or other suitable moiety in the above structures with a covalent bond to L.

[0449] Aspect B 18. The adaptor molecule of aspect B 16, wherein

[0450] A comprises

[0451] Aspect B 19. The adaptor molecule of aspect B 16, wherein

[0452] A comprises

[0453] Aspect B20. The adaptor molecule of any one of aspects Bl -Bl 9, wherein L is selected from

[0454] LI : wherein:

[0455] X101and X102are independently at each occurrence selected from a bond, aryl, heteroaryl, cycloalkyl, heterocycle, NR130, C(R130)2, O, C(O), and S; Rioo, R101, R102, R103, and R104are independently at each occurrence selected from the group consisting of a bond, alkyl, -C(O)-, -C(O)O-, -OC(O)-, -SO2-, -S(O)-, C(S)-, -C(O)NR130-, -NR130C(O)-, -O-, -S-, -NR130-, -C(R130R130)-, -P(O)(OR106))-, -

[0456] R(O)(OR106)-, alkenyl, alkynyl, haloalkyl, alkoxy, aryl, heterocycloalkyl, cycloalkyl, heteroaryl, lactic acid, or glycolic acid, each of which may be optionally substituted with one or more (for example, 1, 2, 3, or 4) substituents independently selected from R140;

[0457] R106is independently at each occurrence selected from the group consisting of hydrogen, alkyl, arylalkyl, heteroarylalkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl;

[0458] R130is independently as each occurrence selected from the group consisting of hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, -C(O)H, -C(O)OH, -C(O)alkyl, -C(O)Oalkyl, -C(O)(cycloalkyl, heterocycloalkyl, aryl, or heteroaryl), -C(O)O(cycloalkyl, heterocycloalkyl, aryl, or heteroaryl), alkenyl, or alkynyl; and

[0459] R140is independently at each occurrence selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, fluoro, bromo, chloro, hydroxyl, alkoxy, azide, amino cyano, -NH(alkyl, cycloalkyl, heterocyloalkyl, aryl, or heteroaryl), -N(independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl), -NHSCMalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl), -N(alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryllSChalkyl, -NHSChalkenyl, -N(alkyl)SO2alkenyl, -NHSChalkynyl, -N(alkyl)SO2alkynyl, haloalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

[0460] Aspect B21. The adaptor molecule of aspect Bl selected from:

[0461] or a pharmaceutically acceptable salt thereof, wherein n is an integer selected from 0 to 12.

[0462] Aspect B22. The adaptor molecule of aspect B 1 selected from:

[0463] or a pharmaceutically acceptable salt thereof, wherein n is an integer selected from 0 to 12.

[0464] Aspect B23. The adaptor molecule of aspect B 1 selected from:

[0465]

[0466] Aspect B24. A pharmaceutical composition comprising an adaptor molecule of any one of aspects B 1 -B23 and a pharmaceutically acceptable carrier or excipient.

[0467] Aspect B25. A chimeric antigen receptor (CAR) system, comprising i) an adaptor molecule of any one of aspects B1-B23, and ii) a CAR comprising a receptor that is capable of binding to T on the adaptor molecule, a hinge domain, and a signaling domain.

[0468] Aspect B26. A cell comprising the CAR system of aspect B25.

[0469] Aspect B27. The cell of aspect B26, wherein the cell is an immune cell, a neuron, an epithelial cell, an endothelial cell, or a stem cell.

[0470] Aspect B28. The cell of aspect B26 or aspect B27, wherein the cell is a T cell.

[0471] Aspect B29. The cell of any one of aspects B26-B28, wherein the adaptor molecule is bound to the CAR.

[0472] Aspect B30. A method of treating a medical disorder in a subject in need thereof comprising administering to the subject a cell of any one of aspects B26-B29.

[0473] Aspect B31. A method of treating a medical disorder in a subject in need thereof comprising: a) providing an adaptor molecule of any one of aspects B1-B23; b) administering to the subject a cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises a receptor that is capable of binding to T on the adaptor molecule, a hinge domain, and a signaling domain; and c) administering the adaptor molecule to the subject.

[0474] Aspect B32. The method of aspect B30 or aspect B31, wherein the medical disorder is a cancer, an autoimmune disorder, or a viral infection.

[0475] Aspect B33. The method of aspect B32, wherein the medical disorder is a cancer.

[0476] Aspect B34. The method of aspect B32, wherein the cancer is selected from the group consisting of lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin’s disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of head and neck, lung cancers, small cell lung cancer, nonsmall cell lung cancer, neuroblastoma, glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung, cervical carcinoma, breast cancer, epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, hematopoietic cancers, testicular cancer, colon cancer, rectal cancer, prostatic cancer, and pancreatic cancer. Aspect B35. The method of any one of aspects B30-B34, further comprising administering a stimulus reactive blocking group that blocks the CAR from binding to T on the adaptor molecule.

[0477] Aspect B35. The method of aspect B35, wherein the stimulus reactive blocking group is reactive to a stimulus selected from light, enzyme activity (e.g., from a protease), binding of a small molecule, pH, reactive oxygen species (ROS), and / or hypoxia.

[0478] Aspect B37. The method of aspect B36, wherein the stimulus reactive blocking group ceases to block the CAR from binding to T on the adaptor molecule upon reaction with the stimulus.

[0479] Aspect B38. A compound of Formula II

[0480] E-LA-TA(II) wherein:

[0481] E comprises an active ester moiety;

[0482] LAcomprises a linker moiety; and

[0483] TAcomprises a ligand for Halo-tag.

[0484] Aspect B39. A compound of aspect B38, wherein TAcomprises a chloroalkane moiety.

[0485] Aspect B40. The compound of aspect B38 or aspect B39, wherein T comprises -(C1-C10 alkyl)-Cl.

[0486] Aspect B41. The compound of any one of aspects B38-B40, wherein T comprises

[0487] Aspect B42. The compound of any one of aspects B38-B41, wherein E comprises an active ester moiety selected from an N-hydroxysuccinimidyl ester, a sulfosuccinimidyl ester, a N- hydroxyphthalimidyl ester, a N-hydroxysulfophthalimidyl ester, an o-nitrophenyl ester, a p- nitrophenyl ester, a 2,4-dinitrophenyl ester, a 3-sulfonyl-4-nitrophenyl ester, a 3-carboxy-4- nitrophenyl ester, a pentafluorophenyl ester, and a sulfonyltetrafluorophenyl ester.

[0488] Aspect B43. The compound of any one of aspects B38-B42, wherein E comprises an active ester moiety selected from 0

[0489] Aspect B44. The compound of any one of aspects B38-B42, wherein wherein LAis selected from LI : wher

[0490] X101and X102are independently at each occurrence selected from a bond, aryl, heteroaryl, cycloalkyl, heterocycle, NR130, C(R130)2, O, C(O), and S;

[0491] Rioo, R101, R102, R103, and R104are independently at each occurrence selected from the group consisting of a bond, alkyl, -C(O)-, -C(O)O-, -OC(O)-, -SO2-, -S(O)-, C(S)-, -C(O)NR130-, -NR130C(O)-, -O-, -S-, -NR130-, -C(R130R130)-, -P(O)(OR106))-, -R(O)(OR106)-, alkenyl, alkynyl, haloalkyl, alkoxy, aryl, heterocycloalkyl, cycloalkyl, heteroaryl, lactic acid, or glycolic acid, each of which may be optionally substituted with one or more (for example, 1, 2, 3, or 4) substituents independently selected from R140;

[0492] R106is independently at each occurrence selected from the group consisting of hydrogen, alkyl, arylalkyl, heteroarylalkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl;

[0493] R130is independently as each occurrence selected from the group consisting of hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, -C(O)H, -C(O)OH, -C(O)alkyl, -C(O)Oalkyl, -C(O)(cycloalkyl, heterocycloalkyl, aryl, or heteroaryl), -C(O)O(cycloalkyl, heterocycloalkyl, aryl, or heteroaryl), alkenyl, or alkynyl; and

[0494] R140is independently at each occurrence selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, fluoro, bromo, chloro, hydroxyl, alkoxy, azide, amino cyano, -NH(alkyl, cycloalkyl, heterocyloalkyl, aryl, or heteroaryl), -N(independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl), -NHSCHalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl), -N(alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl)SO2alkyl, -NHSChalkenyl, -N(alkyl)SO2alkenyl, -NHSChalkynyl, -N(alkyl)SO2alkynyl, haloalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

[0495] Aspect B45. The compound of aspect B38 selected from:

[0496]

[0497] Aspect B46. A method of synthesizing an adaptor molecule of Formula III AA-(C=O)-LA-TA(III) wherein AAis selected from an antibody or antigen recognizing fragment thereof; and all other variables are as defined in aspect B38; the method comprising: reacting a compound of any one of aspects B38-B44 with an antibody or an antigen recognizing fragment thereof to form the adaptor molecule of Formula III.

[0498] Aspect B47. An adaptor molecule of Formula III synthesized according to the method of aspect B46.

[0499] Aspect B48. A pharmaceutical composition comprising an adaptor molecule of aspect B47 and a pharmaceutically acceptable carrier or excipient.

[0500] Aspect B49. A chimeric antigen receptor (CAR) system, comprising i) an adaptor molecule of aspect B47, and ii) a CAR comprising a receptor that is capable of binding to TAon the adaptor molecule, a hinge domain, and a signaling domain.

[0501] Aspect B50. A cell comprising the CAR system of aspect B49.

[0502] Aspect B51. The cell of aspect B50, wherein the cell is an immune cell, a neuron, an epithelial cell, an endothelial cell, or a stem cell.

[0503] Aspect B52. The cell of aspect B50 or aspect B51, wherein the cell is a T cell.

[0504] Aspect B53. The cell of any one of aspects B50-B52, wherein the adaptor molecule is bound to the CAR. Aspect B54. A method of treating a medical disorder in a subject in need thereof comprising administering to the subject a cell of any one of aspects B50-B51.

[0505] Aspect B55. A method of treating a medical disorder in a subject in need thereof comprising: a) providing an adaptor molecule of aspect B47; b) administering to the subject a cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises a receptor that is capable of binding to TAon the adaptor molecule, a hinge domain, and a signaling domain; and c) administering the adaptor molecule to the subject.

[0506] Aspect B56. The method of aspect B54 or aspect B55, wherein the medical disorder is a cancer, an autoimmune disorder, or a viral infection.

[0507] Aspect B57. The method of aspect B56, wherein the medical disorder is a cancer.

[0508] Aspect B58. The method of aspect B57, wherein the cancer is selected from the group consisting of lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin’s disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of head and neck, lung cancers, small cell lung cancer, nonsmall cell lung cancer, neuroblastoma, glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung, cervical carcinoma, breast cancer, epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, hematopoietic cancers, testicular cancer, colon cancer, rectal cancer, prostatic cancer, and pancreatic cancer.

[0509] A number of aspects of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the disclosure. Accordingly, other aspects are within the scope of the following claims.

[0510] By way of non-limiting illustration, examples of certain aspects of the present disclosure are given below.

[0511] EXAMPLES

[0512] The following examples are put forth to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, and methods claimed herein are made and evaluated and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure. Efforts have been made to ensure accuracy concerning numbers (e g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in degrees Celsius or is at ambient temperature, and pressure is at or near atmospheric pressure.

[0513] Synthesis of Representative Compounds of Formula I

[0514] CA-PEGn-BG

[0515] The synthesis began with the acidolysis of commercially available acetazolamide 1 to yield compound 2 in 60% yield. Succinic anhydride was added to compound 2 and heated to facilitate ring opening, resulting in compound 3 in an 82% yield. Compound 3 was either directly coupled to commercially available BG-NH2 forming compound 4 in 21% yield or coupled to a tBu protected PEG amine where n = 6 or n = 10. The tBu group was removed by TFA to yield the carboxylic acid, which was then coupled to BG-NH2 resulting in either CA-PEG6-BG 9 or CA-PEG10-BG 10.

[0516] Experimental Procedures

[0517] 5-Amino-l,3,4-thiadiazole-2-sulfonamide »HC1 (2). Concentrated HC1 (3.75 mL) was added slowly to a suspension of acetazolamide 1 (2.50 g, 11.25 mmol) in EtOH (25 mL). The reaction mixture was heated under reflux for 8 hr, then the solvent was evaporated. The reaction was quenched with sat. NaHCOs until pH was neutral, then extracted with EtOAc (5 x 50 mL). The organic layers were washed with brine, dried over Na2SC>4, filtered and concentrated in vacuo to yield 2 as a powders’ white solid (1.21 g, 60%). The analytical data matches literature reports.1

[0518] 4-()x()-4-((5-sulfani()yl-1.3.4-tliiadiaz()l-2-yl)aniino)butanoic acid (3). Succinic anhydride (277 mg, 2.77 mmol) was added to a solution of compound 2 (500 mg, 2.77 mmol) in DMF (6 mL). The reaction mixture was heated at 100 °C for 22 hr. After reaction completion, the solvent was evaporated in vacuo to yield 3 as a yellow oil (638 mg, 82%) without further purification. The analytical data matches literature reports.1

[0519] N1-(4-(((2-Amino-9H-purin-6-yl)oxy)methyl)benzyl)-2V4-(5-sulfamoyl-l,3,4-thiadiazol-2- yl)succinimide (4). PyAOP (94 mg, 0.18 mmol) and DIPEA (94 pL, 0.54 mmol) were added to a solution of compound 3 (50 mg, 0.18 mmol) in DMF (2 mL) and stirred for 10 min. BG- NH2 (59 mg, 0.22 mmol) was added to the reaction mixture and stirred for 1 hr at RT. The crude mixture was purified by reverse-phase chromatography using a RediSepRf Gold C18 column (30 g media) with a gradient of 5-45% acetonitrile (0.1% TFA) in water (0.1% TFA) to yield 4 (20 mg, 21%) after lyophilization.!H NMR (400 MHz, d6-DMSO): 5 12.97 (s, 1 H), 8.48 (s, 1 H), 8.42 (t, 1 H, J = 5.9 Hz), 8.26 (s, 2 H), 7.41 (d, 2 H, J = 8.1 Hz), 7.21 (d, 2 H, J = 8.0 Hz), 5.46 (s, 2 H), 4.20 (d, 2 H, J = 5.8 Hz), 2.71 (t, 2 H, J = 6.7 Hz), 2.43 (t, 2 H, J = 3.5 Hz).13C NMR (100 MHz, d6-DMSO): 5 172.3, 171.2, 164.7, 161.6, 159.2, 153.7, 140.5, 134.4, 129.4, 127.7, 127.4, 46.4, 26.4, 26.3. HRMS (M+H)+calcd for C19H20O5N10S2 (M+H)+533.1132, found 533.1139. terf-Butyl 23,26-dioxo-26-((5-sulfamoyl-l,3,4-thiadiazol-2-yl)amino)-4,7,10,13,16,19- hexaoxa-22-azahexacosanoate (5). PyAOP (188 mg, 0.36 mmol) and DIPEA (188 pL, 1.08 mmol) were added to a solution of compound 3 (100 mg, 0.36 mmol) in DMF (5 mL) and stirred for 10 min. N'lL-PEGe-COO'Bu (164 mg, 0.40 mmol) was added to reaction mixture and stirred overnight at RT. The solvent was concentrated in vacuo and the crude mixture was purified by flash chromatography on SiCL (gradient 0 - 20% MeOH in DCM) to yield compound 5 (210 mg, 87%).]H NMR (400 MHz, d6-DMSO): 5 13.01 (s, 1 H), 8.30 (s, 2 H), 7.99 (t, 1 H, J = 5.3 Hz), 3.57 (t, 2 H, J = 6.2 Hz), 3.49 (m, 20 H), 3.38 (t, 2 H, J = 5.7 Hz), 3.16 (d, 4 H, J = 5.2 Hz), 2.72 (t, 2 H, J = 6.7 Hz), 2.40 (t, 2 H, 6.2 Hz), 1.38 (s, 9 H).13C NMR (100 MHz, de-DMSO): 8 172.3, 171.2, 170.9, 164.7, 161.6, 80.2, 70.3, 70.2, 70.1, 70.0, 69.9, 69.6, 66.7, 46.3, 39.1, 36.3, 30.7, 29.9, 26.3. HRMS (M+H)+calcd for C25H45N5O12S2 (M+H)+672.2579, found 672.2579. tert-Butyl 35,38-dioxo-38-((5-sulfamoyl-l,3,4-thiadiazol-2-yl)amino)-

[0520] 4, 7, 10, 13, 16,19,22,25, 28, 31-decaoxa-34-azaoctatriacontanoate (6). PyAOP (282 mg, 0.54 mmol) and DIPEA (282 pL, 1.62 mmol) were added to a solution of compound 3 (150 mg, 0.54 mmol) in DMF (5 mL) and stirred for 10 min. NH2-PEGIO-COO’BU (150 mg, 0.26 mmol) was added to reaction mixture and stirred overnight at RT. The solvent was concentrated in vacuo and the crude mixture was purified by flash chromatography on SiO2 (gradient 0 - 10% MeOH in DCM) to yield compound 6 (204 mg, 93%).!H NMR (500 MHz, d6-DMSO): 5 13.02 (s, 1 H), 8.31 (s, 2 H), 7.99 (t, 1 H, J = 5.6 Hz), 3.58 (t, 2 H, J = 6.2 Hz), 3.50 (m, 36 H), 3.39 (t, 2 H, J = 5.9 Hz), 3.33 (s, 4 H), 2.73 (t, 2 H, 6.8 Hz), 2.41 (t, 2 H, 6.2 Hz), 1.39 (s, 9 H).13C NMR (100 MHz, d6-DMSO): 5 172.3, 171.2, 170.9, 164.7, 161.6, 80.2, 70.3, 70.2, 70.1, 70.0, 69.9, 69.6, 66.7, 46.3, 39.1, 36.3, 30.7, 29.8, 26.4. HRMS (M+H)+calcd for C33H61N5O16S2 (M+H)+848.3628, found 848.3627.

[0521] 23,26-Dioxo-26-((5-sulfamoyl-l,3,4-thiadiazol-2-yl)amino)-4,7,10,13,16,19-hexaoxa-22- azahexacosanoic acid (7). Compound 5 (150 mg, 0.22 mmol) was dissolved in a mixture of DCM / TFA (1 :1) and stirred at RT for 3 hr. The solvent was concentrated in vacuo and the crude mixture was purified by flash chromatography on Si CT (gradient 0 - 10% MeOH in DCM) to yield compound 7 (116 mg, 86%).!H NMR (500 MHz, d6-DMSO): 5 12.98 (s, 1 H), 8.31 (s, 2 H), 7.99 (t, 1 H, J = 5.5 Hz), 3.59 (t, 2 H, J = 6.3 Hz), 3.50 (m, 20 H), 3.39 (t, 2 H, J = 5.9 Hz), 3.18 (m, 4 H), 2.73 (t, 2 H, J = 6.9 Hz), 2.43 (t, 2 H, 6.3 Hz).13C NMR (100 MHz, d6-DMSO): 5 173.1 , 172.3, 171.2, 164.7, 161.6, 70.3, 70.2, 70.1 , 70.0, 69.9, 69.6, 66.7, 46.2, 39.1 , 35.2, 30.8, 29.9. HRMS (M+H)+calcd for C21H37O12N5S2 (M+H)+616.1953, found 616.1973.

[0522] 35,38-Dioxo-38-((5-sulfamoyl-l,3,4-thiadiazol-2-yl)amino)-4,7,10,13,16,19,22,25,28,31- decaoxa-34-azaoctatriacontanoic acid (8). Compound 6 (150 mg, 0.18 mmol) was dissolved in a mixture of DCM / TFA (1 :1) and stirred at RT for 3 hr. The solvent was concentrated in vacuo and the crude mixture was purified by flash chromatography on SiO2(gradient 0 - 10% MeOH in DCM) to yield compound 8 (115 mg, 80%).!H NMR (400 MHz, d6-DMSO): 5 12.97 (s, 1 H), 8.26 (s, 2 H), 7.95 (t, 1 H, J = 5.6 Hz), 3.55 (t, 2 H, J = 6.3 Hz), 3.46 (m, 34 H), 3.34 (t, 2 H, J = 5.8 Hz), 3.12 (m, 4 H), 2.68 (t, 2 H, J = 6.8 Hz), 2.43 (t, 2 H, 6.3 Hz).13C NMR (100 MHz, ds-DMSO): 5 173.1, 172.3, 171.2, 164.7, 161.6, 70.3, 70.2, 70.1, 70.0, 69.9, 69.6, 66.7, 46.2, 39.1, 35.2, 30.8, 29.9. HRMS (M+H)+calcd for C29H53O16N5S2 (M+H)+792.3002, found 792.3022.

[0523] Arl-(l-(4-(((2-Amino-9H-purin-6-yl)oxy)methyI)phenyl)-3-oxo-6, 9,12,15,18, 21 -hexaoxa- 2-azatricosan-23-yl)-Ar4-(5-sulfamoyl-l,3,4-thiadiazol-2-yl)succinamide (9). PyAOP (26 mg, 0.05 mmol) and DIPEA (68 pL, 0.39 mmol) were added to a solution of compound 7 (24 mg, 0.039 mmol) in DMSO (3 mL) and stirred for 10 min. BG-NH2 (16 mg, 0.058 mmol) was then added to the reaction mixture and stirred overnight at RT. The product was purified by reverse-phase chromatography using a RediSepRf Gold C18 column (5.5 g media) with a gradient of 5 — 45% acetonitrile (0.1% TFA) in water (0.1% TFA) to yield 9 (6 mg, 18%) after lyophilization. ’H NMR (500 MHz, d6-DMSO): 5 13.00 (s, 1 H), 8.36 (t, 1 H, J = 5.9 Hz), 8.30 (s, 2 H), 7.97 (t, 1 H, J = 5.5 Hz), 7.48 (d, 2 H, J = 8.0 Hz), 7.28 (d, 2 H, J = 8.0 Hz), 5.52 (s, 2 H), 3.62 (t, 2 H, J = 6.3 Hz), 3.50 (m, 20 H), 3.38 (t, 2 H, J = 5.9 Hz), 3.18 (q, 4 H, J = 5.7 Hz), 2.73 (t, 2 H, J = 6.9 Hz), 2.38 (t, 2 H, J = 6.4 Hz). HRMS (M+H)+calcd for C34H49O12N11 S2 (M+H)+868.3076, found 868.3061.

[0524] A1-(l-(4-(((2-Amino-9H-purin-6-yl)oxy)methyI)phenyl)-3-oxo- 6,9,12,15,18,21,24,27,30,33-decaoxa-2-azapentatriacontan-35-yl)-jV4-(5-sulfamoyl-l,3,4- thiadiazol-2-yl)succinamide (10). PyAOP (26 mg, 0.05 mmol) and DIPEA (66 pL, 0.38 mmol) were added to a solution of compound 8 (30 mg, 0.038 mmol) in DMSO (3 mL) and stirred for 10 min. BG-NH2 (21 mg, 0.078 mmol) was then added to the reaction mixture and stirred overnight at RT. The product was purified by reverse-phase chromatography using a RediSepRf Gold Cl 8 column (5.5 g media) with a gradient of 5 — 45% acetonitrile (0.1 % TFA) in water (0.1% TFA) to yield 10 (4 mg, 9%) after lyophilization.]H NMR (400 MHz, de- DMSO): 5 13.01 (s, 1 H), 8.37 (t, 1 H, J = 6.2 Hz), 8.31 (s, 2 H), 7.99 (t, 1 H, J = 5.4 Hz), 7.47 (d, 2 H, J = 8.1 Hz), 7.28 (d, 2 H, J = 8.1 Hz), 5.50 (s, 2 H), 3.62 (t, 2 H, J = 6.5 Hz), 3.50 (m, 34 H), 3.38 (t, 2 H, J = 5.8 Hz), 3.18 (q, 4 H, J = 5.0 Hz), 2.73 (t, 2 H, J = 6.9 Hz), 2.37 (t, 2 H, J = 6.4 Hz). HRMS (M+H)+calcd for C42H65O16N11S2 (M+H)+1043.4100, found 1044.4122.

[0525] BMS37-BG-PEG6-N3

[0526] Beginning with the commercially available 4-pentynoic acid 11 and A- Boc ethylenediamine, the synthesis starts with a standard peptide coupling (72%), followed by the deprotection of the Boc group with HC1 to yield 13 in 91% yield. Then, the commercially available 3- hydroxymethyl-2-methylbiphenyl 14 was functionalized via a Mitsonobu reaction with 4- hydroxy-2,6-dimethoxybenzaldehyde to yield 15 in 61% yield. Wang et al. initiated the last step of the synthesis by deprotecting the Boc group of 12 and immediately subjecting 13 as a crude product to the reductive amination conditions. The attempts of this method were unsuccessful, so we proceeded to separate the steps and have optimized the reductive amination conditions with sodium cyanoborohydride to yield 16 in 41 % yield. With the alkyne-modified BMS-37 in hand, the next step was the synthesis of the BG molecule to be “clicked” onto the alkyne. Benzylguanine was coupled to the commercial carboxylic acid-PEGn-azide 17 via a peptide coupling with PyAOyP, to yield the PEGe 18 or PEG12 19 BGs. We chose PEGe and PEGi 2 lengths to compare the possible effect the length of the adaptor may have on the adaptor function. The final step was the synthesis of the BMS-37-BG-NH2-PEGn-N3 via a CuAAC, which was completed for the PEGe compound 20 in 9% yield.

[0527] Experimental Procedures te / 7- Butyl (2-(pent-4-ynamido)ethyl)carbamate (12). A solution of 4-pentynoic acid 11 (502 mg, 5.11 mmol) and DCC (1.265 g, 6.13 mmol) in anhydrous DCM (12 mL) was cooled to 0 °C and stirred for 30 min. Then, A-Boc ethylenediamine (890 uL, 1.10 mmol) was added dropwise to the solution and was allowed to warm to RT overnight. The precipitated white solid was removed by vacuum filtration, and the filtrate was washed with DCM and concentrated in vacuo. The crude mixture was purified by flash chromatography on SiC>2 (2% MeOH in DCM) to yield the product as a white solid (884 mg, 72%). The analytical data match the previous reports. jV-(2-Aminoethyl)pent-4-ynamide (13). To a solution of 12 (300 mg, 1.25 mmol) in anhydrous DCM (8.3 mL) was slowly added 4 N, HC1 in dioxane (6.7 mL) and allowed to stir at RT for 3 h. The volatiles were removed in vacuo and the crude mixture was purified by flash column chromatography on Si O2 (gradient 10 - 15% MeOH in DCM) to yield the product as a yellow oil (160 mg, 91%). The analytical data match previous reports.3

[0528] 2,6-Dimethoxy-4-((2-methyl-[l,l'-biphenyl]-3-yl)methoxy)benzaldehyde (15). A solution of 4-hydroxy-2,6-dimethoxybenzaldehyde 14 (302 mg, 1.66 mmol) in anhydrous THF (6.6 mL) was cooled to 0 °C, and to it was added triphenylphoshine (523 mg, 2 mmol) and 3- hydroxymethyl-2-methylbiphenyl (297 mg, 1.5 mmol). A solution of DIAD (360 mg, 1.78 mmol) in THF (6.6 mL) was added dropwise to the reaction mixture, which was allowed to warm to RT overnight. The volatiles were concentrated in vacuo and purified by flash column chromatography on SiCh (30% EtOAc in Hex) to yield the product as a white solid (369 mg, 61%). The analytical data match previous reports.4

[0529] A-(2-((2,6-dimethoxy-4-((2-methyl- [ 1,1 '-biphenyl] -3- yl)methoxy)benzyl)amino)ethyl)pent-4-ynamide (16). A solution of 2,6-dimethoxy-4-((2- methyl-[l,T-biphenyl]-3-yl)methoxy)benzaldehyde (15) (150 mg, 0.41 mmol), N-(2- aminoethyl)pent-4-ynamide (13) (68.7 mg, 0.49 mmol), sodium cyanoborohydride (134 mg, 2.13 mmol), and acetic acid (~3 drops) in DMF (3 mL) were allowed to stir at 80 °C for 4 h. The volatiles were then removed in vacuo and the resulting crude solid was purified by flash column chromatography on SiO? (gradient 25 - 75% EtOAc in Hex) to yield 16 as a white solid (82 mg, 41%). The analytical data match previous reports.5

[0530] 2V-(4-(((2-amino-9H-purin-6-yl)oxy)methyl)benzyl)-l-azido-3,6,9,12,15,18- hexaoxahenicosan-21-amide (18). l-azido-3,6,9,12,15,18-hexaoxahenicosan-21-oic acid 17a (80 mg, 0.22 mmol) was dissolved in anhydrous DMSO (1.5 mL), and to it was added PyAOP (115 mg, 0.22 mmol) and DIPEA (115 uL, 0.66 mmol) which was allowed to stir for 15 min at RT. Then, benzylguanine (119 mg, 0.44 mmol) was added to the reaction mixture and allowed to stir overnight at RT. The product was purified by reverse-phase chromatography using a RediSepRf Gold C18 column (15.5 g media) with a gradient of 5-65% acetonitrile (0.1% TFA) in water (0.1% TFA) to yield 18 (85 mg, 61%) after lyophilization. *H NMR (400 MHz, de- DMSO) 5 8.37 (t, 1 H, J = 5.6 Hz), 8.25 (s, 1 H), 7.48 (d, 2 H, J = 7.8 Hz), 7.28 (d, 2 H, J = 7.8 Hz), 5.51 (s, 2 H), 4.28 (d, 2 H, J = 5.7 Hz), 3.50 (m, 24 H), 2.38 (t, 2 H, J = 2.3 Hz).13C NMR (125 MHz, d6-DMSO) 5 173.1, 170.1, 159.5, 158.7, 158.4, 140.2, 134.9, 129.2, 127.7, 126.7, 70.3, 70.2, 70.1, 70.0, 69.9, 69.7, 67.3, 50.5, 42.3, 36.6, 35.2. HRMS (M+H)+calcd for C2sH4i OsN9 (M+H)+632.3151, found 632.3150.

[0531] A-(4-(((2-amino-9 / / -purin-6-yl)oxy)methyl)benzyl)-l-azido-

[0532] 3.6.9.12.15.18.21.24.27.30.33.36-dodecaoxanonatriacontan-39-amide (19). 1-azido-

[0533] 3.6.9.12.15.18.21.24.27.30.33.36-dodecaoxanonatriacontan-39-oic acid 17b (70 mg, 0.11 mmol) was dissolved in anhydrous DMSO (1.1 mL), and to it was added PyAOP (57 mg, 0.11 mmol) and DIPEA (57 uL, 0.33 mmol) which was allowed to stir for 15 min at RT. Then, benzylguanine (59 mg, 0.22 mmol) was added to the reaction mixture and allowed to stir overnight at RT. The volatiles were then removed in vacuo and purified by reverse-phase chromatography using a RediSepRf Gold Cl 8 column (5.5 g media) with a gradient of 5-65% acetonitrile (0.1% TFA) in water (0.1% TFA) to yield 19 as a white solid (86 mg, 88%).]H NMR (500 MHz, CDCh) 5 8.38 (t, 1 H), 7.49 (d, 2 H, J = 7.7 Hz), 7.3 (d, 2 H, 7.6 Hz), 5.53 (s, 2 H), 4.28 (d, 2 H, J = 5.8 Hz), 3.59 (m, 48 H), 2.38 (t, 2 H).13C (125 MHz, CDC13)

[0534] A-(4-(((2-amino-9H-purin-6-yl)oxy)inethyl)benzyl)-l-(4-(3-((2-((2,6-dimethoxy-4-((2- methyl-[l,l'-biphenyl]-3-yl)methoxy)benzyl)amino)ethyl)amino)-3-oxopropyI)-lH-l,2,3- triazol-l-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-amide (20). To a solution of 18 (19.5 mg, 0.031 mmol, 100 mM in DMSO) in tert-butyl alcohol (676 uL) was added (15 mg, 0.031 mmol, 100 mM in DMSO) which stirred for 5 min. In a separate flamed-dried vial, copper(II) sulfate pentahydrate (0.78 mg, 0.003 mmol, 100 mM in water), THPTA (6,70 mg, 0.015 mol, 100 mM in water), and sodium ascorbate (1.20 mg, 0.006 mmol, 100 mM in water) were mixed for 10 min. The copper solution was then added to the above mixture, which was allowed to stir at RT overnight. The reaction mixture was diluted with water / DMSO (1 :1) and the product was purified by reverse-phase chromatography using a RediSepRf Gold Cl 8 column (5.5 g media) with a gradient of 5-95% acetonitrile (0.1% TFA) in water (0.1% TFA) to yield 20 (2.9 mg, 9%). after lyophilization as white solid. The product was confirmed by LC-MS and HPLC analysis.

[0535] BG-PEGn-PSMA

[0536] The synthesis began with the joining of the two / Bu-prolected amino acids 22 and 23 through treatment of one of them with triphosgene, which forms an isocyanate that the other amino acid can react with to form the urea 24. The carboxybenzyl protecting group was then removed through a standard hydrogenation with palladium on carbon and hydrogen gas to afford 25. The resulting free amine was then reacted with an NHS-modified aryl alkyne to yield 26. Pathak et al. had performed this step with TMS -protected alkyne, however we found the use of the protecting group unnecessary as 26 could be isolated in high yields.6Nevertheless, 27 was achieved through the removal of all / Bu groups on each carboxylic acid via a solution of TFA in DCM. With the PSMA ligand in hand, a click reaction was performed with previously synthesized BG-PEG6-N3 (18) and BG-PEG12-N3 (19) to yield the final adaptors 28 and 29. The last step was challenging. Many different test conditions that varied catalysts loading, solvent systems, and order of addition were performed before any final compound could be made in significant amounts that permitted isolation.

[0537] Experimental Procedures

[0538] Tri-teri-butyl ( 95,135')-3,ll-dioxo-l-phenyl-2-oxa-4,l 0,12-triazapentad ecane-9,13,15- tricarboxylate (24). L-glutamic acid di-tertbutyl ester hydrochloride (1.34 g, 1.7 eq, 4.53 mmol) and triethylamine (2.06 mL, 5.5 eq, 14.74 mmol) were dissolved in anhydrous DCM (80 mL). The solution was cooled to -78 °C and triphosgene (460.0 mg, 0.6 eq, 1.55 mmol) in anhydrous DCM (50 mL) was added dropwise. Upon complete addition, the reaction mixture was allowed to warm to room temperature, where it stirred for 30 minutes before H-Lys(Z)- OtBu hydrochloride (1.00 g, 2.68 mmol) and triethylamine (0.37 mL, 1.0 eq, 2.68 mmol) were added. The resulting solution was stirred for 24 hours, under argon, before being diluted with DCM (30 mL) and washed with H2O (3 x 150 mL). The organic layers were combined, dried over Na2SO4, and the volatiles were evaporated in vacuo. The resulting crude oil was purified via flash column chromatography on SiCh (60% hexanes in EtOAc) to yield 24 (66%, 1.11 g). All analytical data matches literature reports.6Di-tert-butyl (((5')-6-amino-l-(tert-butoxy)-l-oxohexan-2-yl)carbamoyl)-L-glutamate (25). Compound 24 (1.10 g. 1.77 mmol) was dissolved in anhydrous MeOH (50 mL) before Pd / C (110.0 mg, 0.58 eq, 10% by mass) was carefully added. The air was vacuumed out from the reaction and replaced with argon three times before hydrogen gas was bubbled through the reaction as it stirred at room temperature, overnight. The reaction mixture was filtered through celite before it was concentrated in vacuo to yield 25 (87%, 750.7 mg) without any further purification. All analytical data matched literature reports.6

[0539] Di-tert-butyl (((.S')- 1 -(tert-butoxy )-6-(4-ethynylbenzamido)-l-oxohexan-2-yI)carbamoyl)- L-glutamate (26). 2,5-dioxopyrrolidin-l-yl 4-ethynylbenzoate (374.3 mg, 1.54 mmol) and 25 (750.0 mg, 1 eq, 1.54 mmol) were dissolved in anhydrous DCM (60 mL). Triethylamine (214.6 pL, 1 eq, 1.54 mmol) was added and the mixture was heated to reflux and allowed to stir overnight. The reaction mixture was then allowed to cool to room temperature, washed with water (2 x 100 mL), dried over NazSCh, and concentrated in vacuo. The resulting crude oil was purified via flash column chromatography on SiCh (60% hexanes in EtOAc) to yield 26 (Tl%, 726.0 mg). ’H NMR (500 MHz, CDCh): 8 7.82 (d, 2 H, J = 8.4 Hz), 7.53 (d, 2 H, J = 8.4 Hz), 6.82 (s, 1 H), 5.21 (s, 2 H), 4.32 (s, 1 H), 4.23 (s, 1 H), 3.45 (m, 2 H), 3.17 (s, 1 H), 2.30 (m, 2 H), 2.06 (m, 1 H), 1.83 (m, 2 H), 1.67 (m, 2 H), 1.45 (s, 9 H), 1.44 (s, 9 H), 1.42 (s, 9 H).13C NMR (125 MHz, CDC13): 8 172.4, 172.3, 166.9, 157.1 , 134.7, 132.1 , 127.2, 125.1 , 83.0, 82.3, 81.9, 80.7, 79.2, 53.2, 53.1, 39.8, 32.9, 31.5, 28.5, 28.1, 28.0, 27.9, 22.7. HRMS (M+H)+calcd for C M hoChN ) (M+H)+616.3592, found 616.3577.

[0540] (((S)-l-carboxy-5-(4-ethynylbenzamido)pentyl)carbamoyl)-L-glutamic acid (27). Compound 26 (360.0 mg, 0.59 mmol) was dissolved in 1: 1 solution of DCM:TFA(20 mL total) and the resulting reaction solution was allowed to stir at room temperature for 2 hours. The volatiles were evaporated in vacuo before redissolving in DCM and precipitating with cold EtzO. The resulting precipitate was filtered, washed with EtzO, and dried to yield 27 (56%, 146.8 mg) without any further purification. All analytical data matched literature reports.6

[0541] (((S)-5-(4-(l-(l-(4-(((2-Amino-9H-purin-6-yl)oxy)methyl)phenyl)-2,5,8,ll,14,17- hexaoxanonadecan- 19-yl)-lH-l, 2, 3-triazoI-4-yl)benzamido)-l- carboxypentyl)carbamoyI)-L-gIutamic acid (28). The azide 18 (20.0 mg, 0.032 mmol) and the alkyne 27 (14.2 mg, 1 eq, 0.032 mmol) were dissolved in a mixture of water (776 pL), DMSO (332 pL), and 2-propanol (111 pL). Sodium ascorbate (63.4 pL of a 100 mM stock in water, 0.2 eq, 63.4 pmol) and CuSCh 5HzO (31.7 pL of a 100 mM stock in water, 0.1 eq, 31.7 pmol) were added and the reaction was stirred at room temperature, overnight. The product was purified by HPLC using an Agilent Zorbax SB-C18 column (P / N 883975-902) with a gradient of 5-65% acetonitrile (0.1% TFA) in water (0.1% TEA) to yield 28 (3%, 1.0 mg) after lyophilization.!H NMR (400 MHz, MeOD) 8 8.35 (s, 1 H), 8.19 (s, 1 H), 7.79 (m, 4 H), 7.39 (d, 2 H, J = 7.8 Hz), 7.23 (d, 2 H, J = 7.8 Hz), 5.51 (s, 2 H), 4.53 (t, 2 H, J = 4.4 Hz), 4.29 (s, 2 H), 4.20 (m, 2 H), 3.84 (t, 2 H, J = 4.8 Hz), 3.63 (t, 2 H, J = 5.8 Hz), 3.45 (m, 24 H), 2.38 (t, 2 H, J = 5.4 Hz), 2.04 (m, 1 H), 1.79 (m, 2 H), 1.58 (m, 4 H), 1.40 (m, 3 H).

[0542] (((S)-5-(4-(l-(l-(4-(((2-amino-9H-purin-6-yl)oxy)methyl)phenyl)- 2,5,8,ll,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yI)-lH-l,2,3-triazol-4- yl)benzamido)-l-carboxypentyl)carbamoyI)-L-glutamic acid (29). The azide 19 (20.1 mg, 0.022 mmol) and the alkyne 27 (10.0 mg, 1 eq, 0.022 mmol) were dissolved in a mixture of water (525 pL) and 2-propanol (75 pL). Sodium ascorbate (44.8 pLof a 100 mM stock in water, 0.2 eq, 44.8 pmol) and CuSCU SHiO (22.4 pL of a 100 mM stock in water, 0.1 eq, 22.4 pmol) were added and the reaction was stirred at room temperature, overnight. The product was purified by reverse-phase chromatography using a RediSepRf Gold Cl 8 column (5.5 g media) with a gradient of 5-65% acetonitrile (0.1% TFA) in water (0.1% TFA) to yield 29 (12%, 3.3 mg) after lyophilization. ’H NMR (500 MHz, MeOD) 5 8.48 (s, 1 H), 8.38 (s, 1 H), 7.91 (m, 4 H), 7.51 (d, 2 H, J = 8.1 Hz), 7.35 (d, 2 H, J = 8.1 Hz), 5.63 (s, 2 H), 4.64 (t, 2 H, J = 4.9 Hz), 4.41 (s, 2 H), 4.30 (m, 2 H), 3.94 (t, 2 H, J = 4.9 Hz), 3.74 (t, 2 H, J = 5.9 Hz), 3.59 (m, 46 H), 3.40 (m, 2 H), 2.49 (t, 2 H, J = 5.9 Hz), 2.41 (m, 2 H), 2.14 (m, 1 H), 1.89 (m, 2 H), 1.67 (m, 4 H), 1.51 (m, 3 H). HRMS (M+H)+calcd for C61H90N12O22 (M+H)+1343.6377, found 1343.6429.

[0543] References for Experimental Examples

[0544] 1. More, K. N.; Lee, J. Y; Kim, D.-Y.; Cho, N.-C.; Pyo, A.; Yun, M.; Kim, H. S.; Kim, H.; Ko. K.; Park, J.-H.; Chang, D.-J., Acetazolamide-based [18F]-PET Racer: In vivo validation of carbonic anhydrase IX as a sole target for imaging of CA-IX expressing hypoxic solid tumors. Bioorganic & Medicinal Chemistry Letters 2018, 28 (5), 915-921.

[0545] 2. Koh, M.; Park, J.; An, H.; Park, S. B., Ratiometric analysis of zidovudine (ZDV) incorporation by reverse transcriptases or polymerases via bio-orthogonal click chemistry. Chemical Communications 2011, 47 (27), 7614-7616.

[0546] 3. Mishra, N. M.; Briers, Y; Lamberigts, C.; Steenackers, H.; Robijns, S.; Landuyt, B.; Vanderleyden, J.; Schoofs, L.; Lavigne, R.; Luyten, W.; Van der Eycken, E. V, Evaluation of the antibacterial and antibiofilm activities of novel CRAMP-vancomycin conjugates with diverse linkers. Org Biomol Chem 2015, 13 (27), 7477-86.

[0547] 4. Guzik, K.; Zak, K. M.; Grudnik, P.; Magiera. K.; Musielak, B.; Tomer, R.; Skalniak, L.; Domling, A.; Dubin, G.; Holak, T. A., Small-Molecule Inhibitors of the Programmed Cell Death- 1 / Programmed Death-Ligand 1 (PD-1 / PD-L1) Interaction via Transiently Induced Protein States and Dimerization of PD-L1. Journal of Medicinal Chemistry 2017, 60 (13), 5857-5867.

[0548] 5. Wang, Y: Zhou, Y: Cao, S.; Sun, Y.; Dong, Z.: Li. C.; Wang, H.; Yao, Y; Yu, H.; Song, X.; Li, M.; Wang, J.; Wei, M.; Yang, G ; Yang, C., In vitro and in vivo degradation of programmed cell death ligand 1 (PD-L1) by a proteolysis targeting chimera (PROTAC). Bioorganic Chemistry 2021, 111, 104833.

[0549] 6. Pathak, R. K.; Basu, U.; Ahmad, A.; Sarkar, S.; Kumar, A.; Sumar, B.; Ansari, S.; Wilczek, K.; Ivan, M. E.; Marples, B.; Kolishetti, N.; Dhar, S., A designer bow-tie combination therapeutic platform: An approach to resistant cancer treatment by simultaneous delivery of cytotoxic and anti-inflammatory agents and radiation. Biomaterials 2018, 187, 117-129.

[0550] The compositions and methods of the appended claims are not limited in scope by the specific compositions and methods described herein, which are intended as illustrations of a few aspects of the claims and any compositions and methods that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the compositions and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative compositions and method steps disclosed herein are specifically described, other combinations of the compositions and method steps also are intended to fall within the scope of the appended claims, even if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitly mentioned herein; however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated.

Claims

WHAT IS CLAIMED IS:

1. An adaptor molecule for adoptive cell therapy of Formula I:T-L-A (I) or a pharmaceutically acceptable salt thereof; wherein:T comprises a tag ligand;L comprises a linker moiety; andA comprises a moiety capable of binding a cell surface protein selected from a folate receptor, a carbonic anhydrase, prostate-specific antigen, and programmed death-ligand 1 (PD-L1).

2. The adaptor molecule of claim 1, wherein T comprises a ligand for SNAP-tag, CLIP- tag, Halo-tag, SpyTag, SnoopTag, mSA2, or Isopep-tag.

3. The adaptor molecule of claim 1 or claim 2, wherein T comprises a benzylguanine, chloropyrimidine, benzylcytosine, biotin, or chloroalkane moiety.

4. The adaptor molecule of any one of claims 1-3, wherein T comprises a chloroalkane moiety.

5. The adaptor molecule of any one of claims 1-4, wherein T comprises -(Ci-Cio alkyl)-Cl.

6. The adaptor molecule of any one of claims 1-5, wherein T comprises7. The adaptor molecule of any one of claims 1 -6, wherein A comprises a moiety capable of binding a folate receptor.

8. The adaptor molecule of claim 7, wherein A comprises a moiety derived from a ligand selected from:wherein the point of attachment of L is provided by replacement of a hydrogen or other suitable moiety in the above structures with a covalent bond to L.

9. The adaptor molecule of claim 7, wherein A comprises10. The adaptor molecule of any one of claims 1-6, wherein A comprises a moiety capable of binding a carbonic anhydrase.

11. The adaptor molecule of claim 10, wherein A comprises a moiety derived from a ligand selected from:wherein the point to attachment of L is provided by replacement of a hydrogen or other suitable moiety in the above structures with a covalent bond to L.

12. The adaptor molecule of claim 10, wherein A comprises13. The adaptor molecule of any one of claims 1-6, wherein A comprises a moiety capable of binding prostate-specific antigen.

14. The adaptor molecule of claim 13, wherein A comprises a moiety derived from a ligand selected from:CO2Ho o wherein the point of attachment to L is provided by replacement of a hydrogen or other suitable moiety in the above structures with a covalent bond to L.

15. The adaptor molecule of claim 13, wherein A comprises16. The adaptor molecule of any one of claims 1-6, wherein A comprises a moiety capable of binding PD-L1.

17. The adaptor molecule of claim 16, wherein A comprises a moiety derived from a ligand selected from:CO2HFwherein the point of attachment to L is provided hy replacement of a hydrogen or other suitable moiety in the above structures with a covalent bond to L.

18. The adaptor molecule of claim 16, wherein19. The adaptor molecule of claim 16, whereinA comprises20. The adaptor molecule of any one of claims 1-19, wherein L is selected from LI:wherein:X101and X102are independently at each occurrence selected from a bond, aryl, heteroaryl, cycloalkyl, heterocycle, NR130, C(R130)2, O, C(O), and S;Rioo, R101, R102, R103, and R104are independently at each occurrence selected from the group consisting of a bond, alkyl, -C(O)-, -C(O)O-, -OC(O)-, -SO2-, -S(O)-, C(S)-, -C(O)NR130-, -NR130C(O)-, -O-, -S-, -NR130-, -C(R130R130)-, -P(O)(OR106))-, -R(O)(OR106)-, alkenyl, alkynyl, haloalkyl, alkoxy, aryl, heterocycloalkyl, cycloalkyl, heteroaryl, lactic acid, or glycolic acid, each of which may be optionally substituted with one or more (for example, 1, 2, 3, or 4) substituents independently selected from R140;R106is independently at each occurrence selected from the group consisting of hydrogen, alkyl, arylalkyl, heteroarylalkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl ;R130is independently as each occurrence selected from the group consisting of hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, -C(O)H, -C(O)OH, -C(O)alkyl, -C(O)Oalkyl, -C(O)(cycloalkyl, heterocycloalkyl, aryl, or heteroaryl), -C(O)O(cycloalkyl, heterocycloalkyl, aryl, or heteroaryl), alkenyl, or alkynyl; andR140is independently at each occurrence selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, fluoro, bromo, chloro, hydroxyl, alkoxy, azide, amino cyano, -NH(alkyl, cycloalkyl, heterocyloalkyl, aryl, or heteroaryl), -N(independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl), -NHSCMalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl), -N(alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryljSCbalkyl, -NHSChalkenyl, -N(alkyl)SO2alkenyl, -NHSCbalkynyl, -N(alkyl)SO2alkynyl, haloalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

21. The adaptor molecule of claim 1 selected from:šndor a pharmaceutically acceptable salt thereof, wherein n is an integer selected from 0 to 12.

22. The adaptor molecule of claim 1 selected from: or a pharmaceutically acceptable salt thereof, wherein n is an integer selected from 0 to 12.

23. The adaptor molecule of claim 1 , selected from:

24. A pharmaceutical composition comprising an adaptor molecule of any one of claims 1-23 and a pharmaceutically acceptable carrier or excipient.

25. A chimeric antigen receptor (CAR) system, comprising i) an adaptor molecule of any one of claims 1-23, and ii) a CAR comprising a receptor that is capable of binding to T on the adaptor molecule, a hinge domain, and a signaling domain.

26. A cell comprising the CAR system of claim 25.

27. The cell of claim 26, wherein the cell is an immune cell, a neuron, an epithelial cell, an endothelial cell, or a stem cell.

28. The cell of claim 26 or claim 27, wherein the cell is a T cell.

29. The cell of any one of claims 26-28, wherein the adaptor molecule is bound to the CAR.

30. A method of treating a medical disorder in a subject in need thereof comprising administering to the subject a cell of any one of claims 26-29.

31. A method of treating a medical disorder in a subject in need thereof comprising: a) providing an adaptor molecule of any one of claims 1-23; b) administering to the subject a cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises a receptor that is capable of binding to T on the adaptor molecule, a hinge domain, and a signaling domain; and c) administering the adaptor molecule to the subject.

32. The method of claim 30 or claim 31, wherein the medical disorder is a cancer, an autoimmune disorder, or a viral infection.

33. The method of claim 32, wherein the medical disorder is a cancer.

34. The method of claim 33, wherein the cancer is selected from the group consisting of lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin’s disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of head and neck, lung cancers, small cell lung cancer, nonsmall cell lung cancer, neuroblastoma, glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung, cervical carcinoma, breast cancer, epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, hematopoietic cancers, testicular cancer, colon cancer, rectal cancer, prostatic cancer, and pancreatic cancer.

35. The method of any one of claims 30-34, further comprising administering a stimulus reactive blocking group that blocks the CAR from binding to T on the adaptor molecule.

36. The method of claim 35, wherein the stimulus reactive blocking group is reactive to a stimulus selected from light, enzyme activity (e.g., from a protease), binding of a small molecule, pH, reactive oxygen species (ROS), and / or hypoxia.

37. The method of claim 36, wherein the stimulus reactive blocking group ceases to block the CAR from binding to T on the adaptor molecule upon reaction with the stimulus.

38. A compound of Formula IIE-LA-TA(II) wherein:E comprises an active ester moiety;LAcomprises a linker moiety; and TAcomprises a ligand for Halo-tag.

39. A compound of claim 38, wherein TAcomprises a chloroalkane moiety.

40. The compound of claim 38 or claim 39, wherein T comprises -(Ci-Cio alkyl)-Cl.

41. The compound of any one of claims 38-40, wherein T comprises42. The compound of any one of claims 38-41, wherein E comprises an active ester moiety selected from an N-hydroxysuccinimidyl ester, a sulfosuccinimidyl ester, a N- hydroxyphthalimidyl ester, a N-hydroxysulfophthalimidyl ester, an o-nitrophenyl ester, a p- nitrophenyl ester, a 2,4-dinitrophenyl ester, a 3-sulfonyl-4-nitrophenyl ester, a 3-carboxy-4- nitrophenyl ester, a pentafluorophenyl ester, and a sulfonyltetrafluorophenyl ester.

43. The compound of any one of claims 38-42, wherein E comprises an active ester moiety selected from O44. The compound of any one of claims 38-43, wherein wherein LAis selected from LI : wherein:X101and X102are independently at each occurrence selected from a bond, aryl, heteroaryl, cycloalkyl, heterocycle, NR130, C(R130)2, O, C(O), and S;Rioo, R101, R102, R103, and R104are independently at each occurrence selected from the group consisting of a bond, alkyl, -C(O)-, -C(O)O-, -OC(O)-, -SO2-, -S(O)-, C(S)-, -C(O)NR130-, -NR130C(O)-, -O-, -S-, -NR130-, -C(R130R130)-, -P(O)(OR106))-, -R(O)(OR106)-, alkenyl, alkynyl, haloalkyl, alkoxy, aryl, heterocycloalkyl, cycloalkyl, heteroaryl, lactic acid, or glycolic acid, each of which may be optionally substituted with one or more (for example, 1, 2, 3, or 4) substituents independently selected from R140;R106is independently at each occurrence selected from the group consisting of hydrogen, alkyl, arylalkyl, heteroarylalkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, or heterocycloalkyl ;R130is independently as each occurrence selected from the group consisting of hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, -C(O)H, -C(O)OH, -C(O)alkyl, -C(O)Oalkyl, -C(O)(cycloalkyl, heterocycloalkyl, aryl, or heteroaryl), -C(O)O(cycloalkyl, heterocycloalkyl, aryl, or heteroaryl), alkenyl, or alkynyl; andR140is independently at each occurrence selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, fluoro, bromo, chloro, hydroxyl, alkoxy, azide, amino cyano, -NH(alkyl, cycloalkyl, heterocyloalkyl, aryl, or heteroaryl), -N(independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl), -NHSCMalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl), -N(alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl jStTalkyl, -NHSCLalkenyl, -N(alkyl)SO2alkenyl, -NHSChalkynyl, -N(alkyl)SO2alkynyl, haloalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

45. The compound of claim 38 selected from:

46. A method of synthesizing an adaptor molecule of Formula IIIAA-(C=O)-LA-TA(III) wherein AAis selected from an antibody or antigen recognizing fragment thereof; and all other variables are as defined in claim 37; the method comprising: reacting a compound of any one of claims 38-45 with an antibody or an antigen recognizing fragment thereof to form the adaptor molecule of Formula III.

47. An adaptor molecule of Formula III synthesized according to the method of claim 46.

48. A pharmaceutical composition comprising an adaptor molecule of claim 47 and a pharmaceutically acceptable carrier or excipient.

49. A chimeric antigen receptor (CAR) system, comprising i) an adaptor molecule of claim 47, and ii) a CAR comprising a receptor that is capable of binding to TAon the adaptor molecule, a hinge domain, and a signaling domain.

50. A cell comprising the CAR system of claim 49.

51. The cell of claim 50, wherein the cell is an immune cell, a neuron, an epithelial cell, an endothelial cell, or a stem cell.

52. The cell of claim 50 or claim 51 , wherein the cell is a T cell.

53. The cell of any one of claims 50-52, wherein the adaptor molecule is bound to the CAR.

54. A method of treating a medical disorder in a subject in need thereof comprising administering to the subject a cell of any one of claims 50-53.

55. A method of treating a medical disorder in a subject in need thereof comprising: a) providing an adaptor molecule of claim 47 ; b) administering to the subject a cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises a receptor that is capable of binding to TAon the adaptor molecule, a hinge domain, and a signaling domain; and c) administering the adaptor molecule to the subject.

56. The method of claim 54 or claim 55, wherein the medical disorder is a cancer, an autoimmune disorder, or a viral infection.

57. The method of claim 56, wherein the medical disorder is a cancer.

58. The method of claim 57, wherein the cancer is selected from the group consisting of lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin’s disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of head and neck, lung cancers, small cell lung cancer, nonsmall cell lung cancer, neuroblastoma, glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung, cervical carcinoma, breast cancer, epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, hematopoietic cancers, testicular cancer, colon cancer, rectal cancer, prostatic cancer, and pancreatic cancer.

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