Abstract
This disclosure is in the field of cancer immunotherapy and relates to all cancer types, including but not limited to cancers of the breast, lung, prostate, pancreas, colon, bladder, brain, head-neck, kidney, esophagus, skin, and blood cells. The embodiments provide methods for selecting and amplifying specific targeting molecules to use in therapy and diagnostic testing. Targets include but are not necessarily limited to architecturally preserved, usually heterogeneous specific surface structures present on individual cancer cells and cancer stem cells but not on non-malignant cells. The embodiments are novel in that they provide these binding molecules for one individual's cancer regardless of the rarity of an individual cancer cell or stem cell and promptly enough to initiate treatment without requiring lengthy immunization or hybridoma production that are current art.
Claims
1. A method of preparing membrane fragments from heterogeneous cancer cell mixtures from an individual patient comprising: a. altered cancer cell morphology caused by the cumulative results of all the DNA mutations, deletions, insertions, and rearrangements in the patient's cancer cells; b. abnormal cell surface molecular arrangements, comprising phospholipids, sphingolipids, lipids, sterols, proteoglycans, carbohydrates, and proteins in said cancer cells; c. the preservation and maintenance of the three-dimensional arrangements of molecules on cancer cell surfaces from an individual patient comprising those that differ from normal cells matched to the same patient; d. said preservation and maintenance is achieved without the use of fixatives, organic solvents, formaldehyde, glutaraldehyde, other cross-linking agents, heat, or other methods of coagulation.
2. Methods comprise producing cancer immunotherapy medication by using said unnatural surface morphology created by cancer DNA modifications, characteristic of an individual cancer, and absent from non-malignant cells matched to the individual patient.
3. Methods comprise preparing and isolating polyclonal anti-cancer binding molecules specific to the abnormal morphology of cancer cells in the individual, regardless of the rarity of the feature.
4. Methods comprise preparing immunotherapy reagents and immunodiagnostics for distinguishing cancer vs. normal cells in any individual patient; a. Methods comprise the selection and purification of polyclonal immunotherapy agents that are unlikely to react with tissues that do not contain malignancy.
5. Methods are prompt and rapid enough to prepare cancer immunotherapy reagents specific for cancer in one individual patient.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0076] FIG. 1A-E. Abnormal structures on cancer cells provide targets for immunotherapy. Cancer-causing DNA mutations comprise but are not limited to protein structures, membrane structures, lipids, sterols, proteoglycans, phospholipids, sphingolipids, and pathologic cell stromal linkages that are not present in non-malignant cells.
[0077] FIG. 2 is a flow chart for the Embodiment 1 protocol to isolate cell surface abnormalities found on cancer cell surfaces but not on normal cell surfaces.
[0078] FIG. 3 is a flow chart summary of steps required to edit the phage display antibody library to remove molecules that bind to normal cells. The figure illustrates examples of membrane proteins, but the process also comprises normal counterparts of cancer cell structures affecting lipids, sterols, proteoglycans, phospholipids, sphingolipids, and stromal linkages.
[0079] FIG. 4 illustrates an amphipathic agent solubilizing a highly abnormal cancer cell membrane to retain surface structures in aqueous solutions.
[0080] FIG. 5 represents an artificial mimic of the extracellular medium designed to preserve the shapes of whole cancer and normal cells.
OBJECTIVES OF THE INVENTION
[0081] AIM 1 of the embodiments is to provide antibody-like molecules that can be used to treat virtually any cancer, without damaging non-malignant cells
[0082] AIM 2 of the embodiments is to provide antibody-like reagents which can serve as a pharmaceutical platform for derivatives which could augment therapeutic effectiveness in the treatment of cancer.
[0083] AIM 3 of the embodiments is to improve available therapy for cancer, regardless of its stage, grade.
[0084] AIM 4 of the embodiments is to provide therapy when standard therapy has either failed or become too toxic to continue.
[0085] AIM 5 of the embodiments is to produce specific anticancer antibody-like molecules quickly enough for them to become an option for primary therapy.
[0086] These and other aims of the invention are clear from the description herein.
BRIEF DESCRIPTION OF THE INVENTION
[0087] The embodiments overcome the disadvantages of the prior art and use the discovery of cancer's abnormal cell morphology to guide methods to prepare therapeutic reagents. The procedures contain sufficient detail so that the skilled practitioner can use them for any type of cancer at any stage or grade. Cancer-specific cell structural aberrations are universal in cancer and distinct from prior art involving neoantigens or other cancer antigens. A significant aspect of the embodiments preserves cancer cell surface morphology as a new process derived from methods to determine three-dimensional protein structures. Systems and methods select and amplify therapeutic antibody-like molecules specific for cancer cell surface structures that do not exist on normal cells.
[0088] The embodiments supplement the universal immune deficit in cancer to compensate for it. Polyclonal immune supplements make the embodiments independent of the number of different malignant species present and the development of new malignant clones. The design solves the problem of rare surface antigens such as those on cancer stem cells by amplifying binding molecules to any desired level. The embodiments remove antibody fragments that react with normal cells, preventing normal cells from being targeted. The embodiments increase the concentration of molecules specific to cancer cell surfaces to improve the ability to select cancer binding fragments from large phage display libraries. The embodiments are superior to targeted therapy because they do not require identifying shared mutated genes or even which type of cell is malignant. Unlike checkpoint inhibitors, the immune supplements do not remove essential controls on the immune system that permit self-recognition. A skilled laboratory can produce cancer binding antibody fragments fast enough to add polyclonal immunotherapy reagents to primary cancer therapy.
[0089] Subtracting out antibodies that bind normal off-target organs increases safety. Because of the increase in safety, the embodiments provide a bridge preventing tumor regrowth during periods when organ toxicity forces patients to discontinue or stop conventional therapy. Another aspect of the embodiments are their speed of implementation. Another aspect is preserving the morphology of cells and cell surface molecules, whether they represent cancer- or non-malignant cells. Other aspects are new uses for rapid methods to enrich cancer cell-specific molecules from the surfaces of cancer cells and to assay cancer and normal cell surface molecules for the preservation of their 3-dimensional structures.
Significant Aspects of Invention Embodiments
[0090] A major aspect of these embodiments is that they preserve the 3-dimensional structures of molecules specific to cancer cell surfaces and absent from non-malignant cells.
[0091] A significant aspect of the embodiments is the prohibition of chemical cross-linking fixatives, organic solvent precipitation, coagulation, physical fixative reagents, or any other conditions that change the 3-dimensional structure of cancer cell surface antigens.
[0092] Another aspect of the embodiments is that therapy with antibody-like molecules does not identify or target cancer cell driver mutations.
[0093] Another aspect of the embodiments is that they do not depend on the cell, tissue, or organ from which cancer originates.
[0094] Another aspect of the embodiments is that they do not depend on stratifying patients based on the presence or absence of specific driver gene mutations or prognostic factors.
[0095] Another aspect if the embodiments is that they comprise rapid production of antitumor binding molecules so that they said molecules can contribute to primary anticancer therapy
DETAILED DESCRIPTION OF THE INVENTION
[0096] Abnormal surface morphology in cancer has never been recognized either as a general basis for therapy or a general distinguishing characteristic. Preserving the structure and arrangements of abnormalities on cancer cell surfaces are essential to produce therapeutic antibody-like molecules. With reference to FIGS. 1-4:
[0097] The aggregate total of DNA mutations, breaks, deletions, insertions, and rearrangements in heterogeneous populations of cancer cells with diverse genetic mutations create abnormal cancer cell surface structures. FIGS. 1A-E illustrate cancer-induced changes in proteins, in the asymmetric distribution of phospholipids, and in other molecules in the inner and outer cell membranes of lipid bilayers. These changes comprise all sites susceptible to cancer, some of which are indicated on the male and female drawings. Mutations responsible for these abnormalities create myriad targets for immunotherapy. Membrane proteins, phospholipids, sphingolipids, proteoglycans, and sterols depend on countless steps and interactions in metabolic pathways, all subject to mutation. Cancer DNA further causes abnormal cell morphology because mutations affecting some membrane proteins and sterols reorganize, harden, or soften membranes. Qui et al. (2020) demonstrated that side chains of membrane proteins cause marked ordering of the membrane around integral proteins. Mutations disrupt this ordering creating potential immunotherapy targets. FIG. 1 comprise examples of typical cancer cell structures with information on how they embed in the membranes. The sample proteins are individually labeled based on silhouettes obtained from the Protein Data Base (PDB). The cell membrane is a lipid bilayer that displays cancer cell associated structures. The lipid bilayer is composed of an inner and outer layer which differ in composition. A small animal cell contains about 1 billion molecules in its cell membrane, with phospholipids being the most abundant. The bilayer also includes sphingolipids and interspersed sterols such as cholesterol (depicted), which generally stiffens the bilayer. Typically, 1300-1500 different phospholipid species distribute differently between the inner vs. the outer plasma membrane and help determine cell shape (Llado et al., 2014.) In FIG. 1A-E, cancer cell DNA disrupts a myriad of normal interactions with diverse molecules. This disruption creates pathologic structures that are not present in normal cells.
[0098] FIG. 1A depicts an invadopodium (1) from a cancer cell surface that breeches extracellular membrane structures (2) and begins cancer spread from its source cell. Invadopodia are known to unmask and distort proteins and release proteolytic enzymes, facilitating invasion and metastasis (Hoshino et al. J. Cell Science 2013). Invadopodia are likely in breast, colorectal, bladder, and head and neck squamous cell carcinoma (Yamaguchi 2012).
[0099] FIG. 1B represents the CD44 cell surface molecule (3) that becomes mostly incompatible with its normal display on the cell surface because of a DNA mutation. Absence of CD44 creates abnormal membrane structures (4) (black). The cancer cell lacking surface CD44 can no longer bind extracellular hyaluronic acid (5) so the cell loses a positional anchor. CD44 variations have been linked to non-Hodgkins lymphomas, colorectal cancer and breast cancer.
[0100] In FIG. 1C, a Src enzyme (6) inside the cell (cytoplasmic) is an enzyme associated with the cell membrane. Here, Src mutates and alters membrane structure (shown in black). The Src mutation changes the cancer cell membrane by altering the distributions of membrane constituents such as phospholipids and sphingolipids (7) and cholesterol (8). (indicated in black). Prostate, lung, breast and colorectal cancers involve Src.
[0101] FIG. 1D. Integrins are transmembrane receptors that mediate cell adhesion. Inactive integrin does not associate with lipid rafts but activated integrin moves laterally using lipid rafts. Cytoplasmic tethering restrains integrin from boarding the rafts. (Leitinger and Hogg, 2001).
[0102] FIG. 1D shows integrin mutation creating distinctive cancer immunotherapy targets because it alters not only integrin (9) but also the cancer cell membrane (indicated by black structures). Integrin mutation and a cholesterol-sphingolipid enriched raft (10) have ferried mutated integrin (9) laterally to a new location, altering cell shape and adhesion reactions with other cells. Melanoma, glioblastoma, and cancers of the breast, prostate, pancreas, ovary, lung, colon, and cervix have abnormal integrins.
[0103] In FIG. 1E, Tetraspannin ordinarily regulates transporting molecules into structurally distinct, small, specialized cell membrane regions. A non-functional Tetraspannin protein (11) causes changes in distributions of (black) membrane components (12), thereby altering cell morphology. Tetraspannins participate in chronic lymphocytic leukemia, lymphoma, melanoma and cancers of the breast, esophagus, kidney, bladder, and lung (non-small cell),
Embodiment 1 (FIG. 2)
[0104] Embodiment 1 takes advantage of cancer cell surface deformations caused by mutations and their effects to alter membrane-proteins. Embodiment 1 selects and amplifies cancer binding molecules for rapid, routine cancer immunotherapy. The most critical factor in using these cell surface formations as immunotherapy targets is to preserve their 3-dimensional structures and arrangements. If cancer cell surface structures become artificially distorted, protocols will select antibodies that recognize the distorted cancer cell surfaces. These antibodies have a high affinity for the distorted antigen but not for the antigen in tumor samples. [0105] 1. Cancer cells comprise structures (1-6) on the same or different cell populations and contributions from the microenvironment (2) such as those from FIG. 1 [0106] 7. Preserving cancer cell structures begins by enriching for the outer cell membrane surfaces. The Membrane Fraction preparation comprises gentle lysis and differential centrifugation at speeds routinely used to prepare cell membrane fractions (Suski et al., 2014), e.g., 25,000 g and 100,000 g. Preservation of abnormal structures such as those comprising (1-6) is monitored by full or partial retention of activity to a panel of fluorescent antibodies known to react with cell surface molecules such as CD24, CD44, CD142, CD144, CD146, CD171, CD220, CD221, ERBB1, SSEA-1. [0107] 8. Combinations of one or more “Structure-Preserving Detergents” disperse the membrane fraction (7). Because of differences among cancers in different organs, it is unlikely that any single detergent will always be the best choice. Five of the most widely used detergents work at concentrations of 1-2%: Dodecyl mannoside, nonyl glucopyranose, octyl glucopyranoside, lauryl maltose neopentyl glycol, and lauryl dimethyl amine N-oxide. X-ray crystallography and other structural studies validate the ability of these detergents to preserve protein three-dimensional structures. (Mus-Veteau I. 2014; Bonnete and Loll, 2017; Stetsenko, 2017). Again, reactivity to a panel of known anti-cell surface antibodies monitors the preservation of three-dimensional structures by the detergents selected for each individual cancer. The binding of fluorescent derivatives of anti-cell surface antibodies is measured by flow cytometry. [0108] 9. The addition of amphipathic stabilizing agents such as biotinylated amphipol A8-35 at about three times the weight of membrane structures stabilizes abnormal versions of surface structures (comprising structures labeled as 1a-2a, 3a, 4a, 5a, and 6a) to safely remove detergent (step 7). Trapping in the amphipathic agent requires adjusting NaCl concentration to 100 mM and detergent to near its critical micellar concentration (0.2 mM for dodecyl maltoside). Optimizing stabilization requires determining the amount of amphipol to maintain abnormal structures in an aqueous solution after removing detergent. Adsorption onto polystyrene beads (Bio-Beads at 4° C. for 3 hrs.) or simple dilution prevents detergent interference, which usually does not cause problems (Zoonens et al., 2014). [0109] 10. Editing a phage display library subtracts antibody-like molecules that bind non-malignant cells (10). Modern phage display technology allows reliable access to extensive antibody libraries. These libraries yield anti-cancer cell surface protein antibodies that permit enormous amplification without lengthy immunizations, without degrading cancer cells, and without changing their cell morphology. [0110] Targeting cancer cell surfaces lowers the number of antibodies required since cell surfaces contain only a small fraction (about 6000) of total cellular proteins. The phage (bacterial virus) genome includes most human antibody binding region genes within its DNA as placed by recombinant DNA technology. When the phage infects bacteria such as e coli, the phage uses bacterial machinery to make (“display”) the binding regions of human antibodies as part of its outer coat. Phage display technology also facilitates removing off-target antibodies (enclosed by dashed line in box), [0111] The phage display library initially comprises 1-100 billion different species, comprising antibody-like fragments that react with normal cells in addition to those that react with malignant cells. Normal cell surface structures trapped in amphipathic agent react with the phage display antibody library to edit it by removing molecules that bind normal cells (11-16), minimizing toxicity to normal cells. Normal examples comprise normal Src kinase membrane complex (11), CLCA1 (12), Multidrug transporter ABCB1 (13), normal integrin (14), normal Tetraspannin CD81(15), and carbonic anhydrase. Because the molecules shown binding structures 11-16 are single chain (ScFv) derivatives, they contain only one binding site and will not cause cross-linking and precipitation in the stabilized environment. The presence of biotin on the amphipathic stabilizing agent enables easy removal of these anti-normal cell molecules. [0112] 17. The edited phage display library (17) recognizes amphipol-trapped cell surface proteins and other molecules, labeled as 1a-6a. After binding to the amphipathic stabilizing agent, the detergent is removed by adsorption to polystyrene beads (Bio-Beads) [0113] 18. Members of the edited phage display library bind to abnormal cancer cell surfaces (1a-6a). It is not necessary to know the identity of the surface molecule or the cell of origin. All that is required is that the surface molecule does not belong within any non-malignant cell environment. [0114] 19. Cancer cell surfaces as biotin-amphipathic derivatives bind to a streptavidin containing solid immunoabsorbent (as in FIG. 3). Phages displaying irrelevant antibody fragments do not stick and wash away. Biotin and streptavidin bind very rapidly, forming the most stable known non-covalent bond between a protein and a ligand. Broad extremes of pH, temperature, organic solvents, and other denaturing agents do not disrupt the complex. The tenacious biotin-streptavidin bond prevents excess amphipol from interfering with subsequent elution of the cancer cell binding molecules. Low pH or trypsin then elutes only the bound antibody phages, which can then reinfect bacteria (usually e. coli). [0115] 20. The number of e coli cells doubles every 20 minutes, and infected cells multiply faster. Moreover, an infected cell produces 1000 progeny bacterial viruses in one hour (Smeal et al., 2017). Bound antibody phages are eluted, used to reinfect more e. coli to make more specific antibody phages. [0116] 21. Repeating these steps three or more times yields large amounts of phage-displayed antibody mixtures at billion-fold levels of amplification. The capacity for virtually unlimited amplification makes it feasible to produce therapeutic antibodies to very rare cancer-specific antigens. The speed of phage display antibody production permits using it for truly personalized individual therapy. Diversification in error-prone e. coli further increases the potential number of antibody-like molecules.
[0117] FIG. 3. Solid-phase immunoadsorption subtracts antibodies to non-malignant cell surfaces and the amphipathic reagent such as biotinylated amphipol A8-35. The phage-display library initially contains 1-100 billion different binding molecules. [0118] 1. Normal cells from tumor margins clear of malignancy, any additional available non-malignant normal tissue, and non-malignant finite cell lines undergo the same procedures as the tumor cells (FIG. 2). Removing phage display antibodies to non-malignant cell surfaces produces the “edited” antibody phage display library used in FIG. 2. This subtraction enriches the library in antibody-like molecules that bind cancer cell surfaces. [0119] 2. The normal cell samples undergo gentle lysis and differential centrifugations to prepare membrane fractions. [0120] 3. The same detergents used for cancer cells (FIG. 2) disperse the cell surface structures. [0121] 4. Biotinylated amphipol then stabilizes non-malignant cell surface structures as shown, substituting for the original cell membrane. The detergent can then be removed by adsorption to polystyrene (Bio-Beads). [0122] 5. Antibody fragments from the phage display library (6) bind the amphipol stabilized structures (5) react with antibody derivatives within the phage display library (6), comprising 1 to 100 billion molecular species. Binding is measured by flow cytometry of fluorescent antibody derivatives. [0123] 6. The phage antibody derivatives (ScFv fragments) in the library comprise 1 to 100 billion binding regions, which differ at the molecular level. Each member of the library has only a single binding site, so it cannot cause cross-linking or precipitation. [0124] 7. Solid streptavidin particles (7) then bind biotin groups on the amphipathic stabilizing agent. [0125] 8. The binding of streptavidin beads immobilizes biotinylated amphipol derivatives of surface molecules from normal cells. Phages that display antibodies to normal cell surface molecules will stick, allowing their removal. The phages that do not stick contain binding molecules specific to cancer cell surfaces and irrelevant antibody fragments. [0126] 9. An “edited” phage display library then will not react with surface proteins from the normal cells.
[0127] FIG. 4. The membrane structure in a cancer cell is no longer regular. Invasive membrane structures that differ from non-malignant cells include invadopodia (invasive actin-rich projections), blebs (membrane bulges), and membrane ruffles. At least one antibody can recognize invadopodia in cancer. (Baik et al., 2019).
[0128] Mutations accumulate and may generate highly abnormal cancer cell membrane structures (10 and grossly malformed cancer cells. Inappropriate mixing of the inner and outer cell membrane components, mutations in biosynthetic pathways, and changes in protein structure produce aberrant cell membranes. The illustration shows an example of an abnormal membrane fragment patterned after known cancer cellular abnormalities. When the protocol in FIG. 2 is applied, pathologic phospholipid and other membrane assemblies (black) disperse on cell lysis and structure-preserving detergents (2). Amphipathic agents such as biotinylated amphipol A8-35 stabilize the abnormal structures. For example, one part of the abnormal structure (3) produces the stabilized aqueous compatible derivative (4). Structure 4 and the remaining other parts of the cancer cell surface select binding antibodies from an edited phage display library as in FIGS. 2 and 3.
Embodiment 2 (FIG. 5)
[0129] To preserve the structure of normal cell and cancer cell antigens, cells are immobilized in microwells on a microplate (“1” in FIG. 5) under conditions that maintain in vivo morphology. Before exposure to a patient derived cancer cell specimen, we generate conditions mimicking cancer cell physiologic environments. First, microwell plates are coated with stromal cells (“2” in FIG. 5.) (such as fibroblasts or smooth muscle cells). Next comes a combination of fibronectin and hyaluron added in sequence (“3” in FIG. 5). Human fibronectin (0.1%) facilitates adhesion through binding to integrin receptors (1-5 mcg/cm2). Hyaluronic acid coating is done by the method of Corradetti et al., 2017. The last addition is animal origin-free recombinant Type 1 human collagen (“4” in FIG. 5) in dilution medium to encourage adhesion.
[0130] After cancer cells are placed on the substrate, the cells are exposed to an edited phage display library. The library has been edited so that it does not bind the substrate or normal cells in the cancer cell preparation.
[0131] Embodiment 2 works if the cancer cell membrane structure is highly abnormal, as in FIG. 1D or of the cancer cell surfaces are fragile. Lysis, binding, detergent, and amphipathic agent suspension are then conducted as in FIG. 2. A competitive ELISA assay tests for distortion after cancer cells are immobilized.
[0132] Embodiments 1 and 2 remove phage display antibody fragments that bind to normal cells under the same conditions used to prepare the immunoabsorbent for cancer cells. The preservation of normal cell morphology is determined by maintaining reactivity to one or more members of a panel of antibodies directed against non-malignant cell surfaces.
[0133] Cancer cells are then exposed to the antibodies that do not bind in either embodiment 1 or 2. Washing removes irrelevant antibodies so antibodies specific for cancer cells can be recovered, amplified, and purified.
[0134] The phage particles that carry the cancer-specific antibodies have an engineered trypsin cleavage site making it convenient to release bound phage particles. Limited trypsin digestion releases bound phages that are still infectious and still produce antibody-like fragments such as ScFv, Fab, FHH, or other forms. The released phage particles then reinfect e-coli. A few cycles of reinfection and re-release yield highly purified anti-cancer cell therapeutics with billion-fold amplification.
[0135] Diversify the anti-cancer antibodies. One advantage of the anti-cancer antibody-like fragments is that they are mixtures (polyclonal), capable of forming large, tight complexes. Another aspect of these mixtures includes recognizing different parts of the same cancer, reacting with varying antigens of cancer in other parts of the same tumor, and in tumor metastases. Antibodies are amplified in error-prone e-coli to diversify the anti-cancer antibodies, increase binding avidity, and produce binding to rare cancer stem cell antigens. Even with the time it takes for affinity maturation and to add effector functions, it is much faster to produce patient-specific anti-cancer cell surface binding antibodies than to produce hybridoma antibodies.
[0136] Production of Cytotoxic Antibodies.
[0137] ScFv fragments are converted to ScFv-Fc fragments according to the prior art. For example, TGEX-SCblue is a commercially available mammalian expression vector designed to convert ScFv phage clones into bivalent ScFv-Fc fusion proteins that can be quickly expressed in HEK 293 cells (Jager et al., 2013). The conversion begins with phagemids with antibody V genes inserted between two SFiI sites. The V-genes are then cut and pasted into a mammalian expression vector by restriction fragment cloning without needing PCR amplification (Yoon et al., 2012). Typical yields are at least 10-100 mg/ml in a few days. The product reproduces the binding of a fully reconstructed monoclonal antibody. Transient transfections are easy in 24- and 48-well plates in parallel.
[0138] The prior art also describes preparing other cytotoxic antibody-like derivatives such as diabodies, Triomab, Fab2, Tandem ScFv, and multi-specific forms.
[0139] Radioactive derivatives or combinations with cytotoxic drugs are also readily prepared according to prior art.
[0140] The immunotherapy anticancer binding molecules may be coadministered with standard cancer treatment.
