WO2021127583A1 - Selective high affinity ligand diagnostics and therapeutics - Google Patents

Abstract

This disclosure provides novel Selective High Affinity Ligands (SHALs) and methods for their use in treating cancer, autoimmune disease, and obesity-related disorders. Also provided are methods of modulating drug sensitivity and metabolism in a subject comprising administration of the SHAL.

Description

SELECTIVE HIGH AFFINITY LIGAND DIAGNOSTICS AND

THERAPEUTICS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119(e) of U.S. Provisional

Application No. 62/952,055, filed December 20, 2019, the contents of which is incorporated by reference in its entirety into the present application.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on December 18, 2020, is named 121684-0120_ST25.txt and is 434 bytes in size.

FIELD OF THE DISCLOSURE

[0003] This disclosure pertains to the development of targeting molecules, inhibitors and immunomodulators. More particularly this disclosure pertains to the development of selective high affinity ligands (SHALs) that can be used in a manner analogous to antibodies, pro-drugs, inhibitors, and/or peptide ligands or antigens as affinity reagents, enzyme inhibitors, pro-drugs and/or immune system activators for the diagnosis and treatment of various diseases.

BACKGROUND

[0004] In general, the chemotherapeutics currently used as anti-cancer drugs are toxic to cells in both normal and cancerous tissues. Consequently, the side effects of such drugs can be as devastating to the patient as the malignant disease itself. Monoclonal antibodies and peptide ligands have been used to improve drug specificity/selectivity. In addition, antibody-drug conjugates that link cytotoxic agent to an antibody or peptide ligand directed against antigens present on malignant cells, but not present on normal cells, have been shown to selectively kill malignant cells. Antibody -based therapies, however, have their own limitations. Antibodies are large macromolecules that often do not effectively penetrate the tumor and gain access to all the malignant cells. They also can induce a life-threatening immune response in the patient that is directed against the therapeutic agent. In addition, antibodies often do not show sufficient specificity for the target ( e.g cancer) tissue and thus are useful in only limited therapeutic regimens.

[0005] Although significant advances have been made in the treatment of malignant disease, curative regimens for most patients have not yet been developed and those that show efficacy in suppressing tumor growth are often associated with toxicities that provide the patient with poor quality of life. Therefore, new strategies for the treatment of most malignant diseases are needed. These strategies should, as their goal, maximize therapeutic effect and minimize toxicity. One approach has involved the use of ligands specific for cell surface receptors or antibodies specific for malignant cell associated antigens as a means of targeting drugs or radioisotopes to the malignant cells. This approach is attractive for treating many malignant diseases because the malignant cells display a variety of tumor-restricted or upregulated antigens and/or receptors on their cell surfaces which are available for targeting. Thus far, antibody/antigen systems have been found to be better than ligand receptor systems because the antigens are more restricted than receptors and in greater abundance on the malignant cell.

[0006] Small molecule chemotherapeutics circumvent some of the disadvantages of antibody or peptide therapeutics, but most of today’s small molecule oncology drugs have other limitations. Following the repeated exposure of patients to many small molecule cancer drugs, their tumors often develop resistance to the drugs by increasing their expression of efflux transporter proteins, such as MDRl/P-gp and BCRP, that work to reduce the concentration of chemotherapeutics inside the tumor cells by rapidly pumping the drugs out of the cell and back into the blood. Increases in tumor cell expression of drug metabolizing enzymes such as the uridine diphosphate-glucuronyltransferase (UGT) and cytochrome P450 (CYP450) enzymes also contribute to this lowering of the concentration of many drugs and their active metabolites in tumor cells by chemically transforming them into a form that can be more easily exported from the cells and eliminated from the body. In the cases where resistance doesn’t develop, the increased expression of these drug metabolizing enzymes requires the drug be administered at significantly higher doses to maintain the intracellular concentration of the drug required to provide tumor cell killing. Because most small molecule therapeutics affect both normal and tumor cells, the requirement for the use of such high doses of the drugs often leads to patients experiencing serious adverse effects. SUMMARY

[0007] The SHALs in this disclosure overcome the limitations of prior therapeutic approaches not only in terms of use as anti-tumor agents and other therapeutic modalities but also as general diagnostics. The disclosure also identifies a group of new target molecules the SHAL binds to in addition to HLA-DR10, thereby expanding the range of cancers the SHAL diagnostics and drugs treat. These new target molecules include the efflux transporters present in bacteria and overexpressed in many cancers whose presence lead to the development of drug resistance by cancer cells and antibiotic resistance in many of the strains of bacteria called “Superbugs”.

[0008] In one aspect provided herein is a Selective High Affinity Ligand (SHAL) molecule of the structure Group A, Group B, or Group C, wherein Group A is of the structure:

(Group A), wherein:

Ri and R3 are each independently

R2 is

Group B is of the structure:

(Group B), wherein: Ri9 is

5 and R20 is

and

Group C is of the structure:

(Group C), wherein: R-21 is

R!!, R23, R2₆ and R27 are each independently

and

R24 and R25 are each independently

wherein each L is independently selected from Li, L2, L3, and L4:

wherein:

R₄ is H, NH2, N(CH₃)₂, CO2, NH(CH₃), NO2 or CF₃;

Rs is H, NH2, NO2 or CH₃;

Ai is a bond, -CH2-, -NH-, -N=N-, -0-, -CH2-CH2-, -CH2-NH-, -CH=NH-, -CH2-O-, -CH=CH- -NHCH2-, -NH=CH-, -OCH2-, phenyl ene, -NHNH-, -NHC(O)-, or -(O)CNH-; R6 is any one of:

R7 is H, Cl, or F; lei and R9 are each independently

A 2 is -NH-, -0-, -CH2-, -NHCH2-; R11 is H, methyl, Cl, NH2,

5 R12 is H, methyl, Cl, NH2,

Ri3 is H, methyl, Cl, NH2, or

Ri4 is methyl, H or NH2; Ri5 is methyl, H or NH2, or

wherein each L1-L4, * denotes attachment to the rest of the ligand L1-L4,

denotes attachment to the SHAL, and W is or OH; and R is a label tag or effector.

[0009] In another aspect, a composition comprising, consisting essentially of, or consisting of the SHAL disclosed herein and a carrier, is provided.

[0010] In another aspect, a method for one or more of: detecting a cancer cell that expresses or comprises atypical expression of Major Histocompatibility Complex Class II (MHC Class II) proteins, inhibiting the growth or proliferation of a cancer cell that express or has atypical expression of MHC Class II, or killing a cancer cell that expresses or has atypical expression of MHC Class II proteins is provided, the method comprising, consisting essentially of, or consisting of contacting the cells with an effective amount of: a SHAL having a structure from Group A, Group B, or Group C, comprising, consisting essentially of, or consisting of two or more ligands from Table 1, or a derivative thereof; the SHAL disclosed herein; or the composition disclosed herein, optionally wherein each cancer cell is independently selected from the group of pancreatic cancer, renal cancer, prostate cancer, liver cancer, stomach cancer, urothelial cancers, lung cancer, ovarian cancer, cervical cancer, esophageal cancer, breast cancer, thyroid cancer, colorectal cancer, bone cancer, laryngeal cancer, head and neck cancer, lymphoma, leukemia, myeloma, glioma, histiocytic sarcoma and melanoma.

[0011] In another aspect, a method of treating cancer, cancer cells or a solid tumor that expresses an MHC class II protein, in a subject in need thereof with the SHAL disclosed herein is provided, the method comprising, consisting essentially of, or consisting of treating the cancer cells or solid tumor in the subject by administering to the subject an effective amount of the SHAL, wherein the cancer cells or solid tumor are selected from one or more of pancreatic cancer, renal cancer, prostate cancer, liver cancer, stomach cancer, urothelial cancers, lung cancer, ovarian cancer, cervical cancer, esophageal cancer, breast cancer, thyroid cancer, colorectal cancer, bone cancer, laryngeal cancer, head and neck cancer, lymphoma, leukemia, myeloma, glioma, histiocytic sarcoma and melanoma.

[0012] In another aspect, a method of treating cancer, cancer cells or a tumor that does not express an MHC class II protein, in a subject in need thereof is provided, the method comprising, consisting essentially of, or consisting of administering to the subject a nanoparticle comprising, consisting essentially of, or consisting of a SHAL of a structure selected from Groups A, B, or C, containing two or more ligands from Table 1, or a derivative of each thereof.

[0013] In another aspect, a method for inducing, enhancing or promoting an anti tumor immune response in a subject in need thereof is provided, the method comprising, consisting essentially of, or consisting of administering to the subject an effective amount of a SHAL having a structure from Group A, Group B, or Group C, comprising, consisting essentially of, or consisting of two or more ligands from Table 1 and/or Table 2, or a derivative thereof.

[0014] In another aspect, a method to treat an MHC class II protein linked autoimmune disease or disorder selected from the group of Table 8 comprising, consisting essentially of, or consisting of Rheumatoid Arthritis, Multiple Sclerosis, Type-1 Diabetes, Grave’s Disease, Hashimoto’s Thyroiditis, Myasthenia Gravia, Celiac Disease, Ulcerative Colitis, Systemic Lupus Erythematosus, or Anklylosing Spondylitis in a subject in need thereof is provided, the method comprising, consisting essentially of, or consisting of administering to the subject an effective amount of a SHAL having a structure from Group A, Group B, or Group C, comprising, consisting essentially of, or consisting of two or more ligands from Table 1 and/or Table 2, or derivatives thereof.

[0015] In another aspect, a method for treating a disease or disorder related to a pathological immune response in a subject in need thereof is provided, the immune response contributing to a disease or disorder of the group of vascular injury and leucocyte recruitment leading to restenosis, allergic asthma, inflammation, and inflammation induced restriction of blood flow in ischemia stroke; the method comprising, consisting essentially of, or consisting of administering to the subject an effective amount of a SHAL having a structure from Group A, Group B, or Group C, comprising, consisting essentially of, or consisting of two or more ligands from Table 1 and/or Table 2, or derivatives thereof.

[0016] In another aspect, a method to inhibit cell growth and proliferation or to kill a cell by inhibiting a GTPase activating protein (GAP) selected from the group of MgcRacGAP, p50RhoGAP and BCR GAP is provided, the method comprising, consisting essentially of, or consisting of contacting the GAP with an effective amount of a SHAL having a structure from Group A, Group B, or Group C, comprising, consisting essentially of, or consisting of one or more ligands from Table 1 and Table 2, or a derivative thereof, thereby inhibiting the GAP.

[0017] In another aspect, a method to inhibit cell growth and proliferation or to kill a cell by directly inhibiting a GTPase enzyme selected from the group of Racl, Rac3, p50Rho, RhoA and Cdc42, the method comprising, consisting essentially of, or consisting of contacting the GTPase enzyme with an effective amount of a SHAL having a structure from Group A, Group B, or Group C is provided, the method comprising, consisting essentially of, or consisting of one or more ligands from Table 1 and Table 2, or a derivative thereof, thereby directly inhibiting the GTPase enzyme.

[0018] In another aspect, a method to inhibit cell growth or proliferation or to kill a cell by inhibiting AcetylCoA carboxylase (ACC) is provided, the method comprising, consisting essentially of, or consisting of contacting ACC with an effective amount of a SHAL having a structure from Group A, Group B, or Group C, comprising, consisting essentially of, or consisting of one or more ligands from Table 1 and Table 2, or a derivative thereof, thereby inhibiting ACC.

[0019] In another aspect, a method to prevent one or more drugs taken up by a mammalian or bacterial cell from being pumped back out of the cell by inhibiting a multidrug resistance protein 1 (P -glycoprotein, MDR1 or P-gp) or breast cancer resistance protein (BCRP) efflux transporter or its ortholog is provided, the method comprising, consisting essentially of, or consisting of contacting the transporter with an effective amount of a SHAL having a structure from Group A, Group B or Group C, comprising, consisting essentially of, or consisting of one or more ligands from Table 1 and/or Table 2, or a derivative thereof, thereby inhibiting the activity of a transporter protein.

[0020] In another aspect, a method to inhibit organic-anion-transporting polypeptide (OATP)-transporter mediated uptake of hormones, hormone conjugates, or growth promoting chemicals that a tumor cell requires to grow and survive is provided, the method comprising, consisting essentially of, or consisting of contacting OATP -transporter with an effective amount of a SHAL having a structure from Group A, Group B, or Group C, comprising, consisting essentially of, or consisting of one or more ligands from Table 1 and/or Table 2, or a derivative thereof, thereby inhibiting the activity of the OATP- transporter protein.

[0021] In another aspect, a method to reduce the required dosage of a drug delivered to a subject in need thereof by inhibiting metabolic UDP-glucuronosyltransferase (UGT) enzyme is provided, the method comprising, consisting essentially of, or consisting of contacting the UGT enzyme with an effective amount of a SHAL having a structure from Group A, Group B, or Group C, comprising, consisting essentially of, or consisting of one or more ligands from Table 1 and/or Table 2, or a derivative thereof, thereby inhibiting activity of the UGT enzyme.

[0022] In another aspect, a method to deliver one or more prodrugs to a cell is provided, the prodrug comprising, consisting essentially of, or consisting of a SHAL having a structure from Group A, Group B, or Group C, comprising, consisting essentially of, or consisting of one or more SHAL ligands from Table 1 and/or Table 2, or a derivative thereof, the method comprising, consisting essentially of, or consisting of binding the SHAL or a derivative thereof to a target protein or the cell.

[0023] In another aspect, a method of delivering to a cell an effective amount of a

SHAL having a structure from Group A, Group B or Group C, comprising, consisting essentially of, or consisting of one or more ligands from Table 1 and/or Table 2, or a derivative thereof is provided, the method comprising, consisting essentially of, or consisting of the two or more ligands binding simultaneously to two or more different sites on a protein, enzyme, or the cell to act as adjuvant to work synergistically with another drug.

[0024] In another aspect, a method to kill or inhibit the growth or proliferation of a cancer cell that expresses an MHC class II protein that is not HLA-DRIO or does not contain a Lym-1 epitope is provided, the method comprising, consisting essentially of, or consisting of contacting the cell with an effective amount of a SHAL having a structure from Group A, Group B, or Group C, comprising, consisting essentially of, or consisting of two or more ligands from Table 1 and/or Table 2, or a derivative thereof, wherein the cancer cell is selected from the group of pancreatic cancer, renal cancer, prostate cancer, liver cancer, stomach cancer, urothelial cancers, lung cancer, ovarian cancer, cervical cancer, esophageal cancer, breast cancer, thyroid cancer, colorectal cancer, bone cancer, laryngeal cancer, head and neck cancer, lymphoma, leukemia, myeloma, glioma, histiocytic sarcoma and melanoma.

[0025] In another aspect, a method of treating cancer cells or a tumor that expresses an MHC class II protein that is not HLA-DR10 or does not contain a Lym-1 epitope, in a subject in need thereof is provided, the method comprising, consisting essentially of, or consisting of administering to the subject an effective amount of a SHAL having the structure from Group A, Group B, Group C, Specimen-Group- Al, Specimen-Group-Bl, or Specimen- Group-Cl, containing two or more ligands from Table 1 and/or Table 2, or a derivative thereof, wherein the cancer is selected from the group of pancreatic cancer, renal cancer, prostate cancer, liver cancer, stomach cancer, urothelial cancers, lung cancer, ovarian cancer, cervical cancer, esophageal cancer, breast cancer, thyroid cancer, colorectal cancer, bone cancer, laryngeal cancer, head and neck cancer, lymphoma, leukemia, myeloma, glioma, histiocytic sarcoma and melanoma.

[0026] In another aspect, a method for treating cancer cells or a tumor that does not expresses an MHC class II protein, in a subject in need thereof is provded, the method comprising, consisting essentially of, or consisting of administering to the subject a nanoparticle comprising, consisting essentially of, or consisting of a SHAL of the structure selected from Group A, B, or C, comprising, consisting essentially of, or consisting of two or more ligands from Table 1 and/or Table 2, or a derivative thereof, thereby treating the cancer cells or tumor that does not express an MHC class II protein.

[0027] In another aspect, a method of treating cells, tissue, organs or tumors that do not express an MHC class II protein, in a subject in need thereof is provided, the method comprising, consisting essentially of, or consisting of administering to the subject a DOTA- tagged or biotin-tagged SHAL of the structure selected from Group A, B, or C, comprising, consisting essentially of, or consisting of two or more ligands from Table 1 and/or Table 2, complexed to a bispecific antibody, diabody or antibody-avidin conjugate that recognizes and binds to the DOTA or biotin on the SHAL and also recognizes and binds to a cell surface receptor or protein that is not an MHC Class II protein targeted by the SHAL

[0028] In another aspect, a method of pre-targeting a SHAL to a cell, tissue, organ or tumor in a subject is provided, the method comprising, consisting essentially of, or consisting of: administering to the subject a bispecific antibody, diabody or antibody-avidin conjugate that recognizes and binds to both: (a) a cell surface receptor or protein; and (b) a DOTA tag or biotin tag on the SHAL, the SHAL comprising, consisting essentially of, or consisting of the structure selected from Group A, B, or C, comprising, consisting essentially of, or consisting of two or more ligands from Table 1 and/or Table 2; followed by administering the SHAL to the subject after a suitable period of time.

[0029] In another aspect, a pre-targeting method for delivering a drug to a cell or tumor in a subject, the cell or tumor expressing an MHC class II protein recognized by a SHAL is provided, the method comprising, consisting essentially of, or consisting of: administering to the subject: (a) a biotin-tagged or DOTA-tagged SHAL complex comprising, consisting essentially of, or consisting of the SHAL of Group A, B, or C, comprising, consisting essentially of, or consisting of two or more ligands from Table 1 and/or Table 2, and (b) a bispecific antibody, diabody or antibody-avidin conjugate or fusion protein that recognizes and binds to both the DOTA tag or biotin tag of the SHAL and the drug; and administering the drug to the subject a suitable period of time after administration of (a) and (b).

[0030] In another aspect, a method to facilitate the delivery of a drug to a normal cell, tissue, organ or cancer cell expressing an MHC Class II protein, of a subject is provided, the method comprising, consisting essentially of, or consisting of: administering to the subject an anti-drug/anti-DOTA or biotin bispecific antibody, diabody, antibody-avidin conjugate, or fusion protein comprising, consisting essentially of, or consisting of both the drug bound thereto and a DOTA-tagged or biotin-tagged SHAL of the structure selected from Group A,

B, or C comprising, consisting essentially of, or consisting of two or more ligands from Table 1 and/or Table 2, thereby delivering the drug into cells expressing MHC Class II proteins targeted by the SHAL.

[0031] In another aspect, a method to kill or suppress the activity of an activated microglia, lymphocyte, dendritic cell or macrophage is provided, the method comprising, consisting essentially of, or consisting of contacting the activated microglia, lymphocyte, dendritic cell or macrophage with an effective amount of a SHAL of structure from Group A, Group B, or Group C, comprising, consisting essentially of, or consisting of two or more ligands from Table 1 and/or Table 2, or a derivative thereof. [0032] In another aspect, a microarray or microtiter plate comprising, consisting essentially of, or consisting of one or more SHAL(s) is provided, each SHAL having a structure independently selected from Group A, Group B, or Group C, or a derivative thereof, comprising, consisting essentially of, or consisting of one or more ligands from Table 1 and/or Table 2 that bind to a MHC class II protein, a transporter, a UGT metabolizing enzyme, a GAP, a GTPase, or an ACC enzyme, and optional instructions for use. In some embodiments, a kit is provided comprising, consisting essentially of, or consisting of the SHAL disclosed herein and instructions for use.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG. 1 : The binding of a biotinylated form of the SHAL cancer therapeutic

SH7139 to microarrays containing tumor biopsy sections from patients diagnosed with diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), Burkitt’s lymphoma (BL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), mucosa-associated lymphoid tissue lymphoma (MALTL) and anaplastic large cell lymphoma (ALCL) show the tumors of each of these types of non-Hodgkin’s lymphoma express the HLA-DR protein targeted and bind the SHAL drug. The biotin in the bound SHAL was detected using streptavidin horse-radish peroxidase reduction of 3,3-diaminobenzidine to produce a colored product. The amount of bound SHAL was determined by densitometric analysis of each tumor section.

[0034] FIG. 2: The binding of a biotinylated form of the SHAL cancer therapeutic

SH7129 to microarrays containing tumor biopsy sections from patients diagnosed with other types of solid cancers show the SHAL MHC-class II target is expressed on at least 16 additional cancers, and the expression is variable as shown also in the histogram. The biotin in the bound SHAL was detected using streptavidin horse-radish peroxidase reduction of 3,3- diaminobenzidine to produce a colored product. The amount of bound SHAL was determined by densitometric analysis of each tumor section.

[0035] FIG. 3 : SHALs can induce an anti -tumor immune response by binding into the antigen binding pocket of HLA-DRs. Because the SHAL bound to HLA-DR looks like a foreign peptide, it can be presented to T-cells and induce the formation of T-Helper cells which stimulate the production of antibodies targeting tumors with the SHALs bound to HLA-DR. SHALs can also act as a small molecule antibody-drug conjugate (ADC) wherein the linked ligands function as both the targeting agent (antibody) and the cell-killing agent (drug) following the selective release of one or more ligands or by metabolism of ligands with prodrug activity to produce active cytotoxic metabolites. Tumor cells killed by this mechanism release tumor antigens that are recognized as being foreign and stimulate the activation of cytotoxic T-cells that target the tumor directly.

[0036] FIG. 4: The classical MHC Class II exogenous antigen presentation pathway produces antibodies in response to foreign antigens. Exogeneous antigen is imported into antigen-presenting cells (APCs), such as dendritic cells, B-cells and macrophages, and then enters the endocytic pathway (encompassing the early endosome, late endosome and lysosome stages) where the antigen is degraded. At the same time MHC Class II molecules complexed with the invariant chain (Ii) move to the endocytic pathway, where the Ii chain is digested, leaving only CLIP bound to the MHC-Class-II molecule. CLIP is then replaced with degraded antigen and then the MHC/antigen complex is exported to the surface of the cell for presentation to CD4+ T-Helper Cells. In autoimmune diseases, recognition of self antigen produces autoantibodies against a constituent of its own tissues. By binding tightly inside the antigen binding pocket of HLA-DRs, SHALs can block self-antigen presentation by MCH Class II cells or, following the SHALs internalization and metabolism, it can kill B- cells to mitigate the production of autoantibodies (boxed).

[0037] FIGs. 5A-5B: To determine if SH7139 or a fragment of the SHAL (SH7117) containing only the Dv and Cb ligands inhibit the conversion of GTP to GDP by the GTPase directly, fast cycling mutants of Racl (FIG. 5 A) and Cdc42 (FIG. 5B) were tested for inhibition in the absence of the GAP proteins. GTP hydrolysis was assayed using the ADPhunter reagent. The inhibition by SH7139 is shown by the filled squares. The inhibition by SH7117 is shown by the open circles. The results show the rapid cycling Racl GTPase activity is inhibited by both SH7139 and SH7117. SH7139 and SH7117 are less effective in inhibiting the conversion of GTP to GDP by the rapid cycling Cdc42 GTPase.

[0038] FIG. 6: Structure of a polyvalent SHAL containing two SH7139 molecules linked together. [0039] FIG. 7: Example of a bispecific antibody used to deliver SHALs into a tumor that does not express MHC Class II proteins targeted by SH7139. These antibodies simultaneously recognize and bind to two different antigens. In the example shown, one arm of the antibody recognizes and binds to an antigen present on the surface of a tumor cell. The other arm recognizes and binds to the DOT A tag on the SHAL SH7139.

[0040] FIG. 8 shows SH7129 binding to different types of nine non-lymphoid solid cancers. SH7129 binding data shown in FIG. 2 were sorted by type for nine of the cancers and the binding to the different tumors within each type were plotted for comparison.

SH7129 binding to the different types of lung, liver, ovarian, laryngeal, gastric, breast and bone cancers were not found to be significantly different. In a number of cases there were two few cases to provide a meaningful comparison. A statistically significant difference was only observed for two types of cervical cancer; the squamous cell carcinomas (SC) bound more SH7129 than the adenocarcinoma (A) type (p = 0.006). Liver cancers: hepatocellular carcinoma (HC), bile duct carcinoma (BDC) and clear cell carcinoma (CCC). Ovarian cancers: serous cystadenocarcinoma (SC), endometrioid (EA), mucinous cystadenocarcinoma (MC), granulosa cell tumor (G), thecoma (T) and undifferentiated adenocarcinoma (U).

Breast cancers: ductal and medullary. Larynx cancer: squamous cell carcinoma (SCC), basal oid squamous cell carcinoma (BSCC) and acinic cell carcinoma (ACC). Gastric cancers: adenocarcinoma (AC) and ring cell carcinoma (RCC). Lung cancers: bronchioloalveolar carcinoma (BC), adenocarcinoma (A), squamous cell carcinoma (SCC), adenosquamous carcinoma (ASC) and neuroendocrine tumor (NT). Thyroid cancers: papillary carcinomas (PC), follicular papillary carcinoma (FC), tall cell papillary carcinoma (TCP), medullary carcinoma (MC), follicular adenoma (FA), colloid adenoma (CA), embryonic adenoma (EA) and clear cell adenoma (CCA). Cervical cancers: squamous cell carcinoma (SC), adenocarcinoma (A) and adenosquamous carcinoma (ASC). Bone cancers: osteosarcoma (OS) and chondrosarcoma (CS).

[0041] FIG. 9: Comparison of SH7129 binding to nine cancers by grade. SH7129 binding data shown in FIG. 2 were sorted by grade for nine of the cancers for which there was grade information, and the binding to the different tumors within each type were plotted for comparison. Statistical analyses of the data indicate there is no correlation between the amount of SH7129 bound and tumor grade in liver, ovarian, gastric, prostate, laryngeal, lung, cervical or pancreatic cancers. The comparison suggested what appears to be a significantly higher level of SH7129 binding to grade III compared to grade II kidney cancers (p =

0.0350), but this result is based on the analysis of only two grade III cases.

[0042] FIG. 10: Concentration-dependent growth inhibition of Raji (HLA-DR(+)) lymphoma cells by SH7129 and SH7139. Data at the 48-hour time point for Raji cells treated with SH7129 (blue line and filled squares) was collected in triplicate in two separate but identical experiments (n=6), while data for the cells treated with SH7139 (open circles and black line) was obtained in quadruplicate (n=4). The percent of non-viable cells in the untreated controls (< 5% over the course of the assays) was subtracted from the values obtained for the treated cells, and the data was fitted to a Boltzmann model to obtain the theoretical curves shown. The results show the replacement of the DOTA effector in SH7139 with biotin in SH7129 has little effect on the cytotoxicity of the SHAL to tumor cells expressing HLA-DR.

DETAILED DESCRIPTION

Definitions

[0043] As it would be understood, the section or subsection headings as used herein is for organizational purposes only and are not to be construed as limiting and/or separating the subject matter described.

[0044] As used in the specification and claims, the singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof.

[0045] As used herein, the term “comprising” is intended to mean that the compositions or methods include the recited steps or elements, but do not exclude others. “Consisting essentially of’ shall mean rendering the claims open only for the inclusion of steps or elements, which do not materially affect the basic and novel characteristics of the claimed compositions and methods. “Consisting of’ shall mean excluding any element or step not specified in the claim. Embodiments defined by each of these transition terms are within the scope of this disclosure. [0046] As used herein, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. The term “about” when used before a numerical designation, e.g., temperature, time, amount, and concentration, including range, indicates approximations which may vary by (+) or (-) 15%, 10%, 5%, 3%, 2%, or 1 %.

[0047] “Optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not.

[0048] As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0049] As used herein, the term “animal” refers to living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term “mammal” includes both human and non-human mammals.

[0050] The term “subject,” “host,” “individual,” and “patient” are as used interchangeably herein to refer to animals, typically mammalian animals. Any suitable mammal can be treated by a method, cell or composition described herein. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig). In some embodiments a mammal is a human. A mammal can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero). A mammal can be male or female. A mammal can be a pregnant female. In some embodiments a subject is a human. In some embodiments, a subject has or is suspected of having a cancer or neoplastic disorder.

[0051] “Eukaryotic cells” comprise all of the life kingdoms except monera. They can be easily distinguished through a membrane-bound nucleus. Animals, plants, fungi, and protists are eukaryotes or organisms whose cells are organized into complex structures by internal membranes and a cytoskeleton. The most characteristic membrane-bound structure is the nucleus. Unless specifically recited, the term “host” includes a eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include simian, bovine, porcine, murine, rat, avian, reptilian and human.

[0052] “Prokaryotic cells” usually lack a nucleus or any other membrane-bound organelles and are divided into two domains, bacteria and archaea. In addition to chromosomal DNA, these cells can also contain genetic information in a circular loop called on episome. Bacterial cells are very small, roughly the size of an animal mitochondrion (about 1-2 pm in diameter and 10 pm long). Prokaryotic cells feature three major shapes: rod shaped, spherical, and spiral. Instead of going through elaborate replication processes like eukaryotes, bacterial cells divide by binary fission. Examples include but are not limited to Bacillus bacteria, E. coli bacterium, and Salmonella bacterium.

[0053] Throughout this application, reference is made to treating or inhibiting the growth or proliferation of a cell such as a cancer cell. It should be understood that treatment or inhibition includes any cell, cell mass, tissue or organ comprising a cancerous or malignant cells. Non-limiting examples include solid tumors, blood cells, lymphnodes, tissues and organs.

[0054] A “composition” typically intends a combination of the active agent, e.g., the

SHAL of this disclosure and a naturally-occurring or non-naturally-occurring carrier, inert (for example, a detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like and include pharmaceutically acceptable carriers. Carriers also include pharmaceutical excipients and additives proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri, tetra-oligosaccharides, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume. Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid components, which can also function in a buffering capacity, include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. Carbohydrate excipients are also intended within the scope of this technology, examples of which include but are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffmose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myoinositol.

[0055] The compositions used in accordance with the disclosure, including cells, treatments, therapies, agents, drugs and pharmaceutical formulations can be packaged in dosage unit form for ease of administration and uniformity of dosage. The term "unit dose" or "dosage" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses in association with its administration, i.e., the appropriate route and regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the result and/or protection desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described herein.

[0056] As used herein, the terms “nucleic acid sequence” and “polynucleotide” are used interchangeably to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi -stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0057] The terms “specific binding” or “preferential binding” refer to that binding which occurs between such paired species as enzyme/substrate, receptor/agonist, antibody/antigen, and lectin/carbohydrate which may be mediated by covalent and/or non- covalent interactions. When the interaction of the two species typically produces a non- covalently bound complex, the binding which occurs is typically electrostatic, and/or involves hydrogen-bonding, and/or hydrophobic/lipophilic interactions. Accordingly, “specific binding” occurs between pairs of species where there is interaction between the two that produces a bound complex. In particular, the specific binding is characterized by the preferential binding of one member of a pair to a particular species as compared to the binding of that member of the pair to other species within the family of compounds to which that species belongs. Thus, for example, a ligand may show an affinity for a particular pocket on an HLA-DR10 molecule that is at least two-fold or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or 10 fold to 1,000,000 fold, greater than its affinity for a different pocket on the same or related proteins.

[0058] The terms “ligand” or “binding moiety”, as used herein, refers generally to a molecule that binds to a particular target molecule and forms a bound complex as described above. The binding can be highly specific binding, however, in certain embodiments, the binding of an individual ligand, such as those used to create SHALs, to the target molecule can be with relatively low affinity and/or specificity. The ligand and its corresponding target molecule form a specific binding pair. Examples include, but are not limited to small organic molecules, sugars, lectins, nucleic acids, proteins, antibodies, cytokines, receptor proteins, growth factors, nucleic acid binding proteins and the like which specifically bind desired target molecules, target collections of molecules, target receptors, target cells, and the like. Ligands of the disclosure are presented in Table 1 and are described further below.

[0059] The term “small organic molecule” refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes natural biological macromolecules ( e.g ., proteins, nucleic acids, etc.) and other man-made polymers ( e.g ., aptamers, peptoids, etc. Preferred small organic molecules range in size from about 300 Da up to about 5000 Da, or from about 300 Da up to 2000 Da, or from about 300 Da up to about 1000 Da.

[0060] The term “ligand library” refers to a collection (e.g., to a plurality) of ligands or potential ligands. The ligand library can be an actual physical library of ligands (e.g., NCI/DTP Open Chemicals Repository, ChemBridge DIVERSet-CL, MayBridge Collection, MedChemExpress Bioactive Screening Libraries, etc.) and/or a database (e.g, a compound database comprising descriptions of a plurality of potential ligands such as the MDL® Available Chemicals Directory, ChemSpider, ZINC 15, and the like). [0061] A “disulfide bond” as used herein a refers to a functional group with the structure R'-S-S-R², wherein R¹ and R² comprise, consist essentially of, or consist of two separate parts of the molecule each comprising, consisting essentially of, or consisting of an SH or thiol group, such as for example a peptide. The linkage is also called an SS-bond or sometimes a disulfide bridge and is usually derived by the coupling of two thiol groups. In biology, disulfide bridges formed between thiol groups in two cysteine residues are an important component of the secondary and tertiary structure of proteins. Disulfide bonds have proven useful in chemistry and biology because they can be broken by exposure to a reducing agent or environment, releasing the R¹ and/or R² or fragments of either or both thereof, wherein the released R¹ and/or R² or fragments of either or both thereof comprise free SH (thiol) groups.

[0062] “Suitable period of time” as used herein, intends any period of time between two actions, for example administration of a SHAL to a subject and administration of a drug or other therapeutic to a subject. The period of time between the two actions may be, for example, 0-1 min, 0-5 min, 0-10, min, 10-30 min, 30-60 min, 60-90 min, 90-120 min, 2-5 hr, 5-10 hr, 10-15 hr, 15-24 hr, 1-2 day, 2-5 day, 5-10 day, 10-20 days, or 20-30 days.

[0063] The term “SHAL” refers to a molecule comprising a plurality of ligands that each bind to a different region of the target molecule to which the SHAL is directed. The ligands are joined together either directly or through a linker or by attachment to a scaffold constructed of linkers in order to position the attached ligands in three-dimensional space so as to maximize the number of intermolecular contacts that can be made between the ligands present in the SHAL and the surface of a target molecule to form a polydentate moiety that typically shows a high avidity and selectivity for the target molecule. In certain embodiments, the SHAL comprises, consists essentially of, or consists of two or more ligands that bind their target with low affinity (e.g., < 10⁶M and/or dissociates within seconds or less) that, when coupled together, form a SHAL that binds the target with high affinity (e.g.,

> 10⁶M, or > 10⁷ M, or > 10⁸ M, or > 10⁹ M, or > 10 ¹⁰ M, or > 10 ¹¹ M and/or dissociates slowly, e.g., hours to days). The binding affinities of the SHALs can be estimated by mass spectrometry of the SHAL-target complexes (see, e.g., Prieto Conway MC, Whittal, RM, Baldwin, MA et al. J. Am. Soc. Mass Spectrom. 17: 967-976, 2006), followed by a more accurate surface plasmon resonance (SPR) spectroscopy (Shuck (1997) Annu. Rev. Biophys. Biomol. Struct., 26: 541-566; Van Regenmortal (2001) Cell Mol Life Sci., 58: 794-800; Rich RL and Myszka DG. J. Mol. Recognition 16: 351-382, 2003) measurement of the SHAL- target binding affinity using for example, an IASYS Plus or BiaCore instrument.

[0064] The term “polydentate” when used with respect to a SHAL indicates that the

SHAL comprises, consists essentially of, or consists of two or more ligands. The ligands typically bind independently and to different sites on the surface of the target molecule the SHAL is designed to recognize.

[0065] The terms “bidentate,” “tridentate”, and so forth when used with respect to a

SHAL refer to SHALs consisting of two ligands, SHALs consisting of three ligands, respectively, and so forth (e.g., tetradentate, pentadentate...).

[0066] The term “polyvalent SHAL” refers to a molecule in which two or more

SHALs (e.g., two or more bidentate, tridentate, and so forth SHALs) are joined together. Thus, for example a bivalent SHAL refers to a molecule in which two SHALs are joined together. A trivalent SHAL refers to a molecule in which three SHALs are joined together, and so forth. A bivalent version of the tridentate SHAL SH7139 is illustrated in FIG. 6).

[0067] A “polyspecific SHAL” comprises, consists essentially of, or consists of 2 or more SHALs joined together where each SHAL is polydentate and either or both SHALs can be either monovalent (i.e., bidentate, tridentate or so forth) or polyvalent so each polyspecific SHAL can bind to 2 or more different targets. For example, a SHAL can be synthesized with two or more ligands that bind in the cavities of HLA-DR and two or more ligands that bind in cavities on CD20 or CD22, or all 3, etc. This polyspecific SHAL could be used to target some cancers, such as lymphomas, that overexpress both HLA-DR and CD receptors.

[0068] The term “virtual in silico” when used, e.g., with respect to screening methods refers to methods that are performed without actual physical screening of the subject moieties. Typically, virtual in silico screening is accomplished computationally, e.g., utilizing computer generated models of the particular molecules (e.g., ligands and protein target) of interest. In certain embodiments, the virtual methods can be performed using physical models of the subject molecules and/or by simple visual inspection and manipulation. [0069] The phrase “target for a SHAL” refers to the moiety that is to be specifically bound by the SHAL. In some embodiments target for a SHAL refers to the protein the SHAL has been designed to bind to, such as an HLA-DR. In other embodiments the target would be the cancer cell that has the target protein on its surface.

[0070] The term “pocket” when referring to a pocket in a protein is a cavity, indentation or depression in the surface of the protein molecule that is created as a result of the folding of the peptide chain into the 3 -dimensional structure that makes the protein functional. A pocket can readily be recognized by inspection of the protein structure and/or by using commercially available protein modeling software (e.g., Autodock, CASTp,

PyMOL, etc ).

[0071] The terms “GTPase-activating protein” or “GTPase-accelerating protein”

(GAP) are a family of regulatory proteins whose members bind to and inhibit activated G proteins by stimulating their GTPase activity, with the result of terminating a signaling event. Examples include MgcRacGAP, p50RhoGAP and BCR GAP. MgcRacGAP refers to Rac GTPase Activating Protein 1, whose functions, nucleotide sequences, and amino acid sequences are known in the art and available, for example, at genecards.org/cgi- bin/carddisp.pl?gene=RACGAPl and uniprot.org/uniprot/Q9H0H5. P50rhoGAP refers to Rho GTPase Activating Protein 1, whose functions, nucleotide sequences, and amino acid sequences are available, for example, at genecards.org/cgi-bin/carddisp.pl?gene=ARHGAPl and uniprot.org/uniprot/Q07960. BCR GAP is short for Breakpoint Cluster Region (BCR) Activator Of RhoGEF And GTPase, whose functions, nucleotide sequences, and amino acid sequences are available, for example, at genecards.org/cgi-bin/carddisp.pl?gene=BCR and uniprot.org/uniprot/P11274, last accessed on December 17, 2020.

[0072] The term “organic-anion-transporting polypeptide” (OATP), as used herein, refers to a membrane transport protein or “transporter” that mediates the transport of mainly organic anions across the cell membrane. Therefore, OATPs are present in the lipid bilayer of the cell membrane, acting as the cell’s gatekeepers. Some OATP transporters, such as the OATP1 family, function both as an influx and efflux transporter.

[0073] As used herein, the term “UDP-glucuronosyltransferase” (UGT) refers to a cytosolic glycosyltransferase that catalyzes the transfer of the glucuronic acid component of UDP-glucuronic acid to a small hydrophobic molecule during phase II metabolism of the molecule.

[0074] The term “self-antigen” refers to any molecule or chemical group derived from an organism which acts as an antigen in inducing antibody formation in another organism but to which the healthy immune system of the parent organism is tolerant.

[0075] The phrase “small-molecule antibody-drug conjugate,” as used herein, refers to a small molecule (i.e., a SHAL) conjugated to a drug in which the small molecule performs the same function as the antibody in an antibody drug conjugate. In a small-molecule antibody-drug conjugate, the SHAL may be responsible for targeting and binding the conjugate to a specific antigen and/or affecting therapy by the SHAL functioning as a prodrug.

[0076] “Normal cell,” as used herein, refers to healthy cells, not experiencing proliferative dysfunction or cancer.

[0077] “Lym-1” as used herein, refers to an antibody that targets a conformational epitope on the beta-subunit of Human Leukocyte Antigen-antigen D Related (HLA-DR) proteins. Four amino acids on the beta-subunit are required for Lym-1 binding to HLA-DR, either an arginine or glutamine at position 70, an arginine at position 71, an alanine at position 74 and a valine at position 85.

[0078] “MHC Class II protein” refers to a class of major histocompatibility complex

(MHC) molecules normally found only on professional antigen-presenting cells such as dendritic cells, mononuclear phagocytes, some endothelial cells, thymic epithelial cells, and B-cell lymphocytes. These cells are important in initiating immune responses. The three human MHC class II protein isotypes that bind and present antigenic peptides (self and foreign) to receptors on T-cell lymphocytes are HLA-DR (Gene ID’s 3122 and 3123), HLA- DP (Gene IDs 3113, 3115, 3116 and 646702) and HLA-DQ (Gene IDs 3117-3120). MHC class II proteins with the same function are found in the dog (Gene IDs 474860-474862 and 481731; UniProtKB IDs I0CHJ4, D1G658, G8XQQ0, G1G668, A0A0K0KQB1, Q8MGV5 and Q1JRY3; IDP-MHC accession # DLA04913-DLA04916, DLA08125, DLA08142, DLA08152, DLA08176, DLA08179 and DLA08276; DLA DRB 1*47:01, DLA DRB 1*80:02), bull (Gene IDs 282528, 282530, 282534, 282535, 506214, and 539241; IDP- MHC accession # BoLA03116, BoLA03138, BoLAlOOl 1, BoLA09949, BoLA09877, BoLA09813, BoLA03234, and BoLA03136; UniProtKB IDs Q9MXT7, D6R0B0, and A0A3Q9XTM6; BoLADRB3*20:ll and BoLA DRB3*133:01), horse (Gene IDs 100052006, 100051944, 100052184, 100060320 and UniprotKB IDs SJX51227, Q30465, A0A346PXV6, Q9BDB6, Q9XRK8, Q9TPW7, G8FSA1-G8FSA4), pig (Gene IDs 100037921, 100135040, 100153386, and 100153387; IDP-MHC accession # SLA06004, SLA06037, SLA06047-SLA06051, SLA06062, SLA06074, SLA06076, SLA09697and SLA05980; UniProtKB IDs B4XKC4, A0A1Y0K5A1, Q8MGW9, Q8SPB1, Q8HX73, Q8MHH2, A7UAU2, and Q31079), sheep (Gene IDs 101109747, 100144754, 101119342, 101120364, 101114758, 101108961,105612264, 100153387, 101120148, 100037921, and 106990179; UniProtKB IDs Q95HC6, Q95HC9, A8IW56, W1I9L1 and Q9TP59; IDP-MHC accession # OLA02247, OLA02786, OLA08669, OLA08675, OLA088677, OLA08679, OLA08693, OLA08710, OLA08718, OLA08841, OLA08842, and OLA02978; Ovar DRB1*15:03) cat (Gene ID 101098301 and UniProtKB IDs C6ZK78, Q8MHQ4-Q8MHQ7), mouse (Gene IDs 14960 and 14969; UniProtKB IDs AAB17689.1, AWY63480.1, AIC84019.1, Q5XQH7, AAB17688.1, BAA14080.1, AWY63489.1, AWY63450, 002900, Q5XQJ3, PI 8469, P04231, AAA39630.1, CAG24025.1, CAG24019.1 and AAD31754.1) and rat (Gene IDs 309621, 294269, and 100911800; UniProtKB IDs Q9TQA5, Q9TQA7, QTUT75, Q5UT73, Q5UT78, F1M7F9, A0A0G2JW17, Q6AYB1 and Q5UT95). Reference herein is made to designations of public databases - for proteins: UniProtKB database: uniprot.org/help/uniprotkb and ncbi.nlm.nih.gov/protein; IDP-MHC database (Major histocompatibility sequences for a number of species): ebi.ac.uk/ipd/mhc/; IDP- IMGT/HLA database (Human major histocompatibility sequences): ebi.ac.uk/ipd/imgt/hla/. For gene sequences: Genbank: ncbi.nlm.nih.gov/genbank/ and Nucleotide database: ncbi.nlm.nih.gov/nucleotide/. All databases last accessed on December 19, 2019.

[0079] “HLA-DR” (Human Leukocyte Antigen-antigen D Related), as used herein, is the MHC class II cell surface receptor encoded by the human leukocyte antigen complex on chromosome 6 region 6p21.31. This receptor (Gene ID’s 3122 and 3123) has two subunits, an invariant alpha subunit and a variable sequence beta subunit. The complex of HLA-DR (Human Leukocyte Antigen - DR isotype) and peptides, generally between 9 and 30 amino acids in length, are presented by antigen presenting cells to the T-cell receptor (TCR) to activate other lymphocytes and induce an immune response. HLA-DP (Gene IDs 3113, 3115, 3116 and 646702) and HLA-DQ (Gene IDs 3117-3120) are two other MHC class II molecules that function as cell surface receptors for self and foreign antigens. These receptors also contain an alpha and beta subunit. Both subunits in these receptors have variants and much less is known about the peptides they bind.

[0080] “HLA-DR10” (DR10) refers to an HLA-DR serotype that contains a beta- subunit that is expressed by the thirty-six known allelic DRB 1*10 variants (e.g. DRB 1*1001, DRB 1*1002, DRB 1*1003, etc) of the DRB 1 gene (Gene IDs 3122 and 3123; Gencard #GC06m032578). Other human HLA-DR serotypes which comprise variants of the same DRB 1 gene (Gene ID 3123) include, but are not limited to HLA-DR1, HLA-DR3, HLA- DR4, HLA-DR7, HLA-DR8, HLA-DR9, HLA-DR11, HLA-DR12, HLA-DR13, HLA-DR 14, HLA-DR15, and HLA-DR16. HLA-DR ortholog proteins with the same function are found in the dog (UniProtKB IDs I0CHJ4, D1G658, G8XQQ0, G1G668, A0A0K0KQB1, Q8MGV5 and Q1JRY3; IDP-MHC accession # DLA04913- DLA04916, DLA08125, DLA08142, DLA08152, DLA08176, DLA08179 and DLA08276; DLA DRB 1*47:01, DLA DRB 1*80:02), bull (IDP-MHC accession # BoLA03116, BoLA03138, BoLAlOOl l, BoLA09949, BoLA09877, BoLA09813, BoLA03234, and BoLA03136; UniProtKB IDs Q9MXT7, D6R0B0, and A0A3Q9XTM6; BoLA DRB3*20:l l and BoLA DRB3* 133:01), horse (UniprotKB IDs SJX51227, Q30465, A0A346PXV6, Q9BDB6, Q9XRK8, Q9TPW7, G8FSA1-G8FSA4), pig (IDP-MHC accession # SLA06004, SLA06037, SLA06047- SLA06051, SLA06062, SLA06074, SLA06076, SLA09697and SLA05980; UniProtKB IDs B4XKC4, A0A1Y0K5A1, Q8MGW9, Q8SPB1, Q8HX73, Q8MHH2, A7UAU2, and Q31079), sheep (UniProtKB IDs Q95HC6, Q95HC9, A8IW56, W1I9L1 and Q9TP59; IDP- MHC accession # OLA02247, OLA02786, OLA08669, OLA08675, OLA088677, OLA08679, OLA08693, OLA08710, OLA08718, OLA08841, OLA08842, and OLA02978; Ovar DRBl* 15:03) cat (UniProtKB IDs C6ZK78, Q8MHQ4-Q8MHQ7), mouse (UniProtKB IDs AAB17689.1, AWY63480.1, AIC84019.1, Q5XQH7, AAB17688.1, BAA14080.1, AWY63489.1, AWY63450, 002900, Q5XQJ3, P18469, AAA39630.1, CAG24025.1, CAG24019.1, P04231 and AAD31754.1) and rat (UniProtKB IDs Q9TQA5, Q9TQA7, QTUT75, Q5UT73, Q5UT78, F1M7F9, A0A0G2JW17, Q6AYB1 and Q5UT95). Reference herein is made to designations of public databases - for proteins: UniProtKB database: uniprot.org/help/uniprotkb and ncbi.nlm.nih.gov/protein; IDP-MHC database (Major histocompatibility sequences for a number of species): ebi.ac.uk/ipd/mhc/; IDP- IMGT/HLA database (Human major histocompatibility sequences): ebi.ac.uk/ipd/imgt/hla/. For gene sequences: Genbank: ncbi.nlm.nih.gov/genbank/ and Nucleotide database: ncbi.nlm.nih.gov/nucleotide/. All databases last accessed on December 19, 2019.

[0081] “DRB1 (Gene ID 3123; Gencard #GC06m032578),” “DRB3 (Gene ID 3125;

Gencard #GC06Mn03715),” “DRB4” (Gene ID 3126; Gencard #GC06Mo03851) or “DRB5 (Gene ID 3127; Gencard # GC06M032519),” as used herein, refer to different paralogs of the beta subunit belonging to the HLA-DR family of MHC class II proteins. Reference herein is made to designations of public databases - for proteins: UniProtKB database: uniprot.org/help/uniprotkb and ncbi.nlm.nih.gov/protein; IDP-MHC database (Major histocompatibility sequences for a number of species): ebi.ac.uk/ipd/mhc/; IDP- IMGT/HLA database (Human major histocompatibility sequences): ebi.ac.uk/ipd/imgt/hla/. For gene sequences: Genbank: ncbi.nlm.nih.gov/genbank/ and Nucleotide database: ncbi.nlm.nih.gov/nucleotide/. All databases last accessed on December 19, 2019.

[0082] “Derivative” in reference to the SHAL, refers to a SHAL with a chemical modification of the original SHAL to which “derivative” refers. Such chemical modifications include any of those known in the art of chemical synthesis and include the addition or removal (for example covalently) of functional groups or moieties described herein. Examples of such functional groups and moieties include any defined herein such as micelle, nanoparticle, label, tag, effector, chelators, radionuclides; or functional groups such as alkyl, cyloalkyl, aryl, heterocycle, heteroaryl, alkoxy, amino, amide, thiol, halo, carboxyl, nitrile, oxo, alkenyl, or alkynyl. Other examples of derivatives include homologues, for example, a functional group such as alkylene, arylene, heteroarylene, cycloalkylene, alkenylene, alkynylene, amino, amidino, O, S, or other single atom may a point of connection between any two atoms of the derivative SHAL corresponding to two bonded atoms of the original SHAL. Derivatives also include stereoisomers, diastereomers, epimers, enantiomers or isotopic variants of the original SHAL. Such stereoisomers of a SHAL may be a substantially pure stereoisomer or mixtures of 2 or more stereoisomers of the SHAL derivatives of this disclosure. Derivatives of a SHAL may include a replacement of one or more of a linker, effector, or ligand in the original SHAL to which “derivative” refers. [0083] “Cytoreductive therapy,” as used herein, is a treatment that is used to reduce the number of cells in a lesion such as a tumor or other malignancy. The process is usually employed to remove as much of a tumor’s bulk as possible before a second treatment is delivered to maximize the tumor’s response to the second treatment. In cases wherein the second treatment may lead to a rapid killing of a large percentage of the cells in the tumor, this “debulking” of the tumor may also be accomplished using cytoreductive surgery to improve the efficacy of the second therapy and also minimize the patient’s likelihood of going into shock and dying when a large tumor mass disintegrates rapidly and dumps the potassium and other contents of all its cells into the bloodstream.

[0084] The phrase “atypical expression,” as used herein, is MHC class II protein expression in cells that do not express the proteins when they are functioning normally ( e.g . non-hematological tumor cells) or in cells that express MHC class II proteins when they are functioning in a way we want to prevent or stop (e.g. activated white blood cells in an autoimmune disease). It can also refer to increased levels of MHC class II expression that are higher than normal for the cell (e.g. leukemias and lymphomas, which are derived from lymphocytes).

[0085] The term “micelle,” refers to an aggregate (or supramolecular assembly) of surfactant molecules dispersed in a liquid as a colloid. A typical micelle in aqueous solution forms an aggregate with the hydrophilic “head” regions in contact with surrounding solvent, sequestering the hydrophobic single-tail regions in the micelle center. Drugs can be trapped inside micelles to facilitate their delivery to tumor cells and minimize systemic exposure to drugs that are highly toxic to both normal and cancer cells.

[0086] The term “carrier,” as used herein, refers a vehicle that aids in the delivery, handling or absorption of the SHAL it acts as a carrier for. SHALs can be mixed with a suitable pharmaceutical carrier (vehicle) or excipient as understood by practitioners in the art. Non-limiting examples of carriers and excipients include starch, milk, sugar, certain types of clay, gelatin, lactic acid, stearic acid or salts thereof, including magnesium or calcium stearate, talc, vegetable fats or oils, gums and glycols.

[0087] A “liposome,” as used herein, is a spherical vesicle having at least one lipid bilayer. The liposome can be used as a vehicle for delivering nutrients, pharmaceutical drugs or other molecules (e.g. antibodies, DNA, RNA, peptides, etc.) into cells. Liposomes can be prepared by disrupting biological membranes (such as by sonication).

[0088] A “nanoparticle,” as used herein, refers to particles between 1 and

500 nanometers (nm) in diameter with a surrounding interfacial layer. The interfacial layer is an integral part of nanoscale particle that gives the nanoparticle its unique properties. The interfacial layer typically comprises, consists essentially of, or consists of ions, inorganic and organic molecules, which may include polymers. Nanoparticles are well known in the art and described in the literature, for example, Salata, et al., Journal of Nanobiotechnology volume 2, Article number: 3 (2004), the entire disclosure of which is hereby incorporated by reference. Biocompatible nanoparticles known in the art that may be used in the present compositions include silver, gold, hydroxyapatite, clay, titanium dioxide, silicon dioxide, zirconium dioxide, carbon, diamond, aluminum oxide, ytterbium trifluoride, albumin, amino acid based polymers, dextran, chitosan, cyclodextrine, cetylpalmitate, and biodegradeable polymers such as poly(lactic-co-glycolic acid) (PLGA), polyethylene glycol poly(lactic-co- glycolic acid) (PEG-PLGA), PLGH, polyalkylcyanoacrylate (PACA), N-(2-hydroxypropyl) methacrylamide (HPMA), polybutylcyanoacrylate (PBCA), methoxypolyethylene polylactic acid (mPEG-PLA), polyethylene glycol polyacrylic acid (PEG-PAA), poly(D,L-lactic-co- glycolic acid)-6/oc&-poly(eth-ylene glycol) (PLGA-b-PEG), polyethylene glycol polycyclic aromatic hydrocarbon (PEG-b-PAH), polyethylene glycol polyglutamic acid (PEG-PGA), polyhydroxybutyrate, poly(ethyleneoxide-modified poly (b-amino ester), D-a-tocopheryl polyethylene glycol succinate (TPGS) nanoparticles.

[0089] A hydrogel is a network of polymer chains that are hydrophilic, sometimes found as a colloidal gel in which water is the dispersion medium. A three-dimensional solid results from the hydrophilic polymer chains being held together by cross-links. Because of the inherent cross-links, the structural integrity of the hydrogel network does not dissolve from the high concentration of water. Hydrogels are highly absorbent (they can contain over 90% water) natural or synthetic polymeric networks. Examples include polyacrylamide, polymacon, silicone hydrogels and of cross-linked polymers such as polyethylene oxide, poly AMPS and polyvinylpyrrolidone.

[0090] As used herein, a “cancer” is a disease state characterized by cells demonstrating abnormal uncontrolled replication and in some aspects, the term is used interchangeably with the term “tumor.” A “solid tumor” is an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors can be benign or malignant, metastatic or non-metastatic. Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors include sarcomas, carcinomas, and lymphomas.

[0091] The term “cancer markers” refers to biomolecules such as proteins that are useful in the diagnosis, prognosis and treatment of cancer. As used herein, “cancer markers” include but are not limited to: prostate specific antigen (PSA), human chorionic gonadotropin, beta-2-microglobulin, alpha-fetoprotein, carcinoembryonic antigen (CEA), bladder tumor antigen, chromogranin, calcitonin, cancer antigen (CA) 125, CA 15-3, CA 19- 9, CA 27.29, cluster of differentiation proteins CD2, CD4, CDlla, CD20, CD22, CD25, CD27, CD30, CD31, CD33, CD34, CD40, CD44, CD47, CD52, CD54, CD58, CD62L,

CD70, CD79a, CD80, CD86, CD105, CD115, CD117, CD120b, CD127, CD134, CD137, CD137L, CD 146, CD152, CD153, CD154, CD178, CD223, CD252, CD253, CD257,

CD258, CD261, CD262, CD264, CD266, CD268, CD269, CD270, CD272, CD273, CD274, CD275, CD276, CD278, CD279, CD309, CD314, CD339, CD357, cadherin 13 (CDH13), D16S422HER-2, phospatidylinositol 3-kinase (PI 3-kinase), HLA-DRs, MUC-1, gastrin, HE4, 5-HIAA, lactate dehydrogenase, neuron specific enolase, nuclear matrix protein 22, prostatic acid phosphatase, somatostatin receptor, thyroglobulin, trypsin, trypsin- 1 complexed with alpha(l)-antitrypsin, estrogen receptor, progesterone receptor, c-erbB-2, bcl- 2, S-phase fraction (SPF), pl85erbB-2, low-affinity insulin like growth factor-binding protein, urinary tissue factor, vascular endothelial growth factor, urokinase plasminogen activator, epidermal growth factor, epidermal growth factor receptor, apoptosis proteins (p53, Ki67), factor VIII, adhesion proteins (sialyl-TN, blood group A, bacterial lacZ, human placental alkaline phosphatase (ALP), alpha-difluoromethylornithine (DFMO), thymidine phosphorylase (dTHdPase), thrombomodulin, laminin receptor, fibronectin, anticyclins, anticyclin A, B, or E, proliferation associated nuclear antigen, lectin UEA-1, and von Willebrand’s factor.

[0092] The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The term also includes variants on the traditional peptide linkage joining the amino acids making up the polypeptide such as those incorporating unnatural a-, b-, and g- amino acids, peptoids, and peptide isosteres.

[0093] The terms “nucleic acid” or “oligonucleotide” or grammatical equivalents herein refer to at least two nucleotides covalently linked together. A nucleic acid of the present invention is preferably single-stranded or double stranded and will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage et al. (1993) Tetrahedron 49(10): 1925) and references therein; Letsinger (1970) J. Org. Chem. 35:3800; Sprinzl et al. (1977) Eur. J. Biochem. 81: 579; Letsinger et al. (1986) Nucl. Acids Res. 14: 3487; Sawai et al. (1984) Chem. Lett. 805, Letsinger et al. (1988) J.

Am. Chem. Soc. 110: 4470; and Pauwels et al. (1986) Chemica Scripta 26: 1419), phosphorothioate (Mag et al. (1991) Nucleic Acids Res. 19:1437; and U.S. Patent No. 5,644,048), phosphorodithioate (Briu et al. (1989) J. Am. Chem. Soc. Ill :2321, O- methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), and peptide nucleic acid backbones and linkages (see Egholm (1992) J. Am. Chem. Soc. 114:1895; Meier et al. (1992) Chem. Int. Ed. Engl. 31: 1008; Nielsen (1993) Nature, 365: 566; Carlsson et al. (1996) Nature 380: 207). Other analog nucleic acids include those with positive backbones (Denpcy et al. (1995) Proc. Natl. Acad. Sci. USA 92: 6097; non-ionic backbones (U.S. Patent Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Angew. (1991) Chem. Inti. Ed. English 30: 423; Letsinger et al. (1988) J. Am. Chem. Soc. 110:4470; Letsinger et al. (1994) Nucleoside & Nucleotide 13:1597; Chapters 2 and 3, ASC Symposium Series 580, “Carbohydrate Modifications in Antisense Research”, Ed. Y.S. Sanghui and P. Dan Cook; Mesmaeker et al. (1994), Bioorganic & Medicinal Chem. Lett. 4: 395; Jeffs et al. (1994) J. Biomolecular NMR 34:17; Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones, including those described in U.S. Patent Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, Carbohydrate Modifications in Antisense Research, Ed. Y.S. Sanghui and P. Dan Cook. Nucleic acids comprising, consisting essentially of, or consisting of one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins et al. (1995),

Chem. Soc. Rev. pp 169- 176). Several nucleic acid analogs are described in Rawls, C & E News June 2, 1997 page 35. These modifications of the ribose-phosphate backbone may be done to facilitate the addition of additional moieties such as labels, or to increase the stability and half-life of such molecules in physiological environments.

[0094] The term “biotin” refers to biotin and modified biotins or biotin analogues that are capable of binding avidin or various avidin analogues. “Biotin”, can be, inter alia, modified by the addition of one or more functional groups or small molecules, usually through its free carboxyl residue. Useful biotin derivatives include, but are not limited to, active esters, amines, hydrazides, fluorescent or luminescent tags, and thiol groups that are coupled with a complimentary reactive group such as an amine, an acyl or alkyl group, a carbonyl group, an alkyl halide or a Michael-type acceptor on the appended compound or polymer.

[0095] Avidin, typically found in egg whites, has a very high binding affinity for biotin, which is aB-complex vitamin (Wilcheck et al. (1988) Anal. Biochem. 171: 1). Streptavidin, derived from Streptomyces avidinii, is similar to avidin, but has lower non specific tissue binding, and therefore often is used in place of avidin. As used herein “avidin” includes all of its biological forms either in their natural states or in their modified forms (e.g., streptavidin, neutravidin, etc.). Modified forms of avidin which have been treated to remove the protein’s carbohydrate residues (“deglycosylated avidin”), and/or its highly basic charge (“neutral avidin”), for example, also are useful in the invention.

[0096] The term “residue” as used herein refers to natural, synthetic, or modified amino acids.

[0097] As used herein, an “antibody” refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

[0098] A typical immunoglobulin (antibody) structural unit is known to comprise, consist essentially of, or consist of a tetramer. Each tetramer is comprises, consists essentially or, or consists of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.

[0099] Antibodies exist as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)’2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)’2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab’)2 dimer into a Fab’ monomer. The Fab’ monomer is essentially a Fab with part of the hinge region (see, Fundamental Immunology, W.E. Paul, ed., Raven Press, N.Y. (1993), for a more detailed description of other antibody fragments). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such Fab’ fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein also includes antibody fragments either produced by the modification of whole antibodies, by expression in vitro or synthesized de novo using recombinant DNA methodologies. Preferred antibodies include single chain antibodies (antibodies that exist as a single polypeptide chain), more preferably single chain Fv antibodies (sFv or scFv) in which a variable heavy and a variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide. The single chain Fv antibody is a covalently linked VH-VL heterodimer which may be expressed from a nucleic acid including VH- and VL- encoding sequences either joined directly or joined by a peptide-encoding linker. Huston, et al. (1988) Proc. Nat. Acad. Sci. USA, 85: 5879-5883. While the VH and VL are connected to each as a single polypeptide chain, the VH and VL domains associate non-covalently. The first functional antibody molecules to be expressed on the surface of filamentous phage were single-chain Fv’s (scFv), however, alternative expression strategies have also been successful. For example Fab molecules can be displayed on phage if one of the chains (heavy or light) is fused to g3 capsid protein and the complementary chain exported to the periplasm as a soluble molecule. The two chains can be encoded on the same or on different replicons; the important point is that the two antibody chains in each Fab molecule assemble post-translationally and the dimer is incorporated into the phage particle via linkage of one of the chains to, e.g., g3p (see, e.g., U.S. Patent No: 5733743). The scFv antibodies and a number of other structures converting the naturally aggregated, but chemically separated light and heavy polypeptide chains from an antibody V region into a molecule that folds into a three-dimensional structure substantially similar to the structure of an antigen-binding site are known to those of skill in the art (see e.g., U.S. Patent Nos. 5,091,513, 5,132,405, and 4,956,778). Particularly preferred antibodies should include all that have been displayed on phage or yeast (e.g., scFv, Fv, Fab and disulfide linked Fv (Reiter et al. (1995) Protein Eng.

8: 1323-1331).

[0100] The term “specifically binds,” as used herein, when referring to a SHAL or to a biomolecule (e.g., protein, nucleic acid, antibody, etc.), refers to a binding reaction that is determinative of the presence of the SHAL or biomolecule in a heterogeneous population of molecules (e.g., proteins and other biologies). Thus, under designated conditions (e.g., binding assay conditions in the case of a SHAL or stringent hybridization conditions in the case of a nucleic acid), the specified ligand or SHAL preferentially binds to its particular “target” molecule and preferentially does not bind in a significant amount to other molecules present in the sample.

[0101] An “effector” refers to any molecule or combination of molecules whose activity it is desired to deliver into and/or localize at a target (e.g., a cell displaying a characteristic marker). Such effectors include, but are not limited to radiolabels, cytotoxins, enzymes, growth factors, transcription factors, drugs, lipids, divalent or trivalent metal ions, etc. In other embodiments an “effector” refers to macromolecular structures such as nanoparticles, liposomes, or micelles that carry, deliver or transport other molecules contained within them into cells, blood vessels or across barriers (e.g., the blood-brain or blood-testis barrier).

[0102] A “reporter” is an effector that provides a detectable signal (e.g., a detectable label). In certain embodiments, the reporter need not provide the detectable signal itself, but can simply provide a moiety that subsequently can bind to a detectable label. [0103] “Microglia-mediated neurodegenerative disease” may include any disease mediated by dysfunction of the microglia. For example, the disease may include neuropathic pain, neuroinflammation, amyloid deposition, tau protein deposition, and the like.

[0104] The term “conservative substitution” is used in reference to proteins or peptides to reflect amino acid substitutions that do not substantially alter the activity (specificity or binding affinity) of the molecule. Typically, conservative amino acid substitutions involve substitution of one amino acid for another amino acid with similar chemical properties (e.g., charge or hydrophobicity). The following six groups each contain amino acids that are typical conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0105] The terms “epitope tag” “affinity tag” or simply “tag” are used interchangeably herein, and usually refers to a molecule or domain of a molecule that is specifically recognized by an antibody or other binding partner. The term also refers to the binding partner complex as well. Thus, for example, biotin or a biotin/avidin complex are both regarded as an affinity tag. In addition to epitopes recognized in epitope/antibody interactions, affinity tags also comprise, consist essentially of, or consist of “epitopes” recognized by other binding molecules (e.g., ligands bound by receptors), ligands bound by other ligands to form heterodimers or homodimers, His6 bound by Ni-NTA, biotin bound by avidin, streptavidin, or anti-biotin antibodies, and the like. Other tags known to those of skill in the art include include chitin binding protein (CBP), maltose binding protein (MBP), polyanionic amino acids such as FLAG-tag, avi-tage, C-tag, Calmodulin-tag, polyglutamate tage, E-tag, HA-tag, His-tag, Myc-tag, NE-tag, RholD4-tag, S-tag, SBP-tag, Softag 1, Softag 3, Spot-tag, Strep-tag, T7-tag, TC tag, Ty tag, V5 tag, VSV tag, Xpress tag, isopeptag, Spy Tag, SnoopTag, DogTag, SdyTag, Spy Tag/Spy Catcher, BCCP, green fluorescent protein tag, halotag, SNAP -tag, CLIP -tag, maltose binding protein-tag, Nus-tag, thioredoxin-tag, Fc-tag, carbohydrate recognition domain or CRDSAT-tag, Strep-tag and glutathione-S-transferase (GST). The poly(His) tag is a widely used protein tag, which binds to metal matrices.

Epitope tags include V5-tag, Myc-tag, HA-tag, Spot-tag, T7-tag and NE-tag. [0106] Epitope tags are well known to those of skill in the art. Moreover, antibodies specific to a wide variety of epitope tags are commercially available. These include but are not limited to antibodies against the DYKDDDDK (SEQ ID NO: 1) epitope, c-myc antibodies (available from Sigma Chemical Co., St. Louis, MO), the HNK-1 carbohydrate epitope, the HA epitope, the HSV epitope, the His4, Hiss, and His₆ epitopes that are recognized by the His epitope specific antibodies (see, e.g., Qiagen Inc., Germantown, MD), and the like. In addition, vectors for epitope tagging proteins are commercially available. Thus, for example, the pCMV-Tagl vector is an epitope tagging vector designed for gene expression in mammalian cells. A target gene inserted into the pCMV-Tagl vector can be tagged with the FLAG ® epitope (N-terminal, C-terminal or internal tagging), the c-myc epitope (C-terminal) or both the FLAG (N-terminal) and c-myc (C-terminal) epitopes.

[0107] “Label” as used herein, refers to a moiety which may aid with the visualization or imaging of the SHAL. Labels may include radioactive isotopes, radiopaque labels, fluorescent or luminescent moieties. Labels include moieties detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.

[0108] A “PEG type linker” refers to a linker comprising a polyethylene glycol

(PEG).

[0109] “Immune cells” includes, e.g., white blood cells (leukocytes) which are derived from hematopoietic stem cells (HSC) produced in the bone marrow, lymphocytes (T cells, B cells, natural killer (NK) cells) and myeloid-derived cells (neutrophil, eosinophil, basophil, monocyte, macrophage, dendritic cells). “T cell” includes all types of immune cells expressing CD3 including T-helper cells (CD4+ cells), cytotoxic T-cells (CD8+ cells), natural killer T-cells, T-regulatory cells (Treg) and gamma-delta T cells. A “cytotoxic cell” includes CD8+ T cells, natural-killer (NK) cells, and neutrophils, which cells are capable of mediating cytotoxicity responses. Cytokines are small secreted proteins released by immune cells that have a specific effect on the interactions and communications between the immune cells. Cytokines can be pro-inflammatory or anti-inflammatory. Non-limiting example of a cytokine is Granulocyte-macrophage colony-stimulating factor (GM-CSF), which stimulates stem cells to produce granulocytes (neutrophils, eosinophils, and basophils) and monocytes. [0110] As used herein, the phrase "immune response" or its equivalent

"immunological response" refers to the development of a cell-mediated response (e.g. mediated by antigen-specific T cells or their secretion products). A cellular immune response is elicited by the presentation of polypeptide epitopes in association with Class I or Class II MHC molecules, to treat or prevent a viral infection, expand antigen-specific B-reg cells,

TCI, CD4+ T helper cells and/or CD8+ cytotoxic T cells and/or disease generated, autoregulatory T cell and B cell “memory” cells. The response may also involve activation of other components. In some aspect, the term “immune response” may be used to encompass the formation of a regulatory network of immune cells. Thus, the term “regulatory network formation” may refer to an immune response elicited such that an immune cell, preferably a T cell, more preferably a T regulatory cell, triggers further differentiation of other immune cells, such as but not limited to, B cells or antigen-presenting cells - non-limiting examples of which include dendritic cells, monocytes, and macrophages. In certain embodiments, regulatory network formation involves B cells being differentiated into regulatory B cells; in certain embodiments, regulatory network formation involves the formation of tolerogenic antigen-presenting cells.

[0111] The term “transduce” or “transduction” as it is applied to the production of chimeric antigen receptor cells refers to the process whereby a foreign nucleotide sequence is introduced into a cell. In some embodiments, this transduction is done via a vector.

[0112] The term “B-cell lymphoma or leukemia” refers to a type of cancer that forms in issues of the lymphatic system or bone marrow and has undergone a malignant transformation that makes the cells within the cancer pathological to the host organism with the ability to invade or spread to other parts of the body.

[0113] As used herein, the term “comprising” is intended to mean that the compositions and methods include the recited elements, but do not exclude others. “Consisting essentially of’ when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the intended use. For example, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives and the like. “Consisting of’ shall mean excluding more than trace elements of other ingredients and sub stantial method steps for administering the compositions disclosed herein. Aspects defined by each of these transition terms are within the scope of the present disclosure.

[0114] As used herein, the term “detectable marker” refers to at least one marker capable of directly or indirectly, producing a detectable signal. A non-exhaustive list of this marker includes enzymes which produce a detectable signal, for example by colorimetry, fluorescence, luminescence, such as horseradish peroxidase, alkaline phosphatase, b- galactosidase, glucose-6-phosphate dehydrogenase, chromophores such as fluorescent, luminescent dyes, groups with electron density detected by electron microscopy or by their electrical property such as conductivity, amperometry, voltammetry, impedance, detectable groups, for example whose molecules are of sufficient size to induce detectable modifications in their physical and/or chemical properties, such detection may be accomplished by optical methods such as diffraction, surface plasmon resonance, surface variation , the contact angle change or physical methods such as atomic force spectroscopy, tunnel effect, or radioactive molecules such as ³² P, ³⁵ S or ¹²⁵1.

[0115] As used herein, the term “purification marker” or “reporter protein” refer to at least one marker useful for purification or identification. A non-exhaustive list of this marker includes His, lacZ, GST, maltose-binding protein, NusA, BCCP, c-myc, CaM, FLAG, GFP, YFP, cherry, thioredoxin, poly(NANP), V5, Snap, HA, chitin-binding protein, Softag 1, Softag 3, Strep, or S-protein. Suitable direct or indirect fluorescence marker comprise FLAG, GFP, YFP, RFP, dTomato, cherry, Cy3, Cy 5, Cy 5.5, Cy 7, DNP, AMCA, Biotin, Digoxigenin, Tamra, Texas Red, rhodamine, Alexa fluors, FITC, TRITC or any other fluorescent dye or hapten.

[0116] As used herein, the term “expression” refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. The expression level of a gene may be determined by measuring the amount of mRNA or protein in a cell or tissue sample. In one aspect, the expression level of a gene from one sample may be directly compared to the expression level of that gene from a control or reference sample. In another aspect, the expression level of a gene from one sample may be directly compared to the expression level of that gene from the same sample following administration of a compound.

[0117] As used herein, “homology” or “identical”, percent “identity” or “similarity”, when used in the context of two or more nucleic acids or polypeptide sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, e.g., at least 60% identity, preferably at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region (e.g., nucleotide sequence encoding the chimeric PVX described herein). Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. The alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST, using default parameters. In particular, preferred programs are BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov/cgi-bin/BLAST. The terms “homology” or “identical,” percent “identity” or “similarity” also refer to, or can be applied to, the complement of a test sequence. The terms also include sequences that have deletions and/or additions, as well as those that have substitutions. As described herein, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is at least 50-100 amino acids or nucleotides in length. An “unrelated” or “non-homologous” sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences disclosed herein. [0118] The phrase “first line” or “second line” or “third line” refers to the order of treatment received by a patient. First line therapy regimens are treatments given first, whereas second or third line therapy are given after the first line therapy or after the second line therapy, respectively. The National Cancer Institute defines first line therapy as “the first treatment for a disease or condition. In patients with cancer, primary treatment can be surgery, chemotherapy, radiation therapy, or a combination of these therapies. First line therapy is also referred to those skilled in the art as “primary therapy and primary treatment.” See National Cancer Institute website at www.cancer.gov, last visited on May 1, 2008. Typically, a patient is given a subsequent chemotherapy regimen because the patient did not show a positive clinical or sub-clinical response to the first line therapy or the first line therapy has stopped.

[0119] It is to be inferred without explicit recitation and unless otherwise intended, that when the present disclosure relates to a polypeptide, protein, polynucleotide, an equivalent or a biologically equivalent of such is intended within the scope of this disclosure. As used herein, the term “biological equivalent thereof’ is intended to be synonymous with “equivalent thereof’ when referring to a reference protein, polypeptide or nucleic acid, intends those having minimal homology while still maintaining desired structure or functionality. Unless specifically recited herein, it is contemplated that any of the above also includes equivalents thereof. For example, an equivalent intends at least about 70% homology or identity, or at least 80% homology or identity and alternatively, or at least about 85%, or alternatively at least about 90%, or alternatively at least about 95%, or alternatively at least 98% percent homology or identity and/or exhibits substantially equivalent biological activity to the reference protein, polypeptide, or nucleic acid. Alternatively, when referring to polynucleotides, an equivalent thereof is a polynucleotide that hybridizes under stringent conditions to the reference polynucleotide or its complement.

[0120] The phrase “equivalent polypeptide” or “equivalent peptide fragment” refers to protein, polynucleotide, or peptide fragment encoded by a polynucleotide that hybridizes to a polynucleotide encoding the exemplified polypeptide or its complement of the polynucleotide encoding the exemplified polypeptide, under high stringency and/or which exhibit similar biological activity in vivo, e.g., approximately 100%, or alternatively, over 90% or alternatively over 85% or alternatively over 70%, as compared to the standard or control biological activity. Additional embodiments within the scope of this disclosure are identified by having more than 60%, or alternatively, more than 65%, or alternatively, more than 70%, or alternatively, more than 75%, or alternatively, more than 80%, or alternatively, more than 85%, or alternatively, more than 90%, or alternatively, more than 95%, or alternatively more than 97%, or alternatively, more than 98% or 99% sequence homology. Percentage homology can be determined by sequence comparison using programs such as BLAST run under appropriate conditions. In one aspect, the program is run under default parameters.

[0121] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) having a certain percentage (for example, 80%, 85%, 90%, or 95%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. The alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST, using default parameters. In particular, preferred programs are BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov/cgi-bin/BLAST.

[0122] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme. [0123] Examples of stringent hybridization conditions include: incubation temperatures of about 25 °C to about 37 °C; hybridization buffer concentrations of about 6x SSC to about lOx SSC; formamide concentrations of about 0% to about 25%; and wash solutions from about 4x SSC to about 8x SSC. Examples of moderate hybridization conditions include: incubation temperatures of about 40 °C to about 50 °C; buffer concentrations of about 9x SSC to about 2x SSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5x SSC to about 2x SSC. Examples of high stringency conditions include: incubation temperatures of about 55°C to about 68°C; buffer concentrations of about lx SSC to about O.lx SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about lx SSC, O.lx SSC, or deionized water. In general, hybridization incubation times are from 5 minutes to 24 hours, with 1, 2, or more washing steps, and wash incubation times are about 1, 2, or 15 minutes. SSC is 0.15 MNaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be employed.

[0124] The term “isolated” as used herein refers to molecules or biologicals or cellular materials being substantially free from other materials. In one aspect, the term “isolated” refers to nucleic acid, such as DNA or RNA, or protein or polypeptide, or cell or cellular organelle, or tissue or organ, separated from other DNAs or RNAs, or proteins or polypeptides, or cells or cellular organelles, or tissues or organs, respectively, that are present in the natural source. The term “isolated” also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Moreover, an “isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. The term “isolated” is also used herein to refer to polypeptides which are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides. The term “isolated” is also used herein to refer to cells or tissues that are isolated from other cells or tissues and is meant to encompass both cultured and engineered cells or tissues.

[0125] As used herein, “treating” or “treatment” of a disease in a subject refers to (1) preventing the symptoms or disease from occurring in a subject that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or the symptoms of the disease. As understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. For the purposes of the present technology, beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (i.e., not worsening) state of a condition (including disease), delay or slowing of condition (including disease), progression, amelioration or palliation of the condition (including disease), states and remission (whether partial or total), whether detectable or undetectable. When the disease is cancer, the following clinical end points are non-limiting examples of treatment: reduction in tumor burden, slowing of tumor growth, longer overall survival, longer time to tumor progression, inhibition of metastasis or a reduction in metastasis of the tumor. In one aspect, treatment excludes prophylaxis.

[0126] The term “contacting” means direct or indirect binding or interaction between two or more. A particular example of direct interaction is binding. A particular example of an indirect interaction is where one entity acts upon an intermediary molecule, which in turn acts upon the second referenced entity. Contacting as used herein includes in solution, in solid phase, in vitro, ex vivo, in a cell and in vivo. Contacting in vivo can be referred to as administering, or administration.

[0127] “Cryoprotectants” are known in the art and include without limitation, e.g., sucrose, trehalose, and glycerol. A cryoprotectant exhibiting low toxicity in biological systems is generally used.

Construction of SHALs.

Linking the Ligands (Binding Moieties) to Produce a Polydentate SHAL.

[0128] Once two more ligands (binding moieties) are identified that bind to different sites on the target, the ligands are linked either directly or through a linker to produce a polydentate SHAL. Where only two ligands are joined the SHAL is bidentate. Where three ligands are joined the SHAL is tridentate, and so forth.

[0129] A number of chemistries for linking molecules directly or through a linker are well known to those of skill in the art. The specific chemistry employed for attaching the ligands (binding moieties) to each other to form a SHAL will depend on the chemical nature of the ligand(s) and the “interligand” spacing desired. Ligands typically contain a variety of functional groups e.g., carboxylic acid (COOH) or free amine (-NH2) groups, that are available for reaction with a suitable functional group on a linker or on the other ligand to bind the ligand thereto.

[0130] Alternatively, the ligand(s) can be derivatized to expose or attach additional reactive functional groups. The derivatization may involve attachment of any of a number of linker molecules such as those available from Pierce Chemical Company, Rockford Illinois, or described in Table 4 herein.

[0131] In certain embodiments, a bifunctional linker having one functional group reactive with a group on a first ligand and another group reactive with a functional group on a second ligand can be used to form the desired SHAL. Alternatively, derivatization may involve chemical treatment of the ligand(s), e.g., glycol cleavage of the sugar moiety of glycoprotein, carbohydrate or nucleic acid with periodate to generate free aldehyde groups. The free aldehyde groups can be reacted with free amine or hydrazine groups on a linker to bind the linker to the ligand (see, e.g., U.S. Patent No. 4,671,958). Procedures for generation of free sulfhydryl groups on polypeptides, such as antibodies or antibody fragments, are also known (See U.S. Pat. No. 4,659,839).

[0132] In certain embodiments, lysine, glutamic acid, aspartic acid or an aminohexanoic acid and polyethylene glycol (PEG) based linkers different length are used to couple the ligands. A number of SHALs have been synthesized using a combination of lysine and PEG to create the linkers (see, e.g., Examples and Table 3). Chemistry of the conjugation of molecules to PEG is well known to those of skill in the art (see, e.g., Veronese (2001) Biomaterials, 22: 405-417; Zalipsky and Menon-Rudolph (1997) pp. 318-341 In: Poly(ethyleneglycol) Chemistry and Biological Applications. J.M. Harris and X. Zalipsky (eds)., Am. Chem. Soc. Washington, D.C.; Delgado et al. (1992) Drug Carrier Syst., 9: 249- 304; Pedley et al. (1994) Br. J. Cancer, 70: 1126-113-0; Eyre and Farver (1991) Pp. 377-390 In: Textbook of Clinical Oncology, Holleb et al. (eds), Am. Cancer Soc., Atlanta GA; Lee et al. (1999) Bioconjug. Chem., 10: 973-981; Nucci et al. (1991) Adv. Drug Deliv., 6: 133-151; Francis et al. (1996) J. Drug Targeting, 3: 321-340). [0133] One advantageous feature of the synthetic scheme used to create these SHALs is that the approach allows the attachment of almost any type of molecule to a third fourth or fifth site on the linker. In the final round of SHAL synthesis, biotin has been attached at this third site to facilitate the in vitro binding studies using an NHS ester derivative of biotin that reacts with the free amine on the SHAL to covalently/permanently bind biotin to the non functional end of the SHAL. The biotin tag makes it possible to quickly measure the binding to the isolated protein target and/or isolated cells containing the protein by surface plasmon resonance and examine the selectivity of the SHAL for binding to whole cells using ELISA or flow cytometry assays and tissue sections using immunohistochemical methods. The use of biotinylated molecules to determine binding affinities for their targets is well known to those of skill in the art (see e.g., Zhu M, Shezifi D, Nimri S and Luo R. Bioradiations, Feb 12, 2013; Patching SG. Biochim. Biophys. Acta - Biomembranes 1838: 43-55; Papalia G and Myszka D. Anal. Biochem. 403: 30-35. 2010). Analyses of protein markers on cells and tissues using ELISA (for examples see: Erdile LF, Smith D, Berd E. J. Immunol. Methods 258: 47-53 (2001); Kohl, TO and Ascoli CA. in Immunoassays, Cold Spring Harbor Protocols 2017, doi: 10.1101/pdb.top093690), flow cytometry (for examples see: van Vloten, JP, Santry LA, McAusland TM et al. Protocol 13: 154-166 (2019); Lee J, Tam H, Adler L, et al. Plos One 12:e0183594 (2017); Pillai V and Dorfman DM. Acta Cytologica 60:336-343, 2016) and immunohistochemical techniques (for examples see: Selves J, Long-Mira, E, Mathieu, M-C, Rochaix, P and Iliel M. Cancers (Basel) 10: 108 (2018); Okimoto T, Tsubata Y, Sutani A, Fuchita H et al. Anticancer Research 34: 2755-2761, 2014) are also well known to those of skill in the art.

[0134] Once the SHAL has been tested and confirmed to bind to the target (e.g.,

HLA-DRs, transporters, enzymes or other proteins to which the SHALs are designed to bind), metal chelators such as DOTA or other effectors can be attached in the final round of SHAL synthesis to enable the delivery of radionuclides or other effectors to cells (e.g., tumor cells, normal cells, or bacteria) bearing the target. Radioimmunotherapy is well known in the art (for examples see: Kairemo KJA, Acta Oncologica 35: 343-355, 1996; Larson SM, Carrasquillo JA, Cheung N-K V, and Press OW, Nature Reviews Cancer 15: 3470360, 2015) as an example of an approach used to kill tumor cells by exposing them to radionuclides delivered to the surface or interior of the cells. The use of nanoparticles as effectors to deliver drugs into tumor or other cells (see e.g., Gridelli C, Chen T, Ko A et al. Drug Design, Development and Therapy 12:1445-1451 2018; Yan Y, Cai T, Xia X et al. Drug Delivery 23:2250-3357, 2016; Sharma G, Sharma AR, Nam J-S et al. Nanobiotechnol. 13:74, 2015; Baptista PV et al., 2018 Frontiers in Microbiology 9: 1-26; Wang L et al., 2017 Int. J. Nanomedicine 12: 1227-1249) and the inhibition of transporters that provide multidrug resistance to oncology drugs in tumors and to antibiotics in bacteria as a means to block or reverse the development of resistance and lower the dosage of drugs required for therapy (see e.g., Nanayakkara AK, Follit CA, Chen G et al. Scientific Reports 8: 967, 2018; Choi C-H, Cancer Cell International 5:302005; Chen H, Shien K, Suzawa K et al. Oncology Letters 14: 4349-4354, 2017; Breedveld P, Beijen JH and Schellens JH, Trends Pharmacol Sci 27: 17-24, 2006; Grossman TH et al., 2015 Antimicrobial Agents and Chemotherapy 59: 1534-41; Mullin S, et al., 2004 Antimicrobial Agents and Chemotherapy 48: 4171-76; Gibbons S et al., J of Antimicrobial Chemotherapy 51 : 13-17; Leitner I et al., 2011 J. of Antimicrobial Chemotherapy 66: 834-839) are both well known to those of skill in the art.

[0135] After retesting the effector-SHAL conjugates to reconfirm their ability to bind to the target, the conjugates exhibiting the best selectivity for their targets can be radiolabeled (e.g., by incorporating into the DOTA chelating group a metal radioisotope such as ^U1ln) and used to test the SFLALs for their tissue biodistribution and clearance rate in test organisms (e.g., mice). Other unique molecules can also be attached to the free amine located at the non-function end of the SFLAL in future studies so these same SFLALs can also be used, for example, to test the utility of pretargeting approaches for radioisotope delivery in cancer therapy or for use whole body imaging of patients to identify the location and size of tumors or to monitor the progression of their disease (e.g. cancer). Radiolabeled drugs and other compounds are routinely used to determine pharmacokinetics of the compounds in animal models and humans (see e.g., Moriya Y, Kogame A, Tagawa Y et al. Drug Metabolism and Disposition 47: 1004-1012, 2019; Zhou X, Pusalkar S, Chowdhury, SK et al. Invest New Drugs 37: 666-673, 2019) and for assessing drug biodistribution (see e.g., Sudo H, Tsuji AB, Sugyo A, et al. Cancer Sci. 110: 1653-1664, 2019; Izquierdo-Sanchez V, Muniz-Hemandez S, Vazquez-Becerra H, et al. Molecules 23: pii: E3138, doi: 10.3390/molecules23123138, 2018) or for whole body imaging (see e.g., Baum RP, Prasad V, Hommann M and Horsch D. Recent Results in Cancer Research 170: 225-241, 2008; Conti PS. JNucl. Med. 46: 1812-8, 2005; Gmeiner Stopar T, Fettich J, Zyer S, et al. Nucl. Med. Commun. 29:1059-65, 2008).

Stepwise Solid-Phase SHAL Synthesis.

[0136] In certain embodiments, SHAL synthesis proceeds by using a stepwise-solid phase synthesis approach. Solid phase synthetic methods are used routinely for the synthesis of peptides (see e.g., Hayata A, Itoh H and Inoue M. J Am. Chem. Soc. 140: 10602-10611, 2018; Varela YF, Vanegas Murcia, M and Patarroyo, ME. Molecules 5: doi: 10.3390/molecules23112877; Hansen AM, Skovbakke SL, Christensen SB et al. Amino Acids 51: 205-218) and are being used more frequently for the synthesis of small molecule drugs (see e.g., Kwon HJ, Kim YJ, and Gong YD, ACS Comb Sci 21: 482-488, 2019; Zimmermann T, Christensen SB, FranzykH, Molecules 23: doi. 3390/molecules23061463). In this approach a linker is attached to the surface of a resin and each additional linker component or ligand is attached onto a growing molecule (SHAL) covalently attached to the resin. After each chemical reaction the resin can be extensively washed to remove the unreacted products. After the addition of the final ligand to the SHAL, the resin with bound SHAL is extensively washed and the SHAL is then released from the resin by treatment with acid. The free amine at the end of the linker scaffold produced by the acid cleavage reaction can then be used as a site for attaching effectors or tags to the SHAL using solution phase carbodiimide chemistry or other reactions. Following the completion of the chemical reaction linking the effector or tag to the SHAL, the SHAL is typically purified away from the unbound effector or tag and other reaction components using a chromatographic method such as high-performance liquid chromatography (HPLC).

[0137] In one approach, DOTA was attached to the SHAL following its synthesis and cleavage from the resin by reacting l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester with the SHAL released from the resin. In another approach, biotin was attached to the SHAL by reacting biotin N-hydroxysuccinimide ester with the SHAL released from the resin. The DOTA and biotin links are extremely stable, so they do not come off the SHAL once they have been attached. Screening SHALs for Affinity and Selectivity.

[0138] In certain embodiments, a group of SHALS comprising, consisting essentially of, or consisting of different ligands (binding moieties) and/or comprising different length linkers is screened to identify those SHALS that have the best affinity and/or selectivity for the target. Such screening assays can be performed in a number of formats including, but not limited to screening for binding to isolated protein targets, screening for binding to cells immobilized on the bottoms of microtiter plates, screening for binding to cells in normal tissue or arrays of tumor biopsy sections, and screening for in vivo binding to the desired target (e.g., HLA-DRs) present on human tumors grown as xenografts in mice or tumor tissues in patients being imaged to monitor disease progression.

[0139] In certain embodiments, the group of SHALs is screened for their inhibition of the uptake or efflux of other drugs or small molecule substrates by cultured cells (or their membrane preparations) transfected with individual transporter genes whose proteins provide these functions (e.g., OATP1, OATP3, P-gP/MDRl, BCRP, etc.). Drugs are routinely assayed using these techniques (see e.g., Heredi-Szabo K, Palm JE, Andersson TB et ah, Eur. J. Pharm. Sci. 49: 773-81, 2013) prior to their being approved for advancement into clinical trials to assess the likelihood they will interfere with the function of other drugs being taken by patients. Inhibitors of the efflux transporters are also of interest for their potential to block tumors and bacteria that have or may at some point develop resistance to drugs. While bacterial cells are structurally very different from mammalian cells in many ways, some of the key transporters that contribute to their developing antibiotic resistance are inhibited by the same compounds that inhibit the mammalian enzymes (Grossman TH et ah, 2015 Antimicrobial Agents and Chemotherapy 59: 1534-41; Mullin S, et ah, 2004 Antimicrobial Agents and Chemotherapy 48: 4171-76; Gibbons S et ah, J. of Antimicrobial Chemotherapy 51: 13-17; Leitner I et ah, 2011 J. of Antimicrobial Chemotherapy 66: 834-839). The methods used to screen for inhibition are well established in the art (see e.g., Dei S, Braconi L, Romanelli MN and Teodori, E. Cancer Drug Resistance 2: 710-43, 2019).

[0140] In other embodiments the group of SHALs are screened for the inhibition of enzymes such as those involved in the regulation of fatty acid synthesis and degradation (e.g., acetylCoA carboxylase), metabolism of drugs (e.g., UGT, CYP450, etc.) and/or the activation or inhibition of GTPases and their activating proteins (GAPs). Assays for the inhibition of acetylCoA carboxylase (see, e.g., Cheng D, Chu CH, Chen L, et al., Protein Expr.

Purification 51:11-21, 2006; Secor J and Cseke C, Plant Physiol. 86: 10-12, 1988), UGTs (see, e.g., Walsky RL, Bauman JN, Bourcier K, et al., Drug Metabolism & Disposition 40: 1051-1061, 2012), CYP450s (see e.g., Perloff ES, Mason AK, Dehal SS, et al., Xenobiotica 39: 99-112, 2009) and GAP proteins and their GTPases (see e.g., van Adrichem, AJ, Fagerholm A, Turunen, L, et al., Combinatorial Chemistry & High Throughput Screening 18: 3-17, 2015) are well known to those skilled in the art.

[0141] In certain embodiments, the group of SHALs is tested for activity in blocking the presentation of self-antigens by an MHC class II protein or suppressing inflammation by their binding more tightly to the antigen binding pockets of HLA-DRs than the natural antigen peptides and/or by killing the HLA-DR expressing B-lymphocytes involved in the production of autoantibodies or the perpetuation of an auto-immune disease (e.g.,

Rheumatoid Arthritis, Multiple Sclerosis, Type-1 Diabetes, Grave’s Disease, Hashimoto’s Thyroiditis, Myasthenia Gravia, Celiac Disease, Systemic Lupus Erythematosus, Anklylosing Spondylitis, etc.). Methods have been developed by others for identifying drugs/compounds that block the binding of peptide antigens to HLA-DRs (see e.g., Mamedov AE, Zakharova MY, Favorova OO, et al., Dokl. Biochem. Biophys. 485:115-118, 2019; Watanabe N, Suzuki Y, Yonezu T, et al., Sci. Reports 7:6798, 2017), assessing their cytotoxicity to lymphocytes (see e.g., Salimi A, Pirhadi R, Jamali Z, et al., Human Exp. Toxicol. 38:1266-1274, 2019), and monitoring the stimulation of T-cells (see e.g., Williams T, Krovi HS, Landry LG et al.,

J. Immunol. Methods 462:65-73, 2018; Kumagai M, Mizukoshi E, Tamai T et al., Liver Int. 38: 1635-1645, 2018) and the production of autoantibodies (see e.g., Harlow L, Fernandez I, Soejima M, et al., Innate Immun. 18:876-885, 2012; Kiefel V, Santoso S, Schmidt S, et al., Transfusion 27: 262-265, 1987; Pedreno M, Sepulveda M, Armangue T, et al., Mult. Scler. Relat. Disord. 28:230-234, 2019; Liu T, Chen B, Yang H, et al., Mult. Scler. Relat. Disord. 28:177-183, 2019).

[0142] In other embodiments, the group of SHALs is screened for their ability to induce an anti-tumor response (e.g., activation of CD4+ and CD8+ T-cells) by binding to the antigen binding pocket of HLA-DRs and being presented to T-cell lymphocytes as a foreign antigen. Methods for detecting the induction of a T-cell response to a chemical stimulus are well known to those skilled in the art (see e.g., Bechara R, Pollastro S, Azoury ME, et al., Front. Immunol. 10:1331, 2019; Martens A, Pawelec G and Shipp C. Methods Mol. Biol. 1913:141-151, 2019).

SHAL Binding to Isolated Targets (e.g., proteins).

[0143] In certain embodiments, NMR spectroscopy (see e.g., Cosman, M, Lightstone

FC, Krishnan VV, et al. Chem. Res. Toxicol. 15, 1218-1228, 2002; Cosman M, Krishnan VV, Balhom R. Methods Mol. Biol. 300: 141-63, 2005), mass spectrometry (see e.g., Lightstone FC, Prieto, MC, Singh, AK et al. Chem. Res. Toxicol. 13:356-362, 2000; Shields SJ, Oyeyemi O, Lightstone FC and Balhom, R. J. Am. Soc. Mass. Spectrom. 14: 460-470, 2003) or surface plasmon resonance (see e.g., Al Olaby, R, Azzazy HM, Harris, R et al. J. Comput. Aided Mol. Design 27: 337-346, 2013) techniques can be used to determine if a SHAL binds to its target protein. The binding affinities of the best SHALs can be estimated by mass spectrometry of the SHAL-target complexes (see, e.g., Prieto Conway MC, Whittal, RM, Baldwin, MA et al. J. Am. Soc. Mass Spectrom. 17: 967-976, 2006), followed by a more accurate surface plasmon resonance (SPR) spectroscopy (Shuck (1997) Annu. Rev. Biophys. Biomol. Struct., 26: 541-566; Van Regenmortal (2001) Cell Mol Life Sci., 58: 794-800; Rich RL and Myszka DG. J. Mol. Recognition 16: 351-382, 2003) measurement of the SHAL- target binding affinity using for example, the IASYS Plus or BiaCore instruments. In order to perform the SPR measurement, biotin can be added to the linker through a third functional group (as described above) and the SHAL can be bound to commercially available streptavidin coated chips. In certain preferred embodiments, only those SHALs exhibiting nM or higher binding affinities can be considered useful. The SHALs exhibiting the greatest affinity can then be tested for their selectivity. Experiments can be performed to test the selectivity of SHAL binding to targets in the presence of the targets and other molecules related to the targets. Thus, for example, where the SHAL is designed to bind to an HLA- DR, the SHAL can be evaluated for its ability to bind target molecules in the presence of tumor cell surface proteins extracted and separated by gel electrophoresis. After treating the gel with the biotinylated SHAL and rinsing out excess unbound SHAL, the location of the bound SHAL can be detected by staining with Rhodamine tagged streptavidin. In certain embodiments, the SHALs that are considered to exhibit reasonable protein selectivity can be those molecules in which 95% or more of the fluorescence is associated with the HLA-DR monomer and multimer peaks. SHAL Binding to Cells in Culture.

[0144] Where the SHAL target is a marker on a cell (e.g., a cancer cell marker) it may be desired to assess the specificity of binding of the SHAL to intact cells. Cell binding studies can be conducted with the biotinylated (or otherwise labeled) SHALs, using for example the fluorescence of bound Rhodamine-tagged streptavidin to confirm the SHALs bind to target cancer cells (e.g., Raji) and do not bind to cancer cells (e.g. Jurkats) lacking the marker (see e.g., DeNardo GL, Natarajan A, Hok, S et al. Cancer Biotherapy & Radiopharmaceut. 23: 783-795). The selectivity of the SHAL binding to specific variants of the marker can be determined by testing the biotinylated SHALs for binding to normal cells expressing different variants of the marker. If the cell marker is an HLA-DR, glass microscope slides containing peripheral blood mononuclear cells (PBMCs) isolated from the blood of normal individuals who express different HLA-DR variants can be treated with the biotinylated SHALs to determine which variants of the HLA-DR marker are recognized and bound by the SHAL. When the biotinylated SHAL binds to the HLA-DR variant marker on the PBMCs, the cells expressing HLA-DR (lymphocytes, macrophages and dendritic cells) are stained a brown or black color when streptavidin conjugated to horseradish peroxidase is added to the SHAL treated cells and the cells are subsequently exposed to a peroxidase substrate. Biotinylated SHALs can also be immobilized on the bottom of wells in streptavidin coated ELISA plates and used to identify their binding to cultured cells added to the plates. When the marker is present, the cells added to the plates (which are suspended in solution) stick to the SHALs and remain attached to the well bottom after rigorous washing. Cells lacking the marker are washed off the plate. The presence of bound cells is detected by adding a stain such as cresyl violet (see e.g., Balhom R, Hok S, Burke PA et al. Clin. Cancer Res. 13:5621s-5628s, 2007).

[0145] In certain embodiments, those SHALs that exhibit at least a 2-fold, at least a

5-fold, or at least a 10-fold increase in the staining intensity of target (e.g., tumor) cells over controls can be selected for further testing and development. In such cases, SPR measurements can be conducted to determine the strength of binding (affinity) of the SHAL to intact cells containing the marker (see e.g., Ogura T, Tanaka Y, and Toyoda H. Anal. Biochem. 508: 73-7, 2016; Schasfoort RBM, Abali F, Stojanovic I, et al. Biosensors 8: 102, 2018). Analogs of the SHALs with the highest affinities can be synthesized with a DOTA molecule attached to the linker, and binding experiments can be conducted using radionuclide-tagged SHALs to obtain additional binding data for the highest affinity SHALs and also attempt to determine if the SHAL is retained on the surface of the cell or is internalized. In cases where DOTA analogs of the SHALs are being developed for radioimmunotherapy, this information can be used to determine the type of radioisotope that should be loaded into the chelator. If the SHAL remains on the surface, the SHAL is typically utilized alone or with effectors that do not require internalization (e.g., alpha emitters such as ⁹⁰Yttrium, etc.). If evidence is obtained to suggest the SHAL is internalized upon binding to the target cells, b-emitters can be incorporated into the DOTA chelators to provide more localized radiation damage. In other embodiments wherein the SHAL is being designed to deliver a pro-drug, other effectors can be added to the SHAL in place of the DOTA that become active when internalized. In other embodiments other effectors, such as divalent or trivalent metal ions (e.g. Fe⁺², Fe⁺³, Cr⁺³, Cu⁺², etc.) can be loaded into the DOTA or other chelating group attached to the SHAL and delivered selectively into tumor or other cells.

Analysis of Cell Selectivity Using Tissue Arrays.

[0146] Tissue array technology can be used to screen SHALs to determine their tissue binding specificity (e.g., malignant and normal tissue reactivity in the case of anti-tumor SHALs). The preparation and use of tissue arrays are well known to those of skill in the art (see, e.g., Kononen et al. (1998) Nat Med., 4:844-847; Torhorst et al. (2001) Am. J. Pathol., 159: 2249-2256; Nocito et al. (2001) Int. J. Cancer, 94: 1-5, and the like). Tissue microarrays are prepared by taking small cores of each individual tissue (or tumor) and assembling these cores into a single paraffin block. Thin sections of the block are cut using a microtome, and individual sections are deposited onto a glass slide such that each slide contains a thin tissue or tumor section from each core in the block. Slides containing these core sections, which are called microarrays, can then be used to screen for SHAL binding using standard immunohistochemistry techniques. Using these microarrays, one can assay hundreds of tissue or tumor biopsy samples for SHAL binding in one experiment rather than having to perform hundreds of different experiments. By having each tissue in the array being exposed to the SHAL under identical conditions (temperature, length of time exposed to the SHAL and other reagents, etc.), the experiment to experiment variation that is often encountered when different samples are screened for binding independently can be minimized.

[0147] In one embodiment, the normal tissue array contains twenty-seven human tissues, which include heart, colon, esophagus, ovary, hypophysis (pituitary), thymus, peripheral nerve, uterine cervix, salivary gland, thyroid, parathyroid, tonsil, lung, stomach, spleen, liver, kidney, small intestine, bone marrow, pancreas, skeletal muscle, adrenal, breast, cerebrum, cerebellum, prostate and skin. The tumor microarrays contained biopsy sections obtained from 24 to 122 different cases of seven different types of non-Hodgkin’s lymphoma (diffuse large b-cell, follicular, anaplastic large cell, MALT, mantle cell, Burkitt’s and small lymphocytic), myeloma, melanoma, ovarian, lung, cervical, pancreatic, gastric, esophageal, breast, kidney, prostate, thyroid, liver, colorectal, bone, bladder, laryngeal and head and neck cancers. Using these and other similar tissue arrays, one can determine the non-specific binding of the SHALs to normal tissue and specific binding to both hematological and non- hematological cancers. Using biotinylated forms of the SHALs, SHAL binding to the various tissues and tumor biopsy sections was tested using the same method described previously for assessing SHAL binding to cells wherein horse-radish peroxidase conjugated streptavidin is used to identify those cells that bind the biotinylated SHALs. In each case, SHAL binding to individual cells within the tissues or tumors is verified by visual inspection (using a microscope).

[0148] In one illustrative approach, slides containing a microarray of biopsy cores taken from 75 different patients diagnosed with ovarian cancer (core diameter 1.1 mm and thickness 4 pm) were purchased from a commercial source (US Biomax, Rockville, MD). Prior to treatment with the SHAL, the slides were deparaffmized using the Leica dewax solution, rehydrated with an alcohol series (100%, 95%, 70% and 30% for 4 min each) followed by antigen retrieval in citrate buffer at pH 6 and 90 °C for 20 min. After performing a 5 min hydrogen peroxide block, the slides were washed three times with BOND Wash Solution, endogenous biotin was blocked using the Ventana Endogenous Biotin Blocking kit (Roche Molecular Diagnostics, Pleasanton, CA), the slides were washed three additional times with BOND Wash Solution, and then stained with biotinylate SHAL (100 pg/ml in PBS, 1% DMSO) for 30 min. Following three washes with BOND Washing Solution, the slides were treated with Streptavidin-horse radish peroxidase (SAHRP) for 30 min, washed 3 times with BOND Wash Solution and once with deionized water, treated with Mixed DAB (3,3-diaminobenzidine) Refine for 10 min, and then washed four times with deionized water, once with BOND Wash Solution and a final deionized water wash as per the BOND Polymer Refine IHC protocol (Histowiz Inc., Brooklyn, NY). The SH7129 stained tumor microarray slides were not counterstained with hematoxylin. The slides were then dehydrated by immersion in an alcohol series (30%, 70%, 95% and 100% for 4 min each), cleared with xylene and mounted with Permount. Representative digital images of each tumor biopsy section were captured from the SHAL treated and control (duplicate slide cut from same core treated with PBS instead of SHAL) slides at 40X magnification. SHAL binding or lack of binding to the cells was confirmed by visual inspection. Cells expressing the HLA-DRs that bind the SHAL showed stain associated with both the membranes and cytoplasm. To estimate the tumor-to-tumor variation in SHAL binding, a digital image containing the array of cores for the two slides (SHAL treated and control without the SHAL) were captured at the same magnification (10X), the images were inverted, and the amount of bound SHAL was determined by densitometric analysis of each tumor section using the program ImageJ 1.42. Integrated density data were collected from a 384-pixel area of each core and from ten blank (background) 384 pixel areas distributed across the slide near or between the cores. Core sections containing voids or tears (missing tissue), lacking a corresponding core in the control slide, or obtained from pigmented tumors were not analyzed. In cases where there were duplicate or triplicate cores for each biopsy on the slides, the data obtained from the analyses of the replicates were averaged. The amount of bound SHAL (per 384-pixel area) was then calculated for each biopsy sample as follows:

Bound SHAL = (IntDensHAL - IntDensHALBkg) - (IntDenNoSHAL - IntDenNoSHALBkg) where IntDensHAL is the integrated density of the biopsy section treated with the SHAL, IntDensHALBkg is the mean of the integrated densities of the ten blank regions of the SHAL treated slide, IntDenNoSHAL is the integrated density of the biopsy section that was processed for staining without the SHAL, and IntDenNoSHALBkg is the mean of the integrated density of the ten blank regions of the control slide processed for staining without the SHAL.

[0149] Visualization of the SHAL treated cells and the biopsy tissue samples in the tumor microarrays and capture of the high-resolution digital images used to identify SHAL binding are performed using a high-throughput slide scanner, such as the Leica AT2. The current digitization technology produces images of the entire glass slide and the attached cells or tissue sections at 40X magnification. With an autoloading capacity of 400 slides, the slides are imaged at a rate of 50 slides per hour. The images can then be either viewed locally or from a web server. Unlike standard web-based still images, which are typically downloaded to be viewed within a browser, the processed images are viewed from the web in an application that allows any region of the image to be magnified up to 40-fold.

In vivo Analysis of Selectivity.

[0150] Of course in vivo selectivity of a SHAL can also readily be determined. This is accomplished by administering the SHAL to a test animal (e.g., a laboratory rat) comprising a cell or tissue that displays the target to which the SHAL is directed. After sufficient time, the animal is sacrificed and the target tissue(s) and normal tissues examined (e.g., histologically) to evaluate the specificity of SHAL binding and amount of SHAL delivered to the target tissue. In certain embodiments, the SHAL is coupled to an imaging reagent that permits non-invasive imaging and thereby permit the evaluation of real time pharmacody nami cs .

[0151] By way of illustration, pharmacokinetic and radiation dosimetric mouse studies can be performed, e.g., on the SHALs illustrated in the Examples, to generate data upon which to select one for clinical trials of pharmacokinetics and radiation dosimetry in patients, using established methods. Pharmacokinetics can be performed in female nude mice bearing Raji human lymphoma xenografts of defined size using established methods (DeNardo et al. (1998) Clin. Cancer Res., 4: 2483-2490; Kukis et al. (1995) Cancer Res., 55: 878-884). Mice can be injected with DOTA-tagged SHALs containing ^U1ln or ⁹⁰Y and mice can be sacrificed, e.g., at each of at least 5 time points to obtain tissue samples for analysis. Initial studies can be conducted at the extremes of early and late time points expected for molecules of this size to determine the optimal time frame over which to collect samples for analysis. Known data for other molecules, e.g., peptides, can be used to define the longest time points. When using ^U1ln or ⁹⁰Y as a tracer, the longest time point would typically be about 5 days. Total body clearances can be determined using a sodium iodide detector system. Blood clearance can be monitored by taking periodic blood samples from the tail veins of the mice. At the time of sacrifice, the xenograft and normal tissues can be removed, weighed and counted in a gamma well counter to provide organ distribution data.

[0152] In order to assess the dose-dependent biodistribution of the SHAL, studies can be conducted at, e.g., 5 dose levels, once again beginning with small and large SHAL amounts to guide selection of the intermediate doses to be studied. Because of the novelty of the SHALs, selection of study time points and dose levels will typically be guided by information previously obtained from similar studies in mice using antibodies (e.g., Lym-1 or Rituximab) and other small molecule drugs (e.g. Vandetanib or doxorubicin).

[0153] In certain embodiments, the ideal pharmacokinetics and dosimetry to achieve with Applicant’s SHALs are those that approach what has been accomplished using sodium iodide (Nal) in the treatment of thyroid tumors. The SHALs should be small enough to gain access to all malignant cells and be readily excreted in the urine. Typically, at least an order of magnitude better target recognition and binding affinity to lymphomas and leukemias than current antibodies will provide the desired tumor cell selectivity. While the rapid clearance of smaller molecules, such as the SHALs, from the circulation might be considered a disadvantage, the remarkable effectiveness of Nal in treating thyroid tumors has shown this “disadvantage” can be turned into an advantage if the reagent has the right combination of affinity and selectivity. If the SHALs are taken up well, target only a specific family of cells (e.g., B lymphocytes and their malignant relatives), bind tightly to their target receptors (e.g., HLA-DRs) with low off-rates and are too large to enter cells that do not express the target receptor, rapid clearance of the SHAL from the system should lead to a substantially lower dose received by normal tissues (relative to malignant or diseased cells) than that obtained using existing targeting antibodies or small molecule drugs.

[0154] Using established methods for radiolabeled (DeNardo et al. (2000) J. Nucl.

Med., 41: 952-958; DeNardo et al. (1999) J. Nucl. Med., 40: 1317-1326; DeNardo et al. (1999) J. Nucl. Med., 40: 302-310; Shen et al. (1994) J. Nucl. Med., 35: 1381-1389; Siegel (1994) J. Nucl. Med., 35: 1213-1216) or unlabeled compounds (Gustafson et al (2006) J. Pharmacol. Exp. Ther. 318: 872-80; Luo et al. (2017) International J. Pharmaceutics. 519: 1- 10) as guidelines, protocols can readily be developed for conducting pharmacokinetic and radiation dosimetry studies in patients with lymphomatous diseases of the B cell type or other cancers. In certain embodiments, a protocol is selected that provides the optimal dose level using information on the therapeutic indices for tumor to normal tissue.

Optimization of SHAL Affinity, Selectivity and Metabolism by Varying the Linker Length and Linker and Ligand Structure.

[0155] SHAL affinity, selectivity and metabolism can be optimized by varying the linker length, the number of ligands, and/or the linker and ligand structure, using computer modeling and experimental studies. Linker lengths can be reduced or increased to improve the SHAL’s affinity for its target. Increasing the number of ligands that bind to sites on the target protein also can be used to increase the SHAL’s affinity. Changes in the individual ligands used to create the SHAL or alterations in individual ligand structure can also be made to improve binding, improve or alter target selectivity and optimize the solubility or clearance of unbound SHAL from the organism. Modifications in the structure of the linker itself can also be considered to facilitate SHAL solubility or clearance, if necessary, from normal tissues and peripheral blood through the incorporation of hydrophilic (e.g., polyethylene glycols or polyamines) or cleavable bonds (e.g., a peptide, disulfide, or other cleavable linker) that attach the chelator or specific ligands to the SHAL.

[0156] If a particular SHAL is observed to exhibit non-specific binding (e.g., to many proteins in the cell extracts or to both Raji and control cells), additional SHALs can be synthesized using different combinations of ligands until a suitably specific SHAL is identified.

Maximization of SHAL Binding Affinity for Rarget Molecule(s).

[0157] Binding affinity of multidentate reagents to protein or cell surface targets can be increased by one to several orders of magnitude by changing and optimizing the length of the linker separating the ligands. Without being bound to a particular theory, it is believed that this increase is related to achieving the optimal separation between the ligands to allow them to bind to their individual sites as well as to providing sufficient rotational flexibility within the linker itself to enable the optimal interaction of each ligand within its binding site (e.g., binding pocket). When the linkers are too long, the binding of the individual ligands takes longer (the on-rate is reduced). When the linkers are too short, not all ligands can bind to their sites simultaneously, a result which can reduce the affinity of the SHAL one thousand fold.

[0158] In certain embodiments, the initial linker length that is chosen for use in the

SHAL is identified by estimating the distance between the two (or more) bound ligands that are to be linked together. Once it has been determined that a particular combination of linked ligands actually binds to the target, additional modeling can be conducted to further refine the length of the linker and optimize the SHALs binding affinity.

[0159] For example, where the target is HLA-DR10, the structure of the HLA-DR10 beta subunit can modeled with both ligands bound in their respective pockets and various length PEG linkers interconnecting the ligands (see, e.g., the Examples herein). From molecular dynamics studies the orientations of the bound ligands can be evaluated to improve the linker design. Further molecular dynamics simulations can be performed to include the linkers and the ligands, thus simulating the polydentate ligands interacting with the target, e.g., as described herein.

[0160] Once the results of these modeling experiments are obtained, an additional set of SHALs can be synthesized with linkers spanning the range of sizes predicted to be optimal, and their binding affinities can be experimentally tested (see e.g., Balhorn et al. (2007) Clin. Cancer Res. 13(Suppl 18): 5621s-5628s).

Optimization of Target Selectivity and Metabolism of SHAL.

[0161] Both computational and experimental methods can also be used to determine if changes in the structure of the individual ligands that are linked together to produce the SHAL improve target selectivity and optimize SHAL metabolism, the generation of ligand derivatives that have a specific activity or provide a specific function, and SHAL clearance from normal tissues and peripheral circulation. This can be accomplished for improving target selectivity, as one example, by examining the types of functional groups present inside a targeted binding pocket and their location relative to functional groups present on the bound ligand.

[0162] Docking and/or molecular dynamics studies can be conducted using different conformations of the ligand and selected ligand analogs to aid the identification of ligand derivatives that fit optimally into each binding site (e.g., pocket). NMR spectroscopy (Lin et al. (1997) J. Organic Chem., 62: 8930-8931), mass spectrometry (e.g. Prieto Conaway et al. (2006) J. Amer. Soc. Mass Spectrometry 17967-976), dual polarization interferometry (Al Olaby, et al. (2013) J. Comput. Aided Molecular Design 27:337 -346), or surface plasmon resonance (Al Olaby, et al. (2013) J. Comput. Aided Molecular Design 27:337 -346) methods can be used to compare and rank the affinities of a subset of the ligand analogs. The particular analogs chosen for analysis are typically selected based on the results provided by computer modeling and the analog’s commercial availability or ease of synthesis. If higher affinity analogs are identified experimentally, a set of new SHALs can be synthesized and tested for their affinity, selectivity in binding to targets, and desirable metabolic properties (e.g., rapid clearance from peripheral circulation, liver and kidney). SHAL selectivity, which requires each of the linked ligands to bind simultaneously to its site on the target protein, is at its highest when the ligands used to create the SHAL bind individually to their sites on the target protein with relatively low affinity (millimolar to micromolar). This ensures the binding of any one ligand in a SHAL to a site on a non-target protein will be sufficiently weak that the SHAL will dissociate rapidly if that protein is not the target and there are no sites for the other ligands to bind.

[0163] In certain embodiments, the small size of the SHAL can result in its being cleared from the tissues too quickly to be effective in delivering a suitable amount of SHAL or effector to the target cells. If this is observed, various approaches can be used to optimize the retention time of the SHAL in the target tissue. One involves incorporating another ligand into the SHAL or using a biotin attachment site on the linker to add another molecule that binds to a different site on the target or to another protein or receptor located near the target on the cell. This has been observed to increase the affinity of the SHAL to picomolar levels (Balhom and Cosman Balhorn, (2014) In: Medical Radiology, Radiation Oncology, Volume Therapeutic Nuclear Medicine, R.P. Baum, ed., pl39-150, Springer-Verlag, Germany), which would reduce the off-rate of the bound molecule dramatically. Alternatively, the effective size of the SHAL can be increased substantially by attaching it to larger, multi-arm PEG molecules or the surface of dendrimers, nanoparticles and/or to other molecules or macromolecular structures. Modes For Carrying Out the Disclosure

[0164] In one aspect, a Selective High Affinity Ligand (SHAL) molecule comprising, consisting essentially of, consisting of, or of the structure Group A, Group B, or Group C is provided, wherein Group A is of the structure:

(Group

A), w Wh11eCr1e cimn:.

R is a label or a tag or an effector, for example, selected from Table 4, L is a ligand, n *1

1-4 and X represents an amide bond between 0=C and N-H groups; Ri and R3 are each independently

Group B is of the structure:

(Group B), wherein: R is a label or a tag or an effector, for example, selected from Table 4, L is a ligand, n H

= 1-4 and represents an amide bond between 0=C and N-H groups;

Ri9 is

Group C is of the structure:

(Group C), wherein:

R is a label or a tag or an effector, for example, selected from Table 4, L is a ligand, n = 1-4 and ^ represents an amide bond between 0=C and N-H groups; R21 is

R22, R23, R26 and R27 are each independently

, and

R24 and R25 are each independently

[0165] In some embodiments, each ligand L is independently selected from Li, L2,

L3, and L4:

wherein:

R₄ is H, NH₂, N(CH₃)2, CO2, NH(CH₃), NO2 or CF₃;

Rs is H, NH2, NO2 or CH₃;

Ai is a bond, -CH2-, -NH-, -N=N-, -0-, -CH2-CH2-, -CH2-NH-, -CH=NH-, -CH2-O-, -CH=CH- -NHCH2-, -NH=CH-, -OCH2-, phenyl ene, -NHNH-, -NHC(O)-, or -(O)CNH-; R6 is any one of:

R7 is H, Cl, or F;

Rs and R9 are each independently

Rio is

Ri2 is H, methyl, Cl, NH2,

Ri3 is H, methyl, Cl, NH2, or

O

A

N ---Ά A W

H Ri4 is methyl, H or NH2; Ri5 is methyl, H or NH2, or

wherein each L1-L4, * denotes attachment to the rest of the ligand L1-L4, denotes attachment to the SHAL, and W is / or OH; and R is a label tag or effector.

[0166] In some embodiments, the SHAL comprises a ligand of 3-(3-((3-chloro-5-

(trifluoromethyl)-2-pyridinyl)oxy)anilino)-3-oxopropanoic acid.

In further embodiments, the SHAL comprises, consists essentially of, consists of, or is of a structure selected from the following:

Tri dentate:

OG(C{CCCCNiR])NCECOCCOCGNC(C{CCCCNC(CC{NC1~CC=CC(OC2=NC=C{G(FXF)F}G~C2Cf)~Cr)=0)-

0)NC{COCCOCCNC{C G(C}C)N5{=0}(C3=CC=C(N=NC4=CC=C{N{C)C)C=C4)C=C3)=b)=0}-0}=0}=0)=0 or

or

ln some embodiments, the R is a hydrogen (for example, in a free amine SHAL) or a label or a tag or an effector, for example, selected from Table 4. In some embodiment, the SHAL comprises the ligand 3-(3-((3-chloro-5-(trifluoromethyl)-2-pyridinyl)oxy)anilino)-3- oxopropanoic acid and the SHAL structure is selected from those as identified above. In some embodiments, the SHAL comprises and/or has one of the structures as identified above which comprises the ligand 3-(3-((3-chloro-5-(trifluoromethyl)-2-pyridinyl)oxy)anilino)-3- oxopropanoic acid.

[0167] In some embodiments, the SHAL comprises, consists essentially of, consists of, or is of a structure selected from specimen group A1 :

SH7129,

SH7139 (D-Lys), SH8043 (L-Lys) and SH8039 (¹³C₆-¹⁵N₂-Lys), and

SH7135.

[0168] In some embodiments, the lysine moiety comprises, consists essentially of, or consists of D-lysine. In some embodiments, the lysine moiety comprises, consists essentially of, or consists of L-lysine. In some embodiments, ¹³C6-¹⁵N2-Lys comprises, consists essentially of, or consists of isotopic lysine comprising, consisting essentially of, or consisting of C-13 and N-15 isotopes.

[0169] In some embodiments, the SHAL comprises, consists essentially of, or consists of a SHAL of Specimen Group A2:

wherein * denotes site of attachment to the nitrogen.

[0170] In some embodiments, the SHAL comprises, consists essentially of, or consists of a SHAL of Specimen Group A3:

MB 1000, and MB 1001

[0171] In some embodiments, the SHAL comprises, consists essentially of, or consists of a SHAL of Specimen Group B1 :

wherein * denotes site of attachment to the nitrogen. [0172] In some embodiments, the SHAL comprises, consists essentially of, or consists of a SHAL of Specimen Group B2:

wherein * denotes site of attachment to the nitrogen.

[0173] In some embodiments, the SHAL comprises, consists essentially of, or consists of a SHAL of Specimen Group B3:

[0174] In some embodiments, the SHAL comprises, consists essentially of, or consists of a SHAL of Specimen Group Cl :

wherein * denotes site of attachment to the nitrogen.

[0175] In some embodiments, the SHAL comprises, consists essentially of, or consists of a SHAL of Specimen Group C2:

[0176] The Ligands (L) maybe selected from any ligand represented in Table 1.

Table 1: Ligands

Table 1 (cont.)

Table 1 (cont.)

Table 1 (cont.)

[0177] In some embodiments, the Ligands (L) from the above Table 1, are attached to the SHAL by way of a peptide (amide) bond or ester bond formed at the Ligand’s carboxylic acid, hydroxyl, or amino terminus.

[0178] In some embodiments, the linker or R2 of SHALs of Group A, R21 and R25 of

SHALs of Group B, and/or R24 of SHALs of Group C, are each independently selected from Table 3:

Table 3: Linkers

Table 3 (cont.)

Table 3 (cont.)

[0179] In one embodiment, the SHAL comprises, consists essentially of, or consists of a SHAL of Specimen-Groups Al, B2, Cl and SH7097, SH7119, SH8003, SH8005 and SH5133 (Bl), wherein the SHAL comprises, consists essentially of, or consists of the Ligand (L) from Table 2:

Table 2. Additional Ligand (L) Structure from Ligand -Core-Structure -2

[0180] In one embodiment, all of the Ligands (L) of the SHAL are the ligand of Table

2

Labels Tags and Effectors

[0181] A variety of labels, detectable labels, tags, and effectors may be attached to the

SHALs, for example, antibodies, peptides, non-natural peptides, pharmaceutical drugs, nucleic acids, other SHALs, or manipulatable tags, for example, magnetic beads or light, pH or frequency activated nanostructures or molecules. Representative examples include those tabulated in Table 4 below. Table 4: Labels, tags and effectors comprising Group R to enable the detection of a SHAL, and/or modulate its activity, and/or facilitate its delivery to the target molecule

Imaging compositions

[0182] In certain embodiments, the SHALs of this invention can be used to direct detectable labels to its target, or a cell or a tissue comprising such target, for example, a tumor site. This can facilitate tumor detection and/or localization. In certain particularly preferred embodiments, the effector component of the SHAL is a “radioopaque” label, e.g., a label that can be easily visualized using x-rays. Radioopaque materials are well known to those of skill in the art. The most common radiopaque materials include iodide, bromide or barium salts. Other radiopaque materials are also known and include, but are not limited to organic bismuth derivatives (see, e.g., U.S. Patent 5,939,045), radiopaque polyurethanes (see U.S. Patent 5,346,981, organobismuth composites (see, e.g., U.S. Patent 5,256,334), radiopaque barium polymer complexes (see, e.g., U.S. Patent 4,866,132), and the like.

[0183] The SHALs can be coupled directly to the radiopaque moiety or they can be attached to a “package” (e.g., a chelate, a liposome, a nanoparticle, a dendrimer, a polymer microbead, etc.) carrying or comprising, consisting essentially of, or consisting of the radiopaque material as described below.

[0184] In addition to radioopaque labels, labels such as those detected by positron emission spectroscopy or MALDI imaging mass spectrometry are also suitable for use in this invention. Other detectable labels suitable for use as the effector molecule component of the SHAL of this invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, green fluorescent protein, Alexa Fluor, acridine, cyanine and oxazine dyes and the like), quantum dots, isobaric mass tags, radiolabels (e.g., ³H, ¹²⁵1, ³⁵S, ¹⁴C, ³²P, ¹⁸F, etc.) or other tags for imaging, enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.

[0185] Various preferred radiolabels include, but are not limited to "Tc, ²⁰³Pb, ⁶⁷ Ga,

⁶⁸Ga, ⁷² As, ^luIn, ^113mIn, ⁹⁷Ru, ⁶²Cu, ⁶⁴Cu, ⁵²Fe, ^52mMn, ⁵¹Cr, ¹⁸⁶Re, ¹⁸⁸Re, ⁷⁷ As, ⁹⁰Y, ⁶⁷Cu, ¹⁶⁹Er, ¹²¹Sn, ¹²⁷Te, ¹⁴²Pr, ¹⁴³Pr, ¹⁹⁸ Au, ¹⁹⁹ Au, ¹⁶¹Tb, ¹⁰⁹Pd, ¹⁶¾y, ¹⁴⁹Pm, ¹⁵¹Pm, ¹⁵³Sm, ¹⁵⁷Gd, ¹⁵⁹Gd, ¹⁶⁶HO, ¹⁷²Tm, ¹⁶⁹ Yb, ¹⁷⁵ Yb, ¹⁷⁷Lu, ¹⁰⁵Rh, and ¹¹¹ Ag.

[0186] Means of detecting such labels are well known to those of skill in the art.

Thus, for example, radiolabels may be detected using photographic film, scintillation detectors, PET/CT scanners, and the like. Fluorescent markers may be detected using a photodetector to detect emitted illumination. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label. Examples of fluorescent markers include green fluorescent protein and firefly luciferin.

Radiosensitizers

[0187] In another embodiment, the effector can be a radiosensitizer that enhances the cytotoxic effect of ionizing radiation (e.g., such as might be produced by ⁶⁰Co or an x-ray source) on a cell. Numerous radiosensitizing agents are known and include, but are not limited to benzoporphyrin derivative compounds (see, e.g., U.S. Patent 5,945,439), 1,2,4- benzotriazine oxides (see, e.g., U.S. Patent 5,849,738), compounds comprising, consisting essentially of, or consisting of certain diamines (see, e.g., U.S. Patent 5,700,825), BCNT (see, e.g., U.S. Patent 5,872,107), radiosensitizing nitrobenzoic acid amide derivatives (see, e.g., U.S. Patent 4,474,814 ), various heterocyclic derivatives (see, e.g., U.S. Patent 5,064,849), platinum complexes (see, e.g., U.S. Patent 4,921,963), and the like.

Radioisotopes and Transition Metals

[0188] In certain embodiments, the effector comprises, consists essentially of, or consists of one or more radioisotopes that when delivered to a target cell, bring about radiation-induced cell death.

[0189] For medical purposes, the most important types of decay are gamma emission, beta decay, alpha decay, and electron capture. The gamma emitted by a radionuclide, such a ^{13 X}I, exits the body, allowing the use of external scintigraphic imaging to determine the biodistribution of radiolabeled antibodies (the optimal energy range for immunoscintigraphy is 100-250 keV). In contrast, beta particles deposit most of their energy within a few millimeters of the point of decay. Beta emissions from radionuclides such as ¹³¹I or ⁹⁰Y that have targeted antigen-positive tumor cells can kill nearby antigen-negative tumor cells through a “crossfire” effect.

[0190] Yttrium-90, a pure beta emitter, has several properties that make it an attractive choice for radioimmunotherapy: 1) a high beta energy (Emax=2.29 MeV; maximum range of particulate energy in tissue=l 1.9 mm) which enables it to kill adjacent tumor cells;

2) metal chemistry, which facilitates the synthesis of radioisotope-antibody conjugates and use of a pretargeting approach; and 3) a sufficiently long physical half-life (2.67 days) for use with intact SHALs, which may take 1-3 days to reach their peak concentration in tumors.

[0191] In one embodiment, the radioisotope is for use in medical imaging including but not limited to Tc-99, Ga-67, Ga-68, In-111, Gd-157, Gd-159, Au-198, Au-199, Ag-111, Yb-169, Yb-175; or for use in radioimmunotherapy, including but not limited to 1-131, Cu- 67, Lu-177, Re-186, Y-90, Bi-212, At-211 or 1-125.

[0192] In certain embodiments, such as the internalization of radiolabeled SHALs by tumor cells, the effector can include an alpha emitter, i.e., a radioactive isotope that emits alpha particles and/or an Auger-electron emitter. Alpha-emitters and Auger-electron emitters have recently been shown to be effective in the treatment of cancer (see, e.g., Bodei et al. (2003) Cancer Biotherapy and Radiopharmaceuticals, 18:861). Suitable alpha emitters include, but are not limited ²¹²Bi, ²¹³Bi, ²¹¹At, and the like.

[0193] Transition metals may also be included. They may be covalently attached to the SHAL directly (e.g. by incorporation into a DOTA or other chelator linked to the SHAL) or via a linker. They may be transition metal complexes. In some embodiments, metal- carbonyl derivatives are attached to the SHAL.

[0194] Table 5 illustrates some radionuclides suitable for radioimmunotherapy. This list is intended to be illustrative and not limiting.

Table 5: Illustrative radionuclides suitable for radioimmunotherapy.

* beta irradiation

** alpha irradiation

RBE, relative biologic effectiveness

Nanoparticles

[0195] In some embodiments, the SHAL is attached to the outside or is contained within a nanoparticle. The nanoparticle may include quantum dots, magnetic beads, or be created using poly(DL-lactide-co-glycolide) (PLGA), albumin, polyethylene glycol-lipid conjugates or other amphiphilic molecules. In some embodiments the nanoparticle is selected from silver, gold, copper, cadmium, hydroxyapatite, clay, titanium dioxide, silicon dioxide, zirconium dioxide, carbon, diamond, aluminium oxide, or ytterbium trifluoride nanoparticles. In some embodiments, the nanoparticle is any well known in the art and described in the literature, for example, those described in Zhen et al., 2017, Oncology Reports 38: 611-624, Dinarvand et al., 2011, International Journal of Nanomedicine 6:877- 895, Farokhzad et al.,2006, Proc. Natl. Acad. Sci. U.S.A. 103: 6315-6320, or Jain et al., 2007, Nanotoday 2: 18-29.

[0196] In some embodiments, the nanoparticle is between about 1 nm and 50 nm in diameter or length, an optimal size for use in imaging, being transported through the blood brain barrier, being taken up the small intestine, or for cell or molecule isolation or separation. In some embodiments, the nanoparticle is between about 50 nm and 100 nm in diameter or length, an optimal size for targeted delivery of drugs and drug cocktails into cancer cells. In some embodiments, the nanoparticle is between about 100 nm and 300 nm in diameter or length, a size that can penetrate through capillary walls.

Effector Molecules

[0197] In various embodiments the effector molecule can be a small molecule, a metal ligand, a radioisotope, an enzyme, a peptide, an enzyme inhibitor, a toxin, an epitope tag, or an antibody. Particularly preferred effectors are those that bind to surface markers on cancer cells or immune cells or those that inhibit biological activities required for normal or cancer cell function. In some embodiment, the effector is selected from Table 4 or any of the radiosensitizer, the radioisotope, the nanoparticle, the chelator, the cytotoxin, the viral particle, or other therapeutic moiety as disclosed herein.

Ligands

[0198] In most embodiments the ligand is an ion, metal atom or a molecule that binds to another molecule. In some embodiments, the SHALs comprise, consist essentially of, or consist of one or more small molecule ligands from Table 1. In other embodiments, a ligand from Table 1 binds to the chelating group in a SHAL or is attached to the SHAL free amine. In some embodiments, the ligand is selected from Table 1 and/or Table 2.

Chelates

[0199] Many of the SHALs described herein contain a chelator or a metal chelating group. The chelator or chelating group is typically coupled to a SHAL through the free amino or carboxyl group at the end of the linker scaffold.

[0200] Chelating groups are well known to those of skill in the art. In certain embodiments, chelating groups are derived from ethylene diamine tetra-acetic acid (EDTA), di ethylene triamine penta-acetic acid (DTP A), cyclohexyl 1,2-diamine tetra-acetic acid (CDTA), ethyleneglycol-0, 0^,-bis(2-aminoethyl)-N,N,N^,,N’-tetra-acetic acid (EGTA), N,N- bis(hydroxybenzyl)-ethylenediamine-N,N’-diacetic acid (HBED), tri ethylene tetramine hexa- acetic acid (TTHA), 1,4,7, 10-tetraazacyclododecane-N,N’-,N”,N”’ -tetra-acetic acid (DOTA), hydroxy ethyldiamine triacetic acid (HEDTA), 1,4,8,11-tetra-azacyclotetradecane- N,N’,N”,N’” -tetra-acetic acid (TETA), l,4,8,ll-tetraazabicyclo[6.6.2]hexadecane-4,ll- diacetic acid (CB-TE2A), substituted DTP A, substituted EDTA, and the like.

[0201] Examples of certain preferred chelators include unsubstituted or, substituted 2- iminothiolanes and 2-iminothiacyclohexanes, in particular 2-imino-4- mercaptomethylthiolane.

[0202] One chelating agent, 1,4,7,10-tetraazacyclododecane-N, N, N”, N’”-tetraacetic acid (DOTA), is of particular interest because of its ability to chelate a number of diagnostically and therapeutically important metals, such as radionuclides and radiolabels.

[0203] Conjugates of DOTA and proteins such as antibodies have been described.

For example, U.S. Pat. No. 5,428,156 teaches a method for conjugating DOTA to antibodies and antibody fragments. To make these conjugates, one carboxylic acid group of DOTA is converted to an active ester which can react with an amine or sulfhydryl group on the antibody or antibody fragment. Lewis et al. (1994) Bioconjugate Chem. 5: 565-576, describes a similar method wherein one carboxyl group of DOTA is converted to an active ester, and the activated DOTA is mixed with an antibody, linking the antibody to DOTA via the epsilon-amino group of a lysine residue of the antibody, thereby converting one carboxyl group of DOTA to an amide moiety. This same approach can be used to conjugate DOTA to the epsilon-amino group of the terminal lysine residue in the SELAL linker scaffold.

[0204] It is noted that the macrocyclic chelating agent 1,4,7,10- tetraazacyclododecane-N,N’,N”,N”’-tetraacetic acid (DOTA) binds ⁹⁰Y and ^U1ln with extraordinary stability. Kinetic studies in selected buffers to estimate radiolabeling reaction times under prospective radiopharmacy labeling can be performed to determine optimal radiolabeling conditions to provide high product yields consistent with FDA requirements for a radiopharmaceutical. It is also noted that protocols for producing Yttrium-90-DOTA chelates are described in detail by Kukis et al. (1998) J. Nucl. Med., 39(12): 2105-2110. [0205] In some embodiments, the chelator is:

Cytotoxins

[0206] The SHALs of this invention can be used to deliver a variety of cytotoxic molecules including therapeutic drugs, an isotope emitting radiation, divalent or trivalent metals (e.g. Fe⁺², Fe⁺³, Cr⁺³, Cu⁺², etc.), molecules derived from plants, fungi, viruses or bacteria, biological proteins, and mixtures thereof. These cytotoxic molecules can be linked directly to the SFLAL or they can be encapsulated into nanoparticles or liposomes linked to SHALs that target cells, tissues or other molecules or macromolecular structures. The cytotoxic drugs can be intracellularly acting cytotoxic drugs, such as short-range radiation emitters, including, for example, short-range, high-energy a-emitters as described above, or enzyme inhibitors.

[0207] Particularly preferred enzymatically active toxins thereof are exemplified by diphtheria toxin (DT), exotoxin A (from Pseudomonas aeruginosa), ricin, abrin, modeccin, alpha-sacrin, Pokeweed antiviral protein S, Pokeweed antiviral protein type II, curcin, restrictocin, phenomycin, and enomycin, for example. Pseudomonas exotoxin A (PE) is an extremely active monomeric protein (molecular weight 66 kD), secreted by Pseudomonas aeruginosa, which inhibits protein synthesis in eukaryotic cells through the inactivation of elongation factor 2 (EF-2) by catalyzing its ADP-ribosylation (catalyzing the transfer of the ADP ribosyl moiety of oxidized NAD onto EF-2).

[0208] Like PE, diphtheria toxin (DT) kills cells by ADP-ribosylating elongation factor 2 thereby inhibiting protein synthesis. Diphtheria toxin, however, is divided into two chains, A and B, linked by a disulfide bridge. In contrast to PE, chain B of DT, which is on the carboxyl end, is responsible for receptor binding and chain A, which is present on the amino end, contains the enzymatic activity (Uchida et al. (1972) Science, 175: 901-903; Uchida et al. (1973) J. Biol. Chem., 248: 3838-3844).

Viral Particles

[0209] In certain embodiments, the effector comprises, consists essentially of, or consists of a viral particle. The SHAL can be conjugated to the viral particle e.g., via a protein expressed on the surface of the viral particle (e.g., a filamentous phage). The viral particle can additionally include a nucleic acid that is to be delivered to the target (prostate cancer) cell. The use of viral particles to deliver nucleic acids to cells is described in detail in O’Keefe, 2013, Mater. Methods 3:174, Ni et al., 2016, Adv. Drug Delivery Reviews 106: 3- 26 and Nayerossadat, et al., 2012, Adv. Biomed. Res. 1:27.

Other Therapeutic Moieties

[0210] Other suitable effector molecules include pharmacological agents or encapsulation systems comprising, consisting essentially of, or consisting of various pharmacological agents. Thus, the SHAL can be attached directly to a drug that is to be delivered directly to the tumor. Such drugs are well known to those of skill in the art and include, but are not limited to, doxorubicin, vinblastine, genistein, diclofenac, and kinase and PARP inhibitors such as lenvatinib, adpelisib, veliparib, lenalidomide, sorafenib, acalabrutinib, axitinib, lorlatinib, noraparib, aplutamide, gilteritinib, and the like.

[0211] Alternatively, the effector molecule can comprise, consist essentially of, or consist of an encapsulation system, such as a viral capsid, a liposome, a nanoparticle or micelle that comprises, consists essentially of, or consists of a therapeutic composition such as a drug, a nucleic acid (e.g., an antisense nucleic acid or another nucleic acid to be delivered to the cell), or another therapeutic moiety that is preferably shielded from direct exposure to the circulatory system and/or facilitate the delivery of the SHAL to a desired cell organelle, cell, tissue or organ, such as across the blood-brain barrier or across the cell membrane. Examples include but are not limited to human serum albumin nanoparticles such as those used to deliver Paclitaxel to breast, ovarian and lung cancers , Nanomaterials 6: 1 lb- 132 or across the blood-brain barrier to gliomas (Gregory et al., 2020, Nat Commun 2020, 11 (1), 5687), Accurin nanoparticles that deliver AZD2811 to acute myelogenous leukemias and other tumors (Ashton et al., 2016; Science Translational Medicine 8(325): 325), BIND-014 nanoparticles that deliver docetaxel to tumors expressing prostate-specific membrane antigen (Hrkach et al., 2012; Sci Transl Med 2012; 4, 128ral39), NC-6004 micellular nanoparticles that deliver cisplatin derivatives to tumors (Kalra et al., 2014; Cancer Research 2014; 74:7003-7013), styrene-maleic acid micelles that deliver doxorubicin into tumors (Greish et al., 2004, J Control Release. 97: 219-30), polymer micelles that deliver cyclic pentapeptides to tumors expressing integrins (Nasongkla et al., 2004, Angew Chem Int Ed Engl 2004, 43 (46), 6323-7), PLGA nanoparticles that deliver anti-PDl antibodies to the spleen for the treatment of melanomas (Ordikhani et al., 2018, JCI insight. 2018 Oct 18;3(20):e l 22700) or inhibitors of TGFb signaling directly to PD-1 expressing cells for treating colorectal tumors (Schmid et al., 2017, Nat Commun 2017, 8 (1), 1747), and liposomal doxorubicin (Rahman et al., 2007, Cancer Biol Ther. 2004; 3:1021-7; Kundranda and Niu, 2015, Drug Design, Development and Therapy 2015; 9: 3767-3777). Means of preparing liposomes attached to antibodies are well known to those of skill in the art (see, for example, U.S. Patent No. 4,957,735, Connor et al. (1985) Pharm. Ther., 28: 341-365) and similar methods can be used for coupling SHALs (see, for example, Au et al. (2019) ACS Central Science 5: 122-144).

Methods of Treating and Detecting Cancer

[0212] In one aspect, a method for one or more of: detecting a cancer cell or tumor that expresses or has atypical expression of one or more of Major Histocompatibility Complex Class II (MHC Class II) proteins, inhibiting the growth or proliferation of a cancer cell or tumor that expresses or has atypical expression of MHC Class II, or killing a cancer cell or tumor that expresses or has atypical expression of MHC Class II proteins is provided, the method comprising, consisting essentially of, or consisting of contacting the cells with an effective amount of: a SHAL having a structure from Group A, Group B, or Group C, comprising, consisting essentially of, or consisting of two or more ligands as disclosed herein, for example, those from Table 1, or a derivative thereof; the SHAL of any embodiment herein; a pharmaceutical composition comprising, consisting essentially of, or consisting of the SHAL of any embodiment herein.

[0213] In some embodiments which may relate to any aspect and/or any method as disclosed herein, each cancer cell or tumor (such as a solid tumor) is independently selected from the group of gastric cancer, pancreatic cancer, renal cancer, prostate cancer, liver cancer, stomach cancer, urothelial cancers, lung cancer, ovarian cancer, cervical cancer, esophageal cancer, breast cancer, thyroid cancer, colorectal cancer, bone cancer, laryngeal cancer, head and neck cancer, lymphoma, leukemia, myeloma, glioma, breast medullary carcinoma, plasma cell myeloma, histiocytic sarcoma and melanoma. In some embodiments, the cancer cell or tumor (such as a solid tumor) is selected from gastric tumor/cancer, breast medullary carcinoma, or plasma cell myeloma. Additionally or alternatively, the cancer cell or tumor (such as a solid tumor) expresses and/or comprises an HLA-DR. In an optional further embodiment, the cancer cell or tumor (such as a solid tumor) does not express and/or does not comprise any one or any two or any three or four of CD80, CD86, CD74 or CD44.

In some embodiments, the cancer cell or tumor (such as a solid tumor) expresses and/or comprises a target of a SHAL ligand as disclosed herein, such as HLA-DR, and any one or any two or any three or four of CD80, CD86, CD74 or CD44. In some embodiments, the cancer cell or tumor (such as a solid tumor) is not an invasive ductal breast cancer and/or a liver cancer. In some embodiments, the cancer cell or tumor (such as a solid tumor) does not express or comprise either or both of the transporters, OATP1B1 and OATP1B3. In other embodiments, the cancer cell or tumor (such as a solid tumor) expresses or comprises either or both of the transporters, OATP1B1 and OATP1B3, at a low level, for example comparing to a normal cell, such as a normal hepatocyte. In some embodiments, the cancer cell or tumor (such as a solid tumor) has been and/or is being concurrently contacted and/or treated with a combined therapy to increase the expression of the target that a SHAL as disclosed herein specifically binds. One non-limiting example is the cancer cell and tumor (such as a solid tumor) has been or is being concurrently contacted and/or treated with IFN-g. Without wishing to be bound by the theory, IFN-g sensitizes the cancer cell or tumor by increasing the expression level of HLA-DR on the cancer cell or tumor and/or by making the cancer cell or tumor which does not express HLA-DR to express HLA-DR. In one aspect, the method also inhibits metastasis of the cancer to the lymph nodes or other organs of the body.

[0214] In some embodiments which may relate to any aspect and/or any method as disclosed herein, the cancer cell does not express HLA-DRIO or an HLA-DR comprising, consisting essentially of, or consisting of a Lym-1 epitope. In some embodiments, the cancer cell does not express one or more MHC Class II proteins. In one aspect, the SHAL binds to and inhibits the activity of a molecule, such as a protein expressed by cancer cells whose function is required for tumor growth and survival. One example of the molecule and/or protein is neuropilin, a transmembrane glycoprotein receptor expressed by many cancers . Binding of the VEGF-A growth factor to cancer cell neuropilin has been shown to promote angiogenesis (Miao et al., 2000, Faseb j 2000, 14 (15), 2532-9), stimulate tumor cell migration and metastasis (Jia et al., 2010, Br J Cancer 2010, 102 (3), 541-52), and suppress the anti-tumor immune response by reducing the production of TGF by Tregs or macrophages (Hansen et al., 2012, J Exp Med 2012, 209 (11), 2001-16). VEGF-A/neuropilin signaling resulting from this interaction has also been reported to confer resistance to chemotherapy (Goel and Mercurio, 2013, Nat Rev Cancer 2013, 13 (12), 871-82; Peng et al., 2018, Drug Discov. Today 2018). Computational docking studies conducted with the DvKBa, the targeting domain of SHALs SH5141 and SH5143, have shown these SHALs bind inside the same cavity on neuropilin where VEGF-A and inhibitors such as EF00229 that block the VEGF-A:neuropilin interaction bind (data not shown). The calculated free energy of DvKBa’s binding to this site on neuropilin (AG = -9.62Kcal/mol) suggests the SHALs affinity for neuropilin is equal to or greater than that of EG00229 (AG = - 9.55Kcal/mol).

[0215] In some embodiments, the contacting is in vitro or in vivo. In some embodiments, the cancer cell is a mammalian cancer cell.

[0216] In some embodiments, the method is to detect cancer cells in biopsy tissue in an image obtained by light transmission or fluorescence microscopy, scanning mass spectrometry (e.g. for example MALDI mass spectrometry) or scanning probe microscopy or in a positron emission tomography scan (PET scan), in a computerized tomography scan (CT scan), in a magnetic resonance imaging scan (MRI scan), in any other medical imaging scan, in a liquid biopsy, in blood or in cerebral or spinal fluid, or in any other bodily fluids, the method comprising, consisting essentially of, or consisting of contacting the biopsy tissue or fluid with a SHAL having a structure from Group A, Group B, or Group C, comprising, consisting essentially of, or consisting of two or more ligands from Table 1, or a derivative thereof further comprising, consisting essentially of, or consisting of any suitable linker from Table 3 or detection label comprising Group R shown in Table 4. In some embodiments, the biopsy tissue or fluid has been preserved, such as formalin-fixed and/or paraffin-embedded, prior to contacting with a SHAL as disclosed herein. [0217] In one aspect, a method of treating cancer cells, a solid tumor or other cells that expresses or has atypical expression of an MHC class II protein, in a subject in need thereof with the SHAL of any embodiments herein is provided, the method comprising, consisting essentially of, or consisting of treating the cancer cells, solid tumor or other cells in the subject by administering to the subject an effective amount of the SHAL. In some embodiments, the cancer cells or solid tumor are selected from one or more of pancreatic cancer, renal cancer, prostate cancer, liver cancer, stomach cancer, urothelial cancers, lung cancer, ovarian cancer, cervical cancer, esophageal cancer, breast cancer, thyroid cancer, colorectal cancer, bone cancer, laryngeal cancer, head and neck cancers, lymphomas, leukemias, myelomas, gliomas, histiocytic sarcomas, and melanomas. Other cells are selected from the group of lymphoctyes, macrophages, dendritic cells, monocytes, NK cells, epithelial cells, endothelial cells, megakaryocyte progenitors, microglia, keratinocytes, and enterocytes. In some embodiments, the cell does not express HLA-DR10 or an HLA-DR comprising, consisting essentially of, or consisting of a Lym-1 epitope. Additionally or alternatively, the cell expresses an HLA-DR.

[0218] In some embodiments, the SHAL inhibits the growth of the tumor or progression of the cancer or kills the cancer cells. In some embodiments, the cancer cells or solid tumor does not express HLA-DRIO or an HLA-DR comprising, consisting essentially of, or consisting of a Lym-1 epitope. In some embodiments, the cancer cells or solid tumor does not express MHC class II proteins.

[0219] In some embodiments which may relate to any aspect and/or any method as disclosed herein, the method further comprises, consists essentially of, or consists of administering to the subject and/or contacting the cancer cell or tumor or other cells with an effective amount of one or more of an anticancer agent for cytoreductive therapy. Anticancer agents include any known in the art of cancer therapy, non-limiting examples include IFN-g, actinomycin-D, alkeran, ara-C, anastrozole, BiCNU, bicalutamide, bleomycin, busulfan, capecitabine, carboplatin, carboplatinum, carmustine, CCNU, chlorambucil, cisplatin, cladribine, CPT-11, cyclophosphamide, cytarabine, cytosine arabinoside, cytoxan, dacarbazine, dactinomycin, daunorubicin, dexrazoxane, docetaxel, doxorubicin, DTIC, epirubicin, ethyleneimine, etoposide, floxuridine, fludarabine, fluorouracil, flutamide, fotemustine, gemcitabine, hexamethylamine, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone, oxaliplatin, paclitaxel, pamidronate, pentostatin, plicamycin, procarbazine, steroids, streptozocin, STI-571, tamoxifen, temozolomide, teniposide, tetrazine, thioguanine, thiotepa, tomudex, topotecan, treosulphan, trimetrexate, vinblastine, vincristine, vindesine, vinorelbine, VP- 16, xeloda, asparaginase, AIN-457, bapineuzumab, belimumab, brentuximab, briakinumab, canakinumab, cetuximab, dalotuzumab, denosumab, epratuzumab, estafenatox, farletuzumab, figitumumab, galiximab, gemtuzumab, girentuximab (WX-G250), herceptin, ibritumomab, inotuzumab, ipilimumab, mepolizumab, muromonab-CD3, naptumomab, necitumumab, nimotuzumab, ocrelizumab, ofatumumab, otelixizumab, ozogamicin, pagibaximab, panitumumab, pertuzumab, ramucirumab, reslizumab, rituximab, REGN88, solanezumab, tanezumab, teplizumab, tiuxetan, tositumomab, trastuzumab, tremelimumab, vedolizumab, zalutumumab, zanolimumab, 5FC, accutane hoffmann-la roche, AEE788 novartis, AMG-102, anti neoplaston, AQ4N (Banoxantrone), AVANDIA (Rosiglitazone Maleate), avastin (Bevacizumab), BCNU, biCNU carmustine, CCI-779, CCNU, CCNU lomustine, celecoxib (Systemic), chloroquine, cilengitide (EMD 121974), CPT -11 (CAMPTOSAR, Irinotecan), dasatinib (BMS-354825, Sprycel), dendritic cell therapy, etoposide (Eposin, Etopophos, Vepesid), GDC-0449, gleevec (imatinib mesylate), gliadel wafer, hydroxychloroquine, IL-13, IMC-3G3, immune therapy, iressa (ZD-1839), lapatinib (GW572016), methotrexate for cancer (Systemic), novocure, OSI-774, PCV, RAD001 novartis (mTOR inhibitor), rapamycin (Rapamune, Sirolimus), RMP-7, RTA 744, simvastatin, sirolimus, sorafenib, SU-101, SU5416 sugen, sulfasalazine (Azulfidine), sutent (Pfizer), TARCEVA (erlotinib HC1), taxol, TEMODAR schering-plough, TGF-B anti-sense, thalomid (thalidomide), topotecan (Systemic), VEGF trap, VEGF-trap, vorinostat (SAHA), XL 765, XL 184, XL765, zamestra (tipifarnib), ZOCOR (simvastatin), cyclophosphamide (Cytoxan), (Alkeran), chlorambucil (Leukeran), thiopeta (Thioplex), busulfan (Myleran), procarbazine (Matulane), dacarbazine (DTIC), altretamine (Hexalen), clorambucil, cisplatin (Platinol), ifosafamide, methotrexate (MTX), 6-thiopurines (Mercaptopurine [6-MP], Thioguanine [6-TG]), mercaptopurine (Purinethol), fludarabine phosphate, (Leustatin), flurouracil (5-FU), cytarabine (ara-C), azacitidine, vinblastine (Velban), vincristine (Oncovin), podophyllotoxins (etoposide (VP-16}and teniposide (VM- 26}), camptothecins (topotecan and irinotecan ), taxanes such as paclitaxel (Taxol) and docetaxel (Taxotere), (Adriamycin, Rubex, Doxil), dactinomycin (Cosmegen), plicamycin (Mithramycin), mitomycin: (Mutamycin), bleomycin (Blenoxane), estrogen and androgen inhibitors (Tamoxifen), gonadotropin-releasing hormone agonists (Leuprolide and Goserelin (Zoladex)), aromatase inhibitors (Aminoglutethimide and Anastrozole (Arimidex)), amsacrine, asparaginase (El-spar), mitoxantrone (Novantrone), mitotane (Lysodren), retinoic acid derivatives, bone marrow growth factors (sargramostim and filgrastim), amifostine, pemetrexed, decitabine, iniparib, olaparib, veliparib, lenvatinib, adpelisib, lenalidomide, acalabrutinib, axitinib, lorlatinib, noraparib, aplutamide, gilteritinib, everolimus, vorinostat, entinostat (SNDX-275), mocetinostat (MGCD0103), panobinostat (LBH589), romidepsin, valproic acid, flavopiridol, olomoucine, roscovitine, kenpaullone, AG-024322 (Pfizer), fascaplysin, ryuvidine, purvalanol A, NU2058, BML-259, SU 9516, PD-0332991, P276-00, geldanamycin, tanespimycin, alvespimycin, radicicol, deguelin, BIIB021, cis-imidazoline, benzodiazepinedione, spiro-oxindoles, isoquinolinone, thiophene, 5-deazaflavin, tryptamine, aminopyridine, diaminopyrimidine, pyridoisoquinoline, pyrrol opyrazole, indolocarbazole, pyrrolopyrimidine, dianilinopyrimidine, benzamide, phthalazinone, tricyclic indole, benzimidazole, indazole, pyrrolocarbazole, isoindolinone, morpholinyl anthracycline, a maytansinoid, ducarmycin, auristatins, calicheamicins (DNA damaging agents), a-amanitin (RNA polymerase II inhibitor), centanamycin, pyrrolobenzodiazepine, streptonigtin, nitrogen mustards, nitrosorueas, alkane sulfonates, pyrimidine analogs, purine analogs, antimetabolites, folate analogs, anthracyclines, taxanes, vinca alkaloids, other kinase inhibitors, other PARP inhibitors, other topoisomerase inhibitors, and hormonal agents.

[0220] In some embodiments, a method for inducing, enhancing or promoting an anti tumor immune response in a subject in need thereof is provided, the method comprising, consisting essentially of, or consisting of administering to the subject an effective amount of a SHAL as disclosed herein. In some embodiments, the SHAL has a structure from Group A, Group B, or Group C, comprising, consisting essentially of, or consisting of two or more ligands from Table 1 and/or Table 2, or a derivative thereof.

[0221] In some embodiments, the immune response comprises, consists essentially of, or consists of activating B-cell lymphocytes, macrophages, dendritic cells or CD4+ or CD8+ T cell lymphocytes to induce an anti-tumor immune response. The anti-tumor immune response may be directed towards cancer cells or tumors selected from the group of: pancreatic cancer, renal cancer, prostate cancer, liver cancer, stomach cancer, urothelial cancers, lung cancer, ovarian cancer, cervical cancer, esophageal cancer, breast cancer, thyroid cancer, colorectal cancer, bone cancer, laryngeal cancer, head and neck cancers, lymphoma, leukemia, myeloma, glioma, histiocytic sarcoma, melanoma, or any other cancer as disclosed herein.

[0222] In some embodiments, the immune response is induced by binding of the

SHAL to an MHC class II protein and the presentation of the SHAL, by the MHC class II protein, to T-cell lymphocytes.

[0223] In another aspect, a method to kill or inhibit the growth or proliferation of a cancer cell that expresses an MHC class II protein that is not HLA-DR10 or does not contain a Lym-1 epitope is provided, the method comprising, consisting essentially of, or consisting of contacting the cell with an effective amount of a SHAL as disclosed herein. In some embodiments, the SHAL has a structure from Group A, Group B, or Group C, comprising, consisting essentially of, or consisting of two or more ligands from Table 1 and/or Table 2, or a derivative thereof. Additionally or alternatively, the cancer cell is selected from the group of pancreatic cancer, renal cancer, prostate cancer, liver cancer, stomach cancer, urothelial cancers, lung cancer, ovarian cancer, cervical cancer, esophageal cancer, breast cancer, thyroid cancer, colorectal cancer, bone cancer, laryngeal cancer, head and neck cancer, lymphoma, leukemia, myeloma, glioma, histiocytic sarcoma, melanoma, or another cancer as disclosed herein.

[0224] In another aspect, a method of treating cancer cells or a tumor that expresses an MHC class II protein that is not HLA-DRIO or does not contain a Lym-1 epitope, in a subject in need thereof is provided, the method comprising, consisting essentially of, or consisting of administering to the subject an effective amount of a SHAL as disclosed herein. In some embodiments, the SHAL has the structure from Group A, Group B, Group C, Specimen-Group-Al, Specimen-Group-Bl, or Specimen-Group-Cl, comprising, consisting essentially of, or consisting of two or more ligands from Table 1 and/or Table 2, or a derivative thereof. Additionally or alternatively, the cancer is selected from the group of pancreatic cancer, renal cancer, prostate cancer, liver cancer, stomach cancer, urothelial cancers, lung cancer, ovarian cancer, cervical cancer, esophageal cancer, breast cancer, thyroid cancer, colorectal cancer, bone cancer, laryngeal cancer, head and neck cancer, lymphoma, leukemia, myeloma, glioma, histiocytic sarcoma, melanoma, or any other cancer as disclosed herein. In some embodiments, the cells are normal cells or cancer cells.

[0225] For the above methods, an effective amount is administered, and administration of the SHAL or composition serves to treat the disease, inhibit cell proliferation or inhibit metastases, treat any symptom or prevent additional symptoms from arising. When administration is for the purposes of treating or preventing or reducing the likelihood of cancer recurrence or metastasis, the SHAL or compositions can be administered in advance of any visible or detectable symptom. Routes of administration include, but are not limited to, oral (such as a tablet, capsule or suspension), topical, transdermal, intranasal, vaginal, rectal, subcutaneous intravenous, intraarterial, intramuscular, intraosseous, intraperitoneal, epidural and intrathecal. The amount and mode of administration can be determined by the treating veterinarian (for the treatment of animals) or physician.

[0226] The methods provide one or more of: (1) preventing the symptoms or disease from occurring in a subject that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression or relapse of the disease or the symptoms of the disease. As understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. For the purposes of the present technology, beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (i.e., not worsening) state of a condition (including disease), delay or slowing of condition (including disease), progression, amelioration or palliation of the condition (including disease), states and remission (whether partial or total), whether detectable or undetectable. Treatments containing the disclosed compositions and methods can be first line, second line, third line, fourth line, fifth line therapy and are intended to be used as a sole therapy or in combination with other appropriate therapies e.g., surgical recession, chemotherapy, radiation. In one aspect, treatment excludes prophylaxis.

[0227] In some embodiments, the SHAL or a derivative thereof binds to one or more of the MHC class II proteins selected from the group of HLA-DRl, HLA-DR3, HLA-DR4, HLA-DR7, HLA-DR8, HLA-DR9, HLA-DRl 1, HLA-DRl 2, HLA-DRl 3, HLA-DR 14, HLA-DRl 5, HLA-DRl 6, HLA-DP and HLA-DQ. [0228] In some embodiments, the SHAL binds to the MHC class II HLA-DR proteins comprising, consisting essentially of, or consisting of a beta subunit selected from one or more of the beta subunits of DRB1, DRB3, DRB4 or DRB5. In some embodiments, the SHAL inhibits the growth of the tumor or progression of the cancer, or kills the cancer cells. In some embodiments, the method further comprises, consists essentially of, or consists of administering to the subject and/or contacting the cancer cell or tumor or other cells with an effective amount of an anticancer agent for cytoreductive therapy. Additionally or alternatively, the method further comprises, consists essentially of, or consists of administering to the subject and/or contacting the cancer cell or tumor or other cells with an effective amount of an agent (such as IFN-g) which is capable of causing expression of the target which the SHAL binds and/or increasing the expression of the target, thereby facilitating the SHAL treatment as disclosed herein. In some embodiments, such expression is in or on a cancer cell or tumor or other target cells optionally in the subject.

Method of Delivering SHALs into Cells that do not Express MHC Class II Proteins

[0229] In some embodiments which may be combined with any aspect and/or embodiments as disclosed herein, it may be desirable in certain situations to deliver SHALs designed to target HLA-DRs into cells that lack the MHC Class II proteins. These include the treatment of tumors or cancer cells that do not express MHC Class II proteins, normal or activated lymphocytes or other normal or dysfunctional mammalian cells that cause disease, or bacteria that have become resistant to antibiotics and other drugs. The delivered SHALs may be used to kill or suppress the growth of the cancer, to inhibit the transporters responsible for the resistance cancer cells and bacteria develop to drugs, or to reduce the dose of other drugs required to achieve a therapeutic response. Nanoparticles (Steen 2018, Biomaterials 179, 209-245; Kalepu 2015, Acta Pharmaceutica Sinica B 5(5): 442-453;

Ashton 2016, Science Translational Medicine 8(325): 325 ; Zhou 2016, Anticancer Res. 36: 1649-1656; Bobo 2016, Pharmaceutical Res 33(10): 2373-87), liposomes (Kalepu 2015, Acta Pharmaceutica Sinica B 5(5): 442-453) and micelles (Kalepu 2015, Acta Pharmaceutica Sinica B 5(5): 442-453) are currently being used to deliver a number of different oncology drugs (doxorubicin, irinotecan, Paclitaxel, BIND-014, cisplatin, AZD2811, etc.) to cancer cells and tumors. Nanoparticles have also been shown to be effective in delivering drugs into bacteria (Wang 2017, Int J Nanomedicine 12: 1227-1249; Baptista 2018, Frontiers in Microbiology 9: 1-26). Antibodies that are internalized by cells following their binding to their target, such as monoclonal antibodies that recognize cell surface proteins other than HLA-DR to which SHALs could be conjugated or linked, or bispecific antibodies, diabodies and antibody-avidin conjugates or fusion proteins that recognize and bind simultaneously to both a cell surface receptor and a DOTA chelating group or a biotin tag (Figure 7), can also be used to deliver SHALs into cells that do not express MHC the Class II proteins targeted by the SHAL. Examples of monoclonal antibodies that have already been developed and could be used for SHAL delivery into cancer cells include Trastuzumab for treating breast, stomach and esophageal cancers expressing the HER2 protein, Brentuximab for treating anaplastic large cell and Hodgkin’s lymphomas that express CD30, Enfortumab for treating many solid cancers expressing Nectin-4, Gemtuzumab for treating acute myelogenous leukemias expressing CD33, Polatuzumab for treating B-cell malignancies expressing CD79B, Sacituzumab for treating solid cancers expressing Trop-2, Brevituximab for treating Hodgkin’s lymphomas expressing CD30, BAT8001 for treating breast cancers expressing HER2, Mirvetuximab for treating ovarian cancers expressing Folate receptor 1, Loncastuximab for treating B-cell lymphomas expressing CD 19, Camidanlumab for treating Hodgkin’s lymphomas expressing CD25, antibodies recognizing B-cell maturation antigen (CD269) for treating multiple myeloma, or antibodies targeting CD239 (B-Cam) for treating ovarian, skin, liver and lung cancers. Antibodies such as L-243 that recognize the alpha subunit of HLA-DR, which is common to all HLA-DR serotypes, could also be linked to these SHALs and used to treat the other melanomas, cervical, ovarian prostate, liver, kidney, bone, breast, esophageal, head and neck, bladder, colorectal, lung, pancreatic, larynx, gastric, gliomas, and thyroid cancers that express HLA-DRs containing beta-subunits not recognized by the SHALs. Examples of bispecific antibodies, diabodies and antibody-avidin conjugates or fusion proteins that could be used for SHAL delivery to other targets include the anti- DOTA/anti-CEA bispecific antibodies (Yazaki et ak, 2013, Protein Engineering Design & Selection 26 (3), 187-193), anti-DOTA/anti-CD45 bispecific antibodies (Orozco et ak, 2017, Blood 130 (Supplement 1): 1355), anti DOTA/anti-GD2 fustion antibodies (Santich et ak, 2020, J. Nuck Med. 61 (Supplement 1): 34), anti-DOTA/anti-HLA-DR diabodies (DeNardo et ak, 2001, Cancer Bi other Radi opharm 16 (6): 525-35) and avidin, streptavidin or neutravidin-antibody conjugates or fusion proteins that have been designed and tested for pretargeted radioimmunotherapy of lymphoma, breast, ovarian, colorectal and other cancers.

[0230] In one aspect, a method of treating cancer cells or a tumor that does not express an MHC class II protein, in a subject in need thereof is provided, the method comprising, consisting essentially of, or consisting of administering to the subject a nanoparticle comprising, consisting essentially of, or consisting of a SHAL as disclosed herein, thereby treating the cancer cells or tumor that does not express an MHC class II protein. In some embodiments, the SHAL is of a structure selected from Groups A, B, or C, comprising, consisting essentially of, or consisting of two or more ligands from Table 1 and/or Table 2, or a derivative of each thereof.

[0231] In some embodiments which may be combined with any aspect and/or embodiments as disclosed herein, methods that can be used to encapsulate SHALs into nanoparticles for the treatment of cancers that do not express MHC Class II proteins (see for example, Lomis et al., 2016, Nanomaterials 6: 116-132; Karmali et al., 2009, Nanomedicine 2009, 5 (1), 73-82; Farokhzad et al., 2006, Proc Natl Acad Sci U S A 2006, 103 (16), 6315- 20; Chen et al., 2010, Mol Ther 18(9): 1650-6), for facilitating the uptake of SHALs delivered into the stomach, intestine or colon (see for example, Date et al., 2016, J Control Release 240: 504-526), or for delivering the SHALs across the blood-brain or blood-testis barrier to gain access to brain or testis cancers (see for example, Cirpanli et al., 2011, Int J Pharm. 201 l;403(l-2):201-206. 62; Snow-Lisy et al., 2011, Drug Deliv and Transl. Res. 2011; 1:351; Battaglia et al., 2014, J Pharm Sci. 2014;103(7):2157-2165; Zhang et al., 2015, Drug Design, Development and Therapy 9: 2089-2100; Chen et al., 2016, Curr Med Chem 2016, 23 (7), 701-13; Kumari et al., 2017, Scientific Reports 2017; 7:6602; Kang et al., 2019, Journal of Drug Targeting 2019; 27: 103-110; Saeedi et al., 2019, Biomedicine & Pharmacotherapy 2019; 111: 666-675; Teleanu et al., 2019, Nanomaterials 2019; 9: 371) are well known in the art.

[0232] In some embodiments, another method for treating cancer cells or tumors that do not express MHC Class II proteins is provided, comprising, consisting essentially of, or consisting of administering to a subject in need a bispecific antibody, diabody or antibody - avidin conjugate or fusion protein comprising, consisting essentially of, or consisting of a bound DOTA-tagged or biotin-tagged SHAL of a structure selected from Groups A, B, or C, comprising, consisting essentially of, or consisting of two or more ligands from Table 1 and/or Table 2, or a derivative of each thereof. The antibody or diabody component that recognizes and binds the complex to tumor cells expressing the target protein also deliver the SHAL that is bound through its DOTA or biotin tag to the anti-DOTA or anti-biotin antibody or the conjugated avidin, streptavidin or neutravidin.

[0233] In other embodiments, a method for reversing or blocking the development of drug resistance in bacteria or fungi infecting a subject is provided, the method comprising, consisting essentially of, or consisting of administering to the subject a nanoparticle comprising, consisting essentially of, or consisting of a SHAL as disclosed herein, which delivers the SHAL into the bacterial or fungal cells wherein the SHAL inhibits the transporter proteins that actively pump antibiotics and other drugs out of the cells, thereby reversing or preventing the development of resistance to drugs that are substrates for the inhibited transporter. In some embodiments, the SHAL is of the structure selected from Group A, B, or C, comprising, consisting essentially of, or consisting of two or more ligands from Table 1 and/or Table 2, or derivatives thereof.

[0234] For the above methods, an effective amount is administered, and administration of the SHAL or composition serves to treat the disease, inhibit cell proliferation or inhibit metastases, treat any symptom or prevent additional symptoms from arising. When administration is for the purposes of treating or preventing or reducing the likelihood of cancer recurrence or metastasis, the SHAL or compositions can be administered in advance of any visible or detectable symptom. Routes of administration include, but are not limited to, oral (such as a tablet, capsule or suspension), topical, transdermal, intranasal, vaginal, rectal, subcutaneous intravenous, intraarterial, intramuscular, intraosseous, intraperitoneal, epidural and intrathecal. The amount and mode of administration can be determined by the treating veterinarian (for the treatment of animals) or physician. They can be combined with other therapies or methods as determined by the treating veterinarian (for the treatment of animals) or physician.

[0235] The methods provide one or more of: (1) preventing the symptoms or disease from occurring in a subject that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression or relapse of the disease or the symptoms of the disease. As understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. For the purposes of the present technology, beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (i.e., not worsening) state of a condition (including disease), delay or slowing of condition (including disease), progression, amelioration or palliation of the condition (including disease), states and remission (whether partial or total), whether detectable or undetectable. Treatments containing the disclosed compositions and methods can be first line, second line, third line, fourth line, fifth line therapy and are intended to be used as a sole therapy or in combination with other appropriate therapies e.g., surgical recession, chemotherapy, radiation. In one aspect, treatment excludes prophylaxis. Method of pretargeting SHALs or other drugs to tumor cells for therapy, imaging, and diagnostic applications

[0236] Radioimmunotherapy (RIT), a technique that uses radiolabeled antibodies to deliver localized radiation to the surface or interior of tumor cells, has shown considerable promise for treating radiosensitive tumors, but the approach has fallen short of expectations primarily due to the unacceptable radiation doses that are received by normal tissues as a consequence of the slow clearance of radiolabeled antibodies from the circulation. Pretargeting RIT approaches using bispecific antibodies, diabodies and antibody-avidin conjugates or fusion proteins have been developed to reduce radiation damage to normal tissue have improved therapeutic indices significantly. Pretargeted RIT (PRIT) methods typically involve administering the unlabeled bispecific antibody, diabody or antibody-avidin complex or fusion protein to the subject first and allowing enough time for its binding to the target cells and the subsequent clearance of the unbound bispecific antibody, diabody or antibody-avidin complex or fusion protein from the circulation. Small molecules comprising, consisting essentially of, or consisting of the radiation source (e.g. radiolabeled peptides or a DOTA chelator loaded with a radionuclide) are then administered and captured by the bispecific antibody, diabody or antibody-avidin complex or fusion protein that remains bound to the tumor cells. Because the radiolabel is attached to a small molecule, it is rapidly cleared from the blood and excreted and the off-target dose of radiation received by normal tissue is reduced significantly. Similar pretargeting methods can also be used for the delivery of radionuclides (e.g. ⁶⁸Ga, ⁹⁰Y, ^U1ln) and other molecular species (e.g. ¹⁸F) to tumors for disease diagnosis or whole-body tumor imaging by positron emission tomography (PET) or other techniques.

[0237] A method of pre-targeting a SHAL to a cell or tumor in a subject, comprising, consisting essentially of, or consisting of: administering to the subject a bispecific antibody, diabody or antibody-avidin conjugate that recognizes and binds to both: (a) a cell surface receptor or protein; and (b) a DOTA tag or biotin tag on the SHAL, the SHAL comprising the structure selected from Group A, B, or C, comprising two or more ligands from Table 1 and/or Table 2; followed by administering the SHAL to the subject after a suitable period of time. In some embodiments, a radionuclide is bound by the DOTA chelating group. In some embodiments, the SHAL is isotopically labeled.

[0238] For the above methods, routes of administration include, but are not limited to, oral (such as a tablet, capsule or suspension), topical, transdermal, intranasal, vaginal, rectal, subcutaneous intravenous, intraarterial, intramuscular, intraosseous, intraperitoneal, epidural and intrathecal. The amount and mode of administration can be determined by the treating veterinarian (for the treatment of animals) or physician. They can be combined with other therapies or methods as determined by the treating veterinarian (for the treatment of animals) or physician.

Method of Delivering Other Drugs Into Cells Expressing the MHC Class II Proteins A SHAL Target

[0239] Antibodies can be developed to recognize and bind to almost any protein or small molecule. Immunological reactions are frequently encountered in patients treated with biological drugs used to treat cancer and other diseases. It’s also not uncommon for anti-drug antibodies to be produced by an individual’s own body when they are treated with certain small molecule drugs (Coleman 1986, Br. J. Clin. Pharmac. 22: 161-165; Amali 2012; Brinch 2009, Antimicrobial Agents and Chemotherapy 53(11): 4794-4800; Arndt and Garratty 2002, Am J Clin Pathol 118: 256-262). Colchicine, digoxin and oxycodone antibodies have been developed for use in treating overdoses (Pentel, 1995, Toxicology Letters 82-83: 801-806; Pravetoni 2012, Journal of Pharmacology and Experimental Therapeutics, 341(1), 225-232). Bispecific antibodies, diabodies or antibody-avidin conjugates or fusion proteins that have been designed to recognize and bind to both a drug and a biotin (or DOTA) tag on a SHAL can be used to facilitate the delivery of immunoreactive, toxic or highly insoluble drugs into cells expressing the MHC Class II proteins the SHALs target (e.g. cancer cells, activated lymphocytes, macrophages or dendritic cells). The delivery of drugs that are bound tightly to antibodies would be expected to significantly reduce or eliminate the drug’s systemic toxicity or immunogenicity due to its inability to enter normal cells. Using human or humanized bispecific antibodies, diabodies or antibody-avidin conjugates or fusion proteins that and bind both the drug and a DOTA or biotin tag on a SHAL, almost any type of drug could be selectively delivered into tumor or other cells expressing the MHC Class II proteins the SHALs target. Methods for isolating, purifying, sequencing and producing recombinant human antibodies are well known to those in the art.

[0240] A pre-targeting method for delivering a drug to a cell or tumor in a subject, the cell or tumor expressing an MHC class II protein recognized by a SHAL, comprising, consisting essentially of, or consisting of: administering to the subject: (a) a biotin-tagged or DOTA-tagged SHAL complex comprising the SHAL of Group A, B, or C, comprising two or more ligands from Table 1 and/or Table 2, and (b) a bispecific antibody, diabody or antibody- avidin conjugate or fusion protein that recognizes and binds to both the DOTA tag or biotin tag of the SHAL and the drug; and administering the drug to the subject a suitable period of time after administration of (a) and (b).

[0241] For the above methods, an effective amount is administered, and administration of the SHAL is provided in an amount effective to achieve the result of the method. The SHAL or compositions can be administered in advance of any visible or detectable symptom. Routes of administration include, but are not limited to, oral (such as a tablet, capsule or suspension), topical, transdermal, intranasal, vaginal, rectal, subcutaneous intravenous, intraarterial, intramuscular, intraosseous, intraperitoneal, epidural and intrathecal. The amount and mode of administration can be determined by the treating veterinarian (for the treatment of animals) or physician. They can be combined with other therapies or methods as determined by the treating veterinarian (for the treatment of animals) or physician. Method of Blocking the Induction or Relapse of an Autoimmune Disease

[0242] There is a well-established strong association between MHC Class II proteins and autoimmune disease (Figure 4 and Table 8). Different HLA-DRs and other MHC class II proteins bind and present different types of peptide antigens to T-cells as part of the process in which the immune system monitors the body for foreign proteins present in bacteria, viruses and toxins. What leads to the induction of an autoimmune disease is the presentation by HLA-DRs of peptides from normal proteins present in the healthy body to T-cell lymphocytes in such a way that it leads to the T-cell lymphocytes triggering an immune response that targets and degrades normal tissues comprising, consisting essentially of, or consisting of the protein from which the peptide was derived. Targeting abnormal overexpression of MHC Class II proteins with SHALs can be used to modulate or treat autoimmune diseases either by blocking or displacing the binding of self-antigens to MHC Class II proteins on B-cells or by killing the B-cell producing the autoantibodies (e.g., the way Rituximab kills B-cells in treating Rheumatoid arthritis).

[0243] The expression of various MHC Class II proteins is predictive of either a predisposition to or a protection against various autoimmune diseases, as detailed in Table 8.

Table 8: Associations between MHC Class II proteins and Autoimmune Diseases

[0244] In one aspect, a method to treat an MHC class II protein linked autoimmune disease or disorder is provided, the disease or disorder selected from the group of Table 8 comprising, consisting essentially of, or consisting of Rheumatoid Arthritis, Multiple Sclerosis, Type-1 Diabetes, Grave’s Disease, Hashimoto’s Thyroiditis, Myasthenia Gravia, Celiac Disease, Systemic Lupus Erythematosus, or Anklylosing Spondylitis in a subject in need thereof, the method comprising, consisting essentially of, or consisting of administering to the subject an effective amount of a SHAL as disclosed herein. In some embodiments, the SHAL has a structure from Group A, Group B, or Group C, comprising, consisting essentially of, or consisting of two or more ligands from Table 1 and/or Table 2, or derivatives thereof.

[0245] In some embodiments, the immune response comprises, consists essentially of, or consists of activity of lymphocytes, macrophages and dendritic cells. In some embodiments, the immune response comprises, consists essentially of, or consists of blocking presentation of self-antigens by an MHC class II protein or suppressing inflammation. In some embodiments, the method comprises, consists essentially of, or consists of killing of B- lymphocytes involved in the production of autoantibodies. In some embodiments, the method comprises, consists essentially of, or consists of killing of T-lymphocytes and/or macrophages and/or dendritic cells involved in the activation or production of helper (CD4+) or killer (CD8+) lymphocytes. In some embodiments, the method comprises, consists essentially of, or consists of the binding of the SHAL to the peptide binding site on HLA-DR thereby preventing the presentation of the self-peptide and the induction of an immune response against proteins comprising, consisting essentially of, or consisting of the self peptide that is present in normal cells. [0246] In some embodiments, a method for treating a disease or disorder related to a pathological immune response in a subject in need thereof is provided, the immune response contributing to a disease or disorder of the group of vascular injury and leucocyte recruitment leading to restenosis, allergic asthma, inflammation, and inflammation induced restriction of blood flow in ischemia stroke; the method comprising, consisting essentially of, or consisting of administering to the subject an effective amount of a SHAL as disclosed herein. In some embodiments, the SHAL has a structure from Group A, Group B, or Group C, comprising, consisting essentially of, or consisting of two or more ligands from Table 1 and/or Table 2, or derivatives thereof.

[0247] In some embodiments, the method comprises, consists essentially of, or consists of administering an effective amount of a second therapy, prior to, subsequent to, or concurrent to the administration of the SHAL as disclosed herein. In some embodiments, the SHAL has a structure from Group A, Group B, or Group C, comprising, consisting essentially of, or consisting of two or more ligands from Table 1 and/or Table 2, or derivatives thereof. The second therapy may be any therapy known in the art for the treatment of one or more of the autoimmune diseases in Table 8.

[0248] In some embodiments, the effector is selected from the group of a therapeutic agent, a detectable agent, a probe or a marker that can be manipulated, or a structure from Group R in Table 4, and optionally the probe or marker that can be manipulated comprises, consists essentially of, or consists of a magnetic particle or a light, pH, or frequency-activated nanostructure or molecule, or a derivative thereof. In some embodiments, the effector is delivered to a cell that does not express HLA-DRIO or an HLA-DR comprising, consisting essentially of, or consisting of a Lym-1 epitope or any MHC class II protein.

[0249] For the above methods, an effective amount is administered, and administration of the SHAL is provided in an amount effective to achieve the result of the method. The SHAL or compositions can be administered in advance of any visible or detectable symptom. Routes of administration include, but are not limited to, oral (such as a tablet, capsule or suspension), topical, transdermal, intranasal, vaginal, rectal, subcutaneous intravenous, intraarterial, intramuscular, intraosseous, intraperitoneal, epidural and intrathecal. The amount and mode of administration can be determined by the treating veterinarian (for the treatment of animals) or physician. They can be combined with other therapies or methods as determined by the treating veterinarian (for the treatment of animals) or physician.

Method of Treating or Suppressing Neurodegeneration

[0250] Microglia, a specialized population of macrophage-like immune cells in the brain and spinal cord, have been linked to the development and progression of Alzheimer’s, Parkinson’s, Multiple Sclerosis and a number of other neurodegenerative diseases (Perry 2010, Nat Rev Neurol (2010) 6:193-201; Bachiller 2018, Frontiers in Cellular Neuroscience 12: 488; Subramanian 2017, Frontiers in Aging Neuroscience

DOI.org/10.3389/fnagi.2017.00176). Following the transformation of resting or quiescent microglia into an activated state, which can be triggered by their exposure to certain cytokines, cell necrosis factors, lipopolysaccharides, or changes in extracellular potassium (from ruptured cells), activated microglia cluster around, initiate and then drive the degradation of neuronal structure and the loss of neuron function through their release of a variety of proinflammatory and potentially neurotoxic substances. Activated microglia have also been reported to accelerate the growth and invasive proliferation of gliomas and other brain tumors (Wu 2017, Front Bioscience 22: 1805-1829). Mounting evidence indicates the microenvironment surrounding microglial cells that infiltrate gliomas induces their activation, and these activated microglia then perform both glioma-supportive and immunosuppressive roles (Wei 2013, Clin and Developmental Immunology Vol. 2013, Article ID 285246).

[0251] Alzheimer’s disease, the most common form of dementia, is caused by neuro- inflammation and the death of neurons. Neuronal death has been shown to be associated with the extracellular deposition of amyloid-b plaques in the brain, which appears to occur as a result of either an increased production, or a lack of clearance, of amyloid-b peptides derived from amyloid precursor protein cleavage and by abnormal inter-neuronal accumulation of hyperphosphorylated tau protein (Bachiller 2018, Frontiers in Cellular Neuroscience 12:

488). The prevailing hypothesis that describes how neuro-inflammation contributes to the development of Alzheimer’s disease is supported by many studies investigating the clustering of microglial cells within amyloid deposits in the human brain and suggests that the primary mechanism that leads to the development of Alzheimer’s disease is the activation of the microglial cell. In the resting or quiescent state, only a small percentage of microglial cells express HLA-DR. In those cells that express, the level of expression is low (McGreer 1987, Neuroscience Letters 79: 195-200; Ponomarev 2005, JNeurosci Res 81(3): 374-89). When events trigger their activation, microglial cells begin expressing high levels of HLA-DR. The activated microglia expressing HLA-DR (Mattiace 1990, Am J Pathol 1990, 136 (5), 1101- 14) then cluster around and transform diffuse deposits of amyloid-b into compact senile (neuritic) plaques. The peptides that comprise, consist essentially of, or consist of the plaques aggregate and are believed to be toxic to neurons.

[0252] Parkinson’s disease is characterized by a loss of dopamine producing neurons in the pars compacta, a region of the brain containing basal ganglia situated at the base of the forebrain and the top of the midbrain. The loss of these neurons reduces the levels of dopamine (a neurotransmitter) in the basal ganglia and leads to motor dysfunction. In Parkinson’s disease, microglia have been shown to contribute to the degeneration of the dopaminergic neurons (McGreer 1987, Neuroscience Letters 79: 195-200; Akiyama 2000, Neurobiol Aging 21 : 383-421). A growing body of evidence suggest that neuro- inflammation mediated by activated microglia also plays a contributory role in the development of Parkinson’s disease. While relatively little is known about the different microglial activation states in Parkinson’s disease, targeting activated microglial cells and suppressing their proinflammatory neurotoxicity has been suggested and is considered a valid approach for future Parkinson’s disease therapy (Subramanian 2017, Frontiers in Aging Neuroscience DOI. org / 10.3389/fnagi .2017.00176).

[0253] In individuals with Multiple Sclerosis, the immune system attacks the protective sheath (myelin) that covers nerve fibers and causes communication problems between the brain and the rest of the body. Brain tissue in patients diagnosed with Multiple Sclerosis contain lesions throughout the white matter that include infiltrating inflammatory lymphocytes and macrophages, blood-brain barrier leakage, destruction of myelin sheaths, oligodendrocyte dysfunction and loss, and axon damage and loss. Monocyte derived macrophages and activated microglia, both of which express HLA-DR at high levels, are believed to contribute to lesion formation by phagocytosing myelin, which leads to extensive sheath damage and oligodendrocyte dysfunction (Hendriks JJ, Teunissen CE, de Vries HE, Dijkstra CD: Macrophages and neurodegeneration. Brain Res Brain Res Rev 2005, 48: 185— 195.). During this process, the activated microglia and macrophages secrete various inflammatory mediators, including cytokines, chemokines, nitric oxide and reactive oxygen species, which all contribute to multiple sclerosis progression.

[0254] Suppression of microglia-mediated inflammation is currently considered to be an important strategy for neurodegenerative disease therapy. One approach that has been proposed for accomplishing this suppression in the treatment of Alzheimer’s, Parkinson’s and Multiple Sclerosis that should also be applicable to the therapy of gliomas is to target and reduce or remove the population of activated microglial cells responsible for the neurodegeneration (or support of glioma proliferation) that occurs during the development and progression of these diseases (van Horssen 2012; Shi 2019; Olmos-Alonso 2016; Subramanian 2017; Wei 2013). In one aspect, a method to treat an HLA-DR or MHC class II protein linked neurodegenerative disease or disorder is provided, the disease or disorder comprising, consisting essentially of, or consisting of Alzheimer’s, Parkinson’s, multiple sclerosis, amyotrophic lateral sclerosis, frontotemporal dementia or other microglia-mediated neurodegenerative diseases in a subject in need thereof, the method comprising, consisting essentially of, or consisting of administering to the subject an effective amount of a SHAL as disclosed herein. In some embodiments, the SHAL has a structure from Group A, Group B, or Group C, comprising, consisting essentially of, or consisting of two or more ligands from Table 1 and/or Table 2, or derivatives thereof.

[0255] In some embodiments, the method comprises, consists essentially of, or consists of the suppression or killing of activated microglia or microglial cells and/or macrophages that contribute to the neuron damage or destruction in Alzheimer’s,

Parkinson’s, Multiple Sclerosis, Amyotropic Lateral Sclerosis, Frontotemporal Dementia or other microglia-mediated neurodegenerative diseases. In some embodiments, the SHAL comprises, consists essentially of, or consists of an effector that is selected from Group R in Table 4. In some embodiments, the SHAL is delivered across the blood-brain barrier in a nanoparticle, liposome, micelle, or hydrogel.

[0256] For the above methods, administration of the SHAL is provided in an amount effective to achieve the result of the method. The SHAL or compositions can be administered in advance of any visible or detectable symptom. Routes of administration include, but are not limited to, oral (such as a tablet, capsule or suspension), topical, transdermal, intranasal, vaginal, rectal, subcutaneous, intravenous, intraarterial, intramuscular, intraosseous,

-Ill- intraperitoneal, epidural and intrathecal. The amount and mode of administration can be determined by the treating veterinarian (for the treatment of animals) or physician. They can be combined with other therapies or methods as determined by the treating veterinarian (for the treatment of animals) or physician.

Methods of Inhibiting Cell Growth by Modulating GTPase Activating Protein (GAP), GTPase Enzyme, or AcetylCo-carboxylase (ACC)

[0257] GTPase-activating proteins or GTPase-accelerating proteins (GAPs) are a family of regulatory proteins whose members can bind to activated G proteins and stimulate their GTPase activity, with the result of terminating the signaling event. The importance of GAPs comes from its regulation of the crucial G proteins. Many of these G proteins are involved in cell cycling, and as such are known proto-oncogenes. The Ras superfamily of G proteins, for example, has been associated with many cancers because Ras is a common downstream target of many growth factors like FGF, or fibroblast growth factor. Under normal conditions, this signaling ultimately induces regulated cell growth and proliferation. However, in the cancer state, such growth is no longer regulated and results in the formation of tumors.

[0258] Thus, in one aspect, a method to inhibit cell growth or to kill a cell by inhibiting a GTPase activating protein (GAP) selected from the group of MgcRacGAP, p50RhoGAP and BCR GAP, is provided, the method comprising, consisting essentially of, or consisting of contacting the GAP with an effective amount of a SHAL as disclosed herein. In some embodiments, the SHAL has a structure from Group A, Group B, or Group C, comprising, consisting essentially of, or consisting of one or more ligands from Table 1 and Table 2, or a derivative thereof, thereby inhibiting the GAP.

[0259] GTPases are a large family of hydrolase enzymes that bind to the nucleotide guanosine triphosphate (GTP) and hydrolyze it to guanosine diphosphate (GDP). GTPase activity itself may be directly inhibited. Racl, Rac3, RhoA and Cdc42 are four examples of GTPase: Racl is short for Rac Family Small GTPase 1, whose functions, nucleotide sequences, and amino acid sequences are available, for example, at genecards.org/cgi- bin/carddisp.pl?gene=RACl and uniprot.org/uniprot/P63000; Rac3 is short for Rac Family Small GTPase 3, whose functions, nucleotide sequences, and amino acid sequences are available, for example, at genecards.org/cgi-bin/carddisp. pl?gene=RAC3 and uniprot.org/uniprot/P60763; RhoA is short for Ras Homolog Family Member A, whose functions, nucleotide sequences, and amino acid sequences are available, for example, at genecards.org/cgi-bin/carddisp. pl?gene=RHOA and uniprot.org/uniprot/P61586; and Cdc42 is short for Cell Division Cycle 42, whose functions, nucleotide sequences, and amino acid sequences are available, for example, at genecards.org/cgi-bin/carddisp. pl?gene=CDC42 and uniprot.org/uniprot/P60953. All last accessed on December 17, 2020. The term p50RhoGAP is used interchangeably with Rho GTPase-activating protein 1 which activates RhoA and other Rho GTPases. Accordingly, as used herein, the term p50Rho refers a Rho GTPase which is activated by p50RhoGAP, for example RhoA.

[0260] Thus, in one aspect, a method to inhibit cell growth and proliferation or to kill a cell by directly inhibiting a GTPase enzyme selected from the group of Racl, Rac3, p50Rho, RhoA and Cdc42, is provided, the method comprising, consisting essentially of, or consisting of contacting the GTPase enzyme with an effective amount of a SHAL as disclosed herein, thereby directly inhibiting the GTPase enzyme. In some embodiments, the SHAL has a structure from Group A, Group B, or Group C, comprising, consisting essentially of, or consisting of one or more ligands from Table 1 and Table 2, or a derivative thereof.

[0261] In some embodiments, cancer is treated in a subject in need thereof by administering to the subject, an effective amount of a SHAL as disclosed herein, thereby killing the cancer cell by inhibiting the activity of GAP, GTPase enzyme or ACC. In some embodiments, the SHAL has a structure from Group A, Group B, or Group C, comprising, consisting essentially of, or consisting of one or more ligands from Table 1 and Table 2, or a derivative thereof.

[0262] Acetyl-CoA carboxylase (ACC) is a biotin-dependent enzyme that catalyzes the irreversible carboxylation of acetyl-CoA to produce malonyl-CoA through its two catalytic activities, biotin carboxylase (BC) and carboxyltransferase (CT). At the juncture of lipid synthesis and oxidation pathways, ACC presents many clinical possibilities for the production of novel therapeutics and the development of new therapies for diabetes, obesity, and other manifestations of metabolic syndrome. [0263] In one aspect, a method to inhibit cell growth or to kill a cell by inhibiting

AcetylCoA carboxylase (ACC) is provided, the method comprising, consisting essentially of, or consisting of contacting ACC with an effective amount of a SHAL as disclosed herein, thereby inhibiting ACC. In some embodiments, the SHAL has a structure from Group A, Group B, or Group C, comprising, consisting essentially of, or consisting of one or more ligands from Table 1 and Table 2, or a derivative thereof.

[0264] In some embodiments, the cell expresses MHC class II proteins. In some embodiments, the cell does not express MHC Class II proteins. In some embodiments, the contacting is in vitro or in vivo.

[0265] In some embodiments, obesity or obesity-related disorders comprising, consisting essentially of, or consisting of type-2 diabetes, non-alcoholic fatty-liver disease, or metabolic syndrome are treated in a subject in need thereof by administering to the subject, an effective amount of a SHAL as disclosed herein, thereby inhibiting the activity of GAP, GTPase enzyme or ACC. In some embodiments, the SHAL has a structure from Group A, Group B or Group C, comprising, consisting essentially of, or consisting of one or more ligands from Table 1 and Table 2, or a derivative thereof.

[0266] In some embodiments, the method of treating vascular complications in diabetes by inhibiting signal transduction pathways activated by Ras-GTPase involved in the development of diabetic vascular dysfunction comprises, consists essentially of, or consists of administering to the subject an effective amount of a second therapy, prior to, subsequent to, or concurrent with the administration of the SHAL comprising, consisting essentially of, or consisting of a structure from Group A, Group B, or Group C, comprising, consisting essentially of, or consisting of one or more ligands from Table 1 and/or Table 2, or a derivative thereof.

[0267] In some embodiments, the method further comprises, consists essentially of, or consists of administering to the subject an effective amount of a second therapy, prior to, subsequent to, or concurrent with the administration of the SHAL having a structure from Group A, Group B, or Group C, comprising, consisting essentially of, or consisting of one or more ligands from Table 1 and Table 2, or a derivative thereof. [0268] In one embodiment, the second therapy is a therapy to treat cancer, for example, actinomycin-D, alkeran, ara-C, anastrozole, BiCNU, bicalutamide, bleomycin, busulfan, capecitabine, carboplatin, carboplatinum, carmustine, CCNU, chlorambucil, cisplatin, cladribine, CPT-11, cyclophosphamide, cytarabine, cytosine arabinoside, cytoxan, dacarbazine, dactinomycin, daunorubicin, dexrazoxane, docetaxel, doxorubicin, DTIC, epirubicin, ethyleneimine, etoposide, floxuridine, fludarabine, fluorouracil, flutamide, fotemustine, gemcitabine, hexamethylamine, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone, oxaliplatin, paclitaxel, pamidronate, pentostatin, plicamycin, procarbazine, steroids, streptozocin, STI-571, tamoxifen, temozolomide, teniposide, tetrazine, thioguanine, thiotepa, tomudex, topotecan, treosulphan, trimetrexate, vinblastine, vincristine, vindesine, vinorelbine, VP- 16, xeloda, asparaginase, AIN-457, bapineuzumab, belimumab, brentuximab, briakinumab, canakinumab, cetuximab, dalotuzumab, denosumab, epratuzumab, estafenatox, farletuzumab, figitumumab, galiximab, gemtuzumab, girentuximab (WX-G250), herceptin, ibritumomab, inotuzumab, ipilimumab, mepolizumab, muromonab-CD3, naptumomab, necitumumab, nimotuzumab, ocrelizumab, ofatumumab, otelixizumab, ozogamicin, pagibaximab, panitumumab, pertuzumab, ramucirumab, reslizumab, rituximab, REGN88, solanezumab, tanezumab, teplizumab, tiuxetan, tositumomab, trastuzumab, tremelimumab, vedolizumab, zalutumumab, zanolimumab, 5FC, accutane hoffmann-la roche, AEE788 novartis, AMG-102, anti neoplaston, AQ4N (Banoxantrone), AVANDIA (Rosiglitazone Maleate), avastin (Bevacizumab) genetech, BCNU, biCNU carmustine, CCI-779, CCNU, CCNU lomustine, celecoxib (Systemic), chloroquine, cilengitide (EMD 121974), CPT -11 (CAMPTOSAR, Irinotecan), dasatinib (BMS-354825, Spry cel), dendritic cell therapy, etoposide (Eposin, Etopophos, Vepesid), GDC-0449, gleevec (imatinib mesylate), gliadel wafer, hydroxychloroquine, IL-13, IMC- 3G3, immune therapy, iressa (ZD-1839), lapatinib (GW572016), methotrexate for cancer (Systemic), novocure, OSI-774, PCV, RAD001 novartis (mTOR inhibitor), rapamycin (Rapamune, Sirolimus), RMP-7, RTA 744, simvastatin, sirolimus, sorafenib, SU-101,

SU5416 sugen, sulfasalazine (Azulfidine), sutent (Pfizer), TARCEVA (erlotinib HC1), taxol, TEMODAR schering-plough, TGF-B anti-sense, thalomid (thalidomide), topotecan (Systemic), VEGF trap, VEGF-trap, vorinostat (SAHA), XL 765, XL184, XL765, zarnestra (tipifarnib), ZOCOR (simvastatin), cyclophosphamide (Cytoxan), (Alkeran), chlorambucil (Leukeran), thiopeta (Thioplex), busulfan (Myleran), procarbazine (Matulane), dacarbazine (DTIC), altretamine (Hexalen), clorambucil, cisplatin (Platinol), ifosafamide, methotrexate (MTX), 6-thiopurines (Mercaptopurine [6-MP], Thioguanine [6-TG]), mercaptopurine (Purinethol), fludarabine phosphate, (Leustatin), flurouracil (5-FU), cytarabine (ara-C), azacitidine, vinblastine (Velban), vincristine (Oncovin), podophyllotoxins (etoposide (VP- 16}and teniposide {VM-26}), camptothecins (topotecan and irinotecan ), taxanes such as paclitaxel (Taxol) and docetaxel (Taxotere), (Adriamycin, Rubex, Doxil), dactinomycin (Cosmegen), plicamycin (Mithramycin), mitomycin: (Mutamycin), bleomycin (Blenoxane), estrogen and androgen inhibitors (Tamoxifen), gonadotropin-releasing hormone agonists (Leuprolide and Goserelin (Zoladex)), aromatase inhibitors (Aminoglutethimide and Anastrozole (Arimidex)), amsacrine, asparaginase (El-spar), mitoxantrone (Novantrone), mitotane (Lysodren), retinoic acid derivatives, bone marrow growth factors (sargramostim and filgrastim), amifostine, pemetrexed, decitabine, iniparib, olaparib, veliparib, lenvatinib, adpelisib, lenalidomide, acalabrutinib, axitinib, lorlatinib, noraparib, aplutamide, gilteritinib, everolimus, vorinostat, entinostat (SNDX-275), mocetinostat (MGCD0103), panobinostat (LBH589), romidepsin, valproic acid, flavopiridol, olomoucine, roscovitine, kenpaullone, AG-024322 (Pfizer), fascaplysin, ryuvidine, purvalanol A, NU2058, BML-259, SU 9516, PD-0332991, P276-00, geldanamycin, tanespimycin, alvespimycin, radicicol, deguelin, BIIB021, cis-imidazoline, benzodiazepinedione, spiro-oxindoles, isoquinolinone, thiophene, 5-deazaflavin, tryptamine, aminopyridine, diaminopyrimidine, pyridoisoquinoline, pyrrol opyrazole, indolocarbazole, pyrrolopyrimidine, dianilinopyrimidine, benzamide, phthalazinone, tricyclic indole, benzimidazole, indazole, pyrrol ocarb azole, isoindolinone, morpholinyl anthracycline, a maytansinoid, ducarmycin, auristatins, calicheamicins (DNA damaging agents), a-amanitin (RNA polymerase II inhibitor), centanamycin, pyrrolobenzodiazepine, streptonigtin, nitrogen mustards, nitrosorueas, alkane sulfonates, pyrimidine analogs, purine analogs, antimetabolites, folate analogs, anthracyclines, taxanes, vinca alkaloids, other kinase inhibitors, other PARP inhibitors, other topoisomerase inhibitors, hormonal agents, and combinations thereof.

[0269] In some embodiments, the second therapy is a therapy known to the skilled artisan for the treatment of diabetes or obesity. [0270] For the above methods, an effective amount is administered, and administration of the SHAL is provided in an amount effective to achieve the result of the method. The SHAL or compositions can be administered in advance of any visible or detectable symptom. Routes of administration include, but are not limited to, oral (such as a tablet, capsule or suspension), topical, transdermal, intranasal, vaginal, rectal, subcutaneous intravenous, intraarterial, intramuscular, intraosseous, intraperitoneal, epidural and intrathecal. The amount and mode of administration can be determined by the treating veterinarian (for the treatment of animals) or physician. They can be combined with other therapies or methods as determined by the treating veterinarian (for the treatment of animals) or physician.

Methods of Modulating Drug Uptake, Retention and Metabolism

[0271] Both efflux transporters MDRl/P-gp and BCRP and bidirectional transporters

OATP1B1 and OATP1B3 are located in the membrane of cancer and normal cells. Efflux transporters are involved in the development of resistance to drugs, as well as in modulating their bioavailability. OATP transporters are bidirectional, and upregulated in cancers to import hormones and growth factors cancer cells need to grow and survive. The metabolizing enzyme uridine 5’-diphospho-glucuronosyltransferase (UGT) helps to remove drugs from the cell by adding a glucuronide to make the drug more soluble and easier to export. During its preclinical testing, the SHAL SH7139 was determined to be an excellent inhibitor of the transporters MDRl, BCRP, OATP1B1 and OATP1B3 and the UGT enzymes UGT1 Al, UGT1 A3 and UGT1 A4. A number of clinical trials have shown other inhibitors of the efflux transporters and UGT enzymes present in cancer cells prevent the development of resistance to the oncology drug and also reduce the dose of drug needed as an approach to reduce the oncology drug’s side effects. Most of these inhibitors, however, have been found to also inhibit other metabolizing enzymes (e.g. CYP450s), which adversely affects the metabolism and clearance of the drug, or be too toxic to the liver and other normal tissues for continued use. Unlike the other transporter inhibitors, toxicology and safety studies show SH7139 is not toxic to the liver or other normal tissues and it does not inhibit CYP450 metabolizing enzymes.

[0272] The development of resistance to antibiotics and other drugs by several multi drug resistant species of bacteria often referred to as “Superbugs” has also been shown to be mediated by efflux transporters present in the membranes of these bacteria (Poole, K. et al., 2005 J. Antimicrobial Chem. 56:20-51). While not all bacterial efflux transporters have the same substrates or are affected by the same inhibitors as the mammalian transporters (Brincat JP et al., 2012 ACS Medical Chemistry Letters 3: 248-251; Singh S, et al. 2017 Frontiers in Microbiology 8: 1868-79), the ABC family of transporters are considered the primary transporters of the microbial efflux system that contributes to the development of resistance to ciprofloxacin, norlfloxacin, ofloxacin, rifampin and other important and frequently used antibiotics (Amin ML 2013 Drug Target Insights 7:27-34; Louw GE et al., 2011 Am. J. Respir. Crit. Care Med. 184: 269-276). In most cases the microbial ABC transporters are also inhibited by the same compounds that inhibit the P-gp/MDRl transporter and prevent or reverse the development of resistance by mammalian cells (Grossman TH et al., 2015 Antimicrobial Agents and Chemotherapy 59: 1534-41; Mullin S, et al., 2004 Antimicrobial Agents and Chemotherapy 48: 4171-76; Gibbons S et al., J. of Antimicrobial Chemotherapy 51: 13-17; Leitner I et al., 2011 J. of Antimicrobial Chemotherapy 66: 834-839).

[0273] In one aspect, a method to prevent one or more drugs taken up by a mammalian or bacterial cell from being pumped back out of the cell by inhibiting a multidrug resistance protein 1 (P -glycoprotein, MDR1 or P-gp) or breast cancer resistance protein (BCRP) efflux transporter, is provided, the method comprising, consisting essentially of, or consisting of contacting the transporter with an effective amount of a SHAL as disclosed herein, thereby inhibiting the activity of a transporter protein. In some embodiments, the SHAL has a structure from Group A, Group B or Group C, comprising, consisting essentially of, or consisting of one or more ligands from Table 1 and Table 2, or a derivative thereof.

[0274] In some embodiments, the uptake of the one or more drugs from the intestine, gut, oral cavity and across the blood-brain and testis barriers will be improved by using the SHAL to inhibit MRDl/P-gp or BCRP transporters in the endothelial cells lining blood vessels or forming the barriers. In some embodiments, the SHAL’s inhibition of the efflux transporters will prevent tumor cells from developing resistance to other drugs (e.g., doxorubicin, Imatinib, thienorphine, crizotinib, topotecan, docetaxel, SN38, paclitaxel, AZD2281, camptotheins, etc.) as has been reported for other transporter inhibitors. In some embodiments, the SHAL’s inhibition of these transporters will reverse the resistance tumor cells have already developed to these drugs. In some embodiments, sensitivity of the cell to the action of the one or more drugs is increased by preventing the one or more drugs from being pumped back out of the cell or by preventing the metabolism of the drug by UGT enzymes (e.g. belinostat, SN38, NU/ICRF 505, etc.).

[0275] In one aspect, a method to inhibit organic-anion-transporting polypeptide (OATP)-transporter mediated uptake of hormones, hormone conjugates, or growth promoting chemicals that a tumor cell requires to grow and survive, is provided, the method comprising, consisting essentially of, or consisting of contacting OATP -transporter with an effective amount of a SHAL as disclosed herein, thereby inhibiting the activity of the OATP -transporter protein. In some embodiments, the SHAL has a structure from Group A, Group B, or Group C, comprising, consisting essentially of, or consisting of one or more ligands from Table 1 and Table 2, or a derivative thereof.

[0276] In one aspect, a method to reduce the required dosage of a drug delivered to a subject in need thereof by inhibiting metabolic UDP-glucuronosyltransferase (UGT) enzyme is provided, the method comprising, consisting essentially of, or consisting of contacting the UGT enzyme with an effective amount of a SHAL as disclosed herein, thereby inhibiting activity of the UGT enzyme. In some embodiments, the SHAL has a structure from Group A, Group B, or Group C, comprising, consisting essentially of, or consisting of one or more ligands from Table 1 and Table 2, or a derivative thereof.

[0277] In one aspect, a method of delivering to a cell an effective amount of a SHAL having a structure from Group A, Group B or Group C is provided, the SHAL comprising, consisting essentially of, or consisting of two or more ligands from Table 1 and/or Table 2, or a derivative thereof, the method comprising, consisting essentially of, or consisting of the two or more ligands binding simultaneously to two or more different sites on a protein, enzyme, or the cell to act as adjuvant to work synergistically with another drug (e.g., for example doxorubicin, Imatinib, thienorphine, crizotinib, topotecan, docetaxel, SN38, paclitaxel, AZD2281, camptotheins, vinblastine, paclitaxel, etc.).

[0278] In some embodiments, the sensitivity of a cell to a drug’s action is increased by reducing the metabolism of the drug and slowing the rate of export of the drug from the cell. [0279] In some embodiments, the method further comprises, consists essentially of, or consists of administering to a subject in need thereof, an effective amount of a second therapy, prior to, subsequent to, or concurrent with administration to the subject of the SHAL having a structure from Group A, Group B, or Group C, comprising, consisting essentially of, or consisting of two or more ligands from Table 1 and/or Table 2, or a derivative thereof. Examples of a second therapy include Ganetespib for ovarian cancer, Venetoclax for non- Hodgkin’s lymphoma, paclitaxel or etoposide for lung cancer, Irinotecan (SN38) for colorectal cancer, diclofenac or naproxen or indomethacin for rheumatoid arthritis, Azitinib for kidney and pancreatic cancer, Belinostat for refractory peripheral T-cell lymphoma, Alvocidib for esophageal and liver cancer, Enasidenib for acute myeloid leukemia, Sorafenib for many solid tumors, Tofacitinib for ulcerative colitis, etc.

[0280] In some embodiments, the method comprises, consists essentially of, or consists of administration of the other drug, prior to, subsequent to or concurrent with the administration of the SHAL or a derivative thereof. In some embodiments, the cell expresses MHC class II proteins. In some embodiments, the cell does not express MHC Class II proteins. In some embodiments, the cell is a normal cell or a cancer cell. In some embodiments, the cancer cell is selected from the group of pancreatic cancer, renal cancer, prostate cancer, liver cancer, stomach cancer, urothelial cancers, lung cancer, ovarian cancer, cervical cancer, esophageal cancer, breast cancer, thyroid cancer, colorectal cancer, bone cancer, laryngeal cancer, head and neck cancer, lymphoma, leukemia, myeloma, glioma, histiocytic sarcoma and melanoma. In some embodiments, the contacting is in vitro or in vivo.

[0281] For the above methods, an effective amount is administered, and administration of the SHAL is provided in an amount effective to achieve the result of the method. The SHAL or compositions can be administered in advance of any visible or detectable symptom. Routes of administration include, but are not limited to, oral (such as a tablet, capsule or suspension), topical, transdermal, intranasal, vaginal, rectal, subcutaneous intravenous, intraarterial, intramuscular, intraosseous, intraperitoneal, epidural and intrathecal. The amount and mode of administration can be determined by the treating veterinarian (for the treatment of animals) or physician. They can be combined with other therapies or methods as determined by the treating veterinarian (for the treatment of animals) or physician.

Methods of Delivering Prodrugs to a Cell

[0282] Prior work related to the design of SHALs and their applications have focused primarily on making SHALs as stable as possible when prepared in formulations and when introduced into the bloodstream. This strategy has been shown to work well as the SHALs remain stable in formulations for more than a year and the SHAL’s ligands are not released in the blood or inside the cells. Only intra-ligand bond cleavage has been observed when the SHALs are metabolized inside the cells, which results in the production of several metabolites that are toxic to tumor and other cells and inhibit their growth and proliferation.

[0283] In one aspect, a method to deliver one or more prodrugs to a cell is provided, the method comprising, consisting essentially of, or consisting of a SHAL as disclosed herein, that simultaneously binds to a target protein on a cell and leads to the internalization of the SHAL. In some embodiments, the SHAL has a structure from Group A, Group B, or Group C, comprising, consisting essentially of, or consisting of two or more SHAL ligands from Table 1 and/or Table 2, or a derivative thereof.

[0284] In some embodiments, wherein the biological activity from the prodrug is derived from the metabolism of one or more of the SHAL ligands from Table 1 and Table 2, to produce fragments having the biological activity.

[0285] In some embodiments, the biological activity from the prodrug is derived from the reduction of a di-sulfide bond to selectively release one or more ligands having the biological activity. In some embodiments, the SHAL ligands target the target protein or cell with the SHAL acting as a compact small-molecule antibody-drug conjugate or ADC.

[0286] In some embodiments, the cell does not express MHC Class II proteins. In some embodiments, the cell is a normal cell or a cancer cell. In some embodiments, the cancer cell is selected from the group of pancreatic cancer, renal cancer, prostate cancer, liver cancer, stomach cancer, urothelial cancers, lung cancer, ovarian cancer, cervical cancer, esophageal cancer, breast cancer, thyroid cancer, colorectal cancer, bone cancer, laryngeal cancer, head and neck cancer, lymphoma, leukemia, myeloma, glioma, histiocytic sarcoma and melanoma. In some embodiments, the contacting is in vitro or in vivo. [0287] For the above methods, an effective amount is administered, and administration of the SHAL is provided in an amount effective to achieve the result of the method. The SHAL or compositions can be administered in advance of any visible or detectable symptom. Routes of administration include, but are not limited to, oral (such as a tablet, capsule or suspension), topical, transdermal, intranasal, vaginal, rectal, subcutaneous intravenous, intraarterial, intramuscular, intraosseous, intraperitoneal, epidural and intrathecal. The amount and mode of administration can be determined by the treating veterinarian (for the treatment of animals) or physician. They can be combined with other therapies or methods as determined by the treating veterinarian (for the treatment of animals) or physician.

Pharmaceutical Compositions

[0288] The SHALs, and/or chelates, and/or chimeric molecules of this invention

(particularly those specific for cancer or other pathologic cells) are delivered by parenteral, topical, oral, or local administration (e.g., injected into a tumor site), aerosol administration, or transdermal administration, for prophylactic, but principally for therapeutic treatment. The pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration. For example, unit dosage forms suitable for oral administration include powder, tablets, pills, capsules and lozenges. It is recognized that pharmaceutical compositions of this invention, when administered orally, can be protected from digestion. This is typically accomplished either by complexing the active component (e.g., the SHAL, the chimeric molecule, etc.) with a composition to render it resistant to acidic and enzymatic hydrolysis or by packaging the active ingredient(s) in an appropriately resistant carrier such as a liposome or nanoparticle. Means of protecting components from digestion are well known in the art.

[0289] The pharmaceutical compositions of this invention are particularly useful for parenteral administration, such as intravenous administration or administration into a body cavity or lumen of an organ. The composition for administration commonly comprises, consists essentially of, or consists of a solution of the SHAL and/or chimeric molecule dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, e.g., buffered saline and the like. These solutions are sterile and generally free of endotoxins, heavy metals, residual solvents and other undesirable matter. These compositions may be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of chimeric molecule in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient’s needs.

[0290] Thus, a typical pharmaceutical composition for intravenous administration would be about 0.02 to 10 mg SHAL per patient per day. Dosages from 0.1 up to about 100 mg per patient per day may be used, particularly when the drug is administered to a secluded site and not into the blood stream, such as into a body cavity or into a lumen of an organ. Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington: The Science and Practice of Pharmacy, Pharmaceutical Press, 22^nd Edition, 2012.

[0291] The compositions comprising, consisting essentially of, or consisting of the present SHALs and/or chimeric molecules or a cocktail thereof (i.e., with other therapeutics) can be administered for therapeutic treatments. In therapeutic applications, compositions are administered to a patient suffering from a disease, e.g., a cancer, in an amount sufficient to cure or at least partially arrest the disease and its complications. An amount adequate to accomplish this is defined as a “therapeutically effective dose.” Amounts effective for this use will depend upon the severity of the disease and the general state of the patient’s health.

[0292] Single or multiple administrations of the compositions may be administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of the SHALs to effectively treat the patient.

[0293] It is known to one of skill in the art that there are some regions of tissues, organs and tumors that are not heavily vascularized or that are protected by cells joined by tight junctions and/or active transport mechanisms which reduce or prevent the entry of macromolecules present in the blood stream. [0294] One of skill in the art will appreciate that in these instances, the therapeutic compositions of this invention can be administered directly to the tumor site. Thus, for example, brain tumors can be treated by administering the therapeutic composition directly to the tumor site (e.g., through a surgically implanted catheter).

[0295] Alternatively, the therapeutic composition can be placed at the target site in a slow release formulation. Such formulations can include, for example, a biocompatible sponge or other inert or resorbable matrix material impregnated with the therapeutic composition, slow dissolving time release capsules or microcapsules, and the like.

[0296] Typically the catheter or time release formulation will be placed at the tumor site as part of a surgical procedure. Thus, for example, where major tumor mass is surgically removed, the perfusing catheter or time release formulation can be placed at the tumor site as an adjunct therapy. Of course, surgical removal of the tumor mass may be undesired, not required, or impossible, in which case, the delivery of the therapeutic compositions of this invention may comprise, consist essentially of, or consist of the primary therapeutic modality.

Kits

[0297] When a SHAL comprising, consisting essentially of, or consisting of a radioactive or other labile effector is used as a diagnostic and/or therapeutic agent, it is frequently impossible to provide a ready -for-use composition of the SHAL to the user, because of the short shelf life of the radiolabeled SHAL and/or the short half-life of the radionuclide used. In such cases the user can carry out the labeling reaction with the radionuclide or the addition of the labile effector in the clinical hospital, physician’s office, or laboratory. For this purpose, or other purposes, the various reaction ingredients can then be offered to the user in the form of a so-called “kit.” The kit is preferably designed so that the manipulations necessary to perform the desired reaction should be as simple as possible to enable the user to prepare from the kit the desired composition by using the facilities that are at their disposal. Therefore the invention also relates to a kit for preparing a composition according to this invention.

[0298] Such a kit according to the present invention preferably comprises, consists essentially of, or consists of a SHAL as described herein. The SHAL can be provided, if desired, with inert pharmaceutically acceptable carrier and/or formulating agents and/or adjuvants is/are added. In addition, the kit optionally includes a solution of a salt or chelate of a suitable radionuclide (or other active agent or effector), and (iii) instructions for use with a prescription for administering and/or reacting the ingredients present in the kit.

[0299] The kit to be supplied to the user may also comprise, consist essentially of, or consist of the ingredient(s) defined above, together with instructions for use, whereas the solution of a salt or chelate of the radionuclide (or other active agent or effector) which can have a limited shelf life, can be put to the disposal of the user separately.

[0300] The kit can optionally, additionally comprise, consist essentially of, or consist of a reducing or conjugating agent and/or, if desired, a chelator or effector, and/or instructions for use of the composition and/or a prescription for reacting the ingredients of the kit to form the desired product(s). If desired, the ingredients of the kit may be combined, provided they are compatible.

[0301] In certain embodiments, the complex-forming reaction with the SHAL can simply be produced by combining the components in a neutral medium and causing them to react. For that purpose the effector may be presented to the SHAL in the form of a chelate or chemically activated effector.

[0302] When kit constituent(s) are used as component(s) for pharmaceutical administration (e.g., as an injection liquid) they are preferably sterile. When the constituent(s) are provided in a dry state, the user should preferably use a sterile physiological saline solution as a solvent. If desired, the constituent s) can be stabilized in the conventional manner with suitable stabilizers, for example, ascorbic acid, gentisic acid or salts of these acids, or they may comprise, consist essentially of, or consist of other auxiliary agents, for example, fillers, such as glucose, lactose, mannitol, and the like.

[0303] While the instructional materials, when present, typically comprise, consist essentially of, or consist of written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips, flash drives), optical media (e.g., CDs, DVDs), written instructions or U-tube or other videos (in, for example, .avi, .mov, .qt, .mkv, mp4, .avchd, .flv, .swf, etc. formats ) located at web or cloud sites accessible through the internet, and the like.

[0304] The following examples are illustrative of procedures which can be used in various instances in carrying the disclosure into effect.

EXAMPLES

Example 1: Procedure used to synthesize a typical SHAL

[0305] The following is an example of a protocol that is used to synthesize a typical

SHAL comprising, consisting essentially of, or consisting of three ligands selected from Table 1 and/or Table 2. The SHAL used as this example is SH8041. The SHAL was synthesized using solid phase chemistry by the stepwise attachment of Fmoc-D-Lys#l(Boc)- OH, Fmoc-AEEA-OH#l (Fmoc-8-amino-3,6-dioxaoctanoic acid), Fmoc-D-Lys#2(Dde)-OH, Fmoc-AEEA-OH#2, Fmoc-L-Val-OH, and Dabsyl chloride to a Wang resin using standard Fmoc (N-9-fluorenylmethoxy carbonyl) chemistry with HBTU (2-( 1 //-benzotriazol-1 -yl)- 1, 1,3,3-tetramethyluronium hexafluorophosphate)/HOBt (Hydroxybenzotriazole)/DIPEA (N,N-Diisopropylethylamine) as the coupling reagents. The side chain of Fmoc-D-Lys#2- (Dde)-OH, was then deprotected with 4% hydrazine in dimethylformamide (DMF) and then coupled to Fmoc-D-Lys#3-(Dde)-OH using the same coupling procedure. After Fmoc deprotection, Fmoc-AEEA-OH#3 was coupled to the a-amine of D-Lys#3. 4-(4-(4- chlorobenzyl) piperazine)-3-nitrobenzenecarboxylic acid (Cb ligand) was then linked to the Fmoc deprotected amine of AEEA-OH#3 using the same coupling procedure. The third ligand 3-[(2-{[3-chloro-5-(trifluoromethyl)pyridine-2yl]oxy}phenyl)amino] propanoic acid was then directly attached to the deprotected e-amine of D-Lys#3. The assembled free amine form of the SHAL was cleaved from the resin, deprotected and subsequently precipitated as a crude solid. The crude product was purified by standard RP-HPLC methods and isolated by lyophilization. DOTA (1,4,7, 10-Tetraazacyclododecane-l,4,7,10-tetraacetic acid) was attached to the free amine on the terminal lysine by dissolving the SHAL in anhydrous DMF, N,N-Diisopropylethylamine (DIEA) and solid DOTA N-hydroxysuccinimide ester. The mixture was mutated for 15 min, and the reaction was monitored by analytical HPLC. Upon completion, the reaction solution was diluted with a small volume of water/acetonitrile (50/50) containing 1% trifluoroacetic acid (TFA) and directly purified by HPLC. The resulting purified SH7129 was lyophilized and then analyzed by LC/MS and NMR spectroscopy to determine its purity and confirm its molecular mass and identity, respectively. The molecular mass of the product was determined to be 2308.8039 Da. The yield of solid SH8041 was 32 mg and the purity as determined by liquid chromatography was 71.4%.

Example 2: Single dose tumor cell growth inhibition screen (10 mM) of SH8041, SH8045 and SH8037

[0306] The structures of the three tested SHALs, SH8041, SH8045 and SH8037, are shown below in Table 6. This assay was performed by the Developmental Therapeutics Program of the National Institutes of Health. The human tumor cell lines of the cancer screening panel are grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine. For a typical screening experiment, cells are inoculated into 96 well microtiter plates in 100 pL at plating densities ranging from 5,000 to 40,000 cells/well depending on the doubling time of individual cell lines. After cell inoculation, the microtiter plates are incubated at 37° C, 5 % CO2, 95 % air and 100 % relative humidity for 24 h prior to addition of experimental drugs. The results are tabulated in Table 6, below.

[0307] After 24 h, two plates of each cell line are fixed in situ with TCA, to represent a measurement of the cell population for each cell line at the time of drug addition (Tz). The SHALs are solubilized in dimethyl sulfoxide at 400-fold the desired final maximum test concentration and stored frozen prior to use. At the time of drug addition, an aliquot of frozen concentrate is thawed and diluted to twice the desired final maximum test concentration (10 mM) with complete medium containing 50 pg/ml gentamicin. Aliquots of 100 pi of 20 pM SHAL are added to the appropriate microtiter wells already containing 100 pi of medium, resulting in the required final drug concentrations.

[0308] Following drug addition, the plates are incubated for an additional 48 h at

37°C, 5 % CO2, 95 % air, and 100 % relative humidity. For adherent cells, the assay is terminated by the q TCA (final concentration, 10 % TCA) and incubated for 60 minutes at 4°C. The supernatant is discarded, and the plates are washed five times with tap water and air dried. Sulforhodamine B (SRB) solution (100 pi) at 0.4 % (w/v) in 1 % acetic acid is added to each well, and plates are incubated for 10 minutes at room temperature. After staining, unbound dye is removed by washing five times with 1 % acetic acid and the plates are air dried. Bound stain is subsequently solubilized with 10 mM trizma base, and the absorbance is read on an automated plate reader at a wavelength of 515 nm. For suspension cells, the methodology is the same except that the assay is terminated by fixing settled cells at the bottom of the wells by gently adding 50 pi of 80 % TCA (final concentration, 16 %

TCA). Using the three absorbance measurements [time zero, (Tz), control growth, (C), and test growth in the presence of the SFLAL (Ti)], the percentage growth is calculated.

Percentage growth inhibition is calculated as:

[(TiTz)/(CTz)] x 100 for concentrations for which Ti>/=Tz

[(TiTz)/Tz] x 100 for concentrations for which Ti<Tz.

Table S

Single dose tumor ceil: grow h inhibition screen (10 mM) of SH8G41 , SHS045 (Sp& men-Group-A2i and SH8037 Gronp-82 having structures shown below

Example 3: Identification of other HLA-DRs that bind to SH7129 and comparison of their amino acid sequences to the sequences of HLA-DR10.

[0309] To identify the HLA-DRs targeted by SH7139 and SH7129, fixed slides of

PBMCs obtained from HLA-typed individuals who express HLA-DRs containing b-subunits from specific DRBl alleles were stained with SH7129, the biotinylated form of SH7139. The biotin in the bound SH7129 was detected using streptavidin horse-radish peroxidase (SAHRP) and the substrate 3,3-diaminobenzidine, and the slides were then counter-stained with hematoxylin to visualize the cells. Cells expressing HLA-DRs that bind SH7129 are stained brown.

[0310] HLA typed PBMCs were obtained from four commercial sources - AllCells

(Alameda, CA) , Cellular Technology Limited (Shaker Heights, OH), Precision for Medicine (New York, NY) and StemCell Technologies (Seattle, WA). Peripheral blood samples from individuals expressing DRB1*10 and DRB1*08 alleles were obtained from 3 healthy volunteers by a collaborator at the Madurai Kamaraj University, Madurai, India.

[0311] A stock solution of SH7129 was prepared by dissolving 10 mg of the dry

SHAL in 1 ml dimethyl sulfoxide. Slides of HLA-typed PBMCs were stained using a Leica BOND RX Automated Slide Stainer (Leica Biosystems Inc, Buffalo Grove, IL). The formalin fixed slides were incubated in citrate buffer at pH 6 and 90°C for 20 min to remove the aldehyde crosslinks (a process called antigen retrieval). After performing a 5 min hydrogen peroxide block, the slides were washed three times with BOND Wash Solution and then stained with SH7129 (100 pg/ml in PBS, 1% DMSO) for 30 min. Following three washes with BOND Washing Solution, the slides were treated with Streptavidin-horse radish peroxidase (SAHRP) for 30 min, washed 3 times with BOND Wash Solution and once with deionized water, treated with Mixed DAB Refine for 10 min, and then washed four times with deionized water, BOND Wash Solution and a final deionized water wash as per the BOND Polymer Refine IHC protocol (Histowiz, Brooklyn, NY). After counterstaining the PBMC slides with hematoxylin, the slides were then dehydrated by immersion in an alcohol series (30%, 70%, 95% and 100% for 4 min each), cleared with xylene and mounted with Permount. Images of the sections were obtained at 40X magnification and the images were processed and analyzed using ImageJ 1.42. Lymphocytes and macrophages expressing specific HLADRs that bind SH7129 are stained brown by the insoluble product that is produced following the oxidation of the DAB substrate by the horse-radish peroxidase conjugated to the streptavidin that bound to the biotin on the SHAL bound to HLA-DR molecules on the lymphocytes and macrophages. Lymphocytes and macrophages in the PBMCs samples obtained from individuals expressing an HLA-DR that does not bind to SH7129 remain unstained.

[0312] The results of the SH7129 staining experiments revealed that in addition to

HLA-DR 10, SH7129 and SH7139 also bind to HLA-DR7, HLA-DR9, HLA-DR11, HLA- DR12, HLA-DR13, HLA-DR 15 and HLA-DR16. The ability of SH7129 and SH7139 to bind to these HLA-DRs was unexpected since these SHALs were designed to bind specifically to HLA-DRIO or other HLA-DRs that contain the four amino acids recognized by the antibody Lym-1 (i.e. the Lym-1 epitope). Based on an amino acid sequence comparison of the peptide binding pockets of these HLA-DRs (Table 7), none contain the Lym-1 epitope and none would bind to Lym-1.

Example 4: Expression of SHAL HLA-DR target in lymphoma subtypes is variable

[0313] To determine which types of lymphoma tumors express the HLA-DRs targeted by SH7139, tumor microarrays (TMAs) containing fixed and paraffin embedded tumor biopsy sections obtained from human patients diagnosed with different B-cell and T- cell lymphomas were obtained from U.S. Biomax (Rockville, MD). A stock solution of SH7129 was prepared by dissolving 10 mg of the dry SHAL in 1 ml dimethyl sulfoxide. Formalin fixed slides containing arrays of tumor biopsy samples from 24-200 different patients diagnosed with a specific type of non-Hodgkin’s lymphoma were stained using a Leica BOND RX Automated Slide Stainer (Leica Biosystems Inc, Buffalo Grove, IL). The formalin fixed slides were deparaffmized using the Leica dewax solution, rehydrated with an alcohol series (100%, 95%, 70% and 30% for 4 min each) followed by antigen retrieval in citrate buffer at pH 6 and 90°C for 20 min. After performing a 5 min hydrogen peroxide block, the slides were washed three times with BOND Wash Solution and then stained with SH7129 (100 pg/ml in PBS, 1% DMSO) for 30 min. Following three washes with BOND Washing Solution, the slides were treated with Streptavidin-horse radish peroxidase (SAHRP) for 30 min, washed 3 times with BOND Wash Solution and once with deionized water, treated with Mixed DAB Refine for 10 min, and then washed four times with deionized water, BOND Wash Solution and a final deionized water wash as per the BOND Polymer Refine IHC protocol (Histowiz, Brooklyn, NY). The slides (which were not counterstained with hematoxylin) were then dehydrated by immersion in an alcohol series (30%, 70%, 95% and 100% for 4 min each), cleared with xylene and mounted with Permount. Tumor cells within the biopsy sections that express HLA-DRs that bind SH7129 are stained brown by the insoluble product produced upon oxidation of the DAB substrate by the horse-radish peroxidase conjugated to the streptavidin. Tumors that do not express HLA- DR do not bind SH7129 and remain unstained. Images of the sections were obtained at 40X magnification with a light microscope and the images were processed and analyzed using ImageJ 1.42. Because the slides were not counterstained with hematoxylin, the amount of bound SH7139 could be determined by densitometric analysis of 384 pixel sections of each captured biopsy image.

[0314] Applicants analyses of biopsy samples obtained from patients diagnosed with seven subtypes of non-Hodgkin’s lymphoma (diffuse large B-cell, follicular, anaplastic large cell, mucosa associated lymphoid tissue, mantle cell, Burkitt’s and small lymphocytic lymphomas) have shown a significant fraction of each subtype tested express the HLA-DRs targeted by SH7139 and bind its biotinylated form SH7129. The percentage of the cancers expressing the target ranged from 28% for mantle cell lymphoma to 100% for anaplastic large cell lymphoma.

Example 5: The SHAL MHC-class II target is expressed on at least 19 other nonlymphoma cancers

[0315] SH7129 was also used to screen a large number of biopsy samples obtained from patients diagnosed with solid cancers to determine if any other types of cancer might also express the target HLA-DRs. Tumor microarrays (TMAs) containing fixed and paraffin embedded tumor biopsy sections obtained from human patients diagnosed with 18 other types of cancer were obtained from U.S. Biomax (Rockville, MD). A stock solution of SH7129 was prepared and the TMA slides were deparaffmized and stained using a Leica BOND RX Automated Slide Stainer (Leica Biosystems Inc, Buffalo Grove, IL) as described in Example 4. Tumor cells within the biopsy sections that express HLA-DRs that bind SH7129 are stained brown by the insoluble product produced upon oxidation of the DAB substrate by the horse-radish peroxidase conjugated to the streptavidin. Tumors that do not express HLA-DR do not bind SH7129 and remain unstained. Images of the sections were obtained at 40X magnification with a light microscope and the images were processed and analyzed using ImageJ 1.42. Because the slides were not counterstained with hematoxylin, the amount of bound SH7139 could be determined by densitometric analysis of 384 pixel sections of each captured biopsy image. [0316] SH7129 binding to biopsy samples from patients diagnosed with eighteen other solid cancers show many of these tumors also express the HLA-DRs targeted by SH7139. Cervical, ovarian, colorectal and prostate cancers bind the most SH7129. Only a few (< 5%) esophageal and head and neck tumors bound the diagnostic. In marked contrast to invasive ductal breast cancers, in which only 4% of the tumors expressed HLA-DR, two thirds of the medullary carcinomas of the breast expressed the target. Within the tumors tested, cell to cell differences in SH7139 target expression, as determined by SH7129 binding, varied by only 2 to 3-fold while expression levels for different tumors varied as much as 10 to 100-fold. The observed binding of SH7129 to these cancers suggest many patients diagnosed with B-cell lymphomas, myelomas, melanomas, ovarian, lung, cervical, breast, pancreatic, gastric, kidney, prostate, thyroid, liver, colorectal, bone, bladder, and laryngeal cancers as well as a small subset of esophageal and head and neck cancers can be considered potential candidates for therapies, such as SH7139, that target HLA-DR.

Example 6: SHAL inhibition of GTPase-activating protein (GAP) activation of GTPase Racl, Rac3 or Cdc42

[0317] Rho GAPs and GTPases are molecular switches that play central roles in the regulation of the actin and microtubule cytoskeletons and gene transcription, and influence adhesion, polarity, motility and invasion, as well as cell-cycle progression and survival. They are also involved in the initiation of cytokinesis, actin disassembly, centromere maintenance, nuclear translocation of STAT transcription factors, regulation of cell migration, phagocytosis and colony-stimulating factor 1 induced motility, or macrophage function.

[0318] A series of in vitro assays were conducted with SH7139, other SHALs

(fragments of SH7139 containing Cb), and the free Cb ligand to determine if the intact drug or its fragments or metabolites inhibit the activation of the GTPases that participate in these functions. Recombinant GAP proteins and their domains were tested for activation of the GTPases Racl, Rac3 and Cdc42 using an ADP Hunter Plus assay (see van Adrichem AJ, el al., 2015, Combinatorial Chem & High Throughput Screening 18: 3-17), which measures the production of GDP, and a malachite green assay (Biomol Green, Enzo Life Sciences) used for phosphate detection. The results are shown in Table 9. Table 9: SHAL inhibition of GTPase-activating protein (GAP) activation of GTPase Racl, Rac3 or Cdc42 activity (IC50, mM)

[0319] The optimal recombinant GAP domain concentrations for use in the assays were determined prior to running the assays: MgcRacGAP (residues 345-618) at 2 nM, BCR GAP (residues 1010-1271) at 200 nM and p50RhoGAP (residues 205-439) at 10 nM. The primary GAP assays were performed with 600 nM GTPase in 15 mM HEPES (pH 7.5), 20 mM NaCl, 1 mM EGTA, 0.02% Tween 20, 0.1 mg/mL bovine serum albumin, 2% DMSO and 150 mM GTP, in the presence or absence of GAP domain protein (2nM MgcRacGAP or 200 nM BCR or 10 nM p50RhoGAP) at room temperature for 2 hr. An ADP Hunter Plus assay (DiscoveRx) was then run to measure the production of GDP using 5 pL volumes (2.5 pi of the protein mixture and 2.5 pi of GTP to kick off the reaction). The concentrations of SHAL or free ligand tested were 0.1, 0.5, 1, 5, 10, 50 and 100 pM. The assays were run in a 384 well format. A malachite green assay (Biomol Green, Enzo Life Sciences) was run using 10 pi volumes for phosphate detection according to the manufacturer’s instructions. The results of the assays (Table 9) show SH7139 and fragments of the SHAL containing the Cb ligand (SH8003, SH8005 and SH7117) all inhibit the activation of the Racl, Rac3 and Cdc42 GTPases by the GAP proteins MgcRacGAP, p50RhoGAP and BCRGAP. Example 7: Inhibition of GTPase Racl and Cdc42 activities by SH7139 and SH7117

[0320] To determine if SH7139 or SH7117 inhibit the conversion of GTP to GDP by

GTPase in the absence of the GAP proteins, fast cycling mutants of Racl and Cdc42 were tested for inhibition without the GAP proteins. GTP hydrolysis (Figure 5) was assayed using the ADP Hunter reagent (see van Adrichem AJ, etal ., 2015, Combinatorial Chem. & High Throughput Screening 18: 3-17). To measure the amount of GTP hydrolysis, the ADP Hunter Plus assay kit (DiscoveRx) was used according to the manufacturer’s instructions at half-volumes. The assays were performed a 384 well format using 600 nM of the kinase in 15 mM HEPES (pH 7.5), 20 mM NaCl, 1 mM EGTA, 0.02% Tween 20, 10 mM MgCk, 0.1 mg/mL bovine serum albumin, 2% DMSO, 150 mM GTP and eight concentrations of SHAL or free ligand (0.1, 0.5, 1, 5, 10, 50, 100 and 200 pM). After incubating 10 pi of the reaction mix and compounds for 2 hrs at room temperature, 5 pi Reagent A and 10 pi Reagent B were added, the mixture was incubated an additional 60 min at room temperature, and the reaction was stopped by the addition of 2.5 pi of Stop solution. Resorufm fluorescence was then measured at excitation at 530 nm and emission at 590 nm using a PHERAstar FS (BMG Labtech) multilabel plate reader.

[0321] These inhibition experiments show isolated/free Ct and Cb ligands do not inhibit Rac 1 or Cdc42 directly. In contrast, the Dv ligand, SH7117 and SH7139 inhibited, albeit weakly, both Racl and Cdc42. Taken together with the results obtained in the GAP inhibition experiments, these results suggest that SH7139 inhibition of GTPase activity occurs both indirectly by inhibition of the associated GAP and directly by preventing the binding of nucleotide. This inhibition appears to result from synergistic contributions from several of its components (Cb, Dv, D-Lys and mini-PEG) being covalently linked together.

Example 8: Inhibition of Uridine diphospho-glucuronosyltransferases (UGTs)

[0322] Drug metabolizing enzymes are an integral part of phase-II metabolism that helps in the detoxification of exogenous, endogenous and xenobiotics substrates. Uridine 5’- diphospho-glucuronosyltransferase (UDP-glucuronosyltransferase, UGT), which catalyzes the addition of glucuronic acid to small hydrophobic molecules, is perhaps the most important of Phase II conjugation enzymes involved in the body’s elimination of the most frequently prescribed drugs. Glucuronidation is also the major pathway for removal oncology drugs, dietary substances, toxins and endogenous substances. UGTs transform their substrates into more polar metabolites, which are better substrates for the ABC transporters, MDR1, MRP and BCRP, than the native drug. UGT-mediated drug resistance, which is observed frequently in ovarian, lung, breast and other cancers, is coordinated with the expression of ABC transporters. This coupling of UGT and multi drug resistance proteins has been intensively studied, particularly in the case of cancer therapy. Multidrug resistance coordinated with glucuronidation has also been described for drugs used in the management of epilepsy, psychiatric diseases, HIV infections, hypertension and hypercholesterolemia (Mazerska, 2016, Pharmacol Ther 2016, 159, 35-55; Cosman et al., 2002, Chem. Res.

Toxicol. 15: 1218-1228; Cosman et al., 2005, Methods Mol. Biol. 300: 141-63).

[0323] The role the UGT enzymes play in regulating the bioavailability and clearance of drugs and the development of multi-drug resistance has made them important biological targets whose inhibition could contribute significantly to reducing the dosages of drugs that need to be prescribed or administered and to preventing or reversing the resistance our cells normal and diseased) develop to many drugs. The majority of oncology and prescription drugs are metabolized by three UGT enzymes, UGT1 Al, UGT1 A3 and UGT1 A4. During the preclinical testing of drugs, assays are routinely run to test the drug for activity in inhibiting these three UGTs as well as UGT1 A6, UGT1 A9 and UGT2B7. This information is then used to determine how the administration of the new drug might impact the metabolism and bioavailability of other drugs being taken by the patient.

[0324] In all diseases, where suppression of cell growth or cell-killing would be beneficial, UGT inhibitors can be used as an adjuvant to modulate the activity of the drugs being used to treat the disease. Using commercially available recombinant forms of the enzymes, SH7139 was tested for activity in inhibiting UGT1 Al, UGT1 A3, UGT1 A4,

UGT1 A6, UGT1 A9 and UGT2B7. To test for inhibition, SH7139 was incubated at seven concentrations (0.78, 1.56, 3.13, 6.25, 12.5, 25 and 50 mM) with purified recombinant forms

TM of each human UGT enzyme (0.25-0.5 mg/ml UGT-expressed Supersomes , Corning Life Sciences, New York, NY), alamethicin (25 pg/mg enzyme) and UDP-glucuronic acid (5 mM) in 50 mM Tris HC1 buffer (pH 7.4) in the presence of a UGT isoform-specific probe substrate (10-50 pM, estradiol for UGT1A1, sulindac sulfone for UGT1A3, trifluoperazine for UGT1 A4, naphthol for UGT1 A6, propofol for UGST1 A9 and naloxone for UGT2B7) at 37°C with gentle shaking (180 rpm) for 30-60 minutes (time varied for different UGTs). The reactions were terminated by adding an aliquot of the reaction mix to one volume of methanol containing an analytical internal standard. The samples were centrifuged at 5000 rpm for 10 min at 4°C, the supernatants were removed, and an aliquot was analyzed by LC- MS/MS to determine the amount of the probe substrate metabolites formed. A decrease in the formation of the metabolites compared to the vehicle control was used to identify inhibition of the UGT enzyme and calculate an ICso value (test compound concentration that produces 50 % UGT inhibition). The results of this set of assays (Table 10A) showed SH7139 is more effective in inhibiting UGT1 A1 and UGT1 A3 than most inhibitors (Table 10B) used as standards in these assays. UGT1 A4 is also inhibited by SH7139, but its ICso is approximately 10-fold higher.

Table 10A: Inhibition by SH7139 of UGT glucuronidation enzymes and efflux and uptake transporters

Table 10B: IC50 for the inhibition of the various UGTs by other inhibitors.

Example 9: Effect of SHALs on ABC transporters involved in clearance

[0325] The ABC (ATP -binding cassette family) efflux transporters MDRl/P-gp

(multidrug resistance protein 1/P -glycoprotein, ABCB1) and BCRP (breast cancer resistance protein, ABCG2) are located in the apical membranes of epithelial cells. In normal and tumor cells the breast cancer resistance protein (BCRP) and the Multidrug resistance protein 1 (MDR1) or P-glycoprotein 1 (P-gp) function as xenobiotic transporters. These transporters also block the absorption of drugs into the intestine, at the blood-testis barrier and the blood- brain barrier, and they enhance excretion of xenobiotics by the kidneys. Multidrug resistance protein 1 (MDR1) or P-glycoprotein 1 (P-gp) is an important protein of the cell membrane that pumps many foreign substances out of cells, including drugs, by which it is involved in regulating the distribution and bioavailability of drugs. Both also play key roles in the development of multidrug resistance because they actively efflux a wide variety of structurally diverse chemotherapeutic and targeted small therapeutic molecules from the cancer cell (Kadkhodayan et ah, 2000, Protein Expr Purif 19, 125-130 (2000); Lightstone et ah, 2000, Chem Res Toxicol 13, 356-362 (2000); Shields et ah, 2003, J. Am. Soc. Mass Spectrom. 14: 460-470; Hajduk et ah, 2003, J Comput Aided Mol Des 17, 93-102).

[0326] Inhibition of these transporters can improve oral absorption, CNS penetration and delivery of anticancer agents to brain tumors or CNS metastases (Hajduk et ah, 1999, J Med Chem 42, 3852-3859) by decreasing the clearance of drugs, which leads to an increase in drug plasma concentrations and greater bioavailability (Hajduk et ah, 2000, J Med Chem 43, 4781-4786 (2000) and JMed Chem 43, 3862-3866 (2000)) of prescription drugs (e.g., Risperidone, Thienorphine, Imipramine, Paroxetine, etc.) and oncology drugs used to treat cancer patients (e.g., Imatinib, Docetaxel, Crizotinib, Paclitaxel, Topotecan, etc.).

[0327] To determine the level of transport inhibition of the efflux transporters MDR1 and BCRP, a vesicular transport assay was conducted using cell membrane vesicles containing either human BCRP (ABCG2/MXR) or human MDR1 (ABCBl/P-gp). SB- BCRP-HEK293 membrane vesicles (Solvo Biotechnology USA, San Francisco, CA) were used for the BCRP assays, and ³H-Estrone-3 -sulfate was used as the probe substrate and Kol34 was the reference inhibitor. SB-MDR1-HEK293 membrane vesicles (Solvo Biotechnology USA, San Francisco, CA) were used to conduct the MDR1 assays, and ³H-N- methyl quinidine was used as the probe substrate and PSC833 was the reference inhibitor. These assays determine the ability of the unlabeled SH7139 (or reference inhibitor) to block the transport of the labeled probe into the membrane vesicles in the presence of MgATP or AMP.

[0328] Prior to conducting the assay, the solubility of SH7139 in the assay buffer

(lOmM Tris pH 7, 250mM sucrose, lOmM MgC12) was determined by preparing a series of drug concentrations (15, 30, 37.5, 45, 52.5, 60, 67.5 and 75mM) in the buffer containing 1.5% DMSO and incubated at 37°C for 15 min. Solubility was assessed by optical microscopy to determine if there were any visible crystals or particles. The highest concentration of SH7139 that remained soluble in this buffer was 67.5mM. This result defined the upper limit of SH7139 solubility that would be tested in the assay.

[0329] A stock solution of SH7139 was prepared in DMSO and seven concentrations of the drug, 45, 12.5, 3.13, 0.78, 0.20, 0.05, O.OImM, were tested in a 96 well plate format. The MDRl/Pgp assay was performed using the MDRl PREDIVEZ Reagent Kit Protocol vl.l (Solvo Biotechnology USA, San Francisco, CA, solvobiotech.com/products/items/sb-predivez-vt-reagent-kit-for-mdrl-p-gp). The BCRP assay was performed using the BCRP PREDIVEZ Reagent Kit Protocol_vl.3 (Solvo Biotechnology USA, San Francisco, CA, solvobiotech.com/products/items/sb-predivez-vt- reagent-kit-for-bcrp). In both assays, the reaction mixtures containing the start reagent (MgATP or AMP) were pre-incubated separately for 15 min at 37°C and the reaction was initiated by adding the start reagent to the reaction mixtures in each well in the assay plate. ATP dependent transport was calculated by subtracting the values measured in the absence of ATP (AMP samples) from those measured in the presence of ATP (MgATP samples). The relative inhibition of the transport of the radiolabeled substrate was determined and used to calculate the IC50. The IC50 values were determined by non-linear regression analysis of the concentration-response curves using the Hill equation. The results of these experiments show SH7139 is a remarkably effective inhibitor of both mammalian efflux transporters P-gp and BCRP.

Example 10: Effect of SHALs on OAT and OATP transporters involved in absorption, distribution and excretion of drugs

[0330] The OAT and OATP transmembrane proteins function by transporting organic anions and cations across the membranes of mammalian cells. OATs transport a wide range of low molecular weight molecules including biogenic amines, drugs, toxins and conjugates of steroid hormones. OAT IB 1 and OAT1B3 are transporters that play important roles in intra- and inter-individual variability of the therapeutic efficacy and the toxicity of many drugs. OATP-mediated transport is ATP- and sodium-independent and mainly focuses on amphipathic molecules with molecular weights of more than 300 kDa (Hajduk et ah, 2000, J Med Chem 43, 4781-4786 (2000) and J Med Chem 43, 3862-3866 (2000); Hajduk et ah,

2003, J Comput Aided Mol Des 17, 93-102; Huth et ah, 2007, Chem Biol Drug Des 70, 1-12; Shuker et ah, 1996, Science 274, 1531-1534). OATPs are capable of bidirectional transport, and several studies have suggested that they work as electroneutral exchangers (Hajduk et ah, 2000, J Med Chem 43, 4781-4786 (2000) and J Med Chem 43, 3862-3866 (2000)). They transport various endo- and xenobiotics, including hormones and their conjugates as well as numerous drugs such as several anticancer agents (Huth et ah, 2007, Chem Biol Drug Des 70, 1-12). OATP1B1 and OATP1B3 are examples of tissue- specific OATPs as both are selectively expressed in the liver where they are localized to the basolateral membrane of hepatocytes (Huth et ah, 2007, Chem Biol Drug Des 70, 1-12; Szczepankiewicz et ah, 2003, J Am Chem Soc 125, 4087-4096). Studies show, however, that the expression of these two OATPs can be altered in cancers. They are downregulated in liver cancers, possibly due to the dedifferentiation of the hepatocellular carcinomas (Huth et ah, 2007, Chem Biol Drug Des 70, 1-12; Carlson et ah, 2007, ACS Chem Biol 2, 119-127). In contrast, they have been shown to lose their liver specificity and be upregulated in many other types of cancers, including the colon, breast and prostate (Huth et ah, 2007, Chem Biol Drug Des 70, 1-12; Shuker et al., 1996, Science 274, 1531-1534 (1996)). Inhibition of OATPs can therefore help mediate the uptake of hormones, hormone conjugates, or growth promoting chemicals that the tumor cell requires to grow and survive.

[0331] SH7139 was tested for inhibition of the OAT1, OAT3, OATP1B1, OATP1B3, and OCT2 influx transporters at 8 concentrations (0.03, 0.1, 0.3, 1, 3, 10, 30 and 100 mM). The appropriate human recombinant CHO cells expressing the OAT1, OAT3, OATP1B1, OATP1B3 or OCT2 transporter were seeded in a 96-well culture plate at -20,000 cells/well and were used on day 3 post-seeding. On the day of assay each concentration of SH7139 was prepared in assay buffer (HBSS-HEPES, pH 7.4) with a final DMSO concentration of 1%, added to the cell plate and pre-incubated at 37°C for 15 minutes. Each concentration was performed in duplicate. Subsequently, substrates (10 mM 6-carboxyfluorescein for OAT1 and OAT3, 5 mM fluorescein methotrexate for OATP1B1 and OATP1B3, and 5 pM ASP+ for OCT2) were added to the plate followed by a 20-minute incubation at 37°C. The plate was then washed with cold assay buffer followed by fluorescence reading for assays with fluorogenic substrates (excitation wavelength 485 nm, emission wavelength 590 nm for OCT2 and 528 nm for OAT1, OAT3, OATP1B1, and OATP1B3). The ICso for reference inhibitors were also determined for comparison and as a positive control (Probenecid for OAT1 and OAT3, Rifampicin for OATP1B1-CHO and OATP1B3, and Verapamil for OCT2). The percent of control is calculated using the following equation.

Control = X 100

[0332] Compound is the individual reading in the presence of SH7139. T1 is the mean reading in the absence of the SH7139. Background is the mean reading in the absence of both SH7139 and the substrate. A decrease in signal represents the inhibition of the transporter activity.

[0333] These inhibition studies have shown that SH7139 is an effective inhibitor of both OATP1B1 and OATP1B3 transporters. The SHAL does not inhibit either of the tested OAT transporters (OAT1 or OAT3) or the OCT2 transporter.

Example 11: Inhibition of Acetyl-CoA-Carboxylase (ACC)

[0334] The inhibition of ACC enzymes is a viable therapeutic target for treating obesity by increasing fatty acid oxidation and suppressing fatty acid synthesis, a combination that may lead to loss of body fat in obese subjects. ACC inhibition can provide a treatment for obesity or obesity-related diseases or metabolic disorders, such as type-2 diabetes, metabolic syndrome and nonalcoholic fatty liver disease. ACC up-regulation has also been recognized in multiple human cancers, promoting lipogenesis to meet the need of cancer cells for rapid growth and proliferation. Therefore, ACC is considered a potent target for cancer intervention, and ACC inhibitors would be potential therapeutic agents for cancer therapy (Seethala etal ., 2006, Anal Biochem 358:257-265).

[0335] The Ct ligand 3-(2-((3-chloro-5-(trifluoromethyl)-2-pyridinyl) oxy)-anilino)-3- oxopropanoic acid), a ligand present in many of the SHALs that have demonstrated antitumor activity, is a structural analog of haloxyfop and several related inibitors of ACC (clodinafop, diclofop, fluazifop, and trifop). Two SHALs containing Ct (SH7133 and SH7097), along with the Ct ligand and haloxyfop, were tested for activity in inhibiting the two human AcetylCoA carboxylase enzymes ACC1 and ACC2, to determine if the Ct ligand or SHALs containing it might be an effective inhibitor of either enzyme. While SHALs inhibiting ACC would have applications in the treatment of cancer or obesity, SHALs that do not inhibit ACC would have other applications that only require their binding to HLA-DRs (e.g. treatment of autoimmune diseases).

[0336] The ligands and two SHALs were tested for ACC inhibition using an

ACC/FAS coupled scintillation proximity assay (SPA) (Id). For this assay, 25 mΐ of recombinant ACC and FAS enzymes and 5 mΐ of compound in 3% DMSO or 3% DMSO as control were added to a 384-well FlashPlate (50 mΐ total well volume) and incubated for 10 minutes, after which the reaction was started by the addition of 20 mΐ of a substrate mixture containing radiolabeled acetyl-CoA and ATP in assay buffer (50 mM Tris-HCl, pH 7.6, 10 mM sodium citrate, 10 mM MgCb, 6 mM NaHCCb, and 100 mM NADPH). A similar study was also done using a low-volume 384-well Image FlashPlate (15 mΐ total well volume), 9.5 mΐ ACC and FAS enzymes in assay buffer and 0.5 mΐ compound in 3% DMSO or 3% DMSO as control. The reaction was started by the addition of 5 mΐ of substrate mixture. The reactions were carried out either at room temperature or at 37 °C for 2 hours and then quenched by the addition of EDTA (10 mM final concentration). The amount of [³H]palmitic acid produced was determined by counting in a TopCount (Perkin-Elmer) for the FlashPlates or by reading in a LeadSeeker (GE Healthcare) for the Image Flashplates. [0337] Since neither the Ct ligand or two SHALs containing Ct inhibit the two human acetyl CoA carboxylase enzymes ACC1 or ACC2 (Table 11), two new SHALs (SH8045 and MB 1000) containing haloxyfop substituted for the Ct ligand have been developed for potential use in treating cancer or obesity or obesity-related diseases such as type-2 diabetes and nonalcoholic fatty liver disease by releasing into a cell haloxyfop (see Scheme 1 below), which is a known inhibitor of ACC (Table 11). The SHALs having the structures shown below may provide a treatment for cancer or obesity or obesity-related diseases such as type- 2 diabetes and nonalcoholic fatty liver disease by releasing into a cell haloxyfop (see Scheme 1 below), which is a known inhibitor of ACC (Table 11). SH8045 has been tested and shown to be cytotoxic to a number of cultured tumor cell lines, including those derived from a leukemia (SR786), renal cell carcinoma, non-small cell lung cancer, colon adenocarcinoma and a central nervous system astrocytoma.

Scheme 1: Release of haloxyfop by SHAL

Table 11: Inhibition of human Acetyl-CoA Carboxylase (ACC) (IC50, mM)

Example 12: Therapeutic applications of the selective high affinity ligand drug SH7139 extend beyond NHL to many other types of solid cancers

[0338] Abstract: Selective High Affinity Ligands (SHALs) are small molecule antibody mimics that can be designed to bind selectively and with high affinity to almost any protein. SH7139, the first of a series of SHAL oncology therapeutics created to target the HLA-DR proteins overexpressed on many B-cell lymphomas, has demonstrated exceptional efficacy in the treatment of Burkitt lymphoma xenografts in mice and a safety profile that may prove to be unprecedented for an oncology drug. Tumor tissue binding studies conducted with SH7129, a biotinylated derivative of SH7139, demonstrate that the HLA-DRs targeted by SH7139 are expressed by more than half of the non-Hodgkin’s lymphoma cases tested to date. SH7129 binding to biopsy samples from patients diagnosed with eighteen other solid cancers show many of these tumors also express the HLA-DRs targeted by SH7139. Cervical, ovarian, colorectal and prostate cancers bind the most SH7129. Only a few (< 5%) esophageal and head and neck tumors bound the diagnostic. In marked contrast to invasive ductal breast cancers, in which only 4% of the tumors expressed HLA-DR, two thirds of the medullary carcinomas of the breast expressed the target. Within the tumors tested, cell to cell differences in SH7139 target expression, as determined by SH7129 binding, varied by only 2 to 3-fold while expression levels for different tumors varied as much as 10 to 100-fold. The observed binding of SH7129 to these cancers suggest many patients diagnosed with B-cell lymphomas, myelomas, melanomas, ovarian, lung, cervical, breast, pancreatic, gastric, kidney, prostate, thyroid, liver, colorectal, bone, bladder, and laryngeal cancers as well as a small subset of esophageal and head and neck cancers can be considered potential candidates for therapies, such as SH7139, that target HLA-DR. Materials and Methods

Synthesis of the biotin derivative of SH7129

[0339] Biotinylated SH7129 was synthesized and purified by AmbioPharm Inc.

(North Augusta, SC) as described previously (DeNardo etal ., 2008, Cancer Biotherapy and Radiopharmaceuticals 23: 783-795; DeNardo et al., 2009, Int J Oncol. 2009; 34: 511-6). Briefly, the SHAL was synthesized using solid phase chemistry by the stepwise attachment to a Wang resin of Fmoc-D-Lys#l(Boc)-OH, Fmoc-AEEA-OH#l (Fmoc-8-amino-3,6- dioxaoctanoic acid), Fmoc-D-Lys#2(Dde)-OH, Fmoc-AEEA-OH#2, Fmoc-L-Val-OH, and Dabsyl chloride using standard Fmoc (N 9 fluorenylmethoxycarbonyl) chemistry with HBTU (2-( lH-benzotriazol- 1 -yl)- 1 , 1 ,3 ,3 -tetramethyluronium hexafluorophosphate)/HOBt (Hydroxybenzotriazole)/DIPEA (N,N-Diisopropylethylamine) as the coupling reagents. The side chain amino group of D-Lys#2-(Dde)-OH was deprotected with 4% hydrazine in dimethylformamide (DMF) and then coupled to Fmoc-D-Lys#3-(Dde)-OH using the same coupling procedure. Fmoc-AEEA-OH#3 was next coupled to deprotected D-Lys#3(Dde) and 4-(4-(4-chlorobenzyl) piperazine)-3-nitrobenzenecarboxylic acid (Cb ligand) was then linked to the deprotected AEEA-OH#3 using the same coupling procedure. The third ligand Ct (3- (2-((3-chloro-5-(trifluoromethyl)-2-pyridinyl) oxy)-anilino)-3-oxopropanoic acid) was then attached to the deprotected e-amine of D-Lys#3. The assembled free amine form of the SHAL was cleaved from the resin, deprotected and subsequently precipitated as a crude solid. The crude product was purified by standard RP-HPLC methods and isolated by lyophilization. Biotin was attached to the free amine on the terminal lysine by dissolving the SHAL in anhydrous DMF, N,N-Diisopropylethylamine (DIEA) and adding solid biotin N- hydroxysuccinimide ester (biotinyl-OSu). The mixture was nutated for 15 min, and the reaction was monitored by analytical HPLC. Upon completion, the reaction solution was diluted with a small volume of water/acetonitrile (50/50) containing 1% trifluoroacetic acid (TFA) and purified by HPLC. The purified SH7129 was lyophilized and then analyzed by LC/MS and NMR to determine its purity and confirm its molecular mass and structure, respectively. Tissue and tumor microarrays

[0340] Normal tissue microarrays (FDA808-1 and FDA808-2) containing fixed and paraffin embedded sections of twenty-seven different tissues obtained from three individuals and tumor microarrays (TMAs) containing fixed and paraffin embedded tumor biopsy sections obtained from patients diagnosed with different non-Hodgkin’s lymphoma subtypes and other solid cancers were obtained from U.S. Biomax (Rockville, MD). An additional set of diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma and SLL/CLL TMAs were prepared and provided by Dr. John G. Gribben, Barts Cancer Institute, Queen Mary University of London, Charterhouse Square, London, UK.

SH7129 staining protocol

[0341] SH7129 was prepared as a stock solution by dissolving 10 mg of the dry compound in 1 ml dimethyl sulfoxide. The formalin fixed normal tissue and tumor microarrays were stained using a Leica BOND RX Automated Slide Stainer (Leica Biosystems Inc, Buffalo Grove, IL) to maximize slide-to-slide uniformity in staining and processing. The fixed slides were deparaffinized using the Leica dewax solution, rehydrated with an alcohol series (100%, 95%, 70% and 30% for 4 min each) followed by antigen retrieval in citrate buffer at pH 6 and 90°C for 20 min. After performing a 5 min hydrogen peroxide block, the slides were washed three times with BOND Wash Solution, endogenous biotin was blocked using the Ventana Endogenous Biotin Blocking kit (Roche Molecular Diagnostics, Pleasanton, CA), the slides were washed three additional times with BOND Wash Solution, and then stained with SH7129 (100 pg/ml in PBS, 1% DMSO) for 30 min. Following three washes with BOND Washing Solution, the slides were treated with Streptavidin-horse radish peroxidase (SAHRP) for 30 min, washed 3 times with BOND Wash Solution and once with deionized water, treated with Mixed DAB (3,3-diaminobenzidine) Refine for 10 min, and then washed four times with deionized water, once with BOND Wash Solution and a final deionized water wash as per the BOND Polymer Refine IHC protocol (Histowiz Inc., Brooklyn, NY). The SH7129 stained tumor microarray slides were not counterstained with hematoxylin. The slides were then dehydrated by immersion in an alcohol series (30%, 70%, 95% and 100% for 4 min each), cleared with xylene and mounted with Permount. Analysis of SH7129 binding to NHL and other solid tumors

[0342] Representative digital images of each tumor biopsy section were captured from the SH7129 stained and control (duplicate slide cut from same core treated with PBS instead of SH7129) slides at 40X magnification. SH7129 binding or lack of binding to the cells was confirmed by visual inspection. Cells expressing the HLA-DRs that bind SH7129 showed stain associated with both the membranes and cytoplasm. Cell-to-cell variation in SH7129 binding was determined by performing densitometric analyses of fifty (50) individual tumor cells from a representative moderate to high SH7129 binding tumor for six of the NHL subtypes using ImageJ 1.42(Schneider et al ., 2012, Nat Methods. 2012; 9:671- 675). An effort was made during the analysis to include cells representing the full range of SH7129 binding. Individual cell data were not obtained for small lymphocytic lymphomas due to Applicant’s inability to accurately define the cell boundaries in these tumors. To estimate the tumor-to-tumor variation in SH7129 binding, an additional lower magnification digital image containing the array of cores for the two slides (SH7129 stained and control without SH7129) were captured at the same magnification (10X), the images were inverted, and the amount of bound SH7129 was determined by densitometric analysis of each tumor section using ImageJ 1.42. Integrated density data were collected from a 384 pixel area of each core and from ten blank (background) 384 pixel areas distributed across the slide near or between the cores. Core sections containing voids or tears (missing tissue), lacking a corresponding core in the control slide, or obtained from pigmented tumors were not analyzed. In cases where there were duplicate or triplicate cores for each biopsy on the slides, the data obtained from the analyses of the replicates were averaged. The amount of bound SH7129 (per 384 pixel area) was then calculated for each biopsy sample as follows:

Bound SH7129 = (IntDensH7i29 - IntDensH7i29Bkg) - (IntDenNoSH7i29 - IntDenNoSH7129Bkg)

[0343] where IntDensm is the integrated density of the biopsy section stained with

SH7129, IntDensH7i29Bkg is the mean of the integrated densities of the ten blank regions of the SH7129 stained slide, IntDenNosm is the integrated density of the biopsy section that was processed for staining without SH7129, and IntDenNoSH7i29Bk_g is the mean of the integrated density of the ten blank regions of the control slide processed for staining without SH7129. [0344] The data were analyzed and plotted using GraphPad Prism 8.1.2. Statistical analyses of two groups of data were performed using an unpaired t-test. Analyses of three or more groups of data in which the standard deviations of the groups were similar were analyzed using a one-way ANOVA test followed by Tukey’s multiple comparison test. Data sets containing three or more groups in which the standard deviations of the groups being compared were different were analyzed using both a Brown-Forsythe and Welch’s ANOVA test followed by Dunnett’s T3 multiple comparisons test. In both cases in which three or more groups were compared, the assumption was made that the data fit a Gaussian distribution based on the observation that the amount of SH7129 bound per biopsy case did roughly fit a Gaussian distribution.

Results

SH7129 binding to normal tissue

[0345] SH7129 binding to normal tissue was evaluated using microarrays containing twenty-seven different tissues obtained from three healthy individuals. Following the staining of the microarrays with SH7129, the slides were not counter-stained with hematoxylin. This enables the detection of very low levels of SH7129 binding that would normally be obscured by the presence of the counter-stain. Cells expressing the targeted HLA-DRs that bind SH7129 are stained brown by the horse-radish peroxidase’s conversion of the 3,3-diaminobenzidine tetrahydrochloride (DAB) substrate to a brown insoluble product. SH7129 binding was observed to tonsil, thymus, spleen, and bone marrow - all tissues that produce or contain large numbers of antigen presenting cells. No binding was observed to breast, cerebrum, colon, hypophysis (pituitary), small intestine, ovary, pancreas, salivary gland, skeletal muscle, thyroid, uterine cervix or peripheral nerve tissue. In each of the three skin samples tested the basal keratinocytes appeared to be stained by SH7129, but examination of the control slides (those stained with hematoxylin and eosin without SH7129) revealed this brown coloration is melanin pigment, not bound SH7129. Some staining was observed in kidney tissue, but the bound SH7129 was limited to the macrophages, dendritic cells and monocytes located between tubules.

[0346] Cells in the zona reticularis of adrenal tissue were stained very lightly with

SH7129, suggesting these cells may also express very low levels of HLA-DR. HLA-DR expression by adrenal cells has been reported previously (Khoury et al., 1987, Am J Pathol. 1987; 127: 580-91; Marx et al., 1997, J Clin Endocrinol Metab. 1997; 82: 3136-40) and it has been suggested that this expression might be induced during the final maturation step for reticularis cells as they become competent to secrete androgens. It has also been suggested the HLA-DR may trigger the induction of apoptosis in these cells via MHC class II mediated programmed cell death as part of the normal process of adrenal cell turnover (Marx et al., 1997, J Clin Endocrinol Metab. 1997; 82: 3136-40). A very low level of SH7129 binding was also observed in cerebellum white matter. Others who have also reported the binding of anti-HLA-DR antibodies to white matter have suggested this binding may be to resting or non-reactive microglia (Styren et al., 1990, Exp Neurol. 1990; 110: 93-104), cells of the central nervous system that function as macrophages. Very light staining of parietal cells was also observed in stomach sections obtained from two individuals (no staining was observed in the section from the third individual). Parietal cells are epithelial cells that have been reported to express HLA-DR in cases of gastritis (Archimandritis et al., 2000, Clin Exp Immunol. 2000; 119: 464-71; Spencer et al., 1986, Gut. 1986; 27: 153-7).

[0347] While SH7129 did not bind to lung, esophagus, prostate, cardiac muscle, and parathyroid tissues obtained from two of the three individuals, the tissue from one individual in each case showed very light staining which is just barely detectable in the captured images. Although these tissues do not normally express HLA-DRs (Dunne et al., 2017, Cancer Immunol Immunother. 2017; 66: 841-50; Arbustini et al., 1994, Am J Pathol. 1994; 145: 310- 21; Bjemeroth et al., 1998, Surgery. 1998; 124: 503-9), the low level of SH7129 binding to the tissues from these individuals may reflect undetected tissue inflammation or very early stage disease. In the lung and esophagus tissue section showing staining, SH7129 binding was localized to the epithelial cells in the alveolar ducts of the lung and the squamous epithelium of the esophagus - cell types that have been shown to express HLA-DRs during inflammatory lung and esophagus injury or disease (Dunne et al., 2017, Cancer Immunol Immunother. 2017; 66: 841-50; Beninati et al., 1993, J Allergy Clin Immunol. 1993; 92: 442- 9; Campbell et al., 1986, Clin Exp Immunol. 1986; 65: 165-71). The prostate case showed a very light staining of the stroma; no binding was observed to the acinar or basal cells.

[0348] In certain cases of chronic immuno-mediated inflammation, such as benign prostatic hyperplasia, prostate stromal cells have been reported to express HLA-DR and function as antigen presenting cells (Penna et al., 2009, J Immunol. 2009; 182: 4056-64; Theyer et al., 1992, . Lab Invest. 1992; 66: 96-107). In the cardiac tissue section from the one individual showing extremely light staining, the binding appeared to be associated with some (not all) of the myocytes - an observation others have reported to occur in association with transplant tissue (Rose ML et al. Transplantation. 1986; 41 : 776-80, Milton AD et al. J Exp Med. 1985; 161: 98-112, and Steinhoff G, et al. European Heart Journal. 1987; 8: 25-8], myocarditis (Caforio AL, et al. J Autoimmun. 1990; 3: 187-200, Seko Y, et al. Circ Res.

1990; 67: 360-7, and Wojnicz R, et al. Eur Heart J. 1998; 19: 1564-72) and other types of cardiovascular disease (Caforia et al., 1990, J Autoimmun. 1990; 3: 187-200; Grundtman et al., 2010, Arthritis Rheum. 2010; 62: 667-73). Thus the very low level of SH7129 staining of these normal tissues are consistent with the observations in previous studies that have shown HLA-DR expression in healthy lung, esophagus, prostate, cardiac tissue, cerebellum and stomach is confined to non-lymphoid cells that begin functioning as antigen presenting cells in response to injury or disease.

[0349] The only other tissue to which SH7129 binding was observed, also at a very low level, was the liver. Hepatocytes in normal liver do not express HLA-DR (Barbatis et al., 1987, J Clin Pathol. 1987; 40: 879-84; Gehring et al., 2007, J Virol. 2007; 81: 2940-9;

Senaldi et al., 1991, J Clin Pathol. 1991; 44: 107-14), but epithelial cells surrounding portal tracts have occasionally been observed to express HLA-DR in tissue obtained from healthy individuals and much more frequently in cases of disease (Terada et al., 1991, Clin Exp Immunol. 1991; 84: 303-7; Terada et al., 1991, Arch Pathol Lab Med. 1991; 115: 993-7). Hepatocytes expressing HLA-DR have only been observed in patients with immune mediated liver disorders (Senaldi et al., 1991, J Clin Pathol. 1991; 44: 107-14). Since the SH7129 binding was observed in the liver sections from all three individuals and appears to be localized specifically to hepatocytes, it is highly unlikely the tissues were obtained from three individuals that all have a liver disorder. It is more likely in this one tissue that the staining may reflect a very low level of SH7129 binding to something other than HLA-DR. SH7139 has been determined recently to inhibit the hepatic transporters OATP1B1 and OATP1B3. These proteins, which are found only in hepatocytes, are so abundant (3.18 pmoles OATP1B1 and 2.73 pmoles OATP1B3 per 10⁶ hepatocytes (Burt et al., 2016, Drug Metab Dispos. 2016; 44: 1550-61)) that they exceed the number of HLA-DRs expressed by the Burkitt lymphoma cell line Raji (2.66 pmoles/10⁶ Raji cells (Roucard et al., 1996, J Biol Chem. 1996; 271: 13993-4000; West et al., 2006, Cancer Biother Radiopharm. 2006; 21: 645-54) and some of the highest expressing ovarian cancer cells (0.5 pmoles/10⁶ cells(Schuster et al, 2017, Proc Natl Acad Sci U S A. 2017; 114: E9942-E51) by 2 to 10-fold, respectively. While the binding affinity of SH7129 has not been determined for either transporter, the IC50 for OATP1B1 (0.29 mM) and OATP1B3 (0.15 mM) inhibition by SH7139 indicate SH7129’s affinity for the transporters will be lower than its affinity for HLA-DR (KD = 23 pM (Balhom et al, 2014, Cancer Research. 2014; 74: 2703)). However, SH7129 would be expected to bind to both transporters under the staining conditions used, and the very weak hepatocyte staining observed in the liver sections may simply reflect the binding of SH7129 to the abundant OATP1B1 and OATP1B3 transporters present in these cells.

[0350] Based on the results of a series of toxicology and safety studies conducted with SH7139 in dogs and rats at doses of the drug up to 4,000 and 12,000 times, respectively, of the anticipated therapeutic dose (data not shown), the low-level of SHAL binding to these normal tissues does not appear to have an adverse impact on their function. No organ or tissue in the treated animals showed any macroscopic or microscopic indication of pathology or toxicity, B-cell lymphocytes expressing low levels of HLA-DR were not adversely affected, the serum chemistry of the treated animals showed no evidence of liver or renal damage, there were no observed abnormalities in electrocardiography parameters (heart rate, RR interval, PR interval, QRS duration, QT interval or QTc interval), and the Functional Observational Battery performed to assess the central nervous system for pharmacological effects showed no indication of an adverse association with SH7139 exposure.

HLA-DR expression by non-Hodgkin’s lymphoma

[0351] Using the same SH7129 staining protocol, tumor biopsy sections obtained from patients diagnosed with seven subtypes of NHL were screened for the expression of HLA-DRs targeted by SH7139. Digital images of the tumor cells in each section were obtained from the stained and control (duplicate slide with no SH7129 treatment) slides, the images were inverted, and the amount of bound SH7129 was estimated by processing the captured images of each tumor and quantifying the amount of brown oxidized DAB product generated by the horse-radish peroxidase using ImageJ1.42. [0352] SH7129 staining of biopsy tissues containing cells expressing HLA-DR show

SH7129 binds to HLA-DR proteins located on the surface of the tumor cells, in the cytoplasm, and near the nucleus where the endoplasmic reticulum is located. Connective tissue is not stained. As shown in Table 12, a significant number of the tested tumors in each of the types of NHL examined were found to bind SH7129. Tumor biopsies obtained from all twenty-four of the anaplastic large cell lymphoma (ALCL) cases examined expressed the targeted HLA-DR and bound SH7129. Nearly every MALT lymphoma (75 of the 80 cases) biopsy sample examined also bound the diagnostic. At the other end of the spectrum, only 28% of the mantle cell and 34% of the follicular lymphomas were observed to express the target and bind SH7129.

Table 12. Solid cancers tested for SH7129 binding as an indicator of their expression of the HLA-DRs targeted by SH7139. SH7129 was used in an IHC-type protocol to stain tumor microarrays containing tumor biopsy sections, and the biotin in the bound SH7129 was detected using Streptavidin-horse radish peroxidase oxidation of DAB.

The stained sections were examined visually to confirm tumor cell binding.

[0353] Within a typical lymphoma biopsy section, the cell to cell variation in SH7129 binding, as measured by image analysis of the horse-radish peroxidase generated oxidation product of DAB deposited in individual tumor cells, was less than 3-fold (Table 13). The level of HLA-DR expression and SH7129 binding by the cells in different patient’s tumors within the same type of NHL, however, differed by as much as 10 to 100-fold. Statistical analyses of SH7129 bound by the seven types of NHL indicate these NHL tumors fall into three groups. Biopsy tissues obtained from patients diagnosed with anaplastic large cell lymphoma (ALCL) exhibited the highest level of SH7129 binding. Diffuse large B-cell lymphomas (DLBCL), small lymphocytic lymphomas (SLL), and mucosa-associated lymphoid tissue (MALT) lymphomas bound intermediate amounts of SH7129. Follicular lymphomas (FL), Burkitt’s lymphomas (BL) and mantle cell lymphomas (MCL) bound the least SH7129 of the types of NHL analyzed.

Table 13. Cell to cell variation in SH7129 binding in a typical NHL tumor. Bound SH7129 was quantified by image analysis for 50 cells representing the full range of binding within a biopsy sample obtained from patients diagnosed with each of six subtypes of NHL. The Level of SH7129 Binding is a subjective descriptor of the overall intensity of SH7129 staining of the tumor tissue relative to other tumors in that subtype. Cell Range in SH7129 Bound is the amount of SH7129 bound by the most intense staining tumor cell divided by the amount of SH7129 bound by the least intense staining tumor cell in the tumor section.

Diffuse Large B Cell LM482E3 High 2.98 Follicular T203B6 Moderate 1.69 Burkitt's LM482D8 Moderate 2.42 MALT LY804E7 High 1.94

Anaplastic Large Cell LM242D5 High 2.75 Mantle Cell MC2B9 Moderate 1.78

Identification of other solid cancers that also express HLA-DRs targeted by SH7139

[0354] Biopsy sections from eighteen additional solid cancers that have been reported by others to express MHC class II proteins were also examined for expression of the HLA- DRs targeted by SH7139 using SH7129 and the same IHC staining protocol. While many (33-100%) of the tumors analyzed in sixteen of these cancers were found to bind SH7129 and express the HLA-DR target (Table 12), only two of the ninety-nine esophageal (2%) and two of the forty head and neck tumors (5%) showed detectable SH7129 binding. The highest percentage of cases showing SH7129 binding was observed for ovarian, lung, cervical, gastric, prostate, myeloma and colorectal cancers. Ovarian, colorectal, prostate and cervical cancers exhibited the highest levels of SH7129 binding of all the solid tumors examined. In marked contrast to the other types of cancer, which had cases representing the full spectrum (low to high) of HLA-DR expression and SH7129 binding, all of the ovarian and all but one of the cervical cancer biopsy sections examined bound moderate to high levels of SH7129. The amount of SH7129 bound by the two esophageal and two head and neck tumors were amongst the lowest of all the cancers tested.

[0355] Similar to the results obtained in the analysis of the different types of NHL, a broad range in the level of SH7129 binding/HLA-DR target expression was observed between individual tumors for many of these solid cancers. The gastric and pancreatic cancer cases showed the least variability in SH7129 binding. Within the ovarian, prostate, melanoma, breast and bone cancers the tumors from a small number of outlier cases expressed extremely high levels of the HLA-DR target. The amount of SH7129 bound by these outliers was nearly twice the amount bound by the lymphoma outliers.

Variation in SH7129 binding by tumor type or grade within the non-hematological cancers

[0356] SH7129 binding to the different types of nine of the non-lymphoid solid cancers analyzed was also compared to determine if a particular type might express more or less of the HLA-DRs targeted by SH7139. With one exception, the level of target HLA-DR expression, as determined by SH7129 binding, by the different types of lung, liver, ovarian, laryngeal, gastric, breast and bone cancers were not found to be statistically different. Within the cervical cancers analyzed, the squamous cell carcinomas (SC) bound more SH7129 than the adenocarcinomas (A) (p = 0.006).

[0357] A comparison of the amount of SH7129 bound to tumors classified as different grades of the disease yielded a similar result. The results suggest there is also no difference in SH7129 binding as a function of tumor grade in liver, ovarian, gastric, prostate, laryngeal, lung, cervical or pancreatic cancers. The comparison suggested what appears to be a significantly higher level of SH7129 binding to grade 3 compared to grade 2 kidney cancers (p = 0.0350), but this result is based on the analysis of only two grade 3 cases. While the difference may prove to be real, such a conclusion cannot be confirmed until a much larger number of cases are examined. It is important to point out that the same is true for many of the cancer types and grades analyzed. The results obtained from the comparisons involving only a few cases per cancer type or grade may not reflect the true variation that is present in the larger population.

Example 13: The small molecule antibody mimic SH7139 targets a family of HLA-DRs expressed by B-cell lymphomas and other solid cancers

[0358] Abstract: HLA-DR expression is often upregulated in B-cell lymphomas and many non-hematological cancers. SH7139, the first of a new class of cancer therapeutics developed for treating non-Hodgkin’s lymphoma and other solid cancers expressing HLA- DR, is unique in that both targeting and multiple anti-tumor activities have been incorporated into the same small molecule. Functioning as an antibody mimic, SH7139 was designed to target a unique structural epitope located within the antigen-binding pocket of HLA-DRIO. Pre-clinical testing of the drug has demonstrated exceptional safety profiles in mice, rats and dogs and remarkable efficacy in treating Raji lymphoma xenografts in mice. SH7129, a biotin derivative of SH7139, has been synthesized for use as a companion diagnostic to pre screen biopsy samples and identify those patients whose tumors should respond to SH7139 therapy. Using an immuno-histochemical type assay to stain peripheral blood mononuclear cells (PBMCs) obtained from individuals expressing specific HLA-DRB1* alleles, SH7129 binding to PBMCs has revealed that other HLA-DRs are also targeted by the drug. Comparisons of the activities of SH7129 and SH7139, which show the biotinylated form of the drug retains the HLA-DR binding selectivity and functionality of SH7139, suggest SH7129 should work well as a companion diagnostic.

Materials and Methods

Materials

[0359] Tumor microarrays (TMAs) containing fixed and paraffin embedded tumor biopsy sections obtained from human patients diagnosed with different B-cell and T-cell lymphomas were obtained from U.S. Biomax (Rockville, MD). Canine B-and T-cell lymphoma biopsy sections were obtained from archived tissues stored within the University of California Davis Comparative Cancer Center. The tissues were collected using routine biopsy procedures performed on client-owned pet dogs with spontaneous lymphomas that were presented to the University of California Davis Veterinary Medical Teaching Hospital. The protocol for collection of tissues was approved by the U.C. Davis Clinical Trial Review Board, and signed owner consent was obtained prior to collection of any patient tissues. The canine tissue sections were formalin fixed and embedded in paraffin, and Hematoxylin and Eosin (H&E) stained sections were used to identify tumor type. Immunophenotyping was performed using monoclonal mouse anti-canine CD3 and CD21 antibodies to determine T- and B-cell immunophenotypes, respectively.

[0360] HLA typed PBMCs were obtained from four commercial sources - AllCells

(Alameda, CA) , Cellular Technology Limited (Shaker Heights, OH), Precision for Medicine (New York, NY) and StemCell Technologies (Seattle, WA). Peripheral blood samples from individuals expressing DRB1*10 and DRB1*08 alleles were obtained from 3 healthy volunteers after written informed consent and following the ethics guidelines of the Madurai Kamaraj University, Madurai, India. The tumor cell lines Raji (CCL-86), Jurkat (TIB- 152), ARH77 (CRL-1621) and Ramos (CRL-1596) were obtained from the American Type Culture Collection (Manassas, VA). Granta-519 (ACC 342) was obtained as a gift from Dr. Joseph Tuscano (U.C. Davis Cancer Center, Sacramento, CA). Lym-1 antibody was provided by Dr. Gerald DeNardo, University of California, Davis).

Synthesis of SH7129

[0361] Biotinylated SH7129 was synthesized and purified by AmbioPharm Inc.

(North Augusta, SC) as described previously (DeNardo etal ., 2008, Cancer Biotherapy and Radiopharmaceuticals 23: 783-795; DeNardo et al., 2009, Int J Oncol. 2009; 34: 511-6). Briefly, the SHAL was synthesized using solid phase chemistry by the stepwise attachment of Fmoc-D-Lys#l(Boc)-OH, Fmoc-AEEA-OH#l (Fmoc-8-amino-3,6-dioxaoctanoic acid), Fmoc-D-Lys#2(Dde)-OH, Fmoc-AEEA-OH#2, Fmoc-L-Val-OH, and Dabsyl chloride to a Wang resin using standard Fmoc (N-9-fluorenylmethoxy carbonyl) chemistry with HBTU (2- ( UT-benzotriazol- 1 -yl)- 1 , 1 ,3 ,3 -tetramethyluronium hexafluorophosphate)/HOBt (Hydroxybenzotriazole)/DIPEA (N,N-Diisopropylethylamine) as the coupling reagents. The side chain of Fmoc-D-Lys#2-(Dde)-OH, was then deprotected with 4% hydrazine in dimethylformamide (DMF) and then coupled to Fmoc-D-Lys#3-(Dde)-OH using the same coupling procedure. After Fmoc deprotection, Fmoc-AEEA-OH#3 was coupled to the a- amine of D-Lys#3. 4-(4-(4-chlorobenzyl) piperazine)-3-nitrobenzenecarboxylic acid (Cb ligand) was then linked to the Fmoc deprotected amine of AEEA-OH#3 using the same coupling procedure. The third ligand Ct (3-(2-((3-chloro-5-(trifluoromethyl)-2-pyridinyl) oxy)-anilino)-3-oxopropanoic acid) was then directly attached to the deprotected e-amine of D-Lys#3. The assembled free amine form of the SHAL was cleaved from the resin, deprotected and subsequently precipitated as a crude solid. The crude product was purified by standard RP-HPLC methods and isolated by lyophilization. Biotin was attached to the free amine on the terminal lysine by dissolving the SHAL in anhydrous DMF, N,N- Diisopropylethylamine (DIEA) and solid biotin N-hydroxysuccinimide ester (biotinyl-OSu). The mixture was nutated for 15 min, and the reaction was monitored by analytical HPLC. Upon completion, the reaction solution was diluted with a small volume of water/acetonitrile (50/50) containing 1% trifluoroacetic acid (TFA) and directly purified by HPLC. The resulting purified SH7129 was lyophilized and then analyzed by LC/MS and NMR to determine its purity and confirm its molecular mass and identity, respectively.

Preparation of Peripheral Blood Mononuclear Cells

[0362] Three 5 ml blood samples were collected from each donor in tubes containing anticoagulant. Peripheral blood mononuclear cells (PBMC) were isolated using the Lymphoprep (BAG, Germany) density gradient method as described previously (Boyum 1968, Scand J Clin Lab Invest Suppl 1968, 97, 7). The samples were centrifuged for 20 min at 1600 rpm and 20°C without applying a brake. The PBMC interface was carefully removed by pipetting and was washed twice with RPMI media, once followed by centrifugation for 15 min at 2000 rpm and a second time for 10 min at 1500 rpm for platelet removal. Smeared slides were prepared and stored in the refrigerator until they could be stained with SH7129. HLA-class II typing was performed based on PCR-SSP and SBT methods as described previously (Olerup and Zetterquist, 1992; van Dijk etal ., 2007).

Quantification of HLA-DR expression in lymphoma cells by flow cytometry using Lym- 1 antibody

[0363] Optimal flow cytometry conditions for the analysis of the expression of HLA-

DR by lymphoma cell lines were determined by examining the binding of Lym-1 to the Burkitf s lymphoma cell line Raji as a function of antibody concentration. Raji cells in exponential growth were washed twice with Dulbecco’s Phosphate Buffered Saline (DPBS) to remove excess media, resuspended in cold fluorescence-activated cell sorting (FACS) buffer (10% FCS in DPBS) and counted. The cell suspension was diluted to a concentration of 20 x 10⁶ cells/ml and 50 mΐ (10⁶ cells) was added to a series of FACS tubes. 50 mΐ of Lym- 1 antibody, a mouse IgG2a antibody at 2X the desired concentration, was added to the tubes and the mix was incubated at 4°C for 30 min on ice. The Lym-1 antibody was tested at final concentrations of 0, 0.1, 1 and 10 mg/ml to identify the lowest concentration required to saturate the Lym-1 binding sites on Raji lymphoma cells and provide maximal cell staining (mean fluorescence intensity). This concentration, 10 pg/ml, was used in the final flow experiments. Mouse IgG2a UPC- 10 (10 pg/ml, Sigma Chemical, M5409) was run as the isotype control. After the 30 min incubation, 2 ml cold FACS buffer was added to each tube and the cells were pelleted by centrifugation at 1500 rpm, rinsed twice with 2 ml cold FACS buffer, and IOOmI of PE-labeled goat anti-mouse IgG (Jackson Laboratories) was added to the pellet. After resuspension of the cells, the mixture was incubated at 4°C for 30 min in the dark. Cold FACS buffer (2 ml) as then added to the tube, the cells were pelleted by centrifugation, and the pellet was resuspended in 200 mΐ FACS buffer and kept on ice in the dark. 10 mΐ of 7-Amino actinomycin D (7-AAD) was added to each sample immediately prior to analysis to identify non-viable cells.

[0364] Five lymphoma derived tumor cell lines Raji, Jurkat, Granta-519, ARH77 and

Ramos were subsequently analyzed for HLA-DR expression based on staining with the anti- HLA-DRLym-1 antibody. All cell lines were grown in RPMI-1640 medium (Corning 10041CV) supplemented with 10% fetal bovine serum (FBS, Coming 35015CV) at 37°C in 5% CO2 and 90% relative humidity. Cells still in their exponential growth phase were washed twice in staining buffer (5mM EDTA, 0.5% BSA) and then resuspended in staining buffer to a final concentration of 20 x 10⁶ cells/ml. Aliquots of cells were then stained with the monoclonal antibody Lym-1 to quantify the expression of HLA-DRs recognized by Lym-1. For this staining, 50 mΐ of cells were added to a FACS tube and either 50m1 of Lym-1 (20 pg/ml) or the isotype control (20 pg/ml) was added to the cells and the tube was incubated at 4°C for 30 min. After adding 2 ml cold staining buffer, the cells were centrifuged at 1500 rpm for 10 min, the cell pellet was washed again using staining buffer, and then the cell pellets were resuspended in 100 mΐ staining buffer. PE-labeled secondary antibody (1:100 dilution) was added, the mixture was incubated at 4°C for 30 min in the dark, 2 ml cold staining buffer was added and the cells were pelleted by centrifugation, the supernatant was removed and the cells were resuspended in 200 mΐ staining buffer. The stained cells were analyzed by flow cytometry using a propidium iodide stained set of samples to determine the gate parameters to use for selecting viable cells and FCS-A/FCS-H plots to identify cell singlets. Stained cells were identified as those cells with <1% events in the isotype control fluorescence histogram.

Staining of PBMC smears and tumor biopsy sections

[0365] A stock solution of SH7129 was prepared by dissolving 10 mg of the dry

SFLAL in 1 ml dimethyl sulfoxide. Tumor microarrays or slides of HLA-typed PBMCs were stained using a Leica BOND RX Automated Slide Stainer (Leica Biosystems Inc, Buffalo Grove, IL). The formalin fixed slides were deparaffmized using the Leica dewax solution, rehydrated with an alcohol series (100%, 95%, 70% and 30% for 4 min each) followed by antigen retrieval in citrate buffer at pH 6 and 90°C for 20 min. After performing a 5 min hydrogen peroxide block, the slides were washed three times with BOND Wash Solution and then stained with SH7129 (100 pg/ml in PBS, 1% DMSO) for 30 min. Following three washes with BOND Washing Solution, the slides were treated with Streptavidin-horse radish peroxidase (SAHRP) for 30 min, washed 3 times with BOND Wash Solution and once with deionized water, treated with Mixed DAB Refine for 10 min, and then washed four times with deionized water, BOND Wash Solution and a final deionized water wash as per the BOND Polymer Refine IHC protocol (Histowiz, Brooklyn, NY). Instead of DAB (3,3- diaminobenzidine), the DAB/Ni substrate (SK-4100 Vector Laboratories, Burlingame, CA) was used in the canine biopsy staining reactions to provide a darker (gray/black) SAHRP product. After counterstaining the PBMC slides with hematoxylin (the tumor microarray slides were not counterstained with hematoxylin), the slides were then dehydrated by immersion in an alcohol series (30%, 70%, 95% and 100% for 4 min each), cleared with xylene and mounted with Permount. Images of the sections were obtained at 40X magnification and the images were processed and analyzed using ImageJ 1.42 (Schneider et al., 2012, Nat Methods. 2012; 9:671-675).

Cytotoxicity assay

[0366] Raji cells (a Burkitt’s lymphoma) were grown and maintained in RPMI-1640 media supplemented with 10% fetal calf serum (FCS), 200 mM L-glutamine, 100 mM sodium pyruvate, 1% nonessential amino acids and 1% penicillin/streptomycin at 37°C in a 5% CO2 atmosphere. Upon reaching exponential growth, the cells (50,000 per well) were plated in fresh media supplemented with 10% FCS into a 96-well plate. A stock solution of SH7129 or SH7139 was added to each well to provide a series of SHAL concentrations ranging from 0 to 7 nM. Each treatment condition was run in triplicate (SH7129) or quadruplicate (SH7139). Following incubation at 37°C for 48 hr, the cells were resuspended by gently pipetting the media containing the cells up and down ten times, and 20m1 of the cell suspension was mixed with 20m1 of Trypan blue (final concentration 0.1%). After mixing the stain and the cells well, the sample was added to a counting slide and the live and dead cells were counted using a Cellometer Auto T4 (Nexcelom Bioscience LLC, Lawrence, MA). Untreated cells continued to multiply during the 48 hr incubation, and the number of non- viable cells remained < 5% over the course of the assays. The extent of cell killing was determined by subtracting the percent of non-viable cells in the untreated control from the percent of non-viable cells in the treated samples. Results were plotted in Excel, and best-fit curves were determined using a Boltzmann Model in which y= l/(l+exp((Half value- (ln(concentration))/slope)) (Verschuuren 2013, YouTube: www.youtube.com/watch?v=fMghQw2Ry2c; 2013). It was not possible to calculate accurate IC50/EC50 values at the 48 hour time point due to the complexity of the SHALs’ mechanism of action (Douglass et al, 2013, J Am Chem Soc. 2013;135:6092-6099; Schoemaker etal ., 1998, J Pharmacokinet Biopharm. 1998; 26:581-593) and because the maximal effect was not reached for either compound. However, rough estimates for EC 50 enabling the comparison of the relative activities of SH7129 and SH7139 were obtained using the Inhibitory Effect Sigmoid Emax Model in PKSolver 2.0 (Zhang et al, 2010, Comput Methods Programs Biomed. 2010;99:306-314).

Computational Analysis and Docking

[0367] The homology model of the b-subunit of HLA-DR10 generated previously for use in designing SH7139 (Balhorn et al., 2007, Clinical Cancer Research 13: 5621-8), together with the coordinates for the invariant a-subunit of HLA-DR1 (1 AQD.pdb (Murthy et al, 1997, Structure. 1997; 5:1385-1396) with endogenous peptide and b-subunit removed), was used as the HLA-DR10 target for the initial docking runs. Crystal structures for the other HLA-DR targets HLA-DR1 (40V5 (Yin etal, 2014, J Biol Chem. 2014; 289:23449-23464) containing DRB1*01:01), HLA-DR3 (1A6A (Ghosh etal, 1995, Nature. 1995; 378:457-462) containing DRB1*03:01), HLA-DR4 (1D5M (Bolin etal, 2000, J Med Chem. 2000; 43:2135-2148) containing DRB 1*04:01), HLA-DR 11 (6CPL (Galperin etal, 2018, Sci Immunol. 2018; 3) containing DRB1*11:01), HLA-DR14 (6ATZ (Scally etal, 2017, Ann Rheum Dis. 2017; 76:1915-1923) containing DRB1*14:02) and HLA-DR 15 (1BX2 (Smith et al, 1998, J Exp Med. 1998; 188:1511-1520) containing DRB 1*15:01) were obtained from the RSCB Protein Databank (www.rscb.org). The structures of the three recognition ligands Ct (3-(2-((3-chloro-5-(trifluoromethyl)-2-pyridinyl) oxy)-anilino)-3-oxopropanoic acid, ZINC4073762), Cb (4-(4-(4-chlorobenzyl) piperazine)-3-nitrobenzenecarboxylic acid, ZINC4013670) and Dv (4-(dimethylamino)azobenzene-4’-sulfonyl-L-valine, ZINC44075892) were obtained from the University of California San Francisco ZINC database (www.zinc.docking.org) as M0L2 files.

[0368] Following removal of the coordinates for the other associated molecules, the

HLA-DR heterodimer crystal structure datasets containing both the a- and b-subunit were used for docking. Each structure was checked for errors, and any missing atoms were inserted using Chimera (www.cgl.ucsf.edu/chimera/). Each HLA-DR structure was examined in Pymol (V2.2.3, Schrodinger LLC; www.pymol.org; Schrodinger, San Diego, CA), its surface electrostatic potential was calculated in Pymol, and SwissDock (www.swissdock.ch, Swiss Institute of Bioinformatics) was then used to dock each ligand (Ct, Dv, Cb) to the entire surface of the protein. The top 250 conformers with the lowest free energy for each of the bound ligands were examined and mapped onto the surface of HLA- DR to identify the sites where each of the Ct, Cb and Dv ligands were predicted to bind. The atoms of each docked ligand were converted to non-bonded spheres and color-coded blue (Ct), red (Dv) or yellow (Cb) in order to distinguish the sites where each ligand was observed to bind to the seven docked HLA-DR structures.

Results

Quantification of Lym-1 binding to different tumor cell lines

[0369] The Lym-1 antibody has been used by others to detect or identify the presence of a subset of HLA-DRs expressed by lymphoma cell lines and tumors, but in many of these studies only semi-quantitative results (- binding or + to ++++ binding) have been reported (Epstein et al, 1987, Cancer Res. 1987; 47:830-840; Tawara etal, 2007, Cancer Sci. 2007; 98:921-928; Funakoshi et al. , 1997, Blood. 1997; 90:3160-3166; Kostelny et al. , 2001, Int J Cancer. 2001; 93:556-565). To determine how closely the amount of bound Lym-1 correlates with HLA-DR expression, flow cytometry was used to quantify Lym-1 antibody binding to five lymphoma cell lines, the results of which were then compared to HLA-DRBl mRNA expression data reported for four of the cell lines by Boegel et al. (Boegel et al. ,

2014, Oncoimmunology. 2014; 3:e954893). Four of the cell lines, Raji (Tawara etal ., 2007, Cancer Sci. 2007; 98:921-928; Hok etal. , 2007, Bioconjug Chem. 2007; 18:912-921; Silver etal., 1980, Immunogenetics. 1980; 10:295-298), ARH-77 (Tawara etal., 2007, Cancer Sci. 2007; 98:921-928; Carlo-Stella etal, 2007, Cancer Res. 2007; 67:3269-3275), Granta-519 (Tawara et al ., 2007, Cancer Sci. 2007; 98:921-928; Amin et al. , 2003, Arch Pathol Lab Med. 2003; 127:424-431) and Ramos (Tawara et al., 2007, Cancer Sci. 2007; 98:921-928; Pagel et al. , 2009, Blood. 2009; 113:4903-4913), have been shown by others to express HLA-DR. Jurkat, a T-cell leukemia line known to lack HLA-DR expression (Holling et al ., 2004,

Blood. 2004; 103:1438-1444), was included as a negative control.

[0370] Based on the Lym-1 binding data obtained by flow cytometry (Table 14), Raji cells (a Burkitt’s lymphoma) expressed the most HLA-DR, followed by ARH-77 (82.1%), Granta-519 (35.3%), Ramos (10.1%) and Jurkat (0.004%) cells. The percentage of cells in each population stained by Lym-1 show that all of the Raji, ARH-77 and Granta-519 cells express HLA-DR, while a subset (approximately 19%) of the Ramos cells do not. Jurkat cells did not bind significant amounts of antibody, confirming previous reports that these T- cell derived leukemia cells do not express detectable levels of HLA-DR (Holling et al., 2004, Blood. 2004; 103:1438-1444). These results, together with the mRNA expression data obtained by Boegel et al. (Boegel et al., 2014, Oncoimmunology. 2014; 3:e954893) and a previous flow cytometry study comparing Lym-1 binding to Raji and Ramos cells (Pagel et al., 2009, Blood. 2009; 113:4903-4913), show Lym-1 binding provides a reasonably accurate measure of the amount of HLA-DR expressed by these lymphoma and leukemia cells (Table 14).

Table 14: Correlation between Lym-1 binding and HLA-DR expression by lymphoma cell lines.

^aMFI: Mean fluorescence intensity ^b Relative number of HLA-DR targets expressed per cell relative to Raji calculated as follows: Relative expression = (Lym-1 MFI/cell for each cell line divided by the Lym-1 MFI/cell for Raji) X 100. ^cPercent DRB1 mRNA expression relative to Raji, Boegel etal. (Boegel etal ., 2014, Oncoimmunology. 2014; 3:e954893) ^dHolling etal. (Holling et al., 2004, Blood. 2004; 103:1438-1444) reported the absence of MHC class II expression in leukemic T cells was due to a lack of expression of the class II transactivator (CIITA), whereas in T-lymphoma cells, expression of CIITA correlated with expression of MHC class II.

SH7129 HLA-DR target selectivity

[0371] Since SH7139 was designed to target the same region of the antigen binding pocket of HLA-DR recognized by Lym-1, the staining of tumor biopsy tissue with SH7129 would be expected to yield comparable results to Lym-1. Human and canine lymphoma biopsy samples were tested to confirm the selectivity of SH7129 binding to tumor sections that have been determined to express or lack HLA-DR (human) or DLA-DR (dog) based on their staining with Lym-1 antibody (Balhorn et al., 2010, Vet Immunol Immunopathol. 2010;137:235-242; Edwards et al., 1985, Immunology. 1985; 55:489-500). Lym-1 was chosen to identify HLA-DR target expression because it recognizes a unique epitope located within the b-subunit of both human HLA-DRs (Epstein et al., 1987, Cancer Res. 1987; 47:830-840; Rose et al., 1996, Cancer Immunol Immunother. 1996; 43:26-30; Rose et al., 1999, Mol Immunol. 1999; 36:789-797) and canine DLA-DRs (Balhorn et al., 2010, Vet Immunol Immunopathol. 2010;137:235-242) that SH7129 and SH7139 were designed to also recognize. The flow cytometry experiments also confirmed Lym-1 binding to lymphoma tumor cells correlates well with the cell’s level of HLA-DR expression.

[0372] In the staining protocol developed for these experiments, the tissue sections were treated either with Lym-1 followed by a secondary biotinylated anti -mouse IgG antibody or with SH7129. The slides were then washed to remove unbound antibody or SH7129 and the bound Lym-1 bound secondary antibody or SH7129 was detected using a streptavidin-horse radish peroxidase (SAHRP) amplification system. Cells expressing HLA- DRs that bind Lym-1 or SH7129 are stained brown or black (if Ni+ is included in the assay) as a result of the deposition of the insoluble tetrazolium product produced by the horse-radish peroxidase oxidation of the substrate. To facilitate the visualization and analysis of Lym-1 and SH7129 binding, the tissue sections were not counterstained. By leaving out the hematoxylin counterstaining step, cells that do not express HLA-DR or DLA-DR remain unstained and are not visible when the biopsy section is imaged. This has enabled the detection of very low levels of SH7129 binding. It has also made it possible to use image analysis to directly quantify the amount of bound SH7129 by integrating the absorbance of the colored insoluble SAHRP product generated during the staining procedure and to compare the relative levels of HLA-DR or DLA-DR target expression for different cells within a section or different biopsy cores in a tumor microarray.

[0373] As expected, human and canine B-cell lymphomas expressing the HLA-DR or

DLA-DR targets that were stained by Lym-1 were also stained by SH7129. The human biopsy sections that bound SH7129 included cases obtained from patients diagnosed with diffuse large B-cell lymphoma (DLBCL), anaplastic large B-cell lymphoma (ALCL), follicular lymphoma (FL) and lymphoplasmacytic lymphoma (LPL). A centroblastic lymphoma (DLBCL variant) that did not bind Lym-1, also failed to bind SH7129. Two canine B-cell lymphomas (Beagle and German Shepherd) bound Lym-1 and SH7129, but another canine B-cell lymphoma (Bouvier) and two T-cell lymphomas (Keeshond and Siberian Husky) tested were not stained by Lym-1 or SH7129.

SH7129 also retains the anti-tumor activity of SH7139

[0374] Previous studies that have shown both SH7129 and ^U1ln-labelled SH7139 uptake and residualization by lymphoma cells requires HLA-DR expression (DeNardo et ah, 2008, Cancer Biotherapy and Radiopharmaceuticals 23: 783-795) not only provide additional support for the selective binding of SH7129 and SH7139 to HLA-DR-expressing cells, but the results also show that the DOTA and biotin tags do not prevent the transport of the HLA- DR:SHAL complex into the tumor cell. Since HLA-DR binding and internalization represent only the first steps required for SHAL prodrug function, an additional experiment was performed to confirm that the replacement of DOTA by biotin does not alter the function of the SHAL once it is internalized. In this experiment, the cytotoxicities of the two SHALs were compared by measuring the uptake of Trypan blue by Burkitt’s lymphoma (Raji) cells after incubation with SH7129 or SH7139. Trypan blue is a blue water-soluble dye that cannot pass through the intact membranes of live cells and is routinely used to stain and identify dead cells.

[0375] Raji cells were harvested in exponential growth phase and treated with

SH7139 or SH7129 at a series of concentrations ranging from 29 pM to 7 nM or with buffer (negative control). The cells were analyzed after a 48-hour incubation using an automated system (Cellometer Auto T4) to determine the number of live (unstained) and dead (stained) cells. Both SH7139 and SH7129 were cytotoxic to Raji cells with -34% (SH7129) to -38% (SH7139) of the cells being killed at a concentration of 2.3 nM SHAL. EC50 values for the SHALs, which are affected by both drug exposure time and the drug’s mechanism of action, could only be roughly estimated for comparison purposes since maximal cell killing was not reached under the experimental conditions used. An analysis of the data using an Inhibitory Effect Sigmoid Emax prediction model provided EC50 estimates for both SH7129 (EC50 - 70 ± 9 nM) and SH7139 (EC50 - 24 ± 16 nM) consistent with observation that the two SHALs induce growth inhibition activity in Raji cells at nM concentrations. The slightly lower inhibitory effect observed for SH7129 may reflect the presence of the hydrophobic biotin tag in SH7129 and the SHAL’s significantly reduced solubility in aqueous solutions compared to that of SH7139, which contains a more hydrophilic negatively charged DOTA chelating group.

Identification of HLA-DR variants targeted by SH7129

[0376] To identify the HLA-DRs targeted by SH7139 and SH7129, fixed slides of

PBMCs obtained from HLA-typed individuals who express HLA-DRs containing b-subunits from specific DRBl alleles were stained with SH7129 using a protocol similar to that used for staining tumor biopsy tissue. The biotin in the bound SH7129 was detected using SAHRP and the substrate 3,3-diaminobenzidine, and the slides were then counter-stained with hematoxylin to visualize the cells. Cells expressing HLA-DRs that bind SH7129 are stained brown.

[0377] The results summarized in Table 15 show that SH7129 binds to PBMCs expressing all but five of the HLA-DRs tested, namely those homozygous for DRB 1*01:01, DRB1*03:01, DRB1*04:07, DRB1*08:02 and DRBl*14. The DRB1*14 donor was homozygous for DRB 1*14 but expressed two different DRB* 14 alleles, DRB 1*14:02,

DRB 1*14:06. PBMCs obtained from two different donors homozygous for DRB 1*12:02 show SH7129 staining, but the cells obtained from both donors yielded smears of sufficiently poor quality that SH7129 binding was designated as probable. Many of the stained cells on each slide appeared to be partially disrupted, taking on an appearance similar to the smudge cells often observed in patients that have been diagnosed with chronic lymphocytic leukemia (CLL), other hematological and solid cancers, infections, or cardiac arrest (Chang et ah,

2016, Asian Pac J Cancer Prev. 2016; 17:1847-1850; Nowakowski et ah, 2009, J Clin Oncol. 2009; 27:1844-1849).

[0378] Donors homozygous for the HLA-DR b-subunits expressed by the DRB 1*09 and DRB 1*16 alleles could not be obtained for inclusion in the study. In order to test for SH7129 binding to HLA-DRs containing these two b-subunits, PBMCs were obtained from individuals with the two heterozygous HLA-DRBl haplotypes DRB1*09:01,DRB1*04:03 and DRB1*16:02, DRB1*04:07. These haplotypes were selected based on an earlier determination that SH7129 does not bind to homozygous DRB1*04:07 PBMCs (Table 15) and the fact that the sequence of the b-subunit DRB 1*04:03 differs from DRB 1*04:07 by only a single amino acid (a valine at position 86 in DRB 1*04:03 that is glycine in DRB 1*04:07) that is buried inside the protein and has had no impact on the binding of SH7129 to other HLA-DRs. Thus, these two heterozygous allele combinations provide cells that express a single DRB1*09:01 or DRB1*16:02 allele as well as aDRBl*04 allele that Applicant already determined does not (DRB 1*04:07) and Applicant expects should not (DRB1*04:03) bind SH7129. The results obtained from the staining of these PBMC samples show SH7129 binds to HLA-DRs containing the b-subunit DRB 1*16:02 and provide a strong indication that SH7129 binds to HLA-DRs containing the b-subunit DRB1*9:01 (Table 15). They also demonstrate, along with the staining of donor LP151’s PBMCs containing a single HLA-DR10 allele (DRB1*10:01, DRB1*14:02), that expression of the HLA-DR target and binding of SH7129 can be detected in PBMCs containing a single allele for an HLA-DR recognized by SH7129. In contrast to tumor cell populations expressing HLA-DR, in which the majority of the cells express the MHC class II protein, only a small percentage of resting (unstimulated) B-cell lymphocytes, macrophages and dendritic cells express HLA-DR in healthy individuals (Edwards et al., 1985, Immunology. 1985; 55:489-500). In B-cell lymphomas and many other types of cancers, HLA-DR expression can be upregulated as much as 10 to 100-fold (Roucard et al., 1996, J Biol Chem. 1996; 271: 13993-4000) .

Table 15. SH7129 binding to HLA-typed PBMCs expressing known DRB1 alleles.

^aHLA-DR10 is used as the reference DRB1 ^b Presence of smudge cells made analysis more difficult.

SHAL ligand docking to HLA-DR targets

[0379] To gain insight into why SH7129 binds to some HLA-DRs and not others, a series of small molecule computational docking experiments were conducted using the structures of three HLA-DRs expressed by the PBMCs that were observed to bind SH7129 (HLA-DR10, HLA-DR11 and HLA-DR15) and four HLA-DRs expressed by PBMCs that did not bind the SHAL (HLA-DR 1, HLA-DR3, HLA-DR4 and HLA-DR 14). Since it was not feasible to dock the entire SH7129 molecule to these HLA-DRs due to SH7129’s large size and flexibility (too many degrees of freedom), SwissDock (Grosdidier et ah, 2011, Nucleic Acids Res. 2011; 39:W270-277; Grosdidier et ah, 2011, J Comput Chem. 2011; 32:2149- 2159) was used to dock each of the Ct, Dv and Cb ligands to the homology model of HLA- DR10 (Balhorn et ah, 2007, Clinical Cancer Research 13: 5621-8) (no crystal structure is available) and to crystal structures of HLA-DRl (40V5 (Yin et ah, 2014, J Biol Chem. 2014; 289:23449-23464) containing DRB 1*01:01), HLA-DR3 (1A6A (Ghosh et ah, 1995, Nature. 1995; 378:457-462) containing DRB1*03:01), HLA-DR4 (1D5M (Bolin et ah, 2000, J Med Chem. 2000; 43:2135-2148) containing DRB 1*04:01), HLA-DRl 1 (6CPL (Galperin et ak, 2018, Sci Immunol. 2018; 3) containing DRB1*11:01), HLA-DRl 4 (6ATZ (Scally et ah, 2017, Ann Rheum Dis. 2017; 76:1915-1923) containing DRB1*14:02) and HLA-DRl 5 (1BX2 (Smith et ah, 1998, J Exp Med. 1998; 188:1511-1520) containing DRB1*15:01). All HLA-DR structures used for docking contained both the invariant b- and variant b subunits. The expectation was that the docking of the three ligands, whose binding to specific sites on HLA-DR provide SH7129’s target recognition (DeNardo et ah, 2008, Cancer Biotherapy and Radiopharmaceuticals 23: 783-795; DeNardo et ah, 2009, Int J Oncol. 2009; 34: 511-6) and contribute to its high affinity binding (Balhorn et ah, 2014, Cancer Research. 2014; 74:

2703), might yield information that helps explain the selectivity of the SHAL’s binding to different HLA-DRs.

[0380] The top 250 ligand conformations with the lowest free energies of binding obtained from each HLA-DRl 0 docking experiment were mapped onto the HLA-DRl 0 surface and used to identify the sites where the three ligands are most likely to bind. Interestingly, all three ligands were predicted to bind to sites on the b-subunit inside the antigen binding pocket of HLA-DR10 that either corresponded to (the Ct and Dv ligands) or were located near (the Cb ligand) the binding sites identified more than a decade ago using a different docking approach and program (DeNardo et ah, 2008, Cancer Biotherapy and Radiopharmaceuticals 23: 783-795). After confirming experimentally by surface plasmon resonance and NMR that the ligands not only bound to HLA-DR10 but also did not compete for binding to the same sites, the docking results in the original study were used to design the scaffold to which the ligands were linked to produce SH7129. This scaffold separated the ligands Ct, Cb and Dv in three-dimensional space in a way that allowed each ligand to simultaneously bind to their sites on HLA-DR10, thereby enabling the creation of a family of SHALs (SH7129, SH7133 and SH7139) that bound selectively and with high affinity to HLA-DR10. Consistent with the original docking study, the results of the current docking experiments predict Ct binds to Site 1 on HLA-DR10 near bn85 and Dv binds to Site 2 in a cavity near PR70 and bA74. A number of Cb conformers are predicted to bind to Site 1 while others are predicted to bind to Site 3 located near bR56 at the other end of the antigen binding cavity.

[0381] The docking experiments conducted with HLA-DR11 and HLA-DR15 provided results similar to those obtained with HLA-DR10 except that all three ligands were more promiscuous with respect to the sites where they were predicted to bind. For HLA- DR15, a number of Ct conformers were predicted to bind inside the antigen binding cavity to all three sites. The majority of the conformers of Dv docked to HLA-DR15 were predicted to bind to Site 2 as in HLA-DR10, but a few also bound in or near Site 1. Similar to Ct, the Cb ligand was predicted to bind to Site 3 as well as to Site 1 and Site 2. In the experiments conducted with HLA-DR11, all three ligands were predicted to bind to Site 1, Dv and Cb were predicted to bind to Site 2, and Ct and Cb conformers were predicted to bind to Site 3. While the predicted binding of each ligand to multiple sites suggest the structures or surface properties of the cavities in HLA-DR15 and HLA-DR11 must differ sufficiently from those in HLA-DR10 to reduce the selectivity of the ligand’s binding to a single specific site as observed with HLA-DR10, these results also indicate SH7129 should be fully capable of binding to the antigen binding pockets of HLA-DR15 and HLA-DR11 when the three ligands are linked together.

[0382] Docking of the Ct, Cb and Dv ligands to HLA-DR1, HLA-DR3, HLA-DR4 and HLA-DR14, the four HLA-DRs expressed by PBMCs that did not bind SH7129, provided very different results. In three of these cases (HLA-DR1, HLA-DR4 and HLA- DR14), the three ligands are all predicted to bind to the same site, Site 1. The positions of the ligands bound to HLA-DR14 are shifted slightly towards Site 2 such that part of the bound molecules extends into Site 2. None of the ligands bind in Site 3. Ligand dockings to HLA- DRS differed from all the others in that Cb is not predicted to bind to any site in the antigen binding pocket. The conformers of Ct bind in between Sites 1 and 2 with parts of the molecule extending into Site 2 and Dv is predicted to bind to Site 2 with part of the Dv molecules overlapping the Ct conformers. Because each of the three ligands in SH7129 would be competing for binding to the same site in these four HLA-DRs, the docking results suggest these SHALs should not be able to bind to HLA-DR with an affinity greater than that provided by the binding of a single ligand (Kd in the millimolar range).

Effects of HLA-DR binding site amino acid sequence on SH7129 binding

[0383] An analysis of the HLA-DR variant amino acid sequences that comprise the peptide antigen binding pocket and the residues that surround the three ligand binding sites also suggest differences that may help explain why SH7129 binds to some HLA-DRs and not others. The b-subunit amino acids pVll, bP3, pL26, bE28, bR30, bU47, bί67, bR71, and bA74 and the a-subunit residues aN62 and aV65 that form the surface of Site 2 where the Dv ligand is predicted to bind to HLA-DRIO vary the most amongst the different alleles and span a long segment of the protein near the center of HLA-DR’ s peptide binding cavity. The replacement of the arginine at position 71 that has been shown to be essential for Lym-1 binding with either a negatively-charged glutamic acid ^R71E) or a hydrophobic alanine (b R71A) did not prevent the binding of SH7129 to HLA-DR 13 (DRBl *13:01) or HLA-DR 15 (DRB1*15:02), respectively. Substitution of the other critical HLA-DRIO residue 70 required for Lym-1 binding, which can be either an arginine or glutamine, with the glutamic acid found in five of the seven HLA-DRs to which SH7129 binds clearly does not inhibit binding either. Only the replacement of the hydrophobic alanine at position 74 in HLA-DR3 (DRB1*03:01) with arginine changed the structure of Site 2 in a way that might be expected to prevent Dv binding. Alanine 74 is located at the bottom of a cavity in Site 2 where Dv is predicted to bind in HLA-DR10, and the arginine that replaces alanine 74 in the crystal structure of HLA-DR3 fills this cavity and projects into the Dv binding site. Since the presence of the arginine side-chain would be expected to interfere with Dv binding, the replacement of alanine 74 with two other bulky amino acids, the uncharged glutamine (PA74Q) in HLA-DR7 (DRB1*07:01) and the negatively charged glutamic acid (bA74E) in HLA-DR9 (DRB1*09:01), both of which bind SH7129, and the glutamic acid (bA74E) in HLA-DR4 (DRB1*04:07), that does not bind SH7129, might be expected to have a similar effect. Unlike the arginine sidechain in HLA-DR3, however, the glutamic acid at position 74 in the crystal structure of HLA-DR4 (Bolin et al., 2000, J Med Chem. 2000; 43:2135-2148) does not project into the cavity at the bottom of Site 2. Both HLA-DR7 and HLA-DR9 bound SH7129, which is consistent with the Dv site being open. But SH7129 did not bind to HLA- DR4. The presence of the additional negative charge provided by the carboxyl group at the bottom of the site would be expected to change the local electrostatics, but HLA-DR9 also contains an E74 residue and SH7129 binds to it.

[0384] The surface residues in Site 1 where the Ct ligand binds to HLA-DR10 (bU78, b N82 and bn85 and aF22, aA52, aS53, aF54 and aE55) and in Site 3 where the Cb ligand binds (bE52, bE53, bQ54, bB55, bR56, bϋ57, bE59 and bU60 and aR76) are located at opposite ends of the antigen binding cavity. The amino acids in both Sites 1 and 3 are highly conserved, with very few exceptions, across the different alleles. Neither the replacement of the Site 1 polar tyrosine at position 78 with either a polar glutamine (bU78(¾ or a negatively charged glutamic acid (bU78E) or the hydrophobic valine at position 85 with a hydrophobic alanine (bΎ85A) appears to affect binding.

[0385] Because the Ct, Dv and Cb ligands of SH7139 bind to three separate sites that span the entire length of HLA-DR’s peptide antigen binding cavity, SH7129 binding is likely to be affected by changes in other surface amino acid residues unique to specific HLA-DRs, many of which have no impact on Lym-1 antibody binding. HLA-DR1 variants containing the DRB 1*01:01 and DRB 1*01:02 b-subunits have two amino acid substitutions (bΎIIE and bR30C) in Site 2 (Dv binding site) that are unique to the HLA-DR1 variants. Both are located in positions that could induce subtle alterations in the shape of the cavity or the hydrophobic nature of its inner surface. In addition to the Site 2 sequence differences between HLA-DR10 and HLA-DR3 (the pR71 K and PA74R substitutions) mentioned earlier, HLA-DR3 variants contain another unique difference - a surface asparagine (bN77) located right next to Site 1 that replaces the threonine present in all other HLA-DRs. The presence of this asparagine could affect the structure of Site 1 or alter the Ct ligand’s interaction with it. The PF13H change in the surface of Site 2 in HLA-DR4 (DRB1*04:07) and the PA74L Site 2 substitution in HLA-DR8 (DRB 1*08:02) are both unique to these HLA-DRs and located in positions that could easily impact SH7129 binding. With respect to HLA-DR14, however, there are no unique differences in surface or buried amino acids located near Site 2 that might explain why SH7129 binds to HLA-DR10 but doesn’t bind to HLA-DR14 (DRB1*14:01 or DRB 1 * 14:06). There were also no unique residues in the sequences that contribute to the formation of the surfaces of the other HLA-DR14 sites. What cannot be ruled out, in HLA- DR14 or any of the other HLA-DR variants that don’t bind SH7129, are changes in residues located outside the binding site cavities that may induce subtle changes in protein packing or the properties of specific regions of the antigen binding cavity that prevent the binding of SH7129 or reduce its affinity sufficiently that its binding might not be detected under the stringent washing conditions used in the staining protocol.

Effects of HLA-DR binding site electrostatic potential on SH7129 binding

[0386] Differences in the surface electrostatic potential energies of the ligand binding sites in the different HLA-DRs may also impact the ability of the polar ligands Ct, Cb and Dv to form stable electrostatic interactions within their HLA-DR binding sites. The b-subunit amino acids that surround Site 2 where Dv is predicted to bind vary more between HLA-DR variants than any other region of the protein. The calculated electron density of this site is significantly lower (providing a more positive environment) in HLA-DRIO and HLA-DR9 than the same site in all the other HLA-DR variants. The primary reason for this difference is due to the replacement of the positively charged arginine at position 70 in HLA-DRIO and HLA-DR9 with a negatively charged aspartic acid or a polar glutamine in the other variants. The other sequence changes (relative to HLA-DRIO) that may affect the electrostatics of the HLA-DR variants that do not bind SH7129 are all found inside the cavity that forms Site 2. These include the bR30C substitution in HLA-DRl variants (a change from a the positive- charged arginine to a polar cysteine having a partial negative charge), the bR71K substitution (a change from an arginine with a strong positive charge to a lysine with a weaker positive charge) and the PA74R substitution (a change from a hydrophobic alanine to highly positive- charged arginine) and the bT77N substitution (a change from a polar threonine containing a partial negative charge to an asparagine containing a partial positive charge) in HLA-DR3 variants, and the bP3H substitution (a change from a hydrophobic phenylalanine to a positive charged histidine) in HLA-DR4.

Example 14: High-Performance Concurrent Chemo-Immuno-Radiotherapy for the Treatment of Hematologic Cancer through Selective High-Affinity Ligand Antibody Mimic-Functionalized Doxorubicin-Encapsulated Nanoparticles

[0387] Abstract: Non-Hodgkin lymphoma is one of the most common types of cancer. Relapsed and refractory diseases are still common and remain significant challenges as the majority of these patients eventually succumb to the disease. Herein, Applicant reports a translatable concurrent chemo-immuno-radiotherapy (CIRT) strategy that utilizes fully synthetic antibody mimic Selective High-Affinity Ligand (SHAL)-functionalized doxorubicin-encapsulated nanoparticles (Dox NPs) for the treatment of human leukocyte antigen-D related (HLA-DR) antigen-overexpressed tumors. Applicant demonstrated that the tailor-made antibody mimic-functionalized NPs bound selectively to different HLA-DR- overexpressed human lymphoma cells, cross-linked the cell surface HLA-DR, and triggered the internalization of NPs. In addition to the direct cytotoxic effect by Dox, the internalized NPs then released the encapsulated Dox and upregulated the HLA-DR expression of the surviving cells, which further augmented immunogenic cell death (ICD). The released Dox not only promotes ICD but also sensitizes the cancer cells to irradiation by inducing cell cycle arrest and preventing the repair of DNA damage. In vivo biodistribution and toxicity studies confirm that the targeted NPs enhanced tumor uptake and reduced systemic toxicities of Dox. Applicant’s comprehensive in vivo anticancer efficacy studies using lymphoma xenograft tumor models show that the antibody-mimic functional NPs effectively inhibit tumor growth and sensitize the cancer cells for concurrent CIRT treatment without incurring significant side effects. With an appropriate treatment schedule, the SHAL-functionalized Dox NPs enhanced the cell killing efficiency of radiotherapy by more than 100% and eradicated more than 80% of the lymphoma tumors. Materials and Methods

[0388] Materials. Methoxy poly(ethylene glycol)-block-poly(D,L-lactic-co-glycolic) acid copolymer (mPEG(3K)-PLGA(30K), AK101; molecular weight ~ (3 + 30) kDa ~ 33 kDa), poly(D,L-lactide)-block-poly(ethylene glycol)-N-hydroxysucci-nimide ester end-cap (PL A( 16K)-PEG( 10K)-NHS, AI068; molecular weight ~ (16 + 10) kDa ~ 26 kDa) and poly(lactide-co-glycolide) rhodamine B end-capped (PLGA-Rhod, AV027; molecular weight = 45-55 kDa) were purchased from Akina, Inc. (West Lafayette, IN). Primary amine- functionalized SHAL (SH7133), DOTA-functionalized SHAL (SH7139), and biotin- functionalized SHAL (SH7129) were provided by SHAL Technologies, Inc. (Livermore,

CA). The synthesis, purification, and characterization of all SHALs were reported previously in refs 21, 22, 29, and 50. The SHALs used in this study were trifluoroacetate salts with the following purities as determined by LC/MS: SH7129, 96.2%; SH7133, 95.4%; SH7139, 95.0%. All SHALs were used without further purification. Doxorubicin hydrochloride salt (Dox HCl, >99%) was purchased from LC Laboratories (Woburn, MA). Dimethyl sulfoxide (DMSO; anhydrous, >99.9%), triethyl-amine (TEA; >99.5%), methanol (HPLC grade, >99.9%), ethanol (200 proof, for molecular biology), dimethylforma-mide (anhydrous, >99.8%), diethyl ether (ACS reagent, >99.9%), acetonitrile (HPLC plus, >99.9%), deionized water (sterile-filtered, BioReagent), dichloromethane (anhydrous, >99.8%), propidium iodide solution (1 mg/mL in water), Triton X-100 (BioXtra), DNase-free RNase (from bovine pancreas), sodium azide (Laboratory grade), and bovine serum albumin (fraction V lyophilized powder) were purchased from Sigma (St. Louis, MO). Alexa Fluor 488-labeled antihuman HLA-DR antibody (clone L243), phycoerythrin-Cy5-labeled streptavidin, phycoerythrin (PE) anti-H2A.X phosphor (Serl39), antibody (clone 2F3) PE-labeled antihuman CD243 antibody (BioLegend, Clone: 4E3.16) and FITC-labeled antihuman p53 antibody (BioLegend, Clone DO-7) were purchased from BioLegend (San Diego, CA). Human BD Fc Block (antihuman CD16/CD32 antibody) was purchased from BD Bioscience (San Jose, CA). Alexa Fluor 488-labeled anti-calreticulin monoclonal antibody (clone: EPR3924) was purchased from Abeam (Cambridge, MA). Endogenous biotin-blocking kit and dead cell apoptosis kit (contain Alexa Fluor 488 Annexin V and propidium iodide solutions) were purchased from Fischer Scientific (Hampton, NH). All reagents, unless specified, were used without further purifications. [0389] Methods. Synthesis of SHAL-Functionalized PEG-PLA. SFLAL- functionalized PEG-PLA was prepared via a primary amine-NHS ester reaction between primary amine-function-alized SHAL (SH7133) and PLA-PEG-NHS ester. Briefly, amine- functionalized SHAL (SH7133, 4 mg, 2.06 pmol) was first dissolved in 0.8 mL of anhydrous DMSO before added to a DMF solution (0.5 mL) contained PLA-PEG-NHS (48 mg, E85 pmol) and triethylamine (1 pL, 7.2 pmol). The mixture was stirred at 20 °C in the dark for 18 h. The reaction was quenched by the addition of 1 : 1 v/v deionized water/methanol (10 pL). The SHAL-functionalized PEG-PLA was purified by precipitation into a large excess of cold 2:3 v/v of methanol/ diethyl ether twice and cold diethyl ether 3 times. The precipitated polymers were collected by centrifugation (4000g, 15 min, 4 °C). After each precipitation step, the collected polymer pallet was dissolved in dichloromethane (1 mL) before reprecipitation. The purified polymer pallet was dry under nitrogen gas in the dark for 2 days. The dried polymer pallet was stored at -20 °C in the dark before further studies.

[0390] The number-average molecule (MN) of the unmodified PLA-PEG-NHS ester and PLGA-PEG- SHAL was 24000 Da (P.D.I. = 1.36) and 28 600 Da (P.D.I. = 1.68), respectively, as determined by gel-permeation chromatography (GPC) used tetrahydrofuran as an elute and used different molecular weight standard polystyrenes (Agilent PS2) as a calibration standard. The GPC analysis was performed by Akina, Inc. (West Lafayette, IN).

[0391] The degree of functionalization of PLA-PEG with SHAL was quantified by

UV-visible spectroscopy. Briefly, SHAL has a strong and characteristic visible absorption band centered at 452 nm (extinction coefficient at 452 nm, eisinm = 21 500 M-l cm-1 in DMSO). The degree of functionalized PLA-PEG (dissolved in a known amount of DMSO) was calculated from the extinction coefficient of SHAL at 452 nm.

[0392] Preparation of SHAL-Functionalized Dox-Encapsulated PEG-PLGA NPs and

Nontargeted Dox-encapsulated PEG-PLGA NPs. Targeted and nontargeted Dox NPs were prepared via nanoprecipitation method. The target drug loading was 5 wt/wt Dox HCl was converted to hydrophobic Dox in situ. Briefly, 1.5 mg of Dox HCl was first dissolved in 30 pL of 1 : 1 v/v TEA/DMSO. The Dox solution was incubated in the dark for 30 min before the preparation of the NPs. For the preparation of 30 mg of SHAL-functionalized Dox- encapsu-lated NPs, 30 mg of mPEG(3K)-PLGA(30K) was first dissolved in 3 mL of acetonitrile before it was added to the Dox solution before the addition of 33.6 pL of SHAL- PEG-PLA solution (5 mg/mL, in anhydrous DMSO). The mixture was vortexed at 2000 rpm for 20 s before it was added slowly (1 mL/min) to 12 mL of deionized water under constant stirring (1000 r.p.m.). The pH of the mixture was about pH 9, as determined by pH paper.

The mixture was stirred under reduced pressure in the dark at 20 °C for 2 h. The Dox- encapsulated NPs were washed 3 times with a 15 mL 30 000 nominal molecular weight cutoff Amicron Ultra ultrafiltration membrane filter (3000g for 15 min). After each wash, the NPs were resuspended in 3 mL of deionized water. At the final purification cycle, the NPs were first resuspended in 1.5 mL (final volume) of deionized water before mixed with 1.5 mL of 2xPBS to give a 10 mg/mL NP solution. Nontargeted Dox NPs were prepared via the same method except SHAL-PEG-PLA was not added to the mPEG(3K)-PLGA(30K) solution before the preparation of the NPs.

[0393] Preparation of Drug-free Rhodamine-Labeled SHAL-Functionalized PEG-

PLGA NPs. Drug-free Rhod-labeled SHAL-functionalized NPs composed of 1 wt/wt% of PLGA-Rhod were prepared via a nanoprecipitation method. For the preparation of 30 mg of SHAL-functionalized Rhod-labeled NPs, 30 mg of mPEG(3K)-PLGA(30K) was first dissolved in 3 mL of acetonitrile contained 0.1 mg/mL of PLGA-Rhod before mixed with 33.6 pL of SHAL-PEG-PLA solution (5 mg/mL in anhydrous DMSO). The mixture was vortexed at 2000 rpm for 20 s before added slowly (1 mL/min) to 12 mL of deionized water under constant stirring (1000 rpm). The mixture was stirred under reduced pressure in the dark at 20 °C for 2 h. The NPs were washed 3 times with a 15 mL 30 000 nominal molecular weight cutoff Amicron Ultra ultrafiltration mem-brane filter (3000g for 15 min). After each wash, the NPs were resuspended in 3 mL of deionized water. At the final purification cycle, the NPs were first resuspended in 1.5 mL (final volume) of deionized water before being mixed with 1.5 mL of 2x PBS to give a 10 mg/mL NP solution.

[0394] Characterization of Nanoparticles. A transmission electron microscopy

(TEM) image of different targeted and non-targeted NPs was recorded used a JEOL 1230 transmission electron microscope operated at 120 kV in the Microscopy Services Laboratory Core Facility at the UNC School of Medicine. Before TEM imaging, NPs samples were diluted to 10 pg/mL with deionized water before added to glow-discharged 400-mesh carbon- coated copper grids (10 pL per grid). After 5 min, extra water was removed from the grid via a filter paper before being stained with 4% uranyl acetate aqueous solution (10 pL per grid) for 20 s. The excess staining solution was removed by filter paper at the edge of the copper grid. The mean number-average diameter (Dn) and particle concentrations of different NP dispersions were determined by an NP-tracking analysis method recorded on a Nanosight NS500 instrument (Malvern, Inc.) in Microscopy Services Laboratory Core Facility at the UNC School of Medicine. All NP dispersions were diluted to 5 pg/mL before the NP tracking analysis. The average number of conjugated SHAL molecules per NP was calculated from the number of PLA-PEG-SHAL used per each mg of NPs and the number of NP per each mg of polymer used. Intensity-average diameter (Dh, also known as hydrodynamic diameter) and mean zeta potential (mean Q of different NP dispersions were determined by dynamic light scattering and an aqueous electrophoresis method using a Zetasizer Nano ZS Instrument (Malvern, Inc.). Before the measurements, NPs were diluted to 1 mg/mL with 1 ^c PBS. All measurements were based on the average of three separate measurements.

[0395] Drug Loading and in Vitro Drug Release Study. The Dox loadings in the targeted and nontargeted NPs were quantified via the spectroscopic method as previously reported. UV- visible spectra of NP dispersions were recorded in a NanoDrop 1000 Microvolume spectrophotometer (Thermo Scientific). A molar extinction coefficient of 1 x 10⁴ M^_1 cm^-1 for Dox at 495 nm (e495nm) was used for the quantification. Drug-free targeted and nontargeted PEG-PLGA NPs (2 mg/mL) showed insignificant visible absorption at 495 nm. The pH-dependent in vitro drug release profile of targeted and nontargeted Dox NPs was recorded under conditions at pH 5.5, 6.5, or 7.0. Briefly, NP solutions at a concentration of 2 mg/mL were split into Slide-A-Lyzer MINI dialysis microtubes with a molecular cutoff of 10 kDa (Pierce, Rockford, IL) and subjected to dialysis against a large excess (2000 times) of 1 x PBS at pH 5.5, 6.5, or 7.0 with gentle stirring at 37 °C in dark.

The concentration of Dox retained in the NPs was quantified by the spectroscopic method through a NanoDrop 1000 Micro-volume spectrophotometer. All measurements were performed in triplicate.

[0396] In Vitro Studies. Jurkat, Ramos, Daudi and Raji cells were obtained from the

Tissue Culture Facility at UNC Lineberger Comprehensive Cancer Center that purchased the cancer cells from the American Type Culture Collection (ATCC). All lymphoblast cancer cell lines were cultured using RPMI-1640 medium (Gibco) supplemented with 10% (v/v) FBS and antibiotic-antimycotic (100 units/mL of penicillin, 100 pg / mL of streptomycin and 0.25 pg/mL of Gibco amphotericin B) in a 37 °C atmosphere supplemented with 5% CO2. The cell density was determined by a hemocytometer.

[0397] Flow Cytometry. Unless specified, viable cells were first washed three times with FACS buffer (0.1 M PBS with 5g/L of BSA, 1 g/L of sodium azide, and 2 mM of EDTA) via centrifugation (600g, 4 min)-redispersion method. The cell density was determined and adjusted to 10 ^c 10⁶ cells/mL and blocked with human Fc blocker (antihuman CD 16/32 antibody, 2 pg/million cells; BD) at 4 °C for 20 min before being stained with desired antibody/antibodies according to the manufacturer’s instructions.

Stained cells were washed three times with FACS buffer before analysis on a Biosafety Level 2 (BSL2) Intellicyt iQue Screener PLUS flow cytometer in the UNC Flow Cytometry Core Facility at the UNC School of Medicine. All cells were analyzed within 2 h (at 4 °C) after staining and were analyzed without fixing. All collected FACS data were analyzed through a FlowJo VIO.0.7 software pad.

[0398] Quantification of HLA-DR Expression. The HLA-DR expression of selected lymphoblast cancer cell lines were determined by FACS binding assay used A4884abeled antihuman HLA-DR antibody (clone L243) according to the manufacturer’s instructions.

[0399] Quantification of MDR-1 (CD243) and Intracellular p53 Expressions. The multidrug resistance protein 1 (MDR-1) antigen and intracellular p53 expressions of Raji, Daudi, and Ramos cells were determined by FACS binding assay used PE-labeled antihuman CD243 (clone 4E3.16) and FITC-labeled antihuman p53 antibody (clone DO-7) according to the manufacturer’s instructions. Briefly, the cells were first labeled with PE-labeled antihuman CD243 and fixed. The fixed cells were permeabilized with Intracellular Staining Perm Wash Buffer (BioLegend) before stained with the FITC-labeled antihuman p53 antibody (clone DO-7).

[0400] Quantification of Free SHAL Binding Affinity. The binding affinity biotin- functionalized free SHAL, SH7129, was quantified by FACS assay. Before the in vitro binding study, FACS buffer-washed cells were first blocked by endogenous biotin-blocking kit (Fisher) according to the manufacturer’s protocol. Blocked cells (1 ^c 10⁶ cells/100 pL) were stained with different concentrations of SH7129 (0-200 nM) in FACS buffer at 20 °C for 30 min. After two washes (2000g, 3 min) with FACS buffer, membrane-bound SH7129 was labeled with phycoerythrin-Cy5-labeled streptavidin (BioLegend) according to the manufacturer’s protocol. The labeled cells were washed twice with FACS buffer before being analyzed on a BSL2 Intellicyt iQue Screener PLUS flow cytometer.

[0401] Cells stained with 200 nM of free SFLAL followed by PE-Cy5 streptavidin were saved, fixed with 10% neutral buffered formalin at 20 °C for 15 min, and washed with PBS before mixed with equal volume of 4',6-diamidino-2-phenylindole (DAPI)-containing ProLong Gold (Invitrogen) for confocal fluorescence imaging. Confocal fluorescence images were recorded using a Zeiss LSM 710 spectral confocal laser scanning microscope in the Microscopy Services Laboratory at the UNC School of Medicine.

[0402] Quantification of Drug-free Rhodamine-Labeled SHAL-Functionalized

PEG-PLGA NPs Binding Affinity. FACS assay quantified the binding affinity rhodamine- labeled SHAL-functionalized PEG-PLGA NPs. Briefly, FACS buffer-washed cells (1 ^c 10⁶ cells/100 pL) were stained with different concentrations of targeted NPs contained a known concen-tration of conjugated SHAL in the dark at 20 °C for 30 min. After two washes (2000g, 3 min) with FACS buffer, membrane-bound SH7129 was labeled by Alexa Fluor 610-R-phycoerythrin streptavidin. The labeled cells were washed twice with FACS buffer before analyzed on a BSL2 Intellicyt iQue Screener PLUS flow cytometer.

[0403] Cells stained with 200 nM of conjugated SHAL were saved, fixed with 10% neutral buffered formalin at 20 °C for 15 min, and washed with PBS before mixed with equal volume of DAPI-containing ProLong Gold (Invitrogen) for confocal fluorescence imaging. Confocal fluorescence images were recorded through a Zeiss LSM 710 spectral confocal laser scanning microscope in the Microscopy Services Laboratory at the UNC School of Medicine.

[0404] In Vitro Dox Uptake Study. The uptake of free Dox and different Dox- encapsulated NPs in Ramos, Daudi, and Raji cell lines was quantified by FACS and CLSM methods. Briefly, variable cells were washed twice with phenol red-free RPMI1640. The cell densities were adjusted to 10 ^c 10⁶ cells/mL (in RPMI-1640), before being incubated with 10 pM (final concentration) of free and encapsulated Dox. After incubation at 37 °C for 1 h (in the dark), the treated cells were washed twice with cold FACS buffer (4 °C). Half of the cells were analyzed in a BSL2 Intellicyt iQue Screener PLUS flow cytometer in the UNC Flow Cytometry Core Facility at the UNC School of Medicine within 60 min. The Dox fluorescence was quantified in a PE-Texas Red channel (excitation at 561 nm, emission at 615-620 nm). The remaining cells were saved and fixed with 10% neutral buffered formalin at 20 °C for 15 min and washed with PBS before being mixed with an equal volume of DAPI-containing ProLong Gold (Invitrogen) for confocal fluorescence imaging. Confocal fluorescence images were recorded through a Zeiss LSM 710 spectral confocal laser scanning microscope in the Microscopy Services Laboratory at the UNC School of Medicine.

[0405] A control Dox uptake study was performed to validate the concept of HLA-

DR targeting. In the control study, Daudi or Raji cells (10 x 10⁶ cells/mL) were first treated with a saturated amount of free SFLAL (SHAL7139, 200 nM) at 37 °C for 1 h to block all HLA-DR antigen, washed, before being further incubated with SHAL-functionalized Dox NPs contained 1 mM of encapsulated Dox at 37 °C for 1 h. The treated cells were washed twice with FACS buffer before being analyzed in a BSL2 Intellicyt iQue Screener PLUS flow cytometer.

[0406] In Vitro Toxicity. The in vitro toxicities of free Dox, different Dox nanoformulations and SHAL in Ramos, Daudi, and Raji cell lines were evaluated by a 3 -(4, 5- dimethylthiazol-2-yr)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) proliferation assay. In the in vitro toxicity study, cells were first washed twice with phenol red-free RPMI1640 before being resuspended in completed media (phenol red-free RPMI1640 supplemented with 10% FBS and antibiotic-antimycotic (100 units/mL of penicillin, 100 pg/mL of streptomycin and 0.25 pg/mL of Gibco amphotericin B). Cells were seeded at 10 ^c 10³ cells per well in 96 well plates before being treated with desired concentrations of free Dox, different Dox nanoformulations, or free/conjugated SHAL at physiological conditions for 72 h. The proliferation of the lymphoma cells was quantified by MTS assay (Promega) according to the manufacturer’s protocol. Briefly, the drug-treated cells (100 pL/well) were incubated 20 pL of MTS/ PMS solution in the dark at physiological conditions for 45 min (Raji cells) to 2.5 h (Daudi and Ramos cells). The cell viabilities were quantified via a plate reader by measuring the absorbance at 495 nm.

[0407] In Vitro Radiosensitizing Study. The radiosensitizing properties of free Dox and different Dox nanoformulations in the presence and absence of free SHAL SH7129 or conjugated SHAL were quantified using an Alexa Fluor 488 Annexin V (AV) and propidium iodide (Pl)-based dead cell apoptosis kit via FACS. In the in vitro study, cells were first treated with therapeutic doses of free Dox or different Dox NPs (contained IC50 of free Dox) for 24 h before being subjected to 5 Gy X-ray irradiation through an X-RAD 320 X-ray irradiator (Precision X-ray Inc., CT) operated at 320 kVp and 12.5 mA. Cells were allowed to grow at physiological conditions for 3 days. Cells in the control groups were treated with different therapeutics at physiological conditions for 4 days. Treated cells were then washed twice with cold PBS (1 x, 4 °C) before being resuspended in annexin-binding buffer (1 x) at a cell density of about 1 ^c 10⁶ cells/mL. A488-labeled Annexin V and PI were added to the cells and incubated in the dark for 15 min before being analyzed by a BSL2 Intellicyt iQue Screener PLUS flow cytometer. Viable cells were defined as AV-PI-, apoptotic cells were defined as AV+PI-, necrotic cells were defined as AV+PI+, and dead cells were defined as AV-PI+.

[0408] DNA Cell Cycle Analysis. The DNA contents of differently treated cells were quantified using a propidium iodide-based FACS assay. Cells were first treated with therapeutic doses of free Dox or different Dox NPs (contained IC50 of free Dox) for 24 h. Cells were then washed twice with cold PBS, and fixed in 70% ethanol at -20 °C for 24 h. Fixed cells were then washed once with PBS before being resuspended in 2 mL staining solution contained 0.1% Triton X-100, 0.4 mg of DNase-free RNase and 40 pL of 1 mg/mL PI solution. After being incubated at 20 °C for 30 min, stained cells were analyzed in a BSL2 Intellicyt iQue Screener PLUS flow cytometer.

[0409] Quantification of dsDNA Breaks. The dsDNA breaks (DBS) induced by Dox treatment, XRT, and their combina-tions were quantified by anti-H2AX-based FACS assay. Briefly, cells were first treated with therapeutic doses of free Dox or different Dox NPs (contained IC50 of free Dox) for 24 h, before being subjected to 5 Gy X-ray irradiation through an X-RAD 320 X-ray irradiator (Precision X-ray Inc., CT) operated at 320 kV and 12.5 mA. After another 24 h, cells were washed twice with cold PBS, fixed with 70% ethanol for 2 h, and washed twice with FACS buffer before being stained with PE-labeled anti-H2AX (BioLegend) for 30 min according to the manufacturer’s protocol. Stained cells were washed twice with FACS buffer before being analyzed in a BSL2 Intellicyt iQue Screener PLUS flow cytometer. Cells in the control groups only received Dox treatment and were fixed and analyzed 48 h after the initial treatments.

[0410] Quantification of Time-Dependent Calreticulin Expression after Different in

Vitro Treatments. The change of calreticulin expression of Raji cells after treatment with Dox (with or without free SHAL) or SHAL-functionalized Dox NPs and X-ray irradiation was quantified using an anti-HLA DR-based FACS assay. In the study, Raji cells were first treated with a subtherapeutic dose of free Dox (with/without free SHAL SH7129 at a molar ratio of SHAL/Dox = 1 :2940) or SHAL-functionalized Dox NPs containing an IC25 of Dox for 24 h before being subjected to 5 Gy X-ray irradiation through an X-RAD 320 X-ray irradiator (Precision X-ray Inc., CT) operated at 320 kVp and 12.5 mA. Treated cells were then incubated at physiological conditions for 24, 72, and 120 h before being stained with the A488-labeled anti-calreticulin monocolonal antibody (Abeam, clone EPR3924) according to the manufacturer’s instructions. In the control groups (without X-ray irradiation), cells were treated with different therapeutics and incubated at physiological conditions for 48, 96, and 144 h before being stained for FACS analysis. Unstained cells were used as a control to demonstrate the background fluorescence from different Dox treatments would not interfere with the FACS study.

[0411] Quantification of Time-Dependent HLA-DR Expression after Different in

Vitro Treatments. The change of HLA-DR expression of Raji cells after treatment with Dox (with or without free SHAL) or SHAL-functionalized Dox NPs and X-ray irradiation was quantified using an anti-HLA DR-based FACS assay. In the study, Raji cells were first treated with a subtherapeutic dose of free Dox (with/without free SHAL SH7129 at a molar ratio of SHAL/Dox = 1 :2940) or SHAL-functionalized Dox NPs containing an IC25 of Dox for 24 h before subjected to 5 Gy X-ray irradiation through a X-RAD 320 X-ray irradiator (Precision X-ray Inc., CT) operated at 320 kVp and 12.5 mA. Treated cells were then incubated at physiological conditions for 24, 72, and 120 h before staining with the A488- labeled antihuman HLA-DR antibody (clone L243) according to the manufacturer’s instructions. In the control groups (without X-ray irradiation), cells were treated with different therapeutics and incubated at physiological conditions for 48, 96, and 144 h before being stained for FACS analysis. Unstained cells were used as a control to demonstrate the background fluorescence from different Dox treatments would not interfere with the FACS study.

[0412] In Vivo Studies. Animals were maintained in the Division of Comparative

Medicine (an AAALAC-accredited experimental animal facility) under sterile environments at the University of North Carolina. All procedures involving experimental animals were performed in accordance with the protocols that the University of North Carolina Institutional Animal Care and Use Committee approved, and they conformed to the Guide for the Care and Use of Laboratory Animals (NIH publication No. 86-23, revised 1985). Athymic nude mice (Nu, also known as Nu/J) were obtained from UNC Animal Services Core (Chapel Hill, NC). The house breed Nu mice were originally obtained from the Jackson Lab. CD1 IGS mice were purchased from Charles River Laboratory (Durham, NC).

[0413] In Vivo Toxicity Study. In vivo toxicity of different small- molecule/conjugated SHAL and small-molecule/encapsulated Dox were evaluated in healthy tumor-free CD1 IGS mice (female, about 12 weeks old, 20-21 g). Mice in the control and treatment groups received the following treatments: (1) PBS (nontreatment control group);

(2) small-molecules SHAL (SH7139, 300 ng/mouse); (3) drug-free SHAL-functionalized NPs (7.5 mg NPs per mouse, 300 ng conjugated SHAL per mouse); (4) small-molecule Dox (10 mg/kg, MTD of free Dox); (5) nontargeted Dox NPs (10 mg encapsulated Dox/ kg); (6) SHAL-functionalized Dox NPs (10 mg encapsulated Dox/kg); (7) small-molecule SHAL (300 ng/mouse) plus small-molecule Dox (10 mg/kg); (8) SHAL NPs (contained 300 ng conjugated SHAL/mouse) plus nontargeted Dox NPs (10 mg encapsulated Dox/kg); and (9) drug-free mPEG- PLGA NPs (15 mg per mouse). 48 h after the i.v. Injection, mice were anesthetized via s.c. Injection of 100 pL of ketamine hydrochloride/xylazine hydrochloride solution (Sigma; St Louis, MO). Circulating blood was collected from the heart. 500 pL of each whole-blood sample was stored in an EDTA-coated tube and stores at 4 °C before blood toxicity study in the Animal Clinical Laboratory Core Facility at the UNC School of Medicine. Key organs (heart, lung, liver, spleen, and kidney) were preserved by 4% (v/v) neutral buffered formalin at 4 °C for 2 days and 40% ethanol at 4 °C for another 2 days before submitting to Animal Histopathology Core Facility at UNC School of Medicine for hematoxylin and eosin (H&E) stain. Representative H&E-stained tissue sections were imaged via an Olympus BX61 optical microscope in Microscopy Services Laboratory at UNC School of Medicine.

[0414] In Vivo Biodistribution Study. The biodistributions of Dox administered as a free drug or encapsulated in the nontargeted or SHAL-functionalized PEG-PLGA NPs were evaluated in Ramos, Daudi and Raji xenograft tumor-bearing Nu mice. Athymic Nu mice were chosen for this biodistribution study due to the absence of mature B cells in the body that would interfere with the in vivo study. Xenograft tumors were inoculated in the flank of male Nu mice via the subcutaneous injection of 2 x 10⁶ Ramos, Daudi or Raji cells in 200 pL of a 1:1 (v/v) mixture of a serum-free RPMI1640/Matrigel solution in the left flank. Each type xenograft tumor group contained 25-30 mice. Ten days after inoculation, mice in each group were randomized and divided into 7 subgroups. Mice in the 7 subgroups received the following treatments: (1) PBS (nontreatment control group); (2, 3) free Dox; (4,5) nontargeted Dox NPs; and (6,7) SHAL-functionalized Dox NPs. All Dox formulations were administered via a single tail-vein i.v. Injection of 3.5 mg/kg of free or encapsulated Dox. Mice in groups (1), (2), (4), and (6) were euthanized via s.c. injection of 100 pL of ketamine hydrochloride/xylazine hydrochloride solution 24 h after administration of therapeu-tics. Xenograft tumor, circulating blood and key organs (liver, kidney, lung, heart and spleen) were preserved. Mice in groups (3), (5) and (7) were euthanized 72 h after administration of therapeutics. Again, xenograft tumor, circulating blood and key organs (liver, kidney, lung, heart, and spleen) were preserved. Ex vivo images were recorded using an IVIS Kinetic imaging system equipped with a light source excited at 575-560 nm and a DsRed emission filter (kem = 575-650 nm). Preserved tumors were fixed in 4% (v/v) neutral buffered formalin at 4 °C for 2 days and 40% ethanol at 4 °C for another 2 days before being submitted to Animal Histopathology Core Facility at UNC School of Medicine for sectioning. Tumor sections were imaged via a Zeiss LSM710 Spectral Confocal Laser Scanning microscope in Microscopy Services Laboratory at UNC School of Medicine.

[0415] In Vivo Anticancer Efficacy Studies. The in vivo anticancer activities of free

Dox, free SHAL SH7129, conjugated SHAL, nontargeted Dox NPs and SHAL- functionalized Dox NPs for chemo-immunotherapy and concurrent CIRT with a short treatment cycle (rest period between treatment = 3-4 days) were comprehensively evaluated in Duadi and Raji xenograft tumor models in Nu mice. Xenograft tumors were established via subcutaneous injection of 2 x 10⁶ Duadi or Raji cells in 200 pL of a 1 : 1 (v/v) mixture of a serum-free RPMI1640/Matrigel solution in the left flank. Each type of tumor model contained 120 female Nu mice (6-7 weeks old, 20-21 g). Four days (for Raji xenograft tumor model) or 7 days (for Daudi xenograft tumor model) postinoculation, mice were randomized and divided into 16 groups (6-7 mice per group) for different treatments. The control and treatment groups are (1) PBS (nontreatment group); (2) free SHAL SH7129; (3) drug-free SHAL-functionalized NPs; (4) free Dox; (5) nontargeted Dox NPs; (6) SFLAL- functionalized Dox NPs; (7) free SHAL plus free Dox; (8) drug-free SHAL NPs plus nontargeted Dox NPs; (9) PBS (nontreatment group) followed by XRT; (10) free SHAL SH7129 followed by XRT; (11) drug-free SHAL-functionalized NPs followed by XRT; (12) free Dox followed by XRT; (13) nontargeted Dox NPs followed by XRT; (14) SHAL- functionalized Dox NPs followed by XRT; (15) free SHAL plus free Dox followed by XRT; and (16) drug-free SHAL NPs plus nontargeted Dox NPs followed by XRT. Mice in the treatment groups received 3 tail vein i.v. Injections of 3.5 mg/kg free/encapsulated Dox and 5 pg/kg of free SH7129 or conjugated SHAL at day 7, 11, and 14 (for Daudi tumor-bearing mice) or day 4, 8, and 11 (for Raji tumor-bearing mice) postinoculation. Mice in the concurrent CIRT groups received 5 Gy X-ray irradiation 24 h after administration of different therapeutics through a Precision X-RAD 320 (Precision X-ray, Inc.) machine operating at 320kVp and 12.5 mA. The source-subject distance of 70 cm and 50 cGy/ min. Only the tumor regions (left flank) of the mice were irradiated, as the remaining parts of the body were lead-shielded. Tumor volume was measured every 3-4 days via a caliper. The bodyweight was measured every week. Tumor volumes were calculated by using the formula of volume (V) = 0.5 x a x b2, where, a and b are the larger and smaller diameters, respectively. Tumor growth for different treatment modalities was monitored until the volume increased to above 1,000 mm3 or loss more than 20% of the initial bodyweight at which point the animals were euthanized by an overdose of carbon dioxide.

[0416] The impact of upregulation of HLA-DR expression (induced by immunogenic cell death) on the in vivo anticancer efficacy of free Dox, free SHAL SH7129, conjugated SHAL, non-targeted Dox NPs and SHAL-functionalized Dox NPs for chemo-immunotherapy was evaluated in a Daudi xenograft tumor model in Nu mice. A longer treatment cycle (6 days rest day between treatments) was used to allow the HLA-DR expression of variable cancer cells return to background level before subsequent treatment. In the in vivo study, the Daudi xenograft tumors were established via subcutaneous injection of 2x 10⁶ Daudi cells in 200pL of a 1 : l(v/v) mixture of a serum-free RPMI1640/Matrigel solution in the left flank in 48 female Nu mice (6-7 weeks old, 20-21 g). Seven days postinoculation, mice were randomized and divided into eight groups (six mice per group) for different treatments. The control and treatment groups are (1) PBS (nontreatment group); (2) free SHAL SH7129; (3) drug-free SHAL-functionalized NPs; (4) free Dox; (5) nontargeted Dox NPs; (6) SHAL- functionalized Dox NPs; (7) free SHAL plus free Dox; and (8) drug-free SHAL NPs plus nontargeted Dox NPs. Mice in the treatment groups received 3 tail vein i.v. Injections of 3.5 mg/kg free/encapsulated Dox and 5 pg/kg of free SH7129 or conjugated SHAL at day 7, 14, and 21 postinoculation. Each tumor volume was measured every 3- 4 days via a caliper.

The bodyweight was measured every week. Tumor volumes were calculated by using the formula of volume(V) = 0.5xa><b2, where a and b are the larger and smaller diameters, respectively. Tumor growth for different treatment modalities was monitored until the volume increased to above 1000 mm3 or the animal lost more than 20% of its initial bodyweight at which point the animals were euthanized by an overdose of carbon dioxide.

[0417] A treatment sequence-dependent in vivo study was performed in a Raji xenograft tumor model in Nu mice to investigate how the radiotherapy schedule affects the treatment efficacy of the SHAL-functionalized Dox NPs. In the in vivo study, Raji xenograft tumors were established via subcutaneous injection of 2 ^c 10⁶ Raji cells in 200 pL of a 1 : 1 (v/v) mixture of a serum -free RPMI1640/Matrigel solution in the left flank in 26 male Nu mice (6-7 weeks old, 25-26 g). Five days postinoculation, mice were randomized and divided into 3 groups (8-9 mice per group) for different treatments. Mice in the treatment groups received 3 tail vein i.v. Injection SHAL-functionalized Dox NPs (contained 3.5 mg/kg encapsulated Dox and 5 pg/kg of conjugated SHAL) at day 5, 9, and 12 postinoculation. In the concurrent CIRT group, mice received 5 Gy X-ray irradiations 24 h after each i.v. administration of the therapeutics. Mice in the sequential CIRT group received three 5 Gy X- ray irradiations at day 17, 20, and 23 postinoculation. In vivo radiotherapy was performed using a Precision X-RAD 320 (Precision X-ray, Inc.) machine operating at 320 kV and 12.5 mA. The source-subject distance was 70 cm, and the dose was administered at a rate of 50 cGy/min. Only the tumor regions (left flank) of the mice were irradiated, as the remaining parts of the body were lead-shielded. Tumor volume was measured every 3-4 days via a caliper. The bodyweight was measured every week. Tumor volumes were calculated by using the formula of volume (V) = 0.5 ^c a ^c b2, where, a and b are the larger and smaller diameters, respectively. Tumor growth for different treatment modalities was monitored until the volume increased to above 1000 mm³ or there was a loss of more than 20% of the initial bodyweight at which point the animals were euthanized by an overdose of carbon dioxide.

[0418] Histopathological Study. Xenograft tumors were estab-lished via subcutaneous injection of 2 x 10⁶ Raji cells in 200 pL of a 1 : 1 (v/v) mixture of a serum-free RPMI1640/Matrigel solution in the left flank. Four days postinoculation, mice were randomized and divided into 16 groups for different treatments. The control and treatment groups are (1) PBS (nontreatment group); (2) free SHAL SH7129; (3) drug-free SHAL- functionalized NPs; (4) free Dox; (5) nontargeted Dox NPs; (6) SHAL-functionalized Dox NPs; (7) free SHAL plus free Dox; (8) drug-free SHAL NPs plus nontargeted Dox NPs; (9) PBS followed by XRT; (10) free SHAL SH7129 followed by XRT; (11) drug-free SHAL- functionalized NPs followed by XRT; (12) free Dox followed by XRT; (13) nontargeted Dox NPs followed by XRT; (14) SHAL-functionalized Dox NPs followed by XRT; (15) free SHAL plus free Dox followed by XRT; and (16) drug-free SHAL NPs plus nontargeted Dox NPs followed by XRT. Mice in the treatment groups received a single tail vein i.v. injections of 3.5 mg/kg free/encapsulated Dox and 5 pg/kg of free SH7129 or conjugated SHAL at day 4 postinoculation. Mice in the concurrent CIRT groups received 5 Gy X-ray irradiation 24 h after administration of different therapeutics through a Precision X-RAD 320 (Precision X- ray, Inc.) machine operating at 320kVp and 12.5 mA. The source-subject distance of 70 cm and 50 cGy/min. Mice were euthanized 24 h to 5 days after the treatment. The tumors were collected and fixed in 4% neutral -buffered formalin for 24 h at 4 °C and then stored in 70% ethanol at 4 °C for 24 h before being submitted to the Animal Histopathology Core Facility at UNC Medical School for sectioning. Caspase 3, and HLA-DR immunohis-tochemistry stains were performed at the Translational Pathology Lab at the UNC Medical School. For quality control purposes, all staining was performed using a biological tissue automatic staining machine. All stained tumor sections were imaged on a Zeiss 710 Spectral CLSM confocal microscope in the Microscopy Services Laboratory Core Facility at the UNC School of Medicine. [0419] Statistical Analysis. Quantitative data were expressed as mean ± SEM. The analysis of variance was completed using a one-way ANOVA in GraphPad Prism 6 software pack. The analysis of survival data was completed using a Log-rank (Mantel-Cox) test in GraphPad Prism 6 software pack. *p < 0.05 was considered statistically significant.

Results

[0420] Fabrication and Characterization of SHAL-Function-alized Dox NPs. SFLAL- functionalized Dox-encapsulated poly(ethylene glycerol)-block-poly(lactide-co-glycolide) (PEG-PLGA) NPs were prepared via nanoprecipitation in basic conditions (pH 9.0). Amine- functionalized SHAL (SH7133) was conjugated to poly-(lactide)-block-poly(ethylene glycerol) N-hydroxysuccinimide ester (PLA(16K)-PEG(10K)-NHS) before preparing the NPs through the amine-NHS ester coupling reaction. The number-average diameter and the intensity-average diameter of the targeted Dox NPs were 50 and 82 nm, as determined by transmission electron microscopy (TEM) and dynamic light scattering (DLS) techniques, respectively. By quantifying the number of NPs formed from each milligram of the polymer mixture using the nanoparticle tracking analysis (NT A) method, Applicant calculated that each NP contained approximately 70 conjugated SHAL molecules, which is an optimal number of targeting ligands for similar diameter NPs. (Saha et al., 2014, Anal. Chem. 2014, 86 (16), 8158) Applicant’s target for Dox loading of the SHAL-functionalized NPs was 5% by weight, while the actual Dox loading was about 2.8% by weight (i.e., the encapsulation efficiency was about 57%). Nontargeted Dox NPs were prepared through the same method in the absence of PLA(16K)-PEG(10K)-SHAL. Drug-free rhodamine (Rhod)-labeled SHAL- functionalized PEG-PLGA NPs were prepared via the same nanoprecipitation method in the presence of 2.5% by weight of Rhod-labeled PLGA(20K) instead of Dox for in vitro binding and imaging studies. Both Dox-encapsulated NPs underwent pH-dependent controlled release in physiological conditions. Approximately 55%, 30%, and 15% of the encapsulated Dox was released at pH 5.5, 6.5, and 7.0 in the first 72 h, respectively. The faster Dox release in the acidic conditions was due to the protonation of the encapsulated Dox (pKl of Dox = 8.2); (Yoo et al., 2004, Controlled Release 2004, 96 (2), 273; Fulop et al., 2013, Int. J. Pharm. 2013, 454 (1), 559; and MacKay et al., 2009, Nat. Mater. 2009, 8 (12), 993) this triggered the conversion of Dox from a hydrophobic form to a hydrophilic salt (Dox HCl). The pH-dependent drug release ensures the majority of encapsulated Dox is released in the acid endosomes of the targeted cells after systemic administration, thus reducing the systemic side effects of Dox.

[0421] SHAL-Functionalized NPs Bind Selectively to HLA-DR-Overexpressed

Lymphoma Cells. The binding affinities of unconjugated “free” SFLAL (the biotin- functionalized tridentate SFLAL (SH7129)) and SHAL-functionalized rhodamine-labeled SFLAL NPs were quantified via a fluorescence-activated cell sorting (FACS) binding assay in four well-established human lymphoma cell lines with varying degrees of HLA-DR expression. Both free SH7129 and SFLAL functionalized NP bound selectively to the HLA- DR-overexpressed Ramos, Daudi, and Raji cells but not to the HLA-DR nonexpressing Jurkat cells. The binding affinities of SHAL-functionalized NPs were significantly higher than the free SH7129 in all three HLA-DR overexpressing cell lines due to the higher avidity of the SHAL-functionalized NPs. The macroscopic dissociation constant (I<d_, Macro) of SHAL-functionalized NPs was calculated as 30 nM in the high HLA-DRIO expression Raji cell line, which is more than 3 -fold lower than that of free SH7129 (K^ Macro ~ 100 nM). The binding of free SHAL and SHAL-functionalized NPs was further confirmed by confocal laser scanning microscopy (CLSM) with a ring pattern of staining that can be observed in the CLSM images of HLA-DR overexpressed Ramos, Daudi, and Raji cells after staining with 200 nM of free SH7129 tagged with PE-Cy 5 -conjugated streptavidin (SH7129-SA) or SHAL functionalized NPs. A further time-dependent CLSM study of the SHAL-functionalized NPs using NPs pretreated Raji cells confirmed the internalization of the SHAL-functionalized NPs in physiological conditions (37 °C), as a patchy staining pattern slowly replaced the sharp ring pattern, which was eventually sequestered in the cytoplasm. Conversely, no significant internalization of the SH7129-SA complex was observed in the treated Raji cells.

Applicant’s results were concordant with previous studies which showed similar cross linking-induced endocytosis phenomena in HLA-DR-overexpressed epidermal cells after incubation with the cross-linked HLA-DR antibody. (Girolomoni et ak, 1990, J. Invest. Dermatol. 1990, 94 (6), 753) They also show the presence of the PE-Cy5-streptavidin conjugated to SH7129 prevents the internalization of free SHAL that has been observed to occur (DeNardo et ak, 2008, Cancer Biotherapy and Radiopharmaceuticals 23: 783-795; Balhom et ak, 2009, Mol. Cancer 2009, 8, 25) as the surface HLA-DR molecules move back into the cells for recycling. (Roche et al., 1993, Proc. Natl. Acad. Sci. U. S. A. 1993, 90 (18), 8581; Pinet et al., 1995, Nature 1995, 375 (6532), 603)

[0422] SHAL Functionalization Enhances the in Vitro Uptake of DOX and Improves

Cytotoxic Effects in the HLA-DR-Overexpressed Lymphoma Cells. The in vitro uptake of free and encapsulated Dox in all three HLA-DR antigen overexpressed lymphoma cell lines was evaluated using the FACS method. Cells from all three lines took up more Dox encapsulated in the SHAL-functionalized NPs than free Dox, and the uptake of the encapsulated Dox was directly proportional to the HLA-DR expression. The uptake of the Dox that was encapsulated in the nontargeted Dox NPs was much lower than that of the free Dox in all three lymphoma cell lines. Co-treatment with free SHAL or drug-free SHAL- functionalized NPs (SHAL/Dox = 1 :2940, which is the molar ratio in SHAL-functionalized Dox NPs) did not significantly affect the uptake of free Dox or nontargeted Dox NPs. Control study indicated free SHAL (SH7139, 200 nM) pretreatment significantly reduced the uptake of SHAL-functionalized Dox NPs in the Daudi and Raji cell lines because free SHAL blocked the binding of the targeted Dox NPs. This validated the concept of HLA-DR targeting. A further CLSM study in the Raji cells confirmed the uptake of both free and encapsulated Dox. This study confirmed the release of encapsulated Dox from SHAL- functionalized Dox NPs; the entry of free Dox into the nucleus was further verified by the CLSM study (including colocalization of Dox fluorescence with 4',6-diamidino-2- phenylindole (DAPI) nuclear stain).

[0423] The in vitro toxicities of different forms of SHAL, Dox, and their combinations in all three HLA-DRIO overexpressed lymphoma cell lines using an MTS cell proliferation assay were determined to start 4 days after the initial treatment. Free Dox had the highest cytotoxicity in all three cell lines, with a half-maximal inhibitory concentration (IC50) of about 0.15 mM. This was expected since the tumor cells were fully exposed to free DOX in the media in the in vitro setting. In comparison, free SHAL and drug-free SHAL- functionalized NPs demonstrated minimal cytotoxic effects (IC50 > 10 pM). The combination of free Dox and free SHAL (Dox/SHAL = 2940:1) did not significantly affect the cytotoxicity of free Dox in any of the three lymphoma cell lines (p = 0.1544 (Ramos), 0.0845 (Daudi), and 0.056 (Raji) vs the nontreatment control group). Although nontargeted Dox NPs (and their combinations with SHAL-functionalized NPs) had significantly lower in vitro cytotoxicity than did free Dox (IC50 = 0.6-3.0 mM, p = 0.0242-0.0385 vs free Dox), the IC50 of SHAL-functionalized Dox NPs was comparable with that of free Dox combined with free SHAL in the Raji cells (IC50 = 0.15 ± 0.02 mM, p = 0.0341) and was only slightly higher than that of free Dox combined with free SHAL in the Daudi cell line. However, the cytotoxicity of SHAL-function-alized Dox NPs was significantly weaker than that of free Dox combined with free SHAL in the Ramos cells, which have a lower HLA-DR expression (p = 0.0416). The in vitro toxicity of the SHAL-functionalized Dox NPs was consistent with the cellular uptake of the targeted NPs and thus with the HLA-DR expression. The internalization of the targeted Dox NPs through endocytosis enhanced the cytotoxicity of the encapsulated Dox NPs, even though some of the encapsulated Dox was released inside the endosomes.

[0424] SHAL-Functionalized Dox NPs Sensitizes HLA-DR-Overexpressed

Lymphoma Cells to Radiation in Vitro. The in vitro radiosensitizing properties of free and encapsulated Dox in all three HLA-DR-overexpressed lymphoma cell lines were evaluated using an annexin V (AV)-propidium iodide (PI) dead cell apoptosis assay. In all three lymphoma cell lines, less than half of the cells remained viable (AV-PI-) after treatment with therapeutic doses of Dox (i.e., IC50 of free Dox at 0.15 mM) for 96 h. The population of necrotic and dead cells (AV+PI+/AV-PI+) varied from about 70% (Raji cells) to about 35% (Daudi cells). In general, SHAL-functionalized Dox showed higher toxicities than did nontargeted Dox NPs in all three types of lymphoma cell lines. The 5 Gy X-ray irradiation effectively led to 45-55% fewer AV-PI- cells than untreated cells. Dox pretreatment (24 h before irradiation) significantly decreased the number of viable cells after 5 Gy irradiation, with less than 5% of the Raji and Daudi cells remaining viable after treatment with free Dox or SHAL-functionalized Dox NPs, followed by radiation. This finding indicates that Dox is an effective radiosensitizer as previously reported. (Bonner et ah, 1990, Int. J. Radiat. Biol. 1990, 57 (1), 55) Due to the poor cellular uptake of the nontargeted Dox NPs, pretreatment with nontargeted Dox NPs led to less significant radiosensitizing effects than treatment with free Dox or SHAL-functionalized Dox NPs. Co-administration of drug-free SHAL NPs with nontargeted Dox NPs (conjugated SHAL: encapsulated Dox = 1 :2940) did not significantly affect the relative number of viable or apoptotic cells after irradiation, which indicates that SHAL alone did not sensitize radiation. [0425] Cell cycle analyses were performed to investigate the radiosensitizing mechanism of free Dox and different Dox nanoformulations. At 24 h after a therapeutic dose of free Dox or SHAL-functionalized Dox NPs (containing the IC50 of Dox), the percentage of cells in the G2/M phase significantly increased in all three lymphoma cell lines, indicating that the treatment induces cell cycle arrest in the radiosensitive G2/M phase. Treatment with free Dox also reduced the population of cells in the more radioresistant S phase. In contrast, treatment with the SHAL-functionalized Dox NPs did not significantly affect the percentage of cells in the S phase. This trend can be explained by slow drug release kinetics and an incomplete release of the encapsulated Dox, which could potentially lower the drug concentration. For the same reasons, in all three lymphoma cell lines, the nontargeted Dox NPs only slightly increased the population of the G2/M phase and did not significantly affect the percentage in the S phase. Control studies revealed that cotreatment with free SHAL (SH7129) or drug-free SHAL NPs with either free Dox or nontargeted Dox NPs did not affect the cell cycle when compared to treatments without free or conjugated SHAL.

[0426] DNA double-strand breaks induced by in vitro treatment with Dox and radiation for the three HLA-DRlO-overex-pressed lymphoma cell lines were quantified using a FACS-based g-H2AC assay. The g-H2AC expressions of all three lymphoma cell lines slightly increased after treatment with therapeutic doses of Dox (i.e., IC50 of free Dox) because cytochrome P450 can metabolize Dox to generate hydroxide radicals, which diffuse into the nucleus and break double-stranded DNA. Also, Dox directly enters the nucleus and binds to double-stranded DNA to form a stable Dox-topoisomerase II complex that prevents proteins from repairing DNA damage. In all three lymphoma cell lines, the increase in the g- H2AX expressions induced by the SHAL-functionalized Dox NPs was comparable to that caused by free Dox, whereas treatment with nontargeted Dox NPs did not significantly affect g-H2AC expression. As in previous studies, 5 Gy X-ray irradiation effectively induced double-stranded breaks and increased the g-H2AC expression in all three lymphoma cell lines. Pretreatment with therapeutic doses of free Dox or SHAL-functionalized Dox NPs (continued therapeutic doses of Dox) significantly increased the g-H2AC expression relative to the treatment group that only received 5 Gy of X-ray irradiation because the Dox- topoisomerase II complex prevented proteins from repairing the DNA damage caused by the X-ray irradiation. [0427] SHAL-Functionalized Dox NPs and X-ray Irradiation Induce Immunogenic

Cell Death and Upregulate HLA-DR Antigen Expression in HLA-DR-Overexpressed Lymphoma Cells. Cytotoxic chemotherapy through XRT and an ICD-inducing agent such as Dox induces ICD, during which dying cancer cells may upregulate antigen expression in the cancer cells that survive. A time-dependent in vitro study was performed to investigate the calreticulin expression of Raji cells after treatment with a subtherapeutic dose of Dox and 5 Gy X-ray irradiation, since the upregulation of calreticulin characterizes ICD. (Fucikova et al., 2011, Cancer Res. 2011, 71 (14), 4821; Kawano et al., 2016, Oncol. Lett. 2016, 11 (3), 2169) The calreticulin expression remained relatively constant 24 h after initial treatment in all treatment groups. The calreticulin expression of all treatment groups received 5 Gy XRT significantly increased 72 h after the irradiation, but it returned to the background level 5 days after the treatment. The calreticulin expression of the Raji cells treated with SFLAL- functionalized Dox NPs followed by 5 Gy XRT was nearly double 72 h after the irradiation but returned to background 2 days later. This confirmed the time-dependent nature of the ICD. Further time-dependent in vitro study was performed to investigate HLA-DR antigen expression in Raji cells after treatment with a subtherapeutic dose of Dox and 5 Gy X-ray irradiation. Untreated Raji cells showed very stable HLA-DR expression (M.F.I. ~ 4.4 x 105). In vitro treatment with a subtherapeutic dose of free Dox (i.e., IC25 of Raji = 80 nM) upregulated the HLA-DR expression 24 h after treatment. The HLA-DR expression reached its maximum (M.F.I. ~ 6.8 x 105, about 55% higher than in the nontreatment group) 3 days after the initial treatment but dropped back to normal 5 days after treatment. The combination of Dox and free SHAL (Dox/SHAL = 2940:1) did not significantly enhance HLA-DR expression. Conversely, in vitro treatment with SHAL-functionalized Dox NPs (continued IC25 of free Dox) showed much quicker and higher upregulation of HLA-DR expression. The HLA-DR expression of the survival fraction was higher than that of the nontreatment group cells 5 days after the initial treatment. Five Gy X-ray irradiation effectively upregulated HLA-DR expression (M.F.I. ~ 5.7 x 105, 24 h after initial treatment, also about 55% higher than in the nontreatment group). As with free Dox, the HLA-DR expression of the surviving cells dropped back to the average level 5 days after the initial treatment. The Dox pretreatment (with either free Dox or SHAL-functionalized Dox NPs) followed by the 5 Gy X-ray irradiation rapidly upregulated HLA-DR expression (45-66% higher than in the nontreatment group) 24 h after irradiation. The HLA-DR expression of the survival fractions of both treatment groups was 95-120% higher than those of the nontreatment group 3 days after irradiation but eventually dropped back to the average level 5 days after irradiation. This time-dependent study confirmed that Dox, X-ray irradiation, and their combination are all sufficient to upregulate HLA-DR expression in HLA-DR- overexpressed lymphoma cells but that the HLA-DR expression of the surviving cells eventually returns to average levels 5 days after treatment. Thus, with an appropriate schedule, the upregula-tion of HLA-DR antigen expression can be utilized to improve the uptake of SHAL-functionalized Dox NPs, both in vitro and in vivo.

[0428] SHAL-Functionalized Dox NPs Improve the in Vivo Uptake and Reduce

Systemic Side Effects of Dox. An ex vivo biodistribution study was performed to quantify the uptake of free Dox as well as Dox nanoformulations in Ramos, Daudi, and Raji tumor xenografts in athymic nude (Nu) mice. The Dox uptake was quantified via a well-established ex vivo fluorescence imaging technique at several time points after intravenous (i.v.) tail vein administration of different therapeutics. Dox fluorescence can be observed in all three different types of tumors when harvested 24 and 72 h postadministration of the therapeutics. In the Raji tumor model, the tumor uptake of Dox delivered through SHAL-functionalized NPs was about 3.5 times higher than that of free Dox (p = 0.0143) at 24 h postadministration , but the uptake of Dox delivered through nontargeted NPs was similar to that of free Dox (p = 0.0539). The amount of Dox retained in the Raji tumor dropped significantly by 72 h postadministration, likely due to the cancer cells clearing the drug through circulation and metabolism. However, the amount of Dox retained in the tumor and delivered through the SHAL-functionalized NPs was still about 100% higher than that found in the group with free Dox. The Daudi tumor model had a very similar tumor uptake trend, but the Daudi tumor took up less of the Dox that was delivered through the SHAL-functionalized NPs than did the Raji tumor, presumably due to the lower HLA-DR expression of Daudi cells. However, in the Ramos tumor model, the tumor uptake of Dox was very similar, whether it was administered as a free drug or as a nanoformulation (p = 0.4341). The low HLA-DR expression could explain this effect in the Ramos cells. Nevertheless, Applicant’s CLSM study on the harvested tumor sections confirmed the selective binding and uptake of the SHAL-functionalized Dox NPs. A ring-stained pattern can be seen in the tumors that were preserved 24 h postadministration of the targeted Dox NPs. In contrast, a diffused pattern of Dox can be observed in tumor sections preserved 72 h postadministration of the targeted Dox NPs, which confirmed the release of the Dox from the NPs.

[0429] A comprehensive in vivo toxicity study was performed to investigate the side effects of different forms of Dox at the maximum tolerated dose (M.T.D.) of Dox (10 mg/kg per week), both with free or conjugated SHAL (15 pg/kg) and without SHAL in healthy CD1 IGS mice. The systemic administration of free Dox-induced significant hematological toxicities. In this study, the lymphocyte and reticulocyte counts were below the reference ranges for healthy mice, likely due to the prolonged systemic exposure to active Dox. The coadministration of free or conjugated SHAL did not incur additional hematological toxicity. In contrast to free Dox, both Dox nanoformulations induced very low toxicities in healthy mice. The postadministration lymphocyte and reticulocyte counts for both nanoformulations were also within the reference ranges for healthy mice. The reduction in side effects can be explained by the kinetics of prolonged Dox release at physiological pH (pH 7.0). The further histopathological study indicated that the administration of free Dox (or of free Dox plus free SHAL) induced significant myocardial toxicity. Substantial lesions and muscular fiber dissociation can be observed in both groups and likely result from prolonged exposure to free Dox after systemic administration. In contrast, the admin-istration of neither of the Dox nanoformulations was associated with observable cardiotoxicity and hepatological toxicity because the Dox-encapsulated NPs could not effectively pass through the sinusoidal endothelium. (Hilmer et ah, 2004, Drug Metab. Dispos. 32(8), 794)

[0430] Immunogenic Cell Death Enhances the in Vivo Anticancer Efficacy of SHAL-

Functionalized Dox NPs for Concurrent Chemo-Immunotherapy and Concur-rent CIRT. Comprehensive in vivo studies were performed in Daudi and Raji xenograft tumor models to investigate the anticancer efficiencies of SHAL-functionalized Dox NPs for chemo- immunotherapy and concurrent CIRT. The in vivo studies involved three short treatment cycles, with a rest period of 3 or 4 days between treatments to ensure that the second treatment started while the previous treatment was still upregulating the HLA-DR expression based on Applicant’s previous in vitro data. In the Daudi tumor model, treatment with a therapeutic dose of free Dox slowed down the tumor growth but only slightly increased the median survival time (M.S.T.) by 3 days versus the nontreatment group. No significant tumor growth inhibition was observed after treatment with the nontargeted Dox NPs, presumably due to the poor uptake of nontargeted NPs. Similar to treatment with an anti- HLA-DR antibody, a single treatment or cotreatment with free SHAL (SH7129, p = 0.0352 vs the nontreatment group) or drug-free SHAL-functionalized NPs (p = 0.0413 vs the nontreatment group) did not significantly affect tumor growth in the immunocompromised mouse xenograft model. However, treatment with SHAL-functionalized Dox NPs (including a therapeutic dose of Dox) significantly slowed down tumor growth compared with the nontreatment control group, resulting in an absolute growth delay (A.G.D.) of about 7 days Although no complete response (CR) or long-term survival after treatment was observed with the SHAL-functionalized Dox NPs, it was calculated that the targeted Dox NPs enhanced the therapeutic efficiency of Dox by about 120%. In vivo fractionated XRT (3 x 5 Gy) significantly delayed the progression of cancer for 35 days.

[0431] Concurrent administration of XRT with free SHAL or drug-free, SHAL- functionalized NPs did not improve the pro-gression of the disease. Concurrent CRT with a therapeutic dose of free Dox prolonged the survival time (M.S.T. = 76 days versus 58 days for the XRT group), but the treatment only increased the regression time by an average of 6 days compared to the XRT treatment only group. Similar to chemotherapy, concurrent CIRT treatment with nontargeted Dox NPs only slightly slowed down the progression of the disease (M.S.T. = 68 days), and no treated mice achieved long-term survival. Concurrent CIRT with SHAL-functionalized Dox NPs effectively controlled the progression of tumor growth, with about 70% of the treated mice achieving a complete response and long-term survival (M.S.T. not reached at >80 days, CR = 70%); this method thus outperformed both concurrent CRT treatments with free Dox (M.S.T. = 76 days, CR = 13%, p = 0.0314) and free Dox plus free SHAL (M.S.T. = 72 days, CR = 14%, p = 0.0279). By comparing the average growth delays (A.G.D.) and normalized growth delays (N.G.D.) of the XRT treatment group and the chemotherapy group treated with SHAL-functionalized Dox NPs, it was calculated that the SHAL-functionalized Dox NPs enhanced the efficiency of XRT by more than 100%. In addition to the superior treatment responses, concurrent CIRT with SHAL-functionalized Dox NPs did not induce any significant adverse effects (e.g., rapid weight loss), whereas significant weight loss after treatment was seen with free Dox plus free SHAL (either with or without further XRT), and about 30% of the treated mice were dead within 10 days of the final treatment.

[0432] The anticancer activities of SHAL-functionalized Dox NPs were further evaluated in the high HLA-DR antigen expressed and highly aggressive Raji xenograft tumor model. As in the Daudi tumor model, treatment with a therapeutic dose of free Dox or nontargeted Dox NPs only slightly delayed the progression of the tumor growth and increased the M.S.T. by only 10 and 7 days (G.D. of the free Dox treatment group = 8 days; A.G.D. of nontargeted Dox NPs treatment group = 5 days), respectively. Overall, the Raji xenograft model was more resistant to chemotherapy with DOX, which only induced transient response followed by rapid tumor progression and death. This is consistent with the highly chemoresistant nature of Raji cells, which has overexpression of MDR1/P- gly coprotein and mutated p53. Therapy combining free Dox and free SHAL or combining nontargeted Dox NPs and drug-free SHAL NPs did not significantly affect the anticancer efficacy, but the combination of free Dox and free SHAL slightly reduced the M.S.T. due to the side effects associated with free drugs (about 25% of the mice were dead 10 days after the final treatment). In contrast, SHAL-function-alized Dox NPs effectively inhibited the progression of tumor growth, with a median progression time of 20 days. By comparing the A.G.D. with that of free Dox, it was calculated that the targeted NPs enhanced the anticancer efficiency of Dox by about 110%. This is consistent with the higher average caspase 3 and HLS-DR expressions observed in the xenograft tumors treated with the SHAL-functionalized Dox NPs compared with that treated with free Dox plus free SHAL. Fractionated XRT (3 ^c 5 Gy) slowed the progression of the disease by an average of 20 days, but the concurrent administration of XRT with free SHAL or drug-free, SHAL-functionalized NPs did not further improve this efficacy. Concurrent CRT with free Dox only slightly slowed down the progression of the tumor (A.G.D. of the free Dox concurrent CRT group and the XRT group were 17 ± 2 days and 13 ± 3 days, respectively), indicating the Raji tumor was resistant not only to chemotherapy but also to radiation. In this model, the radiosensitizing effect of free Dox was limited, presumably due to the poor tumor uptake and rapid clearance of free Dox. For a similar reason, the nontargeted Dox NPs showed a very weak radiosensitizing effect (i.e., the mice survived an average of 3 days longer than in the XRT group). Co administration of free SHAL or drug-free SHAL-functionalized NPs with either free Dox or nontargeted Dox NPs showed no further beneficial effects in concurrent CRT. Conversely, concurrent CIRT with the SHAL-functionalized Dox NPs significantly increased the length of the remission period even in this chemo-/ radioresistant tumor model. At the study end point, 100% of the treated mice were alive with the follow-up of >80 days with 71% of them achieving a complete regression of the tumor. Histopathological study indicated the average caspase 3 expression of tumors treated with the SHAL-functionalized Dox NPs followed by 5 Gy XRT was 23% higher than that treated with free Dox plus free SHAL followed by 5 Gy XRT and 1.6 times higher than that without irradiation. Even in the mice with partial tumor regression, the treatment significantly increased their median remission time (~ 55 days vs ~ 15 days for the other treatment groups that received XRT). The improvement in survival rate was even more dramatic in this chemoresistant Raji tumor model when the group treated with CIRT with SHAL-functionalized Dox NPs (M.S.T. not reached at >80 days) was compared to other treatment groups, including CRT with free DOX (M.S.T. = 48 days, p = 0.0141) and free DOX plus free SHAL (M.S.T. = 52 days, p = 0.0323). Quantitatively, the antibody mimic-functionalized Dox NPs enhanced the efficiency of XRT by more than 100% (E.F. > 2.0). Taken together, concurrent therapy with SHAL-functionalized Dox NPs induced synthetic lethality even in tumors that are resistant to conventional chemotherapy and radiation.

[0433] Additional in vivo studies were performed to investigate the significance of the upregulation of HLA-DR expression induced by ICD in the anticancer effects of SHAL- function-alized Dox NPs. A longer treatment cycle (with a six-day rest period between treatments) in this in vivo study using the Daudi xenograft model to allow the HLA-DR antigen expression of surviving cancer cells to return to the background level before subsequent treatment commenced. As in the shorter treatment cycle treatment schedule, the anticancer activities of the three weekly administrations of free SHAL, SHAL-functionalized Dox NPs, and nontargeted Dox NPs were comparable with those in the nontreatment control group. In contrast to the shorter treatment cycle protocol, the SHAL-functionalized Dox NPs were less effective at inhibiting tumor growth than was free Dox (M.S.T. SHAL- functionalized DoxNPs=42daysvsM.S.T.offreeDox=49days;p= 0.0481). The survival probability of the mice treated with the SHAL-functionalized Dox NPs was similar to that of the nontreatment control group (p = 0.5112). This finding indicates that the ICD-induced upregulation of HLA-DR antigen expression directly enhanced the anticancer activity of the SHAL-functionalized Dox NPs.

[0434] Lastly, treatment sequence-dependent in vivo studies were performed to investigate how the chemo-immunotherapy and XRT treatment sequence affected the anticancer efficacy of SHAL-functionalized Dox NPs. As in the earlier efficacy study using the Raji tumor model, mice in the concurrent CIRT group received three treatments of 5 Gy XRT 24 h after the i.v. administration of SHAL-functionalized Dox NPs. In the sequential CIRT group, mice received three treatments of 5 Gy XRT, 3 days apart, starting 5 days after the final chemo-immunotherapy session. Although the sequential treatment schedule is more widely used than the concurrent schedule in certain clinical situations because of more toxicities associated with concurrent treatment, no significant side effects (e.g., weight loss) were observed in either treatment group in this study. This absence of significant adverse events is likely because the SHAL-functionalized Dox NPs reduce any nonspecific uptake and systemic side effects associated with Dox, as justified by biodistribution and in vivo toxicity studies. However, the concurrent and sequential treatments showed drastically different antitumor effects. As in the earlier in vivo efficacy study, concurrent CIRT effectively inhibited the propagation of tumor growth, with a complete response rate of 78%. In contrast, no mice in the sequential CIRT group achieved a complete response or long-term survival. The sequential treatment only inhibited the tumor growth for approximately 4 weeks (from the date of initial treatment), and the M.S.T. for this group was only 9 days longer than that of the nontreatment control group. This is because systemic administration of SHAL-functionalized Dox NPs followed by 5 Gy XRT significantly increased the HLA- DR expression of cancer cells by about 45% compared without concurrent XRT treatment. The higher HLA-DR expression facilitates the uptake of the SHAL-functionalized NPs in subsequent treatment.

Non-Limiting Embodiments of the Disclosure

[0435] Embodiment 1. A Selective High Affinity Ligand (SHAL) molecule of the structure Group A, Group B, or Group C, wherein Group A is of the structure:

(Group A), wherein:

Ri and R3 are each independently

R2 is

Group B is of the structure:

(Group B), wherein: Ri9 is

and

Group C is of the structure:

(Group C), wherein: R21 is

R22, R23, R26 and R27 are each independently

and

R24 and R25 are each independently

wherein each L is independently selected from Li, L2, L3, and L4:

wherein:

R4 is H, NH2, N(CH₃)₂, CO2, NH(CH₃), NO2 or CF₃;

Rs is H, NH2, NO2 or CH₃;

Ai is a bond, -CH2-, -NH-, -N=N-, -0-, -CH2-CH2-, -CH2-NH-, -CH=NH-, -CH2-O-, -CH=CH-, -NHCH2-, -NH=CH-, -OCH2-, phenyl ene, -NHNH-, -NHC(O)-, or -(O)CNH-; R6 is any one of:

R7 is H, Cl, or F;

Rs and R9 are each independently

A₂ is -NH-, -0-, -CH2-, -NHCH2-;

R11 is H, methyl, Cl, NH2,

Ri2 is H, methyl, Cl, NH2,

Ri3 is H, methyl, Cl, NH2, or

Ri4 is methyl, H or NH2; Ri5 is methyl, H or NH2, or

Ri₆ is

wherein each L1-L4, * denotes attachment to the rest of the ligand L1-L4,

denotes attachment to the SHAL, and W is ^ or OH; and R is a label tag or effector.

[0436] Embodiment 2. The SHAL of Embodiment 1 further comprising a label or tag or effector from Group R from Table 4.

[0437] Embodiment 3. The SHAL of Embodiment 1 having the structure of any of the compounds from Specimen-Group-A2.

[0438] Embodiment 4. The SHAL of Embodiment 1 having the structure of any of the compounds from Specimen-Group-A3.

[0439] Embodiment 5. The SHAL of Embodiment 1 having the structure of any of the compounds from Specimen-Group-B2.

[0440] Embodiment 6. The SHAL of Embodiment 1 having the structure of any of the compounds from Specimen-Group- Specimen-Group-B 3.

[0441] Embodiment 7. The SHAL of Embodiment 1 having the structure of any of the compounds from Specimen-Group- Specimen-Group-C2.

[0442] Embodiment 8. The SHAL of Embodiment 1 or 2 comprising one or more Linker-Molecules having cleavable disulfide bonds “X(SS)” from Table 3 covalently linked to one or more Ligands L from Table 1. [0443] Embodiment 9. The SHAL of any one of Embodiments 1-8 further comprising a micelle, a liposome, a nanoparticle, a hydrogel or a derivative thereof.

[0444] Embodiment 10. A composition comprising the SHAL of any one or more of Embodiments 1-9 and a carrier.

[0445] Embodiment 11. The composition of Embodiment 10, wherein the carrier is a pharmaceutically acceptable carrier.

[0446] Embodiment 12. A method for one or more of: detecting a cancer cell that expresses or comprises atypical expression of Major Histocompatibility Complex Class II (MHC Class II) proteins, inhibiting the growth or proliferation of a cancer cell that express or has atypical expression of MHC Class II, or killing a cancer cell that expresses or has atypical expression of MHC Class II proteins, the method comprising contacting the cells with an effective amount of: a. a SHAL having a structure from Group A, Group B, or Group C, comprising two or more ligands from Table 1, or a derivative thereof; b. the SHAL of any one of Embodiments 1-9; or c. the composition of any one of Embodiments 10 and 11, and optionally wherein each cancer cell is independently selected from the group of pancreatic cancer, renal cancer, prostate cancer, liver cancer, stomach cancer, urothelial cancers, lung cancer, ovarian cancer, cervical cancer, esophageal cancer, breast cancer, thyroid cancer, colorectal cancer, bone cancer, laryngeal cancer, head and neck cancer, lymphoma, leukemia, myeloma, glioma, histiocytic sarcoma and melanoma.

[0447] Embodiment 13. The method of Embodiment 12, wherein the derivatives of the SHALs comprise a label, effector, tag or material from Group R in Table 4.

[0448] Embodiment 14. The method of Embodiment 12 or 13, the SHAL having a structure from Groups A, B or C or a derivative thereof containing two or more ligands from

Table 1 further comprising a micelle, a liposome, a nanoparticle, a hydrogel or a derivative thereof.

[0449] Embodiment 15. The method of any one of Embodiments 12-14 wherein the cancer cell does not express HLA-DRIO or an HLA-DR comprising a Lym-1 epitope. [0450] Embodiment 16. The method of any one of Embodiments 12-14 wherein the cancer cell does not express MHC Class II proteins.

[0451] Embodiment 17. The method of any one of Embodiments 12-16 wherein the contacting is in vitro or in vivo.

[0452] Embodiment 18. The method of any one of Embodiments 12-17 wherein the cancer cell is a mammalian cancer cell.

[0453] Embodiment 19. The method of any one of Embodiments 12-18 to detect cancer cells in biopsy tissue in a positron emission tomography scan (PET scan), in a computerized tomography scan (CT scan), in a magnetic resonance imaging scan (MRI scan), in any other medical imaging scan, in a liquid biopsy, in blood or in cerebral or spinal fluid, or in any other bodily fluids, the method comprising contacting the biopsy tissue or fluid with a SHAL having a structure from Group A, Group B, or Group C, comprising two or more ligands from Table 1, or a derivative thereof further comprising any suitable detection label from Group R from Table 4.

[0454] Embodiment 20. A method of treating cancer cells or a solid tumor that expresses an MHC class II protein, in a subject in need thereof with the SHAL of any one of Embodiments 1-9, comprising treating the cancer cells or solid tumor in the subject by administering to the subject an effective amount of the SHAL, wherein the cancer cells or solid tumor are selected from one or more of pancreatic cancer, renal cancer, prostate cancer, liver cancer, stomach cancer, urothelial cancers, lung cancer, ovarian cancer, cervical cancer, esophageal cancer, breast cancer, thyroid cancer, colorectal cancer, bone cancer, laryngeal cancer, head and neck cancer, lymphoma, leukemia, myeloma, glioma, histiocytic sarcoma and melanoma.

[0455] Embodiment 21. The method of Embodiment 20 wherein the SHAL inhibits the growth of the tumor or progression of the cancer or kills the cancer cells.

[0456] Embodiment 22. The method of Embodiment 20 or 21 wherein the cancer cells or solid tumor does not express HLA-DRIO or an HLA-DR comprising a Lym-1 epitope. [0457] Embodiment 23. The method of any one of Embodiments 20-22 further comprising administering to the subject an effective amount of an anticancer agent for cytoreductive therapy.

[0458] Embodiment 24. A method of treating cancer cells or a tumor that does not express an MHC class II protein, in a subject in need thereof, comprising administering to the subject a nanoparticle comprising a SHAL of a structure selected from Groups A, B, or C, comprising two or more ligands from Table 1, or a derivative of each thereof.

[0459] Embodiment 25. The method of Embodiment 24, wherein the cancer cells or tumor does not express HLA-DR10 or an HLA-DR comprising a Lym-1 epitope.

[0460] Embodiment 26. The method of any one of Embodiments 20-25, wherein the subject is a mammal.

[0461] Embodiment 27. The method of Embodiment 26 where the subject is selected from a human patient or other mammal from the group of a canine, a feline, an equine, a rodent, a bovine, or an ovine.

[0462] Embodiment 28. A method for inducing, enhancing or promoting an anti tumor immune response in a subject in need thereof comprising administering to the subject an effective amount of a SHAL having a structure from Group A, Group B, or Group C, comprising two or more ligands from Table 1 and/or Table 2, or a derivative thereof.

[0463] Embodiment 29. The method of Embodiment 28 wherein the derivative comprises a label, tag, or effector from Group R in Table 4.

[0464] Embodiment 30. The method of Embodiment 28 or 29 wherein the immune response comprises activating B-cell lymphocytes, macrophages, dendritic cells or CD4+ or CD8+ T cell lymphocytes to induce an anti-tumor immune response.

[0465] Embodiment 31. The method of any one of Embodiments 28-30, wherein the anti-tumor response is directed towards cancer cells or tumors selected from the group of: pancreatic cancer, renal cancer, prostate cancer, liver cancer, stomach cancer, urothelial cancers, lung cancer, ovarian cancer, cervical cancer, esophageal cancer, breast cancer, thyroid cancer, colorectal cancer, bone cancer, laryngeal cancer, head and neck cancers, lymphoma, leukemia, myeloma, glioma, histiocytic sarcoma and melanoma. [0466] Embodiment 32. The method of any one of Embodiments 28-31, wherein the immune response is induced by binding of the SHAL to an MHC class II protein and the presentation of the SHAL, by the MHC class II protein, to T-cell lymphocytes.

[0467] Embodiment 33. The method of any one of Embodiments 28-32, the

SHAL further comprising a micelle, a liposome, a nanoparticle, a hydrogel or a derivative thereof.

[0468] Embodiment 34. The method of any one of Embodiments 28-33 where the subject is selected from a human patient or other mammal from the group of a canine, a feline, an equine, a rodent, a bovine, and an ovine.

[0469] Embodiment 35. A method to treat an MHC class II protein linked autoimmune disease or disorder selected from the group of Table 8 comprising Rheumatoid Arthritis, Multiple Sclerosis, Type-1 Diabetes, Grave’s Disease, Hashimoto’s Thyroiditis, Myasthenia Gravia, Celiac Disease, Ulcerative Colitis, Systemic Lupus Erythematosus, or Anklylosing Spondylitis in a subject in need thereof, the method comprising administering to the subject an effective amount of a SHAL having a structure from Group A, Group B, or Group C, comprising two or more ligands from Table 1 and/or Table 2, or derivatives thereof.

[0470] Embodiment 36. The method of Embodiment 35 wherein the immune response comprises activity of lymphocytes, macrophages or dendritic cells.

[0471] Embodiment 37. The method of Embodiment 35 or 36 wherein the immune response comprises blocking presentation of self-antigens by an MHC class II protein or suppressing inflammation.

[0472] Embodiment 38. The method of any one of Embodiments 35-37 comprising killing of B -lymphocytes involved in the production of autoantibodies.

[0473] Embodiment 39. A method for treating a disease or disorder related to a pathological immune response in a subject in need thereof, the immune response contributing to a disease or disorder of the group of vascular injury and leucocyte recruitment leading to restenosis, allergic asthma, inflammation, and inflammation induced restriction of blood flow in ischemia stroke; the method comprising administering to the subject an effective amount of a SHAL having a stmcture from Group A, Group B, or Group C, comprising two or more ligands from Table 1 and/or Table 2, or derivatives thereof.

[0474] Embodiment 40. The method of any one of Embodiments 35-39, the derivatives comprise a label, tag, or effector from Group R in Table 4.

[0475] Embodiment 41. The method of any one of Embodiments 35-40, the

SHAL further comprising a micelle, a liposome, a nanoparticle, a hydrogel or a derivative thereof.

[0476] Embodiment 42. The method of any one of Embodiments 35-41, wherein the subject is a human patient or a mammal selected from a canine, a feline, an equine, a rodent, a bovine, or an ovine.

[0477] Embodiment 43. The method of any one of Embodiments 35-42 further comprising administering an effective amount of a second therapy, prior to, subsequent to, or concurrent to the administration of the SHAL having a structure from Group A, Group B, or Group C, comprising two or more ligands from Table 1 and/or Table 2, or derivatives thereof.

[0478] Embodiment 44. The method of Embodiment 40 wherein the effector is selected from the group of a therapeutic agent, a detectable agent, a probe or a marker that can be manipulated, and a structure from Group R in Table 4, and optionally the probe or marker that can be manipulated comprises a magnetic particle or a light, pH, or frequency- activated nanostructure or molecule, or a derivative thereof.

[0479] Embodiment 45. The method of Embodiment 44 wherein the effector is delivered to a cell that does not express HLA-DRIO or an HLA-DR containing a Lym-1 epitope or any MHC class II protein.

[0480] Embodiment 46. The method of Embodiment 44 or 45, the SHAL further comprising a micelle, a liposome, a nanoparticle, a hydrogel or a derivative thereof.

[0481] Embodiment 47. The method of Embodiment 45 or 46 wherein the cell is a mammalian cell.

[0482] Embodiment 48. The method of Embodiment 47, the mammalian cell selected from a human cell, a canine cell, a feline cell, a rodent cell, an equine cell, a bovine cell, or an ovine cell. [0483] Embodiment 49. The method of any one of Embodiments 45-48, wherein the cell is a cancer cell selected from the group of: pancreatic cancer, renal cancer, prostate cancer, liver cancer, stomach cancer, urothelial cancers, lung cancer, ovarian cancer, cervical cancer, esophageal cancer, breast cancer, thyroid cancer, colorectal cancer, bone cancer, laryngeal cancer, head and neck cancers, lymphoma, leukemia, myeloma, glioma, histiocytic sarcoma and melanoma.

[0484] Embodiment 50. A method to inhibit cell growth and proliferation or to kill a cell by inhibiting a GTPase activating protein (GAP) selected from the group of MgcRacGAP, p50RhoGAP and BCR GAP, comprising contacting the GAP with an effective amount of a SELAL having a structure from Group A, Group B, or Group C, comprising one or more ligands from Table 1 and/or Table 2, or a derivative thereof, thereby inhibiting the GAP.

[0485] Embodiment 51. A method to inhibit cell growth and proliferation of, or to kill, a cell by directly inhibiting a GTPase enzyme selected from the group of Racl, Rac3, p50Rho, RhoA and Cdc42, the method comprising contacting the GTPase enzyme with an effective amount of a SELAL having a structure from Group A, Group B, or Group C, comprising one or more ligands from Table 1 and/or Table 2, or a derivative thereof, thereby directly inhibiting the GTPase enzyme.

[0486] Embodiment 52. A method to inhibit cell growth or proliferation of, or to kill, a cell by inhibiting AcetylCoA carboxylase (ACC) comprising contacting ACC with an effective amount of a SELAL having a structure from Group A, Group B, or Group C, comprising one or more ligands from Table 1 and/or Table 2, or a derivative thereof, thereby inhibiting ACC.

[0487] Embodiment 53. The method of any one of Embodiments 50-52 wherein the cell expresses MHC class II proteins.

[0488] Embodiment 54. The method of any one of Embodiments 50-52 wherein the cell does not express MHC Class II proteins.

[0489] Embodiment 55. The method of any one of Embodiments 50-54 wherein the derivative thereof comprises a label, tag, or effector from Group R in Table 4. [0490] Embodiment 56. The method of any one of Embodiments 50-55, the

SHAL further comprising a micelle, a liposome, a nanoparticle, a hydrogel or a derivative thereof.

[0491] Embodiment 57. The method of any one of Embodiments 50-56 wherein the contacting is in vitro or in vivo.

[0492] Embodiment 58. The method of any one of Embodiments 50-57 wherein the cell is a mammalian cell.

[0493] Embodiment 59. The method of Embodiment 58, wherein the mammalian cell is selected from the group of a canine cell, a feline cell, a rodent cell, an equine cell, a bovine cell, an ovine cell, and a human cell.

[0494] Embodiment 60. The method of any one of Embodiments 50-59 wherein cancer is treated in a subject in need thereof by administering to the subject, an effective amount of the SHAL having a structure from Group A, Group B, or Group C, comprising one or more ligands from Table 1 and/or Table 2, or a derivative thereof, thereby killing the cancer cell by inhibiting the activity of GAP, GTPase enzyme or ACC.

[0495] Embodiment 61. The method of any one Embodiments 50-59 wherein obesity or obesity-related disorders comprising type-2 diabetes, non-alcoholic fatty-liver disease, or metabolic syndrome are treated in a subject in need thereof by administering to the subject, an effective amount of the SHAL having a structure from Group A, Group B or Group C, comprising one or more ligands from Table 1 and/or Table 2, or a derivative thereof, thereby inhibiting the activity of GAP, GTPase enzyme or ACC.

[0496] Embodiment 62. The method of Embodiment 60 or 61 wherein the subject is a human patient or an animal from the group of a canine, a feline, an equine, a rodent, a bovine and an ovine.

[0497] Embodiment 63. The method of any one of Embodiments 60-62 further comprising administering to the subject an effective amount of a second therapy, prior to, subsequent to, or concurrent with the administration of the SHAL having a structure from Group A, Group B, or Group C, comprising one or more ligands from Table 1 and/or Table 2, or a derivative thereof. [0498] Embodiment 64. The method of any one of Embodiments 50-63 wherein the derivative thereof comprises a label, effector, tag, or material from Group R in Table 4.

[0499] Embodiment 65. The method of any one of Embodiments 50-64, the

SHAL further comprising a micelle, a liposome, a nanoparticle, a hydrogel or a derivative thereof.

[0500] Embodiment 66. A method to prevent one or more drugs taken up by a mammalian or bacterial cell from being pumped back out of the cell by inhibiting a multidrug resistance protein 1 (P -glycoprotein, MDR1 or P-gp) or breast cancer resistance protein (BCRP) efflux transporter or its ortholog, comprising contacting the transporter with an effective amount of a SHAL having a structure from Group A, Group B or Group C, comprising one or more ligands from Table 1 and/or Table 2, or a derivative thereof, thereby inhibiting the activity of a transporter protein.

[0501] Embodiment 67. The method of Embodiment 66 wherein the uptake of the one or more drugs from the intestine, gut, oral cavity and across the blood-brain and blood- testis barrier is improved.

[0502] Embodiment 68. The method of Embodiment 66 or 67 to wherein the cell is prevented from developing resistance to a drug different from the one or more drugs.

[0503] Embodiment 69. The method of any one of Embodiments 66-68 wherein sensitivity of the cell to action of the one or more drugs is increased by preventing the one or more drugs from being pumped back out of the cell.

[0504] Embodiment 70. A method to inhibit organic-anion-transporting polypeptide (OATP)-transporter mediated uptake of hormones, hormone conjugates, or growth promoting chemicals that a tumor cell requires to grow and survive, the method comprising contacting O ATP -transporter with an effective amount of a SHAL having a structure from Group A, Group B, or Group C, comprising one or more ligands from Table 1 and/or Table 2, or a derivative thereof, thereby inhibiting the activity of the OATP- transporter protein.

[0505] Embodiment 71. A method to reduce the required dosage of a drug delivered to a subject in need thereof by inhibiting metabolic ETDP-glucuronosyltransferase (UGT) enzyme, comprising contacting the UGT enzyme with an effective amount of a SHAL having a structure from Group A, Group B, or Group C, comprising one or more ligands from Table 1 and/or Table 2, or a derivative thereof, thereby inhibiting activity of the UGT enzyme.

[0506] Embodiment 72. The method of Embodiment 71 wherein the sensitivity of a cell to a drug’s action is increased by reducing the metabolism of the drug and slowing the rate of export of the drug from the cell.

[0507] Embodiment 73. The method of any one of Embodiments 66-72 wherein the cell expresses MHC class II proteins.

[0508] Embodiment 74. The method of any one of Embodiments 66-72 wherein the cell does not express MHC Class II proteins.

[0509] Embodiment 75. The method of any one of Embodiments 66-74 wherein the derivative thereof comprises a label, tag, or effector from Group R in Table 4.

[0510] Embodiment 76. The method of any one of Embodiments 66-75, the

SHAL further comprising a micelle, a liposome, a nanoparticle, a hydrogel or a derivative thereof.

[0511] Embodiment 77. The method of any one of Embodiments 66-76 wherein the cell is a normal cell or a cancer cell.

[0512] Embodiment 78. The method of Embodiment 77 wherein the cancer cell is selected from the group of pancreatic cancer, renal cancer, prostate cancer, liver cancer, stomach cancer, urothelial cancers, lung cancer, ovarian cancer, cervical cancer, esophageal cancer, breast cancer, thyroid cancer, colorectal cancer, bone cancer, laryngeal cancer, head and neck cancer, lymphoma, leukemia, myeloma, glioma, histiocytic sarcoma and melanoma.

[0513] Embodiment 79. The method of any one of Embodiments 66-78 where the contacting is in vitro or in vivo.

[0514] Embodiment 80. The method of any one of Embodiments 66-79 further comprising administering to a subject in need thereof, an effective amount of a second therapy, prior to, subsequent to, or concurrent with administration to the subject of the SHAL having a structure from Group A, Group B, or Group C, comprising one or more ligands from Table 1 and/or Table 2, or a derivative thereof.

[0515] Embodiment 81. The method of any one of Embodiments 66-80 wherein the subject is a human patient or an animal from the group of a canine, a feline, an equine, a rodent, a bovine and an ovine.

[0516] Embodiment 82. A method to deliver one or more prodrugs to a cell, the prodrug comprising a SELAL having a structure from Group A, Group B, or Group C, comprising one or more SELAL ligands from Table 1 and/or Table 2, or a derivative thereof, the method comprising binding the SELAL or a derivative thereof to a target protein or the cell.

[0517] Embodiment 83. The method of Embodiment 82 wherein the biological activity from the prodrug is derived from the metabolism of one or more of the SELAL ligands from Table 1 and/or Table 2, to produce fragments having the biological activity.

[0518] Embodiment 84. The method of Embodiment 83 wherein the biological activity from the prodrug is derived from the reduction of a disulfide bond to selectively release one or more ligands having the biological activity.

[0519] Embodiment 85. The method of any one of Embodiments 82-84 wherein the SELAL ligands target the target protein or cell with the SELAL acting as a compact small- molecule antibody-drug conjugate or ADC.

[0520] Embodiment 86. A method of delivering to a cell an effective amount of a

SELAL having a structure from Group A, Group B or Group C, comprising one or more ligands from Table 1 and/or Table 2, or a derivative thereof, the method comprising the two or more ligands binding simultaneously to two or more different sites on a protein, enzyme, or the cell to act as adjuvant to work synergistically with another drug.

[0521] Embodiment 87. The method of Embodiment 86 further comprising administration of the other drug, prior to, subsequent to or concurrent with the administration of the SELAL or a derivative thereof.

[0522] Embodiment 88. The method of any one of Embodiments 82-87, the derivative thereof comprising a label, effector, tag or material from Group R in Table 4. [0523] Embodiment 89. The method of any one of Embodiments 82-88, the

SHAL or a derivative thereof further comprising a micelle, a liposome, a nanoparticle, a hydrogel or a derivative thereof.

[0524] Embodiment 90. The method of any one of Embodiments 82-89 wherein the cell expresses MHC class II proteins.

[0525] Embodiment 91. The method of any one of Embodiments 82-89 wherein the cell does not express MHC Class II proteins.

[0526] Embodiment 92. The method of any one of Embodiments 82-91 wherein the cell is a normal cell or a cancer cell.

[0527] Embodiment 93. The method of Embodiment 92 wherein the cancer cell is selected from the group of pancreatic cancer, renal cancer, prostate cancer, liver cancer, stomach cancer, urothelial cancers, lung cancer, ovarian cancer, cervical cancer, esophageal cancer, breast cancer, thyroid cancer, colorectal cancer, bone cancer, laryngeal cancer, head and neck cancer, lymphoma, leukemia, myeloma, glioma, histiocytic sarcoma and melanoma.

[0528] Embodiment 94. The method of any one of Embodiments 82-93 wherein the binding to a target protein or cell is in vitro or in vivo.

[0529] Embodiment 95. The method of any one of Embodiments 82-94 wherein the target protein or cell is in a human patient or an animal subject from the group of a canine, a feline, an equine, a rodent, a bovine and an ovine.

[0530] Embodiment 96. A method to kill or inhibit the growth or proliferation of a cancer cell that expresses an MHC class II protein that is not HLA-DRIO or does not contain a Lym-1 epitope, comprising contacting the cell with an effective amount of a SHAL having a structure from Group A, Group B, or Group C, comprising two or more ligands from Table 1 and/or Table 2, or a derivative thereof, wherein the cancer cell is selected from the group of pancreatic cancer, renal cancer, prostate cancer, liver cancer, stomach cancer, urothelial cancers, lung cancer, ovarian cancer, cervical cancer, esophageal cancer, breast cancer, thyroid cancer, colorectal cancer, bone cancer, laryngeal cancer, head and neck cancer, lymphoma, leukemia, myeloma, glioma, histiocytic sarcoma and melanoma. [0531] Embodiment 97. A method of treating cancer cells or a tumor that expresses an MHC class II protein that is not HLA-DR10 or does not contain a Lym-1 epitope, in a subject in need thereof, comprising administering to the subject an effective amount of a SHAL having the structure from Group A, Group B, Group C, Specimen-Group- Al, Specimen-Group-Bl, or Specimen-Group-Cl, containing two or more ligands from Table 1 and/or Table 2, or a derivative thereof, wherein the cancer is selected from the group of pancreatic cancer, renal cancer, prostate cancer, liver cancer, stomach cancer, urothelial cancers, lung cancer, ovarian cancer, cervical cancer, esophageal cancer, breast cancer, thyroid cancer, colorectal cancer, bone cancer, laryngeal cancer, head and neck cancer, lymphoma, leukemia, myeloma, glioma, histiocytic sarcoma and melanoma.

[0532] Embodiment 98. The method of Embodiment 96 or 97 wherein the cells are normal cells or cancer cells.

[0533] Embodiment 99. The method of any one of Embodiments 96-98 wherein the SHAL or a derivative thereof binds to an MHC class II protein selected from the group of HLA-DRl, HLA-DR3, HLA-DR4, HLA-DR7, HLA-DR8, HLA-DR9, HLA-DR11, HLA- DR12, HLA-DRl 3, HLA-DRl 4, HLA-DRl 5, HLA-DRl 6, HLA-DP and HLA-DQ.

[0534] Embodiment 100. The method of any one of Embodiments 96-98 wherein the SHAL binds to the MHC class II protein of HLA-DRs comprising the beta subunit DRB3, DRB4 or DRB5.

[0535] Embodiment 101. The method of any one of Embodiments 96-99 wherein the SHAL binds to the MHC class II protein of HLA-DRs comprising the beta subunit DRB1*01, DRB1*03, DRB1*04, DRB1*07, DRB1*08, DRB1*09, DRB1*11, DRB1*12, DRB1*13, DRB1*14, DRB1*15 orDRBl*16.

[0536] Embodiment 102. The method of any one of Embodiments 96-101 wherein the SHAL inhibits the growth of the tumor or progression of the cancer, or kills the cancer cells.

[0537] Embodiment 103. The method of any one of Embodiments 97-102, further comprising administering to the subject an effective amount of an anticancer agent for cytoreductive therapy. [0538] Embodiment 104. A method for treating cancer cells or a tumor that does not expresses an MHC class II protein, in a subject in need thereof, comprising administering to the subject a nanoparticle comprising a SHAL of the structure selected from Group A, B, or C, comprising two or more ligands from Table 1 and/or Table 2, or a derivative thereof, thereby treating the cancer cells or tumor that does not express an MHC class II protein.

[0539] Embodiment 105. The method of any one of Embodiments 96-104, wherein the derivative thereof comprises a SHAL that comprises a label, effector, tag, or material from Group R in Table 4.

[0540] Embodiment 106. The method of any one of Embodiments 96-105, the

SHAL or derivative thereof further comprising a micelle, a liposome, a nanoparticle, a hydrogel or a derivative thereof.

[0541] Embodiment 107. The method of any one of Embodiments 96-106, wherein the SHAL has the structure of SH7133, SH7129 or SH7139 from Specimen-Group-Al.

[0542] Embodiment 108. The method of any one of Embodiments 96-107, wherein the subject is a mammal.

[0543] Embodiment 109. The method of Embodiment 108 wherein the subject is selected from a human patient or other mammal from the group of a canine, a feline, an equine, a rodent, a bovine, and an ovine.

[0544] Embodiment 110. The method of any one of Embodiments 96-109 where the contacting is in vitro or in vivo.

[0545] Embodiment 111. A method of treating cells or tumors that do not express an MHC class II protein, in a subject in need thereof, comprising administering to the subject a DOTA-tagged or biotin-tagged SHAL of the structure selected from Group A, B, or C, comprising two or more ligands from Table 1 and/or Table 2, complexed to a bispecific antibody, diabody or antibody-avidin conjugate that recognizes and binds to the DOTA or biotin on the SHAL and also recognizes and binds to a cell surface receptor or protein that is not an MHC Class II protein targeted by the SHAL.

[0546] Embodiment 112. A method of pre-targeting a SHAL to a cell or tumor in a subject, comprising: administering to the subject a bispecific antibody, diabody or antibody-avidin conjugate that recognizes and binds to both:

(a) a cell surface receptor or protein; and

(b) a DOTA tag or biotin tag on the SHAL, the SHAL comprising the structure selected from Group A, B, or C, comprising two or more ligands from Table 1 and/or Table 2; followed by administering the SHAL to the subject after a suitable period of time.

[0547] Embodiment 113. A pre-targeting method for delivering a drug to a cell or tumor in a subject, the cell or tumor expressing an MHC class II protein recognized by a SHAL, comprising: administering to the subject:

(a) a biotin-tagged or DOTA-tagged SHAL complex comprising the SHAL of Group A, B, or C, comprising two or more ligands from Table 1 and/or Table 2, and

(b) a bispecific antibody, diabody or antibody-avidin conjugate or fusion protein that recognizes and binds to both the DOTA tag or biotin tag of the SHAL and the drug; and administering the drug to the subject a suitable period of time after administration of the complex containing (a) and (b).

[0548] Embodiment 114. A method to facilitate the delivery of a drug to a normal cell or cancer cell expressing an MHC Class II protein, of a subject, comprising: administering to the subject an anti-drug/anti-DOTA or biotin bispecific antibody, diabody, antibody-avidin conjugate, or fusion protein comprising both the drug bound thereto and a DOTA-tagged or biotin-tagged SHAL of the structure selected from Group A, B, or C comprising two or more ligands from Table 1 and/or Table 2, thereby delivering the drug into cells expressing MHC Class II proteins targeted by the SHAL. [0549] Embodiment 115. The method of any one of Embodiments 111-114, wherein the SHAL has the structure of SH7129 or SH7139 from Specimen-Group- Al. [0550] Embodiment 116. The method of any one of Embodiments 111-115, wherein the subject is a mammal.

[0551] Embodiment 117. The method of any one of Embodiments 111-116 wherein the subject is selected from a human patient or other mammal from the group of a canine, a feline, an equine, a rodent, a bovine, and an ovine.

[0552] Embodiment 118. The method of any one of Embodiments 111-117 where the subject is a human.

[0553] Embodiment 119. A method to kill or suppress the activity of an activated microglia, lymphocyte, dendritic cell or macrophage, comprising contacting the activated microglia, lymphocyte, dendritic cell or macrophage with an effective amount of a SHAL of structure from Group A, Group B, or Group C, comprising two or more ligands from Table 1 and/or Table 2, or a derivative thereof.

[0554] Embodiment 120. The method of Embodiment 119, the method treating a neurodegenerative disease or disorder comprising Alzheimer’s disease, Parkinson’s disease, Multiple Sclerosis, Amyotrophic Lateral Sclerosis, Frontotemporal Dementia or another microglia-mediated neurodegenerative disease, in a subject in need thereof, the method comprising administering to the subject an effective amount of the SHAL, or a derivative thereof.

[0555] Embodiment 121. The method of Embodiment 119, the method treating brain cancer or another cancer by suppressing or killing infiltrating activated microglia, lymphocytes, dendritic cells or macrophages in a subject in need thereof, the method comprising administering to the subject an effective amount of the SHAL, or a derivative thereof.

[0556] Embodiment 122. The method of any one of Embodiments 119-121, the derivative thereof comprising a label, effector, tag or material from Group R in Table 4.

[0557] Embodiment 123. The method of any one of Embodiments 119-122, the

SHAL or a derivative thereof further comprising a micelle, a liposome, a nanoparticle, or a hydrogel. [0558] Embodiment 124. The method of any one of Embodiments 119-123 wherein the activated microglia, lymphocyte, dendritic cell or macrophage expresses an HLA-DR or MHC class II protein.

[0559] Embodiment 125. The method of any one of Embodiments 121-123 wherein tumor cells of the cancer do not express MHC Class II proteins.

[0560] Embodiment 126. The method of any one of Embodiments 121-125 wherein the brain cancer is selected from the group of gliomas, glioblastomas, astrocytomas, chordomas, CNS lymphomas, craniopharyngiomas, medulloblastomas, meningiomas, oligodendrogliomas, schwannomas, ependymomas, pineal tumors, primitive neuroectodermal tumors, and rhabdoid tumors.

[0561] Embodiment 127. The method of any one of Embodiments 121-126 wherein the other cancer is selected from the group of pancreatic cancer, renal cancer, prostate cancer, liver cancer, stomach cancer, urothelial cancer, lung cancer, ovarian cancer, cervical cancer, esophageal cancer, adrenal cancer, testicular cancer, bladder cancer, bile duct cancer, breast cancer, intestinal cancer, thyroid cancer, colorectal cancer, anal cancer, appendix cancer, bone cancer, laryngeal cancer, head and neck cancer, lymphoma, leukemia, myeloma, sarcoma, and melanoma.

[0562] Embodiment 128. The method of any one of Embodiments 121-127 further comprising administration of a drug or drug cocktail, prior to, subsequent to or concurrent with administration of the SHAL or a derivative thereof.

[0563] Embodiment 129. The method of any one of Embodiments 121-128, wherein the subject is a mammal.

[0564] Embodiment 130. The method of any one of Embodiments 121-129 wherein the subject is selected from a human or other mammal from the group of a canine, a feline, an equine, a rodent, a bovine, and an ovine.

[0565] Embodiment 131. A microarray or microtiter plate comprising one or more

SHAL(s) wherein each SHAL has a structure independently selected from Group A, Group B, or Group C, or a derivative thereof, comprising one or more ligands from Table 1 and/or Table 2 that bind to an MHC class II protein, a transporter, a UGT metabolizing enzyme, a GAP, a GTPase, or an ACC enzyme, and optional instructions for use.

[0566] Embodiment 132. A kit comprising a SHAL compound having a structure from Group A, Group B or Group C, or a derivative thereof, comprising one or more ligands from Table 1 and/or Table 2 that binds to an MHC class II protein, a transporter, a UGT metabolizing enzyme, a GAP, a GTPase, or an ACC enzyme, and optional instructions for use.

Equivalents

[0567] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs.

[0568] The present technology illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising,” “including,” “containing,” etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the present technology claimed.

[0569] Thus, it should be understood that the materials, methods, and examples provided here are representative of preferred aspects, are exemplary, and are not intended as limitations on the scope of the present technology.

[0570] The present technology has been described broadly and generically herein.

Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the present technology. This includes the generic description of the present technology with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0571] In addition, where features or aspects of the present technology are described in terms of Markush groups or groups, those skilled in the art will recognize that the present technology is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0572] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.

[0573] Other aspects are set forth within the following claims.

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Claims

WHAT IS CLAIMED IS:

1. A Selective High Affinity Ligand (SHAL) molecule of the structure Group A, Group B, or Group C, wherein Group A is of the structure:

(Group A), wherein:

Ri and R3 are each independently

Group B is of the structure:

(Group B), wherein: Ri9 is

and

Group C is of the structure:

(Group C), wherein: R21 is

R22, R23, R26 and R27 are each independently

and

R24 and R25 are each independently

wherein each L is independently selected from Li, L2, L3, and L4:

wherein:

R4 is H, NH2, N(CH₃)₂, CO2, NH(CH₃), NO2 or CF₃;

Rs is H, NH2, NO2 or CH₃;

Ai is a bond, -CH2-, -NH-, -N=N-, -0-, -CH2-CH2-, -CH2-NH-, -CH=NH-, -CH2-O-, -CH=CH-, -NHCH2-, -NH=CH-, -OCH2-, phenyl ene, -NHNH-, -NHC(O)-, or -(O)CNH-; R6 is any one of:

R7 is H, Cl, or F;

Rs and R9 are each independently

A₂ is -NH-, -0-, -CH2-, -NHCH2-;

R11 is H, methyl, Cl, NH2,

Ri2 is H, methyl, Cl, NH2,

Ri3 is H, methyl, Cl, NH2, or

Ri4 is methyl, H or NH2; Ri5 is methyl, H or NH2, or

Ri6 is

wherein each L1-L4, * denotes attachment to the rest of the ligand L1-L4,

denotes attachment to the SHAL, and W is ^ or OH; and R is a label tag or effector.

2. The SHAL of claim 1, comprising the structure comprising the ligand 3-(3-((3-chloro-5- (trifluoromethyl)-2-pyridinyl)oxy)anilino)-3-oxopropanoic acid, optionally the structure is selected from one or more of the following:

3. The SHAL of claim 1 or 2 further comprising a micelle, a liposome, a nanoparticle, a hydrogel or a derivative thereof.

4. A method for one or more of: detecting a cancer cell that expresses or comprises atypical expression of Major Histocompatibility Complex Class II (MHC Class II) proteins, inhibiting the growth or proliferation of a cancer cell that express or has atypical expression of MHC Class II, or killing a cancer cell that expresses or has atypical expression of MHC Class II proteins, the method comprising contacting the cells with an effective amount of: a. a SHAL having a structure from Group A, Group B, or Group C, comprising two or more ligands from Table 1, or a derivative thereof or b. the SHAL of any one of claims 1-3, and optionally wherein each cancer cell is independently selected from the group of pancreatic cancer, renal cancer, prostate cancer, liver cancer, stomach cancer, urothelial cancers, lung cancer, ovarian cancer, cervical cancer, esophageal cancer, breast cancer, thyroid cancer, colorectal cancer, bone cancer, laryngeal cancer, head and neck cancer, lymphoma, leukemia, myeloma, glioma, histiocytic sarcoma and melanoma.

5. The method of claim 4, wherein the cancer cell does not express HLA-DR10 or an HLA- DR comprising a Lym-1 epitope.

6. The method of claim 4, wherein the cancer cell does not express MHC Class II proteins.

7. A method for inducing, enhancing or promoting an anti-tumor immune response in a subject in need thereof comprising administering to the subject an effective amount of a SHAL having a structure from Group A, Group B, or Group C, comprising two or more ligands from Table 1 and/or Table 2, or a derivative thereof.

8. A method to treat an MHC class II protein linked autoimmune disease or disorder selected from the group of Table 8 comprising Rheumatoid Arthritis, Multiple Sclerosis, Type-1 Diabetes, Grave’s Disease, Hashimoto’s Thyroiditis, Myasthenia Gravia, Celiac Disease, Ulcerative Colitis, Systemic Lupus Erythematosus, or Anklylosing Spondylitis in a subject in need thereof, the method comprising administering to the subject an effective amount of a SHAL having a structure from Group A, Group B, or Group C, comprising two or more ligands from Table 1 and/or Table 2, or derivatives thereof.

9. A method to inhibit cell growth and proliferation or to kill a cell by inhibiting a GTPase activating protein (GAP) selected from the group of MgcRacGAP, p50RhoGAP and BCR GAP, comprising contacting the GAP with an effective amount of a SHAL having a structure from Group A, Group B, or Group C, comprising one or more ligands from Table 1 and/or Table 2, or a derivative thereof, thereby inhibiting the GAP.

10. A method to inhibit cell growth and proliferation of, or to kill, a cell by directly inhibiting a GTPase enzyme selected from the group of Racl, Rac3, p50Rho, RhoA and Cdc42, the method comprising contacting the GTPase enzyme with an effective amount of a SHAL having a structure from Group A, Group B, or Group C, comprising one or more ligands from Table 1 and/or Table 2, or a derivative thereof, thereby directly inhibiting the GTPase enzyme.

11. A method to prevent one or more drugs taken up by a mammalian or bacterial cell from being pumped back out of the cell by inhibiting a multidrug resistance protein 1 (P- glycoprotein, MDRl or P-gp) or breast cancer resistance protein (BCRP) efflux transporter or its ortholog, comprising contacting the transporter with an effective amount of a SHAL having a structure from Group A, Group B or Group C, comprising one or more ligands from Table 1 and/or Table 2, or a derivative thereof, thereby inhibiting the activity of a transporter protein.

12. A method to inhibit organic-anion-transporting polypeptide (OATP)-transporter mediated uptake of hormones, hormone conjugates, or growth promoting chemicals that a tumor cell requires to grow and survive, the method comprising contacting O ATP -transporter with an effective amount of a SHAL having a structure from Group A, Group B, or Group C, comprising one or more ligands from Table 1 and/or Table 2, or a derivative thereof, thereby inhibiting the activity of the OATP -transporter protein.

13. A method to reduce the required dosage of a drug delivered to a subject in need thereof by inhibiting metabolic UDP-glucuronosyltransferase (UGT) enzyme, comprising contacting the UGT enzyme with an effective amount of a SHAL having a structure from Group A, Group B, or Group C, comprising one or more ligands from Table 1 and/or Table 2, or a derivative thereof, thereby inhibiting activity of the UGT enzyme.

14. A method to deliver one or more prodrugs to a cell, the prodrug comprising a SHAL having a structure from Group A, Group B, or Group C, comprising one or more SHAL ligands from Table 1 and/or Table 2, or a derivative thereof, the method comprising binding the SHAL or a derivative thereof to a target protein or the cell.

15. A method to kill or inhibit the growth or proliferation of a cancer cell that expresses an MHC class II protein that is not HLA-DRIO or does not contain a Lym-1 epitope, comprising contacting the cell with an effective amount of a SHAL having a structure from Group A, Group B, or Group C, comprising two or more ligands from Table 1 and/or Table 2, or a derivative thereof, wherein the cancer cell is selected from the group of pancreatic cancer, renal cancer, prostate cancer, liver cancer, stomach cancer, urothelial cancers, lung cancer, ovarian cancer, cervical cancer, esophageal cancer, breast cancer, thyroid cancer, colorectal cancer, bone cancer, laryngeal cancer, head and neck cancer, lymphoma, leukemia, myeloma, glioma, histiocytic sarcoma and melanoma.

16. The method of claim 15, wherein the SHAL or a derivative thereof binds to an MHC class II protein selected from the group of HLA-DRl, HLA-DR3, HLA-DR4, HLA-DR7, HLA-DR8, HLA-DR9, HLA-DRl 1, HLA-DRl 2, HLA-DRl 3, HLA-DRl 4, HLA-DRl 5, HLA-DRl 6, HLA-DP and HLA-DQ.

17. The method of claim 15 or 16, wherein the SHAL binds to the MHC class II protein of HLA-DRs comprising the beta subunits DRB1*01, DRB1*03, DRB1*04, DRB1*07, DRB1*08, DRB1*09, DRB1*11, DRB1*12, DRB1*13, DRB1*14, DRB1*15 or DRB1*16, DRB3, DRB4 or DRB5.

18. A method of treating cells or tumors that do not express an MHC class II protein, in a subject in need thereof, comprising administering to the subject a DOTA-tagged or biotin-tagged SHAL of the structure selected from Group A, B, or C, comprising two or more ligands from Table 1 and/or Table 2, complexed to a bispecific antibody, diabody or antibody-avidin conjugate that recognizes and binds to the DOTA or biotin on the SHAL and also recognizes and binds to a cell surface receptor or protein that is not an MHC Class II protein targeted by the SHAL.

19. A method to kill or suppress the activity of an activated microglia, lymphocyte, dendritic cell or macrophage, comprising contacting the activated microglia, lymphocyte, dendritic cell or macrophage with an effective amount of a SHAL of structure from Group A,

Group B, or Group C, comprising two or more ligands from Table 1 and/or Table 2, or a derivative thereof.

20. The method of claim 19, the method treating a neurodegenerative disease or disorder comprising Alzheimer’s disease, Parkinson’s disease, Multiple Sclerosis, Amyotrophic Lateral Sclerosis, Frontotemporal Dementia or another microglia-mediated neurodegenerative disease, in a subject in need thereof, the method comprising administering to the subject an effective amount of the SHAL, or a derivative thereof.