WO2021127583A1 - Agents diagnostiques et thérapeutiques de ligands sélectifs à haute affinité - Google Patents

Agents diagnostiques et thérapeutiques de ligands sélectifs à haute affinité Download PDF

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WO2021127583A1
WO2021127583A1 PCT/US2020/066237 US2020066237W WO2021127583A1 WO 2021127583 A1 WO2021127583 A1 WO 2021127583A1 US 2020066237 W US2020066237 W US 2020066237W WO 2021127583 A1 WO2021127583 A1 WO 2021127583A1
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shal
group
cancer
cell
hla
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PCT/US2020/066237
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Monique BALHORN
Rodney Balhorn
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Shal Technologies, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/547—Chelates, e.g. Gd-DOTA or Zinc-amino acid chelates; Chelate-forming compounds, e.g. DOTA or ethylenediamine being covalently linked or complexed to the pharmacologically- or therapeutically-active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • 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.
  • HTLs selective high affinity ligands
  • 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.
  • 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 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.
  • 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.
  • efflux transporter proteins such as MDRl/P-gp and BCRP
  • 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”.
  • a Selective High Affinity Ligand 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 5 and R20 is and
  • Group C is of the structure: (Group C), wherein: R-21 is
  • R!!, R23, R2 6 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:
  • R 4 is H, NH2, N(CH 3 ) 2 , CO2, NH(CH 3 ), NO2 or CF 3 ;
  • Rs is H, NH2, NO2 or CH 3 ;
  • 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,
  • 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.
  • composition comprising, consisting essentially of, or consisting of the SHAL disclosed herein and a carrier, is provided.
  • 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 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, colorec
  • MHC Class II Major Histocompatibility
  • 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 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.
  • 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,
  • 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 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.
  • a method for inducing, enhancing or promoting an anti tumor immune response in a subject in need thereof 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.
  • 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.
  • a method for treating a disease or disorder related to a pathological immune response in a subject in need thereof 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.
  • 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, 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.
  • GAP GTPase activating protein
  • 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 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.
  • a method to inhibit cell growth or proliferation or to kill a cell by inhibiting AcetylCoA carboxylase 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.
  • ACC AcetylCoA carboxylase
  • 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, 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.
  • P -glycoprotein, MDR1 or P-gp multidrug resistance protein 1
  • BCRP breast cancer resistance protein
  • 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 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.
  • OATP organic-anion-transporting polypeptide
  • 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, 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.
  • UGT metabolic UDP-glucuronosyltransferase
  • a method to deliver one or more prodrugs to a cell 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.
  • 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.
  • 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, 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 melanom
  • 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, 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, gliom
  • 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, 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.
  • a method of treating cells, tissue, organs or tumors that do not express an MHC class II protein, in a subject in need thereof 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
  • a method of pre-targeting a SHAL to a cell, tissue, organ 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, 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.
  • 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, 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).
  • 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 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.
  • a method to kill or suppress the activity of an activated microglia, lymphocyte, dendritic cell or macrophage 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.
  • 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.
  • a kit is provided comprising, consisting essentially of, or consisting of the SHAL disclosed herein and instructions for use.
  • FIG. 1 The binding of a biotinylated form of the SHAL cancer therapeutic
  • DLBCL diffuse large B-cell lymphoma
  • FL follicular lymphoma
  • BL Burkitt’s lymphoma
  • SLL small lymphocytic lymphoma
  • MCL mantle cell lymphoma
  • MALTL mucosa-associated lymphoid tissue lymphoma
  • ALCL anaplastic large cell lymphoma
  • 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.
  • 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.
  • 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.
  • ADC antibody-drug conjugate
  • 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.
  • APCs antigen-presenting cells
  • Ii invariant chain
  • 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.
  • recognition of self antigen produces autoantibodies against a constituent of its own tissues.
  • 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).
  • 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.
  • FIG. 6 Structure of a polyvalent SHAL containing two SH7139 molecules linked together.
  • 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.
  • 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.
  • SC squamous cell carcinomas
  • A adenocarcinoma
  • Liver cancers hepatocellular carcinoma (HC), bile duct carcinoma (BDC) and clear cell carcinoma (CCC).
  • SC serous cystadenocarcinoma
  • EA endometrioid
  • MC mucinous cystadenocarcinoma
  • G granulosa cell tumor
  • T thecoma
  • U undifferentiated adenocarcinoma
  • 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).
  • 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.
  • 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
  • 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.
  • a cell includes a plurality of cells, including mixtures thereof.
  • 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.
  • 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.
  • animal refers to living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds.
  • mammal includes both human and non-human mammals.
  • 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.
  • 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).
  • 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.
  • a subject is a human.
  • a subject has or is suspected of having a cancer or neoplastic disorder.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • inert for example, a detectable agent or label
  • 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.
  • monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like
  • disaccharides such as lactose, sucrose,
  • compositions used in accordance with the disclosure can be packaged in dosage unit form for ease of administration and uniformity of dosage.
  • 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.
  • solutions 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.
  • nucleic acid sequence and “polynucleotide” are used interchangeably to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides.
  • 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.
  • binding refers 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • small organic molecule refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals.
  • 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.
  • 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).
  • a “disulfide bond” as used herein a refers to a functional group with the structure R'-S-S-R 2 , wherein R 1 and R 2 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.
  • 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 R 1 and/or R 2 or fragments of either or both thereof, wherein the released R 1 and/or R 2 or fragments of either or both thereof comprise free SH (thiol) groups.
  • Suitable period of time 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.
  • 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.
  • the SHAL comprises, consists essentially of, or consists of two or more ligands that bind their target with low affinity (e.g., ⁇ 10 6 M and/or dissociates within seconds or less) that, when coupled together, form a SHAL that binds the target with high affinity (e.g.,
  • 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.
  • 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.
  • SHAL refer to SHALs consisting of two ligands, SHALs consisting of three ligands, respectively, and so forth (e.g., tetradentate, pentadentate).
  • polyvalent SHAL refers to a molecule in which two or more
  • SHALs e.g., two or more bidentate, tridentate, and so forth SHALs
  • 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).
  • 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.
  • 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.
  • 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]
  • 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.
  • 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,
  • GTPase-activating protein or “GTPase-accelerating protein”
  • GAP GTPase Activating Protein 1
  • OATP organic-anion-transporting polypeptide
  • UDP-glucuronosyltransferase 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.
  • 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.
  • small-molecule antibody-drug conjugate 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.
  • 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.
  • Normal cell refers to healthy cells, not experiencing proliferative dysfunction or cancer.
  • Lym-1 refers to an antibody that targets a conformational epitope on the beta-subunit of Human Leukocyte Antigen-antigen D Related (HLA-DR) proteins.
  • HLA-DR Human Leukocyte Antigen-antigen D Related
  • MHC Class II protein refers to a class of major histocompatibility complex
  • MHC myelogenous leukemia
  • Genbank ncbi.nlm.nih.gov/genbank/ and Nucleotide database: ncbi.nlm.nih.gov/nucleotide/. All databases last accessed on December 19, 2019.
  • HLA-DR Human Leukocyte Antigen-antigen D Related
  • 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.
  • TCR T-cell receptor
  • 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.
  • HLA-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).
  • 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 (Uniprot
  • Genbank ncbi.nlm.nih.gov/genbank/ and Nucleotide database: ncbi.nlm.nih.gov/nucleotide/. All databases last accessed on December 19, 2019.
  • 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.
  • Genbank ncbi.nlm.nih.gov/genbank/ and Nucleotide database: ncbi.nlm.nih.gov/nucleotide/. All databases last accessed on December 19, 2019.
  • “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.
  • 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.
  • 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.
  • 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.
  • “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.
  • 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.
  • 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).
  • 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.
  • carrier 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.
  • suitable pharmaceutical carrier vehicle
  • 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.
  • 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).
  • a “nanoparticle,” as used herein, refers to particles between 1 and
  • 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
  • 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.
  • 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.
  • cancer markers refers to biomolecules such as proteins that are useful in the diagnosis, prognosis and treatment of cancer.
  • 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,
  • 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.
  • 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; Sblul 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.
  • 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),
  • 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.
  • 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.
  • “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.
  • residue refers to natural, synthetic, or modified amino acids.
  • 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.
  • 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.
  • Antibodies exist as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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).
  • 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.
  • 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).
  • a “reporter” is an effector that provides a detectable signal (e.g., a detectable label).
  • the reporter need not provide the detectable signal itself, but can simply provide a moiety that subsequently can bind to a detectable label.
  • “Microglia-mediated neurodegenerative disease” may include any disease mediated by dysfunction of the microglia.
  • the disease may include neuropathic pain, neuroinflammation, amyloid deposition, tau protein deposition, and the like.
  • conservative amino acid 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).
  • epitopope 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.
  • 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.
  • 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 glut
  • 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.
  • the HNK-1 carbohydrate epitope the HNK-1 carbohydrate epitope, the HA epitope, the HSV epitope, the His4, Hiss, and His 6 epitopes that are recognized by the His epitope specific antibodies (see, e.g., Qiagen Inc., Germantown, MD), and the like.
  • vectors for epitope tagging proteins are commercially available.
  • 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.
  • Label 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.
  • a “PEG type linker” refers to a linker comprising a polyethylene glycol
  • Immuno 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.
  • GM-CSF Granulocyte-macrophage colony-stimulating factor
  • 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,
  • the term “immune response” may be used to encompass the formation of a regulatory network of immune cells.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 125 1.
  • 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.
  • 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.
  • 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.
  • default parameters are used for alignment.
  • a preferred alignment program is BLAST, using default parameters.
  • 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.
  • the preferred algorithms can account for gaps and the like.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • an equivalent thereof is a polynucleotide that hybridizes under stringent conditions to the reference polynucleotide or its complement.
  • 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.
  • 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.
  • default parameters are used for alignment.
  • a preferred alignment program is BLAST, using default parameters.
  • 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.
  • 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.
  • 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.
  • isolated refers to molecules or biologicals or cellular materials being substantially free from other materials.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • treatment is an approach for obtaining beneficial or desired results, including clinical results.
  • 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.
  • 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.
  • treatment excludes prophylaxis.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • lysine, glutamic acid, aspartic acid or an aminohexanoic acid and polyethylene glycol (PEG) based linkers different length are used to couple the ligands.
  • PEG polyethylene glycol
  • 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.
  • 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).
  • HLA-DRs 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.
  • 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 U1 ln) and used to test the SFLALs for their tissue biodistribution and clearance rate in test organisms (e.g., mice).
  • a metal radioisotope such as U1 ln
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • desired target e.g., HLA-DRs
  • 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.
  • 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).
  • 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.
  • 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.,
  • 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.
  • an anti-tumor response e.g., activation of CD4+ and CD8+ T-cells
  • 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).
  • NMR spectroscopy see e.g., Cosman, M, Lightstone
  • SHAL 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.
  • SPR surface plasmon resonance
  • 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.
  • 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.
  • 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.
  • PBMCs peripheral blood mononuclear cells
  • 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.
  • 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 90 Yttrium, etc.).
  • b-emitters can be incorporated into the DOTA chelators to provide more localized radiation damage.
  • other effectors can be added to the SHAL in place of the DOTA that become active when internalized.
  • 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.
  • 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).
  • 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.
  • 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.
  • the experiment to experiment variation that is often encountered when different samples are screened for binding independently can be minimized.
  • 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.
  • human tissues 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.
  • non-Hodgkin’s lymphoma diffuseuse 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, larynge
  • 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).
  • slides containing a microarray of biopsy cores taken from 75 different patients diagnosed with ovarian cancer were purchased from a commercial source (US Biomax, Rockville, MD).
  • the slides 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.
  • a digital image containing the array of cores for the two slides 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.
  • a SHAL 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.
  • the SHAL is coupled to an imaging reagent that permits non-invasive imaging and thereby permit the evaluation of real time pharmacody nami cs .
  • 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 U1 ln or 90 Y 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 U1 ln or 90 Y 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.
  • 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.
  • 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.
  • target receptors e.g., HLA-DRs
  • 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.
  • a protocol is selected that provides the optimal dose level using information on the therapeutic indices for tumor to normal tissue.
  • 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.
  • hydrophilic e.g., polyethylene glycols or polyamines
  • cleavable bonds e.g., a peptide, disulfide, or other cleavable linker
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 1)
  • R is a label or a tag or an effector, for example, selected from Table 4, L is a ligand, n *1
  • 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
  • Group C is of the structure: (Group C), wherein:
  • R22, R23, R26 and R27 are each independently
  • R24 and R25 are each independently
  • each ligand L is independently selected from Li, L2,
  • R 4 is H, NH 2 , N(CH 3 )2, CO2, NH(CH 3 ), NO2 or CF 3 ;
  • Rs is H, NH2, NO2 or CH 3 ;
  • R6 is any one of:
  • R7 is H, Cl, or F
  • Ri2 is H, methyl, Cl, NH2,
  • Ri3 is H, methyl, Cl, NH2, or
  • 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.
  • the SHAL comprises a ligand of 3-(3-((3-chloro-5-
  • the SHAL comprises, consists essentially of, consists of, or is of a structure selected from the following:
  • 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.
  • 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.
  • 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.
  • the SHAL comprises, consists essentially of, consists of, or is of a structure selected from specimen group A1 :
  • SH7139 (D-Lys), SH8043 (L-Lys) and SH8039 ( 13 C 6 - 15 N 2 -Lys), and
  • 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, 13 C6- 15 N2-Lys comprises, consists essentially of, or consists of isotopic lysine comprising, consisting essentially of, or consisting of C-13 and N-15 isotopes.
  • the SHAL comprises, consists essentially of, or consists of a SHAL of Specimen Group A2: wherein * denotes site of attachment to the nitrogen.
  • the SHAL comprises, consists essentially of, or consists of a SHAL of Specimen Group A3:
  • the SHAL comprises, consists essentially of, or consists of a SHAL of Specimen Group B1 : wherein * denotes site of attachment to the nitrogen.
  • the SHAL comprises, consists essentially of, or consists of a SHAL of Specimen Group B2: wherein * denotes site of attachment to the nitrogen.
  • the SHAL comprises, consists essentially of, or consists of a SHAL of Specimen Group B3:
  • the SHAL comprises, consists essentially of, or consists of a SHAL of Specimen Group Cl :
  • the SHAL comprises, consists essentially of, or consists of a SHAL of Specimen Group C2:
  • the Ligands (L) maybe selected from any ligand represented in Table 1.
  • 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.
  • SHALs of Group B, and/or R24 of SHALs of Group C are each independently selected from Table 3:
  • 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:
  • all of the Ligands (L) of the SHAL are the ligand of Table
  • 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
  • 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.
  • 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.
  • 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.
  • a “package” e.g., a chelate, a liposome, a nanoparticle, a dendrimer, a polymer microbead, etc.
  • 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., DynabeadsTM), 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., 3 H, 125 1, 35 S, 14 C, 32 P, 18 F, 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.
  • fluorescent dyes e.g., fluorescein isothiocyanate, Texas red, rhodamine, green fluorescent protein, Alexa Fluor, acridine
  • Radiolabels include, but are not limited to "Tc, 203 Pb, 67 Ga,
  • 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.
  • the effector can be a radiosensitizer that enhances the cytotoxic effect of ionizing radiation (e.g., such as might be produced by 60 Co or an x-ray source) on a cell.
  • radiosensitizing agents 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.
  • 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.
  • the most important types of decay are gamma emission, beta decay, alpha decay, and electron capture.
  • beta particles deposit most of their energy within a few millimeters of the point of decay.
  • Beta emissions from radionuclides such as 131 I or 90 Y that have targeted antigen-positive tumor cells can kill nearby antigen-negative tumor cells through a “crossfire” effect.
  • 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.
  • 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 212 Bi, 213 Bi, 211 At, and the like.
  • 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.
  • 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.
  • 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.
  • PLGA poly(DL-lactide-co-glycolide)
  • albumin polyethylene glycol-lipid conjugates or other amphiphilic molecules.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the ligand is an ion, metal atom or a molecule that binds to another molecule.
  • the SHALs comprise, consist essentially of, or consist of one or more small molecule ligands from Table 1.
  • a ligand from Table 1 binds to the chelating group in a SHAL or is attached to the SHAL free amine.
  • the ligand is selected from Table 1 and/or Table 2.
  • 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.
  • chelating groups are well known to those of skill in the art.
  • 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-
  • EDTA
  • Examples of certain preferred chelators include unsubstituted or, substituted 2- iminothiolanes and 2-iminothiacyclohexanes, in particular 2-imino-4- mercaptomethylthiolane.
  • chelating agent 1,4,7,10-tetraazacyclododecane-N, N, N”, N’”-tetraacetic acid (DOTA)
  • DOTA 1,4,7,10-tetraazacyclododecane-N, N, N”, N’”-tetraacetic acid
  • U.S. Pat. No. 5,428,156 teaches a method for conjugating DOTA to antibodies and antibody fragments.
  • 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.
  • the macrocyclic chelating agent 1,4,7,10- tetraazacyclododecane-N,N’,N”,N”’-tetraacetic acid binds 90 Y and U1 ln 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:
  • 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.
  • 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.
  • 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.
  • DT diphtheria toxin
  • exotoxin A from Pseudomonas aeruginosa
  • ricin from Pseudomonas aeruginosa
  • abrin from Pseudomonas aeruginosa
  • modeccin from Pseudomonas aeruginosa
  • alpha-sacrin alpha-sacrin
  • Pokeweed antiviral protein S Pokeweed antiviral protein type II
  • curcin Hercin
  • Pseudomonas exotoxin A 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).
  • EF-2 elongation factor 2
  • diphtheria toxin 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).
  • 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.
  • Suitable effector molecules include pharmacological agents or encapsulation systems comprising, consisting essentially of, or consisting of various pharmacological agents.
  • 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.
  • doxorubicin vinblastine
  • genistein genistein
  • diclofenac diclofenac
  • kinase and PARP inhibitors such as lenvatinib, adpelisib, veliparib, lenalidomide, sorafenib, acalabrutinib, axitinib, lorlatinib, noraparib, aplutamide, gilteritin
  • 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.
  • 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
  • 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),
  • 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 doxor
  • 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 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.
  • MHC Class II Major Histocompatibility Complex Class II
  • 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.
  • 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.
  • the cancer cell or 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.
  • the cancer cell or tumor (such as a solid tumor) is not an invasive ductal breast cancer and/or a liver cancer.
  • the cancer cell or tumor (such as a solid tumor) does not express or comprise either or both of the transporters, OATP1B1 and OATP1B3.
  • the cancer cell or 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.
  • 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.
  • the cancer cell and tumor (such as a solid tumor) has been or is being concurrently contacted and/or treated with IFN-g.
  • 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.
  • the method also inhibits metastasis of the cancer to the lymph nodes or other organs of the body.
  • the cancer cell does not express HLA-DRIO or an HLA-DR comprising, consisting essentially of, or consisting of a Lym-1 epitope.
  • the cancer cell does not express one or more MHC Class II proteins.
  • 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.
  • a molecule such as a protein expressed by cancer cells whose function is required for tumor growth and survival.
  • 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 contacting is in vitro or in vivo.
  • the cancer cell is a mammalian cancer cell.
  • 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.
  • scanning mass spectrometry e.g. for example MALDI mass spectrometry
  • PET scan
  • 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.
  • 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 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.
  • 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.
  • 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.
  • the SHAL inhibits the growth of the tumor or progression of the cancer or kills the cancer cells.
  • 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.
  • the cancer cells or solid tumor does not express MHC class II proteins.
  • 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, ifosf
  • a method for inducing, enhancing or promoting an anti tumor immune response in a subject in need thereof comprising, consisting essentially of, or consisting of administering to the subject an effective amount of a SHAL as disclosed herein.
  • 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.
  • 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.
  • 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, hist
  • 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.
  • 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 comprising, consisting essentially of, or consisting of contacting the cell with an effective amount of a SHAL as disclosed herein.
  • 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.
  • 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.
  • 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 comprising, consisting essentially of, or consisting of administering to the subject an effective amount of a SHAL as disclosed herein.
  • 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.
  • 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.
  • the cells are normal cells or cancer cells.
  • 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.
  • 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.
  • 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.
  • treatment is an approach for obtaining beneficial or desired results, including clinical results.
  • 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.
  • 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.
  • 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.
  • the SHAL inhibits the growth of the tumor or progression of the cancer, or kills the cancer cells.
  • 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.
  • 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;
  • 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
  • Antibodies such as L-243 that recognize the alpha subunit of HLA-DR 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.
  • 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.
  • 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, 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.
  • 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.
  • 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
  • another method for treating cancer cells or tumors that do not express MHC Class II proteins 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.
  • a method for reversing or blocking the development of drug resistance in bacteria or fungi infecting a subject 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • treatment is an approach for obtaining beneficial or desired results, including clinical results.
  • 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.
  • Radioimmunotherapy 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.
  • radionuclides e.g. 68 Ga, 90 Y, U1 ln
  • other molecular species e.g. 18 F
  • 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.
  • a radionuclide is bound by the DOTA chelating group.
  • the SHAL is isotopically labeled.
  • 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.
  • oral such as a tablet, capsule or suspension
  • transdermal such as a tablet, capsule or suspension
  • vaginal vaginal
  • rectal subcutaneous intravenous, intraarterial, intramuscular, intraosseous, intraperitoneal, epidural and intrathecal.
  • subcutaneous intravenous, intraarterial, intramuscular, intraosseous, intraperitoneal, epidural and intrathecal 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.
  • 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).
  • 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 SHALs target e.g. cancer cells, activated lymphocytes, macrophages or dendritic cells.
  • 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).
  • 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.
  • 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).
  • a method to treat an MHC class II protein linked autoimmune disease or disorder 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.
  • 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 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.
  • helper CD4+
  • CD8+ killer lymphocytes.
  • 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.
  • a method for treating a disease or disorder related to a pathological immune response in a subject in need thereof comprising, consisting essentially of, or consisting of administering to the subject an effective amount of a SHAL as disclosed herein.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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:
  • 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.
  • 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.
  • 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.
  • 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.).
  • 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.
  • 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).
  • a method to treat an HLA-DR or MHC class II protein linked neurodegenerative 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.
  • 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 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,
  • the SHAL comprises, consists essentially of, or consists of an effector that is selected from Group R in Table 4.
  • the SHAL is delivered across the blood-brain barrier in a nanoparticle, liposome, micelle, or hydrogel.
  • 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,
  • 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.
  • GAP GTPase Activating Protein
  • ACC AcetylCo-carboxylase
  • GTPase-activating proteins or GTPase-accelerating proteins 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 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.
  • 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 comprising, consisting essentially of, or consisting of contacting the GAP with an effective amount of a SHAL as disclosed herein.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • Acetyl-CoA carboxylase 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).
  • BC biotin carboxylase
  • CT carboxyltransferase
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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,
  • the second therapy is a therapy known to the skilled artisan for the treatment of diabetes or obesity.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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 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.
  • 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.
  • 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.
  • 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.
  • the SHAL’s inhibition of these transporters will reverse the resistance tumor cells have already developed to these drugs.
  • 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.).
  • 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 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.
  • 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.
  • 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, 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.
  • 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.
  • 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.).
  • another drug e.g., for example doxorubicin, Imatinib, thienorphine, crizotinib, topotecan, docet
  • 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.
  • 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.
  • 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.
  • the cell expresses MHC class II proteins.
  • the cell does not express MHC Class II proteins.
  • the cell is a normal cell or a cancer cell.
  • 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.
  • the contacting is in vitro or in vivo.
  • 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.
  • a method to deliver one or more prodrugs to a cell 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.
  • 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.
  • 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.
  • 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.
  • the SHAL ligands target the target protein or cell with the SHAL acting as a compact small-molecule antibody-drug conjugate or ADC.
  • the cell does not express MHC Class II proteins.
  • the cell is a normal cell or a cancer cell.
  • 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.
  • the contacting is in vitro or in vivo.
  • 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.
  • compositions of this invention when administered orally, can be protected from digestion.
  • an appropriately resistant carrier such as a liposome or nanoparticle.
  • 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 pharmaceutically acceptable carrier preferably an aqueous carrier.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the therapeutic compositions of this invention can be administered directly to the tumor site.
  • brain tumors can be treated by administering the therapeutic composition directly to the tumor site (e.g., through a surgically implanted catheter).
  • the therapeutic composition can be placed at the target site in a slow release formulation.
  • a slow release formulation 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.
  • the catheter or time release formulation will be placed at the tumor site as part of a surgical procedure.
  • the perfusing catheter or time release formulation can be placed at the tumor site as an adjunct therapy.
  • the delivery of the therapeutic compositions of this invention may comprise, consist essentially of, or consist of the primary therapeutic modality.
  • the various reaction ingredients can then be offered to the user in the form of a so-called “kit.”
  • 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.
  • kits 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.
  • 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.
  • 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.
  • 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.
  • the complex-forming reaction with the SHAL can simply be produced by combining the components in a neutral medium and causing them to react.
  • the effector may be presented to the SHAL in the form of a chelate or chemically activated effector.
  • kit constituent(s) are used as component(s) for pharmaceutical administration (e.g., as an injection liquid) they are preferably sterile.
  • the constituent(s) are provided in a dry state, the user should preferably use a sterile physiological saline solution as a solvent.
  • 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.
  • 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.
  • 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, .
  • 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 (Hydr
  • 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.
  • DIEA N,N-Diisopropylethylamine
  • 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
  • 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.
  • 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 %
  • Percentage growth inhibition is calculated as:
  • 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.
  • 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.
  • SAHRP streptavidin horse-radish peroxidase
  • HLA typed PBMCs were obtained from four commercial sources - AllCells
  • a stock solution of SH7129 was prepared by dissolving 10 mg of the dry
  • 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).
  • SAHRP Streptavidin-horse radish peroxidase
  • 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.
  • 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.
  • 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.
  • TMAs tumor microarrays
  • 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.
  • SH7129 100 pg/ml in PBS, 1% DMSO
  • 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).
  • SAHRP Streptavidin-horse radish peroxidase
  • 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.
  • Applicants analyses of biopsy samples obtained from patients diagnosed with seven subtypes of non-Hodgkin’s lymphoma 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
  • 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
  • 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.
  • 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.
  • 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.
  • MgcRacGAP (residues 345-618) at 2 nM
  • BCR GAP (residues 1010-1271) at 200 nM
  • 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 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
  • 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).
  • 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.
  • PHERAstar FS BMG Labtech
  • 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
  • UGT UGT
  • 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.
  • UGT inhibitors can be used as an adjuvant to modulate the activity of the drugs being used to treat the disease.
  • SH7139 was tested for activity in inhibiting UGT1 Al, UGT1 A3, UGT1 A4,
  • 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).
  • a UGT isoform-specific probe substrate (10-50 pM, estradiol for UGT1A1, sulindac sulfone for UGT1A3, trifluoperazine for UGT1 A4, nap
  • Table 10B IC50 for the inhibition of the various UGTs by other inhibitors.
  • BCRP breast cancer resistance protein
  • MDR1 Multidrug resistance protein 1
  • P-gp P-glycoprotein 1
  • Multidrug resistance protein 1 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).
  • 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.).
  • prescription drugs e.g., Risperidone, Thienorphine, Imipramine, Paroxetine, etc.
  • oncology drugs used to treat cancer patients e.
  • 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
  • 3 H-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 3 H-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.
  • 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).
  • 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.
  • 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,
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • ACC inhibition 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).
  • 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).
  • SHALs containing Ct SH7133 and SH7097
  • SHALs that do not inhibit ACC would have other applications that only require their binding to HLA-DRs (e.g. treatment of autoimmune diseases).
  • ACC/FAS coupled scintillation proximity assay (SPA) (Id).
  • SPA ACC/FAS coupled scintillation proximity assay
  • 25 m ⁇ of recombinant ACC and FAS enzymes and 5 m ⁇ of compound in 3% DMSO or 3% DMSO as control were added to a 384-well FlashPlate (50 m ⁇ total well volume) and incubated for 10 minutes, after which the reaction was started by the addition of 20 m ⁇ 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).
  • 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.
  • Example 12 Therapeutic applications of the selective high affinity ligand drug SH7139 extend beyond NHL to many other types of solid cancers
  • SH7139 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.
  • 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
  • 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 Tissue and tumor microarrays
  • 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).
  • TMAs tumor microarrays
  • 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 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.
  • Bound SH7129 (IntDensH7i29 - IntDensH7i29Bkg) - (IntDenNoSH7i29 - IntDenNoSH7129Bkg)
  • 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
  • IntDenNoSH7i29Bk g is the mean of the integrated density of the ten blank regions of the control slide processed for staining without SH7129.
  • 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.
  • DAB 3,3-diaminobenzidine tetrahydrochloride
  • 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.
  • 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.
  • 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.
  • 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 10 6 hepatocytes (Burt et al., 2016, Drug Metab Dispos.
  • 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.
  • ACL anaplastic large cell lymphoma
  • SH7129 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.
  • 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
  • MCL mantle cell lymphomas
  • Ovarian, colorectal, prostate and cervical cancers exhibited the highest levels of SH7129 binding of all the solid tumors examined.
  • 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.
  • Example 13 The small molecule antibody mimic SH7139 targets a family of HLA-DRs expressed by B-cell lymphomas and other solid cancers
  • 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.
  • SH7129 binding to PBMCs has revealed that other HLA-DRs are also targeted by the drug.
  • TMAs Tumor microarrays 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.
  • HLA typed PBMCs were obtained from four commercial sources - AllCells
  • 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 reagent
  • 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.
  • DIEA N,N- Diisopropylethylamine
  • biotinyl-OSu solid biotin N-hydroxysuccinimide ester
  • PBMC Peripheral blood mononuclear cells
  • 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.
  • DPBS Dulbecco’s Phosphate Buffered Saline
  • FACS cold fluorescence-activated cell sorting
  • Lym- 1 antibody 50 m ⁇ 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.
  • 50 m ⁇ 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 m ⁇ 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 m ⁇ 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.
  • a stock solution of SH7129 was prepared by dissolving 10 mg of the dry
  • 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).
  • 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.
  • FCS fetal calf serum
  • 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.
  • 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.
  • 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.
  • 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).
  • Table 14 Correlation between Lym-1 binding and HLA-DR expression by lymphoma cell lines.
  • Relative expression (Lym-1 MFI/cell for each cell line divided by the Lym-1 MFI/cell for Raji) X 100.
  • c Percent DRB1 mRNA expression relative to Raji, Boegel etal. (Boegel etal ., 2014, Oncoimmunology. 2014; 3:e954893)
  • Holling etal. Holling etal. (Holling et al., 2004, Blood.
  • 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.
  • 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.
  • SAHRP streptavidin-horse radish peroxidase
  • the tissue sections were not counterstained.
  • the hematoxylin counterstaining step 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.
  • 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).
  • DLBCL variant A centroblastic lymphoma that did not bind Lym-1, also failed to bind SH7129.
  • SH7129 also retains the anti-tumor activity of SH7139
  • 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.
  • 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.
  • 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.
  • 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,
  • CLL chronic lymphocytic leukemia
  • 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.
  • 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).
  • 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.
  • HLA-DR structures used for docking contained both the invariant b- and variant b subunits.
  • HLA-DR11 and HLA-DR15 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.
  • 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.
  • 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).
  • 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, b ⁇ 67, 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.
  • HLA-DR7 did not bind to HLA-DR4.
  • 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.
  • HLA-DR1 variants containing the DRB 1*01:01 and DRB 1*01:02 b-subunits have two amino acid substitutions (b ⁇ IIE 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.
  • 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.
  • HLA-DR14 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.
  • 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.
  • 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.
  • 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
  • the PA74R substitution a change from a hydrophobic alanine to highly positive- charged arginine
  • the bT77N substitution a change from a polar threonine containing a partial negative charge to an asparagine containing a partial positive charge
  • the bP3H substitution a change from a hydrophobic phenylalanine to a positive charged histidine
  • 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
  • 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.
  • 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.
  • CIRT concurrent chemo-immuno-radiotherapy
  • NPs bound selectively to different HLA-DR- overexpressed human lymphoma cells, cross-linked the cell surface HLA-DR, and triggered the internalization of NPs.
  • 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).
  • ICD immunogenic cell death
  • 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.
  • ICD immunogenic cell death
  • DMSO dimethyl sulfoxide
  • TAA triethyl-amine
  • 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
  • bovine serum albumin fraction V lyophilized powder
  • 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.
  • GPC gel-permeation chromatography
  • the degree of functionalized PLA-PEG (dissolved in a known amount of DMSO) was calculated from the extinction coefficient of SHAL at 452 nm.
  • 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.
  • 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.
  • PLGA NPs Drug-free Rhod-labeled SHAL-functionalized NPs composed of 1 wt/wt% of PLGA-Rhod were prepared via a nanoprecipitation method.
  • 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.
  • TEM 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.
  • 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 also known as hydrodynamic diameter
  • 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.
  • 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.
  • 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.
  • 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.
  • MDR-1 neutrophil-specific p53
  • CD243 neutrophil-specific p53 p53 p53 p53 p53 p53 p53 p53 p53 p53 p53 p53 p53 p53 p53 p53 p53 p53 p53 p53 p53 p53 p53 p53 p53 p53 p53 p53 p53 p53 p53 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
  • FACS assay quantified the binding affinity rhodamine- labeled SHAL-functionalized PEG-PLGA NPs. Briefly, FACS buffer-washed cells (1 c 10 6 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.
  • Daudi or Raji cells (10 x 10 6 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.
  • 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.
  • 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 10 6 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+
  • dead cells were defined as AV-PI+.
  • 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.
  • 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.
  • DBS dsDNA breaks
  • X-RAD 320 X-ray irradiator Precision X-ray Inc., CT
  • 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.
  • 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.
  • A488- labeled antihuman HLA-DR antibody clone L243
  • 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).
  • mice 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.
  • ketamine hydrochloride/xylazine hydrochloride solution Sigma; St Louis, MO
  • Xenograft tumors were inoculated in the flank of male Nu mice via the subcutaneous injection of 2 x 10 6 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.
  • 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.
  • 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
  • Mice in groups (3), (5) and (7) were euthanized 72 h after administration of therapeutics.
  • xenograft tumor, circulating blood and key organs were preserved.
  • 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.
  • Xenograft tumors were established via subcutaneous injection of 2 x 10 6 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).
  • 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
  • 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.
  • the Daudi xenograft tumors were established via subcutaneous injection of 2x 10 6 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.
  • 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.
  • 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.
  • Xenograft tumors were estab-lished via subcutaneous injection of 2 x 10 6 Raji cells in 200 pL of a 1 : 1 (v/v) mixture of a serum-free RPMI1640/Matrigel solution in the left flank.
  • 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.
  • 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.
  • Statistical Analysis Quantitative data were expressed as mean ⁇ SEM.
  • 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.
  • TEM transmission electron microscopy
  • DLS dynamic light scattering
  • 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.
  • 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.
  • FACS fluorescence-activated cell sorting
  • 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 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.
  • 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).
  • 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.
  • 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.
  • the calreticulin expression remained relatively constant 24 h after initial treatment in all treatment groups.
  • Untreated Raji cells showed very stable HLA-DR expression (M.F.I. ⁇ 4.4 x 105).
  • 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 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.
  • 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.
  • 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.
  • 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.
  • the low HLA-DR expression could explain this effect in the Ramos cells.
  • 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.
  • 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.
  • 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.
  • the Raji xenograft model was more resistant to chemotherapy with DOX, which only induced transient response followed by rapid tumor progression and death.
  • 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.
  • 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:
  • Ri and R3 are each independently
  • Group B is of the structure: (Group B), wherein: Ri9 is
  • 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(CH 3 ) 2 , CO2, NH(CH 3 ), NO2 or CF 3 ;
  • Rs is H, NH2, NO2 or CH 3 ;
  • R6 is any one of:
  • R7 is H, Cl, or F
  • a 2 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
  • Ri 6 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.
  • Embodiment 2 The SHAL of Embodiment 1 further comprising a label or tag or effector from Group R from Table 4.
  • Embodiment 3 The SHAL of Embodiment 1 having the structure of any of the compounds from Specimen-Group-A2.
  • Embodiment 4 The SHAL of Embodiment 1 having the structure of any of the compounds from Specimen-Group-A3.
  • Embodiment 5 The SHAL of Embodiment 1 having the structure of any of the compounds from Specimen-Group-B2.
  • Embodiment 6 The SHAL of Embodiment 1 having the structure of any of the compounds from Specimen-Group- Specimen-Group-B 3.
  • Embodiment 7 The SHAL of Embodiment 1 having the structure of any of the compounds from Specimen-Group- Specimen-Group-C2.
  • 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.
  • 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.
  • Embodiment 10 A composition comprising the SHAL of any one or more of Embodiments 1-9 and a carrier.
  • Embodiment 11 The composition of Embodiment 10, wherein the carrier is a pharmaceutically acceptable carrier.
  • 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.
  • MHC Class II Major Histocompatibility Complex Class II
  • 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.
  • 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.

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Abstract

La présente invention concerne de nouveaux ligands sélectifs à haute affinité (SHAL) et des méthodes pour leur utilisation dans le traitement du cancer, d'une maladie auto-immune et de troubles liés à l'obésité. L'invention concerne également des méthodes de modulation de la sensibilité et du métabolisme d'un médicament chez un sujet comprenant l'administration de SHAL.
PCT/US2020/066237 2019-12-20 2020-12-18 Agents diagnostiques et thérapeutiques de ligands sélectifs à haute affinité WO2021127583A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117777296A (zh) * 2024-02-28 2024-03-29 北京肿瘤医院(北京大学肿瘤医院) B7h3亲和体及其诊疗核素标记物的制备方法与应用

Citations (2)

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Publication number Priority date Publication date Assignee Title
WO2009132020A2 (fr) * 2008-04-21 2009-10-29 The Regents Of The University Of California Ligands polydentés sélectifs à haute affinité et leurs procédés de production
WO2014202775A1 (fr) * 2013-06-21 2014-12-24 Innate Pharma Conjugaison enzymatique de polypeptides

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Publication number Priority date Publication date Assignee Title
WO2009132020A2 (fr) * 2008-04-21 2009-10-29 The Regents Of The University Of California Ligands polydentés sélectifs à haute affinité et leurs procédés de production
WO2014202775A1 (fr) * 2013-06-21 2014-12-24 Innate Pharma Conjugaison enzymatique de polypeptides

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117777296A (zh) * 2024-02-28 2024-03-29 北京肿瘤医院(北京大学肿瘤医院) B7h3亲和体及其诊疗核素标记物的制备方法与应用
CN117777296B (zh) * 2024-02-28 2024-05-28 北京肿瘤医院(北京大学肿瘤医院) B7h3亲和体及其诊疗核素标记物的制备方法与应用

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