Patent Application
20230121248
A sequence listing entitled “Metastasis_Inhibiting_Protein_ST25.txt” is an ASCII text file and is incorporated herein by reference in its entirety. The text file was created on Apr. 21, 2022 and is 42.4 in size.
The disclosure generally relates to biologic products to treat cancer, and more specifically biologic products that promote cancer cell adhesion thereby reducing metastasis and concomitantly enhance CAR-T treatment.
Decades of evidence suggest that alterations in the adhesion properties of neoplastic cells endow them with an invasive and migratory phenotype. Tight junctions (TJs) are present in endothelial and epithelial cells. Tumors arise from such tissues, thus, the role of TJ proteins in the tumor microenvironment is important. In tight junctions, junctional adhesion molecules (JAM) play a key role in assembly of the tight junctions and control of cell—cell adhesion. Reprogramming of immune cells using chimeric antigen receptors (CAR) to allow for target recognition and eradication of tumors is an FDA approved therapy. The CAR-T cells recognize cell-surface proteins, such as CD19 a B-cell surface molecule followed by cancer cell death. CD19 is not a unique marker for tumors, liquid or solid. Metastasis still occurs in patients undergoing CAR-T therapy. There is, therefore, a need in the art to address alternate cancer therapies that concomitantly reduce cancer metastasis and proliferation.
A three-domain, fusion protein is disclosed that includes a detection sequence that associates with cancer cells, a cell adhesion sequence, a signaling sequence configured to signal targeting of an anti-signaling CAR-T cell.
In some embodiments, the detection sequence identifies low pH of cancer cells and inserts the detection sequence into the cancer cell plasma membrane. In some embodiments, the detection sequence corresponds to a pH-low-insertion peptide (pHLIP).
In some embodiments, the cell adhesion sequence is an IgSF protein. In some embodiments, the cell adhesion sequence is selected from JAM-A, JAM-B, JAM-C, and JAM-4. In some embodiments, the cell adhesion sequence is JAM-A. In some embodiments, the cell adhesion sequence is JAM-B. In some embodiments, the cell adhesion sequence is JAM-C. In some embodiments, the cell adhesion sequence is JAM-4. In some embodiments, the cell adhesion sequence is JAM-A. In some embodiments, the cell adhesion sequence increases tumor cell-tumor cell adhesion. In some embodiments, the cell adhesion sequence increases tumor cell adhesion to cellular matrix.
In some embodiments, the fusion protein also includes linking segment located between the cell adhesion sequence and the signaling sequence.
In some embodiments, the signaling sequence is an IgSF protein selected from CD19, CD22, CD133, Her-2, EGFR, and mesothelin. In some embodiments, the signaling sequence is CD19. In some embodiments, the signaling sequence is CD22. In some embodiments, the signaling sequence is CD133. In some embodiments, the signaling sequence is Her-2. In some embodiments, the signaling sequence is EGFR. In some embodiments, the signaling sequence is and mesothelin.
In some embodiments, the fusion protein is selected from SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, and SEQ ID NO. 10. In some embodiments, fusion protein is SEQ ID NO. 4. In some embodiments, fusion protein is SEQ ID NO. 6. In some embodiments, fusion protein is SEQ ID NO. 8. In some embodiments, fusion protein is SEQ ID NO. 10.
In another aspect, a system for treating cancer and reducing cancer metastasis includes a fusion protein described herein and CAR-T cells configured to recognize the signaling sequence.
In another aspect, a method for treating cancer is disclosed that includes providing a therapeutically effective amount of a fusion protein described herein to a patient in need thereof. In some embodiments, the method includes providing a therapeutically effective amount of CAR-T cells that recognize the signaling sequence and induce cancer cell death.
In another aspect, a method for manufacturing a cancer therapy includes expressing a protein from a DNA sequence encoding a detection sequence, a cell adhesion sequence, and a signaling sequence, wherein the signaling sequence is configured to signal targeting of an anti-signaling CAR-T cell. In some embodiments, the DNA sequence is selected from SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, and SEQ ID NO. 9.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided to the Office upon request and payment of any necessary fee.
A detailed description of the invention is hereafter provided with specific reference being made to the drawings in which:
Various aspects are described below with reference to the drawings. The relationship and functioning of the various elements of the aspects may better be understood by reference to the following detailed description. However, aspects are not limited to those illustrated in the drawings or explicitly described below. It should be understood that the drawings are not necessarily to scale, and in certain instances, details may have been omitted that are not necessary for an understanding of aspects disclosed herein, such as conventional fabrication and assembly. Headings are provided for the convenience of the reader and to assist organization of the disclosure and should not be construed to limit or otherwise define the scope of the invention.
Cancer treatment traditionally involves chemotherapy and surgery. Other promising treatments such as genetically engineered T cells called CAR-T (chimeric antigen receptor T cells) target surface proteins such as CD19. Unfortunately, these treatments do not inhibit metastasis. Our biologic consists of 3 components: (1) A fusion protein that detects cancer cells based on their lower pH with the help of pHLIP; (2) JAM binding to other tight junction components with neighboring cells that inhibits the metastasis of cancer cells; (3) A signaling target protein domain, such as CD19, that allows for the recognition of anti-CAR-T (such as anti-CD19-CAR-T) cells to target and eradicate the tumor.
Applicant believes the present disclosure represents the first biologic containing three separate domains with distinct functions targeting cancer while concomitantly reducing metastasis by promoting cancer cell adhesion to other cancer cells.
For example, the present disclosures describes a composition (biologic) having three functional domains: (1) pH-low-insertion peptide (pHLIP) which recognizes the low pH of cancer cells leading to the insertion of the peptide into the plasma membrane; (2) an extracellular domain of JAM proteins that fosters cell—cell interaction (including cellular cohesion); and (3) CD19 to be targeted by CAR-T cells. These compositions target cancer cells, and when coupled with anti-CD19 CAR-T cells, not only promote the death of the cancer cells but also decrease proliferation and metastasis.
The production of a biologic that inhibits metastasis of cancer cells would improve upon currently available targeted cancer treatments. For example, using a fusion protein that would recognize the targeted cell and insert itself into the membrane based on the decreased pH of tumors, which would also allow for the increased interaction between these cancer cells by increasing the tight junctions would decrease the incidence of metastasis. Also, using a targeted signal located on the surface of the membrane of cancer cells that is recognized by existing CAR-T technology would allow for the recognition of a great variety of tumors. Current immune therapy methods are limited due to the need to create individual types of CAR-T cells that recognize different targets such as CD19, CD38 and others, and due to the cost of production. The reason for failure of some of the CAR-T treatments is based on the poor health of the individual, which can result in a low quantity of responsive amount of transformed immune cells.
In our studies, we purified the four members of the Junctional Adhesion Molecule (JAM) protein family JAM-A, —B, —C and 4. We determined that JAMs increase the cell-cell interactions when they are expressed in the tight junction of the cell. We also determined that there is an increase in binding between heterotypic interactions of JAMs compared to homotypic interactions and E-Cadherin. Using the JAM proteins, therefore, will allow for the formation of tight junctions in homotypic or heterotypic interactions that subsequently result in decreasing metastasis in cancer cells. Depending on the patient's target tissue and cancer type the selection of a specific JAM family such as JAM-A, —B, —C or −4 leads to increase tight junction formation with other members of the JAM family.
A peptide sequence known as pH-low-insertion peptide (pHLIP) can recognize changes in pH and insert itself into the membrane of cancer cells. The pHLIP inserts its C-terminus through a membrane under low pH conditions (about 6 to about 6.5). The pHLIP peptide can deliver compounds such as phalloidin which is linked to the C-terminus and is cleaved inside the cells. This results in the immobilization of cytoskeleton and multinucleation due to F actin binding and filament stabilization. Using this pHLIP peptide will allow the fusion biologic to target the cancer cell's low-pH environment, and insert itself into the cancer cell membrane.
Without wishing to be bound to any particularly theory, applicant proposes a method in which a biologic composed of a fusion protein that contains three domains with specific functions will induce cancer cell death while concomitantly reducing cancer cell metastasis. The first domain consists of a pH-sensitive peptide, pHLIP that recognizes and inserts itself into the membrane of cancer cells. The second domain consists of the Junctional Adhesion Molecule (such as JAM-A, —B, —C and −4) which increases the number of tight junctions, allowing for tumor cell-cell interactions that decrease metastasis. The third domain consists of an extracellular region of the target protein that has been seen in many cancers that is a signaling sequence recognizable by a corresponding CAR-T cell. In some embodiments, this signaling sequence is CD19. The signaling sequence (or region) enables CAR-T cells to recognize, bind to, and eradicate the tumor.
To achieve the goal of decreasing metastasis, we created a biologic consisting of a fusion protein N-CD19-GS Linker-JAM-pHLIP peptide-C. This technology is based on the combination of tight junction components that bind tighter than other tight junction components such as claudin proteins. This fusion protein allows the pHLIP peptide to recognize the lower-pH environment produced by cancer cells. When the peptide recognizes the decrease in pH, it incorporates itself into the membrane of the cancer cell. The JAM region of the fusion protein binds to neighboring JAMs, promoting tight junction formation that results in the inhibition of cancer cell metastasis. This approach harnesses the power of tight junctions that allow for the cancer cells to stay within the tumor and not spread throughout the body. The extracellular region of the fusion protein signals recognition by corresponding CAR-T cells that kill (eradicate) the tumor. The novelty of this approach is the introduction of a target site for CAR-T cells on the biologic that can recognize tumors that do not have natively have the extracellular region (CD 19, for example) in or on their membrane surface.
The power of cell adhesion can be harnessed to decrease the incidence of metastasis in tumor cells. Using the tight junction protein junctional adhesion molecules, or JAMs (−A, −B, −C and 4), the incidence of metastasis can be decreased. With the combination of pH-sensitive peptides, pHLIP with JAMs, we can decrease metastasis. Additionally, using CD19 in a fusion with JAMs (−A, −B, −C, and 4) will attract CAR-T cells to target these tumors.
Using the property of cell adhesion, the presently described fusion proteins enable the increase of the tight junction's strength in tumors to prevent metastasis. With the combination of the pHLIP technology, specific tumor cells that increase tumor cell-cell interactions through tight junctions can be enhanced (targeted), resulting in a decrease in metastasis of tumor cells. With the help of extracellular regions (such as CD19), the binding of CAR-T cells can be increased that will target and kill tumor cells.
1. Introduction
The tumor microenvironment (TME) is what surrounds a tumor, including blood vessels, immune cells, fibroblasts, signaling molecules, and extracellular matrix. The tumor and its microenvironment are closely related and constantly interact. Tumor cells achieve these interactions through cell-adhesion and recognition molecules, all members of the immunoglobulin superfamily (IgSF). Among the members of the IgSF are tight junction (TJ) components such as junctional adhesion molecules (JAMs) that act as gates and barriers to control the permeability of the paracellular space. JAMs are an IgSF subfamily that contain four members: A, B, C, and 4. These components are also responsible for compartmentalization of the cellular environment and the separation of tissues. JAM proteins form homotypic and heterotypic interaction among the same family members and may influence other members of the TJ. Contrary to the effects of other TJ components, JAMs are responsible for increased proliferation when downregulated. JAM-A upregulation has been associated with endothelial to mesenchymal transition (EMT). In glioblastoma cells, however, JAM-A may act as a tumor suppressor. In our studies, we determined that JAM homo- and heterotypic interactions are of high binding affinity, resulting in increased cell-to-cell interactions. We also determined that JAMs induced stronger cell adhesion than epithelial cadherin (E-CAD). Harnessing the function of JAMs in the TME may be of importance in translational solutions including those disclosed in the present application.
IgSF proteins play a role in cellular recognition. Tumors often display unique proteins that are naturally targeted by immune cells surveilling the homeostatic landscape. One antigen for cancer immunotherapy is the B-cell-specific surface marker CD19, used because of its expression in B-cell malignancies and lymphomas. An antigen recognizing CD19 (or other cell surface protein), therefore, known as anti-signaling (e.g. CD19) chimeric antigen receptor (CAR)T, is disclosed. It is fused to an intracellular signaling domain capable of activating T-cells to target and eradicate tumors. These CAR-Ts have a surface receptor that works like an antigen recognition domain that recognizes surface receptor targets (such as CD19) leading to activation, cytokine secretion, and cellular proliferation, which in turn lead to tumor eradication. CD19-directed CAR-T cell therapy has been successful in treating several B-cell lineage malignancies, including B-cell non-Hodgkin lymphoma (NHL). CD19-directed CAR-T cell therapy is a FDA-approved treatment that is being expanded to other immune cells and to treat solid tumors. The present disclosure builds on CAR-T cell therapy as a promising future for cancer treatments.
Biologics are powerful treatments that can be made of sugars, proteins, DNA, or composed of whole cells or tissues. Human insulin was the first recombinant biopharmaceutical approved in the United States in 1982. Protein-based therapeutics have been highly successful in the clinic and are recognized for their treatment potential. Based on their pharmacological activity, they can be divided into the categories of: (a) replacing a deficient protein, (b) enhancing a pathway, (c) performing a novel function or activity, (d) interference, and (e) delivering other compounds or proteins. They can be also be classified as non-covalent binding to their respective target, covalent bonding, and non-specific interactions with their respective targets. New engineered proteins—including bispecific mAbs and multi-specific fusion proteins, antibody-drug conjugates, and proteins with optimized pharmacokinetics—are currently under development. There are, however, no conceptually new developments in protein-based biologics. There has been no protein engineering applied to new strategies in decreasing cancer metastasis. Computational designs can be a theoretical approach to practical progress. A paradigm change in the methodologies and understanding of mechanisms is needed to overcome major challenges like the complexity of biological systems, resistance to therapy, and access to targets.
Fusion proteins and fusion peptides as biologics have been described. By joining different proteins that have different beneficial qualities, the potency, stability, and specificity of fusion proteins can be greatly enhanced compared with naturally occurring proteins. PRS-343 is a bispecific fusion protein targeting HER2 and CD137, a costimulatory receptor on T-cells. The PRS-343 architecture is derived from a trastuzumab variant and a CD137 specific anticalin. Anticalins are engineered variants of tear lipocalin and neutrophil-gelatinase-associated lipocalin (NGAL), where loops are randomized by mutagenesis. PRS-343 enables tumor-localized targeting of T-cells. This approach has the potential to provide a more localized activation of the immune system, resulting in higher efficacy and reduced peripheral toxicity. Following this report, the authors initiated a phase I clinical trial with PRS-343 as a first-in-class molecule.
In this application, we disclose a new type of protein-based biologic that aids cancer treatment with CAR-T therapies. We designed a modular three-part biologic that creates new strategies pertaining to pharmacological activity, and activity or function. In this disclosure, we describe the creation of a biologic that consists of three components: (1) a peptide that detects cancer cells based on their lower pH; (2) JAM extracellular domain for binding to other tight junction components in the tumor micro environment that inhibits the metastasis of cancer cells; (3) a signaling target protein domain (e.g. CD19) that allows for the recognition of anti-signaling CAR-T cells (e.g. anti-CD19 CAR-T cells). Our biologic is modular in that the JAM or anti-signaling domains can be exchanged for tumor-specific proteins that modulate cancer cell and tissue types.
Innovations that aid or CAR technology have been developed, such as the secretion of CD19-anti-Her2 bridging protein that allows for T-cell cytotoxicity both in vitro and in vivo. Other examples are the use of bispecific CAR-T to bring two cell types together such as cancer cells and T-cells, and the usage of donor stem cells or induced pluripotent stem cells to produce CAR specific treatments to derive natural killer cells, macrophages that can treat multiple myeloma. While these are very important contributions that target and treat cancer, patient medical needs remain unmet as several patient therapy still results in morbidity often from cancer cell metastasis. In our disclosure, we use a biologic to decrease cellular proliferation and metastasis.
Using JAMs as a part of our cell adhesion domain allows for the formation of cell adhesion homotypic or heterotypic interactions, or both. We recognized that JAM-A binds to other members of the family and coordinates the assembly of the TJ and the interplay with the adherens junction (AJ). The result of strengthening tumor cell—cell interactions subsequently result in decreasing metastasis in cancer cells. In order to introduce the extracellular domain of JAM proteins in the plasma membrane of tumor cells, we considered a peptide sequence known as pH-low-insertion peptide (pHLIP) that recognizes changes in pH and insert itself into the membrane of cancer cells. The pHLIP inserts its C-terminus through a membrane under low pH conditions (about 6.0— about 6.5). We recognized that this peptide has been used for the delivery of therapeutics. In our design, we considered that pHLIP might allow the fusion biologic to target the cancer cell's low pH and insert itself into the membrane, resulting in the extracellular domain of JAMs to be anchored to the cell surface.
The production of a biologic that inhibits metastasis of cancer cells would improve upon currently available targeted cancer treatment. Inhibition of cancer cell proliferation and metastasis is important in controlling tumor growth, but it is also important to sensitize the tumor to therapeutics, decrease proliferation, and ultimately eradicate it. In one embodiment to accomplish this, we used the extracellular domain of human CD19. The human CD19 antigen is a transmembrane protein belonging to the IgSF. CD19 is a biomarker for normal and neoplastic B cells, as well as follicular dendritic cells. CAR-T is an emerging therapy that targets B cell malignancies based on their cell surface display of CD19. We considered that the addition of this extracellular CD19 domain to our biologic would synergistically complement existing CD19 CAR-T therapies for eradicating tumors. Our approach increases tumor cell— cell adhesion, preventing tumor growth and metastasis, while displaying CD19 on the surface of cancer cells. The combination of these three domains will not be restricted to blood malignancies employed in emerging CAR-T therapy but benefit solid tumors as well.
In one embodiment, we created a fusion protein identified hereafter as CM19XA. The number 19 denotes CD19 while the letter A represents JAM-A soluble domain. The advantages of this biologic are that it contains three separate domains with distinct functions that target cancer cells that can be used in any patient, regardless of their health. This biologic helps existing CAR-T cells target a tumor through the recognition of CD19, while decreasing metastasis. Our biologic is modular and CD19 may be exchanged for other cell surface biomarkers such as CD22, CD133, Her-2, EGFR, mesothelin, and others, and the JAM may be any of the four members of the subfamily, whichever is relevant to the tumor tissue of origin.
Recent advances in protein engineering have come from creating multi-functional chimeric proteins containing modules from various proteins. These modules are typically joined via an oligopeptide linker, the correct design of which can contribute to the desired function of new biologically active molecule. Thus, in some embodiments, the peptides include a linking segment located between the cell adhesion sequence and the signaling sequence.
As a component of recombinant fusion proteins, linkers have utility in the construction of stable, bioactive fusion proteins. The general properties of linkers derived from naturally-occurring multi-domain proteins can be considered as the foundation in linker design. Empirical linkers are generally classified into three categories according to their structures: flexible linkers, rigid linkers, and in vivo cleavable linkers. (See Fusion Protein Linkers: Property, Design and Functionality. Xiaoying Chen, Jennica Zaro, and Wei-Chiang Shen. Adv Drug Deliv Rev. 2013 Oct. 15; 65(10): 1357-1369. doi: 10.1016/j.addr.2012.09.039.)
Linkers can also play a contributory role in the engineering of fusion proteins. Linkers can affect protein properties such as expression level, solubility, and biological functions. For linker design and optimization, one of the key factors is the flexibility or rigidity of the linker sequence, which describes the tendency of a linker to maintain a stable conformation, impacting directly the physical distance between domains in a fusion protein. Library methods of design exist or can be easily constructed based on conventional practice and experimental designs depending on the fusion domains and desired function. (See Zliang Huang, Chong Zhang, Xin-Hui Xing. Design and construction of chimeric linker library with controllable flexibilities for precision protein engineering, Methods in Enzymology, Academic Press, Volume 647, 2021, Pages 23-49, ISSN 0076-6879, ISBN 9780128208182, https://doi.org/10.1016/bs.mie.2020.12.004.)
In order to use our biologic to decrease metastasis and to target these cancer cells for destruction by anti-CD19 CAR-T cells, we address the following questions: (1) Does CM19XA target cancer cells specifically? (2) Does CM19XA decrease metastasis by using the JAM components to establish and increase cell—cell interactions? (3) Does CD19 allow for the targeting of anti-CD19 CAR-T cells? (4) Does CM19XA work on other cancer cell lines? Answers to these questions are addressed in the examples below as illustrated by observations in the accompanying figures.
Except in the examples, or where otherwise expressly indicated, all numerical quantities in this description indicating amounts of material or conditions of reaction or use are to be understood as modified by the word “about” in describing the broadest scope of the invention. Practice within the numerical limits stated is generally preferred. The first definition of an acronym or other abbreviation applies to all subsequent uses herein of the same abbreviation and applies mutatis mutandis to normal grammatical variations of the initially defined abbreviation; and, unless expressly stated to the contrary, measurement of a property is determined by the same technique as previously or later referenced for the same property.
Unless indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Throughout this application, where publications are referenced, the disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
It is also to be understood that this invention is not limited to the specific embodiments and methods described below, as specific components or conditions may, of course, vary. Furthermore, the terminology used herein is used only for the purpose of describing particular embodiments of the present invention and is not intended to be limiting in any way.
It must also be noted that, as used in the specification and the appended claims, the singular form “a,” “an,” and “the” comprise plural referents unless the context clearly indicates otherwise. For example, reference to a component in the singular is intended to comprise a plurality of components.
The term “or” is understood to mean “and/or”.
The term “comprising” is synonymous with “including,” “having,” “containing,” or “characterized by.” These terms are inclusive and open-ended and do not exclude additional, unrecited elements or method steps.
The phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. When this phrase appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.
The phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps, plus those that do not materially affect the basic and novel characteristic(s) of the claimed subject matter.
The terms “comprising”, “consisting of”, and “consisting essentially of” can be alternatively used. When one of these three terms is used, the presently disclosed and claimed subject matter can include the use of either of the other two terms.
2. Materials and Methods
2.1. Cloning, Protein Expression, and Purification
We synthesized the E. coli codon-optimized DNA sequence of CM19XA (Twist Bioscience, San Francisco, Calif., USA). CM19XA was supplied by TWIST biosciences cloned in the expression vector pET28a, between restrictions sites Ndel and Xhol. A stop codon was introduced prior to the Xhol. The sequence upstream Ndel was the native sequence of pET28a which includes a 6xHis-tag sequence. pET28a CM19XA is preserved by transformation in the bacterial strain DH5a. Plasmid purification of a single bacterial colony was performed using the Zyppy Plasmid Miniprep Kit from Zymo Research. Sanger sequencing was performed by Genewiz (South Plainfield, N.J., USA) to determine whether the plasmid coding for CD19-JAMA-pHLIP was correct and that there were no mutations present. After the verification of the plasmid sequence, we transformed the plasmid into SHuffle T7 express bacterial cells (New England Biolabs, Ipswich, N.Y., USA) to compare which bacterial strain would give us the highest protein yield. Cells are grown to an OD600 of 1, followed by addition of 0.3 mM IPTG, and maintained at 16° C. for 18 hours. The French Press method was used to lyse the transformed bacterial cells. The resuspended cells were loaded into the Thermo Spectronic French Pressure Cell Press Model FA-078. Lysis was performed at 1500-2000 psi using 30 mL of Wash Buffer consisting of 500 mM NaCl and 30 mM Tris and the lysate was collected in a 50 mL conical tube. Centrifugation was performed on the lysate for 30 min at 10,000 RPM with a F15-8×50cy rotor.
The supernatant was decanted into a 50 mL tube containing Ni-NTA Agarose beads from Prometheus (catalog no. 20-512) and incubated while rotating for 1 h at 4° C. The column was washed with 100 mL of wash buffer containing 30 mM TRIS pH 7.5 and 500 mM NaCl, and 30 mM Imidazole pH 8.0. The supernatant was eluted for 3 min with 300 mM Imidazole, then concentrated using the Microsep Advance with 10 k Omega centrifugal device (reference no. MCP010C41) from Pall Corporation at 10,000 RPM for 10 min until a final volume of 2 mL was reached.
2.2. Size Exclusion Chromatography (SEC)
Size exclusion chromatography was performed using the NGC System (BioRad, Hercules, Calif., USA). The column used was the ENrich™ SEC 65,010×300 mm, 24 mL, prepacked high-resolution SEC 650 column, with a size range of 5650 kDa (BioRad, Hercules, Calif., USA). The protein peak was observed using the BioRad SEC software. The product peaks' positions were compared relative to those of the size exclusion standards from BioRad (catalog no. 151-1901). Protein concentration was determined using the Nanodrop Onec from Thermo Scientific. The running buffer used was PBS, and proteins were also stored in PBS. Purification of JAM-A was performed as described in our previous publication.
2.3. SDS-PAGE Assay
Two μg of boiled MBP, JAM-A, and CM19XA were electrophoresed on 8% SDS-PAGE gel (BioRad). Gel staining was performed using standard protocols.
2.4. Tissue Culture and In Vitro Experiments with CAL27 and A549 Cells
Tongue squamous cell carcinoma cells (Ca127, ATCC CRL-2095) were obtained from American Type Culture Collection (ATCC, Manassas, Va., USA) and cultured according to the guidelines provided by the organization. RPMI, calcium-free with 10% FBS was used for all manipulations and the experimental set-up of CAL27 cells. A549 cells, epithelial cell lung carcinoma, were obtained from ATCC (catalog reference CCL-185).
2.5. Proliferation Assay
The first day of the proliferation assay consisted of 30,000 CAL27 cells seeded on 48-well plates. On the second day (at approximately 16 hours), cells were treated with PBS or proteins at a final protein concentration of 1 μM (MBP-JAMA or CM19XA). After 24 hours, proliferation assays were performed using ATPlite Luminescence Assay System (product number 6016943, PerkinElmer, American Fork, Utah, USA) following the manufacturer's instructions. After 72 hours of mock or anti-CD19 CAR-T killing assay for both CAL27 and A549 cells, we performed the ATPlite Luminescence Assay [49,50].
2.6. Wound Healing Assay
The wound healing assay was performed as follows: on the first day, 15,000 CAL27 cells were seeded on each chamber of the 2-well silicone insert (IBIDI, Gräfelfing, Germany) separated by a silicone gap of 500±100 μm. After 24 hours, proteins were added at a final concentration of 1 μM, JAM-A or CM19XA. Cells were incubated with the treatments for 2 hours at 37° C. Following the treatment, the silicon insert was removed. The wells were rinsed once with PBS and then each well was filled with DMEM F-12 media with 10% FBS (Genesee Scientific, El Cajon, Calif., USA). The closure of the gap (500 μm+/− 100 μm at time zero) was evaluated 16 hours post treatment using an Olympus IX70 microscope (Olympus Life Science, Waltham, Mass., USA). Images were analyzed using cellSens Entry Microscopy Imaging Software by Olympus Life Science. The distance of the gaps was then quantified using ImageJ [511. Data analyzed using FastTrack AI (IBIDI, Grafelfing, Germany)
2.7. Real-Time Cell Invasion
Real-time cell invasion was determined after the various treatments. The xCEL-Ligence RTCA cell monitoring system was used to quantify real-time invasion of cells per the protocol suggested by the manufacturer (ACEA Biosciences, Blue Springs, Mo., USA). The invasion was performed in 16-well CIM plates (n=10 groups per treatment, ACEA Biosciences, Blue Springs, Mo., USA). The tops of the wells were coated using a 1:40 Matrigel concentration (Fisher Scientific, Pittsburgh, Pa., USA). Then, a concentration of 20,000 CAL27 cells was used with 100 μL of 2% FBS RPMI, with and without proteins. The bottom chamber wells were treated with 160 μL of 10% FBS RPMI. Cells were placed in the xCeLLingence RTCA instrument, where the invasion readings were taken 4 times an hour for 24 hours.
2.8. CAR-T Cell Killing Assay
On the first day, 5000 CAL27 or A549 or HUVEC cells/well (catalog C0035C, Thermo Fisher Scientific, Mass., USA) were plated on a 96-well plate. On day 2, cells were incubated for 3 h under the following conditions: 14 wells had no treatment, which was used as a control. In 16 of the wells, 1 μM soluble JAM-A protein was introduced. In 14 of the wells, 1 μM of CM19XA was introduced. On day 2, both mock CAR-T and anti-CD19 CAR-T were dispensed to half of the wells in each treatment. The killing assay was allowed to continue for a total of 72 hours.
2.9. Computer Models of the Biologic
Protein models were produced using UCSF Chimera v. 1.15 package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH P41 RR-01081).
2.10. Statistical Analysis
Student's t-test was performed using GraphPad Prism version 8.0 to generate the graphs to compare control vs. JAM-A and CM19XA.
3. Results and Discussion
3.1. Expression and Purification of CM19XA in E. coli
Extracellular domains of JAM-A were expressed as a fusion with CD19 and pHLIP containing an N-terminal 6x-HIS tag to allow for the use of Nickel NTA purification strategy (
3.2. Proliferation Assay
To determine whether CM19XA targets cancer cells, we used the cell line CAL27 in a proliferation assay. We recognized that the membrane composition of the TJ is simple, consisting of claudin-1, JAM-A, and occludin. We also recognized that the downregulation of JAM-A through siRNA leads to cell proliferation. We confirmed this result and comparing the effects of the soluble extracellular domain of JAM-A and CM19XA in CAL27 cells. The comparison of these two proteins resulted in opposite CAL27 cell behavior in the proliferation assay (
The proliferation assay of CAL27 cells shows that there is a decrease in the proliferation with the addition of CM19XA. The proliferation assay was different for the introduction of JAM-A, where there was an increase of proliferation as compared to no effect with the control. The data discussed above shows that soluble JAM-A decreases tumor cell— cell adhesion, leading to an increase in cellular proliferation. The biologic CM19XA increased cell-to-cell adhesion by increasing JAM protein binding to other TJ proteins either by homotypic or heterotypic interactions. This could be due to JAM-A not inserting itself into the membrane and binding to other JAMs (−A, −B, −C, and 4) in cis that cause an interruption in the cell-to-cell interactions meaning that there is a decrease in TJ trans interactions. In the case of CM19XA, the decrease in proliferation is caused by the pH sensitive region of the biologic inserting itself into the membrane of the cancer cells that allows the JAM-A portion of the biologic to reinforce the binding of native JAM proteins in trans leading to greater tumor cell-to-cell interactions. The phenotype observed, therefore, is consistent with decreased proliferation because CM19XA increases binding of TJ components such as JAMs.
3.3. Wound Healing Assay
In order to validate the ATP proliferation experiments where JAM-A increases CAL27 proliferation and CM19XA decreased CAL27 cell proliferation, we performed a wound healing assay. This assay allow us qualitatively and quantitatively determine whether the JAM-A portion of CM19XA would increase tumor cell—cell interactions, and as a result, decrease cell migration, resulting in a larger observed gap. Cell migration is important in many physiological processes that are heavily regulated. Wound healing and cancer cell migration assays are widely used for the understanding of cues that can increase or decrease cell migration. Thus, we decided to use CM19XA with CAL27 cells to determine the effect this biologic had on cell migration by performing wound healing assays.
The wound healing experiments show the effects of our biologic, CM19XA qualitatively at
3.4. Cell Invasion Assay
Based on the results of the wound healing assay, we determined the effect of CM19XA on cell invasion. Cell invasion recordings were observed to be low for the control containing only 10% FBS. JAM-A resulted in an increase of cell invasion (
Cell invasion recordings were observed to be low for the control containing only 10% FBS. JAM-A resulted in an increase of cell invasion (
3.5. CM19XA Only Targets Cancer Cells
In order to determine whether CM19XA only targeted cancer cells, we used the non-cancerous HUVEC cell line. We determined that addition of soluble JAM-A protein increased cellular proliferation (
To determine whether CM19XA targets other cancer cells, we repeated the cytotoxicity experiments with A549 lung cancer cells. The cytotoxicity cell assay in
3.6. Cytotoxicity Assay
To determine whether the CD19 portion of CM19XA worked as a signal to allow for CAR-T cells to recognize and kill cancer cells, we performed cytotoxicity assays. We used cultured cells as a model system to determine the functionality of extracellular CD19 as a target signal for anti-CD19 CAR-T cells. Cells were observed to grow without problems for mock CAR-T experiments using a CAR-T cell that did not recognize the CD19 portion of CM19XA as expected. There was no effect on the killing of CAL27 cells with the mock CAR-T experiments using control (no protein), JAM-A, and CM19XA (
3.7. Proposed Mode of Action of CM19XA
Without wishing to be bound to any particular theory, we present our biologic, CM19XA that identifies and target cancers according to the decreased pH of the membrane of cancer cells (
The identification of pro- and anti-cancer roles among TJs such as claudins has been puzzling. Similarly, the role of JAM-A and JAM-C in the progression of malignant neoplasm has been described to have a number of contradicting phenotypes. The role of JAM proteins in cancer is, therefore, complex. JAMs function by interacting with other proteins via several mechanisms: direct cell—cell interaction on adjacent cells, stabilization of adjacent cell surface receptors on the same cell, and interactions between JAM and cell surface receptors expressed on adjacent cells. The diverse interactions contribute to both the pro- and antitumorigenic functions of JAM. This paradigm can also be observed in a study that presents evidence that JAM-A knockdown accelerates the proliferation and migration of human keratinocytes. Other research examined the role of JAM-A in multiple myeloma (MM). In vitro JAM-A inhibition impaired MM migration, while in vivo treatment with an anti-JAM-A monoclonal antibody impaired tumor progression. These results could correspond to JAM-A interactions and effects within the same cell or to a signal transduction that is not fully understood. The importance of mechanical transduction from cellular junctions, both TJ and AJ, is poorly understood. Other research highlights the need for further study of this phenomenon. Additionally, a report that JAM-A functions in a tumor-suppressive role by increasing apoptosis and suppressing proliferation in colorectal adenocarcinoma revealed that loss of JAM-A expression increased intestinal epithelial cell proliferation. The relevance of this paradigm may simply indicate that regulation of JAM-A expression in the context of cell proliferation may be tissue- and cell-specific.
Considering that JAM-A is a player that coordinates TJ and Aj's interplay, understanding its function in tumorigenesis and metastasis is germane to the identification and selection of junctional adhesion molecule domain. While assessing invasive breast cancer, data shows that cell lines with the lowest migratory capacity (T47D and MCF-7 cells) express higher levels of JAM-A relative to more migratory lines (MDA-MB-231 cells). Ectopic expression of JAM-A in these highly metastatic cells diminished both cell migration and invasion. On the contrary, silencing of JAM-A expression enhanced the invasiveness of the less migratory lines. Nevertheless, evidence for the opposite phenotypes can also be found. Functional inhibition of JAM-A protein activity inhibits the adhesion and trans-endothelial migration of breast cancer cells. Human nasopharyngeal cancer cells exhibit increased JAM-A levels, which leads to increased endothelial-to-mesenchymal transition. In lung adenocarcinoma, the suppression of JAM-A expression by siRNA inhibited cellular motility and invasiveness, while JAM-A inhibition caused a decrease in colony-forming capability in vitro and an inhibition of tumorigenicity in vivo.
As a final consideration, CM19XA lacks the capability of intracellular signaling. This could be a reason why the results we observed deviate from what could be expected according to the previous discussion. CM19XA is capable of carrying out two functions once inserted in the membrane through pHLIP. First, CD19 attracts CAR-T cells; second, JAM-A interacts with other TJ membrane proteins (trans and cis interactions) and also exhibits self-interaction; both will result in cis and trans interactions. From this point of view, CM19XA can be examined for its role as an adhesion molecule rather than its signal transduction leading to tumor related phenotypes. If our hypothesis is correct, then the idea that regulation of JAM-A expression in the context of cell proliferation may be tissue- and cell-specific will not apply. We imagine that as an adhesion promoting agent CM19XA will be tissue independent. Tissue specificity may require utilizing a different biology (
The modularity of our biologic can address the differences in TJ composition due to tissue-specific expression of its membrane components. CD19 can be used as a recognition signal for anti-CD19 CAR-T cells. In traditional anti-CD19 CAR-T therapies, the cell targets the naturally displayed CD19 of B cell malignancies. The modularity of our biologic will enable the selection of any surface biomarker desired based on the tumor type.
4. Conclusions
We designed and tested CM19XA, a three-domain biologic. We presented evidence that our biologic inserts itself into cancer cells using its pHLIP peptide. The second domain of the biologic, JAM-A, increases cell-to-cell interactions that in turn decrease proliferation and may prevent tumor cells from leaving their niche, inhibiting metastasis. The third domain of the biologic, CD19, is recognized by anti-CD19 CAR-T cells, allowing for targeted cancer cell killing. Our biologic produced similar results in two cell lines, CAL27 and A549 and had no effect on the non-cancer cell line HUVEC, showing that it is cancer specific. This suggests that CM19XA and protein constructs like it may be used as a therapeutic that recognizes multiple cancer cell lines. CM19XA's adhesive properties provide increased cell-to-cell interactions through its JAM domain, and cellular recognition of immune cells through the CD19 domain, resulting in cancer cell killing. We suggest that CM19XA is a new classification of protein-based biologic that pairs with current CAR therapies to recognize cancer cells, increases cell-to-cell interactions that lead to a decrease in proliferation and metastasis, and increases cancer cell killing.
CAR-T is produced from a patient's blood, where the gene for a single receptor is inserted with the purpose of attacking a specific cancer cell. These genetically engineered T-cells are then re-introduced into the patient. Looking forward, CM19XA (and biologics like it) will advance the treatment of cancer by serving as an additional tumor specific mechanism. This biologic can be manufactured at large scale and can be used to target CD19, CD38, or other specific tumor targets using the corresponding CAR-T cell design. Depending on the type of tumor cell to be targeted, the JAM proteins (A, B, C, and 4) can be interchanged. Future work will include in vivo experimentation and characterization of potency, stability, and specificity. Analysis of CM19XA and its derivatives will involve cytokine release syndrome (CRS) grading and other safety measurements as the research progresses.
A nucleic acid sequence useful for expressing a metastasis inhibiting fusion protein of plasmid pET28a is as follows (SEQ ID NO: 1):
The translation product of pET28a from the encoding nucleic acid SEQ ID NO: 1 is SEQ ID NO. 2:
The modularity of fusion protein described herein can be achieved by exchanging CM19XA within the plasmid that expresses the protein in a bacterial host. This process is called sub-cloning. Using Restriction Enzymes Ndel and Xhol CM19XA can be substituted by CM19XB, CM19XC or CM19X4 where XA corresponds to JAM-A, XB corresponds to JAM-B, XC corresponds to JAM-C, and X4 corresponds to JAM-4.
A nucleic acid sequence useful for expressing a metastasis inhibiting fusion protein for encoding CM19XA: CD19-JAM A-pHLIP is as follows (SEQ ID NO: 3):
The translation product from the encoding nucleic acid SEQ ID NO: 3 is SEQ ID NO. 4 (CD19-JAM A-pHLIP):
All proteins are expressed and purified under the same directives of the protocols presented above. The production in small scale and under regular laboratories conditions demonstrate differences among the proteins in regard to yields (mg/L).
A nucleic acid sequence useful for expressing a metastasis inhibiting fusion protein for encoding CM19XB: CD19-JAM B-pHLIP is as follows (SEQ ID NO: 5):
The translation product from the encoding nucleic acid SEQ ID NO: 5 is SEQ ID NO. 6 (CD19-JAM B-pHLIP):
A nucleic acid sequence useful for expressing a metastasis inhibiting fusion protein for encoding CM19XC: CD19-JAM C-pHLIP is as follows (SEQ ID NO: 7):
The translation product from the encoding nucleic acid SEQ ID NO: 7 is SEQ ID NO. 8 (CD19-JAM C-pHLIP):
A nucleic acid sequence useful for expressing a metastasis inhibiting fusion protein for encoding CM19X4: CD19-JAM 4-pHLIP is as follows (SEQ ID NO: 9):
The translation product from the encoding nucleic acid SEQ ID NO: 9 is SEQ ID NO. 10 (CD19-JAM 4-pHLIP):
This application claims the benefit of U.S. provisional application 63/177,703 filed on Apr. 21, 2021, U.S. provisional application 63/274,788 filed Nov. 2, 2021, and U.S. provisional application 63/296,322 filed on Jan. 4, 2022, the disclosures of which are hereby incorporated in their entirety by reference herein.
| Number | Date | Country | |
|---|---|---|---|
| 63296322 | Jan 2022 | US | |
| 63274788 | Nov 2021 | US | |
| 63177703 | Apr 2021 | US |
