WO2016145112A1 - Materials and methods for eliciting targeted antibody responses in vivo - Google Patents
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Definitions
- the disclosure relates generally to medical products and medical procedures, and more specifically to materials and methods for preventing, treating or ameliorating a symptom of a condition characterized by a cell- surface marker, or target, such as cancer.
- a deadly characteristic of cancer cells is their ability to proliferate at uncontrolled rates, invade local tissues, and metastasize to distant sites where they grow anew.
- cancer therapies that effectively target cancer cell growth, invasion or metastasis, either on the market or in development.
- the importance of inhibiting cancer cell proliferation and invasion - at either primary or metastatic sites - is compelling.
- Attempts to identify new targets for therapeutic intervention or to develop the appropriate drugs have been hampered by the inability of in vitro model systems to accurately recapitulate cancer cell proliferation and/or invasion programs as they occur in vivo 1 ' 2.
- ECM extracellular matrix
- the basement membrane normally separates epithelial cells from the underlying type I collagen-rich interstitial matrix (1, 2).
- the epithelium does not establish stable physical contacts with interstitial tissues (1, 2).
- transformed epithelial cells i.e., carcinomas
- carcinoma cells dissolve the intervening basement membrane barrier and establish adhesive interactions with the newly exposed type I collagen fibrillar network (1-5).
- carcinoma cells begin to infiltrate the interstitial matrix, they rapidly adapt themselves to their three-dimensional environment and initiate the proliferative phenotypes that define tumor progression at both primary and metastatic sites (2, 6, 7).
- carcinoma cells do not simply use the surrounding interstitial matrix as a passive substrate, they actively promote increased type I collagen deposition within the peri-tumoral microenvironment as a means to further enhance invasive activity, local growth and cancer stem cell formation (7-12).
- the disclosure provides materials and methods for the discovery, validation, and/or functionalization of unknown molecular biological targets for pharmaceutical intervention and for obtaining specific binding partners, such as antibody products that specifically bind to targets of interest, e.g., cell-surface markers, that are useful therapeutically, prophylactically, and/or diagnostically.
- specific binding partners such as antibody products that specifically bind to targets of interest, e.g., cell-surface markers, that are useful therapeutically, prophylactically, and/or diagnostically.
- the disclosure provides immunogens in a three-dimensional, extracellular matrix that promotes embedded cells to express a unique repertoire of antigens relative to those expressed in standard two-dimensional culture.
- the three-dimensional extracellular matrix provides a cellular microenvironment that promotes the expression of the unique repertoire of cell- surface proteins by the cells embedded in that microenvironment.
- the cells present antigenic cell-surface markers that differ from the markers presented by that cell type when present in two- dimensional culture.
- the difference in cell-surface markers, and hence in antibodies elicited to such markers, is not just a difference without distinction.
- the mouse immune system When injected in vivo, the mouse immune system generates antibodies against cell-surface targets that better recapitulate those antibodies naturally generated in vivo than the antibodies raised against cells in two-dimensional culture.
- the cell-surface marker is a marker of, or associated with, a disease, such as cancer.
- a disease such as cancer.
- Exemplary diseases, disorders or conditions amenable to the disclosed technology include any form of cancer, any form of a fibrotic disease, any form of an
- the marker is associated with a wound, and an immune response elicited by the three-dimensional presentation of the marker is beneficial in wound healing.
- the cell-surface marker is present on its cognate cell within the three-dimensional environment or framework.
- the cell-surface marker may also be associated with a portion of a cell surface, such as a cytosolic membrane or it may be engineered such that it is associated with one or more compounds that yield a three-dimensional structure for the cell- surface marker that mimics the structure of the cell-surface marker when found in vivo.
- the three-dimensional environment or framework is composed of type 1 collagen (the dominant extracellular matrix protein found in humans) or fibrin (the dominant provisional matrix protein localized to tumor or wound sites).
- the elicitation of specific binding partners, e.g., antibodies, to a cell-surface marker is preceded by a tolerizing step in which the host organism is initially exposed to a three-dimensional environment or framework comprising a normal cell exhibiting cell-surface markers characteristic of that normal cell, e.g., a non-cancerous cell.
- a three-dimensional environment or framework comprising a cell exhibiting the cell-surface marker of interest, i.e., the target is administered.
- the disclosure provides a screening platform wherein human carcinoma cells are cultured within aldimine cross-linked, three-dimensional extracellular matrix protein (e.g., type I collagen) hydrogels similar to those found at invasive sites in vivo (16), and the cancer cell-matrix composite is used to generate a library of monoclonal antibodies (mAbs).
- mAbs monoclonal antibodies
- function-blocking or function-activating mAbs are then identified by screening for their ability to suppress carcinoma cell proliferative responses under three-dimensional growth conditions in vitro.
- selected mAbs are then shown to inhibit carcinoma cell proliferation and metastatic activity in xenograft models in vivo.
- the disclosed materials and methods extend beyond the generation of three- dimensional anti-cancer antigens and methods of screening for antibodies blocking an antigen function involved in cancer development or persistence, such as cell proliferation.
- materials and methods for generating three-dimensional antigens of fibrotic disease, an inflammatory disease state, an angiogenic disorder, an infectious disease, or a wound and methods of treating, preventing or ameliorating a symptom of such a disease, disease state or disorder (e.g., wound) comprising administration of an effective amount of a function-blocking or function-activating antibody according to the disclosure, or a function-blocking or function- activating antibody fragment thereof.
- an "antibody” is any form of an antigen-binding protein known in the art, including complete immunoglobulin antibodies of any isotype or sub-isotype, a chimera, a humanized or human antibody, an antibody fragment, a scFv, a diabody, a bi-specific antibody fragment, a tri-specific antibody fragment, a fusion protein with any of a wide variety of therapeutic proteins and/or other moieties, a Fab fragment, a Fab' fragment, a F(ab)2' fragment and any other functional format for specifically binding an antigen presented in a three- dimensional microenvironment, such as in the hydrogels of the disclosure, or in vivo. Any method known in the art is suitable for producing an antibody product of the disclosure, as defined above. For example, an antibody may be elicited or produced in an immunoglobulin antibodies of any isotype or sub-isotype, a chimera, a humanized or human antibody, an antibody fragment, a scFv,
- the antibody may be obtained using an in vitro approach such as phage display, followed by production of the antibody in quantity and, optionally, engineering to form any of the aforementioned antibody products.
- the antibody may be conjugated to a drug and delivered as an antibody-drug conjugate.
- one of fifteen function-blocking monoclonal antibodies was further analyzed and demonstrated an ability to halt carcinoma cell proliferation, inducing apoptosis and exerting global changes in gene expression in vitro.
- the ability of the monoclonal antibody to block carcinoma cell proliferation and metastatic activity was confirmed in vivo and the target antigen identified by mass-spectroscopy as the a 2 subunit of the ⁇ 2 ⁇ integrin, one of the major type I collagen binding receptors in mammalian cells.
- Validating the ability of the in vitro model to predict patterns of antigen expression in the disease setting, immunohistochemical analyses of breast cancer patient tissues verified markedly increased expression of the a 2 subunit in vivo.
- the disclosure provides a method of eliciting an antibody specifically binding a target comprising (a) administering an effective amount of a three-dimensional hydrogel comprising a specific cell type that expresses a biomolecular target molecule; and (b) obtaining an antibody that specifically binds to the target molecule.
- the hydrogel comprises type I collagen, fibrin, or a mixture thereof.
- An exemplary hydrogel comprises type I collagen. Contemplated in most embodiments is the method wherein the type I collagen, fibrin, or a mixture thereof is cross-linked, analogous to the cross-linked state of these molecules in vivo.
- the disclosure provides methods wherein the biomolecular target molecule is a cell-surface protein, e.g.
- the diseased cell is a cancer cell, a fibrotic cell, (e.g. , fibroblasts, pericytes, mesenchymal stem cells, fibrocytes), an inflammatory cell (e.g. , a circulating leukocyte belonging to the neutrophil, eosinophil, mast cell, monocyte/macrophage, or B/T-lymphocyte family), an immune cell (e.g.
- a fibrotic cell e.g. , fibroblasts, pericytes, mesenchymal stem cells, fibrocytes
- an inflammatory cell e.g. a circulating leukocyte belonging to the neutrophil, eosinophil, mast cell, monocyte/macrophage, or B/T-lymphocyte family
- an immune cell e.g.
- a neutrophil a macrophage, a cytotoxic natural killer (NK) cell, a granulocyte, a dendritic cell, a cell from any of various T cell subsets, a B cell) or a cell participating in pathologic angiogenesis, such as an endothelial cell as well as peri- endothelial cell populations (e.g. , pericytes, smooth muscle cells or mesenchymal stem cells).
- exemplary embodiments include methods wherein the diseased cell is a cancer cell or a fibrotic cell.
- the biomolecular target molecule is a2 integrin, a- enolase, calnexin, CD44, filamin, vimentin, or fibrinogen.
- the disclosure provides methods further comprising a subtractive immunization procedure comprising (a) administering an effective amount of a hydrogel comprising a healthy cell that is a counterpart to, or of the same cell type as, the cell associated with a disease, disorder or condition, to a host organism to elicit an antibody response; and (b) delivering an immunosuppressive agent to the host organism.
- the immunosuppressive agent is cyclophosphamide.
- the disclosure provides a method of producing an immunogen comprising (a) obtaining a composition comprising a biomolecular target molecule; (b) combining the composition comprising the biomolecular target molecule and a hydrogel-forming compound; and (c) preparing a three-dimensional hydrogel comprising the composition comprising the biomolecular target molecule.
- the hydrogel comprises type I collagen, fibrin, or a mixture thereof, and in some embodiments the type I collagen, fibrin, or a mixture thereof is cross-linked.
- the composition comprising a biomolecular target molecule is a living cell, such as a diseased cell in a subject such as a human or a non-human animal.
- the biomolecular target molecule is a cell-surface protein, such as methods wherein the cell-surface protein is on the surface of a diseased cell.
- exemplary diseased cells include a cancer cell, a fibrotic cell, a cell involved in pathologic angiogenesis such as an endothelial or peri-endothelial cell involved in pathologic angiogenesis, or a cell involved in a pro-inflammatory disease state such as a leukocyte or blood vessel-associated cell as exemplified by a monocyte (e.g., an Ml macrophage, a dendritic cell, a histiocyte, a Kupffer cell), a granulocyte (e.g., a neutrophil, an eosinophil, a basophil), a T cell, a B cell or a natural killer cell involved in a pro-inflammatory disease state.
- a monocyte e.g., an Ml macrophage, a dendritic cell, a histiocyte,
- the biomolecular target molecule is a2 integrin, a-enolase, calnexin, CD44, filamin, vimentin, or fibrinogen.
- the biomolecular target molecule is not known in advance of performing methods according to the disclosure, such as methods of eliciting an antibody or methods of producing an immunogen.
- compositions such as a cell, that comprises a biomolecular target molecule, such as a cell-surface marker
- a composition comprising a biomolecular target molecule in a three-dimensional hydrogel is a composition, such as a cell, that presents a collection of immunogenic molecules that more closely tracks the molecules presented by that cell type in vivo.
- the disclosure provides a method of identifying an anti-cancer antibody product as functional in vivo comprising (a) contacting a protein capable of cross- linking to form a hydrogel with a cancer cell to produce a seeded hydrogel or hydrogel comprising a cancer cell; (b) incubating the seeded hydrogel or hydrogel comprising a cancer cell; and (c) exposing the seeded hydrogel or hydrogel comprising a cancer cell to an anti-cancer antibody product candidate under conditions suitable for antigen- antibody product binding, wherein binding between the anti-cancer antibody product candidate and the seeded hydrogel or hydrogel comprising a cancer cell identifies the anti-cancer antibody product candidate as an anti-cancer antibody product.
- the cross-linked protein is a cross-linked matrix protein, such as collagen, e.g., type I collagen, fibrin, elastin, or a mixture thereof.
- the extracellular matrix protein contains endogenous aldimine groups to produce the cross-linked protein and/or the protein is modified to generate an aldimine derivative of the protein, thereby allowing the aldimine derivative of the protein to produce the cross-linked protein.
- Exemplary embodiments are contemplated wherein lysyl oxidase or a transglutaminase catalyzes the modification of the protein to produce the aldimine or iso-peptide derivative of the protein.
- the hydrogel further comprises an a2 integrin holoprotein, such as the ⁇ 2 ⁇ ⁇ integrin.
- the hydrogel further comprises the a2 subunit of a2 ⁇ integrin.
- the antibody product is a polyclonal antibody, a monoclonal antibody, an antibody fragment, a hybrid antibody, a chimeric antibody, a CDR-grafted antibody, a single chain antibody, a single chain variable fragment antibody, a Fab antibody fragment, a Fab' antibody fragment, a F(ab')2 antibody fragment, a linear antibody, a bi-body, a tri-body, a tetrabody, a diabody, a peptibody, a bispecific antibody, a bispecific T-cell engaging (BiTE) antibody, or a chimeric antibody receptor.
- the antibody product is a humanized or human antibody product. It will be understood by one of ordinary skill in the art that an antibody product as defined herein also defines an antibody product candidate.
- Another aspect according to the disclosure provides an antibody product produced by the method described above, wherein the antibody product is derived from an anti-a2 integrin antibody.
- the antibody product is derived from an anti-a2 integrin antibody.
- Three monoclonal antibodies specifically binding a2 integrin have been obtained, as exemplified by the 4C3 monoclonal antibody.
- the antibody product is the 4C3 monoclonal antibody.
- a hydrogel comprising (a) a cross-linked protein; and (b) a biomolecular target molecule such as an integrin protein.
- a biomolecular target molecule such as an integrin protein.
- the cross- linked protein is a matrix protein, such as a collagen, e.g. , a type I collagen, type III collagen, type IV collagen, fibrin, elastin, hyaluronic acid, laminin, or a mixture thereof.
- the integrin protein is ⁇ 2 ⁇ integrin and/or the a2 subunit of a2 ⁇ integrin.
- the disclosure provides a hydrogel comprising a cross-linked protein and a biomolecular target, as exemplified by an integrin protein or any protein associated with a disease, disorder or condition of interest.
- a hydrogel comprising a cross-linked protein and a cell comprising a biomolecular target, e.g. , presented on the cell surface, wherein the cell exhibits a disease, disorder or condition.
- Exemplary cells exhibiting a disease, disorder or condition include a cancer cell, a fibrotic cell, an inflammatory cell, an immune cell, and cells associated with pathologic angiogenesis, such as an endothelial cell, a pericyte, a smooth muscle cell or a mesenchymal stem cell.
- FIG. 1 Schematic illustrating MDA-MB-231 breast carcinoma cells, embedded in three-dimensional type I collagen hydrogels (1), used to immunize recipient mice (2).
- Hybridoma cultures were generated (3) and mAbs tested for their ability to inhibt proliferative responses of MDA-MB-231 cells in three-dimensional culture (4).
- the abilities of selected mAbs to inhibit MDA-MB-231 proliferative responses were determined in xenograft models in vivo (5) and the antibody targets identified by immunoaffinity isolation and mass-spectroscopy (6).
- Figure 2 Overview of the subtractive immunization procedure.
- FIG. 3 MDA-MB-231 bone metastasis model.
- FIG. 5 a2 integrin expression in breast carcinoma bone metastases and primary tissues. Biopsies of breast carcinoma bone metastasis were immunostained for a2 integrin expression in a series of 7 patient samples (three shown here with remaining biopsies presented in Fig. 9). Asterisks mark bone tissue.
- FIG. 7 In vitro activity of monoclonal antibody mAb 4C3.
- A MDA-MB-231 cells were seeded in three-dimensional collagen matrices in the absence or presence of mAb 4C3 (10 ⁇ g/ml). Cultures were evaluated by phase contrast or confocal microscopy (red) at day 0 and day 4.
- FIG. 8 MDA-MB-231 cells (lxl0 5 /well) were cultured in 24-well tissue culture plates under two-dimensional conditions in DMEM/10%FCS with or without mAb 4C3 (10 ⁇ g/ml) without affecting cell shape at day 3 (A) or proliferation (B). Results are expressed as the mean + SEM of 3 experiments.
- C MDA-MB-231 cells (lxlO 5 ) were embedded in Matrigel in the presence of a control IgGl or mAb 4C3 (10 ⁇ g/ml each) for 3 days or 4 days without affecting cell shape or cell number. Results are representative of 3 or more experiments.
- FIG. 1 Human squamous cell carcinoma (74B; 2xl0 5 ), ovarian cell carcinoma (ES2; 5xl0 5 ) or fibrosarcoma (HT1080; 2xl0 5 ) cell lines were cultured in three-dimensional type I collagen hydrogels for 2 days in the presence of a control IgGl (10 ⁇ g/ml) or mAb 4C3 (10 ⁇ g/ml). Phase-contrast micrographs highlight the ability of mAb 4C3 to block cell shape changes. Results are representative of 3 or more experiments.
- (B) Cell proliferation in three- dimensional collagen was inhibited as a function of mAb 4C3 concentration as assessed by cellular ATP levels with IC 50 values reported as the mean + SEM (n 3).
- FIG. 10 Anti-carcinoma activity of mAb 4C3 in the chick xenograft model.
- A Vasculature of the chick chorioallantoic membrane (CAM) as visualized following GFP-isolectin B4 (green) infusion by confocal laser microscopy.
- B Perivascular interstitial collagen (blue) in the 11-day-old chick CAM as assessed by second harmonic generation.
- C,D RFP-labeled MDA-MB-231 cells and either mAb 4C3 (0.8 mg/embryo), a vehicle control or control IgGl (0.8 mg/embryo) were introduced intravenously into the chick embryos.
- FIG. 11 Overview of the embryonic chick xenograft model. Shown is a diagram and light microscopic images of the chick embryo and vasculature. Bottom panels show blood vessels (green) and surrounding type I collagen fibrils (blue) as visualized by second harmonic generation microscopy (see also Fig. 10).
- FIG. 12 Peptide mapping of the mAb 8F10 binding sites in an overlapping series of peptides (each 10 amino acids in length) that span the a2 integrin subunit. Asterisks indicate decapeptide epitopes localized within the a-I domain.
- Figure 13 a 2 integrin staining of four additional biopsy specimens of human breast cancer patients with bony metastases. Asterisks indicate bone, and arrows indicate metastatic breast carcinoma cells.
- FIG. 14 Breast tissue biopsy specimens harvested from primary sites highlighting strong a 2 expression in breast carcinoma tissues (black arrows) with additional, but weaker, staining outlining normal myoepithelial cells. In Case 1, the tumor embolus as well as the lymphatic endothelium are positive for a 2 expression.
- CDR Complementarity Determining Regions
- FIG. Immunohistochemistry with mAb 4C3.
- a human breast tumor tissue array was stained with mAb 4C3 (brown) and nuclei counterstained with hematoxylin (blue).
- biomolecular target molecules are frequently found in association with cells in the in vivo environment. Such associations may affect the spatial presentation of such targets, such as cell-surface protein markers, lipoproteins, nucleoproteins, glycoproteins or, indeed, any biomolecule capable of serving as a target.
- a major approach to the identification or recognition of a particular biomolecular target is an immunological approach in which an antibody that specifically binds to a target is elicited and subsequently used in medically relevant procedures such as diagnosis, prophylaxis, therapy or amelioration of a symptom of a disease, disorder or condition.
- the technology disclosed herein takes an unusual approach to antibody elicitation in not seeking to purify a target molecule so as to maximize the likelihood of identifying a target- specific antibody in a conventional antibody screen; rather, the technology retains the target molecule in its complex, natural, in vivo-like, three-dimensional cellular environment to maximize the likelihood that a target- specific antibody, when identified, will also specifically recognize the target in vivo.
- the repertoire of expressed genes and gene products are completely different in standard 2-dimensional culture conditions compared to the three- dimensional microenvironments disclosed herein.
- the cells present on their surface numerous proteins, and the composition of the cell-surface proteins depends on the extracellular
- a method of tolerizing antibody-generating organisms to reduce the presence of undesired antibodies recognizing a cellular antigen that is found on normal cells, and therefore not of interest.
- the likelihood of identifying an antibody recognizing a cancer-specific target molecule is enhanced by first exposing the antibody-generating host organism to a healthy cell of the same type as the cancer cell, and then eliminating antibody-producing cells responding to the healthy cells prior to challenge with the cancer cell.
- This optional tolerization step reduces the complexity in identifying a target- specific antibody while retaining the advantage of using a form of the antigen of interest that mimics its form in vivo, thereby enhancing the opportunity to identify and develop medically useful antibody products.
- human carcinoma cell lines or primary human carcinoma stem cells have been established in three-dimensional extracellular matrices that have been constructed from type I collagen, the most abundant ECM molecule found in humans, or fibrin, the blood clotting protein found surrounding cancer cells at all neoplastic sites (16, 96)] and used as immunogens to generate panels of monoclonal antibodies (Fig. 1).
- the collagen or fibrin hydrogels are constructed from mouse proteins, and the generated panels of monoclonal antibodies are then screened for those that recognize the intact tumor cells by ELISA. Positive clones are then expanded and further screened for functional activity as defined by their ability to inhibit cancer cell invasion or growth in three-dimensional microenvironments. Monoclonal antibodies demonstrating anti-cancer activity represent potential therapeutic agents in their own right and can be used (following immunopurification and mass-spectroscopy) to identify molecular targets of demonstrable utility. To further enrich for tumor- specific antigens, an immunological technique known as subtractive immunization was also used, as described in Example 7 and illustrated in Fig. 2.
- mice are immunized with the normal or healthy counterpart of the human carcinoma cells (e.g., in the case of breast cancer, animals are primed with normal or healthy human mammary epithelial cells) and then treated with the immunosuppressive agent, cyclophosphamide.
- the immunosuppressive agent cyclophosphamide.
- mice prevented from maintaining an immune response against antigens found on the normal human epithelial cells, termed 'tolerized' mice, are then challenged by injection of human carcinoma cells.
- This experimental protocol results in an enhanced immune response directed toward antigens found specifically on the tumor cells.
- the standard immunization protocol using three-dimensional collagen-cancer cell composites i.e., three-dimensional immunogens
- a breast cancer using the MDA-MB-231 cell line (17) as well as the stem cell- enriched, breast carcinoma line, SUM159 (16)], primary human glioblastoma stem cells, pancreatic carcinoma cells, melanoma cells and ovarian carcinoma cells.
- SUM159 (16) the stem cell- enriched, breast carcinoma line
- primary human glioblastoma stem cells pancreatic carcinoma cells
- melanoma cells ovarian carcinoma cells.
- approximately 300 monoclonal antibody-generating hybridoma lines that recognize one or more of these cancer cells have been generated.
- the technology disclosed herein provides the materials and methods for rapidly and reproducibly generating specific binding partners to any of a wide variety of molecular targets, such as cell-surface markers found on cells characterized by a disease, disorder or condition.
- exemplary diseases, disorders or conditions include a cancerous condition, a fibrotic condition, a hypervascularized condition or a pro-inflammatory condition.
- the technology maximizes the efficiency and efficacy of eliciting specific binding partners (e.g. , antibody products) to target molecules of interest by mimicking the in vivo environment of a host organism, such as a human subject or patient, having the disease, disorder or condition.
- the technology provides cells characterized by the disease, disorder or condition in the three-dimensional environment of a hydrogel also comprising type 1 collagen and/or fibrin, and/or elastin.
- the cell presents the target molecule of interest in the form of a cell- surface marker, such as a cancer marker, a marker of fibrotic disease, a marker of pathologic angiogenesis or a marker of pro-inflammatory disease.
- elicitation of the specific binding partner, such as an antibody product is preceded by exposing the host organism to a tolerization step involving administration of a healthy cell otherwise similar or identical to the diseased cell.
- Target molecules may be proteins or peptides, or nucleic acids such as RNA, DNA, or a non-naturally occurring nucleic acid, or lipids, or any other biomolecule capable of contributing to an antigenic determinant specifically recognized by at least one vertebrate antibody.
- the target molecule may be a fused molecule, such as would be found in lipoproteins and nucleoproteins.
- the target molecule may also be derivatized, e.g. , a glycosylated protein or a phosphorylated protein.
- a suitable biomolecular target is bound or associated with the surface of at least one cell type.
- three-dimensional microenvironments may comprise, e.g., cells that, in turn, comprise one or more unknown biomolecular targets that are used in preparing the three-dimensional immunogens used to elicit target- specific antibodies functional in vivo.
- the technology embraces in vitro, three-dimensional microenvironments comprising cells that, in turn, comprise one or more known biomolecular targets, such as a-fetoprotein (AFP) , CA15-3, CA27-29, CA19-9, CA-125, Calcitonin, Calretinin, Carcinoembryonic antigen, CD34, CD99MIC 2, CD 117, Chromogranin, Cytokeratin (various types), Desmin, Epithelial membrane antigen (EMA), Factor VIII, CD31 FLl, Glial fibrillary acidic protein (GFAP), Gross cystic disease fluid protein (GCDFP-15), HMB-45, Human chorionic gonadotropin (hCG), inhibin, keratin (various types), MART-1 (Melan-A), Myo Dl, muscle-specific actin (MSA), neurofilament, neuron- specific enolase (NSE), placental alkaline phosphatase (PLAP), PTP
- AFP a
- Exemplary fibrotic cell markers include a2 macroglobulin, a2 globulin (or haptoglobin), ⁇ globulin, apolipoprotein Al, ⁇ glutamyltranspeptidase, and bilirubin.
- Exemplary fibrotic cell targets include Plasminogen activator inhibitor- 1 (PAI-1); Alpha-2- macroglobulin; Alpha-crystallin B chain; Decorin; Four and a half LIM domains (Fhl2); Major prion protein (CD230) (RaPrP); Alpha- 1, type ICollagen; Smooth muscle aortic alpha-actin; Beta-tropomyosin (TPM2); Collagen, type XII, alpha- 1 (Coll2al); Secreted phosphoprotein 1 (Sppl); Lectin, galactose binding, soluble 1 (Lgalsl); Phosphoprotein enriched in astrocytes 15 (Peal5); Transgelin (Tagln); Lipoprotein lipase (Lpl); Matrix Gla protein (Mgp); Troponin T2, cardiac (Tnnt2); Glypican 3 (GPC3); Glutathione peroxidase 3 (Gpx3); Similar to Loxl protein (Lo
- target molecules are suitable for use in methods according to the disclosure, but it is not necessary to identify a target molecule in advance of efforts to elicit a specifically binding antibody.
- an immunogen could be additional cells (e.g. , cancer-associated fibroblasts, monocytes, T cells (e.g. , CTLs, Tregs)); additional factors (e.g. , cytokines, growth factors); additional manipulations
- transfection or gene targeting in the cells included in the immunogen e.g. , cells with mutated K- Ras following subtractive immunization with wild-type Ras cells
- altered conditions e.g. , hypoxia or altered media conditions
- the disclosure provides for a three-dimensional microenvironment comprising, typically, a cell representative of cells useful in diagnosing a disease, disorder or condition, a cell useful in preventing or treating a disease, disorder or condition, or a cell providing a target useful in obtaining a target- specific binding partner such as an antibody product according to the disclosure.
- methods according to the disclosure use cells comprising a target biomolecule that may either be known in the art or unknown in the art.
- the disclosed methods are useful in selecting specific binding partners that recognize and bind to the form that a target assumes in vivo, but the methods are also useful in providing methods for obtaining target- specific antibody products that specifically bind to target biomolecules and exert a biologic effect (e.g. , function-blocking, function-enhancing or capable of triggering an immune response) that were never identified as having any association to a particular disease, disorder or condition.
- a biologic effect e.g. , function-blocking, function-enhancing or capable of triggering an immune response
- cells embraced by the disclosure include any cell type that causes or manifests a disease, disorder or condition that one would like to prevent, diagnose or treat, or for which symptom amelioration is desired.
- a wide variety of healthy cell types can change to give rise to or exhibit a disease, disorder or condition, and the disclosed technology embraces such cells.
- Exemplary cells include any cell type capable of existing in a cancerous state or giving rise to a cancer, such as Adrenal Cancer, Anal Cancer, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain/CNS Tumors In Adults, Brain/CNS Tumors In Children, Breast Cancer, Breast Cancer In Men, Cancer in Adolescents, Cancer in Children, Cancer in Young Adults, Cancer of Unknown Primary, Castleman Disease, Cervical Cancer, Colon/Rectum Cancer, Endometrial Cancer, Esophagus Cancer, Ewing Family Of Tumors, Eye Cancer, Gallbladder Cancer, Gastrointestinal Carcinoid Tumors, Gastrointestinal Stromal Tumor (GIST), Gestational Trophoblastic Disease, Hodgkin Disease, Kaposi Sarcoma, Kidney Cancer, Laryngeal and Hypopharyngeal Cancer, Leukemia, Leukemia - Acute
- ALL Lymphocytic
- AML Leukemia - Acute Myeloid
- CLL Lymphocytic
- CML Leukemia - Chronic Myeloid
- CMML Myelomonocytic
- Lung Cancer Leukemia in Children, Liver Cancer, Lung Cancer, Lung Cancer - Non-Small Cell, Lung Cancer - Small Cell, Lung Carcinoid Tumor, Lymphoma, Lymphoma of the Skin, Malignant Mesothelioma, Multiple Myeloma, Myelodysplastic Syndrome, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Non-Hodgkin Lymphoma In Children, Oral Cavity and Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer, Pancreatic Cancer, Penile Cancer, Pituitary Tumors, Prostate Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoma - Adult Soft Tissue Cancer, Skin Cancer, Skin Cancer - Basal and Squamous Cell, Skin Cancer
- Additional exemplary cells include any cell types capable of giving rise to or participating in Pulmonary fibrosis, Idiopathic pulmonary fibrosis, Cystic fibrosis, fibrosis of the liver, Cirrhosis of the liver, Endomyocardial fibrosis, Old myocardial infarction, Atrial Fibrosis, Mediastinal fibrosis, Myelofibrosis, Retroperitoneal fibrosis, Progressive massive fibrosis (lungs), Nephrogenic systemic fibrosis (skin), Crohn's Disease, Keloid (skin),
- Scleroderma/systemic sclerosis (skin, lungs), Arthrofibrosis (knee, shoulder, other joints), Peyronie's disease (penis), Dupuytren's contracture (hands, fingers), or some forms of adhesive capsulitis (shoulder).
- Other exemplary cells include cells involved in pathologic angiogenesis, such as endothelial cells, pericytes, smooth muscle cells or mesenchymal cells, as well cells involved in pro-inflammatory disease states such as any of the various leukocyte cell populations (e.g. , hematopoietic stem cells, myeloid leukocytes such as monocytes, macrophages and granulocytes (e.g. , neutrophils, eosinophils, and basophils), and lymphocytes such as T cells, B cells and natural killer cells).
- leukocyte cell populations e.g. , hematopoietic stem cells, myeloid leukocytes such as monocytes, macrophages
- Another group of exemplary cells include cell types involved in inflammatory processes associated with a disease, disorder or condition, including but not limited to, cell types giving rise to or participating in Alzheimer's disease, ankylosing spondylitis, appendicitis, arthritis (including osteoarthritis, rheumatoid arthritis (RA), and psoriatic arthritis), autoimmune diseases (including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE)), asthma, atherosclerosis, bursitis, cancer (e.g., gallbladder carcinoma), colitis, complex regional pain syndrome, Crohn's disease, cystitis, dermatitis, diverticulitis, fibromyalgia, hay fever, hepatitis, inflammatory myopathies, irritable bowel syndrome (IBS), nephritis, Parkinson's disease, periodontitis, phlebitis, reflex sympathetic dystrophy, reflex neurovascular dystrophy, rhinit
- exemplary cells can be incorporated into the three-dimensional immunogens alone or in combination (e.g., cancer cells with endothelial cells or mesenchymal stem cells) disclosed herein and which, in a diseased state in vivo, can be the focus of the immunologically based diagnosis, prevention, treatment or symptom amelioration methods according to the disclosure.
- One further category of exemplary cells are the cells of the vasculature, such as endothelial cells, that are involved in pathologic angiogenesis.
- microenvironment providing context for the (typically) cell-based target molecules used as antigens and as screening tools to identify specifically binding antibodies and antibody products.
- the in vitro, three-dimensional microenvironment mimics the in vivo environment of the target-containing entity (e.g., a cell presenting the target on its surface, a macromolecular complex comprising the target) in at least one important aspect.
- the microenvironment contains an ECM protein with which the target-containing entity is associated in vivo, such as a type I collagen matrix or a fibrin matrix. These matrices may have a single extracellular protein or a mixture of such proteins.
- Additional compositions that may be found in a microenvironment include any compound found associated with the ECM in vivo, such as type III or type IV collagen, elastin, hyaluronic acid and/or laminin.
- the target molecules used to elicit specifically binding antibodies are provided to the antibody-generating organism in a three-dimensional microenvironment that mimics the three-dimensional environment in which the target molecules are found in vivo.
- two levels of mimicry are used to maximize the resemblance of the target molecule used as immunogen to the target molecule found in vivo.
- the first level of mimicry typically involves locating the specific target in its normal in vivo cellular
- a second level of mimicry involves the typical placement of the cell within an ECM-like microenvironment that mimics the in vivo
- the disclosed technology maximizes the likelihood that any specific binding partner elicited in an antibody-generating organism will also recognize the target in its in vivo environment. This significantly increases the likelihood of eliciting, and if desired, constructing a specific binding partner of medical value in diagnosis, prophylaxis, treatment or amelioration of a symptom of a disease, disorder or condition.
- the technology does not limit the type (isotype or sub-isotype) of an antibody elicited using an immunogen according to the disclosure, and the technology does not limit the ultimate form of antibody product that may be derived from such an antibody for use in any of the diagnostic, prophylactic, therapeutic, or symptom-amelioration methods disclosed herein.
- the technology embraces antibody products derived from antibodies elicited in any host organism known in the art, including any vertebrate species, such as man, any domesticated animal or any laboratory animal, e.g., mouse, rat, goat, sheep, cat, dog, horse, or cattle, and camelid antibodies.
- the disclosure contemplates antibody products derived from antibodies identified from libraries that are screened in three-dimensional ECM hydrogels in vitro, such as by using phage screening technologies.
- the antibody may be any type of immunoglobulin known in the art.
- the antibody product is derived from an antibody of isotype IgA, IgD, IgE, IgG, or IgM.
- the antibody product in some embodiments is a monoclonal antibody or is derived from a monoclonal antibody.
- the antibody product is a polyclonal antibody or is derived therefrom.
- the antibody product is derived from an antibody that is a naturally occurring antibody, e.g. , an antibody isolated and/or purified from a mammal, or produced by a hybridoma generated from a mammalian cell.
- the antibody product is a genetically engineered antibody, e.g. , a single-chain antibody, a humanized antibody, a chimeric antibody, a CDR-grafted antibody, a human engineered antibody, a bispecific antibody, a trispecific antibody, and the like. Genetic engineering techniques also provide the ability to make fully human antibodies in a non-human source.
- the antibody product is in polymeric, oligomeric, or multimeric form.
- the antibody product comprises two or more distinct antigen binding region fragments
- the antibody product is considered bispecific, trispecific, or multi- specific, or bivalent, trivalent, or multivalent, depending on the number of distinct epitopes that are recognized and bound by the antibody product.
- the antibody product is an antigen binding fragment of an antibody.
- the antigen binding fragment, or portion may be an antigen binding fragment of any of the antibodies or antibody products described herein, provided that the fragment retains the specific binding property of the whole antibody.
- the antigen binding fragment can be any part of an antibody that has at least one antigen binding site, including but not limited to, a Fab, a Fab', a F(ab') 2 , a dsFv, a sFv, a scFv, a diabody, a triabody, a tetrabody, a bispecific T-cell engager or BiTE, a bis-scFv, a fragment expressed by a Fab expression library, a domain antibody, VhH domains, V-NAR domains, a VH domain, a VL domain, and the like.
- kits comprising a compound suitable for use in preparing a hydrogel, such as type 1 collagen or fibrin, or both compounds, a pharmaceutically acceptable adjuvant, diluent or carrier, and a protocol for preparation and administration of an immunogen according to the disclosure.
- the disclosure provides a new approach to harnessing the power of the immune system to combat diseases, disorders and conditions in a general sense. Accordingly, a wide variety of diagnostic, prophylactic, therapeutic, and symptom-ameliorating methods are provided to administer the antibody products useful in detecting and/or modifying an activity of any of the wide range of target molecules immunologically detectable and suitable for incorporation into the immunogens according to the disclosure.
- An exemplary family of diseases amenable to diagnosis, prophylaxis, or therapy according to the disclosure is the group of cancer diseases.
- Cancers associated with any of the cancer cells identified in the section addressing cells are contemplated as suitable for diagnosis, prophylaxis, or treatment using antibody products elicited using three-dimensional immunogens comprising at least one such cancer cell.
- known cancer cell- surface markers are identified by antibody products ultimately elicited using the marker in a microenvironment mimicking its in vivo environment.
- Vaccines are also contemplated that comprise a hydrogel comprising a cell presenting a biomolecular target molecule of the disclosure.
- Such vaccines will elicit at least one antibody product that specifically binds to a biomolecular target molecule functionally involved in elaboration of a relevant disease process, triggering an immune response against the target molecule.
- Treatment methodologies are also provided wherein a target molecule functionally involved in disease progression is bound by an antibody product elicited according to the disclosure and wherein the bound target molecule is inhibited or prevented from providing the function relevant to disease progression.
- an antibody product specifically binds to a target molecule involved in the presentation of a symptom of a disease, disorder or condition and the specific binding of the antibody product inhibits or prevents the target molecule from providing the function involved in symptom presentation, thereby ameliorating a symptom of a disease, disorder or condition.
- exemplary diseases, disorders or conditions include fibrosis in any of its known forms, pathologic angiogenesis and pro-inflammatory disease states.
- the disclosure comprehends methods of diagnosis, methods of prevention or prophylaxis, methods of treatment, and methods of ameliorating at least one symptom of the disease, disorder or condition.
- type I collagen hydrogels that are naturally cross-linked by lysyl oxidase-derived aldimine bonds (16) to promote carcinoma cells to express a more in vzvo-like display of surface antigens were selected and these hydrogels could be used both as an immunogen for monoclonal antibody (mAb) production as well as a physical platform for functional screening.
- the antibody product can be any form of antibody known in the art, such as a full-length polyclonal antibody or a full-length monoclonal antibody.
- Antibody fragments according to the disclosure retain at least one specific binding characteristic of the parent whole antibody.
- An antibody according to the disclosure can be derived from any class, such as an immunoglobulin G or IgG antibody, and can be of any sub-class, such as an IgGl, IgG2, IgG3, or IgG4 antibody.
- the antibody can be a humanized or human antibody, a chimeric antibody, or a CDR-grafted antibody.
- an antibody fragment according to the disclosure comprises the antigen binding site of the parent antibody and includes, e.g., a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a single-chain antibody, a single-chain F v (i.e., scFv) molecule, a linear antibody, a diabody, a peptibody, a bi-body (bispecific Fab-scFv), a tribody (Fab-(scFv)2), a hinged or hingeless minibody, a mono- or bi-specific antibody, and antibody fusion proteins comprising the antigen binding site of the parent antibody.
- a Fab fragment, a Fab' fragment, a F(ab')2 fragment e.g., a single-chain antibody, a single-chain F v (i.e., scFv) molecule, a linear antibody, a diabody, a peptibody, a bi-body (
- the antibody or antibody fragment as described above may further comprise a second polypeptide covalently bound to the antibody or antibody fragment in a fusion polypeptide, for example an antibody or antibody fragment described above wherein the second polypeptide is a cytotoxic polypeptide.
- the antibody or antibody fragment may also be associated with a non-pro teinaceous cytotoxin.
- the antibody or antibody fragment is labeled.
- the antibody (or fragment) may also contain a sequence conferring additional properties, such as a cellular import function (e.g., Trans Activator of Transcription (TAT) or the HSV70 co-chaperone known as Coat Protein Interacting Protein (CPIP) fusion).
- TAT Trans Activator of Transcription
- CPIP Coat Protein Interacting Protein
- mAb 4C3 The monoclonal antibody designated mAb 4C3 was selected for additional analysis based on its inhibitory activity in our in vitro screen using target cells embedded in three-dimensional type I collagen hydrogels.
- antibody 4C3 did not exert any growth inhibitory effects in standard two- dimensional culture. Based on these results, antibody 4C3 was further characterized as a proof- of-principle prototype to determine whether i) function-blocking activity detected initially in vitro could be extended into in vivo settings, ii) the mAb-reactive antigen could be identified and iii) target antigens discovered using human carcinoma cell-type I collagen composites faithfully predict in vivo patterns of expression in patient samples. As described in Example 4 and shown in Figs.
- mAb 4C3 successfully inhibited the perivascular proliferation of extravasated MDA-MB-231 cells within the three-dimensional type I collagen -rich interstitial matrix of the live chick embryo, an in vivo model xenograft system wherein cancer cell behavior, including invasion, proliferation and metastasis recapitulate those observed in mouse xenograft models (22, 23). Further, the utility of mAb 4C3 to inhibit MDA-MB-231 proliferation in a mouse model was assessed wherein the cancer cells were allowed to metastasize to mouse skeletal tissues, a type I collagen-rich environment relevant to the bone metastatic activity displayed in human patients (17, 26-28).
- the mAb 4C3 target antigen was identified as the a 2 integrin subunit, whose only known partner, the ⁇ integrin chain, forms a heterodimeric complex that serves as a major type I collagen-binding receptor (29, 30) (Fig. 4).
- Peptide mapping characterized the mAb 4C3 epitope within the a-I domain of the a 2 integrin, a metal ion-dependent adhesion site that is responsible for ligand recognition and binding (29, 30) (Fig. 4).
- mice that were bred into a mouse mammary tumor virus-Neu transgenic line, they demonstrated that despite the complete absence of ⁇ 2 ⁇ , tumor initiation was only marginally affected while lung metastatic activity was actually enhanced (49).
- all tissues are rendered a 2 integrin-deficient throughout embryonic and postnatal development.
- the MMTV-New oncogene is expressed, by necessity, in a 2 integrin-null mammary epithelial cells where potential effects of the integrin on tumor transformation and progression are difficult to define (i.e., as opposed to deleting the a 2 integrin in committed carcinoma cells).
- carcinoma cell-type I collagen composites as an antigen for mAb production is not to simply identify collagen-binding ligands, but rather to generate mAbs that interfere with cancer cell behavior in an environment similar to that encountered in vivo. Indeed, these studies indicate that most of the function-blocking mAbs identified in screens performed to date do not target type I collagen receptors (see below), but rather surface molecules with as yet to be characterized mechanisms of action.
- the experimental approach outlined herein allows for the rapid identification of new target antigens in an unbiased fashion as well as the isolation of murine monoclonal antibodies suitable for humanization.
- a human breast carcinoma cell line has been used as a proof- of-concept model, the approach is similarly amenable to the use of primary carcinoma cells or cancer stem cells.
- primary human glioblastoma cancer stem cells have also been used to generate mAb libraries that have also been found to exert inhibitory effects with target identification in process (Table I).
- the phenotypic screening stratagem using either human cancer cell lines, primary cancer cells or even cancer cell-stromal cell composites (71), as well as more complex ECM-supplemented hydrogels to more accurately recapitulate the anticipated changes that occur in connective tissue composition during tumor progression (12, 72), will allow for the identification new targets and therapeutics in neoplastic as well as other disease states (e.g., fibrosis, acute/chronic inflammation, hypervascularization).
- the cell culture conditions may also be manipulated, for example, by application of hypoxic conditions.
- MDA-MB -231 cells (ATCC) were embedded in mouse type I collagen hydrogels (1, 21) and the cell-matrix composite inoculated into 6 week-old Balb/c female mice for
- MDA-MB -231 -reactive mAbs were isolated and screened for anti-proliferative activity in three-dimensional collagen constructs (1, 21).
- Type I collagen was isolated from mouse tail tendons as described (1, 21) and dissolved in 0.2% acetic acid at a final concentration of 2.7 mg/ml. Prior to gelation, the collagen solution was mixed with 10X MEM and 0.34 N NaOH at a ratio of 8: 1: 1 at 4°C with MDA-MB-231 cells (l-5xl0 6 ) suspended in 1ml of this mixture. The carcinoma cell-collagen mixtures were incubated for 1 hour at 37°C to allow for gelation and culture media (MEM supplemented with 10% FCS) added atop the gel.
- MEM MDA-MB-231 cells
- Collagen gel rigidity was assessed in a RFSII rheometer (Rheometrics) using dynamic shear mode, parallel plate geometry and a hydrated chamber as described (2). After a 4-day incubation period, the MDA-MB-231/collagen composites were washed extensively and recovered intact from 12-well plates or, alternatively, after the MDA-MB-231 cells were harvested from the gels by dissolving the collagen hydrogels with collagenase type 3 (Advance BioFactures Corp.). MDA-MB-231/collagen composites or isolated MDA-MB-231 cells were inoculated intraperitoneally into 6 week-old Balb/c female mice, followed by boosts at two-three week intervals for 3 months. Spleens were then removed and somatic cell hybridization performed with P3X63-Ag8.653 mouse myeloma cells as the fusion partner (3).
- MDA-MB-231 cells (lxlO 5 ) were added to 96- well V-bottom PVC plates (Corning) and cell pellets incubated for 1 hour at 4°C with 50 ⁇ of media supernatant from individual hybridoma cultures. After washing, MDA-MB-231 cells were then re-suspended in PBS with a horseradish peroxidase (HRP)-conjugated secondary antibody directed against mouse immunoglobulins (Pierce) for 1 hour at 4°C. Cells were then washed three times with PBS and HRP activity detected with a TMB substrate (Thermo Scientific).
- HRP horseradish peroxidase
- Hybridomas giving rise to anti-MDA-MB -231 -reactive mAbs were sub-cloned by limiting dilution and re-assayed for activity to ensure the isolation of monoclonal populations. Positive hybridomas were then used to generate ascites fluid by injection into mouse peritoneal cavities. The resulting ascites fluid was cleared of debris by centrifugation and antibodies purified by either Melon Gel Purification Resin (Thermo Scientific) or Protein G Resin (Thermo Scientific). Monoclonal antibody (mAb) isotype was determined by Rapid ELISA mouse mAb Isotyping Kit (Pierce).
- a control IgGl mAb (3H5) was raised against dengue virus antigen (4). Following intraperitoneal injection, ascites fluid generated from the hybridoma cell line (ATCC) was purified by Protein G affinity chromatography. Both the control mAb 3H5 and the mAb 4C3 preparations were endotoxin-depleted by DeToxi-Gel column chromatography (Pierce) prior to use.
- MDA-MB-231 cells were embedded in type I collagen (10 5 cells in a final type I collagen concentration of 2.2 mg/ml) or Matrigel (5 mg/ml) in the absence or presence of mAb 4C3 at the indicated concentrations and plated in 24- well plates in MEM/10% FCS.
- mAb 4C3 to affect proliferative responses of human squamous cell carcinoma (74B), ovarian carcinoma (ES2) or fibrosarcoma (HT1080) cells (all obtained from ATCC) was assessed.
- Cell number was quantified by hemocytometry or using a Cell-Titer Glo kit (Promega).
- Caspases 3 and 7 activities were evaluated with a Caspase-Glo 3/7 kit (Promega).
- Total mRNA was collected and purified using RNeasy Mini Kits (QIAGEN) (5). Sample quality was confirmed using a Bioanalyzer 2100 and all samples profiled on Affymetrix Mouse MG-430 PM expression array strips. Expression values for each probe set were calculated using the robust multi-array average (RMA) system (5) and filtered for genes with a fold change greater than 2-fold. Heatmaps of selected gene lists were generated using Gene Cluster 3.0 and Tree View 1.6 (5). Gene ontology analysis was performed using MetaCore from Thomson Reuters (version 6.11, build 41105).
- RFP-transduced MDA-MB-231 were injected with a control IgG or 4C3 into the allantoic vein of 11 -day-old, immune-incompetent chick embryos (6, 22). After a 6-day incubation period, vessel lumens were visualized by injecting chicks with GFP-labeled isolectin- B4. Confocal imaging of second harmonic-generated signals was used to analyze collagen fiber microstructure as described (7, 24). After an additional 1-hour incubation time, embryos were harvested, whole-mount tissue preparations taken distally from the injection site, and carcinoma cells identified by florescent microscopy.
- MDA-MB-231 cells expressing firefly luciferase were injected in an identical fashion with control mAb 3H5 or mAb 4C3 in tandem with the carcinoma cells or 24 hours after the carcinoma cell innoculation.
- eggs were injected i.v. with 100 ⁇ luciferin (40 mg/ml in PBS) 10 minutes prior to removal of the lower chioroallantoic membrane.
- Membranes were washed with PBS and imaged for bioluminescence with a Xenogen IVIS 200.
- Luciferase-labeled MDA-MB-231 cells (lxlO 5 ) were injected via the intracardiac route with either 10 mg/kg of mAb 4C3 or a control IgGl twice-weekly for 4 weeks and tumor progression by whole-body bioluminescent imaging as described (8).
- cells were alternatively injected orthotopically in the 4th mammary gland with or without mAb 4C3.
- MicroCT analysis of bone lesions were imaged at 18- ⁇ isotropic voxel resolution using Explore Locus SP (GE Healthcare Pre-Clinical Imaging) and calibrated three-dimensional images reconstructed (7, 24).
- MDA-MB-231 cells are a well-characterized, triple-negative breast carcinoma cell line whose gene expression profile closely recapitulates that found in human breast cancer tissues (17-19). Further, in a manner similar to human carcinomas expanding in vivo, the cell line undergoes rapid proliferative and tissue-invasive responses when cultured within three- dimensional type I collagen hydrogels in vitro (16, 20, 21). As such, MDA-MB-231 cells were embedded in covalently cross-linked networks of mouse type I collagen with an elastic modulus similar to that found in normal breast tissue [about 150 Pa (11)]. After a 3-day culture period, the human carcinoma cell-mouse matrix composite was then recovered and used as an immunogen to generate a panel of approximately 2500 mAbs (Fig. 1).
- MDA-MB- 231-reactive clones whole cell-based ELISAs were then performed with about 350 of the mAbs scoring positive in initial screens. Each of the reactive clones was then expanded and the individual mAbs tested for their ability to inhibit the proliferative responses of MDA-MB-231 cells in three-dimensional culture (Fig. 1).
- MDA-MB-231 cells When embedded in three-dimensional type I collagen hydrogels, MDA-MB-231 cells rapidly alter their morphology from a spherical to bipolar, mesenchymal cell-like phenotype over the first 48 hours prior to the initiation of proliferative responses (Fig. 7A,B).
- clone 4C3 was one of the more potent IgGl-class antibodies identified, displaying an ability to almost completely block MDA-MB-231 cell shape changes and proliferation in three-dimensional collagen (Fig. 7A,B).
- inhibitory activity was not limited to a "preventative" protocol wherein mAb 4C3 was added at the start of the three-dimensional culture period; addition of the inhibitory mAb 4 days after the initiation of the culture period similarly inhibits carcinoma cell proliferation with an IC 50 of approximately 0.5 ⁇ g/ml (Fig. 7B,C). Furthermore, 4C3 not only blocks MDA-MB-231 proliferative responses, but also initiates apoptosis in three-dimensional culture as assessed by caspase 3 and 7 activation (Fig. 7D).
- mAb 4C3 exerts no inhibitory effects on MDA- MB-231 cell function (Fig. 8).
- the anti-proliferative activity of mAb 4C3 is not confined to MDA-MB-231 carcinoma cells as similar inhibitory effects are observed with human squamous cell carcinoma, ovarian carcinoma and fibrosarcoma cell lines in three-dimensional culture (Fig. 9).
- Monoclonal antibody 4C3 exerts global effects on the MDA-MB-231 transcriptome
- MDA- MB-231 cells were next cultured in three-dimensional type I collagen hydrogels in the presence of either a control IgGl or mAb 4C3 for 48 hours ⁇ i.e., prior to the initiation of proliferative responses) and RNA harvested for gene expression profiling. Under these conditions, mAb 4C3 exerted global effects on gene expression with almost 1200 unique transcripts affected ⁇ i.e., Ill up-regulated and 1004 down-regulated transcripts, respectively, using a 2.0-fold cutoff).
- mAb 4C3 Consistent with its effect on MDA-MB-231 proliferation, GO analysis revealed that mAb 4C3 treatment elicits major alterations in cell cycle, regulation, RNA processing and cell division- related programs (Fig. 7E). Taken together, these results identify mAb 4C3 as a potent regulator of MDA-MB-231 cell function within the confines of a type I collagen-rich ECM.
- Monoclonal antibody 4C3 Prevents Post-Extravasation Carcinoma Growth In Vivo
- extravasated MDA-MB-231 cells initiate proliferative activity in close association with the chick vasculature (Fig. IOC).
- Fig. IOC chick vasculature
- blood vessels colored green
- Fig. 11 blood vessels within chick tissues are surrounded by a dense layer of type I collagen, visualized by second harmonic generation microscopy in Fig. 11.
- mAb 4C3 MDA-MB-231 cell proliferation is inhibited markedly wherein tumor colony formation is readily monitored by both visual inspection and
- mAb 4C3 exerts potent inhibitory effects equivalent to those obtained when the antibody is introduced at the start of the in vivo assay (Fig. 10C,D).
- mouse mammary gland contains only small amounts of type I collagen that is largely confined to periductal regions alone, thus rendering mouse xenograft orthotopic models less useful for analyzing carcinoma cell-type I collagen matrix interactions (25).
- the organic matrix of mouse bone - like that of humans - is largely comprised of type I collagen (17, 26-28). Further, bone is a frequent site of breast cancer metastatic activity in human disease (17).
- ⁇ 2 integrin subunit only forms heterodimeric complexes with the ⁇ integrin to generate the dominant mammalian type I collagen receptor
- ⁇ 2 ⁇ peptide mapping of mAb 4C3 interactions with the a 2 subunit identified a major epitope that lies within the a-I domain of the integrin, the dominant type I collagen recognition site of the ⁇ 2 ⁇ heterodimer (29, 30) (Fig. 4C).
- mAb 4C3 As normal cell trafficking is minimal in adult tissues (except for myeloid cells that do not express ⁇ 2 ⁇ 1), and as all cancer cells must traffic through - and grow within - type I collagen-rich tissues (2), mAb 4C3 is expected to possess qualities that allow it to serve as a broad-acting cancer therapeutic.
- human patients that carry mutations in the a2 integrin that prevents its normal expression are only mildly affected with marginal increases in bleeding tendencies due to the fact that platelets express low levels of the a2 integrin (58).
- mAb 4C3 In addition to mAb 4C3, a second, inhibitory a 2 integrin-reactive mAb that was identified independently in our screen (mAb 8F10) also bound to a distinct, but overlapping, epitope located within the a-I domain (Fig. 12). As expected from its collagen-binding properties, mAb 4C3 inhibits MDA-MB-231 adhesive interactions with type I collagen (Fig. 7C).
- the subtractive immunization procedure involves immunizing mice with the normal cellular counterpart of the human carcinoma (e.g., in the case of breast cancer, animals are primed with normal human mammary epithelial cells) and then treated with the human carcinoma (e.g., in the case of breast cancer, animals are primed with normal human mammary epithelial cells) and then treated with the human carcinoma (e.g., in the case of breast cancer, animals are primed with normal human mammary epithelial cells) and then treated with the
- mice are then prevented from maintaining an immune response against antigens found on the normal human epithelial cells, a process resulting in tolerized mice.
- the tolerized mice are then challenged by injection of human carcinoma cells. This experimental protocol results in an enhanced immune response directed toward antigens found specifically on the tumor cells.
- control counterparts include a healthy, or normal, counterpart in the form of a healthy cell of the same type as a diseased cell, or the extracellular microenvironment from a healthy organism that corresponds to the extracellular microenvironment containing a target for a disease, disorder or condition of interest.
- mAb 4C3 markedly inhibits the ability of MDA-MB-231 cells to maintain proliferative activity within the surrounding ECM (Fig. 10). Similar, if not identical results, are obtained when mAb 4C3 treatment is delayed for 24 hours after cancer cell inoculation to allow extravasation to proceed to completion. Thus, mAb 4C3 exerts potent antiproliferative activity in vivo.
- mice were treated with twice- weekly dosages of 10 mg/kg mAb 4C3 for 4 weeks and tumor progression monitored by luminescent imaging (Fig. 3A).
- Treatment with mAb 4C3 inhibited hindlimb and mandible tumor progression with significant effects on the tumor growth localized to the spinal region (Fig. 3B).
- Fig. 3C spinal nerve damage secondary to vertebral collapse
- Fig. 3D less than 20% of the 4C3-treated mice displayed paralysis during these experiments.
- a technology platform has been validated that allows for the rapid identification of anti-human cancer-neutralizing monoclonal antibodies.
- Isolated murine antibodies can be used as templates for the generation of humanized monoclonals for therapeutic intervention while identified target antigen may be leveraged to direct the synthesis of small- molecule inhibitors.
- the disclosed technology is not only amenable to the use of well-characterized cancer cell lines, but also primary cancer cells, as well as cancer stem cells.
- mAb 4C3 has substantial activity in vivo, indicating its capacity as a therapeutic entity in its own right.
- Table 1 Additional data is presented in Table 1, with those immunogens delivered using subtractive immunization indicated by including "Subtrn" in the immunogen name.
- Column 1 of Table 1 provides the name of the elicited monoclonal antibody
- column 2 identifies the isotype of that antibody (heavy and light chains)
- column 3 discloses the cancer cell-derived immunogen that elicited the antibody
- column 4 identifies the immunoprecipitate as a means of identifying the target of the antibody
- column 5 reveals whether a signal was obtained from a lysate of the relevant cancer cell line using the indicated monoclonal antibody.
- Apparent from Table 1 is the fact that the disclosed technology is capable of eliciting monoclonal antibodies that recognize a variety of targets on cancer cell lines.
- each of the identified antibodies blocked tumor cell proliferation in the live chick xenograft model to a degree comparable to that observed with mAb 4C3. Further, these antibodies were generated by using (or not using) a subtractive immunization protocol. Still further, Table 1 shows that growth-inhibitory monoclonal antibodies were elicited to a series of distinct targets on each of the cancer cell lines used in the three-dimensional hydrogel immunogen (e.g. , a2-integrin, a-enolase, calnexin, CD44, filamin, vimentin, and fibrinogen). Additionally, the data in Table 1 establish that a variety of antibody isotypes (e.g. , IgG, IgM) and sub-isotypes (e.g. , IgGl, IgG2) are amenable to elicitation using the technology disclosed herein.
- IgG, IgM antibody isotypes
- sub-isotypes e.g. , IgGl,
- the technology allows for the preparation of an immunogen in a three-dimensional environment that preserves or mimics the in vivo architecture, thereby maximizing the opportunity to obtain functionally useful (e.g., diagnostically, prophylactically and/or therapeutically useful, or useful in ameliorating a symptom of a disease, disorder or condition) antibodies to any number of immunogenic targets on a wide variety of cell types, including any type of cancer cell, fibrotic cell, or cell involved in either pathologic angiogenesis or inflammatory (pro-inflammatory) diseases, disorders or conditions.
- functionally useful e.g., diagnostically, prophylactically and/or therapeutically useful, or useful in ameliorating a symptom of a disease, disorder or condition
- markers for fibrotic disease include cell- surface markers associated with a variety of cells in addition to fibroblasts.
- pathologic angiogenesis diseases the disclosure comprehends a diseased endothelial cell, smooth muscle cell, pericyte or mesenchymal stem cell.
- leukocytes including any leukocyte type or sub-type.
- human cells comprising human cells are contemplated.
- Antibodies elicited to such immunogens are reasonably expected to exhibit a higher degree of specific binding to the target molecule in vivo because the antibodies were elicited using forms of the target more closely matching the three-dimensional in vivo form of the target than immunogens known in the art.
- the potentially increased complexity of an initial polyclonal antibody response is offset by the realization that once an antibody specifically binding the target of interest is obtained, there is an increased likelihood that such an antibody will bind to the target in vivo, providing the intended beneficial effect on target activity.
- the potentially increased complexity of an initial polyclonal antibody response can be reduced by incorporating the subtractive immunization procedure disclosed herein.
- the disclosed technology has wide applicability in harnessing the immune response to diagnose, prevent, treat or ameliorate the symptom of a wide variety of diseases, disorders or conditions afflicting man, domesticated animals such as livestock or pets, and wild animals.
- This wide applicability to diagnostics, prophylactics, including vaccine development, therapeutics and amelioration of disease symptoms is a result of the broad applicability of the disclosed technology to immunological approaches to disease, disorder or condition diagnosis, prevention or treatment.
- Cox TR et al. (2013) LOX-mediated collagen crosslinking is responsible for fibrosis- enhanced metastasis. Cancer Res 73: 1721-1732. 10. Salo S, et al. (2013) Human bone marrow mesenchymal stem cells induce collagen production and tongue cancer invasion. PLoS One 8:e77692.
- Bos PD et al. (2009) Genes that mediate breast cancer metastasis to the brain. Nature 459: 1005-1009.
- Type I collagen receptor (alpha 2 beta 1) signaling promotes the growth of human prostate cancer cells within the bone. Cancer Res 66:8648-8654.
- Bone marrow-derived myofibroblasts contribute to the mesenchymal stem cell niche and promote tumor growth. Cancer Cell 19:257-272.
- Type I collagen inhibits differentiation and promotes a stem cell like phenotype in human colorectal carcinoma cells.
- Alpha2betal integrin affects metastatic potential of ovarian carcinoma spheroids by supporting disaggregation and proteolysis. J Carcinog 6, 11.
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US20110052596A1 (en) * | 2006-08-11 | 2011-03-03 | Schering Corporation | Antibodies to il-17a |
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WO2010146078A2 (en) * | 2009-06-16 | 2010-12-23 | Bergen Teknologioverføring As | Novel uses of hydroxyproline compositions |
US20120294865A1 (en) * | 2011-05-19 | 2012-11-22 | The Regents Of The University Of Michigan | Integrin Alpha-2 Binding Agents and Use Thereof to Inhibit Cancer Cell Proliferation |
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XU ET AL.: "Three-dimensional polymeric systems for cancer cell studies", CYTOTECHNOLOGY, vol. 54, no. 3, 2007, pages 135 - 143, XP019525901 * |
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