DISEASES AND CANCERS: DIAGNOSIS AND TREATMENT

CROSS-REFERENCE TO RELATED APPLICATIONS

[00001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63/389,169 filed on July 14, 2022, the entire contents of all of which are incorporated herein by reference.

FIELD OF THE INVENTION

[00002] The invention relates generally to methods and systems for treatment of infectious diseases and cancers.

BACKGROUND OF THE INVENTION

[00003] The invention according to the embodiments described herein relates broadly to the fields of cell biology, microbiology and immunology, and more specifically to therapeutic human bispecific antibodies useful in the diagnosis and treatment of infectious diseases and cancers.

[00004] The prophylactic vaccine now in production for protection against the novel coronavirus and COVID-19 vaccine is based on a ribonucleic acid (RNA) - particularly a messenger RNA (mRNA) sequence of the coronavirus that is expected to mount an immune response in the body based on the assumption that the mRNA sequence would make its way into the antigen-presenting cells (APCs) of a vaccine recipient’s immune system and be translated into a protein presented on the surface of the coronavirus, the so-called “spike protein”. Once the spike protein is presented by APCs, it is expected that the recipient’s immune system would build an immune response against the spike protein, thereby developing immunity against the coronavirus and COVID-19.

[00005] Antibodies to regions of the severe acute respiratory syndrome (SARS)-COVID-2 spike protein (the spike protein) are rendered inactive by the accumulation of mutant variants of the spike protein. This is one of the primary issues with the current prophylactic vaccine based on the antigen profile on the spike protein. In order to circumvent this problem, Greaney et al generated ten human antibodies from which they designed three antibody cocktails each with two antibodies. Their findings clearly indicate the complexity of the antigenic profile of this virus and the potential variability in the human host response. [00006] The issue of host responses is further complicated by the variance in host responses due to a native immunosenesence found in some individuals. This is reflected in the need to give two successive shots of the vaccine in order to elicit a full blown antibody response.

[00007] As is known in the art, current prophylactic vaccine will be only partially effective in that about five to thirty percent of the population will not respond and will be still prone to contract the coronavirus. This segment of the population could then be harboring the virus with or without symptoms (asymptomatic), and contributing to disease transmission. This conundrum can be solved only by a second type of vaccine that is a neutralizing antibody (therapeutic) that can detect and destroy the main viral protease in free floating viruses as well as viruses remaining latent inside human cells. This is based on the discovery of a genetic defect associated with the absence of a bispecific immunoglobulin useful in detecting and neutralizing both free and cell bound viruses and also in the treatment of active disease.

[00008] With regard to COVID-19, there are a significant number of individuals who are positive for the virus but asymptomatic, this population may suffer from a loss of mucosal immunity in the gut. This is in agreement with epidemiological data showing increased risk among African-Americans, who are more susceptible to a germ-line deletion that causes an antibody deficiency in the gut. Based on this finding, the current immunization program (prophylactic) involving the use of mRNA will elucidate a sub-group of individuals to be non-responsive because of their inability to mount an immune response to the vaccine. This is a potential hazard since these individuals will harbor the virus without any symptoms while sustaining contagion in the community.

[00009] From open census data, it is projected that about 12.5% of the US population will be non-responders to the vaccine and will need a shot of a neutralizing antibody. By following groups of vaccinated subjects out to twelve weeks following their shots to see if they have developed an antibody in their blood while at the same time screening their fecal cells for the bispecific heterodimeric antibody GIPSITUMAB, candidates for neutralizing antibody shots may be identified.

[00010] It has been reported that in Africa, over 80% of those who are infected by the coronavirus, show no symptoms of disease. This is the same condition that will be faced in the United States because of a subpopulation that has inherited the defect of loss of the natural bispecific antibody, that acts like a firewall against infection. This leads to the need to identify these individuals and administer a different antibody that is directed against human cells infected with the virus.

BRIEF SUMMARY OF THE INVENTION

[00011] An embodiment of the present disclosure includes a method. The method includes collecting a small stool sample from a human patient using noninvasive means. The method further includes isolating the gut cells from the collected stool sample. The method further includes testing the isolated gut cells for the presence of the coronavirus.

[00012] A further embodiment of the present disclosure includes a method for converting membrane-bound antibodies on gastrointestinal progenitor stem cells (G1PC cells) into chimeric secretory antibodies. The method includes isolating gut cells comprising GIPC cells from a stool sample. The method further includes partially differentiating the isolated GIPC cells to exhibit B-lymphocyte behavior. The method further includes converting the membrane-bound antibodies on the partially differentiated GIPC cells into chimeric secretory antibodies comprising an immunoglobulin A and G (IgA and IgG) moieties. The method further includes challenging the chimeric secretory antibodies with antigens, including spike protein, to raise antibodies with anti-infective properties.

[00013] A further embodiment of the present disclosure includes a method for detecting the coronavirus using chimeric secretory antibodies. The method includes isolating gut cells from a stool sample, wherein the isolated gut cells comprise gastrointestinal progenitor stem cells (GIPC cells). The method further includes partially differentiating the isolated GIPC cells to exhibit B-lymphocyte behavior. The method further includes converting the membrane-bound antibodies on the partially differentiated GIPC cells into chimeric secretory antibodies comprising an immunoglobulin A and G (IgA and IgG) moieties. The method further includes challenging the chimeric secretory antibodies with antigens, including spike protein, to raise antibodies with anti-infective properties against the coronavirus. The method further includes detecting the presence of the coronavirus using the raised antibodies.

[00014] A further embodiment of the present disclosure includes a system for isolating and testing gut cells from a stool sample. The system includes a collection device for collecting a small stool sample from a human patient using noninvasive means. The system further includes an isolation unit configured to isolate the gut cells from the collected stool sample. The system further includes a testing unit configured to test the isolated gut cells for the presence of the coronavirus using standard techniques known in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[00015] Figure 1 is a schematic diagram illustrating the steps of a method of one embodiment of the present invention for isolating gut cells for disease testing from a stool sample;

[00016] Figure 2 is a chart illustrating results of cell isolation (followed by DNA extraction) versus direct DNA extraction from whole stool;

[00017] Figure 3 illustrates the comparison of yield after hold times of 0 hours (direct DNA extraction only), 3 hours, and 24 hours at room temperature; and

[00018] Figure 4 is a chart illustrating mean tumor weight after initial Gipsitumab treatment.

DETAILED DESCRIPTION OF THE INVENTION

[00019] For purposes of clarity, the following terms used in this patent application will have the following meanings:

[00020] The terminology used herein is for the purpose of describing example embodiments only and is not intended to be limiting. As used herein, the singular forms “a,” “an,” and “the” may be intended to include the plural forms as well, unless the context clearly indicates otherwise. The terms “comprises,” “comprising,” “including,” and “having,” are inclusive and therefore specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. The method steps, processes, and operations described herein are not to be construed as necessarily requiring their performance in the order discussed or illustrated, unless specifically identified as an order of performance. It is also to be understood that additional or alternative steps may be employed.

[00021] Although the terms first, second, third, etc. may be used herein to describe various elements, components, regions, layers and/or sections, these elements, components, regions, layers and/or sections should not be limited by these terms. These terms may be only used to distinguish one element, component, region, layer or section from another region, layer or section. Terms such as “first,” “second,” and other numerical terms when used herein do not imply a sequence or order unless clearly indicated by the context. Thus, a first element, component, region, layer or section discussed below could be termed a second element, component, region, layer or section without departing from the teachings of the example embodiments.

[00022] Spatially relative terms, such as “inner,” “outer,” “beneath,” “below,” “lower,” “above,” “upper,” and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. Spatially relative terms may be intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures is turned over, elements described as “below”, or “beneath” other elements or features would then be oriented “above” the other elements or features. Thus, the example term “below” can encompass both an orientation of above and below. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly.

[00023] “Substantially” is intended to mean a quantity, property, or value that is present to a great or significant extent and less than, more than or equal to total. For example, “substantially vertical” may be less than, greater than, or equal to completely vertical.

[00024] “About” is intended to mean a quantity, property, or value that is present at ±10%. Throughout this disclosure, the numerical values represent approximate measures or limits to ranges to encompass minor deviations from the given values and embodiments having about the value mentioned as well as those having exactly the value mentioned. Other than in the working examples provided at the end of the detailed description, all numerical values of parameters (e.g., of quantities or conditions) in this specification, including the appended claims, are to be understood as being modified in all instances by the term “about” whether or not “about” actually appears before the numerical value. “About” indicates that the stated numerical value allows some slight imprecision (with some approach to exactness in the value; approximately or reasonably close to the value; nearly). If the imprecision provided by “about” is not otherwise understood in the art with this ordinary meaning, then “about” as used herein indicates at least variations that may arise from ordinary' methods of measuring and using such parameters. In addition, disclosure of ranges includes disclosure of all values and further divided ranges within the entire range, including endpoints given for the ranges.

[00025] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the recited range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

[00026] References to “embodiment” or “variant”, e.g., “one embodiment,” “an embodiment,” “example embodiment,” “various embodiments,” etc., may indicate that the embodiments) or variant(s) of the invention so described may include a particular feature, structure, or characteristic, but not every embodiment necessarily includes the particular feature, structure, or characteristic. Further, repeated use of the phrase “in one embodiment,” or “in an exemplary embodiment,” do not necessarily refer to the same embodiment or variant, although they may.

[00027] As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts. Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[00028] The term “material” is intended to refer to encompass biocompatible materials, including metals, ceramics, plastics, composites, and combinations or hybrids thereof.

[00029] As used in this application the term “layer” is intended to mean a substantially uniform material limited by interfaces between it and adj acent other layers, substrate, or environment.

[00030] The present disclosure pertains generally to a bispecific antibody found on the cell surface of colon cells shed into the fecal stream. This bispecific antibody occurs naturally in bound form. The present disclosure utilizes a technique of converting cells to which the antibody is bound into cells capable of secreting immunoglobulin into a liquid growth medium from which one of ordinary skill in the art can extract the immunoglobulin for administration. [00031] The discovery of the natural bispecific heterodimeric antibody expressed by exfoliated human colon cells has revealed the existence of a natural host response that acts as a “firewall” against cancer and other infections from viruses and bacteria. On an average in normal individuals, about 60% of the shed cells express this antibody. However, the existence of a genetic condition where this antibody is totally absent (among African-Americans generally) as a result of a germline deletion of the deoxyribonucleic acid (DNA) encoding this protein is also recognized. It is also possible individuals with a reduced level of expression of this antibody exist, resulting in elevated susceptibility' to cancer and other infections. The reduced expression of this antibody by certain colon cells referred to herein as gastrointestinal progenitor stem cells (“GIPC cells”) renders this population non-responsive to prophylactic vaccines such as the currently available mRNA-based coronavirus vaccine. This subgroup may be the one that triggered the need for a second follow-up dose of the current vaccine. It is possible that this population may harbor the virus without demonstrating any symptoms and remain a source of infection in the community unless they receive a concurrent second antibody that acts as a neutralizing agent eliminating the reservoir of latent infection. Methods for culturing antibody -producing cells are disclosed in U.S. Patent Publication No. 20080166807, filed October 15, 2007, the entire contents of which are incorporated herein by reference.

[00032] Turning now to the accompanying Figures, there is illustrated the steps of a method of one embodiment of the present disclosure pertaining to isolating gut cells for disease testing from a stool sample. Gut cells isolated according to methods of the present disclosure may comprise GIPC cells, it being a further object of the invention to provide for a method of partial differentiation of GIPC cells toward plasma-like progeny that exhibit B- lymphocyte behavior. In particular, the techniques of the invention may be utilized to convert membrane-bound GIPC antibodies to chimeric secretory antibodies comprising an immunoglobulin A and G (IgA and IgG) moieties capable of challenge with any number of antigens, including but not limited to the spike protein, using techniques familiar to one of ordinary skill in the art. In this manner, chimeric secretory antibodies with anti-infective properties against the coronavirus may be raised much more efficiently than possibly using conventional methods such as hybridomas. Moreover, such antibodies, herein collectively referred to as “GIPSITUMAB”, are broad spectrum and may prove more effective, for example, in the detection of the coronavirus, which may be capable of evading detection by mutating and rendering capsid proteins ineffective as biomarkers of the disease. These and other benefits of the present invention will be clear to one of ordinary skill in the art from the disclosure that follows.

[00033] In certain embodiments, the methods described herein comprise collecting a small stool sample from a human patient using noninvasive means, then isolating the gut cells from the stool sample and testing for the presence of the coronavirus using techniques that will be familiar to those skilled in the art.

[00034] In preferred embodiments, gut cells comprising GIPC cells may be isolated noninvasively and partially differentiated, leading to the conversion of membrane-bound antibodies to GIPSITUMAB, which may then be challenged with the spike protein or other coronavirus antigens in order to isolate GIPSITUMAB useful in diagnosing and treating COVID- 19. In certain embodiments, GIPSITUMAB with broad spectrum responsiveness to the coronavirus may be used to detect latent coronavirus in the gut.

[00035] A simple approach to generate this antibody in vitro as a therapeutic agent for treating COVID-19 patients is also presently disclosed. The approach involves the lineage- directed differentiation of GIPC cells to form a progeny that simulated plasma cells secreting GIPSITUMAB can also be used for treating infectious disease. Furthermore, unlike other bispecific bioengineered antibodies, GIPSITUMAB does not elicit any host versus graft response and is a stable element that can be easily transported and administered. GIPSITUMAB can also be used to identify cells infected by any pathogen and eliminates them before any symptoms set in. In addition, mutant variants of the virus does not impede the ability of this antibody to detect them and destroy them while they reside in the host cells asymptomatically.

[00036] In one embodiment, a cell population isolated as described herein may also comprise GIPC cells comprising membrane bound anti-infective antibodies that may be partially differentiated to produce chimeric secretory antibodies that are IgA/IgG heterodimers, the absence of which may indicate or suggest a pathology or disease condition comprising what is commonly referred to as COVID- 19. In certain embodiments, the IgG moiety binds to an infected cell, leading to cell death, while the IgA moiety recognizes cells infected by destructive microorganisms such as the coronavirus. In one embodiment, a patient population deficient in GIPC cell membrane-bound antibodies as described above comprises a majority of African- American patients, the deficiency being indicative of an immunodeficiency condition unique to African-Americans and possibly increased cancer risk. Further, in certain embodiments a method as described herein further comprises utilizing cells expressing a chimeric IgA/IgG antibody and inducing such cells to produce therapeutic antibodies to viral pathogens such as the coronavirus using known immunological methods. In certain embodiments, certain methods as described herein may be used to produce therapeutic antibodies to the coronavirus, and may be useful in the treatment of individuals deficient in these antibodies.

[00037] In one embodiment, a biomarker comprising the spike protein indicates or suggests the presence of the coronavirus. In one embodiment, the biomarker is not detectable in blood samples prepared according to current clinical methods comprising the invasive collection of patient blood. In one embodiment, the coronavirus is not detectable according to methodologies other than those described herein and may be described as “latent” coronavirus. In one embodiment, a population of patients who are asymptomatic may be considered a coronavirus-negative population may be targets for therapeutic antibodies as described herein. Methods according to such embodiments leverage the detectable expression of coronavirus biomarkers such as the spike protein according to PCR-based methods or measurement of the number of cells expressing the spike protein. In certain preferred embodiments, detection of the spike protein may be achieved by raising responsive GIPSITUMAB for the purposes of broad spectrum coronavirus detection in isolated gut cells according to methods as described herein.

[00038] The methods described herein comprise isolating, transporting and testing cells from stool samples according to established methods, wherein the test used to detect coronavirus in cells isolated from the stool sample are selected from the group consisting essentially of antibody tests, viral identification assays, viral loading tests and PCR tests. In certain embodiments, the methods of the present invention may be used to screen a patient population for coronavirus infection. In other embodiments, the methods of the present disclosure may also be used to monitor disease progression in patients infected with the coronavirus. In certain preferred embodiments, broad spectrum GIPSITUMAB are used to detect the coronavirus in infected gut cells isolated as described herein.

[00039] In addition to coronavirus testing, certain embodiments of the present disclosure comprise detection of a biomarker according to a method or process as described herein, wherein the biomarker is a positive or negative indicator of exposure to coronavirus, such as the presence or absence of the spike protein, respectively. In one embodiment of the present invention, the presence of such a biomarker is an indicator of excessive exposure to the coronavirus of interest as an antigen.

[00040] In one embodiment, the transport medium as described herein may be used for the room temperature preservation of cells from aspirates such as pancreatic, ascites, or pleural effusion aspirates, or for cytopathology. In one embodiment, a method or process as described herein may be employed for dynamic interrogation of colonic cells for detection of coronavirus -infected cells as a measure of viral load. In one embodiment, viral load may be measured according to such a method or process in patients from families lacking in the ability to generate antibodies to coronavirus infection.

[00041] Examples

Fecal Cell Isolation

[00042] A kit may be utilized to isolate several million GIPC cells from a small pellet of fresh stool (0.5 gm). This is used to generate GIPSITUMAB. This technology can also be used to assess the competence of the individual to generate this antibody and identify those who are immunocompromised and hence non-responsive to current coronavirus vaccines. The preservation of gut cells using transport medium enables flexible clinical scheduling of cell isolation and investigation of gastrointestinal genetic and cell surface markers without the need for invasive procedures such as gut biopsies. Additionally, cell archives and complimentary DNA (cDNA) libraries can be constructed for future study. Cells isolated using a procedure as described herein may be subjected to any number of known analytical techniques such as flow cytometry, and progenitor cell lines may be maintained for multiple generations. Cells isolated as described herein may also yield better sample material for the isolation of DNA and conduction of polymerase chain reaction (PCR) amplification for diagnostic methods, as signal degradation due to microbial interference and other complications associated with fecal collection are diminished.

[00043] The kit provides all of the reagents necessary to preserve and isolate several million exfoliated host cells per gram of fecal sample. The transport medium preserves samples up to 5 days at room temperature prior to cell isolation, which utilizes a simple step gradient centrifugation followed by PBS wash. All viable exfoliated cell types are isolated - epithelial, progenitors, and leukocytes.

Applications

[00044] The kit enables researchers and clinicians to investigate gastrointestinal genetic and cell-surface markers without the need for invasive biopsies. Cell archives and cDNA libraries are then easily created. The kit has been used extensively as a cell source for flow cytometry. Progenitor cell lines have been maintained for over 90 generations. For PCR analysis, cell isolation surpasses direct DNA extraction by minimizing signal degradation due to microbial interference and fecal inhibitors of Taq and fluorescence. The kits have been used to isolate cells from human, rat, mouse, cattle, dog, cat, monkey, goat, and sheep samples.

Technique

[00045] The technique involves collecting 0.5 gm (a small pellet) of stool in a tube (containing a sampling scoop and preservative) and transporting the specimen to the laboratory under normal ambient temperature within 5 days. The suspension is filtered, underlayered with a dense cushion medium, and centrifuged at low speed for 10 minutes. Cells are extracted from the interface between the supernatant and cushion (the pellet, which contains clumped, dense, or debris-bound cells, is retained as a backup). The cells are washed repeatedly in PBS, aliquoted, and are ready for analysis or archiving. They can be stained for specific cell surface markers using immunofluorescent antibodies and subjected to flow cytometry. They can be lysed for the extraction of proteins, glycoproteins, mRNA, and genomic DNA.

In Vivo Efficacy of Gipsitumab in Tumor Inhibition

[00046] The in vivo efficacy of Gipsitumab™ in tumor inhibition was tested in nude mice xenografts. In this experiment, mice (n=21) were implanted subcutaneously with HT-29 human adeno-carcinoma cells. After 14 days, the experimental animals (n=6) were treated with six SOOpg intravenous doses (~26pg/g body weight) of Gipsitumab™ on 3 or 4 day intervals. The mean tumor weights over a 32 day period after implant are presented below:

Table 1 Mean tumor weights and growth inhibition over 32 day period Normals not measured on day 4 (value linearly interpolated)

[00047] GIP-C cells are obtained from a patient's stool sample using a Somatic/Stem Cell Sampling & Recovery (“SCSR”) system. SCSR is a technology' for the preservation, transport, and recovery of cells from fecal samples. The SCSR system includes a kit format comprising sampling tubes with transport media, fdters and strainers, and step-gradient cushion used for the separation of the cells in a standard swinging-bucket non-refngerated centrifuge. The technique involves collecting 0.5 gm (a small pellet) of stool in the tube provided (containing a sampling scoop and a preservative) and transporting the specimen to a laboratory under normal ambient temperature within 5 days. The suspension is filtered, underlayered with a dense cushion medium, and centrifuged at low speed for 10 minutes. Cells are extracted from the interface between the supernatant and cushion (the pellet, which contains clumped, dense, or debris-bound cells, is retained as a backup). The cells are washed repeatedly in PBS, aliquoted, and are ready for analysis or archiving. They may be stained for specific cell surface markers using immunofl uorescent antibodies and subjected to flow cytometry. Additionally, they may be lysed for the extraction of proteins, glycoproteins, mRNA, and genomic DNA.

[00048] This configuration allows for transport of the specimen without refrigeration up to 5 days after collection, minimizes the problem of bacterial interference, Taq inhibition, and fluorescence quenching, enables all cell types to be isolated, ensures nontoxic components in the test kit, and cultured and archived cells for years. A typical yield is 20-40 million total cells/gram of stool sample. Of these cells, about 8-1 0% are gastrointestinal progenitor stem cells (GIP-C).

[00049] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present disclosure is not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect of the invention and other functionally equivalent embodiments are within the scope of the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.