Patent Application
20250152627
The instant application contains a Sequence Listing which has been submitted in .xml format and is hereby incorporated by reference in its entirety. Said .xml copy was created on Dec. 5, 2022, is named “018617_01389_ST26.XML”, and is 72,267 bytes in size.
Tumor cells construct a glycocalyx with physical and biochemical attributes that guard against detection and destruction by surveilling immune cells1. Essentially all types of human cancers exhibit changes in glycan synthesis, resulting in their expression at atypical levels or with altered structural attributes, phenomena that were first observed more than six decades ago2-4. Immunomodulatory glycans can reprogram the activities of important immune cell types, including macrophages, dendritic cells, cytotoxic T-cells, and Natural Killer (NK) cells5. Larger macromolecules in the glycocalyx also are proposed to sterically shield molecular epitopes, fundamentally changing how immune cells perceive and interact with tumor cells1. While a multifaceted role for the cancer-associated glycocalyx in immune evasion has become clear, a physical understanding of the glycocalyx remains limited, especially regarding cancer immunotherapy. With the rapid advancement of adoptive cell therapies for cancer, identification of strategies to overcome the immune-protective actions of the cancer cell glycocalyx have become of strong interest.
Cancer cells often generate a glycocalyx with high levels of cell surface mucins, which can directly modulate the tumor immune microenvironment6-10 Mucin-type O-glycans initiate with N-acetylgalactosamine (GalNAc) that is linked to serine or threonine on the mucin polypeptide backbone and elongated into more complex structures through the sequential actions of glycosyltransferases in the Golgi apparatus11-13. Cancer is often associated with dramatically higher expression of cell-surface mucins that bear truncated and more highly sialylated O-glycans9,14. Through the engagement of Siglecs, the macrophage galactose receptor, and other immune-cell receptors, tumor-associated O-glycans have been implicated in the direct suppression of CD4+ T, CD8+ T, and NK cell activities7,15,16, as well as the recruitment and education of tumor-associated macrophages17-19. Mucins and cancer-associated O-glycans also serve as scaffolds for multivalent galectins that can act as negative regulators, or checkpoints, of the immune cell functions20-22. Immune suppression by mucins and mucin-type O-glycans is under active investigation as a mechanism of recalcitrance to cancer immunotherapies.
From a physical perspective, cell-surface mucins are semiflexible biopolymers that form a soft material coating on the tumor-cell surface23,24. Although recent studies of immune modulation by mucins have focused on the biochemical activity of their O-glycans, it has been suggested that the dense grafting of mucin biopolymers on the tumor cell surface may form a steric barrier against immune-cell recognition and cytolytic activities25-17. Direct validation of this possibility has remained elusive due to the challenge of disentangling the biochemical and material effects of specific changes in mucin-type glycosylation27. The dense clustering of O-glycans along the mucin backbone both extends and rigidifies its macromolecular structure11,23,28. Therefore, changes in the number, length, and sialylation (i.e. charge) of O-glycan chains are expected to alter the molecular properties of individual mucins and the ensemble material properties of the mucin-rich glycocalyx23. Given the close, intimate contact that is required for target engagement by effector immune cells, the material thickness of the glycocalyx may be a major determinant of immune evasion. However, the investigation of how the physical dimensions of the glycocalyx change in response to altered states of glycosylation has been challenging due the nanometer-scale precision of measurement that is required to detect the structural changes29.
Clinical interest in Natural killer (NK) cells for adoptive cell therapy has risen swiftly due to their ability to kill cells bearing markers associated with oncogenic transformation30. NK cells have been equipped with chimeric antigen receptors (CAR-NK) to direct attacks against specific tumor antigens, with some CAR-NK therapeutics now in human clinical trials30-33. Compared to CAR-T cells, CAR-NK cells have some significant advantages that may include better safety and more potential for “off-the-shelf” manufacturing of allogeneic therapies. As is the case with other immune therapies, tumors can develop multiple mechanisms to resist attack by NK and CAR-NK cells. Notably, an inverse relationship between NK cell killing and the expression of cell-surface mucins on target cells has been reported27. Truncation of mucin-type O-glycans increases NK cell-mediated antibody-dependent cellular-cytotoxicity, whereas elongation of the O-glycans has been reported to protect tumor cells26,27,34,35. How such changes alter the material properties of the glycocalyx has been previously unknown, and it has been unclear if the effects on NK-cell resistance are caused by specific receptor interactions, changes in the structural attributes of the mucins, or some other consequence of altered glycosylation27. There remains a need for new tools that can probe and measure the material properties of the glycocalyx to understand how these properties may contribute to immune evasion. Further, there is an ongoing and unmet need for improved approaches to overcoming the glycocalyx barrier that impedes anti-cancer immune responses. The present disclosure is pertinent to this need.
The present disclosure provides modified eukaryotic cells comprising one or more plasma membrane-coupled glycocalyx-editing (GE) enzymes. In certain examples, the GE enzyme is a mucinase or a sialidase. Cells that include combinations of plasma membrane-coupled glycocalyx GEs are included in the disclosure. In certain examples the GE enzyme is coupled to the plasma membrane by a transmembrane domain that is a component of the GE enzyme, the GE enzyme is coupled to the plasma membrane by at least two agents that bind to one another, such as a leucine zipper. In one example, the sialidase is coupled to the plasma membrane by a transmembrane domain that is a component of the sialidase, and the mucinase is coupled to the plasma membrane by a leucine zipper that includes a transmembrane domain. In certain examples the modified cells are lymphocytes. In some examples the lymphocytes are T cells, natural killer (NK), natural killer T cells (NKTs), macrophages, or dendritic cells. In certain examples a modified T cell or a modified NK cell is also modified such that it expresses a chimeric antigen receptor (CAR).
Methods of making and using the modified cells are also included. The methods of making the modified cells comprise engineering the cells such that a GE enzyme is coupled to the plasma membrane by a transmembrane domain, a leucine zipper, or a combination thereof. The disclosure includes isolated populations of modified cells.
Methods of using the modified cell a comprise administering the modified cells to an individual in need thereof. In certain examples, the individual in need is an individual who has cancer, or is at risk of developing cancer, or cancer relapse.
Unless defined otherwise herein, 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 disclosure pertains.
Unless specified to the contrary, it is intended that every maximum numerical limitation given throughout this description includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification includes every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.
As used in the specification and the appended claims, the singular forms “a” “and” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value encompasses variations of +/−10%, +/−5%, or +/−1%.
The disclosure includes all polynucleotide and amino acid sequences described herein. Each RNA sequence includes its DNA equivalent, and each DNA sequence includes its RNA equivalent. Complementary and anti-parallel polynucleotide sequences are included. Every DNA and RNA sequence encoding polypeptides disclosed herein is encompassed by this disclosure. Amino acids of all protein sequences and all polynucleotide sequences encoding them are also included, including but not limited to sequences included by way of sequence alignments. Sequences of from 80.00%-99.99% identical to any sequence (amino acids and nucleotide sequences) of this disclosure are included.
The disclosure includes all polynucleotide and all amino acid sequences that are identified herein by way of a database entry. Such sequences are incorporated herein as they exist in the database on the effective filing date of this application or patent.
The disclosure provides modified cells, methods of using the modified cells, methods of making the modified cells, and the modified cells themselves. In general, the cells are modified such they include a plasma membrane-coupled glycocalyx-editing (GE) enzyme. The GE enzyme may be any GE enzyme that is not expressed by the cell without modification, and/or any GE enzyme that is not plasma membrane-coupled absent a described modification. “Plasma-membrane coupled” as used herein means the described protein is connected to the plasma membrane directly or indirectly such that the described protein can modify the glycocalyx of other cells. As such, at least an enzymatically active segment of the described protein is positioned externally relative to the plasma membrane. In one embodiment, the GE enzyme is coupled to the plasma membrane by a transmembrane domain that is a component of the described protein. In another embodiment, a first protein that comprises a transmembrane domain is modified such that that it non-covalently associates with a second protein that is cognate to the first protein. For instance, first and second leucine zipper proteins can be used. One of the leucine zipper proteins comprises a transmembrane domain and is expressed by the modified cell. The other leucine zipper protein is added to the GE enzyme. By combining the GE enzyme comprising a leucine zipper protein that is cognate to the leucine zipper comprised by the modified cell the modified GE enzyme will be coupled to the plasma membrane through the leucine zipper protein pairing. Thus, in an embodiment, a described modified cell is engineered to express a first leucine zipper protein that comprises a transmembrane domain, and a modified GE enzyme that comprises a cognate leucine zipper that is provided to the cells such that the modified GE enzyme is coupled to the plasma membrane by the assembled leucine zipper. In one embodiment, a domain of the GE enzyme that is not a catalytic domain is substituted with a leucine zipper protein to thereby produce an enzymatically active fusion protein comprising a GE catalytic domain and a leucine zipper protein.
In alternative approaches, the described leucine zipper approach may be substituted with other agents. Such agents include but are not necessarily first and second binding agents, wherein the first and second binding agents that are not leucine zipper proteins bind to one another. For instance, an avidin-biotin binding interaction could be used. In embodiments, non-leucine zipper protein tags may be used. In embodiments, one or more epitope tag formats may be used in combination with an antibody or antigen binding segment thereof that specifically binds to the epitope tag.
Representative and non-limiting examples formats that may be used to couple the GE enzyme to the plasma membrane include SpyTag, ALFA-Tag, SNAP-tag, CLIP tag, maltose binding protein, Fc-tag, and Halo-Tag approaches. In other embodiments, the GE enzyme may be coupled to a plasma membrane protein through click chemistry approaches, such as azide-alkyne cycloaddition.
Any suitable GE enzyme can be used. In embodiments, the GE enzyme is a mucinase or a sialidase. In non-limiting embodiments the GE enzyme component of a described protein comprises or consists of the entire protein, or comprises or consists of a catalytic domain. As such, certain amino acids of the GE enzyme may be excluded, changed, or substituted. In embodiments, a signal peptide is excluded from the GE enzyme.
In non-limiting embodiments, the mucinase component comprises or consists of all or a catalytically active segment of secreted protease of C1 esterase inhibitor (StcE), a bacterial protease from Escherichia coli. The amino acid sequence of StcE is known and can be accessed via UniProtKB/Swiss-Prot: O82882.2. A representative full length StcE amino acid sequence is:
mntkmnerwr tpmklkylsc tilaplaigv fsataadnns
pvgqrsgysl pdwivgqevy
vdsgakakvl lsdwdnlsyn rigefvgnvn padmkkvkaw
ngqyldfskp rsmrvvyk.
The italicized sequence is a signal peptide sequence. The bold sequence is the X409 sequence.
In an embodiment, the first 35 amino acids of the StcE protein, which constitute a signal sequence, are deleted. In an embodiment, the X409 lectin domain of StcE is replaced with a leucine zipper to create a fusion between the StcE catalytic domain (amino acids 36-796 of StcE as shown in SEQ ID NO:1) and the leucine zipper. This fusion is then produced recombinantly and tethered to the immune cell surface through coupling with the cognate leucine zipper expressed genetically in the immune cells as a transmembrane fusion protein.
In another embodiment, the mucinase may be BT4244 from Bacteroides thetaiotaomicron or M60-like protease AM0627 from Akkermansia muciniphila.
The amino acid sequence of BT4244 is known and is available under GenBank sequence WP_012419679.1. A representative full length sequence of BT4244 is:
In an embodiment, the first 10 amino acids, which constitute a signal sequence, can be deleted from the BT4244 protein.
The amino acid sequence of AM0627 and is available from GenBank sequence WP_008764444.1. A representative full length sequence of AM0627 is:
In embodiments, the first 34 amino acids, which constitute a signal sequence, can be deleted from the BT4244 protein.
In embodiments, the GE enzyme comprises or consists of all or a segment of a sialidase. Suitable sialidases are known in the art. In one embodiment, the sialidase comprises a neuraminidase. In one embodiment, the sialidase comprises a sequence that is available under GenBank accession number AAA27168.1. A representative amino acid sequence of this protein is:
As discussed above, in certain embodiments, the GE enzyme component may be tethered to the plasma membrane using a leucine zipper approach. The amino acid sequences of leucine zipper protein pairs are known in the art. In embodiments, a first leucine zipper protein may comprises or consist of the sequence MDPDLEIEAAFLERENTALETRVAELRQRVQRLRNRVSQYRTRYGPLGGGK (SEQ ID NO:5). In non-limiting embodiments this sequence is tethered to the GE enzyme. In embodiments, a second leucine zipper protein may comprise or consist of the sequence MDPDLEIRAAFLRQRNTALRTEVAELEQEVQRLENEVSQYETRYGPLGGGK (SEQ ID NO:6). In embodiments, this sequence may be expressed by a modified cell, and as such, it may also comprise a transmembrane domain.
In certain examples, a described protein has a segment joined to another segment using linking amino acids, i.e., a linker. In certain examples, a described protein may comprise linking amino acids that connect a first component to a second component. Suitable amino acid linkers may be mainly composed of relatively small, neutral amino acids, such as glycine, serine, and alanine, and can include multiple copies of a sequence enriched in glycine and serine. In specific and non-limiting embodiments, the linker comprises 3, 4, 5, 6, 7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or more amino acids. In embodiments, a GE enzyme is connected to a leucine zipper using a 35 amino acid linker, a non-limiting example comprises
In non-limiting embodiments, modified cells of the disclosure may display only one plasma membrane coupled GE. In embodiments, modified cells of the disclosure may display more than one plasma membrane coupled GE. In a non-limiting embodiment, a sialidase is coupled to the plasma membrane by a transmembrane domain that is a component of the sialidase, such as in a fusion protein. In a non-limiting embodiment, a mucinase is coupled to the plasma membrane by a leucine zipper. The disclosure also includes a plasma-membrane coupled mucinase that comprises a transmembrane domain, a sialidase that is coupled to the plasma membrane by a leucine zipper, and combinations of the described modifications. Single cells that are modified to have both a plasma-membrane coupled sialidase that comprises a transmembrane domain and to have a mucinase that is coupled to the plasma membrane by a leucine zipper are encompassed by the disclosure.
In embodiments, any suitable plasma-membrane anchor sequence can be used. In embodiments, the transmembrane segment comprises about 20 residues of suitable hydrophobicity so that an α-helix can span the apolar core of a plasma membrane bilayer. Suitable transmembrane domains include, but are not limited to: a member of the tumor necrosis factor receptor superfamily, CD30, platelet derived growth factor receptor (PDGFR, e.g. amino acids 514-562 of human PDGFR; Chestnut et al., 1996, J Immunological Methods, 193:17-27; also see Gronwald et al., 1988, PNAS, 85:3435-3439); nerve growth factor receptor, Murine B7-1 (Freeman et al., 1991, J Exp Med 174:625-631), asialoglycoprotein receptor H1 subunit (ASGPR; Speiss et al. 1985 J Biol Chem 260:1979-1982), CD27, CD40, CD120a, CD120b, CD80 (B7) (Freeman et al., 1989, J Immunol, 143:2714-2272) lymphotoxin beta receptor, galactosyltransferase (e.g., GenBank accession number AF155582), sialyltransferase (E.G. GenBank accession number NM-003032), aspartyl transferase 1 (Asp1; e.g. GenBank accession number AF200342), aspartyl transferase 2 (Asp2; e.g. GenBank accession number NM-012104), syntaxin 6 (e.g. GenBank accession number NM-005819), ubiquitin, dopamine receptor, insulin B chain, acetylglucosaminyl transferase (e.g. GenBank accession number NM-002406), APP (e.g. GenBank accession number A33292), a G-protein coupled receptor, thrombomodulin (Suzuki et al., 1987, EMBO J, 6:1891-1897) and TRAIL receptor. The sequence of the plasma-membrane anchor used in the Examples of this disclosure is
In embodiments, the modified cells comprise any type of eukaryotic cells. In embodiments, the modified cells are mammalian cells. In embodiments, the modified cells are human cells. In embodiments, the cells are leukocytes. In embodiments, the cells are lymphocytes. In embodiments, the modified cells are immune cells. In embodiments, the modified cells are CD4+ T cells, CD8+ T cells (i.e, cytotoxic T cells), Natural Killer T cells (NKTs), i.e., a subset of T-cells that express T-cell receptor αβ chains as well as NK cell markers, or γδ T cells, or cells that are progenitors of T cells, such as hematopoietic stem cells or other lymphoid progenitor cells, such as immature thymocytes (double-negative CD4−CD8−) cells, or double-positive thymocytes (CD4+CD8+). In embodiments, the modified cells are NK cells, including but not limited to the CD56high, CD56low, CD56high/CD16high, CD56high/CD16low, and CD56low/CD16high subsets of NK cells. Such markers and expression levels will be understood by those skilled in the art, and can be used by the skilled artisan to differentiate subsets of NK cells. In embodiments, NK cells modified according to the disclosure are CD3−/CD56+ lymphocytes. In embodiments, the modified cells are macrophages or dendritic cells. In embodiments, the modified cells are primary cells that are obtained from an autologous or allogenic donor. In embodiments, the modified cells are primary cells that are obtained from an autologous or allogenic donor. In the embodiments, the modified cells are immortalized cells grown in culture. In the embodiments, the modified cells are pluripotent stem cells or induced pluripotent stem cells (iPSC). In the embodiments, the modified cells are derived from pluripotent stem cells or iPSC cells.
In embodiments, the modified cells are NK cells or T cells that have also engineered to express a chimeric antigen receptor (CAR). The type of CAR is not particularly limited, but in general is configured to enhance an anti-cancer immune response. Representative CARs are discussed in the Examples.
In one embodiment, a biological sample from an individual is analyzed to determine the presence of cancer cells. The cancer cells if present can be analyzed to characterize the glycocalyx exhibited by the cancer cells. Based at least in part on the characterization of the glycocalyx, a suitable single or combination of GE enzymes can be selected and used to produce and use modified cells according to this disclosure for therapy of the individual.
The disclosure includes isolated modified cells as described herein, and methods of making the modified cells. Compositions used in making the modified cells are also includes within the scope of the disclosure. As such, isolated modified GE enzymes, and expression vectors encoding them, are encompassed by the disclosure.
In embodiments, a genome of one or more cells as described herein may be modified to express any described protein, such as a GE enzyme, or a leucine zipper component, either of which may be modified to include a transmembrane domain. This approach can be performed using any designer nuclease. In embodiments, the nuclease is a RNA-guided CRISPR nuclease. A variety of suitable CRISPR nucleases (e.g., Cas nucleases) are known in the art. In specific and non-limiting embodiments, the Cas comprises a Cas9, such as Streptococcus pyogenes (SpCas9). Derivatives of Cas9 are known in the art and may also be used. Such derivatives may be, for example, smaller enzymes than Cas9, and/or have different proto adjacent motif (PAM) requirements. In a non-limiting embodiments, the Cas enzyme may be Cas12a, also known as Cpf1, or SpCas9-HF1, or HypaCas9. In embodiments, a protein may be administered directly to the cells. For example, a Cas protein and suitable guide RNA(s) may be administered to the cells as ribonucleoproteins (RNPs) using any suitable technique. Alternatively, the Cas protein may be introduced into the cells separately from the guide RNAs. In embodiments, a viral expression vector may be used to introduce sequences encoding one or more of the described proteins. Expression of any of the described proteins can be constitutive or induced. In embodiments, one or more of the described proteins are stably expressed within cells from an introduced genetic element, or from an extra-chromosomal element.
In embodiments, an effective amount of modified cells is administered to an individual in need thereof. In embodiments, an effective amount is an amount of cells that reduces one or more signs or symptoms of a disease and/or reduces the severity of the disease. An effective amount may also inhibit or prevent the onset of a disease or a disease relapse. A precise cellular dosage can be selected by the individual physician in view of the patient to be treated. Dosage and administration can be adjusted to provide sufficient levels of the described modified to maintain the desired effect. Additional factors that may be taken into account include the severity and type of the disease state, age, weight, and gender of the patient, desired duration of treatment, method of administration, time and frequency of administration, drug combination(s), reaction sensitivities, and/or tolerance/response to therapy. The described modified cells and pharmaceutical compositions comprising the modified cells can be administered to an individual in need thereof using any suitable route, examples of which include intravenous, intramuscular, intraperitoneal, intracerobrospinal, and subcutaneous routes. The modified cells may be introduced as a single administration or as multiple administrations or may be introduced in a continuous manner over a period of time.
In embodiments, the individual in need of modified cells of this disclosure has been diagnosed with or is suspected of having cancer. In embodiments, the cancer is a solid tumor or a hematologic malignancy. In embodiments, the cancer is renal cell carcinoma, breast cancer, prostate cancer, pancreatic cancer, lung cancer, liver cancer, ovarian cancer, cervical cancer, colon cancer, esophageal cancer, glioma, glioblastoma or another brain cancer, stomach cancer, bladder cancer, testicular cancer, head and neck cancer, melanoma or another skin cancer, any sarcoma, including but not limited to fibrosarcoma, angiosarcoma, osteosarcoma, and rhabdomyosarcoma, and any blood cancer, including all types of leukemia, lymphoma, and myeloma. In embodiments, the individual in need thereof has cancer cells that exhibit an altered glycocalyx, relative to the glycocalyx of non-cancer cells. In embodiments, cancer cells from the individual overexpress one or more mucins, relative to expression of mucins by non-cancer cells. In embodiments, cancer cells from the individual express one or more different mucines, relative to non-cancer cells. In embodiments, overexpressed mucins by cancer cells comprise galectin-3, GCNT1, or a combination thereof. In embodiments, cancer cells from the individual have an increased thickness of their glycocalyx, relative to the thickness of the glycocalyx of non-cancer cells. In embodiments, cancer cells from the individual have increased mucin surface density, relative to the surface density of non-cancer cells. In embodiments, cancer cells from the individual have increased sialic acid surface density, relative to the surface density of non-cancer cells. In embodiments, the described modified cells may be used prophylactically for any of the described types of cancer.
In embodiments, the described modified cells exhibit an improved activity relative to a control, such as control cells. In embodiments, administering described modified cells to an individual in need thereof exhibits an improved activity relative to a control. In embodiments, the control comprises unmodified cells. In embodiments, the control comprises cells that are modified to display only one GE enzyme, which is compared to modified cells that have more than one plasma-membrane coupled GE enzyme, such as at least two, or only two GE enzymes. In embodiments, the control comprises a different type of cell than the modified cell. In an embodiment, coupling of both a sialidase and a mucinase improves cell mediated cytotoxicity relative to unmodified cells. In an embodiment, the modified cells are NK cells, and exhibit improved cytotoxicity of cancer cells relative to unmodified NK cells. In an embodiment, the improved cytotoxicity comprises a 1-20 fold enhancement in cytotoxicity relative to cytotoxicity of non-modified NK cells. In embodiments, the modified NK cells exhibit at least a 10 fold increased cytotoxicity relative to non-modified NK cells. In an embodiment, using a described leucine zipper approach provides an improved property of modified cells, relative to cells modified using a SpyTag/SpyCatcher approach. This approach is known in the art, for example, from Zakeri et al., Proc Natl Acad Sci USA. 2012 Mar. 20; 109(12): E690-E697, the disclosure of which is incorporated herein by reference. Thus, in embodiments, the described GE enzymes are not coupled to the described cells using an intact or split fibronectin-binding protein. In embodiments, the described GE enzymes are not coupled to a plasma membrane by an antibody.
The modified cells can be used in combination with any other cancer therapy, including but not necessarily limited to chemotherapeutic agents, therapeutic antibodies, including but not limited to checkpoint inhibitors, and adoptive immunotherapies.
The following Examples are intended to illustrate but not limit the disclosure. Certain of the Examples involve a technique referred to herein as Ring-SAIM. In this regard, several imaging strategies have been developed to image the nanoscale dimensions and structural organization of the glycocalyx29,36-38. One of these technologies, called Scanning Angle Interference Microscopy (SAIM), is a localization microscopy technique based on the principles of Fluorescent Interference Contrast Microscopy (FLIC) whereby standing waves of excitation light are used to axially localize fluorescently labelled structures of interest with nanoscale precision24,39-42. To improve the precision of SAIM for glycocalyx materials research, the disclosure includes use of a pair of high-speed, galvanometer-controlled mirrors to generate a revolving circle, or “ring”, of excitation light at defined sample incidence angles, as described in Colville, et al., Scientific Reports 9, 1-13 (2019), the disclosure of which is incorporated herein by reference. The described Ring-SAIM approach improves sample illumination and, thus, the precision of measurement compared to standard SAIM implementations40.
Cell-surface mucins are anchored to the plasma membrane on one end by their transmembrane domain, forming a brush-like structure43-45. Polymer chains are predicted to extend out at higher surface densities due to steric repulsions in a more crowded structure46. In tumors and cell lines that overexpress cancer-associated mucins, high heterogeneity in the cell surface levels is typical47-48. To test how the expression level of mucin affects the material thickness of the glycocalyx, the disclosure provides a cellular model with highly titratable cell-surface levels of the cancer-associated mucin, Muc1. The disclosure provides an expression cassette for Muc1-GFP with 42 tandem repeats (TRs) under the control of a tetracycline-inducible promoter (
Noting that the height and compressibility of a polymer brush are known to scale with the polymer grafting density and polymer chain length49,50, the disclosure provides a description of quantitative relationships that described the thickening of the glycocalyx by increased expression of cell-surface mucins. We first validated that the Ring-SAIM setup could detect nanometer-scale changes in the glycocalyx thickness of live cells using Muc1 constructs of varying size (
The Ring-SAIM measurements in the 1E7 clone indicated how mucin chain length and density would resist close cellular contact with a surface (
This example provides a description of how specific molecular attributes of mucins contribute to the ensemble materials properties of the glycocalyx and, thus, protect against immune cell attack. For bottlebrush copolymers with densely grafted side chains, such as mucins, the size and charge of the side chains can strongly influence the polymer rigidity and persistence length54,55. Thus, we investigated how O-glycan truncation, elongation, and sialylation each contribute to the material thickness of the glycocalyx in 1E7 cells. 0-glycans were truncated in 1E7 cells through CRISPR/Cas9 mediated knockout (KO) of the C1GALT1 gene, which encodes the Core 1 synthase that governs the extension of α-GalNAc (
Utilizing Ring-SAIM, we measured the material thickness of the glycocalyx across the glycoengineered cell panel. At each Muc1-GFP induction level, the glycocalyx was significantly thinner in C1GALT1 and GNE KO cells compared to wild-type 1E7 cells (
Multivalent galectins have been proposed to crosslink and increase the barrier properties of cell-surface mucins56. The disclosure provides data demonstrating specific interactions of galectin-1 and -3 with Muc1-GFP on 1E7 cells (
These observations were consistent with galectins restricting the free chain extension of mucins, presumably through either intermolecular or intramolecular crosslinking. Fluorescence recovery after photobleaching (FRAP) showed that a large percentage of mucins were mobile in the cell-surface brush, indicating that intermolecular crosslinking by galectin was modest (
The described Ring-SAIM measurements and biochemical analyses provided guidance as to how the material and biochemical state of the glycocalyx could be manipulated by tuning mucin density and glycosylation. With this information, the disclosure provides an analysis of how specific glycocalyx states protect against immune cell attack. 1E7 cells at varying Muc1-GFP induction levels and their glycoengineered progenies were co-incubated with NK-92 to assess cytotoxicity. Across the glycoengineered panel, NK-92 mediated cytotoxicity against the mucin-expressing target cells was progressively reduced at higher doxycycline induction levels (
To assess the relationship between resistance to immune cell attack and glycocalyx material thickness, the NK-mediated cytotoxicity reported in
We next tested the ability of NK cells to attack cancer cell lines that endogenously express cell-surface mucins. We benchmarked the Muc1 surface levels of a luminal A breast cancer cell line, ZR-75-1, against the 1E7 cell line. Non-permeabilized cells were probed with conventional Muc monoclonal antibodies and analyzed by flow cytometry. We constructed distributions for Muc surface levels in 1E7 cells at minimal and saturating Muc1-GFP induction (1 ng/mL and 1,000 ng/mL doxycycline) to define standardized gates for low, moderate, and high Muc expression that could be applied to categorize other cell lines. The Muc1 surface levels were highly heterogeneous in the ZR-75-1 cell line and fully overlapped with the 1E7 low, medium, and high expression benchmarks (
Tethering Mucinase to NK Cells can Dramatically Increase their Cytotoxicity Against Mucin-Expressing Target Cells.
Enterohemorrhagic Escherichia coli (EHEC) utilize the mucin-specific metalloprotease, StcE, to breach the gut mucosal barrier and adhere to intestinal cells60. The disclosure includes effector immune equipped with StcE mucinase, which is extendable to other mucinases as further described herein, to breach the mucin barrier on target cells61. The StcE mucinase was highly effective at removing the cell-surface mucin brush from 1E7 cells (
The StcE mucinase possesses a C-terminal lectin domain, referred to as X409, in addition to its catalytic domain12. We tested whether the StcE mucinase would specifically bind to the NK-92 cell surface and remain bound without an additional molecular tether. The recombinant StcE bound stably to the NK-92 cell surface and was retained for prolonged periods even after stringent washing with buffer (
The StcE-equipped NK-92 (StcE-NK) cells were considerably more effective at killing mucin-expressing target cells than the unmodified NK-92 cells (
The disclosure also includes a modular strategy whereby StcE or other glycocalyx-editing (GE) enzymes can be displayed on the NK cell surface, which is considered to be extendable to other types of immune cells as described herein. The X409 lectin domain of StcE was swapped for the EE leucine zipper and the new fusion protein, StcE-EE Zip, was produced recombinantly (
The disclosure includes an analysis of the contact zone between the NK cells and their targets. We observed a localized disruption of the mucin barrier at the interface between StcE-NK cells and target cells. Visible deflection of microvilli on 1E7 cells in the contact zone with both unmodified NK-92 and StcE-NK indicated that both the wild-type and engineered cells could form viable mechanical connections with the target cells (
Surface display of StcE also improved NK attack against cancer cell lines expressing endogenous Muc1. StcE-NK cells attacked the ZR-75-1 cells more aggressively compared to unmodified NK-92 cells (
Chimeric antigen receptors (CAR) engagement in CAR-NK cells can override inhibitory signals deployed by tumor cells and directly trigger the effector cells' intrinsic cytolytic effector functions, as well as the release of pro-inflammatory cytokines63. We next tested whether a mucin barrier would also protect against CAR-NK attack. We engineered the NK-92 cell line to express a humanized anti-HER2 CAR with CD28 and CD3ζ signaling domains (
To test the ability of the glycocalyx to physically resist CAR-NK cell attack, HER2 was overexpressed in the 1E7 cell line (
We evaluated the killing efficiency of StcE-NK, CAR-NK, and StcE CAR-NK cells against cancer cells lines that endogenously express HER2 and cell-surface mucin. We first tested the ovarian cancer cell line, SKOV3, which expresses Muc1 and another large mucin called Podocalyxin66,67. SKOV3 cells had a thick glycocalyx of approximately 120 nm that was reduced to approximately 60 nm following mucinase treatment, confirming that cell-surface mucins were predominant structural elements of the glycocalyx of these cells (
The disclosure includes additional examples showing the modularity of the leucine-zipper based coupling strategy for display of mucinases and glycosidases, individually or in combination, on the immune cell surface. In an embodiment, a fusion protein comprised of the RR leucine zipper, an extracellular ALFA-Tag, a type I transmembrane domain (from PDGFR), and an intracellular superfolder GFP (sfGFP) was genetically encoded and stably expressed in NK-92 cells (
We next demonstrated that the cytotoxicity of NK cells could be enhanced through coupling of more than one type of enzyme on the immune cell surface. We generated recombinant V. cholerae sialidase fused to the EE leucine zipper. The recombinant protein was anchored to the surface of NK-92 cells expressing the membrane-anchored RR leucine (
As an alternative strategy to leucine-zipper based coupling, we also demonstrate that V. cholerae sialidase could be genetically expressed on the immune cells surface. The sialidase was genetically encoded as a fusion with a eukaryotic signal peptide, the PDGFR transmembrane domain, and an intracellular sfGFP tag (
It will be recognized from the foregoing Examples that the present disclosure demonstrates, among other aspects, that NK cell-mediated cytotoxicity shows a strong inverse relationship with the material thickness of the glycocalyx across widely varying states of glycosylation (
In malignant conditions, differential glycosylation frequently results in the generation of truncated O-glycan structures, such as the Tn and sialyl-Tn antigens9. The described results reveal that mucins with truncated O-glycans can still resist close contact with an apposed surface when expressed at high levels. In general, increased mucin surface density can compensate for O-glycan truncation or loss of sialylation to maintain the structural integrity and thickness of the glycocalyx. Thus, the mucin barrier that is characterized in this disclosure may be broadly relevant to cancer types that overexpress mucins, even across the highly heterogeneous patterns of glycosylation that are observed among specific tumor types and individual patients.
The disclosure includes a description of the quantitative relationships that describe the effect of mucin chain length and surface density on glycocalyx thickness. From the perspective of personalized medicine, mucin transcript levels, allele lengths, and surface levels are accessible quantities71,72. Combined with the scaling relations described herein, such metrics could be used to predict the likelihood of a mucin barrier and, thus, patient recalcitrance to immune therapies. Few transferable standards exist for benchmarking and reproducibly categorizing mucin surface levels in clinical or research samples across labs. The disclosure also provides as a potential standard the 1E7 cell line described herein, which allows titration of Muc1 levels with high reliability and reproducibility. The Examples also demonstrate that that StcE mucinase can be directly tethered to the immune cell surface via its X409 lectin domain to provide a performance benefit. The described approach of coupling the NK cells surface with StcE via its X409 lectin domain has some potential drawbacks: First, lectin-mediated affinity may not be sufficient for the prolonged conjugation necessary for in vivo applications. Second, the ligand for X409 on the NK surface is unknown and may be expressed at variable levels. Third, X409 lectin domain is unique to StcE, necessitating the development of alternative coupling strategies for other GE enzymes. To achieve tunable and universal display of GE enzymes on NK (and other type of immune cells) the disclosure demonstrates the GE-NK paradigm via a modular leucine-zipper coupling strategy. GE enzymes that could be coupled with this approach include other mucolytic agents, sialidase, and enzymes that digest other common structural elements in the glycocalyx, such as hyaluronic acid. The disclosure includes GE enzyme surface display encoding a fused membrane anchor. The disclosure includes use of StcE as well as more selective mucinsases, including but not necessarily limited to the above described BT4244 from Bacteroides thetaiotaomicron and M60-like protease AM0627 from Akkermansia muciniphila, which preferentially cleaves mucins with truncated, cancer-associated O-glycans76.
This Example provides a description of the materials and methods used to produce the results described in Examples 1-6.
MCF10A cells were cultured in DMEM/F12 media (Thermo Fisher Scientific) supplemented with 5% horse serum (Thermo Fisher Scientific), 20 ng/mL EGF (Pepro Tech), 10 mg/ml insulin (Sigma), 500 ng/mL hydrocortisone (Sigma), 100 ng/mL cholera toxin (Sigma) and 1× penicillin/streptomycin (Thermo Fisher Scientific) at 37° C. in 5% CO2. HEK293T cells was cultured in DMEM high glucose media (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and 1× penicillin/streptomycin (Thermo Fisher Scientific) at 37° C. in 5% CO2. ZR-75-1 cells and T47D cells were cultured in RPMI media (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and 1× penicillin/streptomycin (Thermo Fisher Scientific) at 37° C. in 5% CO2. SKOV-3 and SK-BR3 cells were cultured in McCoy's 5a Medium Modified (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum and 1× penicillin/streptomycin. NK-92 cells (ATCC CRL-2407) were cultured in α-MEM without ribonucleosides media (Thermo Fisher Scientific) supplemented with 12.5% fetal bovine serum (Thermo Fisher Scientific), 12.5% horse serum (Thermo Fisher Scientific), 0.2 mM Myo-inositol (Sigma Aldrich), 0.1 mM 2-mercaptoethanol (Thermo Fisher Scientific), 0.02 mM folic acid (Millipore Sigma), 100 U/ml recombinant human IL-2 (Pepro Tech), and 1× penicillin/streptomycin (Thermo Fisher Scientific) at 37° C. in 5% CO2.
The following antibodies were used: anti-Human Muc Clone HMPV (555925, BD Biosciences), anti-Human Muc Janelia Fluor 549 (NBP-2-47883JF549, Novus Biologicals), Recombinant anti-GNE antibody (ab189927, Abcam), anti-GCNT1 antibody (ab102665, Abcam), anti-Galectin-3 N-20 (19280, Santa Cruz), anti-Galectin-1 (ab108389, Abcam), mouse anti-3-Actin Clone C4 (47778, Santa Cruz), anti-ErbB2/HER2 Clone 3B5 (ab16901, Abcam), APC conjugated anti-hErbB2/HER2 (FABI129A, R&D systems), APC conjugated anti-Human perforin (Clone dG9, 30811, BioLegend), anti-ErbB2/HER2 Clone 3B5 (ab16901, Abcam), Anti-6× His-tag antibody (ab9108, Abcam). The following secondary antibodies were used: goat anti-mouse IgG DyLight 800 4×PEG conjugate (SA535521, Invitrogen), goat anti-mouse IgG DyLight 680 conjugate (35518, Invitrogen), goat anti-rabbit IgG Alexa Fluor 647 conjugate (A21245, Thermo Fisher Scientific), goat anti-mouse Alexa Fluor 647 conjugate (A28181, Invitrogen), and rabbit anti-goat IgG Alexa Fluor 647 conjugate (A21446, Invitrogen). Lectins used were: CF640R-conjugated PNA (#29063, Biotium), biotin-conjugated MAL-II (B-1265-1, Vector Laboratories). Biotinylated lectins were detected using NeutrAvidin Protein (DyLight 800 conjugated, 22853; DyLight 650 conjugated, 84607; both from Thermo Fisher Scientific). For tetracycline-inducible systems, doxycycline was used for induction (204734, Santa Cruz). For western blot, RIPA lysis buffer (89900, Thermo Fisher Scientific) and Halt Protease and Phosphatase Inhibitor Cocktail (78446, Thermo Fisher Scientific) were used. TD139 (28400, Cayman Chemical) was used to inhibit galectin-3 binding to Muc. For digesting N-glycan, PNGase F (P0704S, New England Biolabs) was used. For detecting dead cell population by NK-mediated killing assay, propidium iodide (P4170, Sigma) and Annexin V conjugated CF647 (#29003, Biotium) were used. For SAIM measurement, MemGlow dye (MemGlow 560: MG02-2; MemGlow 590: MG03-10; MemGlow 640: MG04-02, Cytoskeleton) or CellBrite Steady 550 (#30107-T, biotium) were used to label cell membrane. Myc-Tag (9B11) Mouse mAb conjugated with Alexa Fluor 647 (#2233S, Cell Signaling Technology) was used to detect surface level of a cognate leucine zipper level on NK-92 cells
cDNAs for mOxGFP-tagged Muc1 were previously cloned into a PiggyBac expression system containing a tetracycline-inducible promoter (PCMV-min-tetO7) and a separate puromycin selectin cassette (pPB tetOn PuroR)43,77. In this work, Muc1 cDNAs with approximately 42 native tandem repeats (TRs; Muc1 mO×GFP ACT) or either 0, 10, 21, and 42 perfect TRs of PDTRPAPGSTAPPAHGVTSA (SEQ ID NO:9) (0TR, 10TR, 21TR, 42TR) were used. Lentiviral plasmids for constitutive expression of human GCNT1, LGALS3 (galectin-3), HER2, and an anti-HER2 chimeric antigen receptor (CAR) were prepared by inserting the cDNAs in place of GFP in pLenti CMV GFP Hygro (Addgene #17446), using the BamHI and BsrGI restriction sites. The unmodified human cDNA for GCNT1 was generated by custom gene synthesis (General Biosystems). The human HER2 cDNA was amplified by PCR from the plasmid, HER2 WT (Addgene #16257), with primers that added 5′ BamHI and 3′ BsrGI restriction sites: 5′-TAATCTAGACCCAAGCTTGGGGCAG-3′ (SEQ ID NO:10) and 5′-GCTGTCGACGAGCTCGGTACCAAGCTT-3′ (SEQ ID NO:11). cDNA for human LGALS3 was amplified by PCR from a pET21a expression vector (gift of C. Bertozzi) with primers that added 5′ BamHI and 3′ BsrGI restriction sites: 5′-TAAGCGGATCCGAATTCTATGGCAGACAATT-3′ (SEQ ID NO:12) and 5′-TGCTTTGTACATTATATCATGGTATATGAAGC-3′ (SEQ ID NO:13). cDNA for a previously reported HER2-specific CAR65—including a humanized FRP5 scFV, CD8a hinge, CD28 transmembrane domain, CD28 signaling domain, and CD3ζ signaling domain—was synthesized with a 2A peptide/mTagBFP2 expression reporter by custom gene synthesis (Twist Bioscience) (See Supplemental Table 1 for the complete sequence). A pLentiCRISPR V2 plasmid for knockout of GNE was created by inserting the oligos, 5′-CACCGACATCAAGTTCAAAGAACTC-3′ (SEQ ID NO:14) and 5′-AAACGAGTTCTTTGAACTTGATGTC-3′ (SEQ ID NO:15), into the BsmBI restriction site following standard protocols78. Recombinant GFP nanobody cDNA (PDB #30GO E) was generated by custom gene synthesis (IDT) and cloned into the BamHI and HindIII restriction sites of pTP1112 (Addgene #104158). cDNA for Sialidase with IgK leader sequence, ALFA-Tag, type I transmembrane anchor (PDGFRP) from pDisplay system (Thermo Fisher) and superfold GFP (sfGFP) was synthesized by custom gene synthesis and inserted into a PiggyBac expression system containing a tetracycline-inducible promoter and a separate hygromycin selectin cassette.
MCF10A cells stably expressing the rtTA-M2 tetracycline transactivator were prepared by lentiviral transduction using the pLV rtTA-NeoR plasmid as previously described.38 For preparation of mucin-expressing cell lines, MCF10A rtTA-M2 cells were co-transfected with the PiggyBac hyperactive transposase and PiggyBac plasmids containing ITR-flanked expression cassettes (i.e. pPB tetOn PuroR plasmids) using the NEPA21 Electroporator (NEPAGENE) and subsequently selected with 1 μg/mL puromycin. The stable MCF10A line expressing native Muc mOxGFP ACT was clonally expanded by limiting dilution of single cells in 96-well plates. An individual clone, 1E7, was isolated that exhibited low leaky expression of Muc mOxGFP ACT and titratable Muc surface levels with doxycycline induction.
Progeny of the 1E7 clone overexpressing GCNT1, LGALS3, and HER2 were prepared by transduction with lentiviral particles generated using the respective pLenti CMV Hygro plasmids. Cell selection was with 200 μg/mL hygromycin. Control MCF10A rtTA-M2 cells overexpressing HER2 were similarly prepared. Knockout (KO) of GNE in the 1E7 clone was achieved by lentiviral transduction using the pLentiCRISPR V2 system and selection with 200 μg/mL hygromycin. The selected cells were clonally expanded and individual clones were screened for double knockout of GNE by Sanger sequencing of genomic DNA that was isolated using the Quick-DNA miniprep kit (Zymo, D3024).
KO of C1GALT1 in the 1E7 cells was achieved using the Alt-R CRISPR/Cas9 system (IDT) with homology-directed repair (HDR) templates that introduced an in-frame stop codon and an EcoRI restriction site for screening. CRISPR RNA (crRNA) and HDR template for C1GALT1 KO were: 5′-GTAAAGCAGGGCTACATGAG-3′ (SEQ ID NO:16) and 5′-CTTTGGGAGAAGATTTAAGCCTTATGTAAAGCAGGGCTACTAAGAATTCAGTGG AGGAGCAGGATATGTACTAAGCAAAGAAGCCTTGA-3′ (SEQ ID NO:17). CRISPR/Cas9 editing was conducted according to manufacturer's protocol for the Alt-R system. Briefly, 1E7 cells were transfected with precomplexed crRNA and ATTO550-tracrRNA, along with the HDR template, using electroporation. After 48 hours, transfected 1E7 cells were clonally expanded by limiting dilution in 96-well plates. To screen for successful knockouts, genomic regions encompassing the targeted CRISPR/Cas9 cut sites were amplified by PCR and analyzed by EcoRI restriction digest and Sanger sequencing. PCR primers for C1GALT were 5′-GGAGGATAATAGTTGTAATTCCAGTACCAAAAC-3′ (SEQ ID NO:18) and 5′-TCAAAACCTAGAGAAAAAGGCCAAACAC-3′ (SEQ ID NO: 19). PCR primers for GNE were 5′-AGTGGTTAAGGACTTGAAACTG-3′ (SEQ ID NO:20) and 5′-TCTACTAAGCGGCATCATTG-3′ (SEQ ID NO:21). Sequencing primer for C1GALT1 was 5′-ACACGTCAAAGCTACTTGG-3′ (SEQ ID NO:22) and sequencing primer for GNE was 5′-AGTGGTTAAGGACTTGAAACTG-3′ (SEQ ID NO:23). A NK-92 line expressing the HER2-CAR was prepared by transduction with lentiviral particles generated using the pLenti CMV Hygro plasmid. Cell selection was with 200 μg/mL hygromycin. Selected cells were sorted for high mTagBFP2 signal. A NK-92 line expressing a cognate leucine zipper (RR) was prepared by lentivirus transduction using pTwist SFFV Puro plasmids (Twist Bioscience). Cells were selected with 2 μg/mL Puromycin for 2 weeks or sorted with sfGFP expression to collect higher expression of RR-Zip on the cell surface of NK cells. (See Supplemental Table 2). NK-92 line stably expressing the rtTA-M2 tetracycline transactivator was prepared by lentiviral transduction using the pLV rtTA-NeoR plasmid as previously described.38 For preparation of Sialidase-expressing cell lines, NK-92 rtTA-M2 cells were co-transfected with the PiggyBac hyperactive transposase and PiggyBac plasmids containing ITR-flanked expression cassettes (i.e. pPB tetOn Hygro plasmids) using the NEPA21 Electroporator (NEPAGENE) and subsequently selected with 200 μg/mL hygromycin. The stable NK-92 line expressing Sialidase-PDGFRβ-sfGFP was sorted with higher sfGFP expression.
Cells were plated and induced with 1 g/mL doxycycline for 24 hours before lysis with RIPA buffer. Lysates were separated on NuPAGE 3-8% Tris-Acetate gels or NuPAGE 4-12% Bis Tris gels and transferred to low-fluorescence PVDF membranes (Milipore Sigma, IPFL07810). Primary antibodies were diluted at 1:500 and fluorophore-conjugated or biotinylated lectins were diluted to 2 g/mL in 5% BSA TBST and incubated overnight at 4° C. Secondary antibodies and Neutravidin-DyLight 800 were diluted at 1:1000 or 1 g/mL in 5% BSA TBST and incubated for 1 hour at room temperature. Blots were imaged on a ChemiDoc MP Imaging System (Bio-Rad)
Cells were plated, grown for 24 hours, and then induced with various concentrations of doxycycline for 24 hours. Adherent cells were detached by incubating with Accutase (Innovative Cell Technologies) at 37° C. for 10 minutes. GFP nanobody conjugated Alexa Fluor 647, Neutravidin conjugated DyLight 650, CF640R PNA, Alexa Fluor 647 HPA, and biotin MAL-II were diluted 1:200 in 0.5% BSA PBS and incubated with cells at 4° C. for 30 minutes for each stain. For analysis of Muc1 cell surface expression level, anti-Human Muc Clone HMPV was diluted 1:200 in 0.5% BSA PBS and incubated with cells at 4° C. (for 1 hour. Secondary labelling was with Alexa Fluor 647 conjugated goat anti-mouse, diluted 1:1000 in 0.5% BSA PBS and incubated with cells at 4° C. for 1 hour. For analysis of StcE mucinase binding to NK-92 cells, NK cells were incubated with StcE at 37° C. for 1 hour. Cells were thoroughly washed to remove StcE and coculture with target cells. Anti-6× His-tag antibody was diluted 1:100 in 0.5% BSA PBS and incubated with cells at 4° C. for 1 hour. Secondary labelling was with Alexa Fluor 647 goat anti-rabbit IgG, diluted 1:500 in 0.5% BSA PBS or FluoTag-X2 anti-AFLA conjugated with Alexa Fluor 647 was diluted 1:200 in 0.5% BSA PBS and incubated with cells at 4° C. for 1 hour. To measure cell surface level of leucine zipper or sialidase on NK-92 cells, Myc-tag antibody was diluted 1:10 in 0.5% BSA PBS and incubated with cells at 4° C. for 1 hour. To analyze fluorescent labelled cells, Attune N×T Flow cytometry (Thermo Fisher) was used.
MCF10A cells were plated and induced with 1, 100, and 1,000 ng/mL doxycycline for 24 hours. For endogenous galectins staining of cells, galectin antibodies were diluted 1:50 with 0.5% in 0.5% BSA PBS and incubated on 1E7 clone with 1,000 ng/mL of doxycycline at 4° C. for 1 hour after being fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. For cell-surface galectin-3, TD139 (28400, Cayman Chemical) was diluted to 1, 10, 50 μM in MCF10A media and incubated on 1E7 clone with 1,000 ng/mL of doxycycline at 37° C. for 2 hours. TD139-treated cells were further incubated with anti-galectin-3 antibody at 4° C. for 30 minutes. For GCNT1 imaging, 1E7 clone cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 after induction of 1,000 ng/mL doxycycline. GCNT1 antibody was diluted 1:50 in 0.5% BSA PBS and incubated on samples at 4° C. for 1 hour. For NK-92 cell imaging, NK-92 cells were incubated with 100 nM StcE or StcE variants in NK cell culture media for 1 hour at 37° C. and washed thoroughly with phenol red-free DMEM/F12 supplemented with 5% horse serum, 20 ng/mL EGF, 10 mg/ml insulin, 500 ng/mL hydrocortisone, 100 ng/mL cholera toxin and 1× penicillin/streptomycin. 1E7 cells were plated to glass bottom dishes for 1 days and induced with 1,000 ng/mL doxycycline for 24 hours. StcE-treated NK cells were plated on 1E7 cells at 37° C. for 4 hours. For Hyaluronic acid imaging, cells were incubated with 2.5 pg/mL Hyaluronic Acid Binding Protein Biotinylated (385911, Millipore Sigma) at 4° C. for 1 hour. For other Muc1-GFP clones imaging, two other clones (2E4 and 2G9) were plated onto glass-bottom dishes and induced with 1, 100, 1,000 ng/mL of doxycycline (sc-204734, Santa Cruz) for 24 hours. Secondary antibodies were diluted 1:500 in 0.5% BSA PBS and incubated with cells at 4° C. for 1 hour. Samples were imaged on a LSM 800 confocal microscope using 10× (NA: 0.3 Air), 20× (NA: 0.8 Air), and 63× (NA: 1.4 Water) objectives (Zeiss).
Human LGALS1 and LGALS3 constructs in pET21 were recombinantly expressed in E. coli strain NiCo21 (DE3) (New England Biolabs). Transformed bacteria were grown in LB at 37° C. until an OD600 of 0.6-0.8 was reached. Expression was induced with 0.3 mM IPTG, and protein was produced overnight at 20° C. Cells were harvested by centrifugation at 3,000 g for 20 minutes and lysed by a pressurized homogenizer with cOmplete protease inhibitor Cocktail (Roche) and 1 mg/mL lysozyme (Sigma). The lysate was centrifuged at 20,000 rpm for 45 minutes at 4° C., and the supernatant was incubated with O-lactosyl Sepharose resin for 1 hour at 4° C. before loading into a gravity column. The protein was eluted with 0.1 M β-lactose (Santa Cruz Biotech) and 8 mM DTT (Sigma). The partially purified protein was polished on a HiPrep 16/60 Sephacryl S-100 HR (GE Healthcare Life Sciences) column equilibrated with 0.1 M β-lactose and 8 mM DTT. The final protein was then concentrated by using Amicon Ultra (3 kD MWCO for Galectin-1; 10 kD MWCO for Galectin-3) filters (Millipore Sigma). Conjugation of β-lactose to Sepharose 6B (Sigma) to synthesize the β-lactosyl Sepharose was as previously described79.
StcE mucinase Expression and Purification
The cDNA for StcE-A3560 was synthesized by custom gene synthesis (Twist Bioscience) and inserted into the pET28b expression vector (See Supplemental Table 2). StcE E447D was generated using Q5 Site-Directed Mutagenesis Kit (New England Biolabs) with primers 5′-TCAGTCATGACGTTGGTCATAATTATG-3′ (SEQ ID NO:24) and 5′-ACTCATTCCCCAATGTGG-3′ (SEQ ID NO:25). StcE-ΔX409 was also generated using the Q5 Site-Directed Mutagenesis Kit with primers 5′-TAACTCGAGCACCACCAC-3′ (SEQ ID NO:26) and 5′-ATTTACAGTATAGGTAAGTCCTTC-3′ (SEQ ID NO:27) (See Supplemental Table 2). The general design of StcE EE Zip is as follows62,80. The 35-aa glycine/serine (GGGGS)7 (SEQ ID NO:7) linker and leucine zipper (EE zip) construct was synthesized by custom gene synthesis (Twist Bioscience) and inserted into the pET28b StcE ΔX409 vector using NEBbuilder HiFi DNA Assembly (New England Biolabs) with primers 5′-TGAGATCCGGCTGCTAAC-3′ (SEQ ID NO:28) and 5′-ATTTACAGTATAGGTAAGTCCTTCTG-3′ (SEQ ID NO:29) (See Supplemental Table 2). A cognate leucine zipper (RR) with a IgK leader sequence, HA-tag, Myc-tag, type I transmembrane anchor (PDGFRP) from pDisplay system (Thermo Fisher) was synthesized by custom gene synthesis and inserted into pTwist SFFV Puro lentivirus vector (Twist Bioscience) (See Supplemental Table 2). An alternative leucine zipper (RR) construct with a IgK leader sequence, extracellular ALFA-Tag, type I transmembrane anchor (PDGFRP) from pDisplay system (Thermo Fisher) and superfold GFP (sfGFP) was synthesized by custom gene synthesis and inserted into pTwist SFFV lentivirus vector (Twist Bioscience) (See Supplemental Table 2). Cells were harvested by centrifugation at 3,000 g for 20 minutes, resuspended in lysis buffer (20 mM HEPES, 500 mM NaCl and 10 mM imidazole, pH 7.5) with cOmplete protease inhibitor Cocktail (Roche), and lysed by a pressurized homogenizer. StcE was purified by immobilized metal affinity chromatography (IMAC) on a GE AKTA Avant FPLC system. The lysate was loaded onto a HisTrap HP column (GE Healthcare Life Sciences), washed with 20 column volumes of wash buffer (20 mM HEPES, 500 mM NaCl and 20 mM imidazole, pH 7.5.), and eluted with a linear gradient of 20 mM to 250 mM imidazole in buffer (20 mM HEPES and 500 mM NaCl, pH 7.5.). The elution fractions containing target protein were collected and polished on a HiPrep 26/60 Sephacryl S-200 HR (GE Healthcare Life Sciences) column equilibrated with storage buffer (20 mM HEPES and 150 mM NaCl, pH 7.5). The final protein was then concentrated by using Amicon Ultra 30 kDa MWCO filters (Millipore Sigma)61.
The cDNA for AM0627, BT4244, and Sialidase with 35-aa glycin/serine (GGGGS)7 linker and leucine zipper (EE zip) construct were synthesized by custom gene synthesis (Twist Bioscience) and inserted into the pET28b expression vector (See Supplemental Table 2). Cells were harvested by centrifugation at 3,000 g for 20 minutes, resuspended in lysis buffer (20 mM HEPES, 500 mM NaCl and 10 mM imidazole, pH 7.5) with cOmplete protease inhibitor Cocktail (Roche), and lysed by a pressurized homogenizer. Mucinases was purified by immobilized metal affinity chromatography (IMAC) on a GE AKTA Avant FPLC system. The lysate was loaded onto a HisTrap HP column (GE Healthcare Life Sciences), washed with 20 column volumes of wash buffer (20 mM HEPES, 500 mM NaCl and 20 mM imidazole, pH 7.5.), and eluted with a linear gradient of 20 mM to 250 mM imidazole in buffer (20 mM HEPES and 500 mM NaCl, pH 7.5.). The elution fractions containing target protein were collected. The final protein was then concentrated by using Amicon Ultra 30 kDa MWCO filters (Millipore Sigma).
Recombinant GFP nanobody (PDB #3OGO E) was prepared in chemically competent NiCo21 (DE3) E. coli (NEB) using the pTP1112 plasmid. Transformed bacteria were grown in LB at 37° C. until an OD600 of 0.6 was reached. The cultures were induced with 0.5 mM IPTG overnight at 24° C., harvested, resuspended in B-PER (Thermo Fisher Scientific) and vortexed for cell lysis. The lysates were cleared by centrifugation at 10,000 g for 20 minutes at 4° C. The His-tagged nanobody was purified by IMAC according to standard protocols. Briefly, supernatant diluted into 1×Ni-NTA binding buffer was bound to equilibrated Ni-NTA resin (Qiagen, 30210) for 20 minutes at 4° C., with end-over-end mixing. The resin was added to a spin column, washed thoroughly, and incubated with the Ni-NTA elution buffer for 20 minutes at 4° C., mixing end-over-end. Eluted protein was exchanged into storage buffer (pH 7.4 PBS) using Zeba 7K MWCO desalting columns or by overnight dialysis with 10K MWCO Snakeskin dialysis tubing. Eluted proteins were then sterile filtered and snap-frozen for long-term storage at −80° C. Nanobody-containing constructs were mixed with 0.1% w/v sodium azide prior to snap-freezing. For fluorescent GFP nanobody, purified GFP nanobody was labeled with Alexa Fluor 647 NHS Ester (Thermo Fisher Scientific) per the manufacturer's protocol.
Target MCF10A cells were fluorescently labeled by incubation with 1 M CellTracker Green CMFDA Dye (Invitrogen) in MCF10A growth media for 10 minutes. 3×104 labeled target cells were suspended with varying ratios of NK-92 effector cells in 200 μL of growth media of target cell line in absent of IL-2 and cocultured in an ultra-low attachment U-bottom 96-well plate (Corning) for 4 hours at 37° C. in 5% C02. For all StcE-treated NK cell experiment, unless otherwise indicated, NK cells were incubated with 100 nM StcE at 37° C. for 1 hour. Cells were thoroughly washed to remove StcE and coculture with target cells. After 4 hours, propidium iodide (PI; 20 μg/mL, Sigma) was added to each well for 10 minutes and washed with 0.5% BSA PBS. For different Muc surface level on ZR-75-1, anti-Human Muc Janelia Fluor 549 was diluted 1:200 in 0.5% BSA PBS and incubated with ZR-75-1 cells at 4° C. for 1 hour and sorted the cells with defined standardized gates for low, moderate, and high Muc expression by FACS (Sony MA900 Cell Sorter). Sorted target cells were cocultured with varying ratios of NK-92 effector cells for 4 hours. Annexin V conjugated with CF647 (0.5 g/mL, Biotium) was diluted in HEPES-buffered saline containing 2.5 mM calcium chloride and added to each well for 15 minutes. NK cell cytotoxicity was evaluated by flow cytometry as previously described16,81. At least 1×104 target cells were analyzed after electronic gating on CellTracker Green. Percent cytotoxicity was calculated as 100×(experimental % dead−spontaneous % dead)/(100−spontaneous % dead), where experimental % dead was the percentage of PI positive target cells in NK cocultures and spontaneous % dead was the percentage of PI positive control target cells cultured in the absence of effector cells.
Fluorescence Recovery after Photobleaching
FRAP experiments were performed by using a Zeiss i880 confocal microscope with 63×N.A. 1.4 Oil immersion lens. Five pre-bleached images were firstly taken and photobleaching was performed with 488 nm laser for about 10 seconds to bleach a target area of 2.55 μm×2.55 μm. 1E7 clone (n=11) and 1E7 GCNT1 overexpression (n=10) were used to measure mobile fraction of Muc1-GFP. 10 μM recombinant human galectin-1 was incubated with 1E7 GCNT1 cells for 10 minutes at 37° C. (n=9). Intensity profiles were corrected by normalizing to the time-course decay of GFP signal in non-bleached area by using MATLAB code from Advanced Imaging Center (https://www.mathworks.com/matlabcentral/fileexchange/47327-frap-zip)
Silicon wafers with ˜1900 nm thermal oxide (Addison Engineering) were diced into 7 mm by 7 mm chips, and the oxide layer thickness for each chip was measured with a FilMetrics F50-EXR. Prior to use, the chips were then cleaned with 1:1 methanol and hydrochloric acid for 15 minutes, followed by 1 minutes in a plasma cleaner (Harrick Plasma, PDC-001). The chips were incubated with 4% (v/v) (3-mercaptopropyl)trimethoxysilane in absolute ethanol for 30 minutes at room temperature. After washing with absolute ethanol, the chips were subsequently incubated with 4 mM 4-maleimidobutyric acid N-hydroxysuccinimide ester in absolute ethanol for 30 minutes and rinsed with PBS. 50 μg/mL human plasma fibronectin in PBS was incubated with the functionalized chips overnight at 4° C. for conjugation. Cells were seeded onto the chips at 2×104 cm−2 in full culture medium with varying doxycycline concentrations and incubated for 24 hours. Cells expressing different length of Muc1 were labelled with GFP nanobody conjugated to Alexa Fluor 647 in MCF10A growth media for 10 minutes at 37° C. Cells were labelled with MemGlow dye or CellBrite Steady 550 in serum-free, phenol red-free DMEM/F12 for 10 minutes at 37° C. Sample were then rinsed 3× in PBS, inverted into a 35 mm glass-bottom imaging dish containing imaging buffer (MemGlow dye or CellBrite dye: serum-free, phenol red-free DMEM/F12, Alexa Fluor 647-conjugated GFP nanobody: phenol red-free DMEM/F12 supplemented with 5% horse serum, 20 ng/mL EGF, 10 mg/ml insulin, 500 ng/mL hydrocortisone, 100 ng/mL cholera toxin and 1× penicillin/streptomycin) and imaged at 37° C. For NK-92 cell, the cleaned chips were incubated with 2.5% (v/v) (3-mercaptopropyl)trimethoxysilane, 4.5% (v/v) deionized water and 0.9% (v/v) acetic acid) in methanol overnight at 4° C. After washing with PBS, the chips were reacted with 0.1 mg/mL of maleimide-activated neutravidin protein (31007, Thermo Scientific) with 50 μg/mL fibronectin for 1 hour at room temperature and rinsed with PBS. A biotin-conjugated MAL-II were diluted 1:200 in PBS and incubated with the chips for 1 hour at room temperature. NK-92 cells were seeded onto the chips at 2×104 cm−2 in full culture medium and incubated for 1 hour82.
Scanning angle interference microscopy (SAIM) was conducted on a custom circle-scanning microscope.40 The core of the setup was an inverted fluorescence microscope (Ti-E, Nikon). The excitation lasers (488 nm, Coherent; 560 nm, MPB Communications Inc.; and 642 nm, MPB Communications Inc.) were combined into a colinear beam by a series of dichroic mirrors (Chroma). The combined output beam was attenuated and shuttered by an AOTF (AA Opto-Electronic). The beam was directed onto a pair of galvanometer scanning mirrors (Cambridge Technology). The image of the laser on the scanning mirrors was magnified and relayed to the sample by two 4 f lens systems, a beam expanding telescope and a scan lens/objective lens combination. The beam expander was formed by f=30 mm and f=300 mm achromatic lenses with a m=1 zero-order vortex half-wave plate positioned between them and positioned 2 f from the 300 mm achromatic scan lens (Thorlabs). SAIM experiments were performed with a 60×N.A. 1.27 water immersion objective (Nikon). Fluorescence emission was collected with a quad band filter cube and single band filters (TRF89901-EMv2, Chroma) mounted in a motorized filter wheel (Sutter). Images were acquired with a Zyla 4.2 sCMOS (Andor) camera or an iXon 888 EMCCD (Andor) using the microscope's 1.5× magnifier for a total magnification of 90×. The open-source software Micro-Manager was used for camera and filter wheel control and image acquisition. The circle-scanning galvometers were operated in an autonomous fashion using a custom-designed 16-bit PIC microcontroller, which has been described previously40.
For SAIM, 32 images were acquired at varying incidence angles for the circle-scanned excitation beam. During a typical image acquisition sequence, changes in the scanned incidence angle were triggered by a TTL signal from the camera to the microcontroller. The incidence angles were evenly spaced from 5 to 43.75 degrees with respect to the wafer normal in the imaging media. To obtain the reconstructed height topography of the samples, the raw image intensities, Ij, at each incidence angle, θj, were fit pixelwise by nonlinear least-squares to an optical model:
where H is the unknown sample height and A and B are additional fit parameters. The vortex half-wave plate in the optical setup maintained the s-polarization of the circle-scanned excitation laser. For s-polarized monochromatic excitation of wavelength, λ, the probability of excitation, ƒ(θj, H), for the system is given by:
where the phase shift, ϕ, and the reflection coefficient for the transverse electric wave, rTE, are given by:
where ki is the wavenumber in material i; nSi, nox and nb are the refractive index of the silicon, silicon oxide and sample, respectively; θSi, θox and θb are the angles of incidence in the silicon, silicon oxide and sample, respectively; and dox is the thickness of the silicon oxide layer. The angles of incidence in silicon oxide and silicon were calculated according to Snell's Law. To quantify the glycocalyx thickness of cells, the average height above the silicon substrate was calculated for a 100×100 pixel subregion in each cell. The glycocalyx thickness was reported as the height of the localized GFP nanobody, MemGlow dye or CellBrite dye signal minus the height of the fluorescently labeled fibronectin layer on the silicon substrate. Packages for fitting SAIM image sequences with the above model have been implemented in C++ and Julia.
This reference listing pertains to the Examples above and is not an indication that any reference is material to patentability.
This Example expands on the foregoing Examples and description. Example 8 is illustrated by
Other embodiments of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the disclosure being indicated by the following claims.
This application claims priority to U.S. provisional patent application No. 63/285,947, filed Dec. 3, 2021, the entire disclosure of which is incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/080937 | 12/5/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63285947 | Dec 2021 | US |
