Patent Grant
11560419
Various embodiments disclosed herein relate generally to various recombinant prokaryotic collagen-like or triple helical proteins, methods of manufacture and use.
The concept of drug delivery to tumors holds promise for improved therapeutics, enabling specific delivery of drugs and biologics to pathological tissue, thereby avoiding off-target effects, and reducing the toxicity of currently used therapies. To target these sites, drugs are conjugated to carriers that recognize the tumor microenvironment, such as overexpressed cell-surface and secreted markers.
However, current cancer biomarkers still pose a significant off-target risk due to their expression on healthy cells.
Collagens are major structural proteins present in the extracellular matrix (ECM) of animals. They are defined by a characteristic triple-helix structure that requires a (Gly-Xaa-Yaa), repeating sequence. The residues found in the Xaa and Yaa positions are commonly proline, where Pro in the Yaa position in mammalian collagens is post-translationally modified to hydroxyproline (Hyp) which enhances helical stability. Prokaryotic collagen-like proteins form stable triple helices without the presence of hydroxyprolines and have been shown to be expressed in several bacteria, including Streptococcus pyogenes and filaments on Bacillus anthracis spores.
Fibronectin is a high molecular weight glycoprotein of the ECM and is found throughout the human body in two main forms. Plasma fibronectin, which is produced by hepatocytes in the liver, is secreted in a soluble form into the blood stream, whereas cellular fibronectin (cFn), is produced by a variety of cells, including fibroblasts, and is deposited as an insoluble crosslinked protein in tissues. Humans express over 20 cFn isoforms due to alternative splicing of a single fibronectin gene, FN1, that differ from plasma fibronectin by the inclusion of additional fibronectin type III repeats, extra domains A (EDA/EIIIA), B (EDB/EIIIB) and the variable connecting segments (VCS). Specifically, EDA/cFn and EDB/cFn are expressed during embryogenesis but only at negligent levels in normal adult tissue. However, the expression of both EDA/cFn and EDB/cFn isoforms is substantially upregulated within the tumor microenvironment. Similarly, Tenascin-C (TNC) is a related ECM protein that contains fibronectin type III repeats and has a similar expression pattern to EDA/cFn and EDB/cFn. Thus, EDA- and EDB-containing cFn isoforms, as well as TNC, are attractive biomarkers for targeting the tumor microenvironment.
Antibodies that are able to target EDA and EDB cFn isoforms have been designed, wherein such antibodies are conjugated with pro-inflammatory cytokines to illicit an immune response against cancerous cells. Antibodies, aptamers and small immunoreactive proteins (SIPs) have also been designed to target TNC in tumors. However, antibodies are mono-specific and cannot target more than one tumor-associated ligand at the same time. Additionally, antibody production is costly and time-consuming.
The streptococcal collagen-like protein-1 (Scl1) is a ubiquitous surface adhesin, which is co-expressed with a range of known virulence factors that are regulated by the multiple virulence gene regulator of Group A Streptococcus (GAS). Scl1 is a homotrimeric protein protruding from the GAS surface that contains four structurally distinct regions. The outermost N-terminal variable (V) region is adjacent to a collagen-like (CL) region that consists of a varying number of GlyXaaYaa (GXY) repeats and adopts stable collagen-like triple helices. At the C-terminus, Scl1 contains a linker (L) region which is a series of conserved, direct repeats adjoining the CL region to the cell wall/membrane (WM)-associated region. Functionally, Scl1 has been shown to bind host-cell integrin receptors and plasma components. Scl1 has also been shown to bind to cellular fibronectin, but not plasma fibronectin. Scl1 is also recognized to play a significant role in biofilm formation on abiotic surfaces.
The inventors have discovered that recombinant proteins derived from bacterial collagen-like proteins, such as Scl1, can serve as effective tools for targeting the tumor microenvironment.
Various embodiments recite a recombinant collagen-like protein, wherein the protein includes a binding domain having the capacity to bind to both extra domain A and extra domain B-containing variants of cellular fibronectin. In various embodiments, the collagen-like protein is a prokaryotic collagen-like protein, such as a streptococcal collagen-like protein from a Group A Streptococcus. In various embodiments, the Group A Streptococcus is Streptococcus pyogenes. In various embodiments, the streptococcal collagen-like protein is a variant of Scl1 or Scl2 of S. pyogenes or a combination thereof.
Various embodiments also recite a recombinant collagen-like protein further having a capacity to bind to Tenascin-C.
Various embodiments also recite a recombinant collagen-like protein, wherein the collagen-like protein further recognizes collagen-binding integrin receptors α2β1 and α11β1.
Various embodiments recite a recombinant collagen-like protein having an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
Various embodiments also recite an expression vector including a nucleotide sequence encoding the recombinant collagen-like protein.
Various embodiments also recite a host cell including an expression vector including a nucleotide sequence encoding the recombinant collagen-like protein.
Various embodiments recite pharmaceutical compositions including a recombinant collagen-like protein, wherein the protein includes a binding domain having the capacity to bind to both extra domain A and extra domain B-containing variants of cellular fibronectin.
Various embodiments also recite a method of treating cancer that involves administering a recombinant collagen-like protein, wherein the protein includes a binding domain having a capacity to bind to both extra domain A and extra domain B-containing variants of cellular fibronectin.
Various embodiments also recite a method of treating cancer that involves administering a recombinant collage-like protein having an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
Various embodiments also recite a method of preparing a collagen-like protein including a binding domain having the capacity to bind to both extra domain A and extra domain B-containing variants of cellular fibronectin that involves combining (i) two copies of an integrin recognition sequence GLPGER inserted into the collagen-like (CL) domain of a protein of SEQ ID NO: 4; and (ii) the variable (V) domain from a protein of SEQ ID NO: 3.
In order to better understand various embodiments, reference is made to the accompanying drawings, wherein:
To facilitate understanding, identical reference numerals have been used to designate elements having substantially the same or similar structure or substantially the same or similar function.
The description and drawings presented herein illustrate various principles. It will be appreciated that those skilled in the art will be able to devise various arrangements that, although not explicitly described or shown herein, embody these principles and are included within the scope of this disclosure. As used herein, the term, “or” refers to a non-exclusive or (i.e., and/or), unless otherwise indicated (e.g., “or else” or “or in the alternative”). Additionally, the various embodiments described herein are not necessarily mutually exclusive and may be combined to produce additional embodiments that incorporate the principles described herein.
This disclosure relates to defined isoforms of the extracellular matrix (ECM) protein, cellular fibronectin (cFn) and Tenascin-C (TNC) as biomarkers for the delivery of drugs to the tumor microenvironment. This disclosure further relates to recombinant proteins derived from bacterial collagen-like proteins that may serve as effective tools for targeting the tumor microenvironment
In some embodiments, the bacterial collagen-like protein is derived from a prokaryotic collagen-like protein, such as a Streptococcal collagen-like protein. In some embodiments, the Streptococcal collagen-like protein is selected from a group that includes an Scl1 or Scl2 protein, a variant of an Scl1 or Scl2 protein, or combinations thereof.
In various embodiments, the collagen-like proteins may be designed to have binding capacity for isoforms of the extracellular matrix (ECM) protein, cellular fibronectin (cFn) and Tenascin-C (TNC) that may be found within the tumor microenvironment. In various embodiments, the collagen-like proteins may be designed to have binding capacity for extra-domain A isoforms of cellular fibronectin (EDA/cFn), extra-domain B isoforms of cellular fibronectin (EDB/cFn), TNC isoforms, or combinations thereof.
In various embodiments, the collagen-like proteins may further have binding capacity for integrin receptors. In some embodiments, the collagen-like proteins may be designed to have binding capacity to α2β1 and α11β1 receptors that are upregulated on cancer cells.
In various embodiments, the collagen-like protein may include an rScl1 construct composed of a rod-shaped collagen-like domain (CL) and a globular variable (V) domain, as shown in
In various embodiments, the collagen-like protein may include a recombinant Scl1 hybrid construct characterized by SEQ ID NO: 1. In another embodiment, the collagen-like protein may include a recombinant Scl1 hybrid construct characterized by SEQ ID NO: 2.
In some embodiments, the recombinant Scl1 constructs may be derived from a parental Scl1 sequence. In certain embodiments, the recombinant Scl1 constructs are derived from parental Scl1 sequences of SEQ ID NOS: 3-5. In one embodiment, the parental Scl sequence rScl1.1 of SEQ ID NO: 3 is derived from the scl1 allele from a wild-type M1 serotype strain of group A Streptococcus (GAS). The amino acid sequence of rScl1.1 is the same as the sequence in the genome of M1, however, the rScl1 proteins lack the C-terminally located Linker and Cell Wall Attachment Domains found in the endogenous protein. In another embodiment, the parental Scl sequence rScl2.28 of SEQ ID NO: 4 is derived from the scl2 allele from a wild-type M28 serotype strain of GAS. The amino acid sequence of rScl2.28 is the same as the sequence in the genome of M28, however, the rScl2 proteins lack the C-terminal Cell Wall Attachment Domain found in the endogenous protein. In another embodiment, the parental Scl sequence rScl1.41 of SEQ ID NO: 5 is derived from the scl1 allele from a wild-type M41 serotype strain of GAS. The amino acid sequence of rScl1.41 is the same as the sequence in the genome of M41, however, the rScl1 proteins lack the C-terminally located Linker and Cell Wall Attachment Domains found in the endogenous protein.
In an exemplary embodiment, the rScl.hybrid1 construct (SEQ ID NO: 1) shown in
The hybrid Scl1 proteins substantially described in
In various embodiments, the hybrid Scl1 proteins of the invention may be produced using any suitable expression system, including 6x His-tag expression systems and strep-tag expression systems. In various embodiments, the expression vector comprises a nucleotide sequence encoding the hybrid Scl1 proteins of the invention.
In various embodiments, there is provided a host cell comprising and expressing an expression vector having a nucleotide sequence encoding the hybrid Scl1 proteins of the invention. Suitable host cells include prokaryotic cells, such as Escherichia coli, Streptococcus and Bacillus.
In various embodiments, the collagen-like proteins may be conjugated to a therapeutic agent. In some embodiments, the collagen-like protein may form conjugates with various therapeutic agents used in the treatment of cancer, such as chemotherapeutic agents. Exemplary chemotherapeutic agents include busulfan, improsulfan, piposulfan, benzodepa, carboquone, meturedepa, uredepa, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolomelamine, chlorambucil, chlornaphazine, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, aclacinomycins, actinomycin F(1), anthramycin, azaserine, bleomycin, cactinomycin, carubicin, carzinophilin, chromomycin, dactinomycin, daunorubicin, daunomycin, 6-diazo-5-oxo-1-norleucine, doxorubicin, epirubicin, mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, plicamycin, porfiromycin, puromycin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, fluororacil, tegafur, L-asparaginase, pulmozyme, aceglatone, aldophosphamide glycoside, aminolevulinic acid, amsacrine, bestrabucil, bisantrene, carboplatin, cisplatin, defofamide, demecolcine, diaziquone, elfornithine, elliptinium acetate, etoglucid, etoposide, flutamide, gallium nitrate, hydroxyurea, interferon-alpha, interferon-beta, interferon-gamma, interleukin-2, lentinan, lonidamine, mitoguazone, mitoxantrone, mopidamol, nitracrine, pentostatin, phenamet, pirarubicin, podophyllinic acid, 2-ethylhydrazide, procarbazine, razoxane, sizofiran, spirogermanium, paclitaxel, tamoxifen, teniposide, tenuazonic acid, triaziquone, 2,2′,2″-trichlorotriethylamine, urethan, vinblastine, vincristine, and vindesine.
The present disclosure further relates to pharmaceutical compositions containing the recombinant collagen-like proteins of the invention. In some embodiments, the recombinant collagen-like protein may be formulated in admixture with a pharmaceutically acceptable carrier. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous). In a preferred embodiment, the carrier is a nanoparticle. In a more preferred embodiment, the pharmaceutical composition contains a nanoparticle coated with the recombinant collagen-like protein of the invention.
Another aspect of the present disclosure provides a method of treating cancer that involves administering a therapeutically effective amount of the collagen-like proteins of the invention. In various embodiments, the collagen-like proteins of the invention may be used to treat growing tumors as well as metastatic cancers. Exemplary types of cancer include breast cancer, prostate cancer, melanoma, gastric cancers, colorectal cancer, and head and neck cancers.
rScl proteins are produced in E. coli, and expressed either intracellularly or in the periplasmic space. The extracellular fraction is recovered from culture supernatant following precipitation with ammonium sulphate. Production yields vary between rScl1 constructs, ranging from 1-20 milligrams of protein per liter of culture.
Sequences encoding the rScl.hybrid1 and rScl.hybrid2 proteins were cloned and expressed in an E. coli Strep-tag II system. The hybrid proteins were recovered from E. coli following incubation in a high-sucrose buffer or via cell lysis, and then purified by affinity chromatography, using StrepTactin Sepharose. The proteins were then subjected to dialysis to desired exchange buffers and stored at −20° C.
Recombinant rScl.hybrid1 and rScl.hybrid2 constructs were generated by several sequential manipulations. Both proteins were derived from rScl proteins rScl1.1, rScl1.41 and rScl2.28. Construct rScl.hybrid1 harbors a CL-domain that contains tandem integrin-binding motifs ((GLPGER)2) recloned from the CL-domain of rScl1.41 into the biologically inert CL-domain of rScl2.28, and the V-domain of rScl1.1 with ECM (EDA/cFn, EDB/cFn, and TNC) binding domain.
The rScl.hybrid2 protein is comprised of the CL-domain of rScl.hybrid1 and the rScl2.28 V-domain modified to contain the ECM-binding domain.
Recombinant Scl1 proteins were tested for binding to recombinant EDA and EDB (
Although the various embodiments have been described in detail with particular reference to certain aspects thereof, it should be understood that the invention is capable of other embodiments and its details are capable of modifications in various obvious respects. As is readily apparent to those skilled in the art, variations and modifications can be effected while remaining within the spirit and scope of the invention. Accordingly, the foregoing disclosure, description, and figures are for illustrative purposes only and do not in any way limit the invention, which is defined only by the claims.
This invention was made with government support under NSF Award Number: DGE1144676 awarded by the National Science Foundation. The government has certain rights in the invention.
| Number | Name | Date | Kind |
|---|---|---|---|
| 5882645 | Toth | Mar 1999 | A |
| 6953839 | Hook | Oct 2005 | B2 |
| 7238783 | Hook | Jul 2007 | B2 |
| 7544780 | Hook | Jun 2009 | B2 |
| 7700731 | Hook | Apr 2010 | B2 |
| Entry |
|---|
| An, Bo et al.; “Engineered recombinant bacterial collagen as an alternative collagen based biomaterial for tissue engineering.” Front. Chem. (2014) 2(40) 1-5. |
| McNitt, Dudley H. et al; “Adaptation of the group a Streptococcus adhesion scl1 to bind fibronectin type iii repeates within wound associated extracellular matrix, implications for cancer therapy.” Mol. Microbiol. (2019) 112(3) p. 800-819. |
| Caswell, Claton C. et al; “Scl1, the multifunctional adhesion of group a Streptococcus selectively binds cellular fibrnectin and laminin , and mediates pathgen internalization by human cells.” FEMS Microbiol. Lett. (2010) 3030(1) p. 61-68. |
| Yampolsky, Lev Y. and Stolzfus, Arlin; “The exchangeability of amino acids in proteins.” Genetics (2005) 170 p. 1459-1472. |
| Haikarainen, Teemu et al, “Magnetic properties and structural characterization of iron oxide nanoparticles fromed by Streptococcus suisdpr and four mutants.” J. Biol. Inorg. Chem. (2011) 16 p. 799-807. |
| Seo, Neungseon et al; “An engineered alpha1 integrin binding collagenous sequence.” J. Biol. Chem. (2010) 285(40) p. 31046-31054. |
| Birchler, et al., “Expression of the extra domain B of fibronectin, a marker of angiogenesis, in head and neck tumors”, Laryngoscope, 113(7), pp. 1231-1237. (2003). |
| Bo An, et al., “The influence of specific binding of collagen-silk chimeras to silk biomaterials on hMSC behavior”, Biomaterials, 34(2), pp. 402-412. (Jan. 2013). |
| Bronk, et al., “A multifunctional streptococcal collagen-mimetic protein coating prevents bacterial adhesion and promotes osteoid formation on titanium”, Acta Biomaterialia, 10(7), pp. 3354-3362. (2014). |
| Browning, et al., “Multilayer vascular grafts based on collagen-mimetic proteins”, Acta Biomaterialia, 8(3), pp. 1010-1021. (2014). |
| Caswell, et al., “Identification of the first prokaryotic collagen sequence motif that mediates binding to human collagen receptors, integrins α2β1 and α11β1”, The Journal of Biological Chemistry, 283(52), pp. 36168-36175. (2008). |
| Caswell, et al., “Scl1, the multifunctional adhesin of group A Streptococcus, selectively binds cellular fibronectin and laminin, and mediates pathogen internalization by human cells”, FEMS Microbiology Letters, 303(1), pp. 61-68. (2010). |
| Caswell, et al., “Sell-dependent internalization of group A Streptococcus via direct interactions with the α2β1 integrin enhances pathogen survival and re-emergence”, Molecular Microbiology, 64(5), pp. 1319-1331. (2007). |
| Cosgriff-Hernandez, et al., “Bioactive hydrogels based on Designer Collagens”, Acta Biomaterialia, 6(10), pp. 3969-3977. (2010). |
| Grana, et al., “Pretargeted adjuvant radioimmunotherapy with yttrium-90-biotin in malignant glioma patients: a pilot study”, British Journal of Cancer, 86(2), pp. 207-212. (2002). |
| Gutbrodt, et al., “Antibody-Based Delivery of Interleukin-2 to Neovasculature Has Potent Activity Against Acute Myeloid Leukemia”, Science Translational Medicine, 5(201), 11 pages. (2013). |
| Hemmerle, et al., “The antibody-based targeted delivery of TNF in combination with doxorubicin eradicates sarcomas in mice and confers protective immunity”, British Journal of Cancer, 109(5), pp. 1206-1213. (2013). |
| Ko, et al., “A multimodal nanoparticle-based cancer imaging probe simultaneously targeting nucleolin, integrin αvβ3 and tenascin-C proteins”, Biomaterials, 32(4), pp. 1130-1138. (2011). |
| McNitt, et al., “Surface-exposed loops and an acidic patch in the Scl1 protein of group A Streptococcus enable Scl1 binding to wound-associated fibronectin”, Journal of Biological Chemistry, 293(20), pp. 7796-7810. (2018). |
| Moschetta, et al., “Paclitaxel enhances therapeutic efficacy of the F8-IL2 immunocytokine to EDA-fibronectin-positive metastatic human melanoma xenografts”, Cancer Research, 72(7), pp. 1814-1824. (2012). |
| Oliver-Kozup, et al., “The group A streptococcal collagen-like protein-1, Scl1, mediates biofilm formation by targeting the extra domain A-containing variant of cellular fibronectin expressed in wounded tissue”, Molecular Microbiology, 87(3), pp. 672-689. (2013). |
| Peng, et al., “A Streptococcus pyogenes derived collagen-like protein as a non-cytotoxic and non-immunogenic cross-linkable biomaterial”. Biomaterials 31, pp. 2755-2761. (2010). |
| Peng, et al., “Towards scalable production of a collagen-like protein from Streptococcus pyogenes for biomedical applications”, Microbial Cell Factories, 11:146, 8 pages. (2012). |
| Reardon, et al., “A pilot study: 131I-Antitenascin monoclonal antibody 81c6 to deliver a 44-Gy resection cavity boost”, Neuro-Oncology, 10(2), pp. 182-189. (2008). |
| Spitaleri, et al., “Phase I/II study of the tumour-targeting human monoclonal antibody-cytokine fusion protein L19-TNF in patients with advanced solid tumours”, Journal of Cancer Research and Clinical Oncology, 139(3), pp. 447-455. (2013). |
| Villa, et al., “A high-affinity human monoclonal antibody specific to the alternatively spliced EDA domain of fibronectin efficiently targets tumor neo-vasculature in vivo”, International Journal of Cancer, 122(11), pp. 2405-2413. (2008). |
| Number | Date | Country | |
|---|---|---|---|
| 20200199200 A1 | Jun 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62783843 | Dec 2018 | US |
