MX2015004554A - Production of the immunotoxin igy anti cd133 coupled to an abrin for treating tumors in the central nervous system and/or neoplasms related to carcinogenic stem cells. - Google Patents

Abstract

The carcinogenic stem cells have self-renovation and resistance characteristics to radio and chemotherapy. Antigen CD133 allows carcinogenic stem cells to be identified in osteosarcoma, melanoma, breast cancer, prostate tumors, retinoblastoma, leukemia, non small cell lung cancer, tumors in the central nervous system and larynx and hepatocellular carcinoma. Malignant gliomas represent 70% of the malignant brain tumors. The survival of these patients is poor despite the current treatments. Abrin specifically and irreversibly inhibits the protein synthesis of eukaryotic cells. The advantage of producing an anti CD133 coupled to an abrin is that drugs are selectively released to tumors, reducing toxicity and increasing their power. The present application includes the description and technical data for the production of an immunotoxin of the lgY type coupled to the A chain of the abrin toxin, using techniques of recombinant DNA for the production of the AC133 antigen and the A chain of a brin. This project offers an alternative for eradicating carcinogenic stem cells for developing a specific and affordable therapy that help or replace current therapies, providing a better life quality and expectancy to patients with cancer.

Description

Production of an anti-CD133 IgY Immunotoxin coupled to abrin for the treatment of tumors of the central nervous system and / or neoplasms related to carcinogenic stem cells.

Description of the invention The present invention relates to the field of health, specifically to the area of brain cancer and other neoplasms related to carcinogenic stem cells.

Background Malignant gliomas are the most frequent brain tumors in adults, they are responsible for more than 15,000 deaths in the United States each year [1]. In the National Institute of Neurology and Neurosurgery of Mexico, Glioblastoma Multiforme (GBM) represents 9% of all brain tumors and 45.7% of primary gliomas [2, 3] Despite multiple advances in diagnosis, the prognosis of These tumors are poor; the average survival time of tumors without treatment is only 3 months, due to cerebral edema and increased intracranial pressure.

Even with the best current therapy, which includes radiation, chemotherapy and surgery, the average survival does not exceed more than 14 months [4, 5}. .

Stem cells are defined as cells capable of self-regeneration and multilineal differentiation and due to their survival capacity are subject to the accumulation of multiple mutations that are required for carcinogenesis. [6] Evidence supporting the role of stem cells in carcinogenesis is the observation that both normal and carcinogenic stem cells have several important characteristics. These include: a) the capacity for self-renewal, b) the ability to differentiate, c) active expression of telomerase, d) activation of antiapoptotic pathways, e) increased membrane transport activity, and f) the ability to migrate and metastasize as well that according to recent research, these cells have been attributed tumorigenic capacity, that is, some of the characteristics of malignization of the tumor; infiltration capacity of normal tissue, ability to induce angiogenesis and radio / chemoresistance in all neoplasms [7-9]. In fact; Properties such as anchoring independence, which have been like a hallmark of neoplastic transformation, have recently been reported by several groups as a property of carcinogenic stem cells [10-12] Neural stem cells are characterized as undifferentiated, pluripotent cells, possessing the ability to differentiate neurons, astrocytes and ligolendrocytes [13], have the ability to divide for indefinite periods, deautorregeneration, and the ability to grow in the form of cellular aggregates called neurospheres [ 14] It has been observed that, after being transplanted into the brain of nude mice, they can infiltrate and migrate to other brain areas and differentiate into neuroblasts [15]. Regarding brain tumors, three independent groups showed the existence of a compartment of stem cells in human tumors. These stem cells, like their normal counterpart, are able to form neurospheres in vitro and express markers of neural stem cells such as CD133 and Nestin. So, with only 100 of these cells it is possible to induce tumors by injecting them intracranially into NOD / SCID mice [16,17]. In contrast, 105 CD133 cells - although they grew, were not able to produce a tumor. Tumors produced by CD133 + cells reproduced the phenotypic characteristics and heterogeneity found in the initial tumor [17].

It is believed that the neoplastic stem cells observed in gliomas and later called 'glial stem cells' (CMG) are responsible for the infiltration process and consequent recurrence of glioblastomas multiforme [17]. The first CMG were identified in advanced stages of the glioma, corresponding to glioblastomas multiforme [18]. Even so, the exact time of appearance and the role they play in the early stages of tumor development are unknown, however., it is important to identify and define the time of appearance of CMG in the early stages of the glioma to lay the basis for a future antitumor therapy aimed at eliminating CMG and thus eradicate tumor growth and malignancy. In 2007, Fan et al described tumor cells as the cellular subpopulation capable of regenerating the tumor within a permissive environment [19]. Recent research has shown a close relationship between stem cells and tumor stem cells. Both cell types share the aforementioned characteristics, as well as several common cellular signaling mechanisms: the bcl-2 oncogene, Sonic hedgehog (Shh), the Wnt signaling cascade [20], and the expression of markers such as CD133, the filament intermediate nestin [21] and the transcription factor Sox2 [22] However, differences were observed in terms of proliferation frequency, expression of differentiation markers, chromosomal alterations and tumorigenic capacity.

According to the theory of Holland et al, tumor stem cells could come from neural stem cells with genetic alterations [23] It has been described that the activation of metabolic pathways such as Notch [24], Akt [25] or suppressor gene p53 tumors that intervenes in the generation of tumor stem cells from mother cells.

In addition, it is believed that glial stem cells are the inducers of the angiogenesis process via hypoxia-induced factor (HIF-1) / vascular endothelial growth factor (VEGF) [26] In 19Q2, Dahlstrand et al [21] identified stem cells in glial-type brain tumors. These CMG, after isolating human deglioblastomas and transplanted into the brains of immunosuppressed mice, generated glial tumors [27]. The CMG are a subpopulation within the mass of neoplastic cells, which have characteristics acquired by adaptation to the tumor microenvironment and which possess tumorigenesis capacity much higher than that of the remaining neoplastic cells [9].

Based on this finding, CMGs are currently being considered as a possible therapeutic target. However, after exposing them to radiochemotherapy, the results that have been obtained so far have been negative [7] According to recent research, CMG are resistant to ionizing radiation thanks to a more efficient mechanism of DNA repair and phosphorylation of the protein kinases Chk1 and Chk2 [26]. Resistance to chemotherapeutic drugs is attributed to transporters that pump the chemotherapeutic agents out of the cells [28] characteristics involved in the resistance and recurrence of this type of tumors [29,30]. Recent studies focus on the identification of CMG. Although there is no specific marker for this cell type until today, the expression of the dizzier CD133 has been associated with the identification of CMG of multiform human glioblastomas [18]. In addition to CD133, also known as prominin-1, present in CMG, there are other non-specific markers of these cells, such as nestin, a protein found in neural stem cells present in the subventricular zones, GFAP, Map-2, Neural Tubulin. , Neurofilament, 04, Neurosphere, Noggin, etc. [31, 32]. CD133 has also been found expressed in leukemia, prostate, colon, lung, breast and other cells. [33, 34] Even so, several researchers state that the CD133 is the most reliable marker for the identification of tumor stem cells and, especially, glial cells [35]. As the most recent finding, the expression of CD133 has been associated with the poor prognosis of gliomas [25]. The therapeutic labeling of MGC populations through a molecule such as CD133 represents an unopportune opportunity to eradicate subpopulations of neoplastic cells, drug-resistant tumor initiators.

On the other hand, the immunoglobuiin Y (IgY), the production of polyclonal antibodies in chickens and their extraction from the yolk, is a new biotechnological branch in constant expansion.

IgY is the main circulating antibody in birds. IgY have a general structure similar to the structure of mammalian IgG, with 2 heavy chains (~67-70 kDa) and 2 light chains (22-30 kDa). The molecular weight of the IgY molecule is around 180KDa with 3% carbohydrates. The heavy chains (H) of the IgY's are composed of 4 constant domains and a variable domain, which contains the antigen-binding site. Each H chain contains 2 Nglycosylated sites located in the Cnu2 and Cnu3 domains. IgY avian antibodies have several advantages when compared to the more widely used IgG mammals; among these we can mention that IgY antibodies do not activate the complement, do not bind to protein A and G does not bind to mammalian antibodies for example to rheumatoid factors or human anti-murine antibodies and ultimately bind to the cell surface receptor of Fe. These differences in molecular interactions present great advantages for the application of IgY's in a variety of methods in different areas of research, diagnosis, medical applications and bioteenology [36] The concentration of IgY in the yolk varies between 10 to 20 mg / ml. This variation depends on the genetic line or breed of the bird, such as: Single Comb White Leghorn 2.21 ± 0.44 [SD] mg / ml; SLU-1329 1.95 ± 0.48 mg / ml; and Rhode Island Red 1.68 ± 0.50 mg / ml [37] In addition, the total amount of IgY in the egg is related to the size of the egg and the individual physiological variations, [38]. In addition, IgY's are very stable under normal conditions. In agreement with [39], the authors have stored IgY preparations for more than 10 years at 40 ° C without significant loss in antibody activity. They mention that chicken antibodies also maintain their activity after 6 months at room temperature or 1 month at 37 ° C, this is because the circulating chicken IgY's have a circulating half-life of a month and the normal temperature of a chicken is around 41 ° C.

In an effort to improve the selective cytotoxicity of agents such as toxins, chemotherapeutic agents and radionuclides against malignant cells, these agents have been conjugated to antibodies to produce immunotoxins that recognize antigens associated with the tumor, these conjugates represent a new approach for antineoplastic therapy and are currently under intense investigation . [40-42].

Nevertheless; the efficacy of these agents has been limited, possibly due to: a) limited bioavailability, probably due to the inability to penetrate the hemato-tissue barriers, b) the inability of the immunoconjugate to be endocytosed, since some of these are only active intracellular manner, c) lowcitotoxicity against antigenic variants of low affinity and d) the heterogeneitytumoral [42]. In order to solve these problems, it is necessary to look for new types of conjugates with greater potency and antibodies with greater affinity, contemplating the cost-benefit.

Abrina, isolated from the plant Abrusprecatoriuse s a potent antineoplastic substance [43] that we will use for the construction of the immunotoxin, this toxin is more powerful than the A chain of the ricin toxin and or the diphtheria toxin to eliminateneoplastic cells. The A chain of abrin toxin, which is an acidic peptide chemically different from the A chain of the ricin toxin, is a neutral glycopeptidohydrophobic [43].

These differences in physical and chemical properties may affect the pharmacokinetics of the conjugate. The mechanism of action of the A chain of the Ade abrin toxin is similar to that of the A chain of ricin, since both eliminate neoplastic cells by inactivating ribosomes [44] Given the advantages of this type of immunoconjugate, our intention is to produce antibodies polyclonal and anti CD133 coupled to the A chain of Abrina as immunotoxins in order to eliminate tumor stem cells effectively and selectively.

Description of Figures Figure 1. Design and expression of CD133 recombinantly.

The figure shown corresponds to an electrophoresis of proteins under denaturing conditions SDS-PAGE 15%, where in the first lane the culture of CD133 is observed prior to induction with IPTG and in the second lane the overexpression of the CD133 antigen with an approximate weight of 18kD.

Figure 2. Immunization.

The image corresponds to a denaturing 15% polyacrylamide gel in which the concentrate of the elutions obtained was loaded by HisPur ™ affinity chromatography Ni-NTA Purification Kit (Pierce BiotechnologylL 61105 USA Cat. No. 88229) to subsequently perform the excision of the band corresponding to CD133, process it and with it carry out the corresponding immunization.

Figure 3. Obtaining and purification of IgY antibodies.

The image corresponds to a 12% SDS-PAGE acrylamide gel in which the molecular weight marker is shown, and 4 lanes with fractions of the final product of the IgY antibody purification obtained from the egg yolk by precipitation with polyethylene glycol, where the heavy chain (55-77 kDa) and light chain (23-26 kDa) of the immunoglobulin are observed.

Figure 4. Construction of plasmid pET-28a (+) with the coding sequence of the A chain of Abrina.

The figure corresponds to an agarose gel showing the digestion of plasmid pET-28a (+) containing the insert of the A chain of Abrina. Lane 1 shows the plasmid with the insert of the sequence of the A chain of Abrina, lane 2 shows the digestion of the plasmid digested by the restriction enzymes Hindlll and Xhol.

Figure 5. Expression of Abrina A chain recombinantly The figure corresponds to a 12% SDS-PAGE acrylamide gel in which the molecular weight and two recombinant overexpressions of the A chain of Abrina are shown in the E. coli strain BL21DE3pLysS, the first of which was carried out at a temperature of 30 ° C and the second one at 32 ° C; in both overexpressions, fractions of the cultures prior to induction with IPTG are shown, and 3 fractions after the induction at different times: 2 hours, 4 hours and 24 hours. (O.N.) Figure 6. Purification of Abrina A chain by chromatography by Sephacryl® HR S-200 gel filtration In the 12% SDS-PAGE acrylamide gel, representative fractions of the chromatography peaks are shown by Sephacryl® HR S-200 gel filtration where it can be seen that starting from fraction 69 a protein of approximately 31 kD is observed. which corresponds to the A string of the Abrina.

Figure 7. Cytotoxicity test by MTT in RG2 cells.

In the figure of the dilution 1E-1 to 1E-9 have a p < 0.001, while the dilution 1E-10 has a p = 0.52 and the rest of the dilutions has a p > 0.5 The results were expressed in means, plus DE. Statistical significance was considered with a p < 0.05.

Figure 8. Antibody specificity The image corresponds to a Western Blot where the complete bacterial lysate was used, expressing the CD133 antigen, the anti-CD133 IgY produced as primary antibodies and as a secondary antibody an anti-IgY rabbit IgG conjugated with peroxidase.

Figure 9. Death Induction of CD133 + cells by the IgY immunotoxin.

The image corresponds to 3 graphs of points obtained by flow cytometry where the induction of selective death of CD133 + cells is observed when incubated for 24 hours with the IgY immunotoxin in 3 different glioma samples. The induction of selective death can be observed in more than 50% of the CD133 + carcinogenic stem cells in all cases, without inducing the death of CD133- cells.

Method of use of the Immunotoxin IgY.

The anti-CD133 IgY immunotoxin can be used for the specific elimination of carcinogenic stem cells in various types of neoplasms whose pathology is associated with these cells. Particularly in the case of solid tumors of the central nervous system, it is recommended to use it locally by convection-enhanced delivery technique (CED), a new locoregional drug delivery method of intracranial tumors, which allows direct administration of immunotoxins to brain tumors or surrounding brain tissue infiltrated by tumor cells. Although the specific binding nature will also be possible through systemic, intravenous or oral administration.

Detailed description of the invention: Design and expression of CD133 recombinantly.

For the expression of the CD133 antigen, the cDNA coding for the epitope AC133 (SEQ ID NO: 1), (Bioclone Inc. Cat. RPV0119) as well as the immunodominant sequence AC133-2 (SEQ ID NO: 2), was obtained commercially. corresponds to amino acids 20 to 108 and 73-95 respectively of the human protein CD133, also known as Prominin 1; the codons of both sequences were optimized for an adequate expression of recombinant form in E. coli.

The cDNA was cloned into an expression vector of E. coli called pET28a (Novagen Cat. No. 69337-3), which has a tail of six histidines at its N-terminus which facilitate obtaining the antigen by affinity chromatography once the expression of the protein has been carried out; in addition to having a sequence that codes for a kanamycin resistance gene that allows to select the colonies of interest.

For the production of the CD133 antigen recombinantly, an E. coli strain named BL21DE3pLysS (Novagen Cat. No. 69451-4) was used, which is the most widely used bacterial protein expression system, which offers the deficiency as an advantage of Ion and omp3 proteases; the designation of DE3 is due to the presence of lysogenic profago l containing a chromosomal copy of the T7 RNA polymerase gene inducible by IPTG (lsopropyl-p-D-1-thiogalactopyranoside) under the control of the lacUV5 promoter. pLysS corresponds to the presence of a piásmido with resistance to chloramphenicol and a replicon P15A that codes for lysosim T7, which is a natural inhibitor of T7 RNA polymerase and which is used to suppress the basal expression of it prior to induction, and in this way stabilize the coding of the recombinant proteins contained in the pET plasmids.

For the preparation of the competent E. coli bacteria, the following procedure was carried out: 1. E. coli BL21DE3pLysS colonies were grown in boxes with LB agar medium to obtain individual colonies. 2. A single fresh colony was grown in 5mL of LB medium overnight at a temperature of 37 ° C with continuous agitation at 200rpm. 3. One milliliter of the latter was transferred to 100mL of LB medium and incubated for 3 hours at 37 ° C and 200rpm. A reading of the optical density at 550 nm was made until reaching a D.O. of 0.5 or 0.6. A yield of 4mL of competent bacteria is obtained from each 100mL of medium. 4. The bacteria were centrifuged at 600 x g for 10 minutes at a temperature of 4 ° C and subsequently the bacterial pellet was resuspended in 40 ml of transformation buffer I by gently shaking and incubating on ice for 2 hours. 5. The bacteria were centrifuged at 600 x g for 10 minutes at a temperature of 4 ° C, and the pellet was resuspended in 4mL of transformation buffer II for an initial 100mL culture and incubated on ice for one hour. 6. Once obtained, they were frozen until later use.

The transformation of the competent cells was carried out in the following manner: 1. The competent cells were thawed on ice for approximately 20 minutes. 2. The plasmid DNA was sterilely added to 50 pL of competent cells, and left on ice for one hour. 3. The cells were subjected to a heat stroke in a water bath at 42 ° C for 40 seconds, and then placed back on ice for a period of 10 minutes. 4. 400pL of sterile LB medium was added to each sample and incubated at 37 ° C for one hour. 5. It was centrifuged at 13200 rpm for one minute and the supernatant was discarded. 6. 50 mL of sterile water was added to resuspend the pellet of bacteria. 7. The sample was added in its entirety in Petri dishes by pipetting and distributed with the help of sterile beads. 8. The boxes were incubated overnight at a temperature of 37 ° C.

Once the transformation of the cells was carried out, the expression of the antigen CD133 was carried out by carrying out the following procedure: 1. A BL21 DE3pLysS-CD133 colony was seeded in a Petri dish with half-moon Bertani agar and kanamycin (30pg / mL) and incubated O.N. 37 ° C. 2. A colony was taken from the Petri dish to inoculate 5mL of Luria Bertani medium and kanamycin (30pg / mL) and incubated O.N. 37 ° C with continuous stirring (250 rpm). 3. The culture was centrifuged at 2500 rpm for 4 minutes and the pellet was resuspended in one milliliter of LB medium with kanamycin, to later inoculate 100 mL of LB medium with kanamycin until reaching an optical density of 0.1 at a wavelength of 600 nm. 4. It was incubated at 37 ° C with continuous agitation (250 rpm) until the culture reached an optical density of 0.4 at a l = 600 nm, then IPTG (1mM) was added and incubated at 37 ° C with continuous agitation (250 rpm) for 4 hours and the optical density was determined. 5. The culture was centrifuged at 2500 rpm for 4 minutes, and the supernatant was discarded to preserve the pellets; and if the purification was not carried out immediately, the pellets were frozen at -20 ° C.

Lysate of bacteria. 1. The pellet was resuspended in 5mL of lysis buffer with protease inhibitors. 2. It was sonicated for periods of 30 seconds at an amplitude of 50% until the lysate changed from a whitish coloration to being translucent. 3. It was centrifuged for 4 minutes at 2500 rpm to recover the cells that had not been lysed, resuspend them in lysis buffer and re-sonicate them. 4. The concentration of the lysate was quantified by Pierce ™ BCA ProteinAssay Kit (Pierce BiotechnologylL 61105 USA Cat. No. 23225).

Purification of CD133.

For the purification of the CD133 antigen it was decided to use the immobilized metal affinity chromatography method known as IMAC (Porath, et al., 1975). It is based on the known affinity of transition metal ions, such as Zn + 2, Cu + 2, Ni + 2 and Co + 2 to histidine and cysteine in aqueous solutions and extended to the idea of using metal ions "firmly attached" to a support to fractionate solutions of proteins such as nitrilotriacetic acid (NTA).

The most important application of IMAC is the purification of recombinant proteins expressed in fusion with an epitope containing six or more histidine residues. Due to the high affinity and specificity of the His tail, a single purification step IMAC leads in most cases to the purification of the target protein. The most common form of a His tail consists of six consecutive residues of histidine (H6), which offers a series of six metal-binding sites high enough to change the association / dissociation balance further towards the association side that leads to stable union in most cases.

For the purification of the antigen we used the HisPur ™ Ni-NTA Purification Kit purification kit (Pierce BiotechnologylL 61105 USA Cat. No. 88229) as follows: 1. The columns were equilibrated at room temperature. 2. The mixture of the lysate was made with the equilibrium buffer so that the final volume was twice the volume of the resin. 3. The column was centrifuged at 700 x g for two minutes to remove the storage buffer. 4. The column was balanced with a volume equal to twice the volume of the resin allowing the absorber to penetrate the resin. 5. The column was centrifuged at 700 x g for two minutes to remove the buffer. 6. The protein mixture was added and incubated for 30 minutes at room temperature in an orbital shaker. 7. The column was centrifuged at 700 x g for two minutes and the product was collected. 8. The column was washed with twice the volume of the resin, and centrifuged at 700 x g for two minutes and the product was collected: this process was repeated 3 times.

Immunization.

To prepare the CD133 protein as an antigen, we resorted to the emulsification of the protein bands in polyacrylamide gels for direct injections. This approach is particularly advantageous when the purification of proteins by other means is not practical; An additional advantage of this method is that it improves the immune response, since polyacrylamide helps to retain the antigen in the animal and therefore acts as an adjuvant, since the polyacrylamide is also highly immunogenic. The use of the protein directly in the gel band (without elution) is also useful when only small amounts of protein are available. In some cases it is necessary to purify by affinity the desired antibodies of the resulting anti-serum for the production of specific antibodies.

Chickens can be immunized intramuscularly by vaccination (chest region, left and right, injection volume 0.5-1.0 ml). Depending on the immunogenicity of the antigen, high titers of antibodies (1: 100 000-1: 1,000,000) can be achieved after only one or 3-4 booster immunizations. Normally, a hen lays eggs continuously for approximately 72 weeks, and from then on the capacity decreases. For the immunization of the hens, the elutions obtained from the affinity chromatography were concentrated to subsequently perform a denaturing electrophoresis in 15% polyacrylamide gels, stained with Coomassie blue for half an hour and stained with an acetic acid solution. 10% to identify the 18kD band and proceed to cut and freeze it in liquid nitrogen, pulverize it and resuspend it in 1mL of saline; once the antigen was obtained, a hen (Gallusgallus, HyLineBrown variety) of 14 weeks of age was immunized with the antigen CD133 and another one with PBS, the hens were conditioned in individual cages with "ad livitum" feed. Two weeks prior to the posture period and regular production (1 egg / day), the inoculation stage of the peptides was started with 0.9% NaCl saline solution. The chickens were immunized intramuscularly in several areas of the pectoral region with 0.5 ml of CD133 antigen with an approximate concentration of 200pg / ml and 3 reinforcements were applied at the times indicated in the following table: Table 1. Immunization schedule.

Day 0 14 28 38 56 66 87 Injection 1 ° 2 ° 3 ° 4 ° Preimmune egg yolk * egg (*) The eggs were collected between day 38 and 87 and mixed weekly.

Obtaining and purification of IgY antibodies.

For the purification of the IgY antibodies from the eggs of the immunized hens, the extraction of the total egg yolk IgY was carried out by means of a precipitation procedure. The method involves two important steps. The first is the elimination of the lipids and the second the precipitation of the total IgY from the supernatant of stage one. After dialysis against a buffer (usually PBS) the extracted IgY can be stored at -20 ° C for more than one year. The purity of the extract is around 80%, the total IgY per egg varies from 40 to 80 mg, depending on the age of the hen.

Protocol for the extraction of IgY by means of precipitation by means of polyethylene glycol (PEG).

All steps must be done using latex gloves. 1. The egg shell was carefully broken and the yolk was transferred to a "yolk spoon" in order to remove as much egg white as possible. 2. The yolk was transferred to a filter paper and rolled to remove the remains of egg white, the yolk was broken with a lancet or a similar instrument (tip of the pipette), and poured into a 50 ml tube and the volume It was registered (V1). 3. The volume of the egg yolk of PBS was mixed twice with the yolk (ÅV1 + V2), and 3.5% of PEG 6000 (in grams, powdered) of the total volume was added and stirred, followed by 10 min of incubation in an orbital shaker. This step of the extraction process separates the suspension into two phases. One phase consists of "yolk solids and fatty substances" and an aqueous phase containing IgY and other proteins. 4. The tubes were centrifuged at 4 ° C for 20 min (10,000 rpm according to 13,000 xg, HeraeusMultifuge 3SR +, fixed angle rotor). The supernatant (V3) was poured through a pleated filter and transferred to a new tube. 5. 8.5% PEG 6000 in grams (calculated according to the new volume) was added to the tube, vortexed and placed on an orbital shaker as in step 3. 6. Step 4 was repeated with the difference that the supernatant was discarded. 7. The pellet was carefully dissolved in 1 ml of PBS by means of a glass rod and the vortex. PBS is added to a final volume of 10 ml (V4). The solution is mixed with 12% PEG 6000 (w / v, 1.2 grams) and treated as in step 3 (vortex, orbital shaker). 8. Step 6 was repeated and the precipitate was carefully dissolved in 800 ml of PBS (glass rod and vortex). Wait for the air bubbles to disappear and then transfer the extract to a dialysis capsule. The tube was rinsed with 400 μl of PBS and the volume was added to the dialysis device (V5). 9. The extract was dialyzed overnight in 0.1% saline (1600 ml) and stirred gently by means of a magnetic stirrer. The next morning, the saline solution was replaced by PBS and dialyzed for another three hours. 10. Subsequently, the IgY extract was extracted from the dialysis capsule by a pipette and transferred to 2 ml tubes. The final volume was around 2 mi (V 6). 11. The protein content (mg / ml) of the samples is measured spectrophotometrically at 280 nm (1:50 diluted with PBS) and is calculated according to the Lambert-Beer lcy with an extinction coefficient of 1.33 for IgY 12. It is advisable to store the samples in aliquots at -20 ° C (do not freeze samples at -70 ° C).

Specificity of the antibodies obtained.

In order to corroborate the specificity of anti-CD133 IgY antibodies, the Western Blot technique was used, using as primary antibodies those obtained by PEG precipitation from the egg yolks of the hens immunized with the CD133 antigen and as secondary antibody, an anti-IgY rabbit IgG conjugated with peroxidase (Jackson immunoResearh Laboratories, Inc. CodeNumber 303-035-003); Such procedure was carried out in the following manner: 1. Denaturing electrophoresis was performed to separate the protein mixture where CD133 was located in a semi-pure manner and carry out the transfer to a PVDF membrane 02pm using the Trans-Blot® system (Bio-Rad CatalogNumber 170-4155) at 2.5 A for 10 min. 2. Protein transfer was verified by performing 3 washes of 5 minutes each with bidistilled water and then staining with Poceau red for 5 minutes once the transfer was observed the membrane was washed with water bidet until the staining was removed 3 Nonspecific binding sites were blocked with 5% fat free milk in TBST 1x for one hour at room temperature, and subsequently the membrane was washed to remove residual milk. 4. The membrane was incubated with the primary antibody at a dilution of 1: 200 overnight at 4 ° C on an orbital shaker. 5. The membrane was washed with TBST 1x first for 5 minutes, then for 10 minutes and finally for 15 minutes. 6. The membrane was incubated with the secondary antibody at a dilution of 1: 100,000 for one hour at room temperature on an orbital shaker. 7. The membrane was washed with TBST 1X first for 5 minutes, then for 10 minutes and finally for 15 minutes. 8. The chemiluminescence reaction was performed with the ECL Plus ® Western BlottingDetectionReagentsAmershamBiosciences kit (RPN 2132) according to the manufacturer's recommendations.

Design and expression of the Abrin A chain recombinantly.

The coding sequence for the A chain of Abrina was obtained by bioinformatics (SEQ ID NO: 3), (GenBank: CAA54139.1) and codon optimization was carried out for its expression recombinantly in E. coli.

The cDNA was cloned into an E. coli expression vector designated pET28a (Novagen Cat. No. 69337-3).

The E. coli strain BL21 DE3pLysS (Novagen Cat. No. 69451-4) was used for the production of the Abrina A chain.

The preparation of the competent strains of E. coli, the transformation thereof, as well as the expression of the A chain of Abrina was carried out in the same way as was done with those of the antigen of CD133 and which is described in detail in that section; with the only difference that once the induction with IPTG was performed in the expression of the A chain of Abrina the incubation temperature was 30 ° C.

Lysate of bacteria. 1. The pellet was resuspended in 5mL of lysis buffer with protease inhibitors. 2. It was sonicated for periods of 30 seconds at an amplitude of 50% until the lysate changed from a whitish coloration to being translucent. 3. It was centrifuged for 4 minutes at 2500 rpm to recover the cells that had not been lysed, resuspend them in lysis buffer and re-sonicate them. 4. The concentration of the lysate was quantified by Pierce ™ BCA ProteinAssay Kit (Pierce BiotechnologylL 61105 USA Cat. No. 23225).

Purification of the Abrina A chain.

The purification of the Abrina A chain was performed by chromatography by gel filtration, which separates the proteins with differences in molecular weight; The technique is ideal for the final steps in a purification when the volumes of the sample have been reduced, since this significantly influences the speed and resolution of the gel filtration. The samples are eluted geocratically. The conditions of the shock absorbers vary depending on the type of sample or the requirements for its subsequent purification, analysis or storage stage, since the composition of the buffer does not directly affect the resolution, the proteins are collected in purified form in the chosen buffer .

Sephacryl® High Resolution is a highly versatile gel filtration medium that offers a wide range of fractionation capabilities. The high resolution matrix Sephacryl ® is a crosslinked copolymer of allydextran and N, N-methylene bisacrylamide. This crosslinking provides good rigidity and chemical stability; the distribution of small particles, with narrow selectivity curves, are good characteristics maintaining a high resolution. The hydrophilic nature of the media minimizes non-specific adsorption and maximizes recovery. Excellent resolution and flow characteristics, long-term physical and chemical stability, and ease of handling make Sephacryl® HR the means of choice for routine purification.

The column HiPrep 16/60 was used with Sephachryl S-200 High Resolution, which has a capacity of 1.2 mL of sample. 1. The column was washed with half the total volume of the column, ie 60mL, with distilled water at a flow of 0.5mL / min. 2. The column was equilibrated with two volumes of the total column, or 240mL with a 0.5M sodium phosphate buffer and 0.15M NaCl pH 7.2 and the flow through the column was maintained at 05mL / min 3. Depending on the concentration of the Used, volumes less than 1% of the total volume of the column were introduced, without exceeding a concentration of 70mg / mL. 4. Fractions of approximately 500 pL were collected to subsequently read the absorbance with UV light at 280 nm and construct a chromatogram to identify the fractions in which the protein peaks appeared and their subsequent identification by denaturing SDS-PAGE electrophoresis. 5. To wash the column we used half volume of 0.2M NaOH column MTT cytotoxicity assay in RG2 cells.

The MTT substrate is prepared in a physiologically balanced solution, added to the cells in culture, usually at a final concentration of 0.2-0.5 mg / ml, and incubated for 1 to 4 hours. The amount of formazan (presumably directly proportional to the number of viable cells) is measured by recording the changes in absorbance at 570 n. Viable cells with active metabolism convert MTT into a purple formazan product with a maximum absorbance of 570 nm.

When cells die, they lose the ability to convert MTT to formazan, the formation of color therefore serves as a useful and convenient marker of viable cells. The mechanism Exact cellular reduction of MTT in formazan is not well understood, but probably involves reaction with NADH or similar reduction of molecules that transfer electrons to MTT. (Marshall, et al., 1995) The formazan accumulates as an insoluble precipitate inside the cells, and is also deposited near the cell surface and in the culture medium. The formazan must be solubilized before recording the absorbance readings; Several methods of solubilization include the use of: acidified isopropaool, DMSO, dimeWfofTnamrda. SOS, and combmaoons of detergent and organic solvent. The amount of signal generated depends on several parameters that affect the concentration of MTT, the incubation period, the number of viable cells and their metabolic activity.

MTT test protocol 1. The cells and test compounds were prepared in 96-well plates so that the final volume was 100 pi / well 2. It was incubated for 24 hours with the toxin. 3. 10 mL of MTT solution was added per well to achieve a final concentration of 0.45 mg / mL. 4. It was incubated for 4 hours at 37 ° C. 5. 100 pL solubilization solution was added to each well to dissolve the formazan crystals. 6. It was mixed to ensure complete solubilization. 7. The absorbance was monitored at 570 nm.

Conjugation.

The immunotoxins were constructed using standard protocols (48). The crosslinker Succinimidyk > xycarbonyl-a-methyl-a-. { 2-pyridyldithyo) toluene [SMPT] (ThermoScientific, Rockford, USA), in dimethylsulfoxide to the IgY antibody (2 mg / ml in PBS) at a final concentration of 0.13 mg / ml, mixed gently, and incubated at room temperature for 1 hour. The non-reactive SMPT was removed by desalination. The toxin, at 1 mg / ml in PBS, was degassed, incubated with 2.5 mM dithiothreitol (DTT) for one hour at room temperature and mixed with the activated antibody in relation to 2mg of antibody per g of toxin. After sterilizing by filtration using a 0.22pm filter, the solution was incubated under nitrogen at room temperature for 18 hours. The excess pyridyl disulfide active sites were blocked with 25mg / ml cysteine at room temperature for 6 hours. To purify the unconjugated antibody conjugate and the toxin, the mixture was chromatographed in cigacron 3GA agarose blue.

Induction of selective death by the immunotoxin IgY In order to test the effectiveness of the immunotoxin to induce the death of carcinogenic stem cells, 3 biopsies were collected from patients with glioblastoma multiforme, which were cut into small pieces and disintegrated with trypsin, incubated at 37 ° C in a humid atmosphere. 10 minutes and seeded in DMEMF12 high supplemental serum free glucose with 20ng / ml EGF, 20ng / ml bFGF, 10ng / ml LIF and supplement B27 (MiftenyiBiotec, Usa). 24 hours after sowing, they were incubated for a further 24 hours with 20ng / ml of the immunotoxin. Subsequently, the cells were collected in Falcon tubes for 5 ml polystyrene cytometry and centrifuged at 200 x g for 5 minutes. The supernatant was decanted and the cells were resuspended in the remaining volume (approximately 50mI) and 5mL of the anti-CD133 antibody coupled to alloñcodanin (APC) was added and vortexed for 10 seconds at maximum speed and incubated at room temperature. Darkness. After that time, 1 ml of PBS was added and vortexed for 10 seconds, the tubes were centrifuged for 5 min at 200 xg, the supernatant was decanted and 400 ml of the propidium iodide-PBS staining solution (5pg / ml) was added. ) and incubate for 15 minutes »for its later reading in the FACSCalibur flow cytometer. 10,000 total events were acquired and the percentage of cells in the FL2 channel (for propidium) and FL4 (for CD133 +) was analyzed. The percentages obtained are shown in the dot plots of Figure 9.

Statistic analysis.

The results were expressed as mean ± SD. Statistical significance was considered with a p < 0.05. The distribution of the data (normal and non-normal) was determined by KolmogorovSmirnov and the ANOVA test (one-way analysis of variance) was performed with Tukey's posthoc.

The immunotoxin that was designed is conformed by the A chain of Abrina, a ribosome inactivating protein that specifically and irreversibly inhibits protein synthesis in eukaryotic cells by enzymatically altering the 28S rRNA of the 60S ribosomal subunit with LD50 = 2.8pg / kg in mice. In addition, IgY is stable at pH 4-9 and up to 65 ° C in an aqueous state, and is stable at 4 ° C for a prolonged period.

In this way, it is expected that the conjugate of the A chain of abrin and anti-CD133 IgY will act selectively on CSCs, encouraging the eradication and / or avoiding possible brain tumor recurrence, avoiding the non-specific binding of the toxin to tissues. adjacent healthy.

Given that the production of IgY is a simple and inexpensive technique and the synthesis technology of recombinant Abrina decreases production costs, it is possible that the IgY anti-CD133 immunotoxin is another alternative in the treatment of neoplasms whose malignancy and recurrence is associated to the invasion of carcinogenic stem cells reducing the costs associated with the treatment of these patients.

Preparation of reagents.

Middle Luria Bertani. (1L) 1. 10g of peptone. 2. 5g of yeast extract. 3. 10g of NaCl 4. Drill a liter with deionized water; for solid medium add 15g of agar for each liter of medium. 5 Sterilize the medium at 120 ° C for 20 minutes Transformation Regulator I 1 M Potassium acetate 1M MnC 1M RbCI 1M CaCb Water at 1L Transformation regulator II 1M MOPS pH = 7.0 1M RbCI Glycerol 6ml per each L Water Stock Kanamycin.

Weigh 30mg of kanamycin and add 1mL of water, and then filter (0.2pm filter). Add one mL of antibiotic for each mL of medium.

Inhibitors of EDTA-free proteases Protein analysis by electrophoresis.

To determine the protein composition in both the expression and protein purification stages, protein electrophoresis was carried out denaturingly, loading the volume corresponding to 30pg of protein in the rails and in those cases in which the volume of the sample exceeded the capacity of the electrophoresis gel lane, the maximum volume was applied. For the gels with the CD133 protein a concentration of 15% was used, while in those with the A chain of Abrina a concentration of 12% was used.

Electrophoresis was run at 80V in the concentrator part, and at 120V in the separating part; Once the electrophoresis was done, the gel was stained with Coomassie blue for a minimum of 4 hours, to later perform the destiny for a minimum of 30 minutes or until the background was removed and the protein composition could be clearly appreciated.

Coomassie blue. 200 mL of glacial acetic acid. 0. 5g of Coomassie Blue. 1800 mL of deionized water Mix for one hour and filter with Whatman No.1 paper Fading solution. 45mL of methanol. 45mL of bidistilled water. 10mL of glacial acetic acid.

Acrylamide 30% Bis Acrylamide 0.8% 30g of acrylamide. 0. 8g of acrylamide bis Gauge with deionized water at 100mL and filter.

Buffer for eiectroforesis 10X. 30. 2g of Tris Base. 144g of glycine. 10g of SDS.

Drill a liter with deionized water.

Preparation of reagents for MTT assay Dissolve MTT in Dulbecco's phosphate buffered saline, pH = 7.4 (DPBS) at 5 mg / ml.

Filter the MTT solution through a 0.2 mm filter, protected from light in a sterile container. Store the MTT solution, protected from light, at 4 ° C for frequent use or at -20 ° C for long-term storage.

Choose a suitable container resistant to solvents and work in a ventilated hood. Prepare 40% (vol / vol) of dimethylformamide (DMF) in 2% (vol / vol) of glacial acetic acid.

Add 16% (weight / volume) of sodium dodecyl sulfate (SDS) and dissolve.

Adjust to pH = 4.7 Store at room temperature to avoid precipitation of SDS. If a precipitate forms, heat to 37 ° C and mix to solubilize the SDS.

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Claims (8)

Claims Having described the invention as antecedent, the content of the following claims is claimed as property:

1. - The use and production of IgY antibodies obtained from any bird or recombinantly for the treatment of malignant neoplasms where any antigen or tumor cell receptor is used, alone or coupled to any drug, enzyme, nanoparticle toxin or any other substance susceptible to be coupled to that type of antibodies. Characterized by containing the basic structure of IgY consisting of 2 heavy chains and two light chains. The heavy chain consisting of a variable domain (VH) and four constant domains (CH1, CH2, CH3 and CH4). The two heavy chains are connected by disulfide bridges. The light chain has a variable domain (VL) and a constant domain (CL), where the domain of the antibody fragment (Fab) binds the antigenic epitopes,

2. - The use as therapeutic target or immunization of birds and production of the CD133 antigen recombinantly using the sequences contained in this application. Characterized by containing the use of codons optimized for the reading and expression of the human antigen AC133 and AC133-2 that express the recombinant protein in the most immunodominant regions for the recognition and generation of avian IgY anti CD133 antibodies.

3. - The use or production of the A chain of recombinant abrin that uses the sequences contained in the present description. Characterized for being the safest way to induce the selective death of tumor cells, since it does not contain the B subunit that allows it to bind to carbohydrates. The sequences presented here were optimized for the best expression and recognition by E. coli, cloned in the plasmid pET28a (+) and inserted with the Xhol and Hindlll enzymes.

4. - The production and use in any neoplasia of the Immunotoxin IgY collected to the A chain of Abrina. Characterized by IgY antibodies obtained from the egg yolk of hens immunized with the antigen AC133 and AC133-2 obtained recombinantly and coupled to the A chain of recombinant abrin by SMPT, with recommended use for the treatment and elimination of carcinogenic stem cells of various neoplasms whose malignancy is associated with this type of cells. Its administration can be oral, intravenous or intratumoral under any type of release, administration or infusion.

5. - The production of IgY Antibodies obtained by immunization of any bird or organism producing immunoglobulin Y. Considering that said production has as its objective its use for the treatment of malignant neoplasms according to claim 1, whether said antibodies can be produced by immunization in a polyclonal, monoclonal or recombinant form, obtained from the egg yolk or the serum of the organisms previously immunized.

6. - The use and production of the recombinant CD133 antigens presented here. Either for immunization of immunoglobulin Y producing organisms, cloned and expressed in any expression vector or under any expression system that gives the final antigen presented in this application according to claim 2, and can be expressed in bacterial systems, Cells, in eukaryotic or prokaryotic cells, or by the use of plasmids, viruses, RNA or cDNA or DNA as vectors.

7. -The use and production of the recombinant Abrina A chain presented here Whose purpose is for the treatment of any neoplasm, whether the sequence of said sequence is cloned and expressed in any expression vector or under any expression system that gives as final product the string A of Abrina presented in this application of agreement to claim 3, being able to be expressed in bacterial, cellular systems, in eukaryotic or prokaryotic cells, using plasmids, viruses, RNA or cDNA or DNA as vectors.

8. -Production of the immunotoxin IgY coupled to the A chain of abrin according to claim 4. This immunotoxin is characterized in that the antibody of interest is an aviary antibody of the IgY type, which has advantages over other conventional antibodies in that it has a prolonged conservation of its properties either at room temperature, refrigeration or freezing and high affinity for mammalian antigens. , does not present cross-reactivity, does not fix complement or bind to rheumatoid factors, besides being 3 to 5 times more related to the same antigen as human IgG, which makes it react more quickly against the desired antigen.