Targeting tumor microenvironments with recombinant prokaryotic collagen-like proteins

Abstract

A recombinant collagen-like protein comprising a binding domain having binding capacity for both extra domain A and extra domain B-containing variants of cellular fibronectin. Cancer may be treated BY administering the recombinant collagen-like protein to a patient.

Claims

1. A recombinant collagen-like protein comprising a binding domain having binding capacity for both extra domain A and extra domain B-containing variants of cellular fibronectin, wherein the protein comprises: a recombinant collagen-like (CL)-domain, and a variable (V)-domain comprising an ECM-binding domain from a streptococcal Scl1.1 protein; wherein the recombinant CL-domain is a CL-domain from a streptococcal Scl2.28 protein; wherein the CL-domain from the streptococcal Scl2.28 protein is modified to include an integrin recognition sequence from the CL-domain of the streptococcal Scl1.41 protein.

2. The recombinant collagen-like protein of claim 1, wherein: the protein is a streptococcal collagen-like protein from a Group A Streptococcus, and the Group A Streptococcus is Streptococcus pyogenes.

3. The recombinant collagen-like protein of claim 1, wherein the protein further comprises binding capacity for Tenascin-C.

4. The recombinant collagen-like protein of claim 1, wherein the protein recognizes collagen-binding integrin receptors α.sub.2β.sub.1 and α.sub.11β.sub.1.

5. The recombinant collagen-like protein of claim 1, wherein the protein comprises two copies of an integrin recognition sequence GLPGER.

6. The recombinant collagen-like protein of claim 5, wherein the protein comprises the variable domain of the rScl1.1 sequence of SEQ ID NO: 3.

7. A recombinant collagen-like protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2.

8. A pharmaceutical composition comprising the recombinant collagen-like protein of claim 7; and a pharmaceutically acceptable carrier.

9. The composition of claim 8, wherein the carrier is a nanoparticle.

10. The composition of claim 9, wherein the nanoparticle is coated with the collagen-like protein.

11. A recombinant collagen-like protein comprising a binding domain having binding capacity for both extra domain A and extra domain B-containing variants of cellular fibronectin, wherein the protein comprises: a recombinant collagen-like (CL)-domain, and a variable (V)-domain; wherein: the recombinant CL-domain is a CL-domain from a streptococcal Scl2.28 protein, and the CL-domain from the streptococcal Scl2.28 protein is modified to include two integrin recognition sequences from the CL-domain of the streptococcal Scl1.41 protein.

12. The recombinant collagen-like protein of claim 11, wherein the two integrin recognition sequences each comprise SEQ ID NO: 6.

13. The recombinant collagen-like protein of claim 11, wherein: the variable (V)-domain comprises an ECM-binding domain from a streptococcal Scl1.1 protein; or the variable (V)-domain is a variable domain from a streptococcal Scl1.1 protein.

14. A method of targeting a cancer in a patient in need thereof, comprising administering a recombinant collagen-like protein of claim 7 to the patient.

15. A method of treating cancer in a patient in need thereof, comprising administering a recombinant collagen-like protein of claim 1 to the patient, wherein the collagen-like protein is conjugated with a therapeutic agent useful for the treatment of cancer.

16. A method of targeting a cancer in a patient in need thereof, comprising administering a recombinant collagen-like protein of claim 7 to the patient, wherein the cancer is selected from a group consisting of a tumor or a metastatic cancer.

17. A method of treating cancer in a patient in need thereof, comprising administering a recombinant collagen-like protein of claim 7 to the patient, wherein the cancer is selected from a group consisting of breast cancer, prostate cancer, melanoma, gastric cancer, colorectal cancer, and head and neck cancer, wherein the collagen-like protein is conjugated with a therapeutic agent useful for the treatment of cancer.

18. A method of preparing a collagen-like protein comprising a binding domain having the capacity to bind to both extra domain A and extra domain B-containing variants of cellular fibronectin comprising the steps of combining: (i) two copies of an integrin recognition sequence of SEQ ID NO: 6 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.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In order to better understand various embodiments, reference is made to the accompanying drawings, wherein:

(2) FIG. 1 illustrates a model of rScl hybrid constructs as targeting agents to the tumor microenvironment.

(3) FIGS. 2A and 2B illustrate the binding of rScl hybrid constructs to recombinant rEDA, rEDB and Tenascin-C.

(4) FIG. 3 illustrates the structure of rScl hybrid constructs 1 and 2.

(5) 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.

DETAILED DESCRIPTION

(6) 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.

(7) 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

(8) 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.

(9) 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.

(10) 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 α.sub.2β.sub.1 and α.sub.11β.sub.1 receptors that are upregulated on cancer cells.

(11) 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 FIG. 1. In various embodiments, the rScl1 construct may be designed to bind to at least one isoform of cellular fibronectin, selected from the group consisting of EDA/cFn, EDB/cFn and TNC, and promote human cell attachment through collagen receptors, integrins α.sub.2β.sub.1 and α.sub.11β.sub.1.

(12) 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.

(13) 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.

(14) In an exemplary embodiment, the rScl.hybrid1 construct (SEQ ID NO: 1) shown in FIG. 1 may be designed to have two desired biological functions: (i) 2 copies of the integrin recognition sequence ((GLPGER (SEQ ID NO: 6)) placed into the inert CL domain of rScl2.28 (SEQ ID NO: 4); and (ii) the V domain from the TNC and EDA/EDB-binding protein rScl1.1 (SEQ ID NO: 3). The (GLPGER).sub.2-CL domain was found to promote efficient internalization of group A Streptococcus expressing the rScl1.1 protein, compared to group A Streptococcus that expresses inert rScl2.28. In this embodiment, the V domain of rScl1.1, specifically the loop-region of Scl1.1-V domain, binds to EDA/cFn and to rEDB and TNC. Thus, in this embodiment, the rScl.hybrid1 construct possess both the capacity to bind to TNC, as well as EDA/cFn and EDB/cFn isoforms, highly expressed within and around tumor/cancerous tissues, as well as the capacity to promote internalization via integrin receptors α.sub.2β.sub.1 and α.sub.11β.sub.1.

(15) The hybrid Scl1 proteins substantially described in FIG. 1 differ from current TNC and EDA/EDB targeting technologies due to their capacity to target a combination of biomarkers that include TNC, and both of EDA/cFn and EDB/cFn, as well as recognize collagen-binding integrin receptors α.sub.2β.sub.1 and α.sub.11β.sub.1, enabling both tissue localization and internalization of conjugated drugs, cytokines, and siRNAs. In contrast, antibody therapies are only able to target 1 ligand per antibody.

(16) 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.

(17) 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.

(18) 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.

(19) 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.

(20) 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.

EXAMPLE 1

Production Method of rScl1 Hybrid Proteins

(21) 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.

(22) 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.

EXAMPLE 2

Construction of Recombinant rScl Hybrid Proteins 1 and 2

(23) 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).sub.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.

(24) 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.

EXAMPLE 3

Binding of rScl Proteins to rEDA, rEDB and TNC

(25) Recombinant Scl1 proteins were tested for binding to recombinant EDA and EDB (FIG. 2A), as well as Tenascin-C (FIG. 2B). rScl proteins were immobilized onto Strep-Tactin-coated microplate wells and incubated with rEDA/rEDB or TNC. Primary anti-rEDB or TNC antibodies and HRP-conjugated secondary antibodies were used for ligand detection. The shown graphic bars indicate mean OD.sub.415nm normalized against BSA controls. FIG. 2A shows that both rScl1.1 protein derived from a natural sequence and the engineered rScl.hybrid1 recognize recombinant EDB to a comparable level as recombinant EDA. FIG. 2B shows ELISA binding data between rScl1 proteins and TNC extracted from glioblastoma cancer cells.

(26) 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.

(27) TABLE-US-00001 Amino acid sequence of rScl.hybrid1 SEQ ID NO: 1 EVSSTTMTSSQRESKIKEIEESLKKYPEVSNEKFWERKWYGTYFKEEDFQ KELKDFTEKRLKEILDLIGPTGLAGEKGEAGPQGEKGLPGLTGLPGLPGE RGPRGPKGDRGETGAQGPAGEKGEAGPQGEKGLPGLTGLPGLPGERGPRG PKGDRGETGAQGPAGKAGEAGAKGETGPAGPQGPRGEQGPQGLPGKDGEA GAQGPAGPMGPAGERGEKGEPGTQGAKGDRGETGPVGPRGERGEAGPAGK DGERGPVGPAGKDGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDG LPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQPGKPAPKTPEVPQKPD TAPWSHPQFEK Amino acid sequence of rScl.hybrid2 SEQ ID NO: 2 DEQEEKAKVRTELIQELAQKYPEVSNEKFWERKWYGTYFKFLTYLQEREQ AENSWRKRLLKGIQDHALDGLAGEKGEAGPQGEKGLPGLTGLPGLPGERG PRGPKGDRGETGAQGPAGEKGEAGPQGEKGLPGLTGLPGLPGERGPRGPK GDRGETGAQGPAGKAGEAGAKGETGPAGPQGPRGEQGPQGLPGKDGEAGA QGPAGPMGPAGERGEKGEPGTQGAKGDRGETGPVGPRGERGEAGPAGKDG ERGPVGPAGKDGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDGLP GKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQPGKPAPKTPEVPQKPDTA PWSHPQFEK Amino acid sequence of rScl1.1 SEQ ID NO: 3 EVSSTTMTSSQRESKIKFIEESLKKYPEVSNEKFWERKWYGTYFKEEDFQ KFLKDFTEKRLKEILDLIGKSGIKGDRGETGPAGPAGPQGKTGERGAQGP KGDRGEQGIQGKAGEKGERGEKGDKGETGERGEKGEAGIQGPQGEAGKDG APGKDGAPGEKGEKGDRGETGAQGPVGPQGEKGETGAQGPAGPQGEAGKP GEQGPAGPQGEAGQPGEKAPEKSPEGEAGQPGEKAPEKSKEVTPAAEKPW SHPQFEK Amino acid sequence of rScl2.28 SEQ ID NO: 4 DEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMTKLLTYLQ EREQAENSWRKRLLKGIQDHALDGQDGRNGERGEQGPTGPTGPAGPRGLQ GLQGLQGERGEQGPTGPAGPRGLQGERGEQGPTGLAGKAGEAGAKGETGP AGPQGPRGEQGPQGLPGKDGEAGAQGPAGPMGPAGERGEKGEPGTQGAKG DRGETGPVGPRGERGEAGPAGKDGERGPVGPAGKDGQNGQDGLPGKDGKD GQNGKDGLPGKDGKDGQNGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGK DGKDGQPGKPAPKTPEVPQKPDTAPWSHPQFEK Amino acid sequence of rScl1.41 SEQ ID NO: 5 EDSETATARTKLLEKLTELRSQSQDRVPQTSDITQAYTLWGTSYDSVELY KYLQQIEEYLQKQKYHEEQWKKEITDGLKSGALRGEKGEAGPQGEKGLPG LTGLPGLPGERGPRGPKGDRGETGAQGPVGPQGEKGEAGTPGKDGLRGPQ GDPGAPGKDGAPGEKGDRGETGAQGPVGPQGEKGEAGTPGKDGAPGEKGE KGDRGETGATGAQGPQGEAGKDGAQGPVGPQGEKGETGAQGPAGPQGEKG ETGAQGPAGPQGEAGQPGEKAPEKSPEVTPTPEMPEQPGEQAPEKSKFVT PAPEKPWSHPQFEK Integrin recognition sequence SEQ ID NO: 6 GLPGER