Antibodies and immunoconjugates for individualized therapy

Abstract

The disclosure relates to avian Immunoglobulin Y (IgY) antibodies and antigen binding fragments, variants, multimeric versions, or bispecifics thereof that specifically bind to an antigen (such as a human tumor associated antigen), as well as methods of making and using said antibodies and antigen binding fragments thereof in a variety of therapeutic, diagnostic and prophylactic indications.

Description

[0001] ANTIBODIES AND IMMUNOCONJUGATES FOR

[0002] INDIVIDUALIZED THERAPY

[0003] RELATED APPLICATIONS

[0004] This application claims the benefit of priority to U.S. Provisional Patent Application serial number 63 / 546,877, filed November 1, 2023.

[0005] BACKGROUND

[0006] Antibodies and immunoconjugates have a wide range of applications in therapeutical products. Diverse platforms can be used for the production of antibodies and immunoconjugates for industrial and therapeutic uses including bacteria, yeast, fungi, plant, mammalian, and insect cells. However, these different host systems have several limitations. Some of these limitations are issues with post-translational modifications or high cost. Therefore, there has been a long-felt but unmet need in the art for a method producing and isolating antibodies, such as IgY antibodies, from avian eggs.

[0007] SUMMARY

[0008] While most chickens have lost their capacity to produce diverse polyclonal antibodies, this disclosure provides a superior method of producing and isolating IgY antibodies from eggs derived from native avian species, forming conjugates thereof, and methods of using and producing the same. This disclosure provides an antibody or antigen binding fragment thereof derived from an avian egg that specifically binds to a human tumor associated antigen, wherein the avian egg is injected with a cancer cell and a jasmonate or derivative thereof.

[0009] In one aspect, described herein is a method of producing a population of polyclonal antibodies that specifically bind to an antigen. The method may comprise administering the antigen to a hen or to an avian egg produced; administering a jasmonate compound to the hen or the avian egg; and isolating the population of polyclonal antibodies from the yolk and / or the chorionic amnion membrane of the egg. In some embodiments, the hen is kept under specified pathogen-free (SPF) condition. In some embodiments, the hen is 1 week old, 2 weeks old, 3 weeks old, 4 weeks old, 5 weeks old, 6 weeks old, 7 weeks old, 8 weeks old, 9 weeks old, 10 weeks old, 11 weeks old, 12 weeks old, 13 weeks old, 14 weeks old, 15 weeks old, 16 weeks old, 17 weeks old, 18 weeks old, 19 weeks old, and 20 weeks old. In some embodiments, the antigen is administered to the hen subcutaneously or intramuscularly. In some embodiments, the antigen is administered to the egg’s allantoic chorion membrane. In some embodiments, the antigen is 50 pg to 1,000 pg. In some embodiments, the antigen is 20 pg to 200 pg. In some embodiments, the antigen is a protein expressed on a cancer cell. In some embodiments, the cancer cell is selected from a group consisting of a lung cancer cell, a bronchial cancer cell, a prostate cancer cell, a breast cancer cell, a colorectal cancer cell, a pancreatic cancer cell, an ovarian, a leukemia cancer cell, a lymphoma cancer cell, an esophageal cancer cell, a liver cancer cell, a urinary and / or bladder cancer cell, a renal cancer cell, an oral cavity cancer cell, a pharyngeal cancer cell, a uterine cancer cell and a melanoma cancer cell.

[0010] In some embodiments, the antigen is a tumor associated antigen. In some embodiments, the tumor associated antigen is CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, c-Met; PSMA, Glycolipid F77, EGFRvllI, ganglioside GD-2, NY-ESO-1, MAGE A3S, ADAM9, ALCAM, Alkaline phosphatase, placental like 2 (ALPPL2), Androgen receptor, AXL, Beta- 1,4-N-acetyl-galactosaminyltransferase 1 (B4GALNT1), carcinoembryonic antigen, Carbonic anhydrase IX (CA9), CD74, CD117 (KIT), CD274, CD276, CD44, CD46, CD133, Cadherin 6 (CDH6), Carcinoembryonic antigen related cell adhesion molecule 5 (CEACAM5), CLDN6, claudin 18.2 (CLDN18.2), claudin 18.1 (CLDN18.1), CTAG1B, Delta like canonical Notch ligand 3 (DLL3), Desmoglein 2 (DSG2), EGFR, Epithelial cell adhesion molecule (EPCAM), EphA2, Erb-b2 receptor tyrosine kinase 2 (ERBB2), Erb-b2 receptor tyrosine kinase 3 (ERBB3), Coagulation factor III (F3), Fc receptor like 5 (FCRL5), Fibroblast growth factor receptor 2 (FGFR2), FGFR2-IIIb, Folate hydrolase 1 (FOLH1), folate receptor 1 (FOLR1), globohexaosylceramide (Glono H), glypican-3 (GPC3), Glycoprotein nmb (GPNMB), Guanylyl cyclase 2C (GUCY2C), HER1, HER2, ICAM-1, IGF1R, IL13Ra2, ILl lRa, Integrin subunit alpha 2 (ITGA2), ITGB6, KAAG1, KRAS, L1CAM, LY6 / PLAUR domain containing 3 (LYPD3), MAGE, MAGEA1, MELTF, MET proto-oncogene receptor tyrosine kinase (MET), Mucin 1 (MUC1), mucin 16 (MUC16), Nectin cell adhesion molecule 4 (NECTIN4), NKG2D, PD-1, PD-L1, PROMI, PSCA, Protein tyrosine kinase 7 (PTK7), ROR2, SEZ6, Solute carrier family 34 member 2 (SLC34A2), Solute carrier family 39 member 6 (SLC39A6), Sialyl-Thomsen nouveau antigen (sTN), Somatostatin receptor 5 (SSTR5), Tumor associated calcium signal transducer 2 (TACSTD2), TFRC, Trophoblast glycoprotein (TPBG), Tyrosinase related protein 1 (TYRP1), VTCN1, or WT-1. In some embodiments, the antigen is a viral antigen. In some embodiments, the viral antigen is an HIV antigen or a SARS-CoV2 antigen. In some embodiments, the polyclonal antibodies are an IgY isotype.

[0011] In some embodiments, the jasmonate compound is jasmonic acid, 7-iso-jasmonic acid, 9,10-dihydrojasmonic acid, 9,10-dihydro-isojasmonic acid, 2,3-didehydrojasmonic acid, 3,4- didehydrojasmonic acid, 3,7-didehydrojasmonic acid, 4,5-didehydrojasmonic acid, 4,5- didehydro-7-isojasmonic acid, cucurbic acid, 6-epi-cucurbic acid, 6-epi-cucurbic acid-lactone, 12-hydroxy -jasmonic acid, 12-hydroxy -jasmonic acid-lactone, 11-hydroxy-jasmonic acid, 8- hydroxy -jasmonic acid, homo-jasmonic acid, dihomo-jasmonic acid, 11 -hydroxy-dihomojasmonic acid, 8-hydroxy-dihomo-jasmonic acid, tuberonic acid, tuberonic acid-O-P- glucopyranoside, cucurbic acid-O-P-glucopyranoside, 5,6-didehydro-jasmonic acid, 6,7- didehydro-jasmonic acid, 7,8-didehydro-jasmonic acid, cis-jasmone, dihydrojasmone, or a lower alkyl ester thereof. In some embodiments, the jasmonate compound is contained in a liposome. In some embodiments, the avian egg is an Anatidae egg, a Tinamous egg, a chicken egg, a duck egg, a swan, a quail egg, an ostrich egg, a pheasant egg, a turkey egg, a guinea egg, a guinea fowl egg, or a goose egg. In some embodiments, the avian egg is a Eudromia elegans egg, Lophophorus impejanus egg or Tragopan Satyra egg. In some embodiments, the avian egg shell is glossy. In some embodiments, the avian egg shell prevents the penetration of light having a wavelength of 540nm. In some embodiments, the polyclonal antibodies are further conjugated to a liposome by a linkage selected from the group consisting of a sulfide linkage, a hydrazone linkage, a hydrazine linkage, an ester linkage, an amido linkage, an amino linkage, an imino linkage, a thiosemicarbazone linkage, a semicarbazone linkage, an oxime linkage, a carbon-carbon linkage, or a combination thereof. In some embodiments, the polyclonal antibodies are further conjugated to at least one therapeutic or diagnostic agent. In some embodiments, the therapeutic or diagnostic agent is selected from the group consisting of a drug, a prodrug, a toxin, an enzyme, a biotin, an enhancer molecule, a radionuclide, a cytostatic agent, a photosensitizer, a fibrinolytic agent, an immunomodulator, a cytokine, a hormone, a second antibody or antigen-binding fragment thereof, an antisense oligonucleotide, an RNAi, an anti- angiogenic agent, a pro-apoptosis agent, a dye, a fluorescent agent, a contrast agent, a paramagnetic ion and a photodynamic agent. In some embodiments, the therapeutic agent is selected from the group consisting of aplidin, azaribine, anastrozole, azacytidine, bleomycin, bortezomib, bryostatin-1, busulfan, calicheamycin, camptothecin, 10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin, irinotecan (CPT-11), SN-38, carboplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin, daunomycin glucuronide, daunorubicin, dexamethasone, diethylstilbestrol, doxorubicin, doxorubicin glucuronide, epirubicin glucuronide, ethinyl estradiol, estramustine, etoposide, etoposide glucuronide, etoposide phosphate, floxuridine (FUdR), 3',5'-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, fluorouracil, fluoxymesterone, gemcitabine, hydroxyprogesterone caproate, hydroxyurea, idarubicin, ifosfamide, E-asparaginase, leucovorin, lomustine, mechlorethamine, medroprogesterone acetate, megestrol acetate, melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, phenyl butyrate, prednisone, procarbazine, paclitaxel, pentostatin, PSI-341, semustine streptozocin, tamoxifen, taxanes, taxol, testosterone propionate, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil mustard, velcade, vinblastine, vinorelbine, vincristine, ricin, abrin, ribonuclease, onconase, rapLRl, DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.

[0012] In some embodiments, the jasmonate compound boosts the dormant immunological response in the avian egg and / or enhances the avian egg's ability to produce polyclonal antibodies. In some embodiments, the jasmonate compound is contained in a liposome. In some embodiments, the polyclonal antibodies are further lyophilized. In some embodiments, the polyclonal antibodies are further conjugated with an additional compound. In some embodiments, the additional compound is a camptothecin or camptothecin analog. In some embodiments, the camptothecin has the following chemical structure:

[0013] In some embodiments, the additional compound is a salicylic acid. In some embodiments, the polyclonal antibodies are in the form of a prodrug.

[0014] In another aspect, disclosed here is a population of polyclonal antibodies produced by the method described herein.

[0015] In yet another aspect, disclosed here is a monoclonal antibody or antigen binding fragment thereof derived from the population of polyclonal antibodies described herein.

[0016] In another aspect, disclosed here is a pharmaceutical composition comprising the population of polyclonal antibodies described herein or the monoclonal antibody or antigen binding fragment thereof described herein and a pharmaceutically acceptable carrier.

[0017] In one aspect, disclosed here is a method of treating a disease, the method comprising administering the population of polyclonal antibodies described herein, the monoclonal antibody or antigen binding fragment thereof described herein, or the pharmaceutical composition described herein to a subject in need thereof.

[0018] In some embodiments, the subject is a human. In some embodiments, the disease is cancer. In some embodiments, the cancer is selected from the group consisting of a lung cancer cell, a bronchial cancer, a prostate cancer, a breast cancer, a colorectal cancer, a pancreatic cancer, an ovarian cancer, a leukemia, a lymphoma, an esophageal cancer, a liver cancer, a urinary cancer, a bladder cancer, renal cancer, an oral cavity cancer, a pharyngeal cancer, a uterine cancer and a melanoma. In some embodiments, the method further comprises administering a natural killer (NK) cell. In some embodiments, the disease is a viral infection. In some embodiments, the viral infection is HIV or SARS-CoV2. In some embodiments, the antibody or antigen binding fragment, the pharmaceutical composition, the population of polyclonal antibodies, or the monoclonal antibody or antigen binding fragment thereof is administered by intravenous, intramuscular, intraperitoneal, intravascular, parenteral or subcutaneous administration.

[0019] BRIEF DESCRIPTION OF DRAWINGS

[0020] FIG. 1 is a schematic diagram of a mammalian IgG and an avian IgY.

[0021] FIG. 2 is a photograph of avian eggs from the Tinamous family of birds. The eggs are blue, green, grey and chocolate in color, and are glossy.

[0022] FIG. 3 is a photograph of an avian egg from the Anatidae family of birds.

[0023] FIG. 4A is a microscopy image showing immunohistochemistry staining of a section of a chorioallantoic membrane of an untreated Tinamous egg. Presence of polyclonal antibodies is detected in the intermembrane space.

[0024] FIG. 4B is a microscopy image showing immunohistochemistry staining of a section of a chorioallantoic membrane of an untreated chicken egg. Presence of polyclonal antibodies is not detected in the intermembrane space.

[0025] FIG. 5A is a microscopy image showing immunohistochemistry staining of a section of a chorioallantoic membrane of a Tinamous egg injected with methyl jasmonate. Presence of polyclonal antibodies is detected in the intermembrane space.

[0026] FIG. 5B is a microscopy image showing immunohistochemistry staining of a section of a chorioallantoic membrane of a chicken egg injected with methyl jasmonate. A two-fold increase in formation of polyclonal antibodies is detected in the intermembrane space relative to Tinamous eggs that were untreated with methyl jasmonate.

[0027] FIG. 6A-6G shows region of the chorioallantoic membranes collected from chicken eggs injected with A14 or the vehicle. FIG. 6B shows the effect of increased vascularization compared to the unexposed control and the vehicle control at the same concentration as in the sample without the active ingredient. The other images correspond to sample controls and vehicle at low concentrations and vehicle at high concentration with active ingredient at high concentration. Samples were incubated with active ingredient for 8 days. FIG. 6G shows controls.

[0028] FIG. 7 A is a western blot showing the presence of IgY antibodies in Tinamous eggs that were not induced with methyl jasmonate.

[0029] FIG. 7B is a western blot showing the presence of IgY antibodies in chicken eggs that were induced with methyl jasmonate.

[0030] FIGS. 8A-8C are a series of images of the chorioallantoic membrane of Tinamous eggs that were injected with B16F10 cells. Eggs were injected with B16F10 and assessed 5 days after injection. No leaks or damage to the vasculature were observed indicating the lack of tumor growth and lack of angiogenesis typically observed in tumor growth. The demarcated areas (boxes) correspond to the regions used for qualitative / quantitative analysis of the morphology of the vascular network.

[0031] FIG. 9A-9D are a series of images of the chorioallantoic membrane of Tinamous eggs that were injected with B16F10 cells and treated with methyl jasmonate. Tinamous eggs were injected with B16F10 and methyl jasmonate. On day 14, Tinamous eggs were injected again with B 16F10. No leaks or damage to the vasculature were observed indicating the lack of tumor growth and lack of angiogenesis typically observed in tumor growth. The demarcated areas (boxes) correspond to the regions used for qualitative / quantitative analysis of the morphology of the vascular network.

[0032] FIG. 10A-10B are a series of fluorescence microscopy images showing B16F10 cells treated with IgY produced from gallus gallus and stained with MitoTracker for 24 hours. IgY isolated from Tinamous eggs and from chicken eggs treated with methyl jasmonate were used. B16F10 cells were incubated with either lOuM or lOOuM of IgY. Cells were observed by fluorescence microscopy using MitoTracker. High levels of fluorescence indicate live cells. Low levels of fluorescence indicate cell death. High levels of cell death were observed when B16F10 cells were treated with lOuM of IgY isolated from both sources. The demarcated areas (boxes) correspond to the regions used for qualitative / quantitative analysis of the morphology of the vascular network.

[0033] FIG. 11A is a microscopic image of the peritoneal tissue of a mouse injected with B16F10 melanoma cells. Large dark regions surrounding vasculature indicate diffuse melanoma metastasis.

[0034] FIG. 11B is a microscopic image of the peritoneal tissue of a mouse injected with B16F10 and treated with IgY isolated from Tinamous eggs. FIG. 12 shows sass spectrum on the range of IgY in plasma (m / z 227).

[0035] FIG. 13 shows tandem MS spectrum of daughter ions from the parent ion of m / z 227 with collision energy of 30 V.

[0036] FIG. 14 shows calibration curve used for quantification of IgY present in the plasma.

[0037] FIG. 15 shows profile plasma concentrations versus time of A- 14 and IgY administered at the dose of 897.2 mg / kg Wistar rats with a weight of 275 g.

[0038] FIGs. 16A-16C show high resolution mass spectrum of methyl dihydrojasmonate of m / z 227 and m / z 249 (FIG. 16A), b) cyclodextrine as a group of ions from m / z 648 to 851 (FIG. 16B) and formulation of encapsuled methyl dihydrojasmonate with cyclodextrine of m / z 991 (FIG. 16C).

[0039] FIG. 17 shows the NMR analyses of cyclodextrin, methyl dihydrojasmonate and the formulation A- 14.

[0040] FIG. 18 shows the results of the application of IgY taken from the egg of previously immunized birds.

[0041] FIG. 19 shows molecular structures of the two eggshell pigments (1H and 2H) identifed to date, and the two new pigments (3aH / 3bH and 4H) reported here, isolated as their dimethyl esters (3Me and 4Me, respectively); structure of the literature-known uroerythrin dimethyl ester methyl ether 3aMeOMe is also included.

[0042] FIG. 20A shows normalized UV-Vis spectra of the raw N. maculosa eggshell extract (green trace, EtOAc), extracted biliverdin 2Me (blue trace, MeOH), and extracted uroerythrin 3Me (red trace, MeOH).

[0043] FIG. 20B shows HPLC traces of the raw extract from purple N. maculosa eggshells at two different wavelengths of detection.

[0044] FIG. 20C shows normalized UV-Vis spectra of the raw E. elegans eggshell extract (green trace, EtOAc), and of the two major components: biliverdin 2Me (blue trace, in MeOH) and bilirubin 4Me (orange trace, in MeOH).

[0045] FIG. 20D shows NP-HPLC traces (at 400 nm detection wavelength) of the polar and nonpolar fractions from the E. elegans extract.

[0046] FIGs. 21A-21D show transmission spectra for the variable subtractive mixtures of (FIG. 26A) biliverdin and uroerythrin from N. maculosa and (FIG. 21C) biliverdin and bilirubin from E. elegans eggshells, respectively plotted in their modelled colours. Measured spectral reflectance of those eggshells (solid) is compared to the best predicted spectrum (dashed). All spectra are depicted from 318 to 700 nm. The colour of each spectrum is calculated from its spectrum, and the brightness of those colours was modified to approximate the reflectance of whole eggshells (solid black lines). Intermediate spectra for (FIG. 21B) biliverdin and uroerythrin and (FIG. 21D) biliverdin and bilirubin were plotted within the CIE coordinate space (small black dots) varying from entirely biliverdin (left most large dot) to entirely the novel pigment (rightmost large dot). For comparison, the reflectance values of whole eggshells are plotted (+ symbol) within each coordinate space. Insets (FIG. 21B) and (FIG. 21D): Colour swatch for our best prediction (left) compared against a close-up photograph of the surface colour (right).

[0047] FIG. 22 shows degradation chain of protoporphyrin IX via biliverdin 2H to form uroerythrin 3aH (biotripyrrin b) and formydipyrrinone 5H. The pyrrolic fragments lost in the subsequent products are indicated in blue, the oxygens introduced in the oxidation step in red.

[0048] FIGs. 23A-23F show fetus of chicken with 11 days of development, treated with osteosarcoma. Observe the fetus with 3.0 cm of crow-rump already well developed showing all the body regions formed (FIG. 23A). Highlighting the developing brain (FIG. 23B), showing a division into 4 vesicles. FIG. 23C shows detail of the eyeball showing the pigmented retina. FIG. 23D shows that in the face region, the beak was well developed. The limbs (wings and legs) present the formed and individualized digits (FIG. 23E and FIG. 23F). Throughout the entire length of the fetus, intense vascularization was observed.

[0049] FIG. 24 is a microscopy image showing immunohistochemistry staining of sections of the heart and lung of the chicken in FIG. 23.

[0050] FIG. 25 is a microscopy image showing immunohistochemistry staining of sections of the vertebrae, esophagus, and intestinal loops of the chicken in FIG. 23.

[0051] FIGs. 26A-26E show fetus of chicken with 11 days of development, treated with melanoma. Observe the fetus with 2.7 cm of developing crow-rump (FIG. 26A). Highlighting the developing brain (Fig. 26B), showing a division into 4 vesicles, however they are not as pronounced. FIG. 26C shows detail of the eyeball showing the pigmented retina. The limbs (wings and legs) present the formed and individualized digits (FIG. 26D and FIG. 26E).

[0052] FIG. 27 is a microscopy image showing immunohistochemistry staining of sections of the eye, eye lens, heart, vertebrae, and medulla of the chicken in FIG. 26.

[0053] FIG. 28 is a microscopy image showing immunohistochemistry staining of sections of the heart, lung, and stomach of the chicken in FIG. 26.

[0054] FIG. 29 is a microscopy image showing immunohistochemistry staining of sections of the liver, intestinal loops, kidney, and primitive gonoda of the chicken in FIG. 26.

[0055] FIGs. 30A-30D show fetus of chicken with 11 days of development, treated with canine bone marrow. Observe the fetus with 2.2 cm of crow-rump and its macroscopic characteristics (FIG. 30A). An earlier stage of development is observed. The pronounced brain with a division into 4 vesicles was identified. FIG. 30B shows detail of the eyeball showing the beginning of retinal pigmentation. The wings begin to show the division of the digits (FIG. 30C), however the pelvic limbs are still in the form of buds (FIG. 30D).

[0056] FIG. 31 is a microscopy image showing immunohistochemistry staining of sections of the eye and vertebrae of the chicken in FIG. 30.

[0057] FIG. 32 is a microscopy image showing immunohistochemistry staining of sections of the heart, liver, intestinal loops, and kidney of the chicken in FIG. 30.

[0058] FIG. 33A-33E shows fetus of chicken with 11 days of development, control. Observe the fetus with 3.0 cm of crow-rump and its macroscopic characteristics (FIG. 33A). FIG. 33B shows detail of the pronounced brain, where a division into 4 vesicles was identified. FIG. 33C shows detail of the eyeball showing the pigmented retina. The limbs (wings and legs) present the formed and individualized digits (FIG. 33D and FIG. 33E).

[0059] FIG. 34 is a microscopy image showing immunohistochemistry staining of sections of the eye, lung, vertebrae, and medulla of the chicken in FIG. 33.

[0060] FIG. 35 is a microscopy image showing immunohistochemistry staining of sections of the heart of the chicken in FIG. 33.

[0061] FIG. 36 is a microscopy image showing immunohistochemistry staining of sections of the intestinal loops, liver, kidney, rim / glomerulo, kidney, and primitive gonod of the chicken in FIG. 33.

[0062] FIG. 37 shows melanoma cells spreading in organs of chicken treated with melanoma cells. A and B show that melanoma cells were spread in hens not treated with a jasmonate compound (Methyl jasmonate (MeJA). C to H show that melanoma cells were not spread in hens treated with MeJA (C and D: Treated with 10'7MeJA, E and F: treated with 10'5MeJA, and G and H treated with 10'3MeJA).

[0063] FIG. 38 shows that Melanoma spread all over the embryo that were not treated with a jasmonate compound.

[0064] FIGs. 39A-39C show that the immune response is increased with treatment with a jasmonate compound. FIG. 39A shows that the vessels are surrounded by micro bubbles full of 4.5 jasmonate compounds (didehydromethyl jasmonate, DHMJ) and activating the immune responses towards the antigen. FIG. 39B shows the egg of the same species. On the right position, the egg was laid in captivity without the possibility to build up its nests and adding natural algae that stands between the bio-film that holds the micro vegetal agents and the eggshell. On the left position, the egg was laid in the wild. FIG. 39C shows that the macrophage being activated by the 4,5-didehydrojasmonic acid (4.5 DHMJ).

[0065] FIG. 40 shows that IgY is produced at a higher level with the hens are treated with cancer cells plus a jasmonate compound (Monal) compared to cancer cells alone.

[0066] DETAILED DESCRIPTION

[0067] The present disclosure relates to the generation of avian antibodies that specifically bind to a human tumor associated antigen (TAA). In some embodiments, a TAA is CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, c-Met PSMA, Glycolipid F77, EGFRvllI, GD-2, NY-ESO-1 or MAGE A3.

[0068] The antibodies of the present disclosure are derived from avian eggs that are injected with a cancer cell (or an antigen) and a compound from the jasmonate family or a compound related thereto. Compounds falling within, and related to, the jasmonate family are described extensively in the literature. See e.g., US 8,883,220, 9,592,305, 10,328,155, 10,314,918, and Jarocka-Karpowicz, I. et al., Molecules, 2021, 26(10), 2901; the contents of each of which are fully incorporated by reference herein.

[0069] In some embodiments, the cancer cell is selected from a lung cancer cell, a bronchial cancer cell, a prostate cancer cell, a breast cancer cell, a colorectal cancer cell, a pancreatic cancer cell, an ovarian, a leukemia cancer cell, a lymphoma cancer cell, an esophageal cancer cell, a liver cancer cell, a urinary and / or bladder cancer cell, a renal cancer cell, an oral cavity cancer cell, a pharyngeal cancer cell, a uterine cancer cell, and / or a melanoma cancer cell.

[0070] In some embodiments, the avian egg is produced from the subphylum chordata, and in particular the aves class. In some embodiments, the avian egg is produced from a bird in the superorder palaeognathae or neognathae. In some embodiments, the avian egg is produced from struthioniformes (ostriches, emus, kiwis, and allies), tinamiformes (tinamous), anseriformes (waterfowl), galliformes (fowl, including chickens, ducks, geese, guinea, quail, grouse, pheasant and turkeys), charadriiformes (gulls, button-quails, plovers and allies), gaviiformes (loons), podicipediformes (grebes), procellariiformes (albatrosses, petrels, and allies), sphenisciformes (penguins), pelecaniformes (pelicans and allies), phaethontiformes (tropicbirds), ciconiiformes (storks and allies), cathartiformes (New World vultures), phoenicopteriformes (flamingos), falconiformes (falcons, eagles, hawks and allies), gruiformes (cranes and allies), pteroclidiformes (sandgrouse), columbiformes (doves and pigeons), psittaciformes (parrots and allies), cuculiformes (cuckoos and turacos), opisthocomiformes (hoatzin), strigiformes (owls), caprimulgiformes (nightjars and allies), apodiformes (swifts and hummingbirds), coraciiformes (kingfishers and allies), piciformes (woodpeckers and allies), trogoniformes (trogons), coliiformes (mousebirds), and passeriformes (passerines). In some embodiments, the avian egg is produced from Crypturellus (e.) columbianus, Crypturellus (e.) idoneus, Crypturellus atrocapillus, Crypturellus bartletti, Crypturellus berlepschi,

[0071] Crypturellus boucardi, Crypturellus brevirostris, Crypturellus casiquiare, Crypturellus cinereus, Crypturellus cinnamomeus, Crypturellus duidae, Crypturellus erythropus,

[0072] Crypturellus kerriae, Crypturellus noctivagus, Crypturellus obsoletus, Crypturellus parvirostris, Crypturellus ptaritepui, Crypturellus soui, Crypturellus strigulosus, Crypturellus tataupa, Crypturellus transfasciatus, Crypturellus undulatus, Crypturellus variegatus, Eudromia elegans, Eudromia formosa, Eudromia olsoni, Lophophorus impejanus, Nothocercus bonapartei, Nothocercus julius, Nothocercus nigrocapillus, Nothoprocta cinerascens, Nothoprocta curvirostris, Nothoprocta omata, Nothoprocta pentlandii, Nothoprocta perdicaria, Nothoprocta taczanowskii, Nothura boraquira, Nothura chacoensis, Nothura darwinii, Nothura maculosa, Nothura minor, Nothura parvula, Rhynchotus maculicollis, Rhynchotus rufescens, Taoniscus nanus, Tinamotis ingoufi, Tinamotis pentlandii, Tinamus guttatus, Tinamus major, Tinamus osgoodi, Tinamus solitarius, Tinamus tao or Tragopan Satyra. In a preferred embodiment, the avian egg is produced from Eudromia elegans, Lophophorus impejanus or Tragopan Satyra.

[0073] In some embodiments, the avian egg shell is glossy. In some embodiments, the avian egg shell prevents the penetration of a 540 nm wavelength of light.

[0074] In some embodiments, the avian species is preferably native to the Patagonia region.

[0075] In some embodiments, the avian species being from the Patagonia region affords particular preferable characteristics to the eggs for implementation in the present disclosure.

[0076] This characteristic presents considerable advantages from a development perspective as these T cell retargeting agents share the drug-like properties of human monoclonal antibodies. It is anticipated that their unmodified human sequences and native structure, combined with favorable physicochemical properties, minimize the potential for immunogenicity when administered to patients.

[0077] The disclosure provides IgY polyclonal antibodies that bind tumor associated antigens. These antibodies are collectively referred to herein as IgY antibodies or IgY fragments. Preferably, the IgY antibodies or IgY fragments are specific for at least one human tumor associated antigen. The disclosure also provides chimeric or humanized IgY antibodies that bind tumor associated antigens. Preferably, the chimeric or humanized antibodies have a human constant region of IgGl, IgG2, IgG3 or IgG4 isotype. Preferably, the human constant region is of the IgGl isotype.

[0078] The disclosure also provides monovalent antibodies and / or bispecific antibodies that include at least a binding site that is specific for a tumor associated antigen. Preferably, the monovalent antibodies and / or bispecific antibodies are specific for at least a human tumor associated antigen.

[0079] In some embodiments, the bispecific antibody includes a first arm that binds a first tumor associated antigen and a second arm that binds a second tumor associated antigen. In some embodiments, the first and second tumor associated antigens include, by way of nonlimiting example, CD 19, CD20, CD22, R0R1, mesothelin, CD33 / IL3Ra, c-Met PSMA, Glycolipid F77, EGFRvllI, GD-2, NY-ESO-1 or MAGE A3. In some embodiments, the TAA is an antigen that is expressed on the cell surface of a cancer cell. In some embodiments, the cancer cell is selected from a lung cancer cell, a bronchial cancer cell, a prostate cancer cell, a breast cancer cell, a colorectal cancer cell, a pancreatic cancer cell, an ovarian, a leukemia cancer cell, a lymphoma cancer cell, an esophageal cancer cell, a liver cancer cell, a urinary and / or bladder cancer cell, a renal cancer cell, an oral cavity cancer cell, a pharyngeal cancer cell, a uterine cancer cell, and / or a melanoma cancer cell.

[0080] The compositions and agents described (e.g., the antibodies produced) herein may be used in a variety of modulatory, therapeutic, and therapeutic applications. In any method described herein, such as a modulatory method, therapeutic method, or combination thereof, all steps of the method may be performed by a single actor or, alternatively, by more than one actor. For example, diagnosis may be performed directly by the actor providing therapeutic treatment.

[0081] In one aspect, disclosed herein is a method for treating or delaying progression of a cancer in a subject. The method may comprise administering to the subject an effective amount of an antibody or antigen binding fragment thereof disclosed herein, thereby treating and / or delaying the progression of the cancer in the subject. In some embodiments, the cancer comprises cells aberrantly expressing a tumor associated antigen. In some embodiments, the cancer is an adenocarcinoma, a bile duct (biliary) cancer, a bladder cancer, a bone cancer, a breast cancer, a triple-negative breast cancer, a Her2-negative breast cancer, a carcinoid cancer, a cervical cancer, a cholangiocarcinoma, a colorectal cancer, a colon cancer, an endometrial cancer, an esophageal cancer, a glioma, a head and neck cancer, a head and neck squamous cell cancer, a leukemia, a liver cancer, a lung cancer, a non-small cell lung cancer, a small cell lung cancer, a lymphoma, a melanoma, an oropharyngeal cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a metastatic castration-resistant prostate carcinoma, a renal cancer, a sarcoma, a skin cancer, a squamous cell cancer, a stomach cancer, a testis cancer, a thyroid cancer, a urogenital cancer, or a urothelial cancer.

[0082] Definitions

[0083] Unless otherwise defined, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. For example, the term "a cell" includes a single cell as well as a plurality or population of cells. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization described herein are those well- known and commonly used in the art. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y, (1989)).

[0084] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0085] The term “about,” in some embodiments, encompasses values that are within 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, inclusive, or any range in between (e.g., plus or minus 2%-6%), of a value that is measured. In some embodiments, the term “about” refers to the inherent variation of error in a method, assay, or measured value, such as the variation that exists among experiments.

[0086] The term “activity,” when used with respect to a polypeptide, includes activities that are inherent in the structure of the protein. For example, with regard to an immune cell protein, the term “activity” includes the ability to modulate an inflammatory phenotype of the protein by modulating natural binding protein binding or cellular signaling of the cell (e.g., by engaging a natural receptor or ligand on an immune cell).

[0087] The term “administering” relates to the actual physical introduction of an agent into or onto (as appropriate) a biological target of interest, such as a host and / or subject. A composition may be administered to the cell (e.g., “contacting”) in vitro or in vivo. A composition may be administered to the subject in vivo via an appropriate route of administration. Any and all methods of introducing the composition into the host are contemplated according to the present disclosure. The method is not dependent on any particular means of introduction and is not to be so construed. Means of introduction are well- known to those skilled in the art, and are also exemplified herein. The term include routes of administration which allow an agent to perform its intended function. Examples of routes of administration for treatment of a body which may be used include injection (subcutaneous, intravenous, parenterally, intraperitoneally, intrathecal, etc.), oral, inhalation, and transdermal routes. The injection may be bolus injections or may be continuous infusion. Depending on the route of administration, the agent may be coated with or disposed in a selected material to protect it from natural conditions which may detrimentally affect its ability to perform its intended function. The agent may be administered alone, or in conjunction with a pharmaceutically acceptable carrier. The agent also may be administered as a prodrug, which is converted to its active form in vivo.

[0088] The term “agent” refers to a compound, supramolecular complex, material, and / or combination or mixture thereof. A compound (e.g., a molecule) may be represented by a chemical formula, chemical structure, or sequence. Representative, non-limiting examples of agents, include, e.g., antibodies, small molecules, polypeptides, polynucleotides (e.g., RNAi agents, siRNA, miRNA, piRNA, mRNA, antisense polynucleotides, aptamers, and the like), lipids, and polysaccharides. In general, agents may be obtained using any suitable method known in the art. In some embodiments, an agent may be a “therapeutic agent” for use in treating a disease or disorder (e.g., cancer) in a subject (e.g., a human).

[0089] The term “altered amount” or “altered level” encompasses increased or decreased copy number (e.g., germline and / or somatic) of a biomarker nucleic acid, or increased or decreased expression level in a sample of interest, as compared to the copy number or expression level in a control sample. The term “altered amount” of a biomarker also includes an increased or decreased protein level of a biomarker protein in a sample, e.g., a cancer sample, as compared to the corresponding protein level in a normal and / or control sample. Furthermore, an altered amount of a biomarker protein may be determined by detecting posttranslational modification such as methylation status of the marker, which may affect the expression or activity of the biomarker protein. In some embodiments, the “altered amount” refers to the presence or absence of a biomarker because the reference baseline may be the absence or presence of the biomarker, respectively. The absence or presence of the biomarker may be determined according to the threshold of sensitivity of a given assay used to measure the biomarker.

[0090] The amount of a biomarker in a subject is “significantly” higher or lower than the normal amount of the biomarker, if the amount of the biomarker is greater or less, respectively, than the normal level by an amount greater than the standard error of the assay employed to assess amount, and preferably at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more than that amount. Alternatively, the amount of the biomarker in the subject may be considered “significantly” higher or lower than the normal amount if the amount is at least about two, and preferably at least about three, four, or five times, higher or lower, respectively, than the normal amount of the biomarker. Such “significance” may also be applied to any other measured parameter described herein, such as for expression, inhibition, cytotoxicity, cell growth, and the like.

[0091] The term “altered level of expression” of a biomarker refers to an expression level or copy number of the biomarker in a test sample, e.g., a sample derived from a patient suffering from cancer, that is greater or less than the standard error of the assay employed to assess expression or copy number, and is preferably at least twice, and more preferably three, four, five or ten or more times the expression level or copy number of the biomarker in a control sample (e.g., sample from a healthy subjects not having the associated disease) and preferably, the average expression level or copy number of the biomarker in several control samples. In some embodiments, the level of the biomarker refers to the level of the biomarker itself, the level of a modified biomarker (e.g., phosphorylated biomarker), or to the level of a biomarker relative to another measured variable, such as a control (e.g., phosphorylated biomarker relative to an unphosphorylated biomarker). The term “expression” encompasses the processes by which nucleic acids (e.g., DNA) are transcribed to produce RNA, and may also refer to the processes by which RNA transcripts are processed and translated into polypeptides. The sum of expression of nucleic acids and their polypeptide counterparts, if any, contributes to the amount of a biomarker, such as one or more targets.

[0092] The term “altered activity” of a biomarker refers to an activity of the biomarker which is increased or decreased in a disease state, e.g., in a cancer sample, or a treated state, as compared to the activity of the biomarker in a normal, control sample. Altered activity of the biomarker may be the result of, for example, altered expression of the biomarker, altered protein level of the biomarker, altered structure of the biomarker, or, e.g., an altered interaction with other proteins involved in the same or different pathway as the biomarker or altered interaction with transcriptional activators or inhibitors.

[0093] Unless otherwise specified here within, the terms “antibody” and “antibodies” broadly encompass naturally-occurring forms of antibodies (e.g., IgY, IgG, IgA, IgM, IgE) and recombinant antibodies, such as single-chain antibodies, chimeric and humanized antibodies and multi- specific antibodies, as well as fragments, fusion proteins, and derivatives of all of the foregoing, which fragments and derivatives have at least an antigenic binding site. Antibody derivatives may comprise a protein or chemical moiety conjugated to an antibody. In general, antibody molecules obtained from humans relate to any of the classes IgY, IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgGl, IgG2, and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain.

[0094] Antibodies may be purified by well-known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

[0095] The term "antigen-binding domain" or "binding portion" refers to the part of the immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent stretches within the V regions of the heavy and light chains, referred to as "hypervariable regions," are interposed between more conserved flanking stretches known as "framework regions," or "FRs". Thus, the term "FR" refers to amino acid sequences that are naturally found between, and adjacent to, hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three- dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as "complementaritydetermining regions," or "CDRs." The assignment of amino acids to each domain is in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Eesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature 342:878-883 (1989).

[0096] The term “antigen” as used herein refers to a molecule, moiety, foreign particulate matter, or an allergen, such as pollen, that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body or the avian egg may trigger an immune response. An antigen may be a protein, a peptide, a polysaccharide, a lipid, a nucleic acid, or a cell. Antigens exist on normal cells, cancer cells, parasites, viruses, fungi, and bacteria. Antigens are recognized by antigen receptors, including antibodies and T-cell receptors. Diverse antigen receptors are made by cells of the immune system so that each cell has a specificity for a single antigen. Upon exposure to an antigen, only the lymphocytes that recognize that antigen are activated and expanded, a process known as clonal selection. In most cases, antibodies are antigen- specific, meaning that an antibody can only react to and bind one specific antigen; in some instances, however, antibodies may cross-react to bind more than one antigen. The reaction between an antigen and an antibody is called the antigen- antibody reaction.

[0097] An “isolated” antibody is one that has been identified, separated and / or recovered from a component of its production environment. Preferably, the isolated polypeptide is free of association with all other contaminant components from its production environment. Contaminant components from its production environment are materials that would typically interfere with research, diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified: (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. An isolated polypeptide or antibody may be prepared by a process including at least one purification step. The antibody may be isolated by one of the standard procedures, such as affinity chromatography. If necessary, the antibody is cleaved enzymatically, and the aviary Fc fragments are replaced by human Fc fragments.

[0098] The term "operably linked" as used herein refers to positions of components so described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

[0099] “Prodrug” or “pharmaceutically acceptable prodrug” refers to a compound that is metabolized, for example hydrolyzed or oxidized, in the host after administration to form the compound of the present disclosure (e.g., compounds of formula I). Typical examples of prodrugs include compounds that have biologically labile or cleavable (protecting) groups on a functional moiety of the active compound. Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, or dephosphorylated to produce the active compound. Examples of prodrugs using ester or phosphoramidate as biologically labile or cleavable (protecting) groups are disclosed in U.S. Patents 6,875,751, 7,585,851, and 7,964,580, the disclosures of which are incorporated herein by reference. The prodrugs of this disclosure are metabolized to produce a compound of Formula I. The present disclosure includes within its scope, prodrugs of the compounds described herein. Conventional procedures for the selection and preparation of suitable prodrugs are described, for example, in “Design of Prodrugs” Ed. H. Bundgaard, Elsevier, 1985.

[0100] The term “conjugates" as used herein refers to cell binding agents that are covalently bonded to one or more molecules of a cytotoxic compound. In this regard, "cell binding agent" is a molecule having affinity for a biological target, and may be, for example, an antibody, particularly a monoclonal antibody, or an antibody fragment, and the binding agent functions to direct a biologically active compound to a biological target. In certain embodiments, the conjugate may be designed to target tumor cells through cell surface antigens. The antigen may be a cell surface antigen that is overexpressed or expressed in an abnormal cell type. Specifically, the target antigen may be expressed only on proliferative cells (e.g., tumor cells). The target antigen may be selected on the basis of different expression, usually between proliferative tissues and normal tissues. In the present disclosure, the antibody is bonded to the linker.

[0101] The term “biomarker” refers to a gene or gene product that is a target for modulating one or more phenotypes of interest, such as a phenotype of interest in myeloid cells, such as suppressive myeloid cells, monocytes, macrophages, and / or dendritic cells. In this context, the term “biomarker” is synonymous with “target.” In some embodiments, however, the term further encompasses a measurable entity of the target that has been determined to be indicative of an output of interest, such as one or more diagnostic, prognostic, and / or therapeutic outputs (e.g., for modulating an inflammatory phenotype, cancer state, and the like). In still other embodiments, the team further encompasses compositions that modulate the gene or gene product, including anti-gene product antibodies and antigen-binding fragments thereof. Thus, biomarkers may include, without limitation, nucleic acids (e.g., genomic nucleic acids and / or transcribed nucleic acids), proteins, and antibodies (as well as antigen-binding fragments thereof).

[0102] The terms “cancer” or “tumor” or “hyperproliferative” refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, invasive or metastatic potential, rapid growth, and certain characteristic morphological features. In some embodiments, such cells exhibit such characteristics in part or in full due to the expression and activity of immune checkpoint proteins, such as PD-1, PD- Ll, PD-L2, and / or CTLA-4.

[0103] Cancer cells are often in the form of a tumor, but such cells may exist alone within an animal, or may be a non-tumorigenic cancer cell, such as a leukemia cell. As used herein, the term “cancer” includes premalignant as well as malignant cancers. Cancers include, but are not limited to, a variety of cancers, carcinoma including that of the bladder (including accelerated and metastatic bladder cancer), breast, colon (including colorectal cancer), kidney, liver, lung (including small and non-small cell lung cancer and lung adenocarcinoma), ovary, prostate, testes, genitourinary tract, lymphatic system, rectum, larynx, pancreas (including exocrine pancreatic carcinoma), esophagus, stomach, gall bladder, cervix, thyroid, and skin (including squamous cell carcinoma); hematopoietic tumors of lymphoid lineage including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T- cell lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, hairy cell lymphoma, histiocytic lymphoma, and Burketts lymphoma; hematopoietic tumors of myeloid lineage including acute and chronic myelogenous leukemias, myelodysplastic syndrome, myeloid leukemia, and promyelocytic leukemia; tumors of the central and peripheral nervous system including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; other tumors including melanoma, xenoderma pigmentosum, keratoactanthoma, seminoma, thyroid follicular cancer, and teratocarcinoma; melanoma, unresectable stage III or IV malignant melanoma, squamous cell carcinoma, small-cell lung cancer, non-small cell lung cancer, glioma, gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon carcinoma, and head and neck cancer, gastric cancer, germ cell tumor, bone cancer, bone tumors, adult malignant fibrous histiocytoma of bone; childhood, malignant fibrous histiocytoma of bone, sarcoma, pediatric sarcoma, sinonasal natural killer, neoplasms, plasma cell neoplasm; myelodysplastic syndromes; neuroblastoma; testicular germ cell tumor, intraocular melanoma, myelodysplastic syndromes; myelodysplastic / myeloproliferative diseases, synovial sarcoma, chronic myeloid leukemia, acute lymphoblastic leukemia, Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL), multiple myeloma, acute myelogenous leukemia, chronic lymphocytic leukemia, mastocytosis and any symptom associated with mastocytosis, and any metastasis thereof. In addition, disorders include urticaria pigmentosa, mastocytosises such as diffuse cutaneous mastocytosis, solitary mastocytoma in human, as well as dog mastocytoma and some rare subtypes like bullous, erythrodermic and teleangiectatic mastocytosis, mastocytosis with an associated hematological disorder, such as a myeloproliferative or myelodysplastic syndrome, or acute leukemia, myeloproliferative disorder associated with mastocytosis, mast cell leukemia, in addition to other cancers. Other cancers are also included within the scope of disorders including, but are not limited to, the following: carcinoma, including that of the bladder, urothelial carcinoma, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid, testis, particularly testicular seminomas, and skin; including squamous cell carcinoma; gastrointestinal stromal tumors (“GIST”); hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, hairy cell lymphoma and Burketts lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; other tumors, including melanoma, seminoma, tetratocarcinoma, neuroblastoma and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma;and other tumors, including melanoma, xenoderma pigmentosum, keratoactanthoma, seminoma, thyroid follicular cancer, teratocarcinoma, chemotherapy refractory non-seminomatous germ-cell tumors, and Kaposi's sarcoma, and any metastasis thereof. Other non-limiting examples of types of cancers applicable to the methods encompassed by the present disclosure include human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelio sarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, bone cancer, brain tumor, lung carcinoma (including lung adenocarcinoma), small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease. In some embodiments, cancers are epithelial in nature and include but are not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecologic cancers, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. In some embodiments, the epithelial cancer is non- small-cell lung cancer, nonpapillary renal cell carcinoma, cervical carcinoma, ovarian carcinoma (e.g., serous ovarian carcinoma), or breast carcinoma. The epithelial cancers may be characterized in various other ways including, but not limited to, serous, endometrioid, mucinous, clear cell, Brenner, or undifferentiated. In some embodiments, the cancer is selected from the group consisting of (advanced) non-small cell lung cancer, melanoma, head and neck squamous cell cancer, (advanced) urothelial bladder cancer, (advanced) kidney cancer (RCC), microsatellite instability-high cancer, classical Hodgkin lymphoma, (advanced) gastric cancer, (advanced) cervical cancer, primary mediastinal B-cell lymphoma, (advanced) hepatocellular carcinoma, and (advanced) merkel cell carcinoma.

[0104] The term “coding region” refers to regions of a nucleotide sequence comprising codons which are translated into amino acid residues, whereas the term “noncoding region” refers to regions of a nucleotide sequence that are not translated into amino acids (e.g., 5' and 3' untranslated regions).

[0105] The terms “conjoint therapy” and “combination therapy,” as used herein, refer to the administration of two or more therapeutic agents, e.g., combination of modulators of more than one target, combination of at least one modulator of at least one target and an additional therapeutic agent, such as an immune checkpoint therapy, combination of more than one modulators of one or more targets), and combinations thereof. The different agents comprising the combination therapy may be administered concomitant with, prior to, or following, the administration of the other or others. The combination therapy is intended to provide a beneficial (additive or synergistic) effect from the co-action of these therapeutic agents. Administration of these therapeutic agents in combination may be carried out over a defined time period (usually minutes, hours, days, or weeks depending upon the combination selected). In combination therapy, combined therapeutic agent may be applied in a sequential manner, or by substantially simultaneous application.

[0106] The term “epitope” refers to a determinant or site on an antigen against which an antigen-binding protein (e.g., an immunoglobulin, antibody, or antigen-binding fragment) binds. The epitopes of protein antigens may be either linear epitopes or conformational epitopes. A linear epitope refers to an epitope formed from a contiguous, linear sequence of linked amino acids. Linear epitopes of protein antigens are typically retained upon exposure to chemical denaturants (e.g., acids, bases, solvents, cross-linking reagents, chaotropic agents, disulfide bond reducing agents) or physical denaturants (e.g., thermal heat, radioactivity, or mechanical shear or stress). By contrast, a conformational epitope refers to an epitope formed from non-contiguous amino acids juxtaposed by tertiary folding of a polypeptide. Conformational epitopes are typically lost upon treatment with denaturants. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acids in a unique spatial conformation. In some embodiments, an epitope includes fewer than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 amino acids in a unique spatial conformation. Generally, an antibody, or antigen-binding fragment thereof, specific for a particular target molecule will preferentially recognize and bind to a specific epitope on the target molecule within a complex mixture of proteins and / or macromolecules. In some embodiments, an epitope does not include all amino acids of the extracellular domain of a biomarker protein.

[0107] The term “gene” encompasses a nucleotide (e.g., DNA) sequence that encodes a molecule (e.g., RNA, protein, etc.) that has a function. A gene generally comprises two complementary nucleotide strands (i.e., dsDNA), a coding strand and a non-coding strand. When referring to DNA transcription, the coding strand is the DNA strand whose base sequence corresponds to the base sequence of the RNA transcript produced (although with thymine replaced by uracil). The coding strand contains codons, while the non-coding strand contains anticodons. During transcription, RNA Pol II binds the non-coding strand, reads the anti-codons, and transcribes their sequence to synthesize an RNA transcript with complementary bases. In some embodiments, the gene sequence (i.e., DNA sequence) listed is the sequence of the coding strand.

[0108] The term “inhibit” or “downregulate” includes the decrease, limitation, or blockage, of, for example a particular action, function, or interaction. In some embodiments, cancer is “inhibited” if at least one symptom of the cancer is alleviated, terminated, slowed, or prevented. As used herein, cancer is also “inhibited” if recurrence or metastasis of the cancer is reduced, slowed, delayed, or prevented. Similarly, a biological function, such as the function of a protein, is inhibited if it is decreased as compared to a reference state, such as a control like a wild-type state. Such inhibition or deficiency may be induced, such as by application of an agent at a particular time and / or place, or may be constitutive, such as by a heritable mutation. Such inhibition or deficiency may also be partial or complete (e.g., essentially no measurable activity in comparison to a reference state, such as a control like a wild-type state). In some embodiments, essentially complete inhibition or deficiency is referred to as “blocked.” In one embodiment, the term refers to reducing the level of a given output or parameter to a quantity (e.g., background staining, biomarker signaling, biomarker immunoinhibitory function, and the like) which is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or less than the quantity in a corresponding control. A reduced level of a given output or parameter need not, although it may, mean an absolute absence of the output or parameter. The disclosure does not require, and is not limited to, methods that wholly eliminate the output or parameter. The given output or parameter may be determined using methods well-known in the art, including, without limitation, immunohistochemical, molecular biological, cell biological, clinical, and biochemical assays, as discussed herein and in the examples. The term “promote” or “upregulate” has the opposite meaning.

[0109] The terms “prevent,” “preventing,” “prevention,” “prophylactic treatment,” and the like refer to reducing the probability of developing a disease, disorder, or condition in a subject, who does not have, but is at risk of or susceptible to developing a disease, disorder, or condition.

[0110] As used herein, the term “treatment” refers to clinical intervention designed to alter the natural course of the individual being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of progression, ameliorating or palliating the pathological state, and remission or improved prognosis of a particular disease, disorder, or condition. An individual is successfully “treated,” for example, if one or more symptoms associated with a particular disease, disorder, or condition are mitigated or eliminated.

[0111] The term “small molecule” is a term of the art and includes molecules that are less than about 1000 molecular weight or less than about 500 molecular weight. In one embodiment, small molecules do not exclusively comprise peptide bonds. In another embodiment, small molecules are not oligomeric. Exemplary small molecule compounds which may be screened for activity include, but are not limited to, peptides, peptidomimetics, nucleic acids, carbohydrates, small organic molecules (e.g., polyketides) (Cane et al. (1998) Science 282:63), and natural product extract libraries. In another embodiment, the compounds are small, organic non-peptidic compounds. The term is intended to encompass all stereoisomers, geometric isomers, tautomers, and isotopes of a chemical structure of interest, unless otherwise indicated. The term “subject” refers to an animal, vertebrate, mammal, or human, especially one to whom an agent is administered, e.g., for experimental, diagnostic, and / or therapeutic purposes, or from whom a sample is obtained or on whom a procedure is performed. In some embodiments, a subject is a mammal, e.g., a human, non-human primate, rodent (e.g., mouse or rat), domesticated animals (e.g., cows, sheep, cats, dogs, and horses), or other animals, such as llamas and camels. In some embodiments, the subject is human. In some embodiments, the subject is a human subject with a cancer. The term “subject” is interchangeable with “patient.”

[0112] The term “therapeutic effect” encompasses a local or systemic effect in animals, particularly mammals, and more particularly humans, caused by a pharmacologically active substance. The term thus means any substance intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease or in the enhancement of desirable physical or mental development and conditions in an animal or human. A prophylactic effect encompassed by the term encompasses delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.

[0113] The term “effective amount” or “effective dose” of an agent (including a composition and / or formulation comprising such an agent) refers to the amount sufficient to achieve a desired biological and / or pharmacological effect, e.g., when delivered to a cell or organism according to a selected administration form, route, and / or schedule. As will be appreciated by those of ordinary skill in this art, the absolute amount of a particular agent or composition that is effective may vary depending on such factors as the desired biological or pharmacological endpoint, the agent to be delivered, the target tissue, etc. Those of ordinary skill in the art will further understand that an “effective amount” may be contacted with cells or administered to a subject in a single dose, or through use of multiple doses, in various embodiments. The term “effective amount” may be a “therapeutically effective amount.”

[0114] The terms “therapeutically effective amount” refers to that amount of an agent that is effective for producing some desired therapeutic effect in at least a sub-population of cells in an animal at a reasonable benefit / risk ratio applicable to any medical treatment. Toxicity and therapeutic efficacy of subject compounds may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 and the ED50. Compositions that exhibit large therapeutic indices are preferred. In some embodiments, the LD50 (lethal dosage) may be measured and may be, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more reduced for the agent relative to no administration of the agent. Similarly, the ED50 (i.e., the concentration which achieves a half-maximal inhibition of symptoms) may be measured and may be, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more increased for the agent relative to no administration of the agent. Also, similarly, the IC50 (i.e., the concentration which achieves half-maximal cytotoxic or cytostatic effect on cancer cells) may be measured and may be, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more increased for the agent relative to no administration of the agent. In some embodiments, cancer cell growth in an assay may be inhibited by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100%. In another embodiment, at least about a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% decrease in a solid malignancy may be achieved.

[0115] More generally, the term “EC50” refers to the concentration of an agent, like an antibody or antigen-binding fragment thereof, which induces a response that is 50% of the maximal response, such as hallway between the maximum and baseline response in an in vitro and / or in vivo assay.

[0116] The term “vaccine” refers to a composition for generating immunity for the prophylaxis and / or treatment of diseases.

[0117] Native Birds of Shiny Eggs (abbreviated ANOR):

[0118] As used herein “avian species” or “bird” refers to native species of birds for laying and and production of eggs. Birds include the subphylum chordata, and in particular the aves class. Birds may also include the superorder palaeognathae or neognathae. Birds may include by way of non-limiting example from struthioniformes (ostriches, emus, kiwis, and allies), tinamiformes (tinamous), anseriformes (waterfowl), galliformes (fowl, including chickens, ducks, geese, guinea, quail, grouse, pheasant and turkeys), charadriiformes (gulls, buttonquails, plovers and allies), gaviiformes (loons), podicipediformes (grebes), procellariiformes (albatrosses, petrels, and allies), sphenisciformes (penguins), pelecaniformes (pelicans and allies), phaethontiformes (tropicbirds), ciconiiformes (storks and allies), cathartiformes (New World vultures), phoenicopteriformes (flamingos), falconiformes (falcons, eagles, hawks and allies), gruiformes (cranes and allies), pteroclidiformes (sandgrouse), columbiformes (doves and pigeons), psittaciformes (parrots and allies), cuculiformes (cuckoos and turacos), opisthocomiformes (hoatzin), strigiformes (owls), caprimulgiformes (nightjars and allies), apodiformes (swifts and hummingbirds), coraciiformes (kingfishers and allies), piciformes (woodpeckers and allies), trogoniformes (trogons), coliiformes (mousebirds), and passeriformes (passerines). Bird species may include by way of non-limiting example Crypturellus (e.) columbianus, Crypturellus (e.) idoneus, Crypturellus atrocapillus, Crypturellus bartletti, Crypturellus berlepschi, Crypturellus boucardi, Crypturellus brevirostris, Crypturellus casiquiare, Crypturellus cinereus, Crypturellus cinnamomeus, Crypturellus duidae, Crypturellus erythropus, Crypturellus kerriae, Crypturellus noctivagus, Crypturellus obsoletus, Crypturellus parvirostris, Crypturellus ptaritepui, Crypturellus soui, Crypturellus strigulosus, Crypturellus tataupa, Crypturellus transfasciatus, Crypturellus undulatus, Crypturellus variegatus, Eudromia elegans, Eudromia formosa, Eudromia olsoni, Lophophorus impejanus, Nothocercus bonapartei, Nothocercus julius, Nothocercus nigrocapillus, Nothoprocta cinerascens, Nothoprocta curvirostris, Nothoprocta omata, Nothoprocta pentlandii, Nothoprocta perdicaria, Nothoprocta taczanowskii, Nothura boraquira, Nothura chacoensis, Nothura darwinii, Nothura maculosa, Nothura minor, Nothura parvula, Rhynchotus maculicollis, Rhynchotus rufescens, Taoniscus nanus, Tinamotis ingoufi, Tinamotis pentlandii, Tinamus guttatus, Tinamus major, Tinamus osgoodi, Tinamus solitarius, Tinamus tao or Tragopan Satyra. In a preferred embodiment, the avian egg is produced from Eudromia elegans, Lophophorus impejanus or Tragopan Satyra.

[0119] As used herein “avian egg shell” refers to the outer structure of an egg. Avian egg shell is mainly comprised of calcium carbonate.

[0120] As used herein, “avian egg shell color” refers to pigmentation of the egg shell. The colors can include colors that humans can and can not see. Color is produced through selective absorbance of light at a particular wavelengths by pigments, by nanoscale structures that interact with light (structural color) or by the interaction of pigments and nanoscale structures. Nanostructure is associated with the production of iridescent colors. Iridescence can be produced by diffraction gratings, or when light passes through multiple semi-transparent materials that differ in refractive index, causing light to phase-shift and cancel out particular wavelengths at particular viewing angles. Irridescence is measured by specular and diffuse spectral reflectance on eggshell fragments between 300 and 700nm. Biliverdin IXalpha absorbs light in the near ultraviolet and yellow range and produces blue-green colors. Protoporphyrin IX, which absorbs between 300 and 700 nm and produces brown colors.

[0121] As used herein, “gloss” or “glossy” refers to the specular or mirror-like component of light reflection. Gloss is often produced by smooth or polished surfaces. Light hitting a smooth surface is mostly reflected in the specular direction, causing the material to appear glossy, whereas light hitting a rough surface is scattered in a range of directions by the surface topography, causing the material to appear matte. The refractive index of a surface material can affect gloss. Materials with higher refractive index reflect more light and appear glossier.

[0122] IgY (Immunoglobulin Yolk):

[0123] These define immunoglobulins which are extracted from the egg-yolk of fowl eggs and which correspond to the IgG in the serum of the chickens. These avian immunoglobulins are structurally distinguished from the mammalian IgG primarily by their higher molecular weight resulting from a greater number of constant regions in the Fc fragment.

[0124] Immunoglobulin Y (abbreviated as IgY) is a type of immunoglobulin which is the major antibody in bird, reptile, and lungfish blood. It is also found in high concentrations in chicken egg yolk. As with the other immunoglobulins, IgY is a class of proteins which are formed by the immune system in reaction to certain foreign substances, and specifically recognize them.

[0125] Since chickens can lay eggs almost every day, and the yolk of an immunized hen's egg contains a high concentration of IgY, chickens are gradually becoming popular as a source of customized antibodies for research.

[0126] Cancer cells do not resemble normal cells in terms of morphology and metabolic behavior. Accordingly, efforts are invested worldwide in order to develop anticancer therapies that can kill cancer cells without harming normal cells. These therapies attempt to target differentially expressed functional molecules in cancer and normal, non-transformed cells. For this purpose, a myriad of new small molecular weight synthetic and / or natural inhibitor compounds are being tested, aimed at achieving selective anticancer clinical treatments. Small molecular weight chemicals from plants (phytochemicals) often accomplish multi-targeted anti-cancer activities including cell cycle arrest, inhibition of cell growth, proliferation, and metastasis, and promote apoptosis and cell death. IgY is an antibody found in high concentrations in the egg yolk.

[0127] The IgY is transferred from mother to child via the egg. Oral consumption of the “immune” eggs containing specific IgY antibodies protects the animal against the specific organism(s) with which the hen was stimulated. In other words, it is possible to use IgY as a form of "passive immunization" for various diseases. It is necessary to make the mother bird sensitive to a specific disease and collect the IgY that will be transferred from it to the chick via the egg. IgY has a similar structure as mammalian IgG with some minor differences in the heavy chains. The birds proposed in this work to be used to produce IgY are from the family Tinamidae. These monotonous (grayish) birds lay surprisingly colored eggs (like sky blue, lime green and rich chocolate and so glossy that they can reflect overhead trees and brush. These eggs have a unique structure and iridescence. It is possible that glossy eggs have this characteristic due to defensive causes to the egg, linked mainly to mother-to-child immunization.

[0128] Specific IgY

[0129] In the present context it is defined as the proportion of the entire IgY which recognizes the antigen used for immunization, whilst unspecific IgY defines that proportion of the entire IgY which is formed independently of the immunization by contact of the animals with antigenically acting materials or non-pathogenic and pathogenic causative agents.

[0130] Intact Antibodies

[0131] These shall be understood to be immunoglobulins which are not fragmented, i.e., they possess an Fc fragment and two Fab segments, Fc including the constant regions of the heavy chains and Fab including the variable regions of the heavy and light chains.

[0132] The term "antibody fragment" as used herein is intended to include without limitation, Fv, Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, and multimers thereof, multispecific antibody fragments and Domain Antibodies. Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques Techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the disclosure (see e.g., U.S. Patent Mo. 4,946,778). In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al., 1989 Science 246:1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof.

[0133] As used herein, the term “antigen determinant site” or “epitope” refers to the site on an antigen that is recognized by the antibodies and fragments disclosed herein. The term “epitope” includes any protein determinant capable of specific binding to an immunoglobulin. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. An antibody is said to specifically bind an antigen when the dissociation constant is < 1 micromolar; e.g., < 100 nM, preferably < 10 nM and more preferably < 1 nM.

[0134] Non-covalent interactions occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (Kd) of the interaction, wherein a smaller Kd represents a greater affinity. Immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method entails measuring the rates of antigen-binding site / antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions. Thus, both the “on rate constant” (Kon) and the “off rate constant” (Koff) can be determined by calculation of the concentrations and the actual rates of association and dissociation. (See Nature 361:186-87 (1993)). The ratio of Koff / Konenables the cancellation of all parameters not related to affinity, and is equal to the dissociation constant Kd (See, generally, Davies et al., (1990) Annual Rev Biochem 59:439-473). An antibody disclosed herein is said to specifically bind to a tumor associated antigen, when the equilibrium binding constant (Kd) is < 1 micromolar, < 100 nM, < 10 nM, or < 100 pM to about 1 pM, as measured by assays such as radioligand binding assays or similar assays known to those skilled in the art.

[0135] Bispecific antibodies are antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for a tumor associated antigen. The second binding target is any other antigen, and optionally is a cell-surface protein or receptor or receptor subunit.

[0136] Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol 148(5): 1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab’ portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The “diabody” technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigenbinding sites. Another strategy for making bispecific antibody fragments by the use of singlechain Fv (scFv) dimers has also been reported. See, Gruber et al., J. Immunol. 152:5368 (1994).

[0137] Antibodies with more than two valencies are also contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).

[0138] It can be desirable to modify an antibody disclosed herein with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating diseases and disorders associated with the tumor associated antigen activation and / or activity. For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved internalization capability and / or increased complement-mediated cell killing and antibodydependent cellular cytotoxicity (ADCC). (See Caron et al., J Exp Med., 176:1191-1195 (1992) and Shopes, J. Immunol., 148:2918-2922. (1992)). Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. (See Stevenson et al., Anti-Cancer Drug Design, 3:219-230 (1989)).

[0139] The term “antigen-binding site” or “binding portion” refers to the part of the immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable (“V”) regions of the heavy (“H”) and light (“L”) chains. Three highly divergent stretches within the V regions of the heavy and light chains, referred to as “hypervariable regions,” are interposed between more conserved flanking stretches known as “framework regions,” or “FRs”. Thus, the term “FR” refers to amino acid sequences which are naturally found between, and adjacent to, hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as “complementaritydetermining regions,” or “CDRs”. The assignment of amino acids to each domain is in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)); Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); or Chothia et al., Nature 342:878-883 (1989). The amino acids encompassing the complementarity determining regions (CDRs) are as defined by E.A. Kabat et al., (See Kabat, EA, et al., Sequences of Protein of immunological interest, Fifth Edition, US Department of Health and Human Sendees, US Government Printing Office (1991)). The DNA encoding an antibody disclosed herein also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences [see U.S. Patent No. 4,816,567; Morrison, Nature 368, 812-13 (1994)] or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody disclosed herein, or can be substituted for the variable domains of one antigen-combining site of an antibody disclosed herein to create a chimeric bivalent antibody.

[0140] The antibodies and antibody fragments described herein may be humanized in order to make them better tolerated for use in humans. For example, amino acid residues in the framework regions may be humanized by replacing them with amino acid residues and the human framework regions as long as the replacement does not impair the ability of the antibodies and antibody fragments to bind to the antigen (Vincke C, Loris R, Saerens D, Martinez-Rodriguez S, Muyldermans S, Conrath K., “General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold”, J Biol. Chem. 2008).

[0141] It will be appreciated that murine antibodies or antibodies from other species can be humanized or primatized using techniques well known in the art. See e.g., Winter and Harris Immunol Today 14:43 46 (1993) and Wright et al., Crit. Reviews in Immunol. 12125-168 (1992), The antibody of interest may be engineered by recombinant DNA techniques to substitute the CHI, CH2, CH3, hinge domains, and / or the framework domain with the corresponding human sequence (See WO 92102190 and U.S. Patent Nos 5,530,101; 5,585,089; 5,693,761; 5,693,792; 5,714,350; and 5,777,085). Also, the use of Ig cDNA for construction of chimeric immunoglobulin genes is known in the art (Liu et al., P.N.A.S. 84:3439 (1987) and J. Immunol. 139:3521 (1987)). mRNA is isolated from a hybridoma or other cell producing the antibody and used to produce cDNA. The cDNA of interest may be amplified by the polymerase chain reaction using specific primers (U.S. Pat. Nos. 4,68.3, 195 and 4,683,202). Alternatively, a library is made and screened to isolate the sequence of interest. The DNA sequence encoding the variable region of the antibody is then fused to human constant region sequences. The sequences of human constant regions genes may be found in Kabat et al., (1991) Sequences of Proteins of immunological Interest, N.I.H. publication no. 91-3242. Human C region genes are readily available from known clones. The choice of isotype will be guided by the desired effecter functions, such as complement fixation, or activity in antibody-dependent cellular cytotoxicity. Optional isotypes are IgGl, IgG3 and IgG4. Either of the human light chain constant regions, kappa or lambda, may be used. The chimeric, humanized antibody is then expressed by conventional methods.

[0142] IgY Fragments

[0143] In the present context, these connote Fab fragments of avian immunoglobulins which have no or a small residue of the Fc fragments. For the sake of simplicity, the term "Fab" will hereafter be understood also to mean such fragments as F(ab)2.

[0144] Fab Construct

[0145] The term "Fab construct" is to define synthetic formations of two or more different Fab fragments (bi- or tri-valent constructs, etc.) which recognize one or more different antigen determinant sites (mono-, bi- or tri- specific constructs, etc.) and which may thus satisfy one or several functions, i.e. they act mono-, bi- or tri-functionally.

[0146] IgY Compound

[0147] These are to be understood to be the egg-yolk antibodies (IgY), IgY fragments, Fab constructs or chimeric egg-yolk antibodies extracted from egg-yolk and, if required, purified. They are extracted from the umbilical remanence present in the egg shell along with the antibodies, primarily produced as egg yolk (IgY ) antibodies. The umbilical renaissance mass will be optionally purified, and thus the IgY fragments, Fab constructs or chimeric egg yolk antibodies are understood to be present in the embryonic mass left within the shell. The disclosure also includes an immunoconjugate comprising (1) a binding protein disclosed herein, preferably an antibody or antibody fragment, that has been attached to (2) an effector molecule.

[0148] In one embodiment, the effector molecule is a label, which can generate a detectable signal, directly or indirect. Examples of labels include radioactive isotopes (i.e., a radioconjugate). In another embodiment, the effector molecule is a therapeutic agent. Therapeutic agents include, but are not limited to, antineoplastic agents. In yet another embodiment, the therapeutic agent is a toxin. In some embodiments, the effector molecule, is ascorbic acid, vitamin B5, vitamin D3, a jasmonate (or derivative thereof), a camptotectin, FE118 or any other specific compound disclosed herein.

[0149] A toxin is an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof). Toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

[0150] As used herein, the terms “label” or “labeled” refers to incorporation of a detectable marker, e.g., by incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods). In certain situations, the label or marker can also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g.,3H,14C,15N,35S,90Y, "Tc,n iIn,125I,131I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, p- galactosidase, luciferase, alkaline phosphatase), chemiluminescent, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

[0151] Those of ordinary skill in the art will recognize that a large variety of possible moieties can be coupled to the resultant antibodies and antigen-binding fragments thereof. (See, for example, “Conjugate Vaccines”, Contributions to Microbiology and Immunology, J. M. Cruse and R. E. Lewis, Jr (eds), Carger Press, Mew York, (1989), the entire contents of which are incorporated herein by reference).

[0152] Coupling may be accomplished by any chemical reaction that will bind the two molecules so long as the antibody and the other moiety retain their respective activities. This linkage can include many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding and complexation.

[0153] For example, conjugates of the antibody and effector molecule can be made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro- 2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon- 14-labeled l-isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody (see WO94 / 11026).

[0154] Booster and "Replenishing Immunization

[0155] This term will be mentioned in connection with the immunization of birds. The boosters serve to attain the maximum IgY-titer. Replenishing immunization connotes the renewed antigen exposure by which the maximum IgY-titer is maintained until the end of the laying period.

[0156] Immunoconjugates based on monoclonal antibodies from mice (murine MAK) or from humans (human MAK) are known. Many methods have been established of conjugating signal agents (stains, fluorochromes, diagnostic radionuclides) or active agents (e.g. toxins, therapeutic radionuclides, photosensitizers, prodrugs) to these antibodies. They make possible examinations such as ELISA, RIA, fluorescent microscopy, impulse cytometry, scintigraphies, SPECT (single photon emission computed tomography), PET (positron emission tomography) and photo immunodiagnostics, or treatments such as radiation immunotherapy, immuno toxin therapy or photo dynamic therapy.

[0157] Among the disadvantages of the monoclonal antibody technology are mainly the mono specificity of the antibodies as well as the relatively complex and expensive development and confectioning of the monoclonal antibodies. In general, their confectioning must be carried out in fermenters since production in the ascites of mice has been largely proscribed in many countries.

[0158] Polyclonal immunosera of mammals (sheep, horse, pig, etc.) and immunized / infected donors (e.g. from the blood of HIV infected persons of high p24-antibody titers) are important in various diagnostic processes and passive immunotherapies. From the point of view of improved targeting of complex antigens the monoclonality of these immunosera is useful. However, immunosera of mammals or conventional chickens contain a relatively poorly defined polyspecific mixture of antibodies wherein the desired specific antibodies constitute the minor portion whereas the unspecific and, hence, undesired antibodies constitute the major portion. From the point of view of protecting animals, the production of immunosera from mammals is hardly acceptable, and for that reason it has been largely prohibited in several countries. The production of immunosera from human blood donations is expensive, by no means free of risks (transmission of viruses such as HIV, HCV and, possibly, prions), and is ethically problematic, and, for that reason, it is not practicable for a broad range of applications.

[0159] Polyclonal antibodies of aviary origin, among these egg-yolk antibodies (IgY) are also known. Currently, IgY is being offered by different manufacturers for in vitro diagnostics as an alternative to immunosera from mammals or monoclonal antibodies. Specialized literature also refers to examples of how egg-yolk antibodies can be coupled to biotin, FITC or horse radish peroxidase (POD) in order to render them useful for in vitro diagnostics (Schade et al., 2001 [1]).

[0160] The oral application of aviary antibodies from conventional chickens is also known. The parenteral application in human medicine has hitherto be been largely avoided, on the one hand because in mammals IgY, because of the phylogenic spacing, does not couple either to complement factor Cl or Fc receptors so that it cannot interact effectively with the defensive system of mammals, and, on the other hand, because IgY may cause allergic reactions. Furthermore, the production of IgY from egg-yolks from conventional chickens is not efficient and in respect of purity and affinity the resultant products are of inferior quality. The reason for this is that conventional chickens form numerous unspecific antibodies against other germs, and the response to the target immunogen is thus relatively weak and of short duration. For that reason, in the immunization of conventional chickens the proportion of specific IgY relative to the entire IgY is significantly less than 10% (U.S. Patent 4,550,019; Hansen et al., 1998 [2]; Bouhours et al., 1998 [3]; Gassmann et al., 1990 [4]).

[0161] The closest state of the art is described in German patent specification 19504755 which relates to a passive immunotherapy of HIV infection by IgY. The IgY is targeted at the HIV- core-antigens p24 and pl7 of specified pathogen-free (SPF-) chickens. Passive immunotherapies with human immunosera are known as well. Compared to immunosera and monoclonal antibodies IgY may be produced easily and inexpensively. SPF-chickens under the immunization protocol described in Example 2, after basic immunization and boostering will lay 5 to 7 eggs every week, up to their 16thmonth of life. In this connection it was found that about 15,000 mg of specific IgY could be produced from the 250 to 350 eggs of a SPF-chicken, since, empirically, one egg yolk contains at least 50 mg of IgY of the desired specificity. Moreover, the pollution of SPF-chickens, because of the specified treatment prescribed for these animals, is significantly lower than in common chickens raised for meat and eggs. Consequently, the usual active immunizations, including two prescribed inoculations, against up to nineteen commercially significant infectious diseases common in the raising of conventional chickens are avoided. These immunizations account for the low quality of the IgY compounds of conventional chickens and for the complexity and expense of isolating specific IgY. The keeping of SPF-chickens is free of human and fowl pathogenic germs. For excluding such germs feed, water and immunization status are controlled regularly. No antibiotics are added to the feed in order to prevent concealment of the animals' SPF status.

[0162] From a medical point of view, the advantages of IgY in general reside in the recognition of several antigenic determinants by the specific antibodies, which ensures improved targeting of the target antigen, and in the fact that the antibodies are from fowl the immune system of which differs from that of mammals and humans as regards the antigen recognition. This also enhances the targeting of the target antigen since the aviary immune system recognizes (phylogenetically conditioned) antigenic determinants different from the human immune system. Accordingly, there is reduced competition for epitopes between aviary antibodies in vivo and the naturally present antibodies of a patient. Moreover, the assumed disadvantage of aviary antigens reacting with neither the human complement system nor with Fc receptors, protein A or protein G, may in many cases be shown to be insignificant or even as advantageous.

[0163] It is thus an object of the disclosure to develop and make available alternative products to conventional treatments which are superior in respect of their purity and antigen-targeting to monoclonal antibodies and immunosera from mammals as well as to IgY compounds from conventional chickens.

[0164] Before describing the description of the disclosure, further terms will hereafter be defined as they are understood in the ensuing description of the disclosure as well as in the patent claims.

[0165] "Pretargeting"

[0166] In this context, it embraces applications which cause a boost of signals or effects of the IgY compounds by interposing further antibodies of another species or other suitable molecules (compiled herein also under the generic term "booster molecule) in any phase between antigen connection and signal or active agent.

[0167] "Chimeric Egg- Yolk Antibodies"

[0168] These mean (i) humanized immunoglobulins in which Fab fragments of a chicken are biochemically connected to Fc fragments of human IgG, or (ii) humanized immunoglobulins from birds in which the gene sections of the constant regions of IgY are more or less completely replaced by the constant regions of human IgG.

[0169] "IgY conjugate" In this context, this term embraces compounds of polyclonal IgY preparations (immunological component) and signal agents and / or active agents (diagnostic or therapeutic component) and / or booster molecules.

[0170] In accordance with the disclosure the object is accomplished by polyclonal IgY conjugates from intact egg-yolks antibodies (IgY), IgY fragments, Fab constructs or humanized egg-yolk antibodies from SPF- chickens, preferably from transgenic SPF-chickens. As an immunologically effective component, the antibodies or antibody fragments are optionally to be in combination with or conjugated to at least one other component which may be a signal agent, an active agent of a booster molecule.

[0171] The disclosure relates to IgY conjugates from egg-yolk antibodies (IgY) and their production as diagnostic and therapeutic agents and well as for experimental applications in animals (e.g. mouse, sheep or non-human primates). Its use is not limited to the parenteral application, but, depending upon the application target, it includes external (e.g. in a melanoma) and enteral applications (e.g. cancer of the oesophagus).

[0172] Moreover, the disclosure relates to the confectioning of the IgY conjugates as ready-to- use diagnostics or medicines, either as individual components, as labeling kits or as ready-to- use compounds. Target antigens under consideration are all the proteins, peptides, glycoproteins, nucleic acid, polysaccharides and lipoproteins. Above all, the disclosure relates to IgY conjugates against tumor antigens, hormones, receptor proteins, RNS- and DNS sections, infectious germs, prions or part of the antigenic determinants of these target antigens. Depending upon the complexity of the antigen used for the immunization, the IgY conjugates may contain more or less polyspecific IgY. It should be noted that the term “IgY” can encompass any other antibody or binding fragment thereof and is used as a representative example in this application.

[0173] A further aspect of the disclosure relates to pretargeting methods in which either i) biotinylated IgY connects to the target antigen and serves as an intermediary for labeled avid in or streptavidin, or ii) unconjugated IgY connects to the target antigen and serves as an intermediary for human or humanized anti-IgY conjugates.

[0174] In the case of i) the strong bond between biotin and avidin or streptavidin is used so the biotin component in the IgY conjugate may also be called booster molecule.

[0175] Finally, the present disclosure relates to compounds and applications of the IgY conjugates in accordance with the disclosure by which their diagnostic and / or therapeutic properties may be improved, such as surgical procedures, chemotherapeutic s, immunomodulators and paraimmunity inducers.

[0176] The components related directly to the disclosure, their functions, effects and the application of the IgY conjugates in accordance with the disclosure will hereafter be set forth in greater detail and the solutions will be presented in detail.

[0177] Cancer vaccines

[0178] The immune system’s involvement in the natural history of cancer is no longer a matter of debate. The various immunotherapeutic approaches show that harnessing immune effector mechanisms can control the development of neoplasia. Though this recruitment can be performed by various strategies, the most attractive, in theory, are those that induce active responses, namely, the vaccines. Active immune responses display, by their nature, plasticity, a feature that fits well with the characteristic tumor heterogeneity and pheno- and genotypic evolution in the patient. Thus, inducing active immune responses against tumor cells is a very desirable target in the pursue of cancer treatment improvement. In this context, the unique role of dendritic cells in the immune system physiology, that of initiating adaptive responses, makes these cells the ideal tool and target of cancer vaccines development. Supporting this claim, we have results from different clinical trials where dendritic cell-based vaccines were shown to benefit cancer patients with different types of neoplasia, including advanced melanoma, renal cell carcinoma and glioblastoma.

[0179] A. Pharmaceutical Compositions

[0180] Compositions comprising agents encompassed by the present disclosure, such as antibodies, antigen-binding fragments thereof, cells, and the like, are contemplated without limitation. For example, agents may be used alone or in combination with other agents, such as nucleic acid-based compositions (e.g., messenger RNA (mRNA), cDNA, siRNA, antisense nucleic acids, oligonucleotides, ribozymes, DNAzymes, aptamers, nucleic acid decoys, nucleic acid chimeras, triple helical structures, etc.), protein-based compositions, cell-based compositions, as well as variants, modifications, and engineered versions thereof, are contemplated for use in the methods described herein as well as compositions per se.

[0181] Such compositions may be comprised within pharmaceutical compositions and / or formulations. Such compositions may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the agent, such as an active ingredient, into association with an excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, dividing, shaping and / or packaging the product into a desired single- or multi-dose unit. As used herein, the term “active ingredient” refers to any chemical and biological substance that has a physiological effect in human or in animals, when exposed to it. In the context encompassed by the present disclosure, the active ingredient in the formulations may be any of the agents that modulate a biomarker encompassed by the present disclosure.

[0182] Pharmaceutical compositions encompassed by the present disclosure may be presented as anhydrous pharmaceutical formulations and dosage forms, liquid pharmaceutical formulations, solid pharmaceutical formulations, vaccines, and the like. Suitable liquid preparations may include, but are not limited to, isotonic aqueous solutions, suspensions, emulsions, or viscous compositions that are buffered to a selected pH.

[0183] The agents and other compositions encompassed by the present disclosure may be specially formulated for administration in solid or liquid form, including those adapted for various routes of administration, such as (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; or (5) aerosol, for example, as an aqueous aerosol, liposomal preparation or solid particles containing the compound. Any appropriate form factor for an agent or composition described herein, such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas, is contemplated.

[0184] Depending upon need, the inventive IgY conjugates may be applied in vitro or in vivo. In vivo, the application may take place externally or internally (e.g. in the context of photodynamic therapy as well as aparenterally (for instance in scintigraphy, SPEC, radio immunotherapy or immunotoxin therapy). As used herein, the term parenteral includes intravenous, intraarterial, subcutaneous, intracutaneous, intrathecal, interstitial, intracavemous and intralesional or intratumoral application procedures.

[0185] The disclosure will hereafter be explained and described in greater detail on the basis of detailed examples. The characteristics to be gleaned from the drawings and specification may in other embodiments of the disclosure be applied singly or in any desired combination with each other. The following applications and embodiments merely represent examples which in no manner exhaustively present the possibilities of the inventive IgY conjugates based upon SPF-chickens. A person skilled in the art will thus deduct further embodiments and applications which fall into the ambit of the claims of the present disclosure.

[0186] It will be appreciated that administration of therapeutic entities in accordance with the disclosure will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, PA (1975)), particularly Chapter 87 by Blaug, Seymour, therein. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as Lipofectin™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures may be appropriate in treatments and therapies in accordance with the present disclosure, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration. See also Baldrick P. “Pharmaceutical excipient development: the need for preclinical guidance.” Regul. Toxicol Pharmacol. 32(2):210-8 (2000), Wang W. “Lyophilization and development of solid protein pharmaceuticals.” Int. J. Pharm. 203(1-2): 1-60 (2000), Charman WN “Lipids, lipophilic drugs, and oral drug delivery- some emerging concepts.” J Pharm Sci. 89(8) :967-78 (2000), Powell et al. “Compendium of excipients for parenteral formulations” PDA J Pharm Sci Technol. 52:238-311 (1998) and the citations therein for additional information related to formulations, excipients and carriers well known to pharmaceutical chemists.

[0187] Therapeutic formulations of the disclosure, which include an antibody of the disclosure, are used to treat or alleviate a symptom associated with a cancer, such as, by way of nonlimiting example, leukemias, lymphomas, breast cancer, colon cancer, ovarian cancer, bladder cancer, prostate cancer, glioma, lung & bronchial cancer, colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, urinary bladder cancer, kidney and renal pelvis cancer, oral cavity & pharynx cancer, uterine corpus cancer, and / or melanoma The present disclosure also provides methods of treating or alleviating a symptom associated with a cancer. A therapeutic regimen is carried out by identifying a subject, e.g., a human patient suffering from (or at risk of developing) a cancer, using standard methods. Efficaciousness of treatment is determined in association with any known method for diagnosing or treating the particular immune-related disorder. Alleviation of one or more symptoms of the immune-related disorder indicates that the antibody confers a clinical benefit.

[0188] Methods for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme linked immunosorbent assay (ELISA) and other immunologically mediated techniques known within the art.

[0189] Antibodies directed against a target a tumor associated antigen or other antigen (or a fragment thereof) may be used in methods known within the art relating to the localization and / or quantitation of these targets, e.g., for use in measuring levels of these targets within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like). In a given embodiment, antibodies specific any of these targets, or derivative, fragment, analog or homolog thereof, that contain the antibody derived antigen binding domain, are utilized as pharmacologically active compounds (referred to hereinafter as “Therapeutics”).

[0190] An antibody of the disclosure can be used to isolate a particular target using standard techniques, such as immunoaffinity, chromatography or immunoprecipitation. Antibodies of the disclosure (or a fragment thereof) can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (z.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, P-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include125I,1311,35S or3H.

[0191] Antibodies of the disclosure, including polyclonal, monoclonal, humanized and fully human antibodies, may be used as therapeutic agents. Such agents will generally be employed to treat or prevent a disease or pathology associated with aberrant expression or activation of a given target in a subject. An antibody preparation, preferably one having high specificity and high affinity for its target antigen, is administered to the subject and will generally have an effect due to its binding with the target. Administration of the antibody may abrogate or inhibit or interfere with the signaling function of the target. Administration of the antibody may abrogate or inhibit or interfere with the binding of the target with an endogenous ligand to which it naturally binds.

[0192] A therapeutically effective amount of an antibody of the disclosure relates generally to the amount needed to achieve a therapeutic objective. As noted above, this may be a binding interaction between the antibody and its target antigen that, in certain cases, interferes with the functioning of the target. The amount required to be administered will furthermore depend on the binding affinity of the antibody for its specific antigen, and will also depend on the rate at which an administered antibody is depleted from the free volume other subject to which it is administered. Common ranges for therapeutically effective dosing of an antibody or antibody fragment of the disclosure may be, by way of nonlimiting example, from about 0.1 mg / kg body weight to about 50 mg / kg body weight. Common dosing frequencies may range, for example, from twice daily to once a week.

[0193] Antibodies or a fragment thereof of the disclosure can be administered for the treatment of a variety of diseases and disorders in the form of pharmaceutical compositions. Principles and considerations involved in preparing such compositions, as well as guidance in the choice of components are provided, for example, in Remington: The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

[0194] Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based upon the variable-region sequences of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and / or produced by recombinant DNA technology. (See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). The formulation can also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

[0195] The active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions.

[0196] The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0197] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2- hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and y ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT ™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.

[0198] An antibody according to the disclosure can be used as an agent for detecting the presence of a given target (or a protein fragment thereof) in a sample. In some embodiments, the antibody contains a detectable label. Antibodies are polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab, scFv, or F(ab)2) is used. The term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin. The term “biological sample” is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. Included within the usage of the term “biological sample”, therefore, is blood and a fraction or component of blood including blood serum, blood plasma, or lymph. That is, the detection method of the disclosure can be used to detect an analyte mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of an analyte mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of an analyte protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detection of an analyte genomic DNA include Southern hybridizations. Procedures for conducting immunoassays are described, for example in “ELISA: Theory and Practice: Methods in Molecular Biology”, Vol. 42, J. R. Crowther (Ed.) Human Press, Totowa, NJ, 1995; “Immunoassay”, E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996; and “Practice and Theory of Enzyme Immunoassays”, P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivo techniques for detection of an analyte protein include introducing into a subject a labeled anti-analyte protein antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

[0199] As used herein, the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington’s Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Optional examples of such carriers or diluents include, but are not limited to, water, saline, ringer’s solutions, dextrose solution, and 5% human serum albumin.

[0200] A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration.

[0201] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0202] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0203] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.

[0204] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0205] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0206] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[0207] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0208] In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, poly glycolic acid, collagen, poly orthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0209] It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

[0210] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0211] In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a sustained / controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, poly glycolic acid, collagen, poly orthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.

[0212] In one embodiment, oral or parenteral compositions are formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

[0213] The formulation can also contain more than one active compound as necessary for the particular indication being treated, optionally those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

[0214] The disclosure will be further described in the following examples, which do not limit the scope of the disclosure described in the claims. h) Liposome

[0215] The term “liposome” as used herein, may refer to a type of extracellular vesicle generally ranging in size from about 30 nm to about 150 nm and originating in the endosomal compartment of mammalian cells from which they are trafficked to the cell membrane and released. They may contain a therapeutic drug and function in intercellular communication by being secreted from one cell and taken up by other cells to deliver their cargo.

[0216] Liposomes are the phospholipid-membrane-bound subpopulation of extracellular vesicles derived from the plasma membrane. The main activity of liposomes is cellular communication. Liposomes play an important role as drug delivery tools. The outer phospholipid membrane of Liposome improves drug targeting efficiency. Some of the vital features of liposomes such as biocompatibility, low toxicity, and low immunogenicity make it a more exciting drug delivery system.

[0217] Liposomes are nanoscale extracellular vesicles secreted from several cells. Liposomes formation and biological cargo selection and loading may be regulated via (1) the ESCRT- dependent process and (2) the ESCRT-independent pathway. ILVs are eventually discharged as liposomes into the extracellular environment when MVBs fuse with the plasma membrane. Several mechanisms, including (a) antigen presentation, (b) cell signaling, (c) cell membrane fusion, and (d) pinocytosis or phagocytosis, might lead to the uptake of these liposomes by target cells. Liposomes are overcoming all limitations of polymers and liposomes.

[0218] Drugs can be encapsulated in liposomes, thereby prolonging the drug half-life and improving the stability of drug release. Furthermore, due to their endogenous origin, liposomes are highly biocompatible and can be used as nanocarriers for tissue- specific targeted delivery. Studies show that liposomes were designed with hydrophobic agents such as curcumin, and the results showed that liposomes could carry the hydrophobic agent and also enhanced its antiinflammatory properties. (See Kar R, et al., ACS Biomater. Sci. Eng. 2023, 9, 2, 577-594) Liposomes can not only transport the drugs but also increase their half-life, reduce toxicity, and even overcome various barriers. Rupture of the liposome membrane may be used to allow the entry of functional components into the liposome during drug loading. After the required molecules are loaded into the liposomes, the liposome retains its previous shape. Electroporation sonication, extrusion, and freeze-thaw cycling are some of the methods used to disrupt liposome membranes. (See Kar R, et al., ACS Biomater. Sci. Eng. 2023, 9, 2, 577- 594).

[0219] Liposomes suitable for use in this disclosure can be prepared by conventional methods, see, e.g., Sun, et al. (2010) Mol. Ther. 18: 1606-1614. Likewise, compounds can be encapsulated within liposomes by conventional methods, e.g., incubating the compound with an liposomes preparation in saline at room temperature for several minutes, and separating the liposomes from unencapsulated compound and debris, e.g., by sucrose gradient separation (J Control Release. 2015 December 10; 219: 396-405). EXAMPLES

[0220] The representative examples which follow are intended to help illustrate the disclosure, and are not intended to, nor should they be construed to, limit the scope of the disclosure. Indeed, various modifications of the disclosure and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including the examples which follow and the references to the scientific and patent literature cited herein. It should further be appreciated that the contents of those cited references are incorporated herein by reference to help illustrate the state of the art. The following examples contain important additional information, exemplification and guidance which can be adapted to the practice of this disclosure in its various embodiments and equivalents thereof.

[0221] Example 1: Productions of IgY in SPF chickens

[0222] For producing the IgY, birds are immunized subcutaneously (s.c.) or (preferably) intramuscularly (i.m.), preferably in their 14thweek of life, with 50 - 1,000 pg (preferably 50 - 200 pg) of the target antigen or a fragment thereof and complete Freund adjuvant. Thereafter, each bird receives three boosters, until the maximum IgY titer has been reached, and three replenishments are carried out at 50 - 1,000 pg (preferably 50 - 200 pg) each over almost the entire laying period with the same antigen and incomplete Freund adjuvant, for maintaining the maximum IgY titer. The solution of each injection contains 1 ml of antigen suspension and 0.5 ml of adjuvant. An illustrative immunization schedule for birds of SPF status is as follows:

[0223] Day 0* = 1stimmunization (base immunization) i.m. injection;

[0224] Day 28: = 2ndimmunization (1stbooster) i.m. injection;

[0225] Day 56: = 3rdimmunization (2ndbooster) i.m. injection; IgY production;

[0226] Day 84: = 4thimmunization (3rdbooster) i.m. injection; IgY production;

[0227] Day 168: = 5lhimmunization (1streplen.) i.m. injection; IgY production;

[0228] Day 252: = 6thimmunization (2ndreplen.) i.m. injection; IgY production;

[0229] Day 336: = 7thimmunization (3rdreplen.) i.m. injection; IgY production;

[0230] Day 490: -= end of use for IgY production.

[0231] *) = 14 week of life.

[0232] During a period of 12 to 14 months, the eggs from chickens are kept under specified pathogen-free (SPF) conditions and then collected and processed. The egg-yolk is initially separated from the egg-white and may be processed by standard procedures in accordance with Schade et al. (Chicken Egg Yolk Antibodies, Production and Application; Springer- Verlag, Berlin 2001 [1]). The decisive advantage of chickens kept under SPF is that these animals are not immunologically handicapped either because of immunizations or specific infectious diseases. Accordingly, these animals react with a particularly intensive immune response against the antigen with which they are immunized, so that the proportion of specific antibodies is higher by an order of magnitude (50 - 90% specific IgY) than by immunization of conventional chickens. Moreover, in these animals, following the boosters, the high titer of specific antibodies can be maintained by repeatedly replenishing immunization up to their 16thweek of life. Without wishing to be bound by theory, it is believed that this is of significance for the IgY conjugates in accordance with the disclosure, since by repeated stimulation of the immunological memory the lymphocyte populations change such that antibodies of particularly high affinity are formed. For that reason, the IgY compounds from eggs of SPF-chickens differ from IgY compounds derived from conventional chickens also in terms of their affinity to the specific antigen. On a molecular level, the higher affinity is conditioned by the structure of the hypervariable region of these antibodies. The IgY conjugates produced in accordance with the disclosure from SPF-chickens in the final analysis reproducibly differ on a molecular plane (i.e. by the special structure of their hypervariable regions) from IgY compounds from conventional chickens.

[0233] Example 2: Diagnostic of neuroendocrine tumors

[0234] The diagnostic of neuroendocrine tumors is carried out scintigraphically with radio immunoconjugates. Pheochromocytoma, neuroblastoma, carcinoids and paraganglioma develop from the cells of the neuroendocrine (APUD) system which are distributed throughout the entire organism. Thus, they generally possess, somatostatin receptors by means of which they may be scintigraphically rendered visible with the aid of octreotide (a somatostatin analog) labeled with indium 111. However, somatostatin analogs bond to the receptors at a 1:1 ratio. However, polyclonal IgY recognizes several antigenic determinants of the receptor. This leads to better targeting and diagnostics of improved image rendition. For producing the IgY compound against somatostatin receptors SPF-chickens are again immunized in accordance with Example 1. The specific IgY may be commercially offered as a labeling kit of the kind of Example 3, 4 or 5. It may then be labeled in situ by the user with a gamma emitter (e.g. technetium 99m). 1 - 24 hours following an i.v. application of 100 mg of specific IgY-Tc 99m (10- 1,000 MBq, preferably 50- 200 MBq) planar images are taken in two planes and, if necessary, a SPECT is performed. The neuroendocrine tumors which are usually hard to detect intraoperatively may be easily found during a following operative procedure and resected. Example 3: Treatment of non-Hodgkin-lymphoma

[0235] A further application of the radio immunoconjugates is the treatment of non-Hodgkin- lymphoma. Non-Hodgkin lymphomata of low malignancy respond poorly to chemotherapy. The radio immunotherapy has been shown to be particularly effective in such cases. In most studies, iodine 131 labeled murine monoclonal antibodies were used against CD20, an antigen which is expressed on the cell surface of 95% of all B-cell-lymphomata. However, monoclonal antibodies recognize but one antigenic determinant of this antigen. The bonding ratio is thus only 1:1. By contrast, improved targeting may be achieved by CD20-specific IgY which simultaneously recognizes several epitomes on the antigen. In this manner, the radiation exposure of the bone marrow is reduced, and higher radiation doses may be applied without necessitating a bone marrow transplant. A reduced radiation dose may also be achieved by pretargeting procedures with avidin or streptavidin labeled monoclonal antibodies and radioactively labeled biotin. By pretargeting, for which protection is also sought, the therapeutic effect of a radio immunotherapy with IgY may yet again be improved. To this end, SPF-chickens are immunized with 50 - 10,000 pg (preferably 50 - 200 mg) CD20 as in Example 1. During a laying period of 12 to 14 months, eggs are obtained from which the entire IgY is isolated and purified on CD20 by affinity chromatography. For pretargeting the specific IgY is filtered in a sterile manner, conjugated with avidin (or streptavidin) and lyophilized or dissolved, with further auxiliary agents, in isotonic NaCl (or PBS, pH 7.4). In this case, the labeling kit preferably contains a piercing vial with the IgY-avidin- conjugate (100 pg - 5 mg, preferably 1 mg), a second piercing vial with biotin in a lyophilized or dissolved state (11 - 1,000 pg) and a third piercing vial with an oxidizing agent (e.g. lodogen) for the labeling with iodine 131 (1 -4 GBq); furthermore, further auxiliary agents such as chromatographic column and DC kit. Alternatively, the biotin may be centrally labeled by the manufacturer and be delivered together with the IgY-avidin-conjugate. Treatment is initiated by intravenous application of the IgY-avidin-conjugates in order to label the lymphocytes. The intravenous application of the iodine 131-labeled biotin follows one or several days later. Alternatively, the radio immunotherapy may be carried out with an alpha emitter such as astatine 211.

[0236] Example 4: IgY against estrogen receptor

[0237] About half the mammary carcinoma possesses estrogen receptors and is dependent on hormones. Egg-yolk antibodies against estrogen receptors thus cannot only competitively inhibit the estrogen bonds at the receptors. As a radio immunoconjugate they are also suitable for detecting metastases and for internal radiation therapy. For immunizing the SPF-chickens, peptides with the extra-cellular antigenic determinants of the estrogen receptor are used. The immunization of the SPF-chickens as well as the preparation of the egg and purification of the IgY may be carried out in the manner of Example 1 and the production in accordance with one of the other Examples supra.

[0238] Example 5: IgY against kidney cell carcinoma

[0239] Commercially available antibodies such as UR07 recognize kidney cell carcinoma and do not bind to healthy kidney tissue. Kidney cell carcinoma and their metastases may thus be specifically detected and / or treated by radio immunoconjugates. To identify a suitable antigen, the proteins of homogenized kidney carcinoma cell are electrophoretically opened and isolated. SPF-chickens are immunized with these antigens and the produced egg-yolk antibodies are examined with kidney carcinoma cells and physiological tissue with respect to specificity and cross reactivity. The selected antibodies, which recognize only malignant cells, are conjugated with a therapeutic radionuclide such as iodine 131 or with a cytotoxin such as ricin A and are used therapeutically.

[0240] Example 6: Treatment of HIV infections

[0241] A further use of the produced IgY is seen in the treatment of HIV infections. In a HIV infection, the retroviral genome is incorporated into the chromosomes of the host cell. A HIV infection cannot be cured with conventional replication inhibitors. The HIV replicating host cell expresses HIV structure proteins (p24, gpl20 and gp41) on its cell surface. Antibodies against these structure proteins recognize these cells and bind therewith. If they are conjugated with a radionuclide or another cytotoxin, they can destroy these cells and, therefore, the production sites of HIV. A precondition for effective targeting of the cells is that the number of free cells in the blood is reduced, for instance by a complementary replication inhibition with antiretroviral medications. This opens up novel possibilities of effectively treating the HIV infection. For the reasons mentioned in the introduction, the use of polyclonal antibodies from specific IgY offers advantages over monoclonal antibodies of the kind described in DE 198 09 785. For producing the antibodies SPF-chickens are immunized with HIV p24, HIV-1 gpl20 or HIV-1 gp41 as in Example 1. The eggs kept under defined conditions are collected over a laying period of 12 - 14 months. The egg-yolk separated from the egg-white, and the IgY is isolated by one of the standard procedures. HIV- specific antibodies are purified by affinity chromatography on the specific antigen so that unspecific IgY is removed. The sterile IgY compound is conjugated with a therapeutic radionuclide (e.g., iodine 131 or astatine 211). Commonly, for each therapeutic application 100 pg of specific IgY is labeled with 1 - 4 GBq iodine 131. In case of an HIV infection, because of the relatively low number of infected cells (compared to the number of malignant degenerate cells of lymphoma) substantially lower activities may prove to be effective. The labeling may be carried out by different established procedures. IgY-iodine 131 could preferably be produced by the iodogen method. The yield after conjugation of the radio immunoconjugate as well as the purity of the radio immunoconjugate after separation of unbound radionuclides may be controlled by HPLC or DC. The radio immunoconjugate produced and / or sterile-filtered under sterile provisions may be infused as isotonic solution.

[0242] Example 7: Treatment of autoimmune diseases

[0243] In addition, IgY are to be used for the treatment of autoimmune diseases. CD-4 positive T-cells play an important role in the pathogenesis of autoimmune diseases and in rejection reaction in organ transplantations. With M. Crohn, monoclonal antibodies against the CD4 receptor displayed lupus erythematosus, rheumatoid arthritis, but insignificant effects after skin transplants in mice. In order to bring about an effective depletion of CD4 positive cells, the antibodies can be conjugated with a therapeutic radionuclide, a cytostatic agent or a toxin. SPF- chickens are immunized against CD4 or CD4 fragments according to Example 1; the entire IgY is isolated from the egg-yolk, and the CD4-specific IgY can be purified on CD4 by affinity chromatography. If necessary, the antibodies are cleaved enzymatically, and the aviary Fc fragments are replaced by human Fc fragments. The IgY compound may then be distributed for a (preferably intravenous) clinical application in an unconjugated state or labeled with a radionuclide as an IgY conjugate in accordance with the disclosure.

[0244] Example 8: Treatment of thromboses

[0245] The application of IgY for treating thromboses represent a further use. Thromboses are generally treated with systemically applied fibrinolytics (streptokinase, urokinase, rt-PA). Streptokinase is quickly broken by proteases of the body. Urokinase and rt-PA, however, are quickly metabolized by the liver. It is thus useful to fix fibrinolytics to the thrombus (Bode et al., J. Biol.Chem. 1989; 264(2):944-8; Eijnen et al. Thromb Res 1990; 57(3):333-42). SPF- chickens or transgenic chickens are immunized against human fibrin in the manner described in Example 1. The specific IgY is isolated and purified. If necessary, the antibodies are enzymatically cleaved, and the Fc fragments are separated. Intact antibodies of Fab fragments (5 - 50 mg) are then conjugated with rt-PA (5 - 50 mg), preferably at a ratio of 1:1. These IgY conjugates may be offered in lyophilized state or in sterile solution for purposes of injection in suitably sized packages for treatment of deep crural thromboses, pulmonary embolisms, cerebro-vascular insults, or myocardial infarcts. Alternatively, a second group of SPF-chickens may be immunized against rt-PA (preferably a region outside of the active center). The Fab fragments of the first group may then be hybridized with the Fab fragments of the second group. In this manner, fibrin- specific bifunctional Fab constructs may be produced to which rt-PA is conjugated by way of one branch.

[0246] Example 9: Photosensitizers for photodynamic diagnostics or therapy

[0247] IgY compounds can be conjugated with photosensitizers for photodynamic diagnostics or therapy. The photodynamic therapy represents a minimum invasive procedure chiefly for the treatment of carcinoma of the surface of the skin or mucous membrane, Kaposi sarcoma and melanoma. It is based upon a tumor-selective enrichment of light-sensitive substances which release their photodynamic action (fluorescence or formation of cell-damaging radicals and destruction of the tumor vascularization) by the absorption of light. The success of photodynamic therapy depends significantly upon the relative distribution of the photosensitizer in the malignant and healthy tissue. The fixation of the photosensitizer at the tumor cells may be significantly strengthened, for instance in cases of Esophagus carcinoma or malignant melanoma, by tumor- specific antibodies. Diagnosis or therapy may then take place with a suitable light source. In diagnostic applications violet light causes fluorescence of the malignant degenerate cells. In photodynamic therapy, malignant degenerate tissue becomes necrotic under the impact of red light

[0248] Example 10: Isolation of IgY antibodies from Avian Eggs injected with Cancer Cells and a Jasmonate

[0249] Tumor tissue samples are taken from human patients. Tumor cells are isolated and cultured. In native Tinamous species bred for this study, Tinamous glossy eggs that have native genes for production of the immune process will be harvested. Using a sterile needle, eggs are injected with the tumor cells cultured from the tumor tissue sample.

[0250] The cells are injected into the allantoic chorion membrane of the embryo in the egg, by the method already described by Pereira Lopes JEP, 2009, without embryonic death occurring. Optionally, this injection may contain complementary, associative or catabolic and / or metabolic substance of immunoconjugates as the driving vehicle. The cells are injected into the egg at the incubation temperature.

[0251] A jasmonate or derivative thereof is also injected into the chorion membrane of the egg. This jasmonate may be in its simple form, and either compounded, and either natural and or composed with methyl salicylate 1 nL - 1 mL of jasmonate or its derivative is used. The jasmonate may also be conjugated to one or more compounds of the present application.

[0252] The injection of the cancer cells can be done simultaneously or consecutively with the jasmonate or its derivative. Alternatively, the jasmonate or its derivative can be applied from 1 minute to sixteen days after the cancer cells have been injected into the eggs. IgY targeting the tumor associated antigens of the cancer cells are produced by this process.

[0253] The embryo at birth leaves an embryonic residual mass usually at the site where the egg air sac was located. This mass should be withdrawn within 12 hours, rehydrated with various colloids, and frozen at -3 to -120 degrees. Most IgY will be in the embryonic remnants of birds after hatching. The IgY formed from this process will be purified and lyophilized and placed in vacuum and sealed vials.

[0254] Example 11: Administration of IgY to Patients For Treatment of Cancer

[0255] Once reconstituted by various colloidal culture media, there will be in vitro testing of cell cultures that gave rise to the induction process. The blood of the donor patient should be drawn between 1 ml and 500 ml of blood plasma where a quantity other than 0.000001 kilograms of this IgY formed should be added to fresh plasma where, after homogeneity obtained, the plasma should remain at the temperature and reapplied in the patient, in a partial and / or fractional and total way. The mixture may also be lyophilized and the contents obtained should be maintained at temperature below 26 degrees Celsius to -120 degrees Celsius with cryoprotectant. It may be reconstituted by several culture colloids of different origin, at temperatures above 30 degrees Celsius to 39.7 degrees Celsius and after homogenization of the product, it must be administered to the donor patient and to the cells used in the induction. Optionally, the antibody or antigen binding fragment is conjugated via known synthetic routes to one or more compounds before treatment. These specifically include a jasmonate compound (or conjugate thereof) and / or FL118. Additionally, the antibody or binding fragment is converted to a prodrug before administering to a patient.

[0256] Example 12: Effects of Methyl Jasmonate on Tinamous Egg and Chicken Egg IgY Antibody Production

[0257] It is hypothesized that the chicken eggs and other domestic eggs manufactured for commercial consumption have lost their immunological memory and capacity to mount an efficient immune response. By contrast, Tinamous eggs and pheasant eggs retain their immunological memory and retain their ability to quickly and specifically recognize an ancestral antigen and initiate a corresponding response. For example, untreated polyclonal antibodies are detected in the intermembrane space of the chorioallantoic membrane of untreated Tinamous eggs (FIG. 4A) but are not observed in untreated chicken eggs (FIG. 4B). As such, there is a potential to use Tinamous eggs to yield polyclonal antibodies against various antigens, including tumor associated antigens from humans.

[0258] The effect of methyl jasmonate, a plant stress hormone, on the production of polyclonal antibodies in Tinamous eggs and chicken eggs was studied. Commercial chicken eggs are produced in a controlled environment and as such, the eggshells are only exposed to a controlled set of environmental factors. By contrast, Tinamous species and pheasants lay their eggs in forests and shrublands with environmental factors including algae and fungi. We hypothesized that compounds derived from surrounding plants in the natural environment, such as methyl jasmonate, which contact the eggshells when the egg is laid, could penetrate the outer coating and provide a synergistic boost to their natural immune system. To test this, chicken eggs were injected with 50mM of methyl jasmonate. A boost of polyclonal antibody production was observed in chicken eggs (FIG. 5A) which resembled the natural levels of polyclonal antibody production observed in Tinamous eggs (FIG. 5B). As such, methyl jasmonate can boost the dormant immunological response in chicken eggs and enhance their ability to produce polyclonal antibodies.

[0259] Example 13: Effects of Methyl Jasmonate on IgY Antibody Production in Tinamous Eggs and Chicken Eggs injected with B16F10 melanoma cell line

[0260] B16F10 cells were grown ex vivo and cells were injected in nude mice to create murine melanoma. Similarly, B16F10 cells were also applied to the chest muscles of hens. Eggs were collected from chickens prior to immunization. IgY antibodies from these eggs and serum were used as the pre-immunization control and used to evaluate IgY titer in eggs that were treated with B16F10. On Day 0, the breast tissues of hens were injected intramuscularly with 0.05 mg or 0.5 mg of B16F10 cells in Freund’s complete adjuvant. The total volume of the B16F10 and Freund’s complete adjuvant was ImL, with the adjuvant making up at least half the volume. On Days 14, 28 and 33, the breast tissues of the hens were injected intramuscularly with a booster immunization comprising 0.025 mg or 0.25 mg of B16F10 cells diluted in the same volume of Freund’s complete adjuvant. The total volume of the B16F10 and Freund’ s complete adjuvant was ImL. Antibodies specific to B16F10 reached a maximum level at Day 40. Eggs were collected daily. For prolonged antibody production, hens received a booster immunizations every 60 days. Individual hens react differently to immunization with different antigens. Antibody titers will vary as will the time period in which the titers are stable. Chicken eggs were injected with lOOnM of methyl jasmonate contained in liposomes. Control chicken eggs were injected with saline solution. Vessel network destruction and leakage was observed in control chicken eggs (FIGS. 6A-6G), which was not observed in chicken eggs injected with methyl jasmonate. This suggests that chicken eggs injected with methyl jasmonate were able to mount an immune response against B16F10 antigens, preventing vessel abnormality and leakage. Embryos from control eggs died on Day 5, whereas embryos from eggs injected with methyl jasmonate survived and were able to clear the tumor formation after Day 11.

[0261] Tinamous eggs and chicken eggs were injected with B16F10 and polyclonal IgY antibody production was assessed. Tinamous eggs that were not injected with methyl jasmonate had the ability to produce IgY antibodies as shown by western blot (FIG. 7A and 7B). Unlike chicken eggs, this shows that Tinamous eggs have natural ability to mount an immune response against B16F10 cells and produce IgY antibodies without the use of methyl jasmonate. However, addition of methyl jasmonate may boost the level of antibody production and immune response. Furthermore, on Day 5 following injection with B16F10, no leaks or vessel abnormalities were observed in regions were the B16F10 cells were growing in the egg (FIGS. 8A-8C). Furthermore, Tinamous eggs that were injected with B16F10 cells and boosted with methyl jasmonate showed no leaks, damage to the vasculature or tumor growth (FIGS. 9A- 9D). At Day 14 of egg incubation, eggs were re-injected with B16F10 without methyl jasmonate. Tinamous eggs all hatched and no abnormalities were observed in Tinamous chicks. This suggests that Tinamous egg was able to mount a strong immune response to produce specific polyclonal antibodies to combat the growth of B16F10 cancer growth and to enable the embryo to mature into a chick. It also suggests that methyl jasmonate did not have toxic effects towards embryo growth. Umbilical remains from untreated Tinamous eggs and eggs treated with methyl jasmonate were collected. IgY was purified from these remains and lyophilized for further testing.

[0262] Example 14: Effects of IgY Produced from Tinamous Eggs injected with methyl jasmonate on B16F10 mouse tumors.

[0263] B16F10 cells were grown in the presence of lyophlized IgY from the previous experiment to determine if the IgY produced from Tinamous eggs had specificity towards B 16F10 and had cytotoxic effects. Two different concentrations of IgY isolated from Tinamous eggs were added to the growth media of B 16F10 cells and exhibited the ability to kill B 16F10 cell lines, also suggesting IgY specificity towards B16F10 antigens (FIG. 10A-10B). Nude mice were injected with B16F10 cells to induce tumor formation. Peritoneal tissue demonstrated melanoma metastasis (FIG. 11 A). Lyophilized IgY from the previous experiments was reconstituted and administered to Bl 6F 10 mice with tumors. A control group of B16F10 mice with tumors received a control treatment of saline solution. Mice treated with IgY showed no signs of tumor growth or angiogenesis in the peritoneal tissue by microscopy (FIG. 1 IB) and no signs of tumor growth by Day 18. By contrast, none of the control mice survived. This clearly demonstrates the therapeutic effects of IgY produced from Tinamous eggs that were immunized with B16F10 and were boosted with methyl jasmonate. As such the methodology of producing therapeutic antibodies described in Example 12 could be generally applicable for the production of antibodies towards any type of cancer cell.

[0264] Example 15: Analysis of IgY with A- 14

[0265] Product: rat plasma for LC-MS / MS analysis

[0266] Eight male Wistar rats of mean weight of 275 g, from the animal colony of Sao Paulo State University - UNESP were used. The animals were transferred to the animal facility of the Department of Principles of Natural Assets and Toxicology, Faculty of Pharmaceutical Sciences, UNESP, Araraquara, Brazil, where they were kept under controlled temperature (23 ± 1 ° C ), humidity (55 ± 5 % ) and light conditions (12 / 12h cycle , lights on the 0700 h) and balanced diet and water ad libitum. The IgY compound was administered through previously implanted into the femoral vein cannula and the administration volume was 1.1 mL of the solution A14 10 -1M. Blood sampling was carried out on 2.5, 5.0, 10.0, 20.0, 30.0, and 60.0 minutes via previously implanted in the femoral artery cannula and pooled for each collection time. Surgical procedure for implantation of cannulae. The cannulation of the femoral artery of the animal was performed to collect blood to determine the drug concentrations by LC- MS / MS. During the cannulation of the vein, it was performed the administration of these drugs. To complete this procedure, the animal was anesthetized by intraperitoneal administration of xylazine (5.5 mg / kg) and ketamine (73mg / kg), then shaving the ventral side of the upper part of the hind paw and the region was taken back near the nape where the cannula is externalized. An incision was made on the ventral side of the upper hind leg, which was found by touch the pulse of the femoral artery, and then the neurovascular bundle was exposed. In the beam to the vessel was isolated and cannulated through a small section has the insertion of polyethylene fused to a P-50 polyethylene tubing filled with heparin, and 2 % to avoid coagulation pipe P - 10. With the aid change the cannula is expressed in the cervical region of the animal by intertwining the suture site. The animal was kept for a recovery period of 24 hours and was used within 72 hours of finishing.

[0267] The IgY obtained by immunizing birds and removed from the eggs laid by these birds was lyophilized, suspended with ringer lactate, mixed with the blood plasma of the mice and introduced in the mice.

[0268] Product: IgY

[0269] The antibodies were produced by immunizing birds with the application of Bl 6F 10 cells in the chest region. After applications in the matrices, the eggs are used to obtain the IgY, removing from the yolk. The separation and identification of the immunoglobulin is done by ELISA test. After obtaining a large amount of IgY, we can lyophilize it at -80 ° C for 24 hours in a vacuum with a gradual increase in temperature to 37 ° C.

[0270] RESULTS

[0271] LC-MS / MS analysis

[0272] The LC-MS / MS analyses were performed with reversed phase liquid chromatography coupled to triple quadrupole mass spectrometer. The mass spectrum of IgY with dihydroxy methyl jasmonate in solution presented a protonated (MH+) molecule of m / z 227 shown on Figure 12.

[0273] The structural information was obtained towards tandem MS experiment of daughter ions (MS / MS) from m / z 227, Figure 13. The fragmentation with helium gas at 30V forms the ions suggested on Scheme 1.

[0274] The fragmentation pathway is based on the mass obtained from the product ions that confirms the authenticity of the parent ion. Scheme 1 shows the fragmentation pathway of protonated dihydroxy methyl jasmonate.

[0275] Sehssa® 1 The information from the tandem mass spectrum led us to construct the MRM functions of the quantification channel of 227 -> 153 and the confirmation channel of 227 -> 107. These two MRM are monitored and the integrated area obtained on retention time of 3.4 min is concentration dependent. The calibration curve obtained from the quantification of methyl dihydrojasmonate is presented in Figure 14, and the results from plasma eight rat pooled samples are given below in the pharmacokinetic section.

[0276] Pharmacokinetic Analysis

[0277] The disposition kinetics of A- 14 were assessed after administration of a single IV bolus dose. Pharmacokinetic parameters were calculated using the plasma concentration versus time curve. The pharmacokinetic profile of the compound showed characteristic monocompartiment model chosen after visual inspection and assessment of the correlation of plasma concentration versus time curve. The elimination constant (kel) was calculated from the application of the exponential equation obtained from the correlation of plasma concentration versus time. The elimination half-life (Tl / 2) was calculated using equation 1.

[0278] Equation 1

[0279] The AUCo-t was calculated by the trapezoidal method, where the mean plasma concentration for each time interval is multiplied by this time interval, with subsequent summation of all (Equation 2). Where, Cp is the plasma concentration and t is time.

[0280] Equation 2

[0281] For AUCo-oo equation 3 was applied. Where Cpnis the last plasma concentration versus time and kel concentration or P curve is the slope of the linear regression of the logarithm of plasma concentrations by time, in the elimination phase.

[0282] Equation 3

[0283] The ratio of AUCo-t and AUCo-® was used to verify that the time sampling was sufficient for reliable determination of pharmacokinetic parameters, approprhas to be equal to or greater than 0.8.

[0284] The clearance (Cl) was calculated by equation 4, independent model. The volume of distribution (Vd) was calculated by applying the equation 5. The mean residence time (MRT) through relationship calculating the area under the moment curve (AUMC) AU Co- / with was calculated.

[0285] Equation 4

[0286] Preliminary Pharmacokinetic Analysis of compound A- 14 The profile of plasma concentrations (ng / mL) versus time (min) Compound A- 14 shown in Figure 15. Concentrations obtained at each time are shown in Table 1.

[0287] Table 1. Time vs pool plasma concentration after administration of A-14 at a dose of 897.2 mg / kg to eight Wistar rat weighing.

[0288] The profile obtained only shows a decay rate of the plasma concentrations (correlation coefficient 0.9975), characteristic of monocompartiment model for pharmacokinetic analysis. The pharmacokinetic parameters obtained from the plasma concentration profile are shown in Table 2. The ratio of areas obtained from (0.84) allows the calculations in pharmacokinetics reliably.

[0289] Table 2. Pharmacokinetic Parameters A- 14 administered at the dose of 897.2 mg / kg Wistar rats with a weight of 275 g.

[0290] The compound exhibits a short elimination half-life with high Cl and high volume of distribution characteristic pharmacokinetic parameters of lipophilic compounds, which in general are widely distributed in the body and has an excretion process dependent on its metabolism.

[0291] ESI-HRMS analysis

[0292] The ESI-HRMS analyses of cyclodextrin, methyl dihydrojasmonate and the formulation A-14 were performed using 100.000 of resolution (Figure 16) and corroborate to the proposed structures.

[0293] Genotoxicity:

[0294] Methodology: Evaluation of Mutagenicity Using a Micronucleus Test in Peripheral Blood Cells of Mice.

[0295] The doses of positive control and IgY were evaluated at different concentration after they were administered to the animals by gavage. Groups of 10 Swiss mice of both sexes (25-30 g) were used in each test group and the control animals. The positive control group receive ip cyclophosphamide (50 mg / kg). The negative control group received 0.3 mL of 1% carboxymethylcellulose (CMC) suspension. A group in which the animals received only water was also used. The laminas were pre-stained with acridine orange. After 30 h, the animals were killed to collect their blood. We counted 1000 reticulocytes per animal and recorded the frequencies of micronucleated cells. After the cytological analysis of the laminas containing samples of peripheral blood from the mice treated with the various drugs, we calculated the average frequencies of cell micro nuclei (and the standard deviations) for each treatment group. These results were tested with analysis of variance (ANOVA). When P < 0.05, the average values for the treatments were compared using the Tukey method, calculating the minimum significant difference at a = 0.05.

[0296] Results:

[0297] Average frequency of micronucleated reticulocytes (MNRET) and standard deviation of 1000 cells obtained from mice treated with the positive control (Ctrol+: (cyclophosphamide 50 mg / Kg), negative control (Ctrol- : water) and methyl jasmonate in concentrations 10-2 M and 10-3 M. *p <0.05 (compared to negative control and methyl jasmonate).

[0298] The following tables are reported as the review of reference 2.

[0299] Table 3. Effect in vitro of natural and synthetic jasmonates on normal and cancer cells. CONCLUSION

[0300] The analyses of IgY with A- 14 in 8 pooled rat plasma using LC-MS / MS showed a pharmacokinetic al profile correspondent to a IV administration and the characterization using NMR and HRMS corroborate to the structures proposed

[0301] Example 16: Expanding the eggshell colour gamut: uroerythrin and bilirubin from tinamou (Tinamidae) eggshells

[0302] Birds’ eggs are found in an expansive variety of shapes, sizes, and colourings. These pigments may provide a range of structural, thermoregulatory, UV -protective, and photodependent antimicrobial benefts, there is a universal consensus that all of these colors are generated by only two pigments: the tetrapyrrolic compounds protoporphyrin IX (referred to here as 1H, rusty-brown) and biliverdin IXa (2H, blue-green). We thus hypothesized that the purplish and green hues of the N. maculosa and E. elegans eggshells, respectively, are generated by mixing biliverdin with other, yet unknown, pigments. Terefore, we re-examined these tinamou eggshells, specifcally extracting and identifying their pigments, and analysed their contributions to the observed eggshell coloration.

[0303] The findings support the conclusion that the eggshell colours of both species are generated by the previously identified biliverdin pigment in combination with the previously unknown colorants: uroerythrin for N. maculosa and bilirubin for E. elegans. Notably, the purplish- brown colour of the N. maculosa eggshells did not require the presence of a brown porphyrin.

[0304] Contrary to previous reports that found only biliverdin in N. maculosa and E. elegans eggshells, this study also discovered the orange pigment uroerythrin 3H and the yellow-brown pigment bilirubin 4H, respectively. We can confidently conclude that both of these newly found pigments are genuine eggshell pigments as we experimentally verified that they are not artefacts generated in the extraction process. Additionally, these results are supported by colour mixing models which found that unique combinations of these pigments would generate the unusual surface colours of these eggshells. Furthermore, the excellent colour matches generated by our colour mixing models suggest that the presence of other minor pigments, such as the light-yellow dipyrrolic degradation products seen in the extract of the N. maculosa eggshells, do not contribute to the colour of the eggshells.

[0305] In conclusion, the investigation of the unusually coloured eggshells of two tinamou species revealed, next to the well-known eggshell pigment biliverdin 2H, the presence of two hitherto unrecognized oligopyrrolic eggshell pigments: the orange tripyrrolic uroerythrin 3H (in eggshells of N. maculosa) and the brown-yellow tetrapyrrolic bilirubin 4H (in eggshells of E. elegans), both isolated and identified as their diacids and their dimethyl esters. A colour mixing model supports the conclusion that the eggshell colours of both species are generated by the presence of the previously identified pigment biliverdin in combination with the two previously unknown colourants. Notably, the chocolate-brown coloration of the E. elegans eggshells can be achieved without any contribution of the traditional brown pigment, protoporphyrin IX. The yellow-brown bilirubin and orange uroerythrin thus expand the pallete of the known eggshell pigments. Furthermore, we suggest that the layering of biliverdin with these pigments possessing different abilities to photo-degrade may have an adaptive value for the tinamou species investigated.

[0306] Example 17: Vaccine Production and Application

[0307] The fresh tumor sample obtained from surgical resection is minced and digested with collagenase type VIII (0.56 mg / mL; Sigma-Aldrich, San Luis, MI, USA), under agitation, at 37 °C for 2 h. Cell suspensions are separated from the non-digested fragments using sterile gauze and washed twice in RPML1640. In parallel, a small portion of the tumor is fixed in paraformaldehyde 4% and processed for pathological analysis (hematoxylin / eosin and immunohistochemistry) .

[0308] Peripheral blood mononuclear cells (PBMCs) are obtained from leukapheresis chambers of blood donors by separation over Ficoll-Paque gradient (GE Healthcare). PBMCs (3 x 108) are seeded in 75 cm2flasks and incubated for 2 h at 37 °C and 5% CO2. After incubation, nonadherent cells are removed, and adherent cells are cultured in AIM-V supplemented with GM-CSF (50 ng / mL; Peprotech, Cranbury, NJ, USA) and IL-4 (50 ng / mL; Peprotech). After five days, the cells receive a maturation stimulus with TNF-a (50 ng / mL; Peprotech), and 48 h after activation, are harvested and resuspended in a sterile 5% glucose solution; tumor cells are thawed, washed, and also resuspended in a sterile 5% glucose solution. Both cell suspensions are at a concentration of 1 x 107cells / mL. The two cell suspensions are mixed, and the cells are fused by an electric pulse of 1000 V / cm at 25 pF (applied by a Gene- Pulser II; Bio-Rad, Richmond, CA, USA), after being aligned in an electrical field (62.5 V / cm) for 15 s. Cells are left to rest for 2 min in the electroporation cuvette and transferred to a relaxation buffer (100-mM KCL, 3-mM NaCl, 1.25-mM EDTA, 10-mM PIPES, 0.5-mM ATP, adjusted to pH 6.8), where they are kept for an additional 3 min. The hybrid cell preparation is centrifuged, resuspended in 1 mL of sterile phosphate-buffered saline (pH 7.2), and after irradiation (200 Gy), injected into each patient. The harvested tumor samples are sufficient to produce 1 to 12 vaccine doses (mean 5; median 4). Freshly prepared hybrid cell suspensions are applied once a month intradermically, in 2 points in the forearm, 0.5 mL each, after proper asepsis with alcohol swabs.

[0309] Example 18: Treatment of cancer

[0310] A further application of the radio immunoconjugates is the treatment of cancer. SPF- chickens were immunized with 50 - 10,000 pg (preferably 50 - 200 mg) IgY-avidin-conjugates as in Example 1 and treated with a jasmonate compound. During a laying period of 12 to 14 months, eggs are obtained from which the entire IgY is isolated and purified by affinity chromatography .

[0311] Eggs from birds immunized against specific cancer cells were obtained. The IgY that immunizes the eggs had a high success rate in fighting against cancer, managing to generate well-structured chicks. Images of sections of different organs in the embryo shows no tumors in the fetus of chicken treated with cancer cells and a jasmonate compound. On the other hand, the fetus of chicken treated with cancer cells without a jasmonate compound showed tumor growth.

[0312] FIGs. 23A-23F show fetus of chicken with 11 days of development, treated with osteosarcoma. Observe the fetus with 3.0 cm of crow-rump already well developed showing all the body regions formed (FIG. 23A). Highlighting the developing brain (FIG. 23B), showing a division into 4 vesicles. FIG. 23C shows detail of the eyeball showing the pigmented retina. FIG. 23D shows that in the face region, the beak was well developed. The limbs (wings and legs) present the formed and individualized digits (FIG. 23E and FIG. 23F). Throughout the entire length of the fetus, intense vascularization was observed. FIG. 24 is a microscopy image showing immunohistochemistry staining of sections of the heart and lung of the chicken in FIG. 23. FIG. 25 is a microscopy image showing immunohistochemistry staining of sections of the vertebrae, esophagus, and intestinal loops of the chicken in FIG. 23.

[0313] FIGs. 26A-26E show fetus of chicken with 11 days of development, treated with melanoma. Observe the fetus with 2.7 cm of developing crow-rump (FIG. 26 A). Highlighting the developing brain (Fig. 26B), showing a division into 4 vesicles, however they are not as pronounced. FIG. 26C shows detail of the eyeball showing the pigmented retina. The limbs (wings and legs) present the formed and individualized digits (FIG. 26D and FIG. 26E). FIG. 27 is a microscopy image showing immunohistochemistry staining of sections of the eye, eye lens, heart, vertebrae, and medulla of the chicken in FIG. 26. FIG. 28 is a microscopy image showing immunohistochemistry staining of sections of the heart, lung, and stomach of the chicken in FIG. 26. FIG. 29 is a microscopy image showing immunohistochemistry staining of sections of the liver, intestinal loops, kidney, and primitive gonoda of the chicken in FIG. 26.

[0314] FIGs. 30A-30D show fetus of chicken with 11 days of development, treated with canine bone marrow. Observe the fetus with 2.2 cm of crow-rump and its macroscopic characteristics (FIG. 30A). An earlier stage of development is observed. The pronounced brain with a division into 4 vesicles was identified. FIG. 30B shows detail of the eyeball showing the beginning of retinal pigmentation. The wings begin to show the division of the digits (FIG. 30C), however the pelvic limbs are still in the form of buds (FIG. 30D). FIG. 31 is a microscopy image showing immunohistochemistry staining of sections of the eye and vertebrae of the chicken in FIG. 30. FIG. 32 is a microscopy image showing immunohistochemistry staining of sections of the heart, liver, intestinal loops, and kidney of the chicken in FIG. 30.

[0315] FIG. 33A-33E shows fetus of chicken with 11 days of development, control. Observe the fetus with 3.0 cm of crow-rump and its macroscopic characteristics (FIG. 33 A). FIG. 33B shows detail of the pronounced brain, where a division into 4 vesicles was identified. FIG. 33C shows detail of the eyeball showing the pigmented retina. The limbs (wings and legs) present the formed and individualized digits (FIG. 33D and FIG. 33E). FIG. 34 is a microscopy image showing immunohistochemistry staining of sections of the eye, lung, vertebrae, and medulla of the chicken in FIG. 33. FIG. 35 is a microscopy image showing immunohistochemistry staining of sections of the heart of the chicken in FIG. 33. FIG. 36 is a microscopy image showing immunohistochemistry staining of sections of the intestinal loops, liver, kidney, rim / glomerulo, kidney, and primitive gonod of the chicken in FIG. 33.

[0316] FIG. 37 shows melanoma cells spreading in organs of chicken treated with melanoma cells. A and B show that melanoma cells were spread in hens not treated with a jasmonate compound (Methyl jasmonate (MeJA). C to H show that melanoma cells were not spread in hens treated with MeJA (C and D: Treated with 10'7MeJA, E and F: treated with 10'5MeJA, and G and H treated with 10'3MeJA).

[0317] FIG. 38 shows that Melanoma spread all over the embryo that were not treated with a jasmonate compound.

[0318] Example 19: Activating Immune Response

[0319] FIG. 39A shows that the vessels are surrounded by micro bubbles full of 4.5 jasmonate compounds (didehydromethyl jasmonate, DHMJ) and activating the immune responses towards the antigen. FIG. 39B shows the egg of the same species. On the right position, the egg was laid in captivity without the possibility to build up its nests and adding natural algae that stands between the bio-film that holds the micro vegetal agents and the eggshell. This micro-film is what gives the glossy look into the egg appearance. In captivity, there is no accumulation of bubbles and through the microvision there is no antibodies activated. On the left position, the egg was laid in the wild. FIG. 39C shows that the macrophage being activated by the 4,5-didehydrojasmonic acid (4.5 DHMJ). the active macrophage attack the cancer cell, after being exposed to the cells for 6 hours.

[0320] Standardization of ELISA-PME / Hardjo A - As an example: Average OD of serum samples in dilutions of 1:50, 1:100, 1:500 and 1:1,000, anti-bovine IgG conjugate diluted 1:5000 and antigen (PME / Hardjo) at a concentration of 0.35 pg / pL; diluted 1:2 (0.17pg / pL), 1:4 (0.08 pg / pL) and 1:8 (0.04 pg / pL).

[0321] FIG. 40 shows that IgY is produced at a higher level with the hens are treated with cancer cells plus a jasmonate compound (Monal, didehydromethyl jasmonate) compared to cancer cells alone.

[0322] INCORPORATION BY REFERENCE

[0323] All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0324] EQUIVALENTS

[0325] While specific embodiments of the subject disclosure have been discussed, the above specification is illustrative and not restrictive. Many variations of the disclosure will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the disclosure should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

What is claimed:

1. A method of producing a population of polyclonal antibodies that specifically bind to an antigen, the method comprising: administering the antigen to a hen or to an avian egg produced; administering a jasmonate compound to the hen or the avian egg; and isolating the population of polyclonal antibodies from the yolk and / or the chorionic amnion membrane of the egg.

2. The method of claim 1, wherein the hen is kept under specified pathogen-free (SPF) condition.

3. The method of claim 1 or 2, wherein the hen is 1 week old, 2 weeks old, 3 weeks old, 4 weeks old, 5 weeks old, 6 weeks old, 7 weeks old, 8 weeks old, 9 weeks old, 10 weeks old, 11 weeks old, 12 weeks old, 13 weeks old, 14 weeks old, 15 weeks old, 16 weeks old, 17 weeks old, 18 weeks old, 19 weeks old, and 20 weeks old.

4. The method of any one of claims 1-3, wherein the antigen is administered to the hen subcutaneously or intramuscularly.

5. The method of any one of claims 1-4, wherein the antigen is administered to the egg’s allantoic chorion membrane.

6. The method of any one of claims 1-5, wherein the antigen is 50 pg to 1,000 pg.

7. The method of any one of claims 1-6, wherein the antigen is 20 pg to 200 pg.

8. The method of any one of claims 33-39, wherein the antigen is a protein expressed on a cancer cell.

9. The method of claim 8, wherein the cancer cell is selected from a group consisting of a lung cancer cell, a bronchial cancer cell, a prostate cancer cell, a breast cancer cell, a colorectal cancer cell, a pancreatic cancer cell, an ovarian, a leukemia cancer cell, a lymphoma cancer cell, an esophageal cancer cell, a liver cancer cell, a urinary and / or bladder cancer cell, a renal cancer cell, an oral cavity cancer cell, a pharyngeal cancer cell, a uterine cancer cell and a melanoma cancer cell.

10. The method of any one of claims 1-9, wherein the antigen is a tumor associated antigen.

11. The method of claim 10, wherein the tumor associated antigen is CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, c-Met;PSMA, Glycolipid F77, EGFRvllI, ganglioside GD-2, NY-ESO-1, MAGE A3S, ADAM9, ALCAM, Alkaline phosphatase, placental like 2 (ALPPL2), Androgen receptor, AXL, Beta-l,4-N-acetyl- galactosaminyltransferase 1 (B4GALNT1), carcinoembryonic antigen, Carbonic anhydrase IX (CA9), CD74, CD117 (KIT), CD274, CD276, CD44, CD46, CD133, Cadherin 6 (CDH6), Carcinoembryonic antigen related cell adhesion molecule 5 (CEACAM5), CLDN6, claudin 18.2 (CLDN18.2), claudin 18.1 (CLDN18.1), CTAG1B, Delta like canonical Notch ligand 3 (DLL3), Desmoglein 2 (DSG2), EGFR, Epithelial cell adhesion molecule (EPCAM), EphA2, Erb-b2 receptor tyrosine kinase 2 (ERBB2), Erb-b2 receptor tyrosine kinase 3 (ERBB3), Coagulation factor III (F3), Fc receptor like 5 (FCRL5), Fibroblast growth factor receptor 2 (FGFR2), FGFR2-IIIb, Folate hydrolase 1 (FOLH1), folate receptor 1 (FOLR1), globohexaosylceramide (Glono H), glypican-3 (GPC3), Glycoprotein nmb (GPNMB), Guanylyl cyclase 2C (GUCY2C), HER1, HER2, ICAM-1, IGF1R, IL13Ra2, ILl lRa, Integrin subunit alpha 2 (ITGA2), ITGB6, KAAG1, KRAS, L1CAM, LY6 / PLAUR domain containing 3 (LYPD3), MAGE, MAGEA1, MELTF, MET proto-oncogene receptor tyrosine kinase (MET), Mucin 1 (MUC1), mucin 16 (MUC16), Nectin cell adhesion molecule 4 (NECTIN4), NKG2D, PD-1, PD-L1, PROMI, PSCA, Protein tyrosine kinase 7 (PTK7), ROR2, SEZ6, Solute carrier family 34 member 2 (SLC34A2), Solute carrier family 39 member 6 (SLC39A6), Sialyl-Thomsen nouveau antigen (sTN), Somatostatin receptor 5 (SSTR5), Tumor associated calcium signal transducer 2 (TACSTD2), TFRC, Trophoblast glycoprotein (TPBG), Tyrosinase related protein 1 (TYRP1), VTCN1, or WT-1.

12. The method of any one of claims 1-9, wherein the antigen is a viral antigen.

13. The method of claim 12, wherein the viral antigen is an HIV antigen or a SARS- CoV2 antigen.

14. The method of any one of claims 1-13, wherein the polyclonal antibodies are an IgY isotype.

15. The method of any one of claims 1-14, wherein the jasmonate compound is jasmonic acid, 7-iso-jasmonic acid, 9,10-dihydrojasmonic acid, 9,10-dihydro-isojasmonic acid, 2,3-didehydrojasmonic acid, 3,4-didehydrojasmonic acid, 3,7-didehydrojasmonic acid, 4,5- didehydrojasmonic acid, 4,5-didehydro-7-isojasmonic acid, cucurbic acid, 6-epi-cucurbic acid, 6-epi-cucurbic acid-lactone, 12-hydroxy -jasmonic acid, 12-hydroxy -jasmonic acid- lactone, 11 -hydroxy -jasmonic acid, 8-hydroxy-jasmonic acid, homo-jasmonic acid, dihomojasmonic acid, 11-hydroxy-dihomo-jasmonic acid, 8-hydroxy-dihomo-jasmonic acid, tuberonic acid, tuberonic acid-O-P-glucopyranoside, cucurbic acid-O-P-glucopyranoside, 5,6- didehydro-jasmonic acid, 6,7-didehydro-jasmonic acid, 7,8-didehydro-jasmonic acid, cisjasmone, dihydrojasmone, or a lower alkyl ester thereof16. The method of any one of claims 1-15, wherein the jasmonate compound is jasmonic acid.

17. The method of any one of claims 1-16, wherein the jasmonate compound is contained in a liposome.

18. The method of any one of claims 1-17, wherein the avian egg is an Anatidae egg, a Tinamous egg, a chicken egg, a duck egg, a swan, a quail egg, an ostrich egg, a pheasant egg, a turkey egg, a guinea egg, a guinea fowl egg, or a goose egg.

19. The method of claim 18, wherein the avian egg is a Eudromia elegans egg, Lophophorus impejanus egg or Tragopan Satyra egg.

20. The method of any one of claims 1-19, wherein the avian egg shell is glossy.

21. The method of any one of claims 1-20, wherein the avian egg shell prevents the penetration of light having a wavelength of 540nm.

22. The method of any one of claims 1-21, wherein the polyclonal antibodies are further conjugated to a liposome by a linkage selected from the group consisting of a sulfide linkage, a hydrazone linkage, a hydrazine linkage, an ester linkage, an amido linkage, an amino linkage, an imino linkage, a thiosemicarbazone linkage, a semicarbazone linkage, an oxime linkage, a carbon-carbon linkage, or a combination thereof.

23. The method of any one of claims 1-22, wherein the polyclonal antibodies are further conjugated to at least one therapeutic or diagnostic agent.

24. The method of claim 23, wherein the therapeutic or diagnostic agent is selected from the group consisting of a drug, a prodrug, a toxin, an enzyme, a biotin, an enhancer molecule,a radionuclide, a cytostatic agent, a photosensitizer, a fibrinolytic agent, an immunomodulator, a cytokine, a hormone, a second antibody or antigen-binding fragment thereof, an antisense oligonucleotide, an RNAi, an anti-angiogenic agent, a pro-apoptosis agent, a dye, a fluorescent agent, a contrast agent, a paramagnetic ion and a photodynamic agent.

25. The method of claim 23, wherein the therapeutic agent is selected from the group consisting of aplidin, azaribine, anastrozole, azacytidine, bleomycin, bortezomib, bryostatin- 1, busulfan, calicheamycin, camptothecin, 10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin, irinotecan (CPT-11), SN-38, carboplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin, daunomycin glucuronide, daunorubicin, dexamethasone, diethylstilbestrol, doxorubicin, doxorubicin glucuronide, epirubicin glucuronide, ethinyl estradiol, estramustine, etoposide, etoposide glucuronide, etoposide phosphate, floxuridine (FUdR), 3',5'-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, fluorouracil, fluoxymesterone, gemcitabine, hydroxyprogesterone caproate, hydroxyurea, idarubicin, ifosfamide, L-asparaginase, leucovorin, lomustine, mechlorethamine, medroprogesterone acetate, megestrol acetate, melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, phenyl butyrate, prednisone, procarbazine, paclitaxel, pentostatin, PSI-341, semustine streptozocin, tamoxifen, taxanes, taxol, testosterone propionate, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil mustard, velcade, vinblastine, vinorelbine, vincristine, ricin, abrin, ribonuclease, onconase, rapLRl, DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.

26. The method of any one of claims 1-25, wherein the jasmonate compound boosts the dormant immunological response in the avian egg and / or enhances the avian egg's ability to produce polyclonal antibodies.

27. The method of any one of claims 1-26, wherein the jasmonate compound is contained in a liposome.

28. The method of any one of claims 1-27, wherein the polyclonal antibodies are further lyophilized.

29. The method of any one of claims 1-28, wherein the polyclonal antibodies are further conjugated with an additional compound.

30. The method of claim 29, wherein the additional compound is a camptothecin or camptothecin analog.

31. The method of claim 30, wherein the camptothecin has the following chemical structure:

32. The method of claim 29, wherein the additional compound is a salicylic acid.

33. The method of any one of claims 1-32, wherein the polyclonal antibodies are in the form of a prodrug.

34. A population of polyclonal antibodies produced by the method of any one of claims 1- 33.

35. A monoclonal antibody or antigen binding fragment thereof derived from the population of polyclonal antibodies of claim 34.

36. A pharmaceutical composition comprising the population of polyclonal antibodies of claim 34 or the monoclonal antibody or antigen binding fragment thereof of claim 35 and a pharmaceutically acceptable carrier.

37. A method of treating a disease, the method comprising administering the population of polyclonal antibodies of claim 34, the monoclonal antibody or antigen binding fragment thereof of claim 35, or the pharmaceutical composition of claim 36 to a subject in need thereof.

38. The method of claim 37, wherein the subject is a human.

39. The method of claim 37 or 38, wherein the disease is cancer.

40. The method of claim 39, wherein the cancer is selected from the group consisting of a lung cancer cell, a bronchial cancer, a prostate cancer, a breast cancer, a colorectal cancer, apancreatic cancer, an ovarian cancer, a leukemia, a lymphoma, an esophageal cancer, a liver cancer, a urinary cancer, a bladder cancer, renal cancer, an oral cavity cancer, a pharyngeal cancer, a uterine cancer and a melanoma.

41. The method of claim 39 or 40, wherein the method further comprises administering a natural killer (NK) cell.

42. The method of claim 37 or 38, wherein the disease is a viral infection.

43. The method of claim 42, wherein the viral infection is HIV or SARS-CoV2.

44. The method of any one of claims 37-43, wherein the antibody or antigen binding fragment, the pharmaceutical composition, the population of polyclonal antibodies, or the monoclonal antibody or antigen binding fragment thereof is administered by intravenous, intramuscular, intraperitoneal, intravascular, parenteral or subcutaneous administration.

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